Therapeutic dendrimers

Abstract

Provided herein are dendrimers comprising: a core unit; a fifth generation building block, the building block being a lysine residue or an analog thereof; a first end group comprising a residue of a camptothecin active substance covalently linked to a diethylene glycol-based linker; and a second end group comprising a PEG group. Also provided herein are pharmaceutical compositions comprising the dendrimers, and methods and uses of the dendrimers in the treatment of conditions such as cancer. Also provided are processes for preparing the dendrimers and intermediates.

Description

Technical Field

[0001] The present invention generally relates to the delivery of camptothecin actives via drug-dendrimer conjugates. The dendrimer comprises a core and building blocks, the outermost building blocks comprising a camptothecin active linked via a cleavable linker. The present invention also relates to pharmaceutical compositions and methods of treatment comprising the drug-dendrimer conjugates, as well as processes and synthetic intermediates for preparing the drug-dendrimer conjugates. Background Art

[0002] Formulating pharmaceutically active agents for delivery to their intended site of action can pose a number of significant challenges associated with issues such as poor aqueous solubility, low bioavailability, instability under biological conditions, rapid degradation in vivo, lack of efficacy, lack of targeted site of action, and toxicity.

[0003] Many methods have been developed to try and solve these problems, including the use of composite formulations intended to improve the bioavailability and / or controlled release of active agents in vivo, for example, involving the use of solubilizing excipients, polymer matrices, or encapsulation in liposomes or micelles. However, there are still problems in controlling the release of pharmaceutically active agents. In some cases, the carrier degrades rapidly, releasing the pharmaceutically active agent before it reaches the target organ. In other cases, the release of the pharmaceutically active agent from the carrier is slowed down to the extent that the therapeutic dose of the drug cannot be reached in the body or in the target organ. In addition, such compositions can present stability and manufacturing challenges.

[0004] Oncology drugs are an important class of medicines that have enabled significant advances in cancer treatment in recent decades. However, many oncology agents are associated with severe side effects due to their cytotoxic properties, providing a narrow therapeutic window and limiting the dosage regimens that can be used and potentially also limiting the efficacy of the treatment.

[0005] One class of oncology drugs is camptothecin compounds. Camptothecin and structurally related compounds (such as SN-38) are topoisomerase 1 inhibitors. However, the known problem of SN-38 is that, although the compound has high topoisomerase 1 inhibitory activity, the preparation is hindered by poor water solubility, and high toxicity and rapid clearance will limit clinical use. Irinotecan is a prodrug containing a dipiperidylcarbamate group at the C-10 position, and when used in vivo, irinotecan is metabolized in the liver and plasma to release the active compound SN-38. Irinotecan has been approved for clinical use as a therapy for the treatment of several cancers. Irinotecan has shown activity in leukemia, lymphoma, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, pancreatic cancer, gastric cancer and breast cancer. However, irinotecan still has rapid clearance and significant side effects, the most notable of which is gastrointestinal toxicity, causing diarrhea, vomiting, abdominal pain, anorexia and hematological toxicity, such as neutropenia, leukopenia and thrombocytopenia. Marketed irinotecan carries a black box warning for diarrhea and bone marrow suppression. In addition, irinotecan is metabolized in the liver by carboxylesterase to the active metabolite SN-38, which is then glucuronidated to the inactive SN-38G, however, the conversion rate and enzyme activity are highly variable among patients. Another problem with camptothecin is that the lactone ring is hydrolyzed to the inactive carboxylate form at pH>6, which reduces its potency.

[0006] To further address some of the shortcomings of the active agent, studies have also been conducted to identify improved formulations of irinotecan. For example, is an injectable liposomal irinotecan product containing liposomes with a diameter of approximately 110 nm dispersed in formulation buffer, which is critical for controlling lipid degradation. The liposomal formulation also carries a black box warning for neutropenia and severe diarrhea.

[0007] Many solubilization strategies have been applied to try to overcome these problems. For example, conjugation of Nektar (NKTR-102) (which failed in Phase 3 studies in metastatic breast cancer), Enzon (EZN2208) and Prolynx (PL038) (PEG) to poly (ethylene glycol) provides relatively water-soluble conjugates. Another approach is to use small micelles or liposomes that are bound and unbound to lipophilic carriers, such as NK-012 (Nippon Kayaku), SN2310 emulsion (Ocogenex) and Irinophore (Champions Oncology). Other approaches are SN-38 triblock copolymer (Mahidol University, Thailand), DSPC / cholesterol nanoparticles, Hyaluran-Ironotecan (Alchemia, failed Phase 3 study in colorectal cancer), CRLX101, cyclodextrin-conjugated CPT-11 (Cerulean), and poly-1-hydroxymethylethylenehydroxymethyl-formaldehyde XMT1001 (Mersana), which failed in Phase 2. These approaches have been largely unsuccessful in the clinic due to lack of efficacy or grade 3 and 4 neutropenia.

[0008] PAMAM-SN-38 dendrimers have been studied in vitro for oral delivery (Goldberg et al, J Control Release, 2011, 150 (3), p318-325). England et al. (J Control Release, 2017, 247, p73-85) produced dendrimers conjugated to the C10 position of SN-38 via random sites on the dendrimer and had only about 8% drug loading. Fox et al. (Mol. Pharm., 2009, 6 (5), p1562-1572) described irinotecan conjugated to aspartic acid or glutamic acid surface dendrimers via carboxylates on glycine or alanine, with 4-6% irinotecan loading.

[0009] There remains a need for alternative and / or improved oncology therapies that provide an effective means of delivering extended effective levels of active agents, with low levels of side effects and a good therapeutic window. There is also a need for therapies that provide consistent and controlled release of therapeutic agents. Such therapies can lead to better patient outcomes and better patient compliance.

[0010] There is also a need to deliver the drug in a controlled and consistent manner under realistic manufacturing conditions, providing a good yield of the active ingredient with well-understood and reproducible properties. Summary of the invention

[0011] The present subject matter is based, in part, on the unexpected discovery that camptothecin active substances, such as SN-38, when conjugated to dendrimers, produce drug-dendrimer conjugates that provide improved efficacy and / or pharmacokinetic properties of the drug.

