Uses and formulations of nanoparticles containing camptothecin (CPT)

Abstract

Disclosed herein are compositions, formulations and methods comprising nanoparticles comprising mucic acid polymers conjugated with camptothecin.Another aspect of the disclosure relates to a kit for administering to a subject the DAN-222 formulation described herein, optionally in combination with another therapeutic agent (e.g., PARP inhibitors such as niraparib).In one embodiment, the kit comprises a composition of DAN-222 formulated in a pharmaceutical carrier suitable for administration, for example, by injection or infusion.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e)(1) of U.S. Provisional Patent Application No. 63 / 309,810, filed February 14, 2022, the entire disclosure of which is incorporated by reference herein, including all drawings and text.

[0002] FIELD OF THEINVENTION The present invention relates generally to the formulation and use of nanoparticles comprising camptothecin conjugated to a mucic acid polymer. [Background technology]

[0003] background Breast cancer is the second most common cancer among women in the United States. Despite advances in screening and treatment, breast cancer remains the second leading cause of cancer death among women. Furthermore, the efficacy of various treatments varies widely among breast cancer subtypes at various stages of progression. This creates a complex situation for pathologists and oncologists to diagnose, treat and predict recurrence in breast cancer patients.

[0004] Genomic DNA is frequently challenged by both endogenous and exogenous DNA damaging agents that can result in various types of DNA lesions. These lesions can block DNA replication and transcription and, if not repaired or repaired incorrectly, can result in mutations that threaten the survival of individual cells or even the survival of the entire organism. The inability to accurately repair complex DNA lesions to relieve DNA replication stress not only leads to genomic instability, contributing to the pathogenesis of cancer, but also makes cancer cells more vulnerable to DNA damaging therapeutic agents.

[0005] Homologous recombination (HR) repair, one of several DNA repair pathways, includes a series of interrelated pathways that function to repair DNA double-strand breaks and interstrand crosslinks. Defects in HR repair are observed in various cancers. Breast cancer genes type 1 (BRCA1) and BRCA type 2 (BRCA2) have long been known to code for proteins that play an important role in HR repair, and patients with mutations in one or both of these genes are at higher risk of developing breast, ovarian, prostate, melanoma and pancreatic cancer. Tumor cells with defective HR repair show increased sensitivity to chemotherapeutic agents, suggesting that if HR repair can be therapeutically inactivated, tumor cells that are skilled in HR can be sensitized to chemotherapeutic agents.

[0006] Topoisomerase inhibitors damage DNA by binding to topoisomerase I and preventing the unwinding of DNA required for replication. This stalls replication forks that can be repaired by poly(ADP-ribose) polymerase (PARP). PARP inhibitors have been shown to block DNA repair activity by the PARP enzyme and induce apoptosis in tumor cells. In addition to direct tumor killing, inhibition of PARP also increases the cytotoxicity of chemotherapeutic drugs and enhances the cytotoxicity of DNA damaging agents such as the topoisomerase inhibitor camptothecin (CPT) and its derivatives.

[0007] Unfortunately, although CPT has known antitumor activity, including across a broad range of human cancers, it has poor aqueous solubility, low plasma stability and dose-limiting toxicity. Summary of the Invention [Means for solving the problem]

[0008] Abstract The present invention provides methods, compositions and uses of camptothecin (CPT) conjugated nanoparticles. In particular, the present invention provides a compound of formula (I): [ka] (In the formula, n is the number of ethylene glycol repeating units in the range of 20 to 200 units, and m is the number of MAP-Gly-CPT repeating units in the range of 5 to 200.) The present invention is directed to compositions, formulations and methods of use, including a formulation of nanoparticles comprising 20(S)-camptothecin covalently attached to a mucic acid-based polymeric carrier via a glycyl linking group (MAP-Gly-CPT), comprising:

[0009] In a preferred embodiment, the MAP-Gly-CPT compound is DAN-222, which has the structure of Formula I, and the average number of repeating ethylene glycol repeat units (n) is a number ranging from about 75 to 85. In a preferred embodiment, in the formulations and compositions of the invention, the average number of n is about 79. In certain embodiments, the average number of repeat units (m) of MAP-Gly-CPT is 16±4.

[0010] Camptothecin (CPT), the active ingredient in DAN-222, is a natural pentacyclic quinolone alkaloid isolated from the bark of Camptotheca acuminata. CPT is a topoisomerase I inhibitor that acts by forming a ternary complex with topoisomerase I and DNA during replication and transcription in tumor cells, blocking DNA religation and creating torsional tension downstream of the transcription or replication fork, causing DNA strand cleavage.

[0011] Unfortunately, although CPT has known antitumor activity, including across a broad range of human cancers, it has poor aqueous solubility, low plasma stability and dose-limiting toxicity.

[0012] However, as discovered by the inventors, DAN-222 accumulates in tumors due to an enhanced vascular permeability and retention (EPR) effect that results in greater and longer-lasting inhibition of topoisomerase I, making it more potent than other topoisomerase I inhibitors, including free CPT.

[0013] In certain aspects, the present invention provides a mucic acid polymer (MAP) camptothecin (CPT) conjugate compound of formula (I): [ka] (In the formula, n is a number in the range of 20 to 200, m is a number ranging from 5 to 200. The present invention provides an aqueous formulation comprising nanoparticles made from

[0014] In certain embodiments, n is a number ranging from about 75 to 85. In certain formulations, m is a number ranging from about 12 to 20.

[0015] In certain formulations of the invention, the compound of formula (I) has a molecular weight of about 75,000 Da. In a preferred embodiment, the nanoparticles of the formulation contain an average of two chains containing the compound of formula (I) and have an average molecular weight of about 150,000 Da. The nanoparticles of the formulation can have an average particle size of about 20 nm to 80 nm. Preferably, the nanoparticles have an average particle size of about 30 to 40 nm. In certain embodiments, the formulation has a nanoparticle concentration of about 17 to 23 mg / mL.

[0016] The formulations of the present invention include those having a total concentration of CPT between 1.7 and 3.6 mg / mL. In a preferred embodiment, the formulation has a total concentration of CPT of about 2.6 mg / mL. In a particular embodiment, the formulation contains a compound of formula (I) at a concentration of about 20 mg / mL.

[0017] In certain embodiments, the formulation has a pH between pH 4 and pH 5. In certain embodiments, the formulation has a pH between pH 4 and pH 4.6. In a preferred embodiment, the formulation has a pH of about pH 4.3.

[0018] The formulation of the present invention may further comprise at least one buffer, for example, selected from sodium succinate, sodium citrate, sodium acetate, phosphoric acid, histidine-HCL and sodium phosphate.In a preferred embodiment, the buffer is sodium acetate buffer.The formulation may further comprise at least one tonicity modifier, for example, selected from KCl, NaCl, proline, arginine-HCl, sucrose and glycine.Preferably, the at least one tonicity modifier is NaCl.

[0019] In a preferred embodiment, the formulation contains about 20 mg / mL of a compound of formula (I), about 20 mM sodium acetate, about 200 mM NaCl, and has a pH of 4.3±0.3.

[0020] The present invention also provides a method for producing a formulation comprising a compound of formula (I). In certain embodiments, the method for producing a formulation of a compound of formula (I) comprises the steps of (i) derivatizing camptothecin (CPT) to obtain Gly-CPT as a trifluoroacetic acid (TFA) salt, (ii) synthesizing a parent mucic acid polymer (MAP), (iii) covalently binding Gly-CPT to the parent MAP to obtain a solid amorphous MAP-Gly-CPT, and (iv) using the amorphous MAP-Gly-CPT to prepare an aqueous formulation, thereby producing an aqueous formulation of nanoparticles comprising a compound of formula (I).

[0021] In certain aspects, step (i) of the method includes dissolving CPT, N-(tert-butoxycarbonyl)-Gly-OH (Boc-Gly) and 4-dimethylaminopyridine (DMAP) in methylene chloride (DCM) to form a reaction mixture, and adding diisopropylcarbodiimide (DIC) to the reaction mixture, thereby generating a solution containing the Boc-Gly-CPT intermediate. In certain embodiments, the reaction is carried out at 25-30° C. for about 6 hours.

[0022] In certain methods of the invention, step (i) further comprises removing between 90% and 99% of the DCM from the solution containing the Boc-Gly-CPT intermediate. In a preferred method, the DCM is removed by vacuum distillation. Step (i) may further comprise precipitating the Boc-Gly-CPT intermediate using methanol. Such methods may also comprise washing the precipitated Boc-Gly-CPT intermediate using isopropyl alcohol (IPA).

[0023] In certain aspects, step (i) further comprises treating the Boc-Gly-CPT intermediate with TFA in DCM to obtain a TFA salt of Gly-CPT, hi certain embodiments, the treatment with TFA is carried out for about 2 hours at 25-30°C.

[0024] In a preferred method of producing the formulation, step (ii) comprises adding to a reactor (succinimidyl propionate) 2 -PEG 3500 (di-SPA-PEG 3500 ) and mucic acid monomer (MAM) neutral species of formula (II): [ka] The reaction includes the steps of: charging MAP to a reactor, adding dimethyl sulfoxide (DMSO) to the reactor, initiating the reaction by adding TFA to the reactor, and adding DIPEA to the reaction, thereby obtaining the parent MAP. In one particular embodiment, the reaction is carried out at 21-23° C. for about 3 hours.

[0025] In certain embodiments, the comonomer ratio of DIPEA:MAM used in step (ii) is at least about 2.5. Preferably, the comonomer ratio is at least 2.6.

[0026] In certain embodiments, step (ii) further comprises precipitating the parent MAP from DMSO by adding at least one anti-solvent. An exemplary anti-solvent is isopropyl alcohol (IPA). In a preferred embodiment, the precipitation comprises a filtration step under an inert atmosphere, such as, for example, nitrogen or argon.

[0027] In certain methods of the invention, step (iii) comprises adding parent MAP and DMSO to a reaction vessel, adding Gly-CPT (TFA salt) and (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) to the reaction vessel, and adding DIPEA to the reaction vessel, thereby producing a solution comprising MAP-Gly-CPT. In certain embodiments, the reaction is carried out at 19-25° C. for 16-22 hours.

[0028] Step (iii) may further comprise purifying the amorphous MAP-Gly-CPT from the solution using tangential flow filtration (TFF). Preferably, the solution is diluted with water at pH 3 prior to tangential flow filtration, which uses a polyethersulfone (PESU) TFF filter. In an additional embodiment, a membrane made from regenerated cellulose is used for the TFF.

[0029] In certain methods of the invention, step (iv) comprises adding water having a pH of approximately pH 4 to amorphous MAP-Gly-CPT to produce a nanoparticle (NP) solution, and adding a formulation buffer to the NP solution.

[0030] In certain embodiments, the formulation buffer comprises sodium acetate. Preferably, the formulation buffer further comprises NaCl and has a pH of approximately pH 4.16.

[0031] In certain methods, the NP solution is filtered after the step of adding water having a pH of about pH 4. In certain embodiments, after the step of adding a formulation buffer to the NP solution, the NP solution is concentrated by ultrafiltration.

[0032] The present disclosure also provides a method of treating cancer in a subject using the DAN-222 formulations described herein. An exemplary method is administering to a subject with cancer a compound of formula (I): [ka] wherein n is a number in the range of 20 to 200 and m is a number in the range of 5 to 200. In a preferred method, n is a number in the range of about 75 to 85 and m is a number in the range of about 12 to 20, and the composition has a pH between pH 4 and pH 4.6.

[0033] Preferably, the composition is supplied in an injectable solution.

[0034] In a preferred method, the composition comprises between 2 mg / mL and 3 mg / mL of camptothecin (CPT). In certain embodiments, the dose of the composition is 2 mg / mL to 3 mg / mL of camptothecin (CPT). 2 ~16mg / m 2 An exemplary dose of the composition is about 2 mg / m 2 , about 4mg / m 2 , about 6mg / m 2 , about 8mg / m 2 , about 10mg / m 2 , about 12mg / m 2 , about 14mg / m 2 or about 20 mg / m 2 The compound includes a compound of formula (I).

[0035] In certain methods, multiple doses are provided to a subject during a treatment cycle. Preferably, the doses are provided as weekly doses, i.e., one dose every seven days.

[0036] The exemplary method of treatment further comprises providing at least one dose of poly(ADP-ribose) polymerase (PARP) inhibitor to the subject. The PARP inhibitor can comprise, for example, one of olaparib, rucaparib, niraparib and talazoparib. Preferably, the PARP inhibitor is niraparib. Preferably, the subject receives a dose of niraparib every day during the treatment cycle. In the exemplary method, each dose of niraparib is a dose of 100 mg.

[0037] In exemplary methods of the invention, the cancer is one or more of: breast, ovarian, brain, lung, testis, head, neck, esophagus, lymphoma, central nervous system, peripheral nervous system, bladder, stomach, pancreas, liver, oral mucosa, colorectal, anus, kidney, bladder, urothelium, prostate, endometrium, uterus, fallopian tube, mesothelioma, melanoma, myeloma, leukemia, and Kaposi's sarcoma.

[0038] In a preferred method, the cancer is breast cancer. Breast cancer can include, for example, breast cancer that is homologous recombination repair deficient (HRD)-positive breast cancer or HRD-negative breast cancer. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the formation of engineered nanoparticles of DAN-222.

[0040] [Diagram 2] Figure 2 presents the calculated median tumor volume (MTV) as a function of time in HRD-positive (ER-positive, PR-positive, HER2-positive) ductal breast carcinoma (BT-474 model) by various treatment groups: Vehicle control (circles, top line), Camptothecin (3 mg / kg) iv once weekly for 3 weeks (diamonds, then bottom line), Irinotecan (100 mg / kg) iv once weekly for 3 weeks (triangles, then bottom line), DAN-222 (3 mg / kg) iv once weekly for 3 weeks (squares, bottom line).

[0041] [Diagram 3] Figure 3 presents a Kaplan-Meier plot of time to endpoint (TTE) for animals in each of the treatment groups described in Figure 2. The endpoint is a tumor volume of 2,000 mm3. The treatment groups are: Vehicle control (circles, first line descending from 100%) Camptothecin (3 mg / kg) iv once weekly for 3 weeks (diamonds, second line descending from 100%) Irinotecan (100 mg / kg) iv once weekly for 3 weeks (triangles, third line descending from 100%) DAN-222 (3 mg / kg) iv once weekly for 3 weeks (squares, line not descending from 100%).

[0042] [Figure 4] FIG. 4 shows median tumor volume measurements over time for various treatment groups in an HRD-positive, triple-negative (ER-negative, PR-negative, HER2-negative) breast adenocarcinoma (MDA-MB-436) model. Treatment groups, in order from top to bottom of the location of the right-most data point, are: Vehicle (filled circle) 25 mg / kg oral niraparib once daily for 28 days (light circle) 0.3 mg / kg intravenous DAN-222 once weekly for 4 weeks (light square) 1 mg / kg intravenous DAN-222 once weekly for 4 weeks (dark square) 0.3 mg / kg intravenous DAN-222 once weekly for 4 weeks and 25 mg / kg oral niraparib once daily for 28 days (light triangle) 1 mg / kg intravenous DAN-222 once weekly for 4 weeks and 25 mg / kg oral niraparib once daily for 28 days (light triangle) 3 mg / kg intravenous DAN-222 once weekly for 4 weeks (dark square) intravenous DAN-222 at 3 mg / kg, once weekly for 4 weeks, and oral niraparib at 25 mg / kg, once daily for 28 days (dark triangles).

[0043] [Diagram 5]FIG. 5 shows median tumor volume measurements over time following DAN-222 administration in HRD-positive (ER-negative, PR-negative, HER2-negative [triple negative]) breast adenocarcinoma (MDA-MB-436 model). Treatment groups, in order from top to bottom of the location of the right-most data point, are: Vehicle (filled circles), 40 mg / kg oral niraparib, once daily for 28 days (light circles), 0.3 mg / kg intravenous DAN-222, once weekly for 4 weeks (light squares), 1 mg / kg intravenous DAN-222, once weekly for 4 weeks (dark squares), 0.3 mg / kg intravenous DAN-222, once weekly for 4 weeks, and 40 mg / kg oral niraparib, once daily for 28 days (light triangles), 3 mg / kg intravenous DAN-222, once weekly for 4 weeks (dark squares), 1 mg / kg intravenous DAN-222, once weekly for 4 weeks, and 40 mg / kg oral niraparib, once daily for 28 days (dark triangles).

[0044] [Figure 6] Figure 6 shows median tumor volume over time in the OVCAR3 human ovarian cancer xenograft model with DAN-222 and high dose niraparib. Treatment groups, in order from top to bottom of the location of the right-most data point, are: Vehicle (solid circles), 50 mg / kg oral niraparib, once daily for 28 days (light circles), 1 mg / kg intravenous DAN-222, once weekly for 4 weeks (squares), 1 mg / kg intravenous DAN-222, once weekly for 4 weeks, and 50 mg / kg oral niraparib, once daily for 28 days (triangles), and 3 mg / kg intravenous DAN-222, once weekly for 4 weeks (squares).

[0045] [Figure 7]FIG. 7 shows the concentration of CPT in the plasma of male rats after intravenous dosing of DAN-222 on days 1 and 22. FIG. 7A (left side of the figure) shows the plasma levels of free CPT after intravenous dosing of DAN-222 at three different doses: 1.1 mg / kg (triangles), 3.2 mg / kg (squares) and 9.6 mg / kg (circles). The upper panel shows the free CPT levels after dosing on day 1. The lower panel shows the free CPT levels after repeated dosing on day 22. FIG. 7B (right side of the figure) shows the plasma levels of total CPT after intravenous dosing of DAN-222 at three different doses: 1.1 mg / kg (triangles), 3.2 mg / kg (squares) and 9.6 mg / kg (circles). The upper panel shows the total CPT levels after dosing on day 1. The lower panel shows the total CPT levels after repeated dosing on day 22.

[0046] [Figure 8] Figure 8 shows the stability of DAN-222 in rat, dog and human plasma at physiological pH over time. The three curves show the results for dogs, rats and humans, respectively, in order of the position of the right-most data point.

[0047] [Figure 9A] Figure 9A shows the biodistribution of DAN-222 in various tissues of JIMT-1 xenograft mice. The darker bars show the results for total CPT, and the lighter bars show the results for free CPT.

[0048] [Figure 9B] FIG. 9B shows the biodistribution of DAN-222 in JIMT-1 xenograft mice over time. The leftmost set of six bars shows the amount of total CPT in plasma. The middle set of six bars shows the amount of total CPT in skeletal muscle. The rightmost set of six bars shows the amount of total CPT in tumor tissue.

[0049] [Figure 10] FIG. 10 provides a summary of the biodistribution studies of DAN-222 that were performed.

[0050] [Figure 11] FIG. 11 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over 14 days at 2-8° C.

[0051] [Figure 12] FIG. 12 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over 14 days at 20-25° C.

[0052] [Figure 13] FIG. 13 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over 14 days at 40° C.

[0053] [Figure 14] FIG. 14 presents stability data for DAN-222 formulations as % free CPT and mean particle size under stirring over 24 hours.

[0054] [Figure 15] FIG. 15 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over six freeze / thaw (F / T) cycles.

[0055] [Figure 16] FIG. 16 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 24-hour 0.02% H2O2 stress.

[0056] [Figure 17] FIG. 17 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 24-hour 0.05% H2O2 stress.

[0057] [Figure 18A]FIG. 18A presents stability data for DAN-222 formulations in acetate buffer as trends of % free CPT and mean particle size over one month at 2-8° C.

[0058] [Figure 18B] FIG. 18B presents stability data for DAN-222 formulations in histidine buffer as trends of % free CPT and mean particle size over one month at 2-8° C.

[0059] [Figure 19] FIG. 19 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 4 month period at 2-8° C.

[0060] [Figure 20A] FIG. 20A presents stability data for DAN-222 formulations in acetate buffer as trends of % free CPT and mean particle size over one month at 20-25° C.

[0061] [Figure 20B] FIG. 20B presents stability data for DAN-222 formulations in histidine buffer as trends of % free CPT and mean particle size over one month at 20-25° C.

[0062] [Figure 21A] FIG. 21A presents stability data for DAN-222 formulations in acetate buffer as trends of % free CPT and mean particle size over 7 days at 40° C.

[0063] [Figure 21B] FIG. 21B presents stability data for DAN-222 formulations in histidine buffer as trends of % free CPT and mean particle size over 7 days at 40° C.

[0064] [Figure 22]FIG. 22 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 4 month period at −20° C.

[0065] [Diagram 23] FIG. 23 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 4 month period at −80° C.

[0066] [Figure 24A] FIG. 24A presents stability data for DAN-222 formulations in acetate buffer as % free CPT and mean particle size under stirring over 24 hours.

[0067] [Figure 24B] FIG. 24B presents stability data for DAN-222 formulations in histidine buffer as % free CPT and mean particle size under stirring over 24 hours.

[0068] [Figure 25A] FIG. 25A presents stability data for DAN-222 formulations in acetate buffer as trends of % free CPT and mean particle size over six freeze / thaw (f / T) cycles.

[0069] [Figure 25B] FIG. 25B presents stability data for DAN-222 formulations in histidine buffer as trends of % free CPT and mean particle size over six freeze / thaw (F / T) cycles.

[0070] [Figure 26A] FIG. 26A presents stability data for DAN-222 formulations in acetate buffer as trends of % free CPT and mean particle size under 0.05% H2O2 stress over 24 hours.

[0071] [Figure 26B]FIG. 26B presents stability data for DAN-222 formulations in histidine buffer as trends of % free CPT and mean particle size under 0.05% H2O2 stress over 24 hours.

[0072] [Figure 27] FIG. 27 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over one month at 2-8° C.

[0073] [Figure 28] FIG. 28 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 3 month period at 2-8° C.

[0074] [Figure 29] FIG. 29 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over one month at 20-25° C.

[0075] [Diagram 30] FIG. 30 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over 7 days at 40° C.

[0076] [Diagram 31] FIG. 31 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 3 month period at −20° C.

[0077] [Diagram 32] FIG. 32 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over a 3 month period at −80° C.

[0078] [Diagram 33]FIG. 33 presents stability data for DAN-222 formulations as % free CPT and mean particle size under stirring at 450 rpm for 24 hours.

[0079] [Diagram 34] FIG. 34 presents stability data for DAN-222 formulations as trends in % free CPT and mean particle size over six freeze / thaw (F / T) cycles.

[0080] [Diagram 35] FIG. 35 provides a schematic diagram of a method for producing a DAN-222 formulation of the present invention.

[0081] [Diagram 36] FIG. 36 presents a scheme for generating Gly-CPT.

[0082] [Figure 37A] FIG. 37A presents the first portion of the workflow used in generating a DAN-222 formulation of the present invention.

[0083] [Figure 37B] FIG. 37B presents the second portion of the workflow used in producing a DAN-222 formulation of the present invention.

[0084] [Figure 38] FIG. 38 shows a synthetic scheme for preparing mucic acid polymers (MAP).

[0085] [Figure 39] FIG. 39 presents the workflow used in generating a DAN-222 formulation of the present invention.

[0086] [Diagram 40] FIG. 40 presents a synthetic scheme for preparing amorphous MAP-Gly-CPT.

[0087] [Figure 41A] FIG. 41A presents the first part of a summary flow chart of the synthesis of amorphous MAP-Gly-CPT, including control points.

[0088] [Figure 41B] FIG. 41B presents the second part of a summary flow chart of the amorphous MAP-Gly-CPT synthesis method, including control points.

[0089] [Diagram 42] FIG. 42 presents a scheme for preparing DAN-222 solutions / formulations.

[0090] [Figure 43A] FIG. 43A presents the first part of a summary flowchart of the DAN-222 FBDS process, including control points.

[0091] [Figure 43B] FIG. 43B presents the second part of a summary flowchart of the DAN-222 FBDS process, including control points.

[0092] [Diagram 44] Figure 44 presents results showing the effect of the comonomer ratio of MAM:diSPA-PEG on the molecular weight (MW) of MAP. Circles indicate a ratio of 1.01:1, squares indicate a ratio of 1.03:1, and triangles indicate a ratio of 1.10:1.

[0093] [Diagram 45] FIG. 45 shows the chemical structures of the di-TFA salt of mucic acid monomer (MAM) and the neutral species of MAM.

[0094] [Figure 46]FIG. 46 presents the results of a first series of experiments (screening 1) conducted to investigate the combined effect of various comonomer ratios and equivalents of TFA and DIPEA on the rate of polymerization of mucic acid monomers (MAM) to mucic acid polymers (MAP). Results are shown as molecular weight of polymer over time. Reaction conditions are shown in the table below the graph. Circles indicate results obtained using reaction conditions shown in column 1-A of the table. Squares indicate results obtained using reaction conditions shown in column 1-B of the table. Triangles indicate results obtained using reaction conditions shown in column 1-C of the table. Diamonds indicate results obtained using reaction conditions shown in column 1-D of the table.

[0095] [Figure 47] FIG. 47 presents the results of a second series of experiments using various conditions for the preparation of mucic acid polymers (MAP). The results are shown as the molecular weight of the polymer over time. The reaction conditions are shown in the table below the graph. Circles indicate results obtained using the reaction conditions shown in column 2-A of the table. Squares indicate results obtained using the reaction conditions shown in column 2-B of the table. Triangles indicate results obtained using the reaction conditions shown in column 2-C of the table. Diamonds indicate results obtained using the reaction conditions shown in column 2-D of the table.

[0096] [Figure 48] Figure 48 shows the molecular weight of MAP as a function of time for scale-up of the MAP synthesis reactions. Three reactions were performed at 5 g scale (triangles, squares, and upper set of circles) and one reaction was performed at 50 g scale (lower set of circles).

[0097] [Figure 49] Figure 49 shows the drug loading and grafting efficiency for the synthesis of MAP-gly-CPT conjugates as a function of CPT equivalents using PyAOP as the coupling reagent. The letters inside the circles refer to the columns in Table 9.

[0098] [Figure 50] FIG. 50 provides a summary of the production variations between lots of DAN-222 formulations produced using the methods disclosed herein.

[0099] [Figure 51] FIG. 51 presents the compatibility results for IV bag administration of DAN-222 in a low dose (0.022 mg / mL) formulation.

[0100] [Figure 52] FIG. 52 presents the compatibility results for IV bag administration of DAN-222 in a high dose (0.400 mg / mL) formulation.

[0101] [Figure 53] FIG. 53 presents a scheme for a dose escalation safety and pharmacology study of DAN-222 in subjects with metastatic breast cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] Detailed Description The present invention provides methods, compositions and uses of camptothecin (CPT)-conjugated nanoparticles. In particular, the present invention is directed to compositions, formulations and methods of use, including compositions comprising DAN-222, a polymer-small molecule conjugate formulated and assembled as a well-designed nanoparticle (NP). The polymer is a linear modified mucic acid-polyethylene glycol (PEG) copolymer. The small molecule active pharmaceutical ingredient is camptothecin (CPT), a topoisomerase I inhibitor. CPT is conjugated to the polymer via a glycyl linking group. DAN-222, as a polymer construct, consists of multiple copies of a primary repeat unit assembled as a linear polymer. The repeat unit of this polymer consists of modified mucic acid attached to a nominally 3,500 Da linear PEG derivative. The chemical structure of the MAP-Gly-CPT polymer-drug conjugate repeat unit of DAN-222 is represented by formula (I): [ka] where n is the number of ethylene glycol repeat units (which for a 3,500 Da PEG is on average n=79) and m is the number of repeat units of the MAP-Gly-CPT polymer-drug conjugate (which for the MAP parent polymer is on average m=16±4).

[0103] Formula (I) represents the polymer-drug conjugate of DAN-222 (MAP-Gly-CPT). The polymer repeat unit consists of a modified mucous acid moiety attached to a bis-propanoyl PEG unit. Each PEG unit has a nominal molecular weight of 3,500 Da ((C 2 H 4 O) 79 On average, each polymer chain consists of 16 repeats of such PEG-MAP units, each of which can be conjugated to up to two Gly-CPT molecules. When Gly-CPT is conjugated to MAP, the reaction is carried out such that approximately 90% of the available sites are reacted.

[0104] The average molecular weight of the MAP parent polymer, consisting of 16 repeating units, is approximately 65,000 Da. The target molecular weight range for this material is 50,000-80,000 Da. The molecular weight of the 65,000 Da MAP polymer with 90% CPT loading is approximately 75,000 Da.

[0105] As shown in Figure 1, DAN-222 self-assembles into NPs when formulated in aqueous media. When formulated as NPs, each NP is expected to consist of approximately two MAP-Gly-CPT chains. Thus, the average DAN-222 NP has a molecular weight of approximately 150,000 Da.

[0106] Failure to accurately repair complex DNA damage and resolve DNA replication stress leads to genome instability, which contributes to the pathogenesis of cancer.For example, defects in homologous recombination (HR) repair have been observed in various cancers.Breast cancer genotype 1 (BRCA1) and breast cancer genotype 2 (BRCA2) have long been known to code for proteins that play an important role in HR repair, and mutations in one or both of these genes place patients at higher risk of developing breast, ovarian, prostate, melanoma and pancreatic cancer.

[0107] At the same time, this inability to repair also makes cancer cells more vulnerable to DNA-damaging therapeutic agents.For example, tumor cells with defective HR repair show increased sensitivity to chemotherapeutic agents, suggesting that if HR repair can be therapeutically inactivated, tumor cells that are skilled at HR can be sensitized to chemotherapeutic agents.Topoisomerase inhibitors such as CPT damage DNA by binding to topoisomerase I, which prevents DNA unwinding required for replication, and replication forks are stalled.This can prevent cancer cells from replicating and growing in subjects.

[0108] Camptothecin (CPT), the active ingredient in DAN-222, is a natural pentacyclic quinolone alkaloid isolated from the bark of Camptotheca acuminata. CPT is a topoisomerase I inhibitor that acts by forming a ternary complex with topoisomerase I and DNA during replication and transcription in tumor cells, which blocks DNA religation and creates torsional tension downstream of RNA polymerase, causing DNA strand cleavage.

[0109] Treatment of many brain disorders, such as brain cancer and brain metastasis, requires that therapeutic molecules be delivered to the brain. Direct delivery of therapeutic agents to the brain brings severe risks to subjects (e.g., destroying the skull) and cannot be practical to carry out continuously, as is necessary for most chemotherapy treatments. However, systemic delivery (e.g., through the bloodstream) does not efficiently deliver molecules to the brain, because of the blood-brain barrier (BBB), a tightly bound layer of endothelial cells that lines the blood vessels of the brain. A similar permeability barrier, known as blood-tumor barrier (BTB), exists in certain solid tumors.

[0110] Breast cancer frequently metastasizes to the brain, and these brain metastases can be treated by chemotherapy molecules used to treat breast cancer, if the therapeutic agent can be delivered to the brain (through the BBB) or tumor (through the BTB) in sufficient concentration. One such chemotherapy agent is CPT.

[0111] Unfortunately, although CPT is known to have antitumor activity across a wide range of human cancers, it has poor aqueous solubility, low plasma stability and dose-limiting toxicity.Furthermore, as described, the BBB and / or BTB can inhibit the ability of cells to take up CPT.

[0112] However, as discovered by the present inventors, DAN-222 accumulates in tumors due to the enhanced vascular permeability and retention (EPR) effect, which results in greater and longer-lasting topoisomerase I inhibition, making it more potent than other topoisomerase I inhibitors, including free CPT. The EPR effect depends on the difference in vascular structure between healthy tissues and tumors. DAN-222 nanoparticles can encapsulate CPT and pass through the leaky walls of tumor neovasculature while not passing through the tougher healthy vascular walls. Furthermore, encapsulation in DAN-222 nanoparticles protects CPT from conversion of its active lactone form to the inactive carboxylate form in serum, thereby preventing rapid blood clearance of NP-encapsulated CPT.

[0113] For example, as described herein, the antitumor activity of DAN-222 was evaluated in several tumor xenograft studies, including cell lines derived from both breast (BT474, MDA-MB-23, MBA-MB-436) and ovarian (OVCAR3) tumors. DAN-222 had significantly greater efficacy and sustained tumor inhibition in all tumor xenograft models tested compared to other topoisomerase I inhibitors, including free CPT and irinotecan (a topoisomerase I inhibitor approved by the FDA for cancer treatment). The biodistribution of DAN-222 compared to irinotecan was evaluated in wild-type mice, demonstrating significantly lower exposure of DAN-222 compared to irinotecan in bone marrow.