[0012] Accordingly, in a first aspect, there is provided a dendrimer comprising:

[0013] i) a core unit (C); and

[0014] ii) building units (BU), each building unit being a lysine residue or an analog thereof;

[0015] wherein the core unit is covalently linked to two building blocks via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block;

[0016] The dendrimer is a five-generation building block dendrimer;

[0017] wherein the building blocks of different generations are covalently linked to each other via an amide bond formed between a nitrogen atom present in one building block and a carbon atom of an acyl group present in another building block;

[0018] The dendrimer further comprises:

[0019] iii) a plurality of first terminal groups (T1) comprising a camptothecin active substance residue covalently linked to a diacyl linker of the formula,

[0020] wherein A is C optionally interrupted by O, S, NH or N(Me) 2 -C 10 alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine; and

[0021] iv) a plurality of second end groups (T2) comprising PEG or PEOX groups;

[0022] wherein at least half of the outer building blocks have one nitrogen atom covalently linked to a first terminal group and have one nitrogen atom covalently linked to a second terminal group;

[0023] or a pharmaceutically acceptable salt thereof.

[0024] In some embodiments, the core unit is formed from a core unit precursor comprising two amino groups.

[0025] In some embodiments, the core unit is:

[0026]

[0027] In some embodiments, the building blocks are each:

[0028]

[0029] wherein the acyl group of each building block provides a covalent attachment point for linkage to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linkage to a subsequent generation building block, a first terminal group, or a second terminal group.

[0030] In some embodiments, the building blocks are each:

[0031]

[0032] In some embodiments, the dendrimer has five complete generations of building blocks.

[0033] In some embodiments, the diacyl linker is selected from the group consisting of

[0034]

[0035] In some embodiments, the diacyl linker is:

[0036]

[0037] In some embodiments, the residue of the camptothecin active substance is the residue of SN-38 linked to the diacyl linker via the C-10 or C-20 position.

[0038] In some embodiments, the residues of SN-38 are:

[0039]

[0040] In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0041]

[0042] and is covalently linked to the diacyl linker via an oxygen atom present on the lactone ring; and wherein the diacyl linker is

[0043] Where A is C interrupted by O, S, NH or N(Me) 2 -C 10 Alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine;

[0044] In some embodiments, each first terminal group (T1) is:

[0045]

[0046] In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0047]

[0048] in

[0049] R 1 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ;

[0050] R 2 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ;

[0051] Each R 3 are independently selected from hydrogen and C 1-6 alkyl;

[0052] wherein the residue of the camptothecin active substance is covalently linked to the diacyl linker via an oxygen atom present on the benzene ring; and wherein the diacyl linker is

[0053] Where A is C 2 -C 10 Alkylene.

[0054] In some embodiments, the second end group comprises a PEG group having an average molecular weight of at least 750 Daltons.

[0055] In some embodiments, the second end group comprises a PEG group having an average molecular weight in the range of 1500 to 2500 Daltons.

[0056] In some embodiments, the second end group comprises a PEG group having an average molecular weight in the range of 1800 to 2200 Daltons.

[0057] In some embodiments, the PEG group is a methoxy-terminated PEG.

[0058] In some embodiments, the dendrimer has 28 to 32 surface units comprising an outer building block attached to a first end group and to a second end group.

[0059] In some embodiments, at least 40% of the nitrogen atoms present in the outer building units are each covalently bonded to the first terminal group; and at least 40% of the nitrogen atoms present in the outer building units are each covalently bonded to the second terminal group.

[0060] In some embodiments, the five-generation building block is a full generation, and wherein the building block of the outer generation provides 64 nitrogen atoms for covalent attachment to a first terminal group or a second terminal group, wherein 28 to 32 first terminal groups are covalently attached to one of the nitrogen atoms, and wherein 28 to 32 second terminal groups are each covalently attached to one of the nitrogen atoms.

[0061] In some embodiments, no more than one fifth of the nitrogen atoms present in the exo building block are unsubstituted.

[0062] In some embodiments, the dendrimer is any of the example dendrimers.

[0063] In some embodiments, the % release of camptothecin active species from the dendrimers in PBS at pH 7.4 and 37°C after 6 hours of incubation ranges from 10 to 50%.

[0064] In some embodiments, the dendrimers are combined with additional active substances.

[0065] In some embodiments, the additional active agent is an immunotherapeutic agent.

[0066] In some embodiments, the additional active substance is a PD-1 or PD-L1 inhibitor.

[0067] In some embodiments, the immunotherapeutic agent is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0068] In some embodiments, the immunotherapeutic agent is pembrolizumab.

[0069] In some embodiments, the additional active agent is a PARP inhibitor.

[0070] In some embodiments, the PARP inhibitor is olaparib.

[0071] In some embodiments, the additional active agent is an EGFR inhibitor.

[0072] In some embodiments, the EGFR inhibitor is an EGFR antibody.

[0073] In some embodiments, the EGFR antibody is cetuximab.

[0074] In another aspect, a composition comprising a plurality of dendrimers or pharmaceutically acceptable salts thereof is provided,

[0075] wherein dendrimer is as defined herein,

[0076] The average number of first end groups per dendrimer in the composition is in the range of 24 to 32, and

[0077] The average number of second end groups per dendrimer in the composition is in the range of 24 to 32.

[0078] In some embodiments, the average number of first end groups per dendrimer is in the range of 28-32, and / or the average number of second end groups per dendrimer is in the range of 28-32.

[0079] In some embodiments, the composition is a pharmaceutical composition, and wherein the composition comprises a pharmaceutically acceptable excipient.

[0080] In some embodiments, the dendrimer or pharmaceutical composition is used to treat cancer.

[0081] In another aspect, a method of treating cancer is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a dendrimer as described herein or a pharmaceutical composition as described herein.

[0082] In another aspect, there is also provided use of a dendrimer as described herein or a composition as described herein in the preparation of a medicament for treating cancer.

[0083] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, and cervical cancer.

[0084] In some embodiments, the therapeutically effective amount of the dendrimer is between 2 and 50 mg / m 2 within the range of body surface area.

[0085] In some embodiments, the dendrimers are administered in combination with additional anti-cancer drugs.

[0086] In some embodiments, the additional anti-cancer drug is an immunotherapeutic agent.

[0087] In some embodiments, the immunotherapeutic agent is a PD-1 or PD-L1 inhibitor.

[0088] In some embodiments, the immunotherapeutic agent is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0089] In some embodiments, the immunotherapeutic agent is pembrolizumab.

[0090] In some embodiments, the additional anti-cancer drug is a PARP inhibitor.

[0091] In some embodiments, the additional anti-cancer drug is olaparib.

[0092] In some embodiments, the additional anti-cancer drug is an EGFR inhibitor.

[0093] In some embodiments, the EGFR inhibitor is an EGFR antibody.

[0094] In some embodiments, the EGFR antibody is cetuximab.

[0095] In some embodiments, the dendrimer is any of the example dendrimers.

[0096] In some embodiments, administration of the dendrimer provides at least 2-fold greater therapeutic drug exposure (AUC) of the camptothecin active agent compared to direct administration of an equivalent dose of free camptothecin active agent.

[0097] In some embodiments, administration of the dendrimer provides enhanced clinical efficacy compared to administration of an equivalent dose of free camptothecin active agent.

[0098] In some embodiments, administration of the dendrimer reduces the occurrence of side effects compared to administration of the same dose of free camptothecin active agent.

[0099] In another aspect, there is provided a process for producing a dendrimer as described herein, comprising:

[0100] a)

[0101] a1) reacting a camptothecin active substance intermediate with a dendrimer intermediate or a salt thereof under amide coupling conditions, wherein the camptothecin active substance intermediate is

[0102]

[0103] wherein X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group;

[0104] The dendrimer intermediate comprises:

[0105] i) a core unit (C); and

[0106] ii) building units (BU), each building unit being a lysine residue or an analog thereof;

[0107] wherein the core unit is covalently linked to two building blocks via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block;

[0108] The dendrimer is a five-generation building block dendrimer;

[0109] wherein the building blocks of different generations are covalently linked to each other via an amide bond formed between a nitrogen atom present in one building block and a carbon atom of an acyl group present in another building block;

[0110] The dendrimer further comprises:

[0111] iv) a plurality of second end groups (T2), each second end group comprising a PEG or PEOX group;

[0112] wherein at least half of the outer building blocks have a nitrogen atom covalently linked to a second terminal group and have an unsubstituted nitrogen atom available for reaction with the first intermediate; and

[0113] a2) subjecting the product of step a1) to deprotection conditions to remove the protecting group PG;

[0114] or

[0115] b)

[0116] b1) reacting a surface unit intermediate with a dendrimer intermediate or a salt thereof under amide coupling conditions, wherein the surface unit intermediate is:

[0117] PEG / PEOX Group

[0118] wherein PEG group is a group containing PEG, and PEOX is a group containing PEOX;

[0119] X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group;

[0120] The dendrimer intermediate comprises:

[0121] i) a core unit (C); and

[0122] ii) a building unit (BU), each building unit being a lysine residue or an analogue thereof;

[0123] wherein said core unit is covalently linked via an amide bond to two building units, each amide bond being formed between a nitrogen atom present in said core unit and a carbon atom of an acyl group present in a building unit;

[0124] said dendrimer intermediate is a fourth-generation building unit dendrimer intermediate;

[0125] wherein building units of different generations are covalently linked to each other via an amide bond formed between a nitrogen atom present in one building unit and a carbon atom of an acyl group present in another building unit;

[0126] and wherein the nitrogen atoms present in the outer building units of said dendrimer intermediate are unsubstituted; and

[0127] b2) when said surface unit intermediate contains a protecting group PG, subjecting the product of step b1) to deprotection conditions to remove PG.

[0128] In a further aspect, there is provided an intermediate for the production of a dendrimer, said dendrimer being

[0129]

[0130] wherein X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group.

[0131] In a further aspect, there is provided an intermediate for the production of a dendrimer, said dendrimer being

[0132] PEG / PEOX group

[0133] wherein the PEG group is a PEG-containing group and PEOX is a PEOX-containing group;

[0134] X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group.

[0135] It should be understood that further aspects, embodiments and examples are described herein, which may include one or more of the above-described embodiments or features. Description of the Drawings

[0136] Figure 1 Showing the effects of the vehicle, irinotecan and the dendrimer of the example on the body weight of mice bearing SW620 tumors in a colon cancer model study.

[0137] Figure 2 The efficacy of vehicle, irinotecan and example dendrimers against SW620 tumor xenografts in mice is shown.

[0138] Figure 3 Display from Figure 2 Kaplan-Meier survival curves for selected data from the shown data.

[0139] Figure 4 Shown are the effects of vehicle, irinotecan and example dendrimers on body weight of HT-29 tumor bearing mice in a colon cancer model study.

[0140] Figure 5 The efficacy of vehicle, irinotecan and example dendrimers is shown against HT-29 cell line xenografts in mice.

[0141] Figure 6 show Figure 5 Kaplan-Meier survival curves for the data shown.

[0142] Figure 7 Shown is the effect of irinotecan and example dendrimers on body weight of mice bearing MDA-MB-231 tumors in a breast cancer model study.

[0143] Figure 8 The efficacy of irinotecan and example dendrimers is shown against MDA-MB-231 cell line xenografts in mice.

[0144] Fig. 9 show Figure 8 Kaplan-Meier survival curves for the data shown.

[0145] Fig.10 Shown are the effects of intravenous and intraperitoneal administration of vehicle and example dendrimers on body weight in CAPAN-1 tumor-bearing mice in a pancreatic cancer model study.

[0146] Fig.11 The efficacy of vehicle and example dendrimers administered intravenously and intraperitoneally is shown against CAPAN-1 cell line xenografts in mice.

[0147] Fig.12 The SN-38 material was shown to be released from the dendrimers in plasma.

[0148] Fig.13 The release of SN-38 material from dendrimers in PBS / DMA was shown over several hours.