[0114] Furthermore, DAN-222 is designed not only to show efficacy, but also to reduce or eliminate the undesirable risks and side effects associated with free CPT. The DAN-222 construct is designed to limit the acute toxicity of CPT by binding CPT to nanoparticle polymers, thereby controlling the release of active CPT moieties. The release of free CPT is caused by hydrolysis, and its release can be controlled in part by the nanoparticle construct and also by using the DAN-222 formulations described herein.

[0115] The present invention also includes compositions and methods that combine DAN-222 with poly(ADP-ribose) polymerase (PARP) inhibitors, such as niraparib. Recently, PARP has emerged as a new target in cancer treatment. PARP inhibitors exploit genomic instability and defects in DNA repair pathways. Four PARP inhibitors, olaparib, rucaparib, niraparib and talazoparib, are currently approved by the US Food and Drug Administration (FDA). Of these, olaparib and talazoparib are approved for HER2-negative locally advanced mBC or mBC with germline BRCA1 / 2 mutations. Currently, PARP inhibitors are considered the most important therapeutic agent for BRCA mutations found in TNBC and BRCA-mutated estrogen receptor-positive breast cancer.

[0116] As explained, topoisomerase inhibitors such as CPT damage DNA by binding to topoisomerase I and preventing the unwinding of DNA required for replication. This stalls replication forks that can be repaired by poly(ADP-ribose) polymerase (PARP). PARP inhibitors have been shown to block DNA repair activity by the PARP enzyme and induce apoptosis in tumor cells. Thus, inhibition of PARP also enhances the cytotoxicity of chemotherapeutic drugs and DNA damaging agents such as topoisomerase inhibitors such as CPT, in addition to direct tumor killing.

[0117] In certain embodiments, the present invention includes formulations useful for treating cancer, cancer metastasis, and / or brain and central nervous system disorders. In certain embodiments, the cancer is selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary cancer, gastric cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer, gastric cancer, and pancreatic cancer. In preferred embodiments, the cancer is breast cancer or ovarian cancer. In certain embodiments, the cancer is HER2-negative metastatic breast cancer (mBC). In certain embodiments, the HER2-negative mBC is homologous recombination deficient (HRD) positive or negative HER2-negative mBC. In additional embodiments, the cancer is brain metastasis of breast tumor. Abbreviations and Definitions

[0118] Reference to an alcohol, aldehyde, amine, carboxylic acid, ketone, or other similarly reactive functional group also includes protected analogs thereof. For example, a reference to hydroxy or alcohol includes those substituents, with hydroxy protected by acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), b-methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl, MMT), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (the most preferred of which include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM) and triisopropylsilyl (TIPS) ethers), ethoxyethyl ether (EE). Reference to amines includes those substituents, where the amine is protected by BOC glycine, carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts) or sulfonamide (nosyl and Nps) groups. Reference to substituents containing a carbonyl group includes those substituents, where the carbonyl is protected by an acetal or ketal, acylal or diathane group.Reference to substituents containing a carboxylic acid or carboxylate group also includes those substituents in which the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert-butyl ester, ester of 2,6-disubstituted phenol (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl ester, orthoester or oxazoline. Abbreviation ASCO American Society of Clinical Oncology AUC0-last Area under the curve from time 0 to the time of the last measurable concentration BOR Best Overall Efficacy BRCA breast cancer genes BRCA1 breast cancer gene type 1 BRCA2 breast cancer gene type 2 CAP College of American Pathologists CBR clinical usefulness rate CI Confidence Interval CL Clearance CL / F Clearance after oral administration Cmax Maximum plasma concentration Cmin minimum plasma concentration CPT Camptothecin CR Complete response CRA Clinical Research Monitor CT Computed Tomography CTC Circulating tumor count CYP3A4 Cytochrome P450 3A4 DAN-222: Mucinous polymer nanoparticles containing 20(S)-camptothecin DAS dose-limiting toxicity analysis population DCR Disease control rate DLT dose-limiting toxicity DOR Duration of response EAS evaluation analysis population ECG Electrocardiogram ECOG Eastern Cooperative Oncology Group eCRF Electronic Case Report Form EDC Electronic Laboratory Record Collection EOT End of administration EPR Increased vascular permeability / retention FAS full analysis set FDA Food and Drug Administration GCP: Standards for conducting clinical trials of pharmaceuticals G-CSF Granulocyte Colony Stimulating Factor HER2 human epidermal growth factor receptor 2 HNSTD Maximum dose at which no severe toxicity occurs HR Homologous recombination HRD Homologous recombination repair deficiency ICF Informed Consent Form ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use ILD interstitial lung disease IRB / IEC Institutional Review Board / Independent Ethics Committee IV Intravenous MAD Maximum Administered Dose mBC Metastatic breast cancer MDS / AML Myelodysplastic syndrome / acute myeloid leukemia MedDRA Glossary of Medical Regulatory Terms MRI Magnetic Resonance Imaging MTD maximum capacity NCI CTCAE National Cancer Institute Common Terminology Criteria for Adverse Events Event NP Nanoparticles ORR Objective Response Rate PARP poly(ADP-ribose) polymerase PD Pharmacodynamics PEG Polyethylene glycol PFS Progression-free survival rate PK Pharmacokinetics PPAS Protocol Permit Analysis Set PR partial response PRES Reversible Posterior Leukoencephalopathy QD Every Day QW Every week RECIST v1.1 Response Evaluation Criteria in Solid Tumors, Version 1.1 RP2D Recommended Dose for Phase 2 Clinical Trials SAE Serious Adverse Event SAS Safety Analysis Set SAP Statistical Analysis Plan SD Stable disease SRT Safety Review Team t1 / 2 terminal elimination half-life TEAEs Treatment-emergent adverse events TNBC Triple-negative breast cancer ULN normal upper limit V distribution volume Distribution volume after oral administration of Vz / F

[0119] When values ​​are expressed as approximations by use of the descriptor "about", it will be understood that the specific value forms another embodiment. In general, the use of the term "about" indicates approximations that may vary depending on the desired properties sought to be obtained by the disclosed subject matter and should be interpreted in the specific context in which it is used based on its function. Those skilled in the art may interpret this as a matter of convention. In some cases, the number of significant figures used in a specific value may be one of the non-limiting ways of determining the extent of the term "about". In other cases, the scale used in a series of values ​​may be used to determine the intended range available for the term "about" for each value. All ranges, when present, are inclusive and combinable. That is, when values ​​are stated in a stated range, they include every value within that range.

[0120] For clarity, it should be understood that certain features of the present disclosure described herein in the context of separate embodiments may also be provided in combination with a single embodiment. That is, unless clearly incompatible or specifically excluded, each individual embodiment is considered combinable with any other embodiment, and such combinations are considered to be separate embodiments. Conversely, various features of the present disclosure described for brevity in the context of a single embodiment may also be provided individually or in any subcombination. Finally, although an embodiment may be described as part of a series of steps or as part of a more general structure, each of the steps may also be considered as an independent embodiment in itself, combinable with other embodiments.

[0121] As used herein, a "pure isomer" compound or an "isomerically pure" compound is substantially free of other isomers of the compound. The term "pure isomer" compound or "isomerically pure" means that the compound contains at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, at least 99.5% by weight, at least 99.6% by weight, at least 99.7% by weight, at least 99.8% by weight, or at least 99.9% by weight of the compound having the specified structure. In certain embodiments, the weight is based on the total weight of all isomers of the compound.

[0122] As used herein, a "stereoisomerically pure" compound or a "stereoisomerically pure" compound is substantially free of other stereoisomers of the compound. That is, the composition is substantially free of isomers that differ at any chiral center. When a compound has multiple chiral centers, a substantially majority of the composition contains a compound that has the same stereochemistry at all chiral centers. The term "stereoisomerically pure" compound or "stereoisomerically pure" means that the compound contains at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, at least 99.5% by weight, at least 99.6% by weight, at least 99.7% by weight, at least 99.8% by weight, or at least 99.9% by weight of the compound with the specified stereochemistry. In certain embodiments, the weight is based on the total weight of all stereoisomers of the compound.

[0123] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, the "S" form of a compound is substantially free of the "R" form of the compound, and is therefore in enantiomeric excess over the "R" form. The term "enantiomerically pure" or "pure enantiomer" means that the compound contains at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, at least 99.5% by weight, at least 99.6% by weight, at least 99.7% by weight, at least 99.8% by weight, or at least 99.9% by weight of its enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0124] The compounds described herein may also contain one or more isotopic substitutions. For example, H is: 1 H, 2 H (D or deuterium) and 3 H may be in the form of any isotopic element, including T or tritium; 12 C.13 C and 14 It may be in any isotopic form, including C; N is 14 N and 15 It may be in any isotopic form, including N; O is 16 O and 18 It may be in any isotopic form, including O;

[0125] The articles "a" and "an" may be used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an analogue" means one analogue or more than one analogue.

[0126] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State government, or a corresponding agency in a country other than the United States, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, or more particularly in humans.

[0127] "Pharmaceutically acceptable salts" refers to salts of the compounds of the present invention that are pharma- ceutically acceptable and that possess the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and inorganic or organic base addition salts. Specifically, such salts include: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylsulfonic acid, 5 ... or (2) salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion, or coordinated to an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. Salts further include, by way of example only, salts of non-toxic organic or inorganic acids, such as sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and, if the compound contains a basic functional group, salts of hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc. The term "pharmacologically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like.See, e.g., Berge, et al., J. Pharm. Sci. (1977) 66(1): 1-79.

[0128] "Solvate" refers to a form of a compound associated with a solvent or water (also referred to as "hydrate"), usually by solvolysis. This physical association involves hydrogen bonding. Common solvents include water, ethanol, acetic acid, and the like. The compounds of the present invention may be prepared, for example, in crystalline form, and may be solvated or hydrated. Suitable solvates include pharma- ceutically acceptable solvates, such as hydrates, and further include both stoichiometric and non-stoichiometric solvates. In certain instances, a solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates.

[0129] The procedures described herein include standard solvents and catalysts known in the art. Although certain compounds (e.g., acids, bases, coupling agents) are listed in this disclosure, it will be apparent to one of skill in the art that different reagents may be used.

[0130] Exemplary solvents include, but are not limited to, chlorinated solvents (e.g., dichloromethane, chloroform, 1,2-dichloroethane), ethers (e.g., diethyl ether, tert-butyl methyl ether, cyclopentyl methyl ether, diglyme, 1,4-dioxane, 2-methyltetrahydrofuran), alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), alkanes (e.g., pentane, hexane, heptane), glycols (e.g., ethylene glycol, polyethylene glycol), polar aprotic solvents (e.g., dimethylacetamide, acetonitrile, dimethylsulfoxide, dimethylformamide, acetone, N-methyl-2-pyrrolidone), and polar protic solvents (e.g., water, ethanol, acetic acid, propionic acid).

[0131] Exemplary acids include, but are not limited to, inorganic acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid) and organic acids (e.g., acetic acid, malonic acid, methanesulfonic acid, propionic acid, thioacetic acid, p-toluenesulfonic acid, tribromoacetic acid, trichloroacetic acid, trifluoroacetic acid).

[0132] Exemplary bases include, but are not limited to, amino bases (e.g., 1,4-diazabicyclo[2.2.2]octane, diethylamine, triethylamine, N,N-diisopropylethylamine, lithium amide, lithium bis(trimethylsilyl)amide, morpholine, piperidine), alkoxides (e.g., barium tert-butoxide, lithium tert-butoxide, sodium methoxide), hydroxides (e.g., tetrabutylammonium hydroxide, sodium hydroxide, potassium hydroxide), organometallic bases (e.g., n-butyllithium, tert-butyllithium, butylmagnesium chloride), pyridines (e.g., 4-dimethylaminopyridine, 2,6-lutidine, pyridine), carbonates (e.g., lithium carbonate, sodium carbonate, magnesium carbonate, potassium carbonate), and hydrides (e.g., sodium hydride, calcium hydride, potassium hydride).

[0133] Exemplary coupling agents include, but are not limited to, carbodiimide reagents (e.g., N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclopentylcarbodiimide), additives for carbodiimide reagents (e.g., 1-hydroxy-7-azabenzotriazole, 6-chloro-1-hydroxybenzotriazole, N-hydroxysuccinimide, 1-hydroxy-2-pyridinone, 6-chloro-N-hydroxy-2-phenylbenzimidazole, ethyl 2-cyano-2-(hydroxyimino)acetate), anhydride-based or anhydride-forming reagents (e.g., di-tert-butyl carbonate, acetic anhydride, 2-ethoxy-1-ethylbenzodiimide, ethyl acetate, etc.), and / or esters thereof (e.g., ethyl acetate, ethyl acetate, etc.). methoxycarbonyl-1,2-dihydroquinoline, ethyl chloroformate), acylazoles (e.g., carbonyldiimidazole), acid halide generating reagents (e.g., thionyl chloride, phosgene, cyanuric chloride, benzyltriphenylphosphonium dihydrogen trifluoride), phosphonium salt coupling reagents (e.g., benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), tetramethylaminium reagents (e.g., 2-(2-oxo-1(2H)-pyridyl-1,1,3,3-tetramethyluronium tetrafluoroborate)), aminium reagents (e.g., 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate), oxymauran salts (oxymauran salts uranium salts (e.g., 1-((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)(morpholino)methylene)pyrrolidinium hexafluorophosphate), uranium antimonate salts (e.g., benzotriazol-l-yloxuy-N,N-dimethyl-methaniminium hexachloroantimonate), organophosphorus reagents (e.g., diethyl cyanophosphonate, 1-oxo-chlorophosphorane, 2-propanephosphonic anhydride), triazine-based reagents (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine), organosulfur reagents (e.g., pentafluorophenyl-4-nitrobenzenesulfonate), pyridinium reagents (e.g., 2-chloro-1-methylpyridinium iodide), and polymer-bound reagents (e.g., polymer-supported N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline).

[0134] As used herein, the term "isotopic variant" refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an "isotopic variant" of a compound can be, for example, an isotope of deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15( 15 In such isotopically substituted compounds, the following atoms, if present, may be altered, thus, for example, any hydrogen may be replaced by any other hydrogen, such as: 2 H / D, where either carbon is 13 C, or any nitrogen may be 15 It is understood that the atom may be N, and the presence and location of such atoms may be determined within the skill of the art. Similarly, the present invention can include the preparation of isotopic variations using radioactive isotopes, for example, in cases where the resulting compounds can be used for drug and / or substrate tissue distribution studies. The radioactive isotope tritium, i.e. 3 H, and carbon-14, i.e. 14 C are particularly useful for this purpose in view of their ease of incorporation and the means of detection already in place. 11 C. 18 F, 15 O and 13 Compounds substituted with positron emitting isotopes, such as N, may be prepared and may be useful in positron emission tomography (PET) studies to examine substrate receptor occupancy. All isotopic variations of the compounds provided herein, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0135] "Stereoisomers": It should also be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space are referred to as "isomers". Isomers that differ in the arrangement of their atoms in space are referred to as "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers" and stereoisomers that are non-superimposable mirror images of one another are termed "enantiomers". When a compound has an asymmetric center, e.g., an atom such as a carbon atom is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric center and are described by the R and S ordering rules of Cahn and Prelog or by the way the molecule rotates the plane of polarized light and are designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers, respectively). Chiral compounds can exist either as individual enantiomers or as mixtures of both enantiomers. A mixture containing equal proportions of the enantiomers is called a "racemic mixture."

[0136] "Tautomer" refers to a compound that is an interconvertible form of a particular compound's structure, differing in the position of hydrogen atoms and electrons. Thus, two structures can be in equilibrium through the transfer of n electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci and nitro forms of phenylnitromethane, which are also formed by treatment with acid or base. Tautomeric forms can be relevant to obtaining optimal chemical reactivity and biological activity of a compound of interest.

[0137] "Subjects" to which administration is contemplated include, but are not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or geriatric adults)), and / or non-human animals, e.g., mammals such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cows, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non-human animal.

[0138] Disease, disorder and condition are used interchangeably herein.

[0139] As used herein, and unless otherwise specified, the terms "treat," "treating," and "treatment" contemplate actions taken while a subject is afflicted with a particular disease, disorder, or condition, which actions reduce the severity of the disease, disorder, or condition, or delay or slow the progression of the disease, disorder, or condition ("therapeutic treatment"), and the above terms also contemplate actions taken before a subject begins to suffer from a particular disease, disorder, or condition ("prophylactic treatment").

[0140] Generally, the "effective amount" of a compound refers to an amount sufficient to induce desired biological response, for example, to treat cancer.As recognized by those skilled in the art, the effective amount of the compound, composition or formulation of the present invention can vary depending on factors such as the desired biological end point, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the age, weight, health and condition of the subject.The effective amount includes therapeutic treatment and prophylactic treatment.

[0141] As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to realize a therapeutic benefit in the treatment of a disease, disorder, or condition, or to delay or minimize one or more symptoms associated with a disease, disorder, or condition. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent, alone or in combination with other therapies, that realizes a therapeutic benefit in the treatment of a disease, disorder, or condition. The term "therapeutically effective amount" can include an amount that improves the overall therapy, reduces or avoids the symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent, i.e., causes a reduction in the amount and / or severity of symptoms associated with the disorder. For example, in the case of cancer metastasis to the brain, administration of a therapeutically effective amount of nanoparticles or nanoparticle formulations described herein results in a reduction and / or reversal of the symptoms of metastasis, e.g., regression of metastatic tumors. The therapeutically effective amount for treating brain metastasis will vary depending on the type and extent of brain damage, and may also vary depending on the overall condition of the subject.

[0142] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease, disorder, or condition, or to prevent one or more symptoms associated with a disease, disorder, or condition, or to prevent its recurrence. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in the prevention of a disease, disorder, or condition. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis, or an amount that enhances the prophylactic efficacy of another prophylactic agent.

[0143] Aqueous suspensions can contain the compound in the mixture with additives suitable for the manufacture of aqueous suspensions.Such additives can be suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing agents or wetting agents (such as natural phosphatides, such as lecithin), or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as condensation products of polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, such as polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0144] DAN-222 Formulation The chemical structure of the MAP-Gly-CPT polymer-drug conjugate repeat unit of DAN-222 is represented by formula (I): [ka] is presented. Formula (I) represents the polymer-drug conjugate of DAN-222 (MAP-Gly-CPT). The polymer repeat unit consists of a modified mucic acid moiety attached to a bis-propanoyl PEG unit. As shown in Figure 1, DAN-222 self-assembles into nanoparticles (NPs) when formulated in aqueous media. When formulated as NPs, each NP is expected to consist of approximately two MAP-Gly-CPT chains.

[0145] In formula (I), (n) is the number of repeating ethylene glycol units. In certain embodiments, in exemplary formulations of the invention, (n) is a number ranging from 5 to 200. Preferably, in formulations of the invention, (n) is, on average, about 79 for the MAP-Gly-CPT molecules of formula (I) in the formulation.

[0146] In formula (I), (m) is the number of repeating units of MAP-Gly-CPT in the chain of the polymer-drug conjugate. In certain embodiments, (m) is a number between 20 and 200. In preferred formulations of the invention, each polymer chain consists of an average of 16±4 repeating such PEG-MAP units, each of which may be conjugated to a maximum of two Gly-CPT molecules.

[0147] When Gly-CPT is conjugated to MAP, the reaction is carried out such that conjugation occurs at between about 75% and 100% of the sites on MAP available for reaction with Gly-CPT. In certain embodiments, about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the available sites react and are conjugated to CPT. In preferred embodiments, at least about 90% of the available sites react and are conjugated to CPT.

[0148] As described, DAN-222 self-assembles into nanoparticles (NPs) when formulated in an aqueous formulation.

[0149] In certain embodiments, the nanoparticle size in the formulation of the present invention is in the range of about 20 to about 80 nm in diameter. Exemplary nanoparticle diameters are about: 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, or 80 nm. In a preferred embodiment, the nanoparticle size is in the range of about 30 nm to about 40 nm in diameter.

[0150] In certain aspect embodiments, the values ​​of (m) and (n) are selected such that, after assembly of the DAN-222 chain(s) into a nanoparticle, the size of the nanoparticle is in the range of 10 to 900 nm, or any integer value therebetween, e.g., 20 nm to 80 nm.

[0151] In certain embodiments, the formulations of the invention comprise a DAN-222 nanoparticle concentration between 5 mg / mL and 40 mg / mL, between 10 mg / mL and 30 mg / mL, or between 15 mg / mL and 25 mg / mL. In preferred embodiments, the nanoparticle concentration is between 17 mg / mL and 23 mg / mL.

[0152] In certain embodiments, the formulations of the present invention comprise DAN-222 nanoparticles having a polydispersity index (PDI) of less than or equal to 0.9. In certain embodiments, the nanoparticles have a PDI of less than or equal to 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1.

[0153] In certain embodiments, the formulations of the invention contain a total concentration of CPT between 0.5 mg / ml and 10 mg / ml, between 1 mg / ml and 9 mg / ml, between 2 mg / ml and 8 mg / ml, between 3 mg / ml and 7 mg / ml, between 4 mg / ml and 6 mg / ml, between 1 mg / ml and 4 mg / ml, between 1.5 mg / ml and 3.8 mg / ml, between 2 mg / ml and 3 mg / ml, between 2.5 mg / ml and 3.0 mg / ml, or between 1.7 mg / ml and 3.6 mg / ml. In a preferred embodiment, the formulations of the invention contain a total concentration of CPT of about 2.6 mg / ml.

[0154] In certain embodiments, the formulation comprises a compound of formula (I) in a concentration of between 5 mg / ml and 100 mg / ml, between 4 mg / ml and 77 mg / ml, between 10 mg / ml and 90 mg / ml, between 10 mg / ml and 80 mg / ml, between 15 mg / ml and 50 mg / ml, between 15 mg / ml and 40 mg / ml, between 15 mg / ml and 30 mg / ml, between 15 mg / ml and 25 mg / ml, between 13 mg / ml and 28 mg / ml, or about 20 mg / ml.

[0155] In certain aspects, the exemplary formulations of the present invention have a pH between 3 and 6. In additional embodiments, the formulations have a pH between pH 3.5 and pH 5.5, or between pH 4 and pH 5. In certain aspects, the formulations have a pH between pH 4 and pH 4.6. In a preferred aspect, the formulations have a pH of about pH 4.3. The inventors have found that formulations with lower pH, for example, between pH 3 and pH 5, exhibit less loss of CPT during storage, and the stability of CPT is improved when the pH value of the formulation is lower. However, formulations with a pH below pH 4 may cause discomfort to patients.

[0156] The formulation of the present invention may further comprise at least one buffer selected from sodium succinate, sodium citrate, sodium acetate, phosphoric acid, histidine-HCL and sodium phosphate.In a preferred embodiment, the buffer is sodium acetate buffer.The formulation may further comprise at least one tonicity modifier selected from KCl, NaCl, proline, arginine-HCl, sucrose and glycine.Preferably, the at least one tonicity modifier is NaCl.

[0157] In a preferred embodiment, the formulation contains about 20 mg / mL of a compound of formula (I), about 20 mM sodium acetate, about 200 mM NaCl, and has a pH of 4.3±0.3.

[0158] Zeta potential is a measure of surface charge. In certain inventive DAN-222 formulations, the zeta potential is between -5mV and +5mV. In preferred embodiments, the inventive DAN-222 formulations exhibit a near-neutral zeta potential.

[0159] In certain embodiments, the osmolality of the DAN-222 formulations of the invention is between 300 mOsm / kg and 600 mOsm / kg. In preferred embodiments, the osmolality of the DAN-222 formulations is between about 336 and about 504 mOsm / kg. In certain embodiments, this range is representative of the osmolality of the formulation buffer, since the DAN-222 molecule has little effect on the osmolality results.

[0160] The molecular weight of the DAN-222 nanoparticles in the aqueous formulations described herein can be determined by gel permeation chromatography (GPC) using PEO standards with nominal molecular weights of 100 kDa and 200 kDa. The molecular weight of the DAN-222 conjugates is as follows: MW コンジュゲート =MW ポリマー / (1-T) (In the formula, MW ポリマーis the molecular weight of the MAP used in the synthesis of the DAN-222 conjugate, and T is the therapeutic agent (CPT) loading, expressed as a decimal, calculated from the molecular weight of the polymer (i.e., MAP) used in the synthesis of the conjugate, adjusted for drug (CPT) loading as follows:

[0161] In a preferred formulation of the invention, the average molecular weight of the MAP parent polymer, consisting of an average of 16 repeating units, is approximately 65,000 Da. The target molecular weight range for this material is 50,000-80,000 Da. The molecular weight of a 65,000 Da MAP polymer with 90% CPT loading is approximately 75,000 Da. In certain embodiments, the molecular weight of the MAP-Gly-CPT molecule is between 30 kDa and 100 kDa, between 40 kDa and 90 kDa, between 50 kDa and 80 kDa, or between 60 kDa and 70 kDa. In a preferred aspect, the nanoparticles of the formulation contain an average of two chains comprising the compound of formula (I) and have an average molecular weight of about 150,000 Da. In additional embodiments, the nanoparticles contain one, two, three, four, five, or six chains of the compound of formula (I).

[0162] Chains per particle (SpP), as used herein, refers to the number of mucine polymer ("MAP") therapeutic agent (CPT) conjugate molecules present in a particle or nanoparticle.For determining SpP, a particle or nanoparticle is an entity that has at least one MAP-Gly-CPT chain / molecule, which behaves as a single unit in any aqueous solution, such as water at neutral pH, PBS at pH 7.4, or in any formulation administered to a patient, at a concentration suitable for administration to humans.For calculating chains per particle, a MAP-Gly-CPT molecule is a single MAP-Gly-CPT conjugate chain.In the preferred formulation of the present invention, DAN-222 nanoparticles contain, on average, two MAP-Gly-CPT conjugate chains.

[0163] The methods disclosed herein provide for the evaluation of nanoparticles, where the particles contain one or more MAP-Gly-CPT molecules. In general, the methods involve providing a sample containing a plurality of the particles, and determining a mean value for the number of MAP-Gly-CPT molecules per particle in the sample, thereby evaluating the particle preparation. The value for the sample of particles is a function of the values ​​obtained for a plurality of particles. As discussed above, SpP is defined as the number of MAP-Gly-CPT molecules that self-assemble into a particle or nanoparticle, i.e., SpP = [MAP-Gly-CPT molecules] / P (or NP), where [MAP-Gly-CPT molecules] is the number of MAP-Gly-CPT chains / molecule and P (or NP) is a single particle (or nanoparticle).

[0164] In certain embodiments, the method further comprises a step of comparing the determined value with a reference value. This comparison can be performed in several ways. For example, depending on the comparison or decision made in the method, a decision or step is taken, for example, a production parameter in the process of making the particles is changed, the sample is classified, selected, approved or discarded, released or withheld, processed into a drug product, shipped, moved to another location, formulated, for example, formulated with another substance, for example, an additive, labeled, packaged, released in commerce, or sold, or offered for sale. For example, based on the result of the decision or upon comparison with a reference standard, the batch from which the sample is taken can be processed, for example, exactly as described.

[0165] To calculate the number of MAP-Gly-CPT chains per particle, the size of the particle is determined, e.g., by the molecular weight of the self-assembled particle by light scattering, and the size of the individual polymers, e.g., by the molecular weight of the individual polymers by light scattering, and, e.g., by the CPT loading on a weight percent basis. Using these values, SpP can be calculated as follows: SpP=MW 粒子 / (MWコンジュゲート ) (In the formula, MW 粒子 is the molecular weight of the particle, and MW コンジュゲート is the following: MW コンジュゲート =MW ポリマー / (1-T%) is the molecular weight of the MAP-Gly-CPT molecule calculated as follows: MW ポリマー is the molecular weight of MAP and T% is the loading of therapeutic agent (CPT) expressed as a decimal, e.g., 10% loading would be T%=0.1).

[0166] Polymer molecular weight distribution and particle dispersity: MAPs are synthesized to have a range of molecular weights. Molecules of different molecular weights provide different contributions to particle size and number of chains per particle.

[0167] Particle shape: The particle shape is considered to be approximately spherical. Self-assembly is presumed to be driven by hydrophobic regions generated by the therapeutic agent on the MAP-Gly-CPT molecule. Methods for Producing DAN-222 Formulations

[0168] The present invention provides a method for producing the DAN-222 formulation described herein. The drug substance, DAN-222, consists of 20(S)-camptothecin (CPT) covalently attached to a mucic acid-based polymer carrier via a glycyl linking group (MAP-Gly-CPT). During manufacture, MAP-Gly-CPT is isolated as an amorphous solid. In aqueous solution, the MAP-Gly-CPT polymer-drug conjugate self-assembles into nanoparticles (NPs).

[0169] In certain embodiments, an exemplary method for producing a DAN-222 formulation comprises four major steps: (i) derivatization of camptothecin (CPT) to obtain Gly-CPT (as the trifluoroacetic acid (TFA) salt), (ii) synthesis of the parent mucous acid polymer (MAP), (iii) covalent attachment of the derivatized camptothecin to the parent polymer to obtain a solid amorphous MAP-Gly-CPT polymer-drug conjugate, and (iv) aqueous formulation of MAP-Gly-CPT to form nanoparticles (NPs).

[0170] Examples 5-8 provide details and data regarding exemplary methods for producing DAN-222 and DAN-222 formulations of the present invention.

[0171] Figure 35 presents a scheme for an exemplary method of manufacturing DAN-222. Starting materials are shown in dark boxes, and manufacturing steps performed under GMP conditions for Phase 1 clinical trial drug substance are shown with text or surrounded by box 3401.

[0172] Briefly, in an exemplary method, in step (i), the production of derivatized CPT is a two-step process whereby the hydroxyl group of CPT is esterified with a tert-butoxycarbonyl protected glycine linker (Boc-Gly), which is then isolated by removal of the Boc protecting group to yield glycine-linked CPT (Gly-CPT) as a trifluoroacetic acid (TFA) salt.

[0173] As described in Example 7, the present inventors have discovered several improvements in the process for producing Gly-CPT as a trifluoroacetic acid (TFA) salt.

[0174] For this Boc-Gly-CPT synthesis step compared to the previous method, it was found that the reaction proceeded more smoothly under more dilute conditions at higher reaction temperatures. In addition, a new aqueous extraction step was added to improve the process to remove impurities. In the earlier synthesis, DCM was removed to obtain a crude solid. In contrast, we used vacuum distillation to remove only 95% of the DCM amount, and added a much lower percentage of methanol at a higher temperature for the final precipitation. An additional wash with isopropyl alcohol (IPA) was found to improve the isolation performance.

[0175] For the deprotection and isolation of Gly-CPT, a fairly small amount of methyl tert-butyl ether (MTBE) was used for the subsequent precipitation step, and the subsequent washing of the filter cake with a MTBE:DCM mixture was performed with a low ratio of MTBE:DCM.

[0176] In an exemplary method for producing the DAN-222 formulation, in step (ii), the parent polymer, mucic acid polymer (MAP), is produced by step-growth polymerization of two comonomers, MAM and di-SPA-PEG (a linear PEG molecule with a succinimide-activated propionyl ester at each end). These comonomers are dissolved in dimethyl sulfoxide (DMSO) and the reaction is initiated by the addition of N,N-diisopropylethylamine (DIPEA), resulting in the amidation of the free amines at each end of the MAM monomer. When the molecular weight (MW) of the MAP is expected to be at or near the target value, the reaction is quenched with water and the MAP is isolated by dialysis against DMSO and water and then lyophilized.

[0177] As described in Example 7, the present inventors have discovered several improvements in the methods for producing the MAP necessary for use in the preparation of DAN-222.

[0178] The process of step (ii) aims to control the reaction rate to bring the polymer MW close to a plateau over time. Although conditions for MAP production to grow MW nearly linearly with time can be identified, this strategy requires precise in-process monitoring of MW and quenching of the reaction. Alternatively, given that polymerizations have been shown to follow known models for stepwise growth of polymers, a strategy to stall the reaction over time was developed based on polymerization theory (modified Carothers equation and Flory-Shulz distribution). This strategy achieves a plateau of growth by exploiting the stoichiometric imbalance of the comonomers, and the reaction slows down as the MAP approaches the target MW. Examples of polymerizations in which the MW of the MAP at later time points is controlled by the stoichiometric ratio of MAM to diSPA-PEG in the reaction mixture are shown in Figures 44 and 46-48.

[0179] The inventors found that increasing the ratio of MAM:diSPA-PEG produced polymers with smaller MW: it was desired that the monomer starting material be prepared with a high degree of functionalization as specified herein, and that the reaction be carried out under anhydrous conditions to maintain the stability of the activated SPA group on the diSPA-PEG to protect against premature termination of the amidation reaction, allowing for a well-controlled stoichiometric ratio of comonomers at or near the target.

[0180] Over the course of the development of step (ii), several modifications were made to this process to make it more robust, scalable, safer, and / or reduce impurity levels. These modifications included (i) using MAM as a di-TFA salt rather than a neutral species, (ii) stoichiometry of MAM, di-SPA-PEG, TFA, and DIPEA, and (iii) using dialysis rather than precipitation for purification.

[0181] To reduce potential variability, it was decided to isolate the MAM as a neutral species (lacking the TFA counterion) to allow for more precise addition of TFA as a subsequent reaction step prior to polymerization.

[0182] The development of the MAM modification was undertaken as a result of reactions carried out using MAMs isolated as di-TFA salts or as neutral species (isolated with traces of TFA) to explore the effect of the presence or absence of TFA in the monomer starting material on the reaction behavior. Although the TFA group does not participate in the actual polymerization reaction, it was found to significantly affect the solubility of the MAMs in DMSO and therefore the reaction rate. Unlike the di-TFA salts, which are generally soluble in DMSO, the neutral species was found to be only minimally soluble in DMSO. Examination of the polymerization of the neutral species with di-SPA-PEG showed that despite the poor solubility of the neutral species, the reaction proceeded without the addition of TFA, albeit at a generally linear rate, independent of the comonomer ratio. In contrast, polymerization of the neutral species with di-SPA-PEG by adding approximately 1-2 equivalents of TFA to the reaction prior to initiation with DIPEA resulted in restoration of the desired plateau behavior.

[0183] Based on the results obtained from the initial experiments, it was decided to explore the development of reaction conditions using MAM isolated as a neutral species, and the subsequent addition of TFA that would allow the polymer MW to plateau at or near the target.

[0184] Furthermore, we conducted a series of experiments to investigate the combined effect of various comonomer ratios and equivalents of TFA and DIPEA on the rate of reaction in step (ii). The results showed that decreasing the amount of DIPEA (about 2.5 equivalents) decreased the reaction rate, while decreasing the ratio of MAM:diSPA-PEG to 1:1 led to an improvement in the MW of the MAP product. Increasing the TFA concentration led to an improved logarithmic plateau in the MW value, rather than a linear plateau. The combined results of the initial screening suggested that the comonomer ratio, equivalents of TFA and DIPEA, and reaction time were sufficient to achieve the target MAP polymerization degree.

[0185] Polymerization conditions were identified that allowed for a plateau in MW over a wide time frame and resulted in MAP with a target MW range of 50,000-80,000 Da, as described in Example 7. A slight increase in DIPEA (2.5 vs. 2.6 equivalents) was evaluated for the scale-up reaction and resulted in similar rates compared to those observed in the small-scale experiments.

[0186] To address the situation of scaling up MAP production, step (ii) can include precipitating MAP from DMSO using IPA under an inert atmosphere such as argon or nitrogen. Use of such a procedure to isolate MAP, as described in Example 7, results in a product with low levels of impurities that is a dry or reasonably dry solid that is easily recovered.

[0187] Other exemplary methods for generating MAPs include those provided in U.S. Patent Application Publication No. US2021 / 0170049A1, which is incorporated by reference herein.

[0188] In an exemplary method for producing a DAN-222 formulation, in step (iii), the synthesis of the amorphous MAP-Gly-CPT polymer-drug conjugate includes the use of EDC / NHS coupling chemistry. However, as described in Example 7 herein, in order to reduce the excess of starting materials and reagents in the reaction, particularly the excess of Gly-CPT, it is preferable to use the more active (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) coupling reagent to prepare MAP-Gly-CPT.

[0189] To minimize the process risk of high molecular weight species (HMWS) formation, high drug loading levels are used to minimize the number of unreacted carboxylic acid groups on the MAM. Additionally, running the reaction at increased dilution significantly reduces the possibility of the end groups of the polymer chain participating in the reaction. Development reactions were run at increased dilution for both 2.5 g and 4 g scales with 1.0, 1.1, and 1.2 equivalents of Gly-CPT as described in Example 7. Using the modified reaction conditions, all reactions proceeded well and did not show the formation of HMWS.

[0190] In an exemplary method for producing a DAN-222 formulation, in step (iv), preparation of MAP-Gly-CPT NPs requires dissolution of an amorphous polymer-drug conjugate in an aqueous medium, whereby the hydrophobic nature of CPT drives the self-assembly of the conjugate into NPs. The initially developed process involved dissolution of the amorphous MAP-Gly-CPT material in water at pH 4.0, addition of concentrated saline (9% NaCl, pH 4.0), concentration using a centrifugal ultrafiltration device, and 0.22 micron sterile filtration.

[0191] Over the course of the development of step (iv), several modifications were made to the process, including (i) changing the concentrated formulation buffer, (ii) using centrifugal ultrafiltration for TFF concentration, and (iii) a conditioning filtration step, making the process more robust, scalable, safer, and / or reducing impurity levels.

[0192] Although MAP-Gly-CPT NPs showed good stability in unbuffered saline solution (0.9% NaCl, pH 4.0), nanoparticles are pH sensitive and robust drug product formulations require a buffer system to protect against pH fluctuations and declines. Thus, in preferred embodiments, the addition of a concentrated formulation buffer (e.g., 200 mM sodium acetate, 2 M NaCl, pH 4.16) is used rather than adding concentrated saline. An exemplary formulation using such a buffer contains 20 mM sodium acetate, 200 mM NaCl (pH 4.3±0.3). As described in Example 7, no effect on particle properties (e.g., size, zeta potential, filterability) was observed using such a buffer to generate a DAN-222 formulation, and the product showed ampoule stability.

[0193] In certain embodiments, step (iv) comprises a concentration step. The concentration process may comprise centrifugal ultrafiltration. In a preferred embodiment, the concentration process comprises TFF ultrafiltration. A scaled-up version of centrifugal ultrafiltration requires longer processing times and generally cannot be performed on a large scale when compared to TFF ultrafiltration.

[0194] In certain embodiments, step (iv) comprises a single final filtration step. In a preferred embodiment, step (iv) comprises an intermediate conditioning filtration step, which can allow for an uninterrupted and risk-reduced final sterile filtration. During the development of the production of the DAN-222 formulation of the present invention, it was observed that the final sterile filtration process is often difficult, and multiple sterile filters are usually required to complete the filtration.

[0195] To reduce this process risk, the amorphous MAP-Gly-CPT material can be dissolved in water at pH 4.0 and filtered to condition the material to remove some of the product-associated particulates. A concentrated formulation buffer can then be added to perform a TFF step to formulate and concentrate the FBDS, allowing the final 0.22 micron sterile filtration to proceed without the risk of extended filtration times. Methods of Treatment, Formulations, Kits and Routes of Administration

[0196] Also provided are therapeutic compositions and formulations comprising DAN-222 as disclosed herein.Such formulations usually comprise nanoparticles and pharma-ceutically acceptable carriers.Supplementary active compounds can also be incorporated into such formulations.

[0197] In certain embodiments, the present invention includes formulations useful for treating cancer, cancer metastasis, and / or brain and central nervous system disorders. In certain embodiments, the cancer is selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer, liver cancer, salivary gland cancer, gastric cancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer, stomach cancer, and pancreatic cancer.

[0198] In a preferred embodiment, the cancer is breast cancer or ovarian cancer. In a particular embodiment, the cancer is HER2-negative metastatic breast cancer (mBC). In a particular embodiment, the HER2-negative mBC is homologous recombination deficient (HRD) positive or negative HER2-negative mBC.

[0199] Inability to accurately repair complex DNA damage and resolve DNA replication stress leads to genome instability, which contributes to the pathogenesis of cancer.For example, defects in homologous recombination (HR) repair have been observed in various cancers.Breast cancer genotype 1 (BRCA1) and breast cancer genotype 2 (BRCA2) have long been known to code for proteins that play an important role in HR repair, and mutations in one or both of these genes put patients at higher risk of developing breast, ovarian, prostate, melanoma and pancreatic cancer.

[0200] At the same time, this inability to repair also makes cancer cells more vulnerable to DNA-damaging therapeutic agents.For example, tumor cells with defective HR repair show increased sensitivity to chemotherapeutic agents, suggesting that if HR repair can be therapeutically inactivated, tumor cells skilled in HR can be sensitized to chemotherapeutic agents.Topoisomerase inhibitors such as CPT bind to topoisomerase I and prevent the unwinding of DNA required for replication, thereby damaging DNA and stalling replication forks.This can prevent cancer cells from replicating and growing in subjects.

[0201] Camptothecin (CPT), the active ingredient in DAN-222, is a natural pentacyclic quinolone alkaloid isolated from the bark of Camptotheca acuminata. CPT is a topoisomerase I inhibitor that acts by forming a ternary complex with topoisomerase I and DNA during replication and transcription in tumor cells, which blocks DNA religation and creates torsional tension downstream of RNA polymerase, causing DNA strand cleavage.

[0202] Treatment of many brain disorders, such as brain cancer and brain metastasis, requires that therapeutic molecules be delivered to the brain. Direct delivery of therapeutic agents to the brain brings severe risks to subjects (e.g., destroying the skull) and cannot be practical to carry out continuously, as is necessary for most chemotherapy treatments. However, systemic delivery (e.g., through the bloodstream) does not efficiently deliver molecules to the brain, because of the blood-brain barrier (BBB), a tightly bound layer of endothelial cells that lines the blood vessels of the brain. A similar permeability barrier, known as blood-tumor barrier (BTB), exists in certain solid tumors.

[0203] Breast cancer frequently metastasizes to the brain, and these brain metastases can be treated with chemotherapy molecules used to treat breast cancer, if the therapeutic agent can be delivered in sufficient concentration to the brain (through the BBB) or tumor (through the BTB). One such chemotherapy agent is CPT.

[0204] Unfortunately, although CPT is known to have antitumor activity, including across a wide range of human cancers, it has poor aqueous solubility, low plasma stability and dose-limiting toxicity.Furthermore, as described, the BBB and / or BTB can inhibit the ability of cells to take up CPT.

[0205] However, as discovered by the present inventors, DAN-222 accumulates in tumors due to the enhanced vascular permeability and retention (EPR) effect resulting in greater and longer-lasting topoisomerase I inhibition, making it more potent than other topoisomerase I inhibitors, including free CPT. The EPR effect depends on the difference in vasculature of tumors versus healthy tissues. DAN-222 nanoparticles encapsulate CPT and allow it to pass through the leaky walls of tumor neovasculature while not passing through the tougher walls of healthy blood vessels. Furthermore, encapsulation in DAN-222 nanoparticles protects CPT from conversion from its active lactone form to its inactive carboxylate form in serum, thereby preventing rapid blood clearance and toxicity to non-malignant tissues.

[0206] In certain embodiments, an exemplary method of the invention for treating cancer in a subject comprises administering to a subject having cancer a compound of formula (I): [ka] (In the formula, n is a number in the range of 20 to 200, m is a number ranging from 5 to 200. The method includes providing at least one dose of a composition comprising:

[0207] In an exemplary method, n is a number ranging from about 75 to 85, m is a number ranging from about 12 to 20, and the composition has a pH between pH 4 and pH 4.6.

[0208] In a preferred method, the composition contains between 2 mg / mL and 3 mg / mL of camptothecin (CPT). In a preferred method, the dosage of the composition is 2 mg / mL to 3 mg / mL of camptothecin (CPT). 2 ~16mg / m 2 For example, the dosage of the composition may be between about 2 mg / m 2 , about 4mg / m 2 , about 6mg / m 2 , about 8mg / m 2 , about 10mg / m 2 , about 12mg / m 2 , about 14mg / m 2 or about 20 mg / m 2 In certain embodiments, the composition may comprise about 2.6 mg / m 2 The compound includes a compound of formula (I).

[0209] The DAN-222 formulations and compositions described herein can be administered to a subject by any suitable route, including, but not limited to, inhalation, topical, intranasal, oral, parenteral (e.g., intravenous, intraperitoneal, intravesical or intrathecal) or rectal, in a vehicle containing one or more pharma- ceutically acceptable carriers, the proportions of which depend on the solubility and chemical properties of the compound, the selected route of administration and standard practice. Administration of DAN-222 described herein can be performed using any method known in the art. For example, administration can be transdermal, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intrathecal, intracapsular, intraperitoneal, intraventricular, intrathecal, intranasal, by aerosol, by suppository, or by oral administration. The pharmaceutical compositions of DAN-222 described herein can be for administration by injection, or for oral, pulmonary, intranasal, transdermal or intraocular administration.

[0210] In a preferred embodiment, a composition containing a compound of formula (I) is provided to a subject as a sterile injectable solution.

[0211] Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intrapulmonary, intravenous, intraarterial, intraocular, intracranial, subthecal or subcutaneous administration (including formulations encapsulated in micelles, liposomes or drug-releasing capsules (active agent incorporated within a biocompatible coating designed for slow release)); ingestible formulations; formulations for topical use, such as eye drops, creams, ointments and gels; and other formulations such as inhalants, aerosols and sprays. Dosages of the compositions of the present disclosure will vary depending on the extent and severity of the need for treatment, the activity of the composition being administered, the general health of the subject, and other considerations well known to those skilled in the art.

[0212] In additional embodiments, the DAN-222 compositions described herein are delivered to or near the site of injury or metastasis. Such localized delivery allows for non-systemic delivery of the composition, which reduces the burden of the composition on the body compared to systemic delivery. Localized delivery can be achieved, for example, by injection, or by the use of various medically implanted devices, including but not limited to stents and catheters, or by inhalation, phlebotomy or surgery. Methods for coating, implanting, embedding and otherwise attaching desired drugs to medical devices such as stents and catheters are established in the art and are contemplated herein.

[0213] The composition may be provided under any suitable dosing regimen. For example, the composition may be provided as a single dose or in repeated doses. The repeated doses may be provided at intervals such as 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. The repeated doses may be provided within a certain period of time. For example, the repeated doses may be provided for 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. The composition may be provided repeatedly for a specified period of time. For example, and without limitation, the composition may be provided for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months or longer.

[0214] In a preferred embodiment, the composition comprising the compound of formula (I) is provided to the subject as a sterile injectable solution in a dose administered once per week. In a preferred embodiment, the composition comprising the compound of formula (I) is provided to the subject as a sterile injectable solution in a dose administered once per week for a four week treatment cycle.

[0215] An exemplary method of the invention includes providing one or more doses of a composition comprising a compound of formula (I) in conjunction with providing at least one dose of a poly(ADP-ribose) polymerase (PARP) inhibitor.

[0216] Exemplary PARP inhibitors for use in the methods of the present invention include, by way of example, olaparib, rucaparib, niraparib and talazoparib. Preferably, the PARP inhibitor is niraparib.

[0217] In a preferred embodiment, a dose of niraparib is provided to the subject daily during a treatment cycle. In certain embodiments, repeated doses of niraparib may be provided at intervals such as 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. Repeated doses may be provided within a certain period of time. For example, repeated doses may be provided for 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. Niraparib may be repeatedly provided for a specified period of time. For example, and without limitation, niraparib may be provided for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months or longer.

[0218] In a preferred embodiment, the method of the present invention comprises providing each dose of niraparib as a dose between 25 mg and 200 mg. In a preferred embodiment, the dose is between about 50 mg and 100 mg. In a preferred embodiment, the dose is a 100 mg dose.

[0219] In certain methods, the subject is provided with a weekly dose of a composition comprising a compound of formula (I) and a daily dose of niraparib during the treatment cycle.

[0220] In certain methods, the cancer is one or more of breast, ovarian brain, lung, testis, head, neck, esophagus, lymphoma, central nervous system, peripheral nervous system, bladder, stomach, pancreas, liver, oral mucosa, colorectal, anal, kidney, bladder, urothelial, prostate, endometrium, uterus, fallopian tube, mesothelioma, melanoma, myeloma, leukemia, and Kaposi's sarcoma.

[0221] In a preferred embodiment, the cancer is breast cancer. In certain embodiments, the breast cancer is a homologous recombination repair deficient (HRD) positive or HRD-negative breast cancer.

[0222] The therapeutic compositions disclosed herein are useful for treating, inter alia, cancer, cancer metastasis, and brain and central nervous system disorders. Thus, a "therapeutically effective amount" of a composition comprising DAN-222 is any amount that alleviates symptoms or stimulates, for example, tumor regression. For example, the dosage of DAN-222 can vary from about 0.1 to 1.0 mg per kg of body weight, or from about 0.5 mg to 2.0 mg per kg of body weight, or from about 1 to 5 mg per kg of body weight, or from about 1 mg per kg of body weight to about 10 mg per kg of body weight, or more (or any integer value therebetween).

[0223] Dosage may be provided to subject with administration frequency of, for example, hourly, twice daily, once daily, twice weekly, once weekly, twice monthly, once monthly, depending on, for example, body weight, administration route, disease severity, etc. Thus, a therapeutically effective amount can include multiple administrations of the same or different amounts of DAN-222. In certain embodiments, a single administration of DAN-222 is a therapeutically effective amount.

[0224] In certain embodiments, the DAN-222 formulation is administered at a dosage of 1-20 mg of DAN-222 per square meter of the subject's body surface area (or any integer or decimal value therebetween), typical of chemotherapy dosages.

[0225] Various pharmaceutical compositions and their preparation and use techniques are known to those skilled in the art in light of the present disclosure.For detailed listing of suitable pharmacological compositions and the techniques for administering them, reference can be made to textbooks such as Remington's Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman and Gilman's The Pharmacological Basis of Therapeutics," McGraw-Hill, 2005; University of the Sciences in Philadelphia (eds.), "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins, 2005; and University of the Sciences in Philadelphia (eds.), "Remington: The Principles of Pharmacy Practice," Lippincott Williams & Wilkins, 2008.

[0226] DAN-222 nanoparticles and DAN-222 nanoparticle formulations described herein can be suspended in a physiologically compatible carrier for administration. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with DAN-222 and any other components of the formulation and is not harmful to its recipient. Those skilled in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include water (e.g., water at pH 4), phosphate buffered saline, Hank's buffered salt solution + / - glucose (HBSS) and multiple electrolyte solutions (e.g., Plasma-Lyte™ A (Baxter), etc.).

[0227] The volume of DAN-222 suspension administered to a subject will vary depending on the site of administration, the treatment goal, and the number concentration of DAN-222 in the solution. Usually, the amount of nanoparticles administered will be a therapeutically effective amount. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of DAN-222 and / or another drug, such as a PARP inhibitor, required to treat a specific disorder, i.e., to cause a reduction in the amount and / or severity of symptoms associated with the disorder. For example, in the case of cancer metastasis, administration of a therapeutically effective amount of the DAN-222 formulation described herein results in a reduction and / or reversal of symptoms of metastasis, such as the regression of metastatic tumors. The therapeutically effective amount will vary depending on the type and extent of damage caused by cancer, and may also vary depending on the overall condition of the subject.

[0228] The disclosed therapeutic compositions and formulations can also include pharma- ceutically acceptable substances, compositions, or vehicles, i.e., carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials. These carriers can, for example, stabilize the DAN-222 nanoparticles and / or promote retention of the DAN-222 nanoparticles in the body. Each carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of substances which can serve as pharma- ceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; polyols such as glycerin, sorbitol, and mannitol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0229] Another aspect of the disclosure relates to a kit for administering to a subject a DAN-222 formulation described herein, optionally in combination with another therapeutic agent (e.g., a PARP inhibitor such as niraparib). In one embodiment, the kit includes a composition of DAN-222 formulated in a pharmaceutical carrier suitable for administration, e.g., by injection or infusion.

[0230] The composition may be formulated for a single daily dose. The composition may be formulated for multiple doses per day, for example, 2, 3, 4, 5, 6 or more doses per day.

[0231] The composition may be administered to a subject according to any dosing schedule. The composition may be administered once per day. The composition may be administered multiple times per day.

[0232] The method of treating a subject comprises the step of providing the composition of the present invention to the subject.Providing may comprise the step of administering the composition to the subject.The composition can be administered by any suitable route or means, such as orally, intravenously, enterally, parenterally, dermally, bucally, topically (including transdermally), by injection, infusion, intranasally, intrapulmonary, and by or on an implanted medical device (e.g., a stent or a drug-eluting stent or a balloon equivalent).

[0233] The composition may be provided as a single unit dose. The composition may be provided as divided doses.

[0234] The composition may be provided under any suitable dosing regimen. For example, the composition may be provided as a single dose or in repeated doses. The repeated doses may be provided, provided that they are spaced apart by intervals such as 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. The repeated doses may be provided within a certain period of time. For example, the repeated doses may be provided for 1 day, 2 days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks or longer. The composition may be provided repeatedly for a specified period of time. For example, and without limitation, the composition may be provided for 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months or longer. EXAMPLES

[0235] Aspects of the present invention are illustrated in the examples presented below. Example 1 Antitumor activity of DAN-222 in vivo

[0236] The antitumor activity of DAN-222 has been evaluated in several tumor xenograft studies, including HRD-positive and HRD-negative cell lines derived from both breast (BT-474, MDA-MB-231, MBA-MB-436) and ovarian (OVCAR3) tumors.

[0237] Camptothecin, the active ingredient in DAN-222, is a topoisomerase I inhibitor that acts by forming a ternary complex with topoisomerase I and DNA during replication and transcription in tumor cells, which blocks DNA religation and creates torsional tension at the replication fork or downstream of the RNA polymerase transcription complex, leading to DNA strand cleavage. Results from tumor xenograft studies have shown that DAN-222 is likely to be more effective as a cancer treatment than other topoisomerase I inhibitors due to its accumulation in tumors through the enhanced vascular permeability and retention (EPR) effect, which results in stronger and more prolonged topoisomerase I inhibition.

[0238] As described below, the antitumor activity of DAN-222 was evaluated in several tumor xenograft studies, including HRD-positive and HRD-negative cell lines derived from both breast (BT-474, MDA-MB-231, MBA-MB-436) and ovarian (OVCAR3) tumors. DAN-222 achieved significantly greater efficacy with sustained tumor inhibition compared to free CPT and the CPT derivative irinotecan alone.

[0239] Additionally, the effect of DAN-222 treatment in combination with orally administered niraparib tosylate (niraparib) was evaluated in HRD-positive MDA MB-436 and HRD-negative OVCAR3 xenograft models, respectively. Combining DAN-222 with a PARP inhibitor demonstrated significantly enhanced tumor inhibition compared to either agent alone.

[0240] Efficacy data from these studies showing stronger antitumor responses compared to vehicle controls or either agent alone provide evidence of the complementary efficacy of DAN222 in vivo as an antitumor agent, as well as information regarding effective doses and dosing schedules. Study ER2019-09-1B: HRD-positive (ER-positive, PR-positive, HER2-positive) ductal carcinoma (BT-474 model)

[0241] In study ER2019-09-1B, DAN-222 was evaluated as a single agent in the BT-474 mouse xenograft model of human breast cancer.

[0242] Figure 2 presents the calculated median tumor volume (MTV) as a function of time, by treatment group. Figure 3 shows the endpoint (TTE, 2,000 mm 3 ) data are presented showing the percentage of animals in each group that remained on study over time. As depicted in Figures 2 and 3, animals in this study were dosed with either DAN-222, unconjugated (free) CPT, or the CPT derivative, irinotecan.

[0243] The results of the study, presented in Figures 2 and 3, demonstrate that dosing with DAN-222 (3 mg / kg) IV once weekly for three weeks sustained tumor growth inhibition throughout and after treatment in BT-474 model animals receiving DAN-222, when compared to tumor growth inhibition of vehicle control, unconjugated CPT, or irinotecan. Study ER2020-07-1: HRD-positive (ER-negative, PR-negative, HER2-negative [triple negative]) breast adenocarcinoma (MDA-MB-436 model)

[0244] Study ER2020-07-1 examined the antitumor activity of a range of doses of DAN-222 (0.3-3 mg / kg) administered weekly for 4 weeks as a single agent and in combination with a low dose of niraparib (25 mg / kg / day) in an HRD-positive (MD-MB-436) xenograft model. The antitumor activity of a range of doses of DAN-222 (0.3-3 mg / kg) administered weekly for 4 weeks was also examined in combination with a higher dose of niraparib (50 / 40 mg / kg / day) in an HRD-positive (MD-MB-436) xenograft model.

[0245] Figures 4 and 5 present median tumor volume measurements over time for study ER2020-07-1, represented by treatment group.

[0246] As shown in Figure 4, DAN-222 demonstrated significant single-agent activity in the MDA-MB-436 study with weekly treatment for 4 weeks with up to 3 mg / kg DAN-222, resulting in tumor growth inhibition or tumor regression. Specifically, DAN-222 (0.3, 1 and 3 mg / kg) induced 11 partial regressions (0.3 mg / kg, n=1; 1 mg / kg, n=6; 3 mg / kg, n=4) and 4 complete regressions (3 mg / kg, n=4), with 3 tumor-free survivors observed at the 3 mg / kg dose level.

[0247] As shown in Figures 4 and 5, combining DAN-222 with low dose (25 mg / kg) or high dose (50 / 40 mg / kg) niraparib produced a more robust antitumor response when compared to either agent alone. When combined with either high or low dose niraparib, the dose response of DAN-222 continued until complete regression.

[0248] Study ER2020-07-01 also found that the combination of DAN-222 (3 mg / kg) with high-dose niraparib (50 / 40 mg / kg) was deemed intolerable based on limited safety endpoints (i.e., mortality and body weight). Efficacy was not measured. Study ER2020-07-2: HRD-negative (ER-positive, PR-positive) ovarian adenocarcinoma cell line (OVCAR3 model)

[0249] Study ER2020-07-2 evaluated the effect of DAN-222 administration in combination with orally administered niraparibut tosylate (niraparib) in an HRD-negative OVCAR3 xenograft model.

[0250] A combination dosing regimen consisting of daily high-dose niraparib (50 mg / kg) and weekly injections of up to 3 mg / kg DAN-222 was evaluated, with the results for low-dose niraparib shown in Figure 6. As shown in Figure 6, the OVCAR3 study demonstrated that the combination of DAN-222 and niraparib showed efficacy regardless of HRD status. Furthermore, DAN-222 administered as a single agent at 3 mg / kg had a complete response with complete responses in all seven animals, and all seven animals were tumor-free survivors. Furthermore, the combination of 1 mg / kg DAN222 with high-dose (50 mg / kg) niraparib resulted in one partial response and six complete responses, including four tumor-free survivors. Example 2 Safety Pharmacology Testing

[0251] Camptothecin exposure and toxicokinetic (TK) parameters were evaluated in rats and dogs following either a single dose or repeated dosing of DAN-222 in studies that are part of a toxicology program. Rats and dogs were selected as pharmacologically relevant rodent and non-rodent species to evaluate the toxicology of DAN-222 and to select a safe starting dose for initial human studies. Because DAN-222 is a conjugated product, the toxicokinetic (TK) evaluation included evaluation of both total (conjugated and unconjugated) and free (unconjugated) CPT.

[0252] To evaluate the nonclinical safety profile of DAN-222 in support of a weekly dose clinical IV dosing regimen and to establish a safe clinical starting dose for Phase 1 clinical trials, studies in the toxicology program consisted of dose-ranging and repeat-dose toxicity studies in rats and beagles. With the exception of the dose-ranging studies in rats and beagles, all studies were conducted in compliance with Food and Drug Administration regulations for Good Laboratory Practice (GLP) for nonclinical studies (21 CFR, §58).

[0253] The effects of DAN-222 on essential organ function (eg, cardiovascular system, respiratory system, and central nervous system) were evaluated in end-point repeated-dose general toxicity studies conducted in rats and dogs. Testing in rats

[0254] In Study NC2020-08-1, a single acute IV dose established the maximum tolerated dose (MTD) at 9 mg / kg, with doses above the MTD (18 mg / kg) resulting in death. In each case, a reduction in the number of hematopoietic cells in the bone marrow was considered to be the cause of death or debilitation. At doses up to the MTD, dose-dependent effects on body weight were observed at 3 mg / kg and higher, and at doses of at least 9 mg / kg, effects on food consumption were observed that correlated with mean body weight loss.

[0255] In Study NC2020-08-3, a GLP 4-week IV repeat dose toxicity study conducted in Sprague-Dawley rats administered DAN-222 at 1 to 9.6 mg / kg, there were no test article-related effects on activity, autonomic, excitability, neuromuscular, physiological or sensorimotor observations in male animals tested with a Functional Observational Assessment on Days 1, 8 and 22. Adverse treatment-related findings observed in both males and females at 9.6 mg / kg included weight loss corresponding to reduced food consumption, and changes in bone marrow cytology associated with microscopic findings of reduced cellularity in the bone marrow. Hematological findings associated with DAN-222 treatment included reduced red blood cell mass and reduced white blood cell counts at 3.2 mg / kg and higher doses. Decreased lymphocyte counts were also observed at 9.6 mg / kg. In males, testicular degeneration was observed at doses of at least 3.2 mg / kg, which correlated with decreases in mean testicular and epididymal weights.

[0256] Based on the findings, the NOAEL was considered to be 3.2 mg / kg CPT for females and 1.1 mg / kg for males.

[0257] Based on the nature and reversibility of the findings, and the absence of DAN-222-associated deaths, a severely toxic dose (STD) was not observed in 10% of animals. 10 ) could not be determined. 10 estimated to be >9.6 mg / kg. Dog testing

[0258] In study NC2020-08-2, acute IV administration of a single dose established the MTD in dogs to be 1.5 mg / kg. When DAN-222 was administered at a dose above the MTD (2.6 mg / kg), animals were euthanized early due to deterioration in body condition due to gastrointestinal degeneration and atrophy. Clinical observations noted prior to early euthanasia included decreased activity, suspected dehydration, hunched posture, and coldness to the touch, and both animals experienced weight loss for several days after the first dose. Animals dosed to the MTD showed clinical observations related to gastrointestinal stress that correlated with decreased food consumption and weight loss, but increases in these endpoints over the course of the study suggested a trend toward recovery.

[0259] In Study NC2020-08-4, a GLP 4-week repeat-dose IV toxicity study conducted in beagle dogs administered 0.32 to 0.96 mg / kg DAN-222, all electrocardiogram (ECG) assessments were qualitatively and quantitatively normal at all dose levels tested. No abnormalities in rhythm or waveform shape were observed based on comparison of pre- and post-dose ECG recordings. No treatment-related effects on heart rate, QRS duration, QT or QTc intervals were observed based on comparison of days 1 and 22 (pre-dose and 2 hours post-dose) and at the end of the recovery period.

[0260] Treatment-related adverse findings included decreased mean body weight, decreased body weight gain, decreased food consumption, and clinical observations of decreased activity and hunched posture were observed in the high-dose (0.96 mg / kg) male group.

[0261] A decrease in thymic cortical cell density was observed in both males and females at the high dose (0.96 mg / kg) that correlated with a decrease in thymus weight.

[0262] In the 0.64 mg / kg female and 0.96 mg / kg male groups, dose-dependent decreases in white blood cell parameters, red blood cell mass parameters, platelets and reticulocytes were all observed, which correlated with microscopic findings of decreased bone marrow cellularity and increased myeloid:erythrocyte (M:E) ratio and percentage of lymphocytes in the bone marrow. Perivascular mixed cell infiltrates were detected in the liver at the end of recovery in 0.96 mg / kg males and females and were considered adverse.

[0263] The NOAEL was considered to be 0.32 mg / kg for females and 0.64 mg / kg for males. Because reversal of bone marrow cellularity and correlative effects on hematology and bone marrow cytology were generally observed, the HNSTD was considered to be 0.64 mg / kg in both sexes, which is approximately 12.8 mg / m in humans. 2 is converted into Genetic Toxicology

[0264] A single in vitro GLP genotoxicity study was conducted to evaluate the potential mutagenic activity of DAN-222 in a bacterial reverse mutation assay (Study T2020-08). No evidence of mutagenicity was observed at any of the dose levels tested in this study (1.0, 5.0, 10, 50, 100, 500, 1000 or 5000 μg / plate). Local tolerance

[0265] The tolerability of IV administration of the clinically investigated formulation was evaluated as part of toxicity studies in rats and dogs. DAN-222 Preclinical Safety Summary

[0266] The above studies formed part of a comprehensive pharmacology and toxicology program designed to evaluate the potential efficacy and toxicity of DAN-222 in relevant models when administered using clinical routes of administration and dosing schedules that mimic the intended clinical regimen. These completed studies provide information to characterize the toxicological properties of DAN-222 sufficiently to understand the severity and reversibility of toxicities associated with DAN-222 use and to inform the selection of a safe clinical starting dose. Additionally, these toxicity studies provided information to enable monitoring for potential safety signals in the clinical setting.