[0149] Fig.14 The release of SN-38 material from dendrimers in PBS / DMA was shown over several days.

[0150] Fig.15 and 17 The efficacy of vehicle, irinotecan, cetuximab and example dendrimer 2f alone and in combination is shown against HT-29 cell line xenografts in mice.

[0151] Fig.16 Shows Fig.15 Kaplan-Meier survival curves for the data shown in .

[0152] Fig.18 Display from Fig.17 Kaplan-Meier survival curves for selected data and vehicle are shown.

[0153] Fig.19 The efficacy of vehicle, olaparib, irinotecan and Example dendrimer 2f alone and in combination is shown against HT-29 cell line xenografts in mice. DETAILED DESCRIPTION

[0154] General Definition

[0155] Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meanings as commonly understood by one of ordinary skill in the art (eg, chemistry, biochemistry, medicinal chemistry, polymer chemistry, etc.).

[0156] As used herein, the term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be understood to provide clear support for both meanings or either meaning.

[0157] As used herein, unless stated to the contrary, the term "about" means + / - 20%, more preferably + / - 10% of the specified value.

[0158] As used herein, the terms “a”, “an” and “the” include both the singular and plural forms, unless the context clearly indicates otherwise.

[0159] As used herein, the term "subject" refers to any organism susceptible to a disease or condition. For example, the subject can be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cattle, horses, pigs), a companion animal (e.g., dogs, cats), or a laboratory animal (e.g., mice, rabbits, rats, guinea pigs, hamsters). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human. In one embodiment, the subject is a non-human animal.

[0160] As used herein, the term "treating" includes alleviating symptoms associated with a particular condition or disorder. For example, as used herein, the term "treating cancer" includes alleviating symptoms associated with cancer. In one embodiment, the term "treating cancer" refers to a decrease in the size of a cancerous tumor. In one embodiment, the term "treating cancer" refers to an increase in progression-free survival. As used herein, the term "progression-free survival" refers to the length of time during and after treatment of cancer that a patient has the disease, i.e., cancer, but the disease does not recur or symptoms increase.

[0161] As used herein, the term "preventing" includes preventing a particular condition or disorder. For example, as used herein, the term "preventing cancer" refers to preventing the onset or duration of symptoms associated with cancer. In one embodiment, the term "preventing cancer" refers to slowing or stopping the progression of cancer. In one embodiment, the term "preventing cancer" refers to slowing or preventing metastasis.

[0162] As used herein, the term "therapeutically effective amount" refers to a dendrimer administered in an amount sufficient to alleviate or prevent to some extent one or more symptoms of the condition or disorder being treated. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease or disorder, or any other desired change in a biological system. In one embodiment, the term "therapeutically effective amount" refers to a dendrimer administered in an amount sufficient to cause a reduction in the size of a cancerous tumor. In one embodiment, the term "therapeutically effective amount" refers to a dendrimer administered in an amount sufficient to cause an increase in progression-free survival. As used herein, the term "effective amount" refers to an amount of a dendrimer that is effective to achieve the desired pharmacological effect or therapeutic improvement without undue adverse side effects or to achieve the desired pharmacological effect or therapeutic improvement with a reduced side effect profile. The therapeutically effective amount can be determined, for example, by routine experimentation, including but not limited to a dose escalation clinical trial. The term "therapeutically effective amount" includes, for example, a prophylactic effective amount. In one embodiment, a prophylactic effective amount is an amount sufficient to prevent metastasis. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to the metabolism of the compound as well as any of the subject's age, weight, general condition, condition being treated, severity of the condition being treated, and the judgment of the prescribing physician. In any individual case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using routine experimentation.

[0163] As used herein, the term "alkyl" refers to a monovalent straight chain (i.e., straight chain) or branched chain saturated hydrocarbon group. In one embodiment, the alkyl group contains 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, the alkyl group contains 1 to 6 carbon atoms (i.e., C 1-6Examples of the alkyl group include methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, sec-butyl, tert-butyl), pentyl and hexyl).

[0164] As used herein, the term "alkylene" refers to a divalent straight chain (i.e., straight chain) or branched saturated hydrocarbon group. In one embodiment, the alkylene group contains 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkylene group contains 2 to 6 carbon atoms (i.e., C 2-6 Examples of alkylene groups include, for example, -CH 2 CH 2 -、-CH 2 CH 2 CH 2 -、-CH 2 CH(CH 3 )-、-CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH(CH 3 )CH 2 -wait.

[0165] Suitable salts of dendrimers include those formed with organic or inorganic acids or bases. As used herein, the phrase "pharmaceutically acceptable salts" refers to pharmaceutically acceptable organic or inorganic salts. Exemplary acid addition salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, citric acid, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronic acid, sucrose salts, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, and pamoates (i.e., 1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, such as those of potassium and sodium, alkaline earth metal salts, such as those of calcium and magnesium, and salts with organic bases, such as dicyclohexylamine, N-methyl-D-glucamine, morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di- or tri-lower alkylamines, such as ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or mono-, di- or trihydroxy lower alkylamines, such as mono-, di- or triethanolamine. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate ions, succinate ions or other counterions. Counterions may be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, pharmaceutically acceptable salts may have more than one charged atom in their structure. Cases where multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions. It will also be understood that non-pharmaceutically acceptable salts also fall within the scope of the invention, as these may be used as intermediates in the preparation of pharmaceutically acceptable salts or may be used during storage or transport.

[0166] Those skilled in the art of organic and / or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are referred to as "solvates". For example, complexes with water are referred to as "hydrates". As used herein, the phrase "pharmaceutically acceptable solvate" or "solvate" refers to the association of one or more solvent molecules with a compound of the invention. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

[0167] As used herein, the term "dendrimer" refers to a molecule containing a core and a dendritic motif attached to the core. Each dendritic motif is composed of multiple generations of branched building blocks, resulting in a branched structure with an increasing number of branches with each generation of building blocks. "Dendrimers", including drug-dendrimer conjugates, may include pharmaceutically acceptable salts or solvates as defined above.