[0267] In acute dose-ranging toxicity studies, a maximum tolerated dose (MTD) was defined (9 mg / kg DAN-222 in rats and 1.5 mg / kg DAN-222 in dogs); doses above the MTD resulted in death and reduced hematopoietic cell numbers due to bone marrow toxicity in rats, or early euthanasia due to gastrointestinal degeneration and atrophy in dogs.

[0268] In a Good Laboratory Practice (GLP) repeated-dose toxicity study, animals were dosed once weekly for 4 weeks, mimicking one dosing cycle of DAN-222 to support weekly dosing in clinical trials. In rats, adverse effects observed upon treatment included decreased food consumption and weight loss, bone marrow toxicity, and testicular degeneration in males, with no observed adverse effect levels (NOAELs) of 3.2 mg / kg for females and 1.1 mg / kg for males (19.2 mg / m, respectively). 2 and 6.6 mg / m 2 In dogs, adverse effects observed following treatment included decreased body weight and food consumption, hematological changes, and bone marrow toxicity, with a NOAEL of 0.32 mg / kg in females and 0.64 mg / kg in males (6.4 mg / m 2 and 12.8 mg / m 2 ) and the maximum dose not causing serious toxicity (HNSTD) was 0.64 mg / kg (12.8 mg / m 2 ) was.

[0269] The proposed first-in-human (FIH) dose of DAN-222, determined by the maximum recommended starting dose (MRSD), is based on the HNSTD determined in dogs, which is considered the most relevant animal species tested. In a 4-week toxicity study in dogs, the HNSTD was 0.64 mg / kg. A dose of 0.64 mg / kg in dogs is equivalent to 12.8 mg / m 2 The MRSD is therefore approximately 2 mg / m based on the six-fold safety factor applied to the HNSTD, in accordance with ICH S9 recommendations. 2 It becomes. Example 3 PK parameters of DAN-222

[0270] CPT exposure and other PK parameters are evaluated in the species used in toxicity studies (i.e., rats and dogs). The distribution of CPT following DAN-222 dosing in naive mice and mouse tumor models was also evaluated. Since DAN222 is a conjugated product, the PK evaluation included evaluation of both total (conjugated and unconjugated) and free (unconjugated) CPT. Additionally, the stability of DAN-222 was tested in human, dog, and rat plasma using in vitro methods.

[0271] After weekly IV injections of DAN-222 in rats, exposure to free and total CPT (area under the plasma concentration-time curve from time 0 to the last observation time [AUC tlast

[0014] increased with DAN-222 dose levels from 1.1 mg / kg to 9.6 mg / kg on days 1 and 22. Exposure to free CPT was low (approximately 0.1%-0.2% of total CPT exposure). The terminal half-life of total CPT was approximately 15-21 hours. Clearance and volume of distribution ranged from 14.9-24.2 mL / h / kg and 404-647 mL, respectively. The data suggest that clearance is low relative to hepatic blood flow (0.4%) and that the volume of distribution indicates minimal distribution to tissues.

[0272] Similarly, the PK of DAN-222 was characterized in beagle dogs after repeated weekly dose administration at dose levels of 0.32, 0.64, and 0.96 mg / kg of DAN-222. AUC tlast For , exposure to total and free CPT increased with dose level in an approximately dose-proportional manner. On days 1 and 22, clearance and volume of distribution ranged from 16.4 to 18.7 mL / h / kg and 563 to 649 mL / kg, respectively. The terminal half-life of total CPT was approximately 23.2 to 26 hours. Exposure to total CPT increased in a dose-proportional manner. There was no accumulation of total or free CPT.

[0273] Calculated mean plasma free and total CPT concentrations were similar between male and female rats. Figures 7A and 7B present representative graphs of mean plasma free and total CPT concentrations in male rats after IV administration of DAN-222 on days 1 and 22.

[0274] The stability of DAN-222 in plasma from rats, dogs, and humans was assessed using an in vitro method based on the half-life of conjugated CPT in each matrix. Figure 8 presents the results of these studies, which show the stability of DAN-222 over time, expressed as the percentage of CPT conjugated, in rat, dog, and human plasma.

[0275] As shown in Figure 8, the half-lives of DAN-222 in rat and dog plasma were 35.3 and 45.5 hours, respectively. Compared with human plasma, where the stability of DAN-222 was calculated to be 35.7 hours, these results indicate that DAN-222 is sufficiently stable in rat and dog plasma, and the in vivo behavior of this compound in animals is expected to be similar to that in humans, which supports the suitability of the selected species as a model in toxicity testing.

[0276] In single-dose studies conducted in rats or dogs, systemic exposure to free and total CPT increased with dose level in both males and females, with exposure to free CPT representing only a small percentage (i.e., <1%) of total CPT exposure. In pivotal toxicity studies conducted in rats and dogs, animals received four weekly IV injections of DAN-222 (rats ranging from 1.1 to 9.6 mg / kg and dogs ranging from 0.32 to 0.96 mg / kg) and TK was evaluated after the first and fourth doses. Over the evaluation days, terminal half-lives of total and free CPT ranged from 15 to 21 hours and 14 to 26 hours in rats and 23 to 26 hours and 24 to 42 hours in dogs, respectively. In general, increases in systemic exposure to total and free CPT with increasing dose levels occurred in an approximately dose-proportional manner. Again, systemic exposure to free CPT represented only a small percentage of total CPT exposure, with AUC tlast With respect to CPT, free CPT exposure accounted for approximately 0.1%-0.2% or <0.1% of total CPT exposure in rats or dogs, respectively. Furthermore, exposure was similar between males and females of both species, and no significant accumulation of total or free CPT was observed following repeated dosing of DAN-222 in any of the dose groups evaluated.

[0277] Biodistribution of CPT to target organs was assessed in female mice after a single dose of DAN-222, including one study in naive CD-1 mice and two studies in immunodeficient mice bearing human breast cancer xenografts. Figures 9A-9B present the results of these studies, showing the biodistribution of free and total CPT from 2 to 48 hours in the liver, spleen, kidney, heart, and bone marrow of tumor-bearing mice (Figure 9A), as well as total CPT levels in plasma, skeletal muscle, and tumor tissue (Figure 9B).

[0278] FIG. 10 presents a mouse biodistribution study and a table summarizing the results.

[0279] These biodistribution studies, performed in naive and tumor-bearing mice, demonstrated limited exposure to free CPT in healthy tissues (e.g., liver, heart, spleen, and kidney), with only low quantifiable levels of total CPT contained in NPs but not released as free CPT in bone marrow. Biodistribution of CPT following DAN-222 dosing in both naive and tumor-bearing mice demonstrated reduced exposure in bone marrow compared to irinotecan. In tumor-bearing mice, increased CPT accumulation was observed in tumors 2-48 hours after a single dose of DAN-222, while levels were reduced in plasma, liver, spleen, heart, and skeletal muscle from the contralateral flank over the same time period.

[0280] Overall, free CPT and total CPT were detected in most organs tested (e.g. liver, heart, spleen and kidney).However, compared with irinotecan, DAN-222 administration significantly reduced the amount of biodistribution to bone marrow.The level of free CPT was less than 1% of the injected dose per gram of tissue in the organs tested.In tumor-bearing mice, accumulation of CPT was observed in tumor tissue but not in normal tissue, demonstrating the presence of EPR effect.

[0281] Overall, the data from studies characterizing the PK parameters and distribution of CPT following DAN-222 administration support the proposed clinical dosing schedule. Example 4 Favorable formulation development

[0282] To determine the preferred clinical formulation of DAN-222, three replicate experimental steps were performed as part of a preliminary formulation development screen. The screen was designed to determine early indications of material stability and critical quality attributes associated with DAN-222. A number of critical quality attributes (CQAs) were evaluated throughout the three formulation development steps, described below, to determine the preferred formulation of DAN-222. After each step, the best performing formulation was selected for evaluation in the subsequent step. In this manner, the preferred formulation of DAN-222 was determined.

[0283] The study was divided into three steps performed under accelerated stability conditions: (1) univariate buffer and pH selection, (2) addition of key additives to the top two buffers from step 1, and (3) top buffers / additives from step 2 at various pH values.

[0284] As described, DAN-222 is a nanoparticle composed of chains of MAP-Gly-CPT (i.e., camptothecin conjugated to a hydrophilic copolymer). MAP-Gly-CPT consists of a 50-80 kDa mucic acid-polyethylene glycol (PEG) copolymer (MAP) conjugated to multiple CPT molecules via glycine linkers. Upon dissolution in aqueous media, the MAP-Gly-CPT polymer chains spontaneously organize into nanoparticles containing hydrophobic CPT in a core surrounded by hydrophilic MAP. The mass of the nanoparticles averages about 180 kDa and the diameter is 40-60 nm (see FIG. 1). The target CPT loading of the formulated nanoparticles is 10-12% by weight, with an average of two MAP-Gly-CPT chains per nanoparticle.

[0285] The physicochemical properties of the DAN-222 drug substance require its delivery as a parenteral solution. Thus, drug development was focused on generating a drug product formulation for intravenous infusion. The formulation must meet several criteria: sterility; acceptable stability during manufacturing of the bulk drug substance and drug product; acceptable stability over long-term storage; and acceptable stability during handling and administration. Because aqueous solutions are required for the formation of DAN-222 nanoparticles, formulation design was focused on identifying excipients that achieve both isotonicity and stability within the desired pH range (e.g., buffered pH). Intentionally, no uncommon or novel excipients were evaluated. All excipients are available in compendial grades and are listed in the FDA's Inactive Ingredients Database.

[0286] The primary concern for the stability of the DAN-222 formulated bulk drug substance (FBDS) and DAN-222 sterile solution for injection is the release of free CPT from the nanoparticles. This measurement (free CPT as a percentage of total CPT) constitutes the key purity assay for DAN-222 and is the clearest indicator of product stability. The DAN-222 construct is designed to limit the acute toxicity of CPT by binding it to the nanoparticle polymer, thereby controlling the release of this active moiety. The release of free CPT is caused by hydrolysis and can be controlled by optimized formulation, avoidance of high temperatures during manufacturing and fill / finish, and keeping the drug substance and drug product frozen during long-term storage. As shown by the results of these studies, the release of free CPT from DAN-222 was controlled by optimized formulation, minimization of high temperatures during manufacturing and fill / finish, and evaluation of the feasibility of keeping the drug substance (DS) and drug product (DP) frozen or lyophilization.

[0287] Nanoparticle size and polydispersity (PDI) are additional factors that can affect the pharmacokinetics and in vivo activity of a product. In addition, solution properties including pH, viscosity, solubility and particle formation are all important to control to ensure a safe and effective product that can be manufactured and administered. As part of three studies, nanoparticle size, polydispersity and solution properties were evaluated for several different formulations exposed to a variety of conditions. Screening methods and materials DAN-222 Active Pharmaceutical Ingredient

[0288] The active pharmaceutical ingredient (API) investigated in these studies was DAN-222 manufactured under development conditions by Abzena (Bristol, PA). The material used in steps 1 and 3 of the screening was batch ABZ-2583 / SKT-757-010-LYO / 45 kDa. The material used in step 2 was batch SKT-757-009-Lyo / 53 kDa / 11.79% CPT. Formulation parameters

[0289] For each stability study, the formulations had fixed parameters of 20 mM buffer concentration, 0.5 mL fill volume, and 10 mg / mL API concentration.

[0290] Tables 1-3 present the various formulations investigated in steps 1-3 of the preliminary formulation development screen. The formulation codes are used in discussing the results obtained from the three steps of screening. [Table 1] [Table 2] [Table 3]

[0291] In all three stages of the early formulation development study, each of the above formulations was subjected to various stress conditions of temperature, agitation, freeze-thaw and forced oxidation designed to evaluate critical manufacturing process risk factors and various temperature risk factors on product stability that may affect the critical quality attributes of DAN-222.

[0292] Solution properties, including pH and visible particle formation, are also important for the product. Through these formulation studies, pH was found to be the most important formulation property for nanoparticle purity. At near-neutral and higher pH, the release rate of CPT from the nanoparticles increased. At lower pH (e.g., pH 4-5), a significant decrease in the rate of CPT loss from the nanoparticles was observed. At a pH below 4 (i.e., pH 3), the release rate of CPT was slower than at pH 4. However, below pH 4, pain during injection is a concern. A pH of 4.3 was chosen to achieve a low release rate of CPT at both freezing and refrigerated temperatures (2-8°C) and to reduce the risk of patient discomfort during injection of this moderately acidic solution. Furthermore, it was found that the formation of visible particles in the solution occurs when the amount of free CPT reaches a critical level (almost 30% of the total CPT) under stress conditions.

[0293] Nanoparticle size and polydispersity index (PDI) are additional product quality attributes that can affect the in vivo behavior of the product. Particle size and polydispersity can also provide evidence of aggregation, which can lead to poor product performance and potential safety issues for patients. Accelerated Stability Testing

[0294] The solid MAP-Gly-CPT conjugate (amorphous DAN-222DS) was reconstituted in bulk with Milli-Q (MQ) water adjusted to pH 4 with phosphoric acid and stirred vigorously at ambient temperature for approximately 12 h without the formation of bubbles.

[0295] Steps 1 and 3 utilized conjugated material from lot SKT-757-010-LYO, and step 2 utilized conjugated material from lot SKT-757009-Lyo. For step 3, after reconstitution, an aliquot of bulk MAP-Gly-CPT in MQ water at pH 4 was withdrawn for concentration measurement by absorbance (pre-0.45 μm filtration) with SOP-0001-CPTRP. After reconstitution, the solution was filtered using a 0.45 μm vacuum filter to remove turbidity. After filtration, the bulk formulated MAP-Gly-CPT in water at pH 4 was divided into appropriate aliquots according to Tables 1, 2, and 3. The concentrated (10x) stock solutions of either buffer alone (step 1 / 3) or buffer / additive / surfactant combination (step 2 / 3) were slowly added to the pH 4 aqueous solution at a 1 / 10 ratio with gentle stirring to obtain a target formulation of approximately 10 mg / mL. For step 3, after filtration and spiking with 10x buffer / additives, an aliquot of each of the formulated MAP-Gly-CPT moieties was withdrawn for concentration measurement by absorbance (measured after 0.45 μm filtration / spiking) with SOP-0001-CPTRP. All absorbance concentration measurements were performed in conjunction with the time 0 measurement. No adjustments were made to sample concentrations after measurement.

[0296] After all formulation preparation was completed, the formulations were sterile filtered in a sterile biological safety cabinet (BSC) using a 0.22 μm syringe barrel filter and filled into final container closures with a fill volume of 0.5 mL. A 100 μL aliquot (step 2) and a 1 mL aliquot (step 3) of each formulation were taken and lyophilized overnight for concentration analysis based on weight of solids. After filling, the containers were sealed, then removed from the BSC, stoppered / crimped and labeled. Samples were stored at the specified temperature or exposed to acute stress conditions such as agitation, freeze / thaw and forced oxidation. Analysis at time 0 was performed.

[0297] Samples in the agitation study were agitated on an orbital shaker at 450 rpm for 6 and 24 hours. For comparison, the same set of samples was also incubated at room temperature without agitation (step 1). A 1-day limited 25°C sample was used as a static control for steps 2 and 3. Forced oxidation samples were incubated with various concentrations of H according to Table 4. 2 O 2 and analyzed immediately after 1, 5 and 24 hours (steps 1 and 2). Freeze / thaw samples were exposed to continuous cycles of uncontrolled freezing and thawing from -80°C to ambient temperature and sampled at 3x F / T and 6x F / T (step 1) or 6x F / T (steps 2 / 3).

[0298] The accelerated stability study included incubation of the formulation candidate at frozen (-80°C and -20°C), refrigerated (2-8°C), stressed (20-25°C) and accelerated (40°C) storage temperatures. The storage temperature portion of the study was conducted over a one week period. After the initial screening, additional extended time points were added until additional sample volumes were available. All additional time points were drawn from the original vials and were not sterilized.

[0299] Table 4 summarizes the acute stress conditions used for the stability evaluation of the formulations. [Table 4] Table 5 summarizes the analytical methods used in the screening. [Table 5]

[0300] Visual inspection was performed under a white light source against black and white backgrounds. pH analysis was performed using an Orion 2 Star® pH meter, calibrated with three pH standard solutions (pH 4, 7, and 10) with a calibration slope of 95% or greater. Samples were not temperature regulated and were equilibrated to ambient temperature prior to measurement. Osmolality was measured using a Model 2020 Freezing Point Osmometer (Advanced Instruments).

[0301] For the RP-HPLC assay, the free CPT sample was treated with HCl to convert the carboxylate form of CPT to the lactone form. If CPT was not converted to the lactone form, it would elute earlier (approximately 2-4 min) along with the polymer-bound CPT. The area of ​​the free CPT peak between 5.6-7.0 min would increase as more CPT was lost from the polymer by hydrolysis. All CPT samples were first treated with NaOH to release CPT from the parent polymer, and then the carboxylate form of CPT was converted to the lactone form by HCl. The amount of these species was measured by monitoring the peak absorbance at 370 nm.

[0302] Dynamic Light Scattering (DLS) was used to characterize particle size based on the scattering of visible light resulting from the difference in refractive index between the dispersed colloid and the dispersion medium. DLS measures the fluctuations of scattered light as a function of time by vibrating particles suspended in a liquid. Brownian motion caused by collisions with the solvent molecules of the liquid causes the vibrations. Step 1 Results

[0303] Step 1 of the accelerated stability study evaluated the effect of specific buffers and pH on the performance of DAN-222. Since development data to date indicates that DAN-222 shows improved stability at lower pH, low pH buffer systems were evaluated, including succinate, citrate, acetate, and histidine. A slightly basic (pH 7.4) phosphate buffer was included in the evaluation as a control (P7.4 in Tables 1 and 2). Visual Inspection Results

[0304] At time 0, and for up to 7 days at 2-8°C and 20-25°C, all formulations (listed in Table 1) were free from visible particles (FFVP). After 3 days at 40°C, the control (P7.4) had visible particles present (PP). After 7 days at 40°C, both formulation W4 and the control (P7.4) had visible particles present, formulation H5 was essentially free of visible particles (PFP), and all other formulations were free of visible particles. After 3 months at both -20°C and -80°C, all formulations were free of visible particles.

[0305] After 24 hours of stirring at 450 rpm, all formulations were free of visible particles (FFVP). After six consecutive uncontrolled cycles of freezing and thawing from -80°C to ambient temperature, all formulations were free of visible particles, except the control (P7.4), which formed a gel-like matrix until it reached room temperature and returned to a liquid state at room temperature. After 24 hours of exposure to either 0.02% or 0.05% peroxide, all formulations were free of visible particles, except the control (P7.4), which had visible particles (PP) present. pH and Osmolality Results

[0306] At time 0, the pH and osmolality of all formulations (listed in Table 1) were within the target range, except for formulation W4. W4 received a 1.4 unit pH improvement after filtration using a PALL Acrodisc® 25 mm w / 0.2 μm Supor® syringe filter (PALL catalog number 4612). All osmolality values ​​were within the target range relative to the theoretical values. After 7 days at 2-8° C., all formulations showed similar pH and osmolality values ​​to those observed at time 0. After 7 days at 20-25° C., all formulations showed equivalent pH and osmolality values ​​to those observed at time 0. After 7 days at 40° C., all formulations showed similar pH and osmolality values ​​to those observed at time 0, except H5. H5 showed a -1.1 pH unit decrease in pH after 7 days at 40° C. After 3 months at -20° C. and -80° C., all formulations showed similar pH values ​​to those observed at time 0, except W4. W4 showed a 1.0 unit improvement in pH at both temperatures, highlighting the importance of adding buffer to maintain pH. All formulations showed similar osmolality values ​​to those observed at time 0.

[0307] After stirring at 450 rpm for 24 hours, all formulations exhibited pH and osmolality values ​​similar to those observed at time 0 and to the static control. After three consecutive uncontrolled freeze-thaw cycles from -80°C to room temperature, all formulations exhibited pH and osmolality values ​​comparable to those observed at time 0. For six consecutive uncontrolled freeze-thaw cycles from -80°C to room temperature, pH and osmolality data were not collected.

[0308] After the addition of 0.02% peroxide and subsequent monitoring for 24 hours, all formulations exhibited pH and osmolality values ​​similar to those observed at time 0. No pH or osmolality data was collected at the 5 hour time point. After the addition of 0.05% peroxide and subsequent monitoring for 24 hours, all formulations exhibited pH and osmolality values ​​comparable to those observed at time 0. No pH or osmolality data was collected at the 5 hour time point. RP-HPLC and DLS results

[0309] At time 0, all formulations (listed in Table 1) except the control (P7.4) showed low free CPT percentages. The phosphate control showed a larger increase in free CPT (+3.37%) at time 0. All formulations showed particle sizes ranging from 41.0 to 51.7 nm and similar PDIs ranging from 0.293 to 0.438.

[0310] As shown in FIG. 11, after 14 days at 2-8° C., formulations S4, C4, and A4 at pH 4 showed relatively small changes in free CPT release compared to that observed at time 0 (+0.51% to +0.58%). Formulations W4 and H5 showed slightly higher free CPT release compared to time 0, ranging from +0.73% to +0.93%. The control formulation (P7.4) showed a larger burst release of CPT after 14 days at 2-8° C., improving by 9.39% compared to time 0. After 14 days at 2-8° C., all low pH formulations showed no significant changes in particle size and PDI compared to time 0, except for formulation S4 on day 7. The control formulation (P7.4) showed a slight improvement in particle size compared to time 0 (±9 nm).

[0311] As shown in Figure 12, after 14 days at 20-25°C, formulations S4, C4 and A4 at pH 4 showed a slight increase in the release of free CPT compared to that observed at time 0 (from +1.60% to +1.76%). Formulations W4 and H5 showed a slightly higher release of free CPT compared to that observed at time 0, ranging from +2.34% to +3.30%. The control formulation (P7.4) showed a significant change in CPT release after 14 days at 20-25°C, improving by 34.7% compared to that observed at time 0.

[0312] As shown in Figure 12, after 14 days at 20-25°C, the low pH formulations showed little change in particle size and PDI compared to that observed at time 0. The citrate-containing formulations showed a slight increase in particle size after 7 and 14 days (from +10.3 nm to +13.9 nm). The control formulation (P7.4) showed a significant increase in particle size compared to time 0 (+140.1 nm).

[0313] As shown in FIG. 13, after 14 days at 40° C., the low pH formulations showed improved release of free CPT (+3.73% to +4.43%) compared to that observed at time 0, with a more significant change from time 0, but significantly less release than the high pH formulations. Formulations W4 and H5 showed slightly higher free CPT release compared to time 0, ranging from +4.16% to +5.23%. The control formulation (P7.4) showed a significant increase in CPT release (+32.47%) after 3 days at 40° C. compared to time 0. The control formulation was not analyzed at later time points due to the presence of heavy particulate matter.

[0314] After 3 days at 40° C., most formulations showed significant particle aggregation. The data suggests particle aggregation (±17.4 nm compared to time 0), but the low pH formulation did not have any visible particles after 14 days at 40° C. The control formulation (P7.4) showed significant particle aggregation after 3 days at 40° C. (+1476.3 nm) compared to that observed at time 0.

[0315] After 3 months at -20°C, all formulations showed little significant change in the amount of free CPT released compared to that observed at time 0 (from +0.17% to +0.24%) and at day 1 at 2–8°C (from +0.0% to +0.07%).

[0316] After 3 months at -20°C, all formulations showed no significant changes in particle size and PDI compared to those observed at time 0.

[0317] After 3 months at −80°C, all formulations showed little significant change in the amount of free CPT released compared to that observed at time 0 (+0.14% to +0.21%) and at day 1 at 2–8°C (±0.06%).

[0318] After 3 months at -80°C, all formulations showed no significant changes in particle size and PDI compared to those observed at time 0.

[0319] As shown in Figure 14, after stirring at 450 rpm for 24 hours, all formulations except for the control formulation P7.4 showed no significant change in the amount of free CPT released compared to the static control. The control formulation (P7.4) showed a slight increase in the amount of free CPT released (+1.56%) compared to the static control.

[0320] After stirring at 450 rpm for 24 hours, all formulations showed no significant change in particle size and no visible particles when compared to the static control.

[0321] After six consecutive uncontrolled cycles of freezing and thawing from -80°C to room temperature, all formulations showed a percentage of free CPT similar to that observed at time 0 (+0.10% to +0.39%). There were no significant differences between three or six consecutive freeze-thaw cycles.

[0322] As shown in Figure 15, after six successive uncontrolled cycles of freezing and thawing from -80°C to room temperature, all formulations showed no significant change in particle size compared to that observed at time 0. The high pH control formulation showed gelation after freezing and thawing until the material reached room temperature.

[0323] As shown in FIG. 16, after 24 hours of addition of 0.02% peroxide, all low pH formulations showed little change in CPT release compared to that observed at time 0 (+0.28% to +0.32%). The significant difference in CPT release between W4 and H5 (+0.95% vs. +0.53%) suggests a protective effect of buffering versus no buffering. The control formulations at high pH showed a significant increase in free CPT release compared to that observed at time 0 (+16.58%). After 24 hours of addition of 0.02% peroxide, all formulations except control formulation P7.4 showed little change in particle size compared to that observed at time 0. The particle size averages confirm that both physical and chemical degradation due to oxidation are correlated with the increase in pH. The high pH control formulation showed significant visible particle formation after the first hour of peroxide addition.

[0324] As shown in FIG. 17, after 24 hours of addition of 0.05% peroxide, all low pH formulations showed little change in CPT release compared to that observed at time 0 (+0.34% to +0.40%). The significant difference in the increase in CPT release between W4 and H5 (+3.05% vs. +1.05%) suggests a protective effect with buffer versus no buffer. The control formulation at high pH showed a significant increase in free CPT release (+29.76%) compared to that observed at time 0. After 24 hours of addition of 0.05% peroxide, all formulations except control formulation P7.4 showed little change in particle size compared to that observed at time 0. The particle size averages confirm that both physical and chemical degradation due to oxidation are correlated with the increase in pH. The high pH control formulation showed significant visible particle formation and a significant increase in particle size (+282.4 nm) after the first hour after addition of peroxide. Step 2 Results

[0325] In step 2 of the accelerated stability study, the two best performing buffer systems from step 1 were selected and used to evaluate the impact of specific excipients on the performance of the DAN-222 product. Common compendial excipients such as sugars, salts, amino acids and surfactants were screened. Visual Inspection Results

[0326] All formulations were free of visible particles (FFVP) at time 0 and up to 1 month at 2-8°C and 7 days at 20-25°C. The control (P7.4) had visible particles (PP) after 14 days at 20-25°C, but all other formulations were free of visible particles up to 1 month. After 7 days at 40°C, all formulations were free of visible particles except the control (P7.4). Visible particles were present in the control (P7.4) after 1 day at 40°C. All formulations tested were free of visible particles up to 2 months at -20°C and -80°C.

[0327] All formulations were free of visible particles (FFVP) after 24 hours of stirring at 450 rpm, six consecutive uncontrolled cycles of freezing and thawing from -80°C, and 24 hours of exposure to 0.05% peroxide. pH and Osmolality Results

[0328] At time 0, all formulations showed pH values ​​within ±0.38 pH units of the target and osmolality values ​​within ±87 mOsm / kg of the theoretical buffer osmolality. After 1 month at 2-8°C, all formulations showed similar pH values ​​(range ±0.17 pH units) and osmolality values ​​(range ±23 mOsm / kg) compared to those values ​​at time 0. After 1 month at 20-25°C, all formulations showed similar pH values ​​(range ±0.19 pH units) and osmolality values ​​(range ±25 mOsm / kg) compared to those observed at time 0. After 7 days at 40°C, all formulations showed a slight decrease in pH compared to time 0. The acetate formulations showed a decrease in the range ±0.26 pH units and the histidine formulations showed a decrease in the range ±1.09 pH units. All formulations exhibited osmolality values ​​similar to those observed at time 0, within a range of ±24 mOsm / kg.

[0329] After 2 months at -20°C to -80°C, all formulations showed similar pH values ​​(range ±0.09 pH units) and osmolality values ​​(range ±19 mOsm / kg) compared to those observed at time 0. After 24 hours of stirring at 450 rpm, all formulations showed similar pH values ​​(range ±0.17 pH units) and osmolality values ​​(range ±24 mOsm / kg) compared to those observed at time 0 and to those observed in the static control. After six consecutive, uncontrolled freeze-thaw cycles from -80°C to room temperature, all formulations showed similar pH values ​​(range ±0.18 pH units) and osmolality values ​​(range ±13 mOsm / kg) compared to those observed at time 0.

[0330] After 24 hours with the addition of 0.05% peroxide, all formulations exhibited similar pH (within a range of ±0.18 pH units) and osmolality values ​​(within a range of ±24 mOsm / kg) compared to those observed at time 0. RP-HPLC and DLS results

[0331] At time 0, all formulations except the control (P7.4) showed low free CPT percentage values ​​(ranging from 0.54% to 0.67%). The phosphate control showed a higher amount of free CPT (2.35%) at time 0. All formulations showed particle sizes ranging from 54.0 nm to 65.1 nm.

[0332] After 14 days at 2-8°C, all acetate and histidine formulations showed little change in free CPT compared to time 0 (+0.31% to +0.47% vs. +0.43% to 0.62%). The control (P7.4) showed a greater burst release of CPT (+9.13%) after 1 month at 2-8°C compared to the amount released at time 0.

[0333] As shown in Figures 18A-18B, after one month at 2-8°C, select low pH formulations tested showed little significant change in free CPT compared to time 0 (+0.44% to +0.81%) in both acetate buffer (Figure 18A) and histidine buffer (Figure 18B). The control (P7.4) showed a large burst release of CPT (+14.72%) after one month at 2-8°C compared to the amount released at time 0. All formulations showed no significant change in particle size and PDI compared to time 0. Most formulations showed a slight decrease in particle size.

[0334] As shown in Figure 19, after 4 months at 2–8 °C, both A4N and W4N exhibited an increase in free CPT (from +1.96% to +2.09%) and a decrease in particle size (from -18.7 nm to -22.6 nm) compared to that observed at time 0.

[0335] As shown in Figures 20A-20B, after 7 days at 20-25°C, all acetate and water formulations showed an increase in free CPT from +0.86% to +1.14% compared to time 0, while after 1 month at 20-25°C, select acetate and water formulations tested showed an increase in the amount of free CPT released from +2.54% to +3.18% (Figure 20A). After 7 days at 20-25°C, histidine formulations showed an increase in free CPT from +1.00% to +1.45% compared to the amount of free CPT at time 0, while select histidine formulations tested after 1 month at 20-25°C showed an increase in free CPT from +2.59% to +3.12% compared to the amount of free CPT at time 0 (Figure 20B). The control formulation (P7.4) showed significant changes in CPT release after 7 days (+24.71%) and 1 month (+38.14%) at 20-25°C. After 14 days at 20-25°C, the acetate, water and histidine containing formulations showed a slight to moderate decrease in particle size (-4.2 nm to -22.8 nm) compared to time 0. The selected formulations tested after 1 month at 20-25°C continued to show a trend towards smaller particle size (-14.0 nm to -21.6 nm) compared to time 0. The control formulation (P7.4) showed little to no change in particle size after 14 days at 20-25°C compared to time 0.

[0336] As shown in Figures 21A-21B, after 7 days at 40°C, all low pH formulations showed a large increase in the amount of free CPT released (+4.05% to +5.74%) compared to the amount of free CPT present at time 0. The results for acetate buffer are shown in Figure 21A. The results for histidine buffer are shown in Figure 21B. The control formulation (P7.4) showed a significant change in the amount of CPT released (+34.78%) after 7 days at 40°C. After 7 days at 40°C, the acetate, water and histidine containing formulations showed a slight decrease in particle size (-0.2 nm to -16.9 nm) compared to the size observed at time 0. The control formulation (P7.4) showed a significant increase in particle size (+990.3 nm) after 3 days at 40°C compared to that observed at time 0 and was not tested at additional time points.