[0168] As used herein, the term "building block" refers to a branched molecule that is a lysine residue or its analog having three functional groups, one functional group for attachment to the core or the previous generation building block and at least two functional groups for attachment to the next generation building block or the surface of the dendrimer-forming molecule.

[0169] As used herein, the term "attached" refers to the connection between chemical components through covalent bonding. The term "covalent bonding" is used interchangeably with the term "covalently linked".

[0170] Dendrimer

[0171] In a first aspect, there is provided a dendrimer comprising:

[0172] i) a core unit (C); and

[0173] ii) building units (BU), each building unit being a lysine residue or an analog thereof;

[0174] wherein the core unit is covalently linked to two building blocks via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block;

[0175] The dendrimer is a five-generation building block dendrimer;

[0176] wherein the building blocks of different generations are covalently linked to each other via an amide bond formed between a nitrogen atom present in one building block and a carbon atom of an acyl group present in another building block;

[0177] The dendrimer further comprises:

[0178] iii) a plurality of first terminal groups (T1) comprising a camptothecin active substance residue covalently linked to a diacyl linker of the formula,

[0179] wherein A is C optionally interrupted by O, S, NH or N(Me) 2 -C 10 Alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine; and

[0180] iv) a plurality of second end groups (T2) comprising PEG or PEOX groups;

[0181] wherein at least half of the outer building blocks have one nitrogen atom covalently linked to a first terminal group and have one nitrogen atom covalently linked to a second terminal group;

[0182] or a pharmaceutically acceptable salt thereof.

[0183] The core unit (C) of the dendrimer is covalently linked to two building blocks via an amide bond, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block; thus, the core unit can be formed, for example, from a core unit precursor comprising two amino groups. Any suitable diamino-containing molecule can be used as a core unit precursor. In some embodiments, the core unit is:

[0184]

[0185] and can be formed, for example, from a nuclear unit precursor:

[0186]

[0187] It has two reactive (amino) nitrogens.

[0188] The building block (BU) is a lysine residue or an analog thereof and can be formed from a suitable building block precursor, for example, lysine or a lysine analog containing an appropriate protecting group. The lysine analog has two amino nitrogen atoms for binding to subsequently generated building blocks and an acyl group for binding to previously generated building blocks or nuclei.

[0189] Examples of suitable building blocks include

[0190]

[0191] wherein the acyl group of each building block provides a covalent attachment point for linkage to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linkage to a subsequent generation building block, a first terminal group, or a second terminal group.

[0192] In some preferred embodiments, the building blocks are each:

[0193]

[0194] wherein the acyl group of each building block provides a covalent attachment point for linkage to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linkage to a subsequent generation building block, a first terminal group, or a second terminal group.

[0195] In some preferred embodiments, the building blocks are each:

[0196]

[0197] wherein the acyl group of each building block provides a covalent attachment point for linkage to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linkage to a subsequent generation building block, a first terminal group, or a second terminal group.

[0198] Outermost building unit (BU outer ) can be formed from lysine or lysine analog building blocks, as used for other generations of building blocks (BU) as described above. The outermost generation building block (BU outer ) is the generation of a building block which is the outermost generation of the core of the dendrimer, i.e., in the outermost generation building block (BU outer ) to which no additional generation of building blocks is connected.

[0199] It will be appreciated that the dendritic motifs of the dendrimers can be synthesized to the desired number of generations, for example by linking the building blocks (BU) accordingly. In some embodiments, each generation of building blocks (BU) can be formed from the same building blocks, for example, the building blocks of all generations can be lysine building blocks. In some other embodiments, one or more generations of building blocks can be formed from building blocks that are different from other generations of building blocks.

[0200] The dendrimer is a five-generation building block dendrimer. A five-generation building block dendrimer is a dendrimer having a structure comprising five building blocks covalently linked to each other, for example in the case where the building block is lysine, it may comprise the following substructures:

[0201]

[0202] In some embodiments, the dendrimer has five complete generations of building units. For a core with two reactive amine groups, such a dendrimer would contain 62 building units (i.e., core unit + 2BU + 4BU + 8BU + 16BU + 32BU). However, it should be understood that due to the nature of the synthetic method used to prepare the dendrimer, one or more reactions performed to prepare the dendrimer may not be fully completed. Therefore, in some embodiments, the dendrimer may contain incomplete generations of building units. For example, a population of dendrimers can be obtained in which the dendrimers have a distribution of the number of building units per dendrimer. In some embodiments, a population of dendrimers is obtained, wherein the average number of building units per dendrimer is at least 55 or at least 56 or at least 57 or at least 58 or at least 59 or at least 60. In some embodiments, a population of dendrimers is obtained in which at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 55 or more building units. In some embodiments, a population of dendrimers is obtained wherein at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers have 60 or more building blocks.

[0203] Each reactive (amino) group of the core represents a conjugation site for a dendrimer comprising a building block.

[0204] In some embodiments, each generation of building blocks in each dendrimer (X) may be represented by the formula [BU] 2 (b-1), where b is the number of generations. A tree primitive (X) with five complete generations of building blocks is represented by [BU] 1 -[BU] 2 -[BU] 4 -[BU] 8 -[BU] 16 .

[0205] Camptothecins

[0206] The dendrimer comprises a plurality of first end groups (T1), the first end groups comprising a camptothecin active substance residue covalently linked to a diacyl linker of the formula,

[0207] wherein A is C optionally interrupted by O, S, NH or N(Me) 2 -C 10 Alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine.

[0208] Camptothecin is a topoisomerase 1 inhibitor with the following structure:

[0209]

[0210] A family of structurally related compounds that also have topoisomerase inhibitory activity has also been identified. In one embodiment, the camptothecin active substance is a compound having the following substructure:

[0211]

[0212] Examples of camptothecin active substances (whose residues may form part of the first terminal group) include SN-38, irinotecan (CPT-11), topotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, and rubitecan. In some embodiments, the residue of the camptothecin active substance is linked to the diacyl linker via the C-10 or C-20 position. In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0213]

[0214] In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0215]

[0216] Where R 1 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ; R 2 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ; Each R 3 are independently selected from hydrogen and C 1-6 In some embodiments, the first terminal group comprises a residue of a camptothecin active substance, which is a residue of SN-38. SN-38 has the following structure:

[0217]

[0218] In some embodiments, the camptothecin active substance residue is a residue of SN-38 linked to the diacyl linker at position C-10 or C-20. In some preferred embodiments, the residue of SN-38 is

[0219]

[0220] In other embodiments, the residues of SN-38 are

[0221]

[0222] Upon in vivo administration, dendrimers typically release the camptothecin active substance (eg, SN-38).