[0337] As shown in FIG. 22, after 2 months at −20° C., all formulations tested showed little significant change in the amount of free CPT released compared to the amount of free CPT present at time 0 (+0.03% to +0.09%). The control formulation (P7.4) showed a slight decrease in free CPT compared to time 0 (−0.32%). After 4 months at −20° C., the formulations tested showed a slight increase in the amount of free CPT released compared to the amount of free CPT present at time 0 (+0.27% to 0.44%). After 2 and 4 months at −20° C., all formulations showed no significant change in particle size and PDI compared to time 0. Most formulations showed a slight decrease in particle size.

[0338] As shown in Figure 23, after 2 and 4 months at -80°C, all formulations tested showed little significant change in the amount of free CPT released compared to the amount of free CPT present at time 0 (+0.01% to +0.08% and +0.11% to 0.15%, respectively). After 2 months, the control formulation (P7.4) showed a slight decrease in free CPT (-0.43%) compared to the amount of free CPT present at time 0. After 2 and 4 months at -80°C, all formulations showed no significant change in particle size and PDI compared to time 0. Most formulations showed a slight decrease in particle size.

[0339] As shown in Figures 24A-24B, after 24 hours of stirring at 450 rpm, all formulations except the control showed no significant change in the amount of free CPT released compared to the amount of free CPT present at time 0. The control formulation (P7.4) showed a +5.11% increase in free CPT compared to time 0. After 24 hours of stirring at 450 rpm, all formulations showed a slight decrease in particle size compared to time 0 (from -2.7 nm to -16.3 nm). Results for formulations in acetate buffer are shown in Figure 24A and results for formulations in histidine buffer are shown in Figure 24B.

[0340] As shown in Figures 25A-25B, after six consecutive uncontrolled cycles of freezing and thawing from -80°C to room temperature, all formulations except the control showed a similar percentage of free CPT compared to the amount of free CPT present at time 0 (+0.14% to +0.28%). The control formulation (P7.4) showed a +2.16% increase in free CPT compared to time 0. After six consecutive cycles of uncontrolled freezing and thawing from -80°C to room temperature, all formulations showed a slight trend toward a decrease in particle size compared to time 0 (+2.7nm to -20.0nm). Results for formulations in acetate buffer are shown in Figure 25A, and results for formulations in histidine buffer are shown in Figure 25B.

[0341] As shown in Figures 26A-26B, at 24 hours after the addition of 0.05% peroxide, all acetate and water formulations showed little change in CPT release compared to time 0 (+0.30% to +0.72%) (Figure 26A). The histidine formulation showed a slightly larger increase in CPT release compared to time 0 (+0.72% to +1.18%) (Figure 26B). The control formulation at high pH showed a significant increase in free CPT release compared to time 0 (+47.64%) (Figure 26B). At 24 hours after the addition of 0.05% peroxide, all formulations except the control showed little change in particle size compared to the size observed at time 0. The control formulation (P7.4) showed a significant increase in particle size 5 hours after the addition of peroxide (+1304.3 nm) (Figure 26B). Step 3 Results

[0342] In step 3 of the accelerated stability study, the top performing buffer system / additive combinations from step 2 were selected for robustness testing at pH 4.0, 4.5 and 5.0. Visual Inspection Results

[0343] At time 0 and up to 1 month at 2-8° C. and 20-25° C., all formulations were free of visible particles (FFVP). After 1 day at 40° C., all formulations were free of visible particles except for the formulation at pH 5, which had visible particles present (PP).

[0344] After one month at -20°C and -80°C, all formulations were free of visible particles.

[0345] After stirring at 450 rpm for 24 hours and uncontrolled cycling of freezing and thawing from -80°C six times, all formulations were free of visible particles. pH and Osmolality Results

[0346] At time 0, all formulations exhibited pH values ​​within ±0.14 pH units of the target and osmolality values ​​within ±34 mOsm / kg of the theoretical buffer osmolality. After 1 month at 2-8°C, all formulations except W4N exhibited similar pH values ​​(within a range of ±0.22 pH units) and osmolality values ​​(within a range of ±15 mOsm / kg) compared to those observed at time 0. W4N exhibited a decrease of 0.54 pH units compared to time 0.

[0347] After 1 month at 20-25°C, all formulations showed similar pH values ​​(range ±0.24 pH units) and osmolality values ​​(range ±16 mOsm / kg) compared to those observed at time 0. After 7 days at 40°C, all formulations showed similar pH values ​​(range ±0.09 pH units) and osmolality values ​​(range ±5 mOsm / kg) compared to those observed at time 0. After 1 month at -20°C to -80°C, all formulations showed similar pH values ​​(range ±0.12 pH units) and osmolality values ​​(range ±10 mOsm / kg) compared to those observed at time 0.

[0348] After 24 hours of stirring at 450 rpm, all formulations exhibited similar pH values ​​(range of ±0.04 pH units) and osmolality values ​​(range of ±5 mOsm / kg) compared to those observed at time 0. After six consecutive, uncontrolled freeze-thaw cycles from -80°C to room temperature, all formulations exhibited similar pH values ​​(range of ±0.12 pH units) and osmolality values ​​(range of ±10 mOsm / kg) compared to those observed at time 0. RP-HPLC and DLS results

[0349] At time 0, all formulations showed low percentage free CPT values ​​(ranging from 0.65% to 0.74%) and particle sizes ranging from 42.2 nm to 47.3 nm.

[0350] As shown in Figure 27, after one month at 2-8°C, all low pH formulations showed a slight increase in free CPT (+0.33% to +0.80%) compared to the amount of free CPT present at time 0. Formulations containing buffer and salt showed a lower percentage of free CPT than their respective pH counterparts containing only buffer (-0.12% on average). Furthermore, the lower the pH of the formulation, the lower the percentage of free CPT. All formulations showed no significant changes in particle size and PDI compared to those values ​​observed at time 0.

[0351] As shown in Figure 28, after 3 months at 2-8°C, all formulations showed an increase in free CPT (from +1.34% to +2.23%) compared to the amount of free CPT present at time 0. Formulations containing buffer and salt showed a lower percentage of free CPT than their respective pH counterparts containing only buffer (-0.28% on average). Furthermore, the lower the pH of the formulation, the lower the percentage of free CPT. All formulations showed no significant change in particle size and PDI (-1.0 nm to +6.45 nm) compared to their values ​​observed at time 0.

[0352] As shown in FIG. 29, after 7 days at 20-25° C., all formulations showed an increase in free CPT (+0.65% to +1.12%) compared to the amount of free CPT present at time 0. Formulations containing buffers and salts showed a lower percentage of free CPT than their respective pH counterparts containing only buffers (-0.30% on average). After 1 month at 20-25° C., all low pH formulations showed a larger increase in free CPT (+2.14% to +4.18%) compared to the amount of free CPT present at time 0. Formulations containing buffers and salts showed a lower percentage of free CPT than their respective pH counterparts containing only buffers (-0.58% on average). Furthermore, the lower the pH of the formulation, the lower the percentage of free CPT detected. After 1 month at 20–25°C, all formulations showed no significant changes in particle size and PDI compared to those values ​​observed at time 0 (−1.6 nm to 4.5 nm).

[0353] As shown in Figure 30, after 7 days at 40°C, all formulations showed a greater increase in the amount of free CPT released (+3.06% to +5.00%) compared to the amount of free CPT present at time 0. All formulations showed a slight change in particle size (±15.9 nm) after 7 days at 40°C compared to time 0.

[0354] As shown in Figure 31, after 1 month at -20°C, all formulations showed neither significant changes in the amount of free CPT released (-0.02% to +0.11%) nor significant changes in particle size (-2.1 nm to +9.6 nm) compared to their values ​​at time 0. After 3 months at -20°C, the amount of free CPT released was slightly increased in the formulations at pH 4.5 and 5.0 compared to that at pH 4.0. Compared to time 0, the percentage of free CPT increased from 0% to 0.27% in the seven different formulations. There was no significant change in particle size (-5.6 nm to +7.4 nm) in the formulations compared to time 0.

[0355] As shown in Figure 32, after 3 months at -80°C, all formulations showed no significant changes in the amount of free CPT released (+0.05% to +0.15%) and no significant changes in particle size (-6.0 nm to 5.8 nm) compared to their values ​​at time 0.

[0356] As shown in Figure 33, after 24 hours of stirring at 450 rpm, all formulations showed little significant change in the amount of free CPT released (+0.07% to +0.39%) compared to the amount of free CPT present at time 0, with the higher pH formulations showing greater release of CPT compared to the lower pH formulations. All formulations showed no significant change in particle size compared to the size at time 0.

[0357] As shown in FIG. 34, after six consecutive uncontrolled cycles of freezing and thawing from −80° C. to room temperature, all formulations showed no significant change in the amount of free CPT released or in particle size compared to their values ​​at time 0. Conclusions from steps 1-3

[0358] Screening of formulation buffers (step 1) showed clear advantages in chemical (CPT loss), physical (particle size and PDI) and solution properties when using low pH based formulation buffers. At 2-8°C, the low pH formulations showed minimal CPT loss of approximately 0.03% per day, while the control (P7.4) showed a 20-fold higher CPT loss of approximately 0.6% per day. All formulations showed no significant changes in physical or solution stability, regardless of pH. At 20-25°C, the pH 4.0 formulations showed approximately 0.03% CPT loss per day, while the pH 5 formulations showed approximately 0.2% CPT loss per day. The control (P7.4) showed a 30-fold higher CPT loss of approximately 3.6% per day. All formulations showed no significant changes in physical or solution stability, regardless of pH. At 40°C, the low pH formulation showed a higher CPT loss rate of about 0.6% per day and an increase in particle size after 3 days. The control (P7.4) showed a significant CPT loss rate of about 32.0% per day and rapid visible particle formation after 1 day. At -20°C and -80°C, all formulations except the control (P7.4) showed minimal CPT loss of about 0.01% per day. The phosphate control showed a CPT loss of about 0.04% per day. All formulations showed no significant change in physical or solution stability.

[0359] After 24 hours of stirring at 450 rpm, all formulations showed minimal impact on either chemical or physical stability. PDI improved slightly after stirring. Freeze / thaw cycles showed minimal impact on particle structure and chemical stability in all groups except for the control (P7.4), where gelation was observed upon short-term thawing. No significant differences were found for chemical or physical stability between three or six consecutive freeze / thaw cycles. At low pH, there was no significant change in physical stability, and minimal change in chemical stability after oxidative stress. A significant decrease in CPT was observed with increasing pH and peroxide levels. The control (P7.4) showed rapid and high physical structure changes after oxidative stress, including visible particle formation.

[0360] The sodium acetate buffer system showed several enhanced advantages compared to other pH 4 systems, including reduced chemical and physical degradation, and as a result, sodium acetate was selected as the lead buffer system to be used in Step 2. Histidine was also evaluated in Step 2 as a mid-pH option.

[0361] In screening formulation excipients (Step 2), a wide range of salts, sugars, amino acids, and surfactants were screened in both acetate buffer at pH 4.0 and histidine buffer at pH 5.0. At 28°C, formulations at pH 4.0 and 5.0 showed minimal CPT loss of approximately 0.03% per day and no significant change in physical or solution stability. Groups of excipients within the same buffer system showed no significant differences in chemical, physical, or solution stability. At 20-25°C, the CPT loss per day was approximately 0.1% to 0.2% at both pH 4.0 and pH 5.0, depending on the excipient. The mean particle size decreased with increasing temperature exposure, plateauing at approximately day 7. There was no significant change in solution stability at either pH. At 40°C, the CPT loss per day increased to approximately 0.6%, and particle size variation was observed after 3 days at both pH values. There was no significant change in solution properties. At -20°C and -80°C, formulations at pH 4.0 and 5.0 showed minimal CPT loss of approximately 0.01% per day and no significant change in physical or solution stability.

[0362] After stirring at 450 rpm for 24 hours, all formulations except the control (P7.4) showed minimal impact on physical stability as evidenced by only a small decrease in particle size. Furthermore, no significant effect on either chemical stability or solution properties was observed by visual inspection. A slight improvement in PDI was observed in some additive groups, but this was less pronounced in the pH 4.0 formulations. Freeze / thaw cycles showed minimal effect on particle structure and chemical stability in the pH 4.0 and 5.0 formulations. After addition of peroxide, all low pH formulations showed no significant change in physical stability and a slight effect on chemical stability. A greater decrease in CPT was observed in the pH 5.0 formulations compared to the pH 4.0 formulations.

[0363] The use of salt as an additive showed the added benefit of controlling the CQAs of the product compared to other additives. Specifically, salt was observed to help control consistent polydispersity across the various stress groups. Sodium chloride was selected to move forward to step 3.

[0364] In the formulation validation screen (Step 3), acetate formulations with and without NaCl were tested at pH 4.0, 4.5, and 5.0. At 2-8°C, the daily CPT decline was approximately 0.03%-0.05% at pH 4.0-5.0. There were no significant changes in physical or solution stability at all pH values ​​up to one month. The W4N formulation showed a decline of 0.54 pH units compared to time 0 after one month at 2-8°C, highlighting the importance of using a buffered system. At 20-25°C, the daily CPT decline was approximately 0.1%-0.2%, depending on pH and the presence of salt. The inclusion of salt demonstrated greater protection against CPT decline compared to buffer alone at the same pH group for all three pH values. No significant changes in physical or solution properties were observed at all pH values ​​with or without salt up to one month. At 40°C, the CPT loss improved to about 0.6% per day at all pH values. After 3 days, particle size variation was observed, with the particle size often decreasing in average size followed by an increase in size. The inclusion of salt at elevated temperatures could minimize the increase in particle size. After 1 day, the formulation at pH 5.0 showed the presence of visible particles, while the formulations at pH 4.0 and 4.5 showed no visible particles for up to 1 week. At -20°C and -80°C, all formulations showed minimal CPT loss of about 0.02% to 0.03% per day and no significant changes in physical or solution stability.

[0365] Agitation stress at 450 rpm for 24 hours had minimal impact on chemical stability (CPT drop of 0.1% per day) and physical stability. Freeze / thaw cycles also had minimal impact on product quality in all formulations, regardless of pH or the presence of salt. A minimal increase in PDI was observed in some groups, more pronounced in the salt-free group.

[0366] For most stress groups, a significant advantage was observed from the inclusion of NaCl over buffer alone, with data for pH 4.0 indicating increased stability compared to pH 4.5 and 5.0.

[0367] The results of this initial formulation development study led to the selection of a target Phase 1 clinical formulation and drug substance concentration of 20 mg / mL DAN-222 in 20 mM sodium acetate (equivalent to 6.8 mM anhydrous sodium acetate, 13.2 mM acetic acid), 200 mM sodium chloride, pH 4.3.

[0368] Follow-up formulation development stability studies were performed using several development lots of DAN-222 material using the proposed clinical formulation and API concentration. The results of these studies indicate that the product is robust and will maintain product quality when exposed to the various conditions expected during manufacturing and storage. The formulation of this GLP toxicology batch is identical to the proposed Phase 1 clinical formulation batch for the DAN-222 FBDS and drug product. Currently, at the same proposed storage temperature for the Phase 1 clinical drug product (-20±5°C), the stability trends indicate that the product is very stable with respect to its critical quality attributes under these storage conditions. Example 5 DAN-222 Formulated Bulk Drug Substance (FBDS) Characteristics

[0369] This example presents the methods and assays used to develop and characterize DAN-222FBDS, which can be used to generate a sterile solution of DAN-222 for injection. Characteristics of DAN-222

[0370] The drug substance, DAN-222, consists of a chain of 20(S)-camptothecin (CPT) covalently attached to a mucic acid-based polymer carrier via a glycyl linking group (MAP-Gly-CPT). During manufacturing, MAP-Gly-CPT is isolated as an amorphous solid. In aqueous solution, the MAP-Gly-CPT polymer-drug conjugate self-assembles into nanoparticles (NPs). The drug substance properties listed herein refer to either amorphous MAP-Gly-CPT or DAN-222 formulated bulk drug substance (FBDS), as specified. A listing of the development batches from which these properties are derived is provided in Table 6. [Table 6]

[0371] Once the concentrated MAP-Gly-CPT is purified and isolated by ultrafiltration / diafiltration, it is lyophilized to yield a solid intermediate. The isolated conjugate material is a pale yellow to yellow amorphous, low density solid.

[0372] DAN-222FBDS is obtained by dissolving amorphous MAP-Gly-CPT in water for injection adjusted to pH 4.0, followed by addition of formulation buffer concentrate to meet the desired buffer concentration (pH 4.3). The solution is concentrated to the desired strength by ultrafiltration. Once formulated, DAN-222FBDS is a clear to slightly opalescent, colorless to pale yellow liquid.

[0373] A series of tests were performed to determine the effect on the pH of the water caused by dissolving MAP-Gly-CPT in purified water. Samples were tested at ambient temperature using a Thermo Scientific Orion 2 Star benchtop pH meter equipped with an Orion 8103BN probe. Two different amorphous MAP-Gly-CPT development batches (SKT-874-052-2 NDMaterials derived from SKT-874-067-1.2CPT and SKT-874-067-1.2CPT-25GLYO) were evaluated. Amorphous MAP-Gly-CPT from these batches was dissolved in sterile filtered Milli-Q® water at various concentrations. Dissolution of MAP-Gly-CPT slightly reduces the pH of the water. This may be due to the presence of some free carboxylic acid moieties that are not conjugated to CPT. DAN-222FBDS solutions, as formulated, are buffered with a target pH of 4.3±0.3.

[0374] The solubility of DAN-222 in water at ambient temperature was evaluated. Amorphous MAP-Gly-CPT from development batches SKT-874052-2ND-1.2CPT and SKT-874-067-1.2CPT-25G-LYO was dissolved in sterile filtered Milli-Q® water. Solubility was determined by creating a saturated mixture in a clear glass vial above the solubility limit and visually assessing the effect of successive water additions.

[0375] Since DAN-222 incorporates a hydrophilic polymer backbone, its behavior at high concentrations differs from that of soluble crystalline material. No significant differences were observed in the solubility of the two batches tested. Starting at 400 mg / mL, the material does not completely dissolve and the mixture is heterogeneous. The solid appears to absorb the first aliquot of water and exhibits a homogeneous translucent gel-like state at approximately 265 mg / mL. Complete dissolution is obtained at approximately 230 mg / mL, resulting in a clear yellow, highly viscous solution that is barely able to flow. Furthermore, the viscosity decreases with the addition of water.

[0376] The viscosity of two different development batches of DAN-222FBDS was measured at ambient temperature using a Brookfield DV-II+Pro viscometer with a Brookfield CPA-40Z plate. The plate speed was set at 50 revolutions per minute (rpm) for both samples, and the torque rate was measured. Two batches, SKT-874-069 and SKT-874-073, were formulated in 20 mM sodium acetate, 200 mM sodium chloride, pH 4.3 (formulation buffer). Results showed that the viscosity was concentration dependent. The formulated solutions (target 20 mg / mL) were slightly more viscous than water (similar to the viscosity of whole milk) and could be easily removed from the vial by syringe.

[0377] MAP-Gly-CPT was isolated as an amorphous solid after concentration and lyophilization prior to formulation as a drug substance. This low density solid material is brittle and somewhat sponge-like, and does not show any obvious signs of crystalline structure. X-ray powder diffraction (XRPD) diffractograms of MAP-Gly-CPT and its precursors, mucic acid polymer (MAP) and glycylcamptothecin (TFA salt, Gly-CPT), were obtained using a Rigaku SmartLab X-ray diffractometer. Data collection was performed with the instrument in a Grabb-Brentano reflection geometry using an X-ray beam as the source.

[0378] The XRPD results demonstrate that the Gly-CPT isolated for use in the synthesis of DAN-222 exhibits a clearly crystalline morphology. In contrast, neither MAP nor MAP-Gly-CPT display any characteristic of ordered crystalline nature. The broadly scattered nature of the diffractograms of both compounds indicates little crystalline organization. Although broad peaks at approximately 19°, 24°, 27°, and 36° theta in the diffractograms of both MAP and MAP-Gly-CPT suggest some potential "disordered" crystallinity, the patterns support the visual assessment that both materials are amorphous. The XRPD pattern for MAP-Gly-CPT is very similar to that of MAP, strongly suggesting that the structure of this solid is dominated by the properties of MAP and that any tendency of the CPT moiety to crystallize is inhibited by its binding to MAP. Development of standards for DAN-222FBDS

[0379] This section presents the rationale for the specifications for the DAN-222 formulated bulk drug substance (FBDS). Each method and initial specification is based on a combination of process capability demonstrated through process development and the capabilities of qualified and / or compendial analytical methods appropriate to the phase.

[0380] The color specification for DAN-222FBDS is a colorless to yellow liquid ≦Y3. This specification incorporates the color range observed in acceptable batches tested and used during development, including batches used in dose range-finding and IND-enabling GLP toxicology studies. The color of the first GMP drug substance batch (Lot 21-M007) is consistent with this limit. The potential for a yellow solution is based on the presence of 20(S)-camptothecin (CPT), which is known to be deep yellow in color. In solid form, both amorphous MAP-Gly-CPT and free CPT are yellow and can impart a light yellow hue to the solution. A color that is more intense than the Y3 color standard (e.g., Y2 or Y1) is an indication of potential degradation.

[0381] The clarity specification for DAN-222FBDS is a clear to slightly opalescent liquid that is ≦standard III. This specification incorporates the range of clarity observed in acceptable batches tested and used during development, including batches used in dose range-finding and GLP toxicology studies. The clarity of the first GMP drug substance batch (Lot 21-M007) is consistent with this limit. DAN-222FBDS is generally clear, but visual assessment may be affected by lighting, viewing conditions, and reflections and refractions from the container.

[0382] Identification of DAN-222FBDS was achieved using a specific combination of four individual analytical results. This multiplex analytical approach is necessary to ensure that DAN-222FBDS, amorphous MAP-Gly-CPT, and free 20(S)-camptothecin (CPT) can be reliably differentiated. The four analytical methods for identification used to differentiate DAN-222FBDS, amorphous MAP-Gly-CPT, and free CPT in solution are (i) ultraviolet-visible spectroscopy (UV-Vis) identification, (ii) appearance, (iii) free CPT percentage (expressed as a percentage of total CPT) measured by reversed-phase high performance liquid chromatography (RP-HPLC), and (iv) weight % of total CPT by RP-HPLC.

[0383] Identification of DAN-222FBDS samples by UV-Vis evaluates two different wavelength ratios (absorbance ratio at 252nm / 356nm; and absorbance ratio at 295nm / 356nm) to indicate identity. These absorbance ratios were found to be unique to CPT-containing compounds. This specification justifies the use of DAN-222FBDS development products as reference standards to establish these absorbance ratios and tolerance ranges.

[0384] As mentioned above, the appearance analysis distinguishes between the amorphous MAP-Gly-CPT (solid) sample and the liquid MAP-Gly-CPT nanoparticle (DAN-222FBDS) sample. This specification is adequate and sufficient to further prove the identity of the liquid FBDS compared to the amorphous MAP-Gly-CPT solid.

[0385] Finally, limits for % free total CPT and % wt total CPT by RP-HPLC are used to distinguish liquid samples of CPT from DAN-222FBDS. These tests are also used to demonstrate that the ratio of CPT present relative to the total drug substance correlates with the ratio expected for DAN-222FBDS. These levels are proven to be reasonable based on the boundary values ​​of the DAN-222FBDS specification presented herein.

[0386] Purity is assessed by the % free CPT (relative to total CPT) in the DAN-222FBDS samples. This value is derived from the calculated concentrations of free and total CPT in the sample. The "% free total CPT" value provides the clearest indication of drug substance stability during storage and use. The DAN-222 construct is designed to limit the acute toxicity of CPT by binding it to a nanoparticle (NP) polymer, thereby controlling the release of this active moiety. The release specification for % free total CPT in the drug substance is ≦3%. The stability specification for % free total CPT in the drug substance is ≦5%. These boundaries are much lower than previous studies using CPT administered directly as a free drug. In two separate 3-week mouse xenograft studies (ER2019-09-1B, ER2019-09-1C), CPT itself was well tolerated dosed at 3 mg / kg, which is comparable to the 9 mg CPT / m 2 has a human equivalent dose of

[0387] Furthermore, these boundaries allow for significantly lower exposure to free CPT for DAN-222 compared to the tolerated human equivalent dose of CPT at the proposed clinical dosing level. For example, a potential high dose cohort for Phase 1 clinical trials would be 16 mg CPT / m 2 In the proposed release standard, the total amount of free CPT is 0.48 mg / m 2 and well below the tolerated dose. The proposed stability specification for ≤5% free CPT is 0.8 mg / m 2 These free CPT exposures are the same as those above for 9 mg / m 2 This is more than an order of magnitude lower than the tolerated human equivalent dose of CPT.

[0388] Assessment of the ratio of total CPT mass to total drug substance mass (wt% total CPT) provides an indication of the performance and control of the MAP-Gly-CPT synthesis process. This criterion is assessed for drug substance manufacturing but will not be repeated for drug product release as the combined assessment of % free total CPT, CPT concentration and %CPT related substances is sufficient to address drug product quality.

[0389] The total CPT weight percent specification is 11-15%. This specification encompasses the mid to high end of the range of CPT loadings explored during process development. The high end of this range is derived from the theoretical maximum CPT loading of MAP-Gly-CPT, assuming that Gly-CPT is conjugated to each available site on the MAP polymer chain. The low end of this specification is at the CPT loading level explored in animal models and successfully demonstrated during process development.

[0390] Measurement of the total CPT concentration in DAN-222FBDS provides the basis for weight-based dosing of DAN-222 in the clinic. The assay value of the total CPT concentration represents the sum of all CPT species present in the vial, including CPT covalently attached to the nanoparticle polymer backbone; free CPT in solution; and possible minor contributions from unreacted Gly-CPT. The drug substance specification range for the total CPT concentration is based on the acceptable total CPT weight % in MAP-Gly-CPT. The specification boundary for the CPT concentration is derived from the minimum acceptable DAN-222FBDS nanoparticle concentration multiplied by the minimum acceptable total CPT weight % value, and the maximum acceptable FBDS nanoparticle concentration multiplied by the maximum acceptable total CPT weight % value. These calculations result in a specification range of 1.7 mg / mL to 3.6 mg / mL. Moreover, this target range of CPT concentration is based on the dose levels desired in Phase 1 clinical trials and incorporates the range of CPT concentrations used in GLP toxicity studies.

[0391] Purity was also assessed by integrating as many as eight individual impurity peaks in the "total CPT" RP-HPLC profile observed thus far in the development batches, believed to represent impurities related to CPT, but which may also include process-related impurities.

[0392] As noted above, eight small impurity peaks were detected by UV absorbance that elute on either side of the unconjugated CPT peak in the "total CPT" RP-HPLC chromatogram (retention times between approximately 4 and 8.5 minutes). For the initial Phase 1 study material, these CPT-related substances have not yet been identified and their response factors determined. However, these impurities were present in the batches used for both the dose range finding and the IND-enabling GLP toxicology study. The specification requires reporting any peaks with an area greater than 0.05% of the total CPT peak area. Any individual peak area must be ≤2% of the total peak area, and the sum of these peak areas must be ≤3% of the total peak area. Note that these limits are measured relative to the CPT content, not the entire drug substance. Considering the weight percent of the total CPT, the individual impurity limits are approximately 0.3 area% of the DAN-222 drug substance, and the sum of the impurity limits is approximately 0.45 area% of the DAN-222 drug substance.

[0393] These boundaries are justified by the presence of these CPT-related substances in the DAN-222 FBDS batches for dose range finding and GLP toxicology. In dose range finding studies, the maximum tolerated dose (MTD) in dogs was 1.5 mg / kg (30 mg CPT / m 2 In a pivotal GLP toxicity study, the highest non-serious toxicity dose (HNSTD) in dogs was determined to be 0.64 mg / kg (12.8 mg CPT / m 2 Using the six-fold safety factor applied to the HNSTD, the proposed initial clinical dose for DAN-222 was determined to be 2 mg CPT / m 2 The total amount of CPT-related substances in the dose range-finding batch and the GLP toxicology FBDS batch is 3% and 2%, respectively, and the human equivalent dose is 1m 2 These values ​​correspond to the proposed initial dose of the DAN-222 drug product (2 mg CPT / m 2 ) exceeds the content of CPT-related substances.

[0394] Nanoparticle concentrations were determined gravimetrically by lyophilizing samples of FBDS to dryness and correcting for non-volatile components of the formulation. Given the planned Phase 1 clinical protocol dosing and the target range for CPT loading (total CPT wt%), a specification of 17-23 mg / mL was selected to provide the desired fill and extractable volumes of the drug product. In-process and stability data indicate that DAN-222 FBDS remains stable in solutions formulated at concentrations as low as 5 mg / mL and as high as 40 mg / mL. Concentrations outside this range may be feasible but have not yet been thoroughly explored.

[0395] The particle size specification for DAN-222FBDS is 20-80 nm. This specification incorporates the particle size range observed in the batches tested and used during development. The lower limit of the specification is set to ensure that a minimum amount of drug substance undergoes renal and hepatic clearance. The upper limit is intended to allow efficient tumor penetration and uptake of the nanoparticles into tumor cells. The nanoparticle sizes for the dose range finding batch and the GLP toxicology batch were 37 nm and 31 nm, respectively.

[0396] The specification for the polydispersity index (PDI) of DAN-222FBDS is ≦0.9. This specification incorporates the range of PDIs observed in the batches tested and used during development. The PDIs for the dose range finding batch and the GLP toxicology batch were 0.463 and 0.346, respectively.

[0397] Zeta potential is a measure of surface charge. The specification for zeta potential of DAN-222FBDS is -5mV to +5mV. This specification incorporates the range of zeta potentials observed in batches tested and used during development. All DAN-222FBDS development batches exhibited a near neutral zeta potential. This value is typically slightly negative, although it is possible for measurements to return a slightly positive charge. The zeta potentials measured for the dose range finder batch and the GLP toxicology batch were -0.751mV and -0.399mV, respectively.

[0398] The specification for osmolality of DAN-222FBDS is 336-504 mOsm / kg. This specification for osmolality is based on results from replicate measurements of various development batches of DAN-222FBDS, including GLP toxicology batches. This range is representative of the osmolality of formulation buffers, as the DAN-222 molecule has little effect on osmolality results. The proposed specification range is representative of osmolality boundaries for drug substance and drug product using comparable buffer formulations.

[0399] The pH specification for DAN-222FBDS is 4.3±0.3. This pH specification was established based on a series of formulation development studies to determine the optimal pH for stability of DAN-222FBDS. By design, free CPT is slowly released from DAN-222 at elevated pH levels. Controlling the pH within pH 4.3±0.3 provides consistent stability for handling and use of the dosage form, as well as reasonable boundaries for storage of drug substance and drug product. The proposed specification is within the normal range for parenteral drug substance and drug product.

[0400] The standard for endotoxins (≤ 6 endotoxin units (EU) per mg of CPT) is set by the United States Pharmacopoeia <85> Based on the permissible endotoxin exposure for 1 hour in accordance with the FDA. The recommended endotoxin limit for parenteral products dosed by patient surface area is 100EU / m, where m is the maximum proposed dose per square meter over 1 hour. Claiming a higher dose than expected would result in a CPT value of 6.25EU / mg, which is rounded to 6EU / mg CPT for the standard.

[0401] Specifications for bioburden limits for aerobic and anaerobic bacteria, yeasts, and molds are set out in the United States Pharmacopeia. <61> The respective assigned values ​​(≤10 colony forming units (CFU) / 10 mL) are set to both demonstrate microbial control of the process and to ensure that the potential microbial load is within the removal capacity of the filtration equipment if sterile filtration of the final drug product is performed immediately prior to filling into vials. Example 6 Method for manufacturing DAN-222 formulated bulk drug substance (FBDS)

[0402] This exemplary DAN-222FBDS manufacturing process consists of four major steps: (i) derivatization of camptothecin (CPT) to obtain Gly-CPT (as the trifluoroacetic acid (TFA) salt), (ii) synthesis of the parent mucic acid polymer (MAP), (iii) covalent attachment of the derivatized camptothecin to the parent polymer to obtain a solid amorphous MAP-Gly-CPT polymer-drug conjugate, and (iv) aqueous formulation of MAP-Gly-CPT to form nanoparticles (NPs).