[0223] The residue of the camptothecin active substance is covalently linked to a diacyl linker of the formula

[0224] wherein A is C optionally interrupted by O, S, NH or N(Me) 2 -C 10 alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine. In some embodiments, the diacyl linker is:

[0225] Where A is C interrupted by O, S, NH or N(Me) 2 -C 10 alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine, and N-methylpyrrolidine; in some embodiments, the diacyl linker is:

[0226] Where A is C interrupted by O, S, NH or N(Me) 2 -C 10 In some embodiments, the diacyl linker is:

[0227] Where A is C interrupted by O, S, NH or N(Me) 2 -C 6 In some embodiments, the diacyl linker is:

[0228] Where A is C interrupted by O, S, NH or N(Me) 2 -C 6 In some embodiments, the diacyl linker is:

[0229] Where A is C not interrupted by O, S, or NH 2 -C 6 In some embodiments, the diacyl linker is:

[0230] Where A is a straight chain C not interrupted by O, S, or NH 2 -C 6 In some embodiments, the diacyl linker is:

[0231] wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine. In some embodiments, the diacyl linker is selected from the group consisting of

[0232]

[0233] In some embodiments, the diacyl linker is selected from the group consisting of

[0234]

[0235] In some embodiments, the diacyl linker is:

[0236]

[0237] The residue of the camptothecin active substance is typically covalently linked to the diacyl linker via an ester bond formed between an oxygen atom present as part of the side chain of the camptothecin active substance and a carbon atom of an acyl group present as part of the diacyl linker. The other acyl group of the diacyl linker forms an amide bond with a nitrogen atom present in the outer building block. In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0238]

[0239] and is covalently linked to the diacyl linker via an oxygen atom present on the lactone ring; and wherein the diacyl linker is

[0240] Where A is C interrupted by O, S, NH or N(Me) 2 -C 10 alkylene, or wherein A is a heterocycle selected from the group consisting of tetrahydrofuran, tetrahydrothiophene, pyrrolidine and N-methylpyrrolidine; in some embodiments, the residue of the camptothecin active substance has the following substructure:

[0241]

[0242] and is covalently linked to the diacyl linking group via an oxygen atom present on the lactone ring; and the diacyl linking group is

[0243]

[0244] In some embodiments, each first terminal group (T1) is:

[0245]

[0246] In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0247]

[0248] in

[0249] R 1 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ;

[0250] R 2 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ;

[0251] Each R 3 are independently selected from hydrogen and C 1-6 alkyl;

[0252] and the residue of the camptothecin active substance is covalently linked to the diacyl linker via an oxygen atom present on the benzene ring; and wherein the diacyl linker is

[0253] Where A is C 2 -C 6 In some embodiments, the residue of the camptothecin active substance has the following substructure:

[0254] Where R 1 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ; R 2 Selected from the group consisting of: hydrogen, C 1-6 Alkyl, -OR 3 and -C 1-6 Alkyl-N(R 3 ) 2 ; Each R 3 are independently selected from hydrogen and C 1-6alkyl; and the residue of the camptothecin active substance is covalently linked to the diacyl linker via an oxygen atom present on the benzene ring; and wherein the diacyl linker is

[0255]

[0256] In some embodiments, each first terminal group (T1) is:

[0257]

[0258] The present inventors have discovered that controlled and consistent release of camptothecin actives can be achieved through the combination of specific cleavable linkers with specific hydroxyl groups present in the camptothecin structure, resulting in good biological activity and good pharmacokinetic properties. For example, the C-20 position of a camptothecin active is more sterically hindered than the C-10 position, and a C-20 construct is thermodynamically less favorable than a corresponding C-10 construct. However, linker groups have been identified that release the drug from a dendrimer at a desired rate, that can be conjugated to a camptothecin active in high yield, and that can also be used to achieve good levels of loading of a camptothecin active onto a dendrimer.

[0259] The second end group

[0260] The dendrimer comprises a plurality of second end groups (T2), each of which comprises a PEG or PEOX group. The second end group T2 is a pharmacokinetic modifier. A pharmacokinetic modifier is an agent that can modify or regulate the pharmacokinetic properties of a dendrimer or a pharmaceutically active agent (i.e., a camptothecin active substance) delivered by the dendrimer. A pharmacokinetic modifier can regulate the absorption, distribution, metabolism, excretion, and / or toxicity of the dendrimer of the pharmaceutically active agent. A pharmacokinetic modifier (T2) can affect the release rate of the pharmaceutically active agent by slowing down or increasing the rate at which the active agent is released from the dendrimer through chemical (e.g., hydrolysis) or enzymatic degradation pathways. A pharmacokinetic modifier (T2) can change the solubility curve of the dendrimer, increasing or decreasing the solubility of the dendrimer in a pharmaceutically acceptable carrier. A pharmacokinetic modifier (T2) can help the dendrimer deliver the pharmaceutically active agent to a specific tissue (e.g., a tumor). Pharmacokinetic modifiers (T2) can prolong the half-life of the pharmaceutically active agent by reducing the clearance rate of the dendrimer.