[0403] Figure 35 presents a scheme for an exemplary method of manufacturing DAN-222FBDS. The starting material is shown in the shaded box at the top of the drawing, and the manufacturing steps performed under GMP conditions for the Phase 1 clinical trial drug substance are shown with text or surrounded by box 3401. Step 1: Derivatization of camptothecin to obtain Gly-CPT

[0404] FIG. 36 shows the derivatization of CPT to synthesize Gly-CPT.

[0405] Gly-CPT is isolated as the TFA salt. All reactions and chemical workups are carried out in glassware using equipment and techniques common to synthetic chemistry procedures. No specialized equipment or catalysts are required for the synthesis of Gly-CPT. Because both CPT and Gly-CPT are highly potent compounds, the synthetic method is controlled under safe handling procedures consistent with good industrial safety practices.

[0406] Figures 37A-37B present a summary flow chart of the Gly-CPT synthesis method, including control points. The flow chart begins in Figure 37A and continues in Figure 37B. A glass reactor is charged with methylene chloride (DCM) and 3 equivalents of N-(tert-butoxycarbonyl)-Gly-OH (Boc-Gly) to a Boc-Gly concentration of approximately 0.06 g Boc-Gly / mL. The solution is stirred for 5-10 minutes, 4-dimethylaminopyridine (DMAP; 2 equivalents) and 20(S)-camptothecin (CPT; 1 equivalent) are added, and the reaction mixture is stirred at 25-30°C for approximately 10 minutes. The reaction is cooled to 0-2°C and 3 equivalents of diisopropylcarbodiimide (DIC) are slowly added, maintaining the temperature at 0-2°C. The reaction is warmed to 25-30°C and stirred for approximately 6 hours.

[0407] The reaction mixture is filtered through a Büchner funnel fitted with a filter cloth to remove any suspended solids and the filter is washed with DCM (approximately 0.5x the original DCM volume, here represented as V). The filtrate containing the desired product is transferred to a new glass vessel and extracted twice with water (each extraction is approximately 0.5x V). The organic layer is concentrated under vacuum in a rotary evaporator to remove 50-70% of the original total volume. The distillate is transferred to a new vessel and methanol (MeOH; approximately 0.6x V) is added to precipitate the intermediate product. The reaction is cooled to 5-10 °C and stirred at 5-10 °C for approximately 1 h. The reaction is filtered through a Büchner funnel fitted with a filter cloth and washed with MeOH (approximately 0.2x V). The wet compound is dispensed from the filter into a new vessel and isopropyl alcohol (IPA; approximately 0.5x V) is added and the reaction is stirred for approximately 30 min. The reaction mixture is filtered through a Büchner funnel with a liner. The collected solid is washed with IPA (approximately 0.2x V), dispensed from the filter, and dried under vacuum at ambient temperature for at least 4 hours to remove excess solvent. The purified Boc-Gly-CPT solid intermediate is stored at ambient temperature in a low density polyethylene (LDPE) bag placed inside a secondary high density polyethylene (HDPE) container.

[0408] The desired intermediate Gly-CPT is obtained by deprotection of Boc-Gly-CPT using trifluoroacetic acid (TFA) as the deprotecting agent. A reactor is charged with DCM and Boc-Gly-CPT to a Boc-Gly concentration of approximately 0.5 g Boc-Gly-CPT / mL. The reaction is stirred for 5-10 minutes. An equal volume of TFA is added and the reaction is stirred for approximately 2 hours at 25-30°C.

[0409] The reaction is cooled to 0-5 °C and approximately 1.5 volumes of methyl tert-butyl ether (MTBE) is added, maintaining the temperature at 0-5 °C. The reaction is stirred at 0-5 °C for approximately 30 minutes. The resulting suspension is filtered through a lined Büchner funnel under nitrogen and the filter cake is washed with a 2:1 MTBE:DCM mixture (approximately 2x the amount used to dissolve Boc-Gly-CPT, here represented as V') and MTBE (approximately 2x V'). This step is repeated with somewhat shorter stirring times and the isolated solid is dried under nitrogen for approximately 1 hour and then dried under vacuum at ambient temperature for at least 4 hours to remove excess solvent. The purified glycyl-camptothecin (TFA salt; Gly-CPT) solid is stored at ambient temperature in an LDPE bag in a secondary HDPE container.

[0410] For the initial Phase I production, three Gly-CPT-TFA batches were produced with batch sizes of 145-150 g. Step 2: Synthesis of the parent MAP polymer

[0411] MAM and di-SPA-PEG 3500 The preparation of MAP from copolymerization with is shown in Figure 38. All reactions are carried out in glass vessels using equipment and techniques common to synthetic chemistry procedures. No specialized equipment or catalysts are required for the synthesis of MAP, although care must be taken to remove as much water as is practical from the reaction mixture.

[0412] Figure 39 presents a summary flow chart of the MAP synthesis method, including control points. Add 1 equivalent of (succinimidyl propionate) to the reactor. 2 PEG 3500 (di-SPA-PEG 3500 ) and 1.07 equivalents of mucine acid monomer (MAM). The reactor is purged with argon and dimethyl sulfoxide (DMSO) is added to disperse the di-SPAPEG. 3500 The concentration of di-SPA-PEG was approximately 0.17 g. 3500 / mL. Trifluoroacetic acid (TFA) (1.8 equiv.) is added and the reaction is stirred for 10-15 min. N,N-diisopropylethylamine (DIPEA) (2.6 equiv.) is added and the reaction is stirred for approximately 3 h at 21-23 °C. These parameters are designed to ensure that the resulting MAP molecular weight is within the target range (50,000-80,000 Da).

[0413] The MAP is precipitated with isopropyl alcohol (IPA) (approximately 2x the total DMSO volume). The reaction is cooled to 0±5° C. Approximately 5 volumes of IPA are added and the reaction is stirred at 0±5° C. for approximately 50 minutes. The polymer is filtered through a Buchner funnel under argon, washed twice with IPA (each wash is approximately 3x the total DMSO volume) and twice with diethyl ether (each wash is approximately 3x the total DMSO volume), dispensed from the filter into a pyrex® drying dish, and dried under vacuum at ambient temperature for at least 12 hours to remove excess solvent. The solids are stored at ≦−20° C. in 1000 mL wide-mouth glass jars with HDPE-lined stoppers.

[0414] For Phase I, MAP was manufactured under GMP conditions in three batches with an average isolated yield of approximately 185 g of copolymer. Step 3: Synthesis of amorphous MAP-Gly-CPT polymer-drug conjugates

[0415] Amorphous MAP-Gly-CPT drug substance showed no instability when exposed to typical laboratory lighting conditions, however, CPT is known to be light sensitive, therefore all manufacturing steps in the presence of CPT were performed with a red lighting filter in place.

[0416] Conjugation of MAP to Gly-CPT to synthesize MAP-Gly-CPT is shown in Figure 40. MAP-Gly-CPT is isolated as an amorphous solid. Nanoparticles (NPs) are formed upon reconstitution of amorphous MAP-Gly-CPT in aqueous media. All reactions and chemical workups are carried out in glass vessels or polymeric single-use bags using equipment and techniques common to synthetic chemistry procedures. No uncommon specialized equipment or catalysts are required for the synthesis of amorphous MAP-Gly-CPT. Because CPT is a highly potent compound, the synthetic method is controlled under safe handling procedures consistent with good occupational safety practices.

[0417] Figures 41A-41B present a summary flow chart of the amorphous MAP-Gly-CPT synthesis method, including control points. The flow chart begins in Figure 41A and continues in Figure 41B. Charge the reactor with 1 equivalent of MAP and purge with argon. Add dimethylsulfoxide (DMSO) to a MAP concentration of approximately 0.01 g MAP / mL. Add glycyl-camptothecin (TFA salt; Gly-CPT; 1.2 equivalents) and 2.2 equivalents of (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and stir the reaction for 5-10 minutes. Add N,N-diisopropylethylamine (DIPEA; 2.5 equivalents) and stir the reaction at 19-25°C for 16-22 hours.

[0418] The reaction is quenched by slow addition of cold pH 3.0 water (compounded from water for injection (WFI), approximately 0.4x the total amount of DMSO) to maintain the reaction temperature below 30°C. The reactor temperature is set to 10°C and additional cold pH 3.0 water (approximately 2.6x the total amount of DMSO) is charged to the reactor and stirred for approximately 30 minutes. The solution is transferred to a stirred polymer bath system with a polyethylene film bath liner and an additional 1x volume of cold pH 3.0 water is added. The total amount of water added is intended to reduce the concentration of DMSO to approximately 20%, as the TFF membrane used for further purification is stable at this DMSO concentration. If immediate purification is not desired at this point, the solution may be stored in a cold room and protected from light.

[0419] The solution is purified by ultrafiltration / diafiltration using tangential flow filtration (TFF) techniques. The process is carried out using a Sartorius FlexAct system with a Sartocon self-contained filter loop assembly holding a 10,000 Da molecular weight cut-off TFF membrane. Alternatively, the FlexAct system can be carried out using a separate Sartocon Hydrosart cassette mounted in a conventional TFF membrane holder. The process begins with an ultrafiltration step during which the total volume of the product solution is reduced by approximately half. At this point, diafiltration is initiated against at least 8 diavolumes of pH 3.0 water to significantly reduce the DMSO content and remove low molecular weight water soluble impurities. Once the diafiltration step is complete, further ultrafiltration is performed to concentrate the solution to approximately 0.04 g / mL of MAP-Gly-CPT. The concentrated solution is frozen and lyophilized for at least 48 hours to obtain the purified MAP-Gly-CPT drug substance intermediate as an amorphous solid. The solids are stored at ≦−20° C. in wide-mouth glass bottles with HDPE-lined stoppers.

[0420] In the event of a failure of the TFF membrane system that allows the MAP-Gly-CPT drug substance to flow into the permeate collection vessel, an opportunity exists to reprocess the permeate. This is done by installing a new TFF membrane unit, pumping the permeate containing the product back into the FlexAct system, and concentrating this solution by ultrafiltration. Once concentration is complete, an additional diafiltration against pH 3 water is performed prior to concentration and lyophilization.

[0421] For Phase I, MAP-Gly-CPT was produced in two batches with a batch size of 250 g MAP, with an average yield of approximately 250 g of conjugate. Step 4: Preparation of MAP-Gly-CPT NP solution (DAN-222)

[0422] The aqueous formulation of MAP-Gly-CPT as a NP solution (DAN-222 formulated bulk drug substance (FBDS)) is shown in FIG.

[0423] All steps are carried out in glass containers or polymeric single-use bags using equipment and techniques common to synthetic chemistry procedures. No unconventional specialized equipment or catalysts are required to formulate MAP-Gly-CPT NP solutions. Because CPT is a highly potent compound, the formulation process is controlled under safe handling procedures consistent with good industrial safety practices.

[0424] Figures 43A-43B present a summary flow chart of the DAN-222FBDS process, including control points. The flow chart begins in Figure 43A and continues in Figure 43B. DAN-222FBDS is formulated in 20 mM acetate, 0.9% sodium chloride buffer, pH 4.3±0.3. To achieve this buffer concentration, a 50 L pallet tank with mixer is charged with WFI, and sufficient sodium chloride, sodium acetate, and glacial acetic acid to obtain a 10× concentration of the desired final buffer.

[0425] A 100 L reactor is washed with water and acetone, then heated to 120° C. under a positive argon flow to sterilize the vessel. The reactor is charged with solid amorphous MAP-Gly-CPT. 3 PO 4 Add water for injection (WFI) adjusted to pH 4.0 with 500 mL of water to achieve a MAP-Gly-CPT concentration of approximately 0.005 g / mL MAP-Gly-CPT and stir the mixture for at least 12 hours. Transfer the MAP-Gly-CPT NP solution to a vessel fitted with a polypropylene vessel liner via tubing fitted with a 0.45 μm filter. Rinse the 100 L reactor thoroughly and transfer the NP solution back to the reactor and stir. Measure the amount of NP solution in the reactor and calculate the appropriate amount of 10X formulation buffer to add slowly to the reactor to avoid splashing. Stir the resulting solution for 2-3 hours and transfer to a HDPE vessel fitted with a polypropylene vessel liner.

[0426] The calculated concentration of MAP-Gly-CPT in the tank is determined by adjusting the total charge of amorphous MAP-Gly-CPT by the residual solvent value obtained from the material. This calculation gives the target weight of permeate to be removed during concentration by ultrafiltration. A Sartorius Flex-Act system equipped with a Sartocube ECO tangential flow filtration cassette with a MW cutoff of 10,000 Da is used. The solution is concentrated by ultrafiltration until the target weight of permeate is reached. The retentate is transferred to a 20 L Flexboy bag, sampled for in-process control (MAP-Gly-CPT concentration), and stored at 2-8 °C until analysis is complete. The target concentration range for DAN-222 FBDS is 17-23 mg / mL MAP-Gly-CPT. Once that range is reached, the solution is prepared in a 20 L Flexboy bag for sterile filtration, sampling, and bottling into FBDS containers. If the concentration is too low, use the measured volume and concentration to calculate the additional amount of permeate to be removed and return the solution for further ultrafiltration. If the concentration is found to be outside the specified range, calculate and add the appropriate amount of 1X formulation buffer to be added and gently agitate the vessel to blend.

[0427] Transfer of DAN-222 FBDS to storage containers is performed in a biosafety cabinet (BSC) for improved microbial control. The solution is transferred to sterile 4 L HDPE bottles using a MasterFlex pump and a sterile tubing set fitted with a 0.22 μm Sartopore 2 MidiCaps® filter. After sampling for release, stability and retention purposes, the 4 L bottles are filled. Prior to filling each bottle, two or three 5 mL samples are dispensed into sterile Falcon tubes, two of which serve as satellite samples for each bottle. Following this sampling, each tared bottle is filled with approximately 3 L of solution, stoppered, tightly sealed and weighed to determine the net weight of the contents. All FBDS containers containing samples are labeled.

[0428] DAN-222FBDS containers will be stored at a controlled temperature of ≦-70°C, except for samples undergoing immediate analysis.

[0429] For Phase I, DAN-222FBDS was made as a single batch of amorphous MAP-Gly-CPT with a batch size of 400 g. From the resulting formulation, 17.4 kg (17.4 L) of DAN-222FBDS was obtained for further use, and 751 mL of DAN-222FBDS was taken as a sample for release, stability, and as a satellite sample for characterization at the drug product filling facility. Example 7 Improved Manufacturing of DAN-222 Formulated Bulk Drug Substance (FBDS)

[0430] Process development for GMP manufacturing of DAN-222 formulated bulk drug substance (FBDS) has progressed and improved from its initial development to the exemplary method outlined in Example 6. The current process consists of four GMP steps. First, 20(S)-camptothecin (CPT) is derived by linking glycine to the 20-hydroxyl group of CPT (Gly-CPT). Second, parent mucic acid polymer (MAP) is synthesized by conjugation of the comonomer mucic acid monomer (MAM) with (succinimidyl propionate). 2 In the third step, the derivatized CPT is covalently attached to the parent polymer to obtain an amorphous MAP-Gly-CPT polymer-drug conjugate. Finally, the conjugate is formulated in an aqueous medium to form a nanoparticle (NP) solution.

[0431] Following initial process validation for producing DAN-222(FBDS), process development efforts continued, resulting in process development, scale-up, and manufacturing of DAN-222FBDS for IND-enabling and Phase 1 clinical trials. Step 1: Derivatization of camptothecin to obtain Gly-CPT - Development

[0432] The generation of derivatized CPT is a two-step process whereby the hydroxyl group of CPT is esterified with a tert-butoxycarbonyl-protected glycine linker (Boc-Gly), which is then isolated by removal of the Boc protecting group to give glycine-linked CPT (Gly-CPT) as the trifluoroacetic acid (TFA) salt.

[0433] The initial process in step 1 involves dissolving CPT, Boc-Gly, and 4-dimethylaminopyridine (DMAP) in dichloromethane (DCM). The reaction is initiated by the addition of diisopropylcarbodiimide (DIC) and the intermediate is isolated by precipitation with methanol (MeOH). To remove the Boc group, the intermediate is stirred with a mixture of trifluoroacetic acid (TFA) and DCM. The Gly-CPT product is isolated by precipitation as the TFA salt with methyl tert-butyl ether (MTBE). During early development, Gly-CPT was synthesized on a research scale of approximately 1 gram per synthesis.

[0434] This development process resulted in changes during synthesis and isolation, primarily related to optimization of solvent volumes and reaction temperatures at initial GMP scale. Although the quantities of this highly potent intermediate required for initial clinical trials were relatively small, after continued development including a 10 gram synthesis, it was decided to perform an almost 15-fold scale-up synthesis (150x relative to research scale) for initial GMP production.

[0435] Over the course of the development of Step 1, several modifications were made to the process to make it more robust, scalable, safer, and / or have lower impurity levels. A summary of these changes is presented in Table 7. These changes primarily address reaction concentrations and solvent volumes, reaction temperatures, and several additional steps to improve the purity of intermediates. [Table 7-1] [Table 7-2]

[0436] For the Boc-Gly-CPT synthesis step, the relative ratios of reactants remained constant, but the reaction was found to proceed more smoothly when carried out under more dilute conditions at a higher reaction temperature. A new aqueous extraction step was added to improve the removal of process impurities. Whereas in previous syntheses, DCM was removed to obtain a crude solid, a vacuum distillation was performed to remove only 95% of the DCM amount, and a significantly lower percentage of methanol was added for the final precipitation at a higher temperature. An additional wash with isopropyl alcohol (IPA) was found to improve the isolation performance.

[0437] For the deprotection and isolation of Gly-CPT the same reagent ratios were used as in the previous synthesis, however for the subsequent precipitation step a significantly smaller volume of methyl tert-butyl ether (MTBE) was used and post-washing of the filter cake with a mixture of MTBE:DCM was performed with a lower ratio of MTBE:DCM.

[0438] During the development of an analytical method for the release of the Gly-CPT intermediate, it was discovered that the level of residual MTBE was not reduced by vacuum drying, suggesting that this intermediate crystallizes as a solvate. 1 H-NMR analysis showed that similar levels of MTBE were present in that batch as well.

[0439] It was also found that even with drying agents (magnesium sulfate and sodium sulfate) and azeotropic distillation under mild conditions, residual water could not be completely removed from the final product. In both cases, the conditions caused the decomposition of the glycyl esters. Given that the subsequent conjugation of Gly-CPT with MAP is sensitive to the presence of excess water, future process development for this step will include methods to reduce or eliminate the presence of water in the intermediate product. Step 2: Synthesis of the parent MAP polymer – Development

[0440] The production of the parent polymer, mucic acid polymer (MAP), is a step-growth polymerization of two comonomers, MAM and diSPA-PEG, a linear PEG molecule with a succinimide-activated propionyl ester at each end. These comonomers are dissolved in dimethylsulfoxide (DMSO) and the reaction is initiated by the addition of N,N-diisopropylethylamine (DIPEA), resulting in the amidation of the free amines at each end of the MAM monomer. When the molecular weight (MW) of the MAP is expected to be at or near the target value, the reaction is quenched with water and the MAP is isolated by dialysis from DMSO and water and then lyophilized.

[0441] The first process aims to control the reaction rate to bring the polymer MW close to a plateau over time. Although conditions for MAP production to grow MW nearly linearly with time can be identified, this strategy requires precise in-process monitoring of MW and quenching of the reaction. Alternatively, given that polymerizations have been shown to follow known models for stepwise growth of polymers, a strategy was developed based on polymerization theory (modified Carothers equation and Flory-Shulz distribution) to stall the reaction over time. This strategy achieves a plateau of growth by exploiting the stoichiometric imbalance of comonomers, and the reaction slows down as the MAP approaches the target MW. An example of a polymerization in which the MAP MW at a later time point is controlled by the stoichiometric ratio of MAM to diSPA-PEG in the reaction mixture is shown in Figure 44.

[0442] As shown in Figure 44, using a plateau growth strategy and increasing the ratio of MAM:diSPA-PEG results in polymers with lower MW.

[0443] Based on the above observations, it was hoped that monomer starting materials with a high degree of functionalization as specified herein would be prepared, and that the reaction would be carried out under anhydrous conditions to maintain the stability of the activated SPA groups on the diSPA-PEG to protect against premature termination of the amidation reaction, allowing for good control of the comonomer stoichiometry at or near the target.

[0444] Over the course of the development of Step 2, several modifications were made to this process to make it more robust, scalable, safer, and / or reduce impurity levels. These modifications included (i) using MAM as a di-TFA salt instead of a neutral species, (ii) the MAM, di-SPA-PEG, TFA, and DIPEA are stoichiometric, and (iii) using dialysis instead of precipitation for purification.

[0445] Initial development batches of MAP were made using a MAM starting material that was intended to be isolated as a di-TFA salt. Subsequent isolation of the MAM with a consistent amount of counterion, TFA, proved difficult. Analysis of the first few MAM development batches revealed that the TFA:MAM equivalents varied from batch to batch by an order of magnitude of about 25% (e.g., 1.5:1 in practice vs. 2:1 as a goal). This variation would likely lead to significant imprecision in the amount of MAM added to the reaction, and therefore significant variation in the MW of the resulting polymer, given the sensitivity to comonomer ratios noted above. To reduce potential variability, it was decided to isolate the MAM as a neutral species (no TFA counterion), allowing for more precise addition of TFA as a subsequent reaction step prior to polymerization. Figure 45 presents the chemical structure of the MAM starting material for comparison.

[0446] To study the effect on reaction behavior of the presence or absence of TFA in the monomer starting material, reactions were carried out using MAMs isolated as di-TFA salts or as neutral species (isolated with only traces of TFA). Although the TFA group does not participate in the actual polymerization reaction, it was found to significantly increase the solubility of the MAMs in DMSO and therefore the reaction rate. Unlike the di-TFA salts, which are generally soluble in DMSO, the neutral species was found to be only minimally soluble in DMSO. Examination of the polymerization of the neutral species with di-SPA-PEG showed that despite the poor solubility of the neutral species, the reaction proceeded without the addition of TFA, albeit at a generally linear rate, independent of the comonomer ratio. In contrast, polymerization of the neutral species with di-SPA-PEG by adding approximately 1-2 equivalents of TFA to the reaction prior to initiation with DIPEA resulted in restoration of the desired plateau behavior.

[0447] Based on the results obtained from the initial experiments, it was decided to explore the development of reaction conditions using MAM isolated as a neutral species, and the subsequent addition of TFA that would allow the polymer MW to plateau at or near the target.

[0448] During initial development studies with the new MAM monomer and subsequent addition of TFA, several small-scale (~250 mg diSPA-PEG) screening reactions were performed to identify conditions that appeared to enable the desired plateau behavior of MAP polymerization. Results from two sets of multiple experiments (screening 1 and screening 2) are presented as examples to demonstrate key findings that informed the selection of final reaction conditions.

[0449] An initial set of experiments (Screening 1) was conducted to explore the combined effect of various comonomer ratios, as well as equivalents of TFA and DIPEA, on the rate of the reaction. The reaction conditions explored in Screening 1 are presented in the lower portion of Figure 46, where italicized items highlight the differences in conditions. The MW values ​​of MAP using the conditions specified in Screening 1 are also shown in the upper portion of Figure 46.

[0450] The results shown in Figure 46 show that decreasing the amount of DIPEA (approximately 2.5 equivalents) slowed down the reaction rate. As expected, decreasing the MAM:diSPA-PEG ratio to 1:1 increased the MW. Furthermore, increasing the TFA was found to improve the MW plateau. Combined with the results of the initial screen, reaction 1-B shows favorable behavior, suggesting that the comonomer ratio, TFA and DIPEA equivalents, and reaction time are sufficient measures to achieve the targeted MAP polymerization.

[0451] Based on the above observations, a second panel of conditions (Screen 2) was evaluated to further explore the effect of TFA and DIPEA levels on the rate of reaction at slightly reduced comonomer ratios and to optimize the conditions. The reaction conditions explored in Screen 2 are presented in the lower portion of Figure 47, where italicized items highlight the differences in conditions. The MW values ​​of MAP using the conditions specified in Screen 2 are shown in the upper portion of Figure 47.

[0452] As shown in the results in Figure 47, a slight decrease in the comonomer ratio was found to result in a slight increase in MW. Furthermore, increasing the TFA concentration also improved the reaction plateau, while decreasing the DIPEA concentration slowed the reaction rate. As a result of Screen 2, conditions were identified for small-scale polymerization (Reaction 2-C) that allowed for a plateau in MW and resulted in MAPs with a target MW range of 50,000-80,000 Da over a wide time frame.

[0453] The dependence of MW on comonomer ratio and equivalents of TFA and DIPEA was reproduced at the 1 g scale and yielded similar trends, although a slight decrease in rate was observed for all reactions. This behavior was likely due to slightly less stringent anhydrous conditions under which the reactions were carried out. As a result, for the scale-up reactions, a slight increase in DIPEA (2.6 vs. 2.5 equivalents) was evaluated, yielding rates similar to those observed in the small-scale experiments.

[0454] The decision to change the purification process from dialysis and freeze-drying to precipitation was made to allow for shorter processing times and to avoid the need for large volumes of buffer and long processing times during isolation of MAP. During the development of MAP production, it was observed that large volumes of dialysis media and long drying times in the freeze dryer were required to isolate the polymer as a solid. It was predicted that with increasing scale, there would most likely be increased volume requirements, longer processing times and / or the development of a tangential flow filtration (TFF) method.

[0455] To mitigate these process risks, the feasibility of isolating MAP as a solid by precipitation was evaluated, which would eliminate the need for dialysis and lyophilization during scale-up and allow for a significant reduction in process time. Precipitation was performed from DMSO by addition to several anti-solvents. Of the various solvents evaluated, isopropyl alcohol (IPA) proved to be promising. Precipitation of the polymer by direct addition of IPA to the polymerization reaction mixture was also attempted and found to work equally well. MW analysis of MAP after precipitation and dialysis showed that the chromatograms, as well as the resulting MW and PDI, were comparable. Furthermore, the precipitated MAP 1H-NMR analysis showed very low levels of impurities (<0.1% by mass). However, filtration under vacuum of precipitated MAP in ambient atmosphere resulted in a sticky solid material that was difficult to remove from the filter. Filtration under a nitrogen or argon head circumvented this behavior and gave a reasonably dry solid that could be easily removed from the filter for further drying.

[0456] Based on the above observations, it was decided to proceed with the IPA precipitation procedure with an argon head during filtration to isolate MAP in subsequent scale-up and GMP batches.

[0457] Following process development, scale-up reactions were performed to confirm the optimized reaction conditions and the resulting reaction kinetics. As previously described, a slight increase in DIPEA was used for the scale-up production compared to the conditions developed during the production of the development batches. In addition, a slight rather than a complete plateau towards the upper molecular weight limit (80,000 Da) of MAP was targeted, rather than the midpoint (65,000 Da), to allow for a wider time window (several hours) for quenching and to eliminate the risk of not reaching the desired MW.

[0458] Scale-up reactions were performed at 5g and 50g scales using the previously identified conditions (1.07:1 MAM:diSPA-PEG, 1.8 equivalents TFA, 2.6 equivalents DIPEA). Three development runs were performed at 5g scale and proceeded for 5 hours to monitor the progress of the long reaction times. These three runs showed good reproducibility and favorable reaction kinetics, provided information on the target quench of the reaction at approximately 3.5 hours, and demonstrated the achievement of a final MAP MW within the target MW range. To aid in GMP manufacturing, a 50g scale reaction was also performed and quenched by the addition of IPA at 3.5 hours. As expected, the large scale reaction proceeded at a similar rate and resulted in a MAP MW within the target range. Additionally, direct precipitation of the reaction mixture by slow addition of IPA afforded the polymer as a white precipitate, which was successfully isolated by filtration under argon atmosphere and dried to obtain a solid.

[0459] MW data for scale-up of the MAP reaction is presented in Figure 48. GMP production was performed at 165 g, approximately a 3-fold scale-up of 50 g, as described in Example 6. Step 3: Synthesis of amorphous MAP-Gly-CPT polymer-drug conjugates - Development

[0460] The generation of the polymer-drug conjugate, MAP-Gly-CPT, involves the reaction of a carboxylic acid on the parent MAP polymer with a derivatized CPT amine to form an amide bond. The initial process utilizes EDC / NHS chemistry to effect this covalent coupling according to the conditions listed in Table 8 (reaction dilutions are italicized). [Table 8]

[0461] MAP is dissolved in DMSO and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS) and Gly-CPT are added. The reaction is initiated by the addition of DIPEA. After completion of the reaction, MAP-Gly-CPT is isolated by dialysis against DMSO and water at pH 3.0, then lyophilized.

[0462] Over the course of the Step 3 development, several modifications were made to this process to make it more robust, scalable, safer, and / or reduce impurity levels. The first was the use of EDC / NHS-based coupling and PyAOP-based coupling. The second was the use of dialysis and TFF purification.

[0463] Initial development batches of MAP-Gly-CPT were made using EDC / NHS coupling chemistry, which requires a moderate (2-3 fold) excess of Gly-CPT starting material and reagents to achieve the desired drug loading level. As a result, it was found that large volumes of dialysis media and long processing times were required to adequately remove unreacted starting material and other impurities from the product.

[0464] To reduce the excess of starting materials and reagents in the reaction, especially that of Gly-CPT, the use of the more active (7-azabenzotriazol-1yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) coupling reagent was evaluated to prepare MAP-Gly-CPT. Given the increased reactivity, it was expected that PyAOP would allow for higher coupling efficiency and therefore reduced levels of Gly-CPT in the reaction compared to the previous EDC / NHS route.

[0465] An initial series of reactions was carried out to explore the coupling efficiency with PyAOP over a range of Gly-CPT equivalents added to the reaction. As expected, nearly quantitative grafting efficiency (>80%), defined as the efficiency of incorporation of Gly-CPT into the MAP-Gly-CPT conjugate material, was achieved across all conditions. In addition to high coupling efficiency, the achieved drug loading levels showed a linear relationship with the equivalents of Gly-CPT added to the reaction, demonstrating significant control over drug loading at this process step. The equivalents (eq.) of Gly-CPT and the resulting weight percent (wt.%) of CPT and grafting efficiency (percent) of representative conjugation reactions are presented in Table 9. [Table 9]

[0466] The grafting efficiency and linear relationship are shown in Figure 49. The data shown in the table for samples A-F are consistent with the results shown in Figure 49.

[0467] However, subsequent development batches revealed sometimes unexpected observations related to the reaction itself and / or to the NPs derived from the conjugate material linked to PyAOP. In one experiment, MAP-Gly-CPT linked to PyAOP formed NPs with larger than normal diameter (~70 nm) that were also difficult to filter. In another attempt, gel formation was observed during the reaction. It was speculated that crosslinked conjugate material could be formed by possible reaction of the amine end groups of the MAP polymer with activated carboxylic acid groups on other polymer chains, resulting in the generation of atypical high molecular weight species (HMWS). Although preliminary evaluations seemed to confirm such a phenomenon, this observation has yet to be investigated in detail. Further modifications to this process were explored to enable the use of PyAOP for Gly-CPT coupling.

[0468] To minimize the process risk of HMWS formation, it was decided to target a high drug loading level to minimize the number of unreacted carboxylic acid groups. The reaction was also modified to run at high dilution, which is known to significantly reduce the likelihood that the end groups of the polymer chains will participate in the reaction. Additional development reactions were run at high dilution on both the 2.5 g and 4 g scales with 1.0, 1.1, and 1.2 equivalents of Gly-CPT. Using the modified reaction conditions, the reactions all proceeded well and did not show the formation of HMWS. It was concluded that these changes were sufficient to minimize the formation of HMWS.

[0469] A comparison of the initial and subsequent modified reaction conditions for PyAOP-based coupling is shown in Table 10, with reaction dilutions in italics. [Table 10]

[0470] Based on the above observations, it was decided to proceed with a modified PyAOP-based coupling procedure in subsequent scale-up and GMP batches in order to reduce the excess of starting materials and reagents, thereby reducing the buffer volumes and processing times required during purification of the conjugate.