[0261] In some embodiments, the second terminal group comprises a PEG group. The PEG group is a polyethylene glycol group, i.e., a group comprising the formula -CH 2 CH 2O-repeating units. The PEG material used to produce the dendrimers of the present invention typically contains a mixture of PEGs with some variation in molecular weight (i.e., ±10%), so where a molecular weight is specified, it is usually an approximation of the average molecular weight of the PEG composition. For example, the term "PEG ～2100 ” refers to a polyethylene glycol (PEG) having an average molecular weight of about 2100 Daltons, i.e., ± about 10% 1890 To PEG 2310 ). The term "PEG ～2300 ” refers to a polyethylene glycol (PEG) having an average molecular weight of about 2300 Daltons, i.e., ± about 10% 2070 To PEG 2530 ). Three methods are commonly used to calculate the MW average: number average molecular weight, weight average molecular weight, and z average molecular weight. As used herein, the phrase "molecular weight" refers to the weight average molecular weight that can be measured using techniques well known in the art, including but not limited to NMR, mass spectrometry, matrix-assisted laser desorption ionization time of flight (MALDI-TOF), gel permeation chromatography or other liquid chromatography techniques, light scattering techniques, ultracentrifugation, and viscometry.

[0262] In some embodiments, the second end group comprises a PEG group with an average molecular weight of about 200 to 5000 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of at least 750 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of 1000 to 2500 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of 1500 to 2500 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of 1900 to 2300 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of 2100 to 2500 daltons. In some embodiments, the second end group comprises a PEG group with an average molecular weight of about 1900, about 2000, about 2100, about 2200, about 2300, about 2400 or about 2500 daltons.

[0263] In some embodiments, the polydispersity index (PDI) of the PEG group is between about 1.00 and about 1.50, between about 1.00 to about 1.25, or between about 1.00 to about 1.10. In some embodiments, the PEG group has a polydispersity index (PDI) of about 1.05. The term "polydispersity index" refers to a measure of the distribution of molecular weights in a given polymer sample. The polydispersity index (PDI) is equal to the weight average molecular weight (M w ) divided by the number average molecular weight (M n) and indicates the distribution of individual molecular masses in a batch of polymers. The polydispersity index (PDI) has values ​​equal to or greater than 1, but will be closer to 1 as the polymers approach uniform length and average molecular weight.

[0264] In the case where the second end group comprises a PEG group, the PEG group can be straight chain or branched. If desired, an end-capped PEG group can be used. In some embodiments, the PEG group is a methoxy-end-capped PEG.

[0265] In one embodiment, the second end group comprises a PEOX group. The PEOX group is a polyethyloxazoline group, i.e., a group comprising repeating units of the formula

[0266]

[0267] PEOX groups are so named because they can be prepared by polymerization of ethyl oxazoline. The PEOX materials used to produce the dendrimers of the present invention typically contain a mixture of PEOX with some variation in molecular weight (i.e., ±10%), so where a molecular weight is specified, it is typically an approximation of the average molecular weight of the PEOX composition. In some embodiments, the second end group comprises a PEOX group having an average molecular weight of at least 750 Daltons, at least 1000 Daltons, or at least 1500 Daltons. In some embodiments, the second end group comprises a PEOX group in the range of an average molecular weight of 750 Daltons to 2500 Daltons or 1000 Daltons to 2000 Daltons. If desired, end-capped PEOX groups may be used. In some embodiments, the PEOX group is a methoxy-end-capped PEOX.

[0268] The second terminal group may be linked to the external building block via any suitable means.In some embodiments, the linker is used to link a PEG group or a PEOX group to the external building block.

[0269] The second end group is typically attached via the use of a second end group precursor containing a reactive group that can react with an amine group, such as a reactive acyl group (which can form an amide bond) or an aldehyde (which can form an amine group under reductive amination conditions).

[0270] In some embodiments, each of the second terminal groups comprises a covalent bond to the PEG linker (L1) via an ether bond formed between a carbon atom present in the PEG group and an oxygen atom present in the PEG linker, and each second terminal group is covalently attached to the building block via an amide bond formed between a nitrogen atom present in the building block and a carbon atom of an acyl group present in the PEG linker. In some embodiments, each of the second terminal groups is PEG group

[0271] And wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of about 1750 to 2500 Daltons.

[0272] In some embodiments, each of the second end groups comprises a covalent bond to the PEOX linking group (L1′) via an ether bond formed between a nitrogen atom present in the PEOX group and a carbon atom present in the PEOX linking group, and each second end group is covalently attached to the building block via an amide bond formed between a nitrogen atom present in the building block and a carbon atom of an acyl group present in the PEOX linking group. In some embodiments, each of the second end groups is PEOX group.

[0273] In the dendrimers of the present invention, at least half of the external building units have one nitrogen atom covalently linked to the first end group and one nitrogen atom covalently linked to the second end group. Thus, the dendrimer can be considered to have a controlled stoichiometry and / or topology. For example, the dendrimer is typically prepared using a synthesis process that allows a high degree of control over the number and arrangement of the first and second end groups present on the dendrimer. The dendrimer can be synthesized using orthogonal protecting groups to allow the end groups to be conjugated to the external building units in a predetermined or controlled manner. In some embodiments, at least two-thirds of the external building units have one nitrogen atom covalently linked to the first end group and one nitrogen atom covalently linked to the second end group; in some embodiments, at least 75%, at least 80%, at least 85%, or at least 90% of the external building units have one nitrogen atom covalently linked to the first end group and one nitrogen atom covalently linked to the second end group; in some embodiments, each functionalized external building unit contains one first end group and one second end group.

[0274] In some embodiments, the dendrimer comprises a surface unit comprising an external building block attached to a first terminal group and a second terminal group, the surface unit having the following structure:

[0275]

[0276] Among those embodiments, in some examples, the PEG group is a methoxy-terminated PEG having an average molecular weight of about 1500 to 2500 Daltons.

[0277] The building block is a lysine residue or an analog thereof. Lysine has an α-nitrogen atom (a nitrogen attached to a carbon atom that is part of the carbonyl group in lysine) and an ε-nitrogen atom (a nitrogen attached to a carbon atom that is part of the carbonyl group in lysine).

[0278] In many cases, dendrimer populations that have been surface-functionalized with dendrimers contain a random stoichiometry and topology of functional groups. For example, the reaction of a dendrimer population containing, for example, 64 reactive surface groups with one or more reactive functional groups can produce a diverse population of functionalized dendrimer products, some of which contain a greater number of functional groups than others. A wide distribution of dendrimer products with different surface topologies can also be obtained in the presence of multiple different surface groups available for reaction with the reactive functional groups.