[0471] The decision to change the purification method from dialysis with TFF to ultrafiltration / diafiltration was based on volume requirements and practicality. Scaled-up versions of dialysis require significantly longer processing times and larger solution volumes and are generally not practical at the scale required for GMP batches. Precipitation was also evaluated with several anti-solvents but did not initially show promise. Instead, the focus was on attempting to develop a TFF process to purify the conjugate. Polyethersulfone (PESU) TFF membranes were found to be compatible with aqueous solutions containing up to 10% DMSO, so it was decided to dilute the reaction mixture 10-fold with water at pH 3.0 before performing TFF (subsequently, PESU membranes were found to tolerate up to 20% DMSO in water and for GMP production the dilution was reduced accordingly). TFF was performed on several 4g batches of MAP-Gly-CPT using scaled-down TFF membranes and cartridges equivalent to those used in the scaled-up GMP configuration. A diafiltration volume of 8 was found to be sufficient to reduce DMSO and impurities to acceptable levels.

[0472] Based on the above observations, it was decided to proceed with TFF purification to isolate MAP-Gly-CPT in subsequent scale-up and GMP batches.

[0473] Following process development, scale-up reactions were performed to confirm modified reaction conditions and purification methods. Two scale-up reactions were performed at 25 g scale to support GMP manufacturing using previously identified conditions (1.2 eq. Gly-CPT, 2.2 eq. PyAOP, 2.5 eq. DIPEA, 100 mL DMSO / g MAP). These duplicate runs showed good reproducibility and achieved drug loading within the target range for MAP-Gly-CPT. Furthermore, TFF purification performed comparable to the development run, with sufficiently low levels of DMSO and impurities after diafiltration of 8 volumes of pH 3.0 water. The solid conjugate was successfully isolated by lyophilization.

[0474] GMP manufacturing was carried out at 250 g, a standard one order of magnitude scale-up from 25 g, as described in Example 6. Step 4: Preparation of MAP-Gly-CPT NP solution (DAN-222)

[0475] Preparation of MAP-Gly-CPT NPs requires dissolution of an amorphous polymer-drug conjugate in an aqueous medium, whereby the hydrophobic nature of CPT drives the self-assembly of the conjugate into NPs. The initially developed process involved dissolving the amorphous MAP-Gly-CPT material in water at pH 4.0, adding concentrated saline (9% NaCl, pH 4.0), concentrating using a centrifugal ultrafiltration device, and 0.22 micron sterile filtration.

[0476] Over the course of Step 4 development, several modifications were made to this process, including (i) changing the concentrated formulation buffer, (ii) using centrifugal ultrafiltration instead of TFF concentration, and (iii) a conditioning filtration step, making the process more robust, scalable, safer, and / or reducing impurity levels.

[0477] While MAP-Gly-CPT NPs showed good stability in unbuffered saline solution (0.9% NaCl, pH 4.0), FBDS is pH sensitive and a robust drug product formulation requires a buffer system that protects against pH fluctuations and deterioration during storage. Based on the formulation development efforts described above, it was decided to change from the addition of concentrated saline to the addition of a concentrated formulation buffer (200 mM sodium acetate, 2 M NaCl, pH 4.16) to support GLP toxicity studies and subsequent preparations. The resulting target DP formulation for MAP-Gly-CPT NPs is 20 mM sodium acetate, 200 mM NaCl (pH 4.3 ± 0.3). No impact on particle properties (e.g., size, zeta potential, filterability) was observed after the change in formulation buffer, indicating that FBDS is stable enough to be considered for in-process and in-use stability.

[0478] The decision to change the concentration process from centrifugal ultrafiltration to TFF ultrafiltration was based on practicality. A scaled-up version of centrifugal ultrafiltration requires longer processing times and generally cannot be performed at the scale sizes required for our GMP batches. Several 4 g MAP-Gly-CPT NP batches were subjected to TFF, again using the same membrane and cartridge types used in the scaled-up GMP configuration. Concentration by TFF yielded the targeted MAP-Gly-CPT concentration and had no effect on particle properties.

[0479] Based on the above observations, it was decided to proceed with TFF concentration for preparing MAP-Gly-CPT NPs in subsequent development, scale-up and GMP batches.

[0480] The decision to change the filtration process from a single final sterile filtration at the end of FBDS production to the use of an intermediate conditioning filtration step was made to allow for an uninterrupted and risk-reduced final sterile filtration. During the development and production of FBDS, it was observed that the final sterile filtration process was often difficult and that multiple sterile filters were usually required to complete the filtration.

[0481] To reduce this process risk, a 0.45 micron filtration step was used to condition the amorphous MAP-Gly-CPT material after dissolving it in water at pH 4.0 to remove some product-associated particulates. The final 0.22 micron sterile filtration can proceed without risk of extended filtration times during the subsequent spike of concentrated formulation buffer and the TFF step to formulate and concentrate the FBDS. The formulation including this conditioning filtration step resulted in a relatively easy and rapid final sterile filtration.

[0482] Based on the above observations, it was decided to proceed with a conditioning filtration step for preparing MAP-Gly-CPT NPs in subsequent scale-up and GMP batches.

[0483] Following process development, a formulation scale-up was performed to validate the optimization method. Two scale-up formulations of MAP-Gly-CPT NP were performed at 25 g scale to support GMP manufacturing using the scale-up conjugate material described above. Dissolution, formulation, and subsequent concentration by TFF performed comparable to the development run, achieving target levels of formulation components and MAP-Gly-CPT concentrations. Additionally, the final sterile filtration performed well.

[0484] GMP manufacturing was performed at 400 g, a 16-fold scale-up of 25 g, as described above. This somewhat larger than normal process scale-up was assessed as low risk, since formation of MAP-Gly-CPT NPs has been shown to be consistent and reproducible at various scales.

[0485] Lot 21-M007 was the first batch of DAN-222FBDS manufactured under GMP conditions as Phase 1 clinical trial material. This lot was manufactured in accordance with the original manufacturing process described in Example 6. The manufacturing differences between this lot and the lots used in the dose range finding (Lot SKT-757-077-NPS) and toxicity (Lot SKT-874-031-NPS) studies are summarized in Figure 50.

[0486] The main differences are in the type of monomer and isolation method used for MAP, the coupling agent and purification method used for the synthesis of MAP-Gly-CPT, and finally, the formulation buffer and concentration and filtration method used for MAP-Gly-CPT NPs (DAN-222FBDS). Analytical data on these batches demonstrates that the quality of GMP DAN-222FBDS and DAN-222 drug products is comparable to that used in IND-enabling toxicity studies. Example 8 Development of DAN-222 sterile solutions and drug products for injection

[0487] DAN-222 sterile solution for injection is supplied as a clear to slightly opalescent, colorless to pale yellow solution. This drug product is stored frozen at -20±5°C and thawed prior to administration.

[0488] DAN-222 sterile solution for injection is delivered as a nanoparticle construct consisting of a hydrophilic polymer backbone conjugated to multiple units of the active moiety 20(S)-camptothecin via a glycyl linker. When dissolved in aqueous media, this polymer conjugate spontaneously forms nanoparticles consisting of a hydrophobic active moiety surrounded by a hydrophilic polymer. Upon administration, 20(S)-camptothecin is slowly released from the nanoparticle construct.

[0489] Following the formulation screening process described above, DAN-222 Sterile Solution for Injection was selected. DAN-222 Sterile Solution for Injection is available as a single strength with a target concentration of 20±3 mg / mL of DAN-222. At this concentration, DAN-222 Sterile Solution for Injection delivers 2.6±0.3 mg / mL of 20(S)-camptothecin active ingredient upon dosing.

[0490] The composition of the DAN-222 sterile solution for injection is presented in Table 11. [Table 11]

[0491] No reconstitution is required for administration of DAN-222 Sterile Solution for Injection and no reconstitution diluent is supplied.

[0492] The physicochemical properties of the DAN-222 drug substance require its delivery as a parenteral solution. Thus, drug development focused on generating a drug product formulation for intravenous infusion. The formulation had to meet several criteria: sterility; acceptable stability during manufacturing of the bulk drug substance and drug product; acceptable stability over long-term storage; and acceptable stability during handling and administration. Because the formation of DAN-222 nanoparticles requires an aqueous solution, formulation design focused on identifying excipients that achieve both isotonicity and stability within the desired pH range (e.g., buffered pH).

[0493] The use of anhydrous sodium acetate and acetic acid at their respective concentrations (6.8 mM and 13.2 mM) is important in controlling the pH of the drug product. These additives were selected for their ability to buffer the formulation in a pH range conducive to product stability, their ability to maintain a consistent pH over a wide range of temperatures, and their common use in pharmaceutical products. These additives were selected for use based on data obtained in the formulation development screening study of multiple buffer systems described in the previous examples.

[0494] This combination of 20 mM concentration of acetate buffer system is a common composition used in the pharmaceutical industry. Thus, the respective concentrations of each additive target a pH of 4.3, which provides acceptable stability characteristics for the drug, as demonstrated in formulation development studies.

[0495] The use of 200 mM sodium chloride in the formulation serves as an isotonicity agent to prevent the solution from becoming hypotonic. Injection of a hypotonic solution can cause pain and safety issues upon delivery. Sodium chloride was also chosen because it is a common excipient in the pharmaceutical industry. It was also observed in formulation screening studies that the presence of sodium chloride appears to provide additional stability to the formulated product.

[0496] Phosphate is used in trace amounts to adjust the pH of the nanoparticle solution during manufacturing of the DAN-222 drug substance to provide a low pH environment conducive to the stability of DAN-222 in solution. The acetate buffer concentration is designed to target a final pH of 4.3 when added to the slightly more acidic aqueous solution during final formulation.

[0497] During its manufacture, the DAN-222 drug substance solution is formulated in its entirety as the DAN-222 formulated bulk drug substance (FBDS). Thus, the composition of the DAN-222 drug product solution in the vial is identical to that of the DAN-222 FBDS. This approach to drug product manufacturing was undertaken to minimize the handling volume and associated product risk of performing additional formulation or compounding operations at the drug product vial filling facility. This approach also provides a stable formulation for storage and handling of the DAN-222 FBDS prior to filling.

[0498] The physicochemical properties for the DAN-222 drug product are the same as those of the drug substance and have been discussed in detail in the previous examples. Additional properties are detailed below. Compatibility

[0499] Formulation stability studies were performed with a vial / stopper configuration equivalent to that selected for the GMP drug product. There was no evidence of incompatibility or loss of material on these surfaces at any point during the development of DAN-222. Furthermore, these studies provide evidence of the suitability of these vials during long-term storage frozen at -20 ± 5°C. From a practical standpoint, the use of sterile filled glass vials with rubber stoppers and crimp cap seals is the standard packaging configuration in pharmaceutical manufacturing. Packaging and shipping considerations for liquid filled vials are well established and easy to configure.

[0500] All polymeric components used in the manufacture of DAN-222 sterile solution for injection are pharmaceutical grade and suitable for use in parenteral filling operations.

[0501] The compatibility of a representative development batch of the DAN-222 drug product was also evaluated by challenging it with various clinical dosing components. The DAN-222 drug product formulation was found to be compatible with a wide range of clinical dosing components and dose preparation procedures. DAN-222FBDS lot SKT-757-077-NPS (the development lot used in the dose-ranging animal study) was reformulated by dialysis to match the Phase 1 clinical formulation of the drug product. This reformulated drug substance was filled into Type 1 borosilicate glass vials with rubber stoppers to replicate the clinical drug product container closure system. The drug was withdrawn from the vial using a needle-equipped syringe for injection into an IV bag.

[0502] Drug product compatibility was evaluated using commercially available 100 mL IV bags containing either 0.9% (w / v) saline or 5% (w / v) dextrose. IV bag materials evaluated were polyvinyl chloride (PVC), polyolefin (PO) and dual polyethylene-polypropylene (PE-PP). An IV administration set with a 0.22 μm PES (polyethersulfone) in-line filter was used for each diluent and bag material combination. In compliance with clinical protocols, drug product was added to IV bags to achieve a target CPT concentration in the IV bag of 0.022 mg / mL (low dose) and 0.400 mg / mL (high dose) to encompass the planned range of IV clinical dose concentrations (0.025-0.36 mg / mL) that may be observed in the IV bag. The high dose configuration requires the use of more than one vial of drug product per IV bag.

[0503] To perform the clinical suitability test, a 25 gauge, 1 / 2 inch needle, silicone-free syringe was used to inject the drug product into each bag to be tested. The bags were placed on a laboratory cart and pushed around the laboratory for 0.5 hours to represent transporting the IV bag from the clinical pharmacy preparation area to the patient for administration. The drug product was continuously held in the IV bag under airless conditions for 12 hours at ambient room temperature (approximately 22-26°C) to represent the worst case holding period. The total time the product was held in the IV bag prior to simulated injection was 12.5 hours (0.5 hour simulated transport and 12 hours airless hold). The IV bag was protected from light during the entire process.

[0504] Samples were taken after passage through an IV administration set with a 25G needle. In-process sampling was done at the IV bag port site with a syringe and expelled through a 25 gauge needle. IV bag port samples were taken and analyzed for informational purposes only immediately following DP injection into the bag; after a 0.5 hour transport event; and after a subsequent 12 hour retention in the IV bag prior to simulated injection. Samples were analyzed for visual appearance (to look for visible particulates), CPT concentration (to determine possible adsorption to the IV bag and administration set by comparing actual CPT concentration with calculated theoretical CPT concentration), % total CPT as free CPT (purity), sub-visible particles (HIAC), particle size, polydispersity index, pH, and osmolality.

[0505] Results are presented in Figure 51 for IV bag administration at the low dose (0.022 mg / mL) and in Figure 52 for the high dose (0.400 mg / mL) format. These results demonstrate that the drug product is stable under the conditions tested and is recovered following simulated infusion through an administration set after a 0.5 hour simulated transport and 12 hour hold period. All samples demonstrated acceptable levels of product adsorption, with 90-110% of the theoretical target CPT concentration remaining in solution over the course of the study. Based on these results, suitability testing supports the use of the drug product as specified in the clinical protocol.

[0506] The drug product will be injected into the IV bag using a silicone-free syringe and an IV administration set with a 0.22 μm filter will be used to match the compatibility test conditions during clinical trials. As found in this study, the acceptable IV bag diluents are 0.9% (w / v) saline and 5% (w / v) dextrose. The use of 100 mL IV bags is specified. Acceptable IV bag materials of construction are PVC, PO, and PE-PP. For product compatibility, the maximum acceptable drug product retention in the IV bag is 12.5 hours at room temperature. Microbiological properties

[0507] DAN-222 sterile solution for injection is supplied in Type 1 borosilicate glass vials with chlorobutyl rubber stoppers and aluminum crimp stoppers. Each vial is for single use and no preservatives are added to the formulation.

[0508] DAN-222 is not stable to terminal sterilization by heat. The polyethylene glycol component of MAP can undergo free radical degradation when exposed to sterilizing levels of gamma radiation. Therefore, microbial safety of both the drug substance and the drug product is provided by sterile filtration. The DAN-222 FBDS is passed through a 0.22 μm filter when transferred to sterile HDPE bottles for storage. Sterilization of the drug product is achieved by passing the FBDS through two 0.22 μm sterile filters in series immediately prior to filling into vials under aseptic conditions.

[0509] Container closure integrity qualification testing (CCIT) was performed on selected container closure systems to ensure continued protection from microbial contamination. CCIT by definitive vacuum decay analysis was determined at the stability time points at the end of the study. Example 9 A DAN-222 Dose Escalation Study of the Safety and Pharmacology of DAN-222 in Subjects With Metastatic Breast Cancer

[0510] This example presents a protocol for a dose escalation study of the safety and pharmacology of DAN-222 in subjects with metastatic breast cancer using DAN-222. This study investigates the use of DAN-222 in subjects presenting with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (mBC).

[0511] This is an open-label, multi-center, dose-escalation study designed to evaluate the safety, tolerability, and pharmacokinetics (PK) of DAN-222 administered intravenously (IV), followed by dose escalation of DAN-222 in combination with niraparib. The clinical program is designed to evaluate DAN-222 as monotherapy and in combination with a PARP inhibitor, which is expected to enhance efficacy without significantly increasing toxicity.

[0512] Within the scope of this study, there are two stages. Stage 1 has two parts. Part A analyzes dose escalation of DAN-222 alone. Part B analyzes dose escalation of DAN-222 in combination with niraparib 100mg. Stage 2 includes the expansion of three separate HER2-negative mBC cohorts: one arm for DAN-222 alone in subjects with homologous recombination repair deficient (HRD) positive or HRD-negative tumors, and one cohort for DAN-222 in combination with niraparib in subjects with either HRD-positive or HRD-negative tumors. DAN-222 is administered IV on days 1, 8, 15, and 22 of a 28-day cycle. Niraparib doses are taken orally once daily on days 1-28. Subjects receive treatment in a defined cycle of 4 weeks. Subjects who demonstrate acceptable safety and evidence of clinical benefit (SD, PR, or CR) will continue to receive DAN-222 until objective disease progression or unacceptable toxicity. Initial evaluations will include MRI / CT scans at 6-week intervals through the first 6 cycles, then at 8-week intervals from week 24 onwards.

[0513] The study design is depicted in Figure 53. Subjects who demonstrate acceptable safety and evidence of clinical benefit (stable disease [SD], partial response [PR], or complete response [CR]) will continue to receive DAN-222 until objective disease progression or unacceptable toxicity is confirmed. Dose Escalation Stage 1

[0514] Stage 1, Part A - The dose escalation phase of the study will evaluate the safety, tolerability, and PK of DAN-222 as a single agent administered by IV infusion. Using a conventional 3+3 design, subjects will be evaluated for DLTs. If no dose-limiting safety signals are detected during the 28-day DLT window, the study will progress to sequentially higher dose levels.

[0515] Stage 1, Part B - The starting dose of DAN-222 administered IV QW in combination with niraparib 100 mg will begin after the monotherapy has passed the DLT safety window at the same dose level or higher (e.g., 2 mg / m 2 DAN-222 + 100 mg niraparib is 2 mg / m 2 (Combination dose escalation will begin only after the single agents have passed the DLT safety window). Combination dose escalation will follow the same dose escalation intervals as the single agent escalation using a traditional 3+3 design, and subjects will be evaluated for DLTs. Escalation will proceed to the maximum dose level identified in Part A or until a safety threshold is met. If no dose-limiting safety signal is detected during the 28-day DLT window, the study will progress to successively higher dose levels.

[0516] Enrollment into dose escalation cohorts based on a 3+3 dose escalation design will be conducted in compliance with the following rules: Initially, a minimum of three subjects will be enrolled in each cohort, but only if the first two enrolled subjects do not experience a protocol-defined DLT, in which case enrollment into the cohort will cease. If none of the first three DLT-evaluable subjects experience a DLT, enrollment of the next cohort at the next higher dose level will proceed with an increase in dose interval of ≦100% of the previous dose. If one of the first three DLT-evaluable subjects experiences a DLT, this cohort will expand to six subjects. Before any dose escalation decision is made, all subjects will be evaluated for DLT. If a DLT is observed in <17% of subjects in a given cohort (e.g., one of six DLT-evaluable subjects is observed to have a DLT), enrollment of the next dose escalation cohort will continue. The increase in interval between successive cohorts will be ≦50% of the previous dose level. If a DLT is observed in >= 17% of subjects in a given cohort, further enrollment and dose escalation at that dose level will be halted and the dose will be declared to have exceeded the MTD. Additional subjects will also be enrolled in the same dose escalation cohort to further describe safety, PK, pharmacodynamics (PD) or efficacy at that dose level.

[0517] The MTD is defined as the highest dose level resulting in a DLT in <17% of at least 6 subjects.

[0518] Additional intermediate dose cohorts between the two dose levels, which are demonstrated not to exceed the MTD, will be evaluated to further characterize dose-dependent toxicity or pharmacodynamic changes.

[0519] If the dose level above the MTD is ≧25% higher than the previously tested dose level, an additional dose cohort of at least 6 subjects will be evaluated at the intermediate dose level to determine the MTD.

[0520] Safety information from the intermediate dose level cohorts will be used to guide dose escalation decisions by the SRT; for example, if the frequency of DLT-defining events observed in the intermediate dose level cohorts is higher than expected based on the escalation cohort data, the SRT may recommend interrupting or discontinuing further dose escalation.

[0521] Enrollment of cohorts evaluating intermediate dose levels in either Part A or Part B may occur simultaneously with enrollment of dose escalation cohorts in the other part to identify the MTD. If the MTD is not exceeded at any dose level, the highest dose administered in the study will be declared the MAD.

[0522] Dose escalation decisions will be made based on the recommendations of the SRT and in consultation with the study investigator. Relevant demographics, AEs, laboratory tests, dose administration and available PK data will be reviewed prior to each dose escalation decision.

[0523] Based on review of real-time safety data and available preliminary PK data, dose escalation may be stopped or modified by the SRT if deemed appropriate. Dose escalation will continue until a dose level is reached at which DLT is observed in ≥17% of ≥6 subjects.

[0524] Dose interruptions and / or reductions will be performed at the investigator's discretion after Cycle 1 for subjects enrolled in Stage 1 (dose escalation) and at any time for subjects enrolled in Stage 2 (dose expansion) due to toxicity of any grade deemed intolerable by the subject.

[0525] Toxicity at each dose will be recorded before administration of subsequent doses and graded according to NCI CTCAE v5.0. All dose reductions will be based on the most severe previous toxicity and will follow the reduction schedule listed in Table 12 below. [Table 12]

[0526] In the event of a dose interruption, the next dose will follow the subject's original calendar schedule. Cycle timing will not be delayed due to the interruption in dosing, and tumor assessments will be performed according to this schedule regardless of whether study drug was discontinued. If the dose of DAN-222 is reduced, any re-escalation will be discussed with the medical monitor.

[0527] For subjects in stage 1, the dose of DAN-222 may be increased to a higher dose level found to be safe during the dose escalation phase after the subject has completed 12 weeks of tumor evaluation and in consultation with a medical monitor.

[0528] Subjects who do not experience a DLT during the DLT observation period are eligible to receive additional cycles of investigational drug with DAN-222. Subjects who demonstrate acceptable safety and evidence of clinical benefit (SD, PR, or CR) may continue to receive DAN-222 until objective disease progression or unacceptable toxicity.

[0529] Any dose delays not attributable to study drug administration may not require discontinuation of study drug administration but must be approved by the medical monitor. Dose reductions of DAN-222 may be permitted if it is determined that clinical benefit can be maintained.

[0530] Subjects who complete study treatment without disease progression will continue to be monitored, including with regularly scheduled tumor evaluations, until discontinuation of post-treatment follow-up or initiation of an alternative therapy or study.

[0531] All adverse events, including DLTs, will be reported and graded according to NCI CTCAE v5.0 unless otherwise indicated. Dose-limiting toxicities will be handled according to clinical practice and monitored until resolution. Adverse events meeting DLT criteria will be reported to the sponsor as soon as possible.

[0532] For dose escalation purposes, the DLT evaluation period for Stage I - Parts A and B will be defined as the first 28 days of dosing (i.e., from Day 1 of Cycle 1 to Day 28 of Cycle 1).

[0533] The criteria for dose-limiting toxicity will be as follows: (i) any treatment-related Grade 3 or Grade 4 non-hematological clinical (i.e., non-laboratory) AEs (excluding fatigue, nausea, vomiting, diarrhea, and electrolyte imbalances that can be controlled by supportive care); (ii) any treatment-related Grade 3 or Grade 4 non-hematological laboratory abnormalities (if medical intervention is required to treat the subject or the abnormality results in hospitalization or the abnormality persists for ≥ 7 days); (iii) any treatment-related hematological toxicity, specifically defined as Grade 3 or Grade 4 thrombocytopenia for ≥ 7 days, or Grade 3 or Grade 4 neutropenia for ≥ 7 days, or Grade 3 or Grade 4 anemia with bleeding or requiring platelet transfusion; Grade 3 or Grade 4 neutropenia for ≥ 7 days, or Grade 4 neutropenia with infection or febrile neutropenia; or Grade 3 or Grade 4 anemia for ≥ 7 days, or Grade 4 neutropenia requiring transfusion.

[0534] Exceptions to DLTs include grade 3 laboratory abnormalities that are asymptomatic and deemed not clinically significant.

[0535] The determination of whether a subject is evaluable for DLT assessment will be made according to the following rules: Subjects who initially receive study drug and remain in the study through the DLT assessment period are considered DLT evaluable. Subjects who receive at least one dose and have a DLT are evaluable. Subjects without DLTs must receive at least four doses to be evaluable. Subjects who discontinue study drug before completion of the DLT assessment period for reasons other than a DLT are not considered evaluable for dose escalation and MTD determinations and will be replaced by an additional subject at the same dose level. Subjects with pre-existing conditions that prevent assessment of a DLT during the DLT assessment period may be replaced at the discretion of the Medical Monitor. Stage 2

[0536] The RP2D for DAN-222 monotherapy may be selected based on one or more of the following: (i) the MAD or MTD (the highest dose level resulting in a DLT rate of <17% in at least 6 subjects); (ii) PK and / or PD results; (iii) the occurrence, nature and severity of toxicity occurring after the DLT evaluation period (i.e., the first 28 days of dosing); and (iv) antitumor activity.

[0537] The dose expansion stage of the study is designed to obtain additional safety, tolerability, PK and preliminary clinical activity data with investigational drug administered at the RP2D dose. Initiation of an expansion cohort is at the discretion of the sponsor. The sponsor, in consultation with the investigator and SRT, will continually evaluate all available safety data from the expansion cohort to assess the tolerability of the dose levels tested. The dose levels of DAN-222 tested in the expansion stage will never exceed the highest dose level that qualifies as the MTD for a given schedule in the dose escalation stage. Additionally, for each expansion cohort, data will be continually reviewed for excessive toxicity or fatal events leading to the potential early cessation of enrollment.

[0538] Expansion cohorts will further evaluate antitumor activity, safety and pharmacodynamics. Enrollment into one or more expansion cohorts will commence following the above review and recommendation by the SRT after consultation with the investigator. After defining the RP2D, dose escalation of single-agent DAN-222 will accept 20 subjects for subjects with HER2-negative mBC and for subjects with either HRD-positive or HRD-negative status. After selecting the RP2D dose of DAN-222 in combination with niraparib, two expansion cohorts of HRD-positive (n=20) and HRD-negative (n=20) subjects will be initiated.

[0539] Single agent expansion cohorts may be initiated simultaneously with ongoing dose escalation in the combination arm. In such an event, the dose of the expansion cohort will be adjusted based on the SRT review of cumulative safety, laboratory and efficacy data. Additional subjects may be enrolled per cohort based on cumulative data, and further cohorts with protocol amendments may be added. Data from the expansion cohorts will provide further support for RP2D selection per SRT review. Test subjects

[0540] The study will enroll approximately 96 subjects. The stage 1 dose escalation study, Part A, single-agent DAN-222, will include approximately 18 subjects. The stage 1 dose escalation study, Part B, combination of DAN-222 with niraparib, will include approximately 18 subjects.

[0541] During the expansion phase, enrollment in this trial is as follows: Group A: DAN-222 single agent in subjects with HER2-negative mBC with HRD-positive or HRD-negative tumors, n=20; Group B: DAN-222 in combination with niraparib in subjects with HER2-negative mBC with HRD-positive tumors, n=20; and Group C: DAN-222 in combination with niraparib in subjects with HER2-negative mBC with HRD-negative tumors, n=20.

[0542] In the dose escalation stage (Parts A and B), subjects who do not complete the DLT evaluation period for reasons other than a DLT will be considered not DLT-evaluable and will be replaced. In the dose expansion phase, subjects who do not have at least one post-baseline radiological tumor assessment for reasons other than discontinuation due to toxicity or death due to disease progression will be considered not evaluable for efficacy and may be replaced. Eligibility

[0543] Subjects must have histologically proven metastatic HER2-negative breast cancer that has progressed after two prior lines of therapy (including adjuvant therapy if progression occurred within the past 12 months; aromatase inhibitors would be considered a line of therapy) or for which standard therapy has proven ineffective or intolerable or is deemed inappropriate. HER2 positivity is defined by 3+ staining by standard of care fluorescent in situ hybridization (FISH) and / or immunohistochemistry (IHC) according to the American Society of Clinical Oncology / College of American Pathologists (ASCO / CAP) Clinical Practice Guideline Focused Update.

[0544] mBC must be at least 2 weeks or 5 half-lives (whichever is longer) from any prior therapy. Treatments may include chemotherapy, immunotherapy, and / or radiation therapy. In addition, recovery from all reversible toxicities, except for alopecia, to grade ≤1 must be achieved at the time of study enrollment.

[0545] Subjects must have measurable disease per RECIST v1.1. Subjects must be female and 18 years of age or older. Subjects must have an ECOG performance status ≦2.

[0546] Subjects who have previously undergone treatment for brain metastases (surgery, whole brain or stereotactic brain radiation therapy) will be allowed, provided that their lesions have been stable for at least 4 weeks, the subject has not been taking steroids for at least 7 days prior to the first dose of study drug administration, and any neurological symptoms have returned to baseline (no evidence of progression by imaging using the same imaging modality for each assessment, either MRI or CT scan).

[0547] Subjects with brain metastases should not be required to use enzyme-inducing antiepileptic drugs (e.g., carbamazepine, phenytoin, or phenobarbital) within 14 days prior to the first dose of study drug and during the study. Use of newer antiepileptic drugs that do not cause enzyme-inducing drug-drug interactions is permitted.

[0548] Subjects must have normal organ and bone marrow function defined as follows: absolute neutrophil count ≥ 1.5x10 without growth factor support in the past 7 days 9 / L; platelets ≥ 100x10 without growth factor support in the past 7 days 9 / L; hemoglobin ≥ 9 g / dL and no blood transfusion within the past 4 weeks; total bilirubin ≤ 1.5 x upper limit of normal (ULN) (except in cases of Gilbert's disease); AST (SGOT) / ALT (SGPT) ≤ 2.5 x ULN (≤ 5 x ULN in cases of liver metastases); and for subjects with creatinine levels above institutional normal, creatinine < 1.5 x ULN, or creatinine clearance ≥ 50 mL / min (calculated using the Cockroft-Gault formula).

[0549] Subjects of childbearing potential must have a negative serum pregnancy test within 72 hours prior to the first dose of study medication, or must agree to refrain from any activity that could result in conception from the screening visit until 120 days after the last dose of study medication, or must be of no potential for conception. Not of childbearing potential is defined as the following (non-medical reasons): subjects 45 years of age or older who have not had menses for >1 year; at least 2 years of amenorrhea without hysterectomy and oophorectomy, and follicle-stimulating hormone (FSH) levels in the postmenopausal range at the time of the pre-study (screening) evaluation; or subjects who have had a hysterectomy, bilateral oophorectomy, or tubal ligation. Documented hysterectomy or oophorectomy must be confirmed by medical records of the actual procedure or confirmed by ultrasound. Tubal ligation must be confirmed by medical records of the actual procedure, or subjects must be willing to use two appropriate barrier methods throughout the study, beginning at the screening visit and continuing through day 120 after the last dose of study treatment.

[0550] Stage 2 inclusion will additionally require documentation of DNA repair deficiency status (e.g., BRCA1, BRCA2, ATM, BARD1, BRIP1, CDK12, CHEK2, FANCA, NBN, PALB2, RAD51, RAD51B, RAD51C, RAD51D, RAD54L) verified by central laboratory plasma testing or archival tumor tissue or germline testing from any Clinical Laboratory Improvement Act (CLIA)-approved laboratory.

[0551] Subject exclusion criteria include: (i) any significant medical condition or laboratory abnormality that, in the discretion of the clinician, would place the subject at unacceptable risk if the subject were to participate in the study and that is not otherwise listed below; and (ii) for the DAN-222 and niraparib combination cohort, subjects may not have a known sensitivity to FD&C Yellow No. 5 (tartrazine). Subjects will also be excluded if they have had an allergic reaction to irinotecan, topotecan, or govitecan. Excluded subjects also include those who have had or have received coadministration of cytochrome P450 3A4 (CYP3A4) enzyme inducers or inhibitors within 2 weeks prior to the first day of study drug administration. Subjects taking medications known to prolong the QT interval or associated with torsades de pointes are also excluded, unless the subject can safely discontinue these medications or change to comparable medications that do not significantly prolong the QT interval at least 5 half-lives or 7 days (whichever is longer) before the first dose of DAN-222. Subjects with a history of myelodysplasia or known additional malignancies that have progressed or required aggressive treatment within the past 3 years are excluded. Exceptions include non-melanoma skin cancer and carcinoma in situ. Subjects diagnosed with carcinomatous meningitis are also excluded. Excluded subjects also include subjects who are pregnant, breastfeeding, or planning to conceive a child within the planned duration of the study, starting from the screening visit and ending 120 days after the last dose of investigational drug administration. Thus, excluded subjects also include subjects who are unable to comply with the study procedures or who do not wish to use adequate contraception. Exclusions further include subjects with uncontrolled intercurrent illnesses, including but not limited to ongoing or active infections, symptomatic congestive heart failure, unstable angina, arrhythmias, or other conditions that limit compliance with study requirements.

[0552] Similarly, subjects with a rate-corrected QT interval (QTc) prolongation of >470 milliseconds at screening will be excluded. If a subject has a QT interval prolongation that, upon the investigator's evaluation, is determined to be due to the pacemaker (i.e., the subject has no other cardiac abnormalities), the subject may be eligible to participate in the study after consultation with the medical monitor.

[0553] Finally, excluded subjects will also include those who, in the opinion of the investigator, are unable to safely participate in and complete a minimum of four cycles of treatment, or who have any serious social, psychosocial or medical condition or clinical laboratory abnormality that may affect compliance with the protocol or interpretation of the results. Estimated Study Duration and Study Completion

[0554] The planned duration of the study is 3 years. Subject participation includes screening, dosing, and follow-up. Screening can occur up to 30 days prior to the first dose of study drug, during which subject eligibility and baseline characteristics are determined. Subjects with confirmed progressive disease will exit the study at an earlier time point. In subjects with SD, PR, or CR, dosing with DAN-222 will continue until unacceptable drug-related toxicity occurs or disease progresses. After dosing, subjects will be observed for disease progression and new anticancer therapy, or until withdrawal of consent or termination of the study, whichever occurs first. The planned duration of individual subject dosing with study drug is approximately 12 months.

[0555] The end of the study will be defined as the date when the last subject visit occurs or when the last data point required for statistical analysis or protocol-defined safety monitoring is received from the last subject, whichever occurs later. DAN-222 and Niraparib Administration

[0556] DAN-222 is supplied by the sponsor and is a polymer-small molecule conjugate NP formulated as a sterile aqueous solution.

[0557] Both DAN-222 drug substance and drug product will be manufactured under current good manufacturing practice. DAN-222 drug product will be a sterile, clear to slightly opalescent, colorless to pale yellow, preservative-free liquid for IV infusion. DAN-222 will be supplied in single-use vials containing 3.1 mL of a solution of DAN-222 containing 2.6 mg / mL CPT. The formulation consists of an aqueous solution of 20 mM sodium acetate and 200 mM sodium chloride, pH adjusted to 4.3. Vials will be stored frozen at -20°C ± 5°C and will be thawed immediately prior to use at either 2-8°C or ambient temperature. Labeling will comply with applicable regulatory agency requirements.

[0558] DAN-222 is administered IV once weekly. The dose of DAN-222 for each subject depends on its dose level assignment. Dose is based on the subject's actual body weight at baseline (cycle 1, day 1, or during screening). If the subject's body weight changes >10% during the course of the study, the body surface area and drug dose are recalculated. Recalculation of drug dose on each dosing day, regardless of the percentage of body weight change, is also permitted according to the institution's dosing policy.

[0559] DAN-222 will be administered to subjects via IV infusion using an IV bag. Testing has shown that DAN-222 is compatible with PVC extension sets and non-silicone polypropylene syringes. DAN-222 will be administered in an environment with ready access to trained emergency medical personnel and facilities equipped to respond to and manage medical emergencies.

[0560] Subjects should be well hydrated throughout DAN-222 administration and are encouraged to drink at least 2 liters of water per day. DAN-222 is infused over 1 hour ± 15 minutes. Infusion may be slowed or interrupted if the subject experiences infusion-related symptoms. For the first infusion, subjects are observed for 90 minutes after infusion for any infusion-related reactions. After each subsequent DAN-222 dose, the dosing time may be progressively shortened by up to 30 minutes, up to a minimum infusion time of 30 minutes. In the absence of infusion-related adverse events, the post-dose observation time may also be progressively shortened by up to 30 minutes, up to a minimum observation time of 30 minutes.

[0561] Subjects undergoing inter-subject dose escalation or rechallenge will receive an initial high-dose infusion of DAN-222 over a minimum of 1 hour ± 15 minutes.

[0562] Any overdose or incorrect administration of DAN-222 will be documented on the Study Drug Administration eCRF. Adverse events related to overdose or incorrect administration of study drug will be recorded on the Adverse Events eCRF.

[0563] Niraparib (2-{4-[(3S)-piperidin-3-yl]phenyl}-2H indazole 7-carboxamide 4-methylbenzenesulfonate hydrate) (1:1:1) is an orally available, potent, and highly selective inhibitor of PARP1 and PARP2. The excipients of niraparib are lactose monohydrate and magnesium stearate.

[0564] Niraparib is provided as a 100 mg capsule and is administered orally once daily (QD) continuously, beginning on day 1 of cycle 1. The daily dose administered is one 100 mg capsule. Subjects are instructed to take their dose at the same time each day, preferably in the morning. Subjects must swallow the capsule and not chew it. Niraparib capsules are dispensed to subjects on day 1 of cycle 1, and every day 1 of a 28-day cycle thereafter, until the subject discontinues study drug administration. Study Drug Administration - Day 1

[0565] Prior to dosing, subjects will be asked to complete all medical history, physical and laboratory assessments. Assessments will be performed as outlined in the Schedule of Assessments (Table 13).

[0566] In addition, vital signs will be measured approximately 5 minutes (± 5 minutes) before and within 15 minutes (± 5 minutes) after the start of the infusion, and then every 30 minutes (± 10 minutes) for the next 2 hours. Vital signs may be monitored continuously after this point until the subject is stabilized and as clinically indicated.

[0567] Subjects enrolled in the dose escalation cohorts will require clinical monitoring following DAN-222 administration on Day 1 of Cycle 1 for 8 hours post-infusion during PK assessments. Ongoing investigational drug administration

[0568] DAN-222 is administered once weekly. Subjects return to the clinic for safety evaluations and to receive infusions of DAN-222. Evaluations will be performed as indicated in the Schedule of Evaluations (Table 13). Maintenance Dosing

[0569] Subjects who continue to tolerate DAN-222 and do not have progression of malignancy may continue on study drug. Subjects will return to the clinic once a week for safety evaluations and to receive infusions of DAN-222. End of administration

[0570] The End of Treatment (EOT) visit must occur within 28 days of discontinuation and prior to the initiation of any new anticancer treatment / regimens. All subjects who discontinue study drug for any reason should undergo the evaluations outlined in the Schedule of Evaluations (Table 13). Early withdrawal

[0571] Regardless of when they completed dosing, all subjects should return to the clinic for an EOT visit evaluation. Adverse events will be evaluated until resolution, return to baseline, or stable, as assessed by the investigator. [Table 13-1] [Table 13-2] [Table 13-3] [Table 14] [Table 15]

[0572] Physical examination, vital signs and performance status

[0573] At the screening visit, a thorough physical examination is performed. The thorough physical examination includes evaluation of the head, eyes, ears, nose, and throat; and the cardiovascular, dermatological, musculoskeletal, respiratory, gastrointestinal, and nervous systems. Changes from baseline are recorded at each subsequent physical examination. New or worsening abnormalities are recorded as adverse events, if appropriate. Limited physical examinations are performed at other visits to assess changes from baseline abnormalities, any new abnormalities, and to evaluate patient-reported symptoms. New or worsening abnormalities are recorded as adverse events, if appropriate.

[0574] Vital signs, including blood pressure, heart rate, respiratory rate, and temperature, will be monitored and recorded at screening and at the times outlined in the Schedule of Assessments (Table 13). In addition to the time points outlined in the Schedule of Assessments, vital signs will be monitored when clinically indicated, and changes will be reported on the appropriate eCRF page if there are any clinically significant new or worsening changes since the last examination. Performance status, as measured by the ECOG scale, will be performed to quantify the subject's overall health and ability to perform activities of daily living. Cardiac function

[0575] A baseline 12-lead ECG will be performed prior to the first infusion (i.e., day 1) of every third 4-week cycle. The ECG should be performed prior to blood draws for PK. Subjects will be in supine position and rest for approximately 5 minutes prior to recording of the ECG. Neurological examination

[0576] At the screening visit, a neurological examination will be performed to record any abnormalities in the cranial nerves, motor system, reflexes, coordination, sensory system, and neuropsychological findings (e.g., speech, cognition, and emotion). A symptomatic neurological evaluation will then be performed prior to infusion. Subjects will be specifically asked about changes in their neurological status since the previous examination, as specified in the Schedule of Assessments (Table 13). Neurological Imaging

[0577] Subjects with neurological signs or symptoms will undergo brain MRI screening during the screening period of the study to exclude central nervous system metastases. If MRI is contraindicated, a brain CT scan may be performed instead. If detected during screening, subsequent brain scans will be performed at the scheduled imaging interval. Disease evaluation

[0578] Subjects will be assessed for disease response by the investigator at the time points indicated in the assessment schedule.Disease assessment will be assessed according to RECIST v1.1.As part of disease assessment, physical examination will include assessment of the presence and extent of lymphadenopathy, hepatomegaly and splenomegaly. Baseline Imaging

[0579] MRI or CT scans of the chest, abdomen, and pelvis are required at screening along with appropriate imaging of all other disease sites (including brain and bone) to confirm eligibility to have measurable disease per RECIST v1.1 and / or to establish a baseline. MRI or CT performed after the subject's last line of therapy but before signing the ICF may be used for confirmation of eligibility if it is performed within 28 days prior to the screening visit and no other anti-cancer treatments have been administered. If an MRI or CT is performed >28 days prior to the screening visit, the MRI or CT scan must be repeated to establish a new baseline. The MRI or CT should be performed as close as possible to the screening visit.

[0580] Subjects who are unable to undergo contrast-enhanced MRI or CT scans due to contrast allergy or impaired renal function may be evaluated using MRI or CT scans without contrast.

[0581] The same radiological assessment modality used at baseline is preferably used for all response assessments. For consistency across various time points, imaging should use the same protocol (e.g., MRI or CT using the same contrast protocol). After starting administration of the study drug

[0582] The first on-study imaging assessment will be performed 6 weeks (42 ± 7 days) from the date of the first dose of study drug. If disease progression is unclear, a confirmatory image is required after <4 weeks. If there is a SD or better response, subsequent tumor imaging will be performed every 6 weeks (42 ± 7 days), or more frequently if clinically indicated and at the time of suspected disease progression. After the 6-month radiological assessment, subjects will undergo imaging every 8 weeks (56 ± 7 days). Imaging should not be delayed due to delayed cycle initiation or extended intervals between combination treatment cycles. The same modality (i.e., CT or MRI) should be used for a given subject throughout the study.

[0583] Subjects with CR or PR will undergo confirmation of response by repeat tumor imaging evaluation at the next scheduled scan (ie, 6 weeks later). Clinical Laboratory Analysis

[0584] Screening and other laboratory evaluations will be performed according to the Schedule of Evaluations. The Investigator may choose to repeat any abnormal tests once to rule out laboratory or sample collection errors. The evaluations listed in Table 16 will be performed in the local laboratory at the time points indicated in the Schedule of Evaluations (Table 13). [Table 16]

[0585] laboratory analysis

[0586] Blood analyses will be performed to evaluate PK and PD markers for DAN-222. Generally, sample testing and analysis will follow a schedule of evaluations. However, allocation of samples to specific tests may be altered if sample material is limited.

[0587] Clinical biospecimens (e.g., serum) are sent from the clinical site to a central laboratory for sample processing, archiving, and distribution to specialized laboratories or to Dantari, Inc. All samples, and any derivatives derived from these samples, may be stored for up to five years to address exploratory scientific questions related to the treatment or disease being studied.

[0588] Multiple specialty laboratories may be utilized for a particular analysis. Pharmacokinetic evaluation (plasma)

[0589] Estimation of DAN-222 in plasma will be analyzed using liquid chromatography with mass spectroscopic detection (LC-MS-MS) specific for DAN-222. Stage 1 (dose escalation): Pharmacokinetic evaluation (plasma)

[0590] Plasma samples for PK analysis are obtained at the following time points: In cycle 1, day 1 samples are collected pre-dose and 30 minutes, 2, 4, 6, 8, 24, 48, 96, and 120 hours after the end of the infusion. PK samples collected pre-dose, 30 minutes, and 2 hours are collected ±5 minutes after the scheduled end of the infusion. PK samples collected at 4 hours+ are collected ±15 minutes after the end of the infusion. In cycle 2, day 1 samples are collected pre-dose and 30 minutes (±5 minutes) and 4 hours (±15 minutes) after the end of the infusion. From cycle 3 onwards, day 1 samples are collected pre-dose and 30 minutes (±5 minutes) after the end of the infusion. In addition, PK samples are collected 4 hours (±15 minutes) after the end of the infusion in cycle 3 and every three cycles thereafter.

[0591] PK samples after discontinuation or early termination will be taken at any time during the visit. Additional PK samples may be taken in the event of significant toxicity or variable drug exposure.

[0592] Post-dose PK samples should be collected from a site other than the site of study drug administration. For example, if the study drug is administered via a vein in the arm, post-dose PK samples should be collected from a vein in the opposite arm. Stage 2 (dose expansion): Pharmacokinetic evaluation (plasma)

[0593] Plasma samples for PK analysis will be obtained at the following time points: Cycle 1, Day 1 samples will be collected pre-dose, 0.5 hours (± 5 minutes), and 4 hours (± 15 minutes) after the end of the infusion. Cycle 2, Day 1 samples will be collected pre-dose, and 0.5 hours (± 5 minutes) after the end of the infusion. Cycle 3 and every three cycles thereafter, Day 1 samples will be collected pre-dose, 0.5 hours (± 5 minutes), and 4 hours (± 15 minutes) after the end of the infusion.

[0594] PK samples after discontinuation or early termination will be collected during the visit. Additional PK samples may be collected in the event of significant toxicity or variable drug exposure.

[0595] Post-dose PK samples should be collected from a site other than the site of study drug administration. For example, if the study drug is administered via a vein in the arm, post-dose PK samples should be collected from a vein in the opposite arm. Pharmacodynamic evaluation (plasma)

[0596] Primary evaluation by the central laboratory will include measurement of circulating tumor counts (CTCs). Statistical considerations

[0597] This is an open-label clinical trial and data will generally be analyzed using descriptive statistics. Data will be tabulated and analyzed for subject enrollment and disposition, demographic and baseline characteristics, past and concomitant medications, efficacy and safety measures. Sample Size Considerations

[0598] Approximately 18-36 subjects will be enrolled in the study during the dose escalation stage (Stage 1), and approximately 60 subjects will be enrolled during the dose expansion stage (Stage 2). The overall sample size for this study is estimated to be between 18-96 subjects.

[0599] The dose escalation part of the study will split a standard 3+3 dose escalation design into two parts. Part A will be used to explore the MTD of DAN-222 as a single agent, and Part B will be used to explore the MTD of DAN-222 in combination with niraparib. There will be up to five pre-planned dose cohort levels per part, with a total of 18 DLT-evaluable subjects projected per part. Subjects not evaluable for DLT will be replaced.

[0600] The sample size of 20 evaluable subjects in each arm (Arm A, Arm B, and Arm C) of Stage 2 is based on practical considerations and clinical judgment to obtain sufficient information for the overall study objectives, including characterization of safety, initial clinical efficacy, and PK data for DAN-222 administered as monotherapy or in combination with niraparib in the selected population. For example, if there are 4 responders in a group of 20 evaluable subjects (i.e., 20% ORR), the 95% exact CI for the ORR is (5.7%, 43.7%). Similarly, if the ORR is 45% (9 responders out of 20 evaluable subjects), the 95% exact CI is (23.1%, 68.5%). statistical methods

[0601] Unless otherwise stated, data is summarized by stage and dose level (Stage 1) or cohort (Stage 2) as appropriate, and all data is listed for all subjects. Data is also presented comprehensively within each stage. Data may also be pooled to support additional aggregate analysis, as described elsewhere herein.

[0602] Descriptive summaries for categorical variables include numbers and percentages. Continuous variables are summarized using standard summary statistics (N, mean, standard deviation, median, minimum and maximum). Medians and 25th and 75th percentiles (where assessable) are presented for survival data. 95% CIs around points are presented where appropriate. Pharmacodynamic analysis

[0603] All subjects in the FAS will be assessed for the reduction in circulating tumor cell counts at Week 4 compared to the baseline visit. Pharmacokinetic analysis

[0604] The PK Analysis Set (PKAS) includes all subjects in the FAS who had PK blood samples collected during the study and who had measurable concentrations of DAN-222. For subjects with sufficient data, the following PK parameters will be estimated from individual concentration-time profiles using non-compartmental analysis methods: total exposure (AUC 0-last );C max ;C min (trough concentration), and time to minimum plasma concentration; if data permit, CL, V and t 1 / 2 ; as well as the PK parameters of unbound CPT.

[0605] If sufficient data are available, parameters will be calculated using data from doses 1 and 5. Additional analyses may be performed if deemed useful and appropriate.

[0606] Summary statistics for pharmacokinetic parameters are presented by dose group and may include mean, standard deviation, geometric mean and standard deviation, median, 25th to 75th percentiles, minimum and maximum. All concentrations below the limit of detection or quantification or missing data will be so indicated in the listing of concentration data. Concentrations below the limit of detection will be treated as 0 in the summary statistics. Efficacy Analysis

[0607] Response assessments will be performed by the investigator based on RECIST v1.1. Efficacy summaries will be based on the EAS unless otherwise specified. For the FAS and PPAS, selected summaries may also be presented. Objective response rate

[0608] ORR is defined as the proportion of subjects identified as having a best overall response (BOR) of complete response (CR) or partial response (PR) according to RECIST v1.1. The proportion of subjects with ORR in the EAS will be reported narratively, with 95% CI based on the Clopper-Pearson exact method. Overall responses will be summarized at each visit. BOR will also be summarized. Secondary analyses of ORR and BOR may be repeated in the FAS and PPAS. Additional Secondary Efficacy Endpoints

[0609] PFS: Progression-free survival is defined as the time from first dose of study drug to first documentation of progressive disease (per RECIST v1.1) or death from any cause. Subjects who do not experience progressive disease and are alive are censored at the time of their last evaluable tumor assessment. Subjects who do not experience progressive disease and start a new anticancer therapy are censored at the time of initiation of the new anticancer therapy or at the last evaluable tumor assessment prior to that time. For any additional analyses performed in the FAS, subjects without an evaluable tumor assessment after baseline are censored at the time of receipt of first study drug. Subjects who are lost to follow-up for evaluation of progressive disease are censored at the time of their last evaluable tumor assessment. Analysis of PFS is based on the Kaplan-Meier method.

[0610] DCR: Disease control rate is defined as the percentage of subjects with a BOR of CR, PR or SD. Analysis of DCR will be performed in the same way as ORR.

[0611] CBR: Clinical benefit rate is defined as the percentage of subjects with a BOR of CR, PR, or documented SD for at least 6 months duration. Analysis of CBR will be performed in the same manner as ORR.

[0612] DOR: Duration of response includes subjects with an objective disease response (PR or CR) and is defined as the time from the first tumor assessment confirming the subject's objective disease response to the time of disease progression or death from any cause. Subjects who do not experience disease progression or death at the time of analysis are censored using the same rules as described for PFS. Analysis of DOR is performed using the Kaplan-Meier method. Homologous recombination repair deficiency analysis

[0613] Special attention is paid to the subset of subjects who may have a clear underlying DNA repair pathway deficiency (HR, nucleotide excision repair or mismatch repair deficiency) and a clear drug response. There may be situations where a decision is made to stop taking biomarker samples or not or to stop blood or tumor analysis, either for practical or sample quality reasons. In such situations, the number of samples may be insufficient to perform a complete data analysis, and thus the available data for the FAS is listed and summarized. In addition, exploratory efficacy analysis may also be performed based on the subgroups identified using HRD and / or other biomarkers. Safety analysis

[0614] All safety analyses will be performed in the FAS. Adverse events will be coded according to the current version of MedDRA. The severity of adverse events will be graded according to the US NCI CTCAE, v5.0.

[0615] TEAEs: Treatment-emergent adverse events are defined as any adverse event occurring (or worsening of a pre-existing medical condition) after the first dose of study drug through 30 days after the last dose of study drug. Events, including TEAEs, adverse events leading to dose reduction / interruption, adverse events related to the study drug, serious adverse events, adverse events leading to discontinuation of the study drug, and fatal adverse events, are summarized by system organ class and preferred term for each treatment group. A summary of NCI CTCAE grade 3 or higher adverse events, the most common adverse events by preferred term, and adverse events by association with study drug administration are presented.

[0616] For stage 1, the incidence of DLTs will be summarized. For stages 1 and 2, the incidence of infusion-related responses will also be summarized. Laboratory test result values ​​and changes from baseline will be summarized by visit. Laboratory test values ​​will be graded according to NCI CTCAE for applicable tests. Shifts from baseline grades in toxicity grades will be summarized. Shifts from baseline in ECOG performance status will also be summarized. Vital signs, ECG and concomitant medication data will be summarized. Adverse Event Management

[0617] Dose interruptions and modifications may be performed at the investigator's discretion after cycle 1 for subjects enrolled in Stage I (dose escalation) and may be performed at any time in Stage 2 (dose expansion). The dose interruption and reduction instructions provided in this section are intended to serve as recommended guidelines to ensure subject safety while allowing subjects without signs or symptoms of progression to continue dosing. In addition to these guidelines, more conservative drug interruptions or dose reductions are permitted for management of adverse events at the discretion of the administering investigator if determined to be in the subject's best interest and to ensure the subject's safety.

[0618] Subjects who discontinue niraparib due to intolerance may remain in the study and receive continued treatment with DAN-222, at the investigator's discretion.

[0619] After Cycle 1, subjects may temporarily discontinue all study drug for up to 28 consecutive days if they experience toxicity deemed related to study drug administration and necessitating interruption of study drug administration. Subjects who miss a scheduled study drug dose for >28 consecutive days will be discontinued from the study.

[0620] DAN-222 dosing must be interrupted for any treatment-related non-hematological CTCAE Grade 3 or Grade 4 event. Subjects may resume DAN-222 at a reduced dose level once resolution to Grade ≦1, unless prophylaxis is deemed feasible (Table 17). DAN-222 dose interruption / modification criteria for hematologic toxicity are based on blood counts and the guidance in Table 17 below. Use of granulocyte colony-stimulating factor (G-CSF) is permitted if clinically indicated, per current ASCO guidelines. Reasons for interruption, reduction, or discontinuation of study drug should be documented in the eCRF. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5]

[0621] Test Screening

[0622] Subjects who fully consent to participate in the study will undergo a screening assessment within 28 days (4 weeks) prior to administration of the first infusion of DAN-222 (unless otherwise specified). The screening period will begin on the day the subject signs an IRB / IEC approved informed consent form...

Claims

1. Compounds of formula (I): 【Chemistry 8】 [In the formula, n is a number in the range of 20 to 200, m is a number ranging from 5 to 200.

1. A method for producing a formulation of (i) derivatization of camptothecin (CPT) to obtain Gly-CPT as a trifluoroacetic acid (TFA) salt; (ii) synthesizing a parent mucic acid polymer (MAP); (iii) covalently binding said Gly-CPT to said parent MAP to obtain solid amorphous MAP-Gly-CPT; and (iv) preparing an aqueous formulation of said amorphous MAP-Gly-CPT, thereby producing an aqueous formulation of nanoparticles comprising said compound of formula (I). A method comprising:

2. Step (i) dissolving CPT, N-(tert-butoxycarbonyl)Gly-OH (Boc-Gly) and 4-dimethylaminopyridine (DMAP) in methylene chloride (DCM) to form a reaction mixture; and adding diisopropylcarbodiimide (DIC) to the reaction mixture, thereby producing a solution containing the Boc-Gly-CPT intermediate; The method of claim 1 , comprising:

3. 3. The method of claim 2, wherein said step (i) further comprises removing between 90% and 99% of said DCM from said solution containing said Boc-Gly-CPT intermediate.

4. 4. The method of claim 3, wherein the DCM is removed by vacuum distillation.

5. 4. The method of claim 3, wherein said step (i) further comprises precipitating said Boc-Gly-CPT intermediate using methanol.

6. 6. The method of claim 5, wherein said step (i) further comprises washing the precipitated Boc-Gly-CPT intermediate using isopropyl alcohol (IPA).

7. 7. The method of claim 6, wherein said step (i) further comprises treating said Boc-Gly-CPT intermediate with TFA in DCM to obtain Gly-CPT.

8. Step (ii) is Into a reactor, (succinimidyl propionate) ２ PEG ３５００ (di-SPA-PEG ３５００ ) and mucic acid monomer (MAM) neutral species of formula (II): 【Chemistry 9】 A step of inputting adding dimethylsulfoxide (DMSO) to the reactor; initiating the reaction by adding TFA to the reactor; and DIPEA was added to the reaction, Thereby, the step of obtaining the parent MAP The method of claim 1 , comprising:

9. 9. The method of claim 8, wherein the comonomer ratio of DIPEA:MAM used in step (ii) is at least about 2.

5.

10. 10. The method of claim 9, wherein the comonomer ratio is at least 2.

6.

11. 9. The method of claim 8, wherein step (ii) further comprises precipitating the parent MAP from the DMSO by adding at least one anti-solvent.

12. The method of claim 11 , wherein the at least one anti-solvent is IPA.

13. 12. The method of claim 11, wherein the precipitating step is followed by a filtration step under an inert atmosphere.

14. Step (iii) is adding the parent MAP and DMSO to a reaction vessel; adding the Gly-CPT (TFA salt) and 7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) to the reaction vessel; and adding DIPEA to the reaction vessel, thereby producing a solution containing MAP-Gly-CPT; The method of claim 1 , comprising:

15. 15. The method of claim 14, wherein step (iii) further comprises purifying amorphous MAP-Gly-CPT from the solution using tangential flow filtration (TFF).

16. 16. The method of claim 15, wherein the solution is diluted with water at a pH of 3 prior to the tangential flow filtration, and the tangential flow filtration uses a polyethersulfone (PESU) TFF filter.

17. Step (iv) adding water having a pH of approximately pH 4 to the amorphous MAP-Gly-CPT to produce a nanoparticle (NP) solution; and Adding a formulation buffer to the NP solution. The method of claim 1 , comprising:

18. 20. The method of claim 17, wherein the formulation buffer comprises sodium acetate.

19. 20. The method of claim 18, wherein the formulation buffer comprises NaCl and has a pH of approximately pH 4.

16.

20. 20. The method of claim 18, wherein after the step of adding water having a pH of approximately pH 4, the NP solution is filtered.

21. 21. The method of claim 20, wherein after the step of adding the formulation buffer to the NP solution, the NP solution is concentrated by ultrafiltration.

22. 1. A formulation comprising nanoparticles, the nanoparticles comprising a mucic acid polymer (MAP) camptothecin (CPT) conjugate compound of formula (I): 【Chemistry 10】 [In the formula, n is a number in the range of 20 to 200, m is a number ranging from 5 to 200. wherein the formulation is formulated as an aqueous formulation.

23. 23. The formulation of claim 22, wherein n is a number in the range of about 75 to 85.

24. 24. The formulation of claim 23, wherein m is a number ranging from about 12 to 20.

25. 25. The formulation of claim 24, wherein the compound of formula (I) has a molecular weight of about 75,000 Da.

26. 26. The formulation of claim 25, wherein the nanoparticles of the formulation contain an average of two chains comprising the compound of formula (I), and the nanoparticles have an average molecular weight of about 150,000 Da.

27. 27. The formulation of claim 26, wherein the nanoparticles of the formulation have an average particle size of about 20 nm to 80 nm.

28. 28. The formulation of claim 27, wherein the nanoparticles have an average particle size of about 30-40 nm.

29. 29. The formulation of claim 28, wherein the formulation has a nanoparticle concentration of about 17-23 mg / mL.

30. 25. The formulation of claim 24, wherein the formulation has a total concentration of CPT between 1.7 and 3.6 mg / mL.

31. 31. The formulation of claim 30, wherein the formulation has a total concentration of CPT of about 2.6 mg / mL.

32. 32. The formulation of claim 31, wherein the formulation comprises the compound of formula (I) in a concentration of about 20 mg / mL.

33. 25. The formulation of claim 24, wherein the formulation has a pH between pH 4 and pH 5.

34. 34. The formulation of claim 33, wherein the formulation has a pH between pH 4 and pH 4.

6.

35. 35. The formulation of claim 34, wherein the formulation has a pH of about 4.

3.

36. 36. The formulation of claim 35, wherein the formulation further comprises at least one buffering agent selected from sodium succinate, sodium citrate, sodium acetate, phosphoric acid, histidine-HCl, and sodium phosphate.

37. 37. The formulation of claim 36, wherein the at least one buffering agent is a sodium acetate buffer.

38. 38. The formulation of claim 37, wherein the formulation further comprises at least one tonicity modifier selected from KCl, NaCl, proline, arginine-HCl, sucrose and glycine.

39. 39. The formulation of claim 38, wherein the at least one tonicity modifying agent is NaCl.

40. 40. The formulation of claim 39, wherein the formulation comprises about 20 mg / mL of the compound of formula (I).

41. 41. The formulation of claim 40, wherein the formulation comprises about 20 mM sodium acetate, about 200 mM NaCl, and has a pH of 4.3±0.

3.

42. A method of treating cancer in a subject, comprising administering to a subject having cancer a compound of formula (I): 【Chemistry 11】 [In the formula, n is a number in the range of 20 to 200, m is a number ranging from 5 to 200. providing at least one dose of a composition comprising:

43. n is a number ranging from about 75 to 85; m is a number ranging from about 12 to 20; The composition has a pH between pH 4 and pH 4.

6.

43. The method of claim 42.

44. 44. The method of claim 43, wherein the composition is supplied in an injectable solution.

45. 45. The method of claim 44, wherein the composition comprises between 2 mg / mL and 3 mg / mL camptothecin (CPT).

46. The dose of the composition is 2 mg / m ２ ~16mg / m ２ 43. The method of claim 42, comprising the compound of formula (I) between:

47. The dose of the composition is about 2 mg / m ２ , about 4mg / m ２ , about 6mg / m ２ , about 8mg / m ２ , about 10mg / m ２ , about 12mg / m ２ , about 14mg / m ２ or about 20 mg / m ２ 43. The method of claim 42, comprising the compound of formula (I):

48. 43. The method of claim 42, wherein multiple doses are provided to the subject during a treatment cycle.

49. 49. The method of claim 48, wherein the multiple doses are provided as weekly doses.

50. Each dose is 2 mg / m ２ ~16mg / m ２ 50. The method of claim 49, comprising the compound of formula (I) between:

51. 43. The method of claim 42, wherein the method further comprises providing at least one dose of a poly(ADP-ribose) polymerase (PARP) inhibitor.

52. 52. The method of claim 51, wherein the PARP inhibitor is selected from olaparib, rucaparib, niraparib, and talazoparib.

53. 53. The method of claim 52, wherein the PARP inhibitor is niraparib.

54. The dosage of the composition comprising the compound of formula (I) is 2 mg / m ２ ~16mg / m ２ 54. The method of claim 53, comprising the compound of formula (I) between:

55. 55. The method of claim 54, wherein multiple doses of the composition comprising the compound of formula (I) are provided to the subject during a treatment cycle.

56. 56. The method of claim 55, wherein the multiple doses of the composition are provided as weekly doses.

57. 57. The method of claim 56, wherein a dose of niraparib is provided to the subject daily during the treatment cycle.

58. 58. The method of claim 57, wherein each dose of niraparib is a 100 mg dose.

59. 43. The method of claim 42, wherein the cancer is one or more of breast, ovarian, brain, lung, testicle, head, neck, esophagus, central nervous system, peripheral nervous system, bladder, stomach, pancreas, liver, oral mucosa, anus, kidney, bladder, urothelium, prostate, endometrium, uterus or fallopian tube cancer, or is colorectal cancer, mesothelioma, melanoma, myeloma, leukemia, lymphoma or Kaposi's sarcoma.

60. 60. The method of claim 59, wherein the cancer is breast cancer.

61. 61. The method of claim 60, wherein the breast cancer is a homologous recombination repair deficient (HRD) positive or HRD-negative breast cancer.

62. 43. The method of claim 42, wherein the cancer is brain metastasis of breast cancer.

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