[0279] In the context of the present invention, in some embodiments, the dendrimer has a controlled stoichiometry and / or a controlled topology with respect to a first end group and a second end group. For example, in some embodiments, the α-nitrogen atom of the outer building unit is linked to the first end group, and the ε-nitrogen atom of the outer building unit is linked to the second end group. In other embodiments, the ε-nitrogen atom of the outer building unit is linked to the first end group, and the α-nitrogen atom of the outer building unit is the second end group.

[0280] The dendrimer scaffolds, intermediates, and processes of the present invention allow for the incorporation of high loads of camptothecin active agents into the dendrimer. Such dendrimers are also thought to promote the release of therapeutically effective levels of the camptothecin active agent over an extended period following administration, and can thus reduce the desired frequency and / or number of administrations.

[0281] The drug loading (% w / w) can be calculated by multiplying the molecular weight of the camptothecin active agent (e.g., SN-38) by the number of camptothecin active molecules loaded onto the dendrimer and dividing by the total molecular weight of the construct. In some embodiments, the camptothecin active agent loading of the dendrimer is at least 10%, at least 11%, at least 12%, at least 13% w / w, or between 10% and 20%, or between 12% and 15%.

[0282] In some embodiments, the dendrimer has 24 to 32, 26 to 32, 28 to 32, 30 to 32, 24 to 30, 26 to 30, 28 to 30, 26 to 30, 26 to 28, or 28 to 30 surface units, the surface units comprising outer building units linked to the first end group and to the second end group.

[0283] In some embodiments, 26 to 32, or 27 to 32, or 28 to 32 first end groups are covalently linked to the nitrogen atoms present on the outer building units. In some embodiments, 26 to 32, or 27 to 32, or 28 to 32 first end groups are covalently linked to the α-nitrogen atoms present on the outer building units.

[0284] In some embodiments, 26 to 32, or 27 to 32, or 28 to 32 second terminal groups are covalently linked to nitrogen atoms present on the outer building blocks. In some embodiments, 26 to 32, or 27 to 32, or 28 to 32 second terminal groups are covalently linked to ε-nitrogen atoms present on the outer building blocks.

[0285] In some embodiments, at least 40% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group. In some embodiments, at least 45% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group. In some embodiments, about 50% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group;

[0286] In some embodiments, at least 40% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group. In some embodiments, at least 45% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group. In some embodiments, about 50% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group;

[0287] In some embodiments, at least 40% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group; and at least 40% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group. In some embodiments, at least 45% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group; and at least 45% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group. In some embodiments, about 50% of the nitrogen atoms present in the outer building units are each covalently linked to the first terminal group; and about 50% of the nitrogen atoms present in the outer building units are each covalently linked to the second terminal group.

[0288] In some embodiments, the five-generation building block is a full generation, and wherein the building block of the outer generation provides 64 nitrogen atoms for covalent attachment to a first terminal group or a second terminal group, wherein 26 to 32 first terminal groups are covalently attached to one of the nitrogen atoms, and wherein 26 to 32 second terminal groups are each covalently attached to one of the nitrogen atoms.

[0289] In some embodiments, no more than one fourth of the nitrogen atoms present in the exo building unit are unsubstituted. In some embodiments, no more than one fifth of the nitrogen atoms present in the exo building unit are unsubstituted. In some embodiments, no more than one sixth of the nitrogen atoms present in the exo building unit are unsubstituted. In some embodiments, no more than one eighth of the nitrogen atoms present in the exo building unit are unsubstituted. In some embodiments, no more than one tenth of the nitrogen atoms present in the exo building unit are unsubstituted.

[0290] In some embodiments, no more than 20 nitrogen atoms present in an Exo building unit are unsubstituted. In some embodiments, no more than 10 nitrogen atoms present in an Exo building unit are unsubstituted. In some embodiments, no more than 5 nitrogen atoms present in an Exo building unit are unsubstituted. In some embodiments, no more than 3 nitrogen atoms present in an Exo building unit are unsubstituted. In some embodiments, no more than 2 nitrogen atoms present in an Exo building unit are unsubstituted. In some embodiments, no more than 1 nitrogen atom present in an Exo building unit is unsubstituted. In some embodiments, substantially all nitrogen atoms present in an Exo building unit are substituted.

[0291] It should be understood that, in addition to the camptothecin active substance and the PEG or PEOX group, additional end groups can be connected to the dendrimer. Therefore, in some embodiments, the dendrimer comprises one or more third end groups. In some embodiments, the third end group comprises the residue of another therapeutic agent, such as a therapeutic agent that is not a camptothecin active substance. For example, the third end group can comprise the residue of other therapeutic agents, and the other therapeutic agent is a chemotherapeutic agent, a chemosensitizer or an immunomodulator. The residue of another therapeutic agent can be connected via, for example, a linker (e.g., a cleavable linker). At least half of the external building block has a nitrogen atom covalently connected to the first end group and has a nitrogen atom covalently connected to the second end group; in some embodiments, when the dendrimer comprises one or more third end groups, the third end group can be connected to the nitrogen atom of the external building block, and the external building block is not covalently connected to the first or second end group.

[0292] In some embodiments, the α-nitrogen atom of the outer building block is linked to the third terminal group. In some embodiments, the ε-nitrogen atom of the outer building block is linked to the third terminal group.

[0293] Conjugate

[0294] In some embodiments, the dendrimer has: a core unit formed from a core unit precursor comprising two amino groups; building blocks, each of which is

[0295]

[0296] wherein the acyl group of each building block provides a covalent attachment point for linking to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linking to a subsequent generation building block, a first terminal group, or a second terminal group; the diacyl linking group is selected from the group consisting of:

[0297]

[0298] and a residue of a camptothecin active substance, which is a residue of SN-38 linked to the diacyl linker via the C-10 or C-20 position.

[0299] In some embodiments, the dendrimer has: a core unit

[0300] Building blocks, each of which is

[0301] wherein the acyl group of each building block provides a covalent attachment point for linking to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linking to a subsequent generation building block, a first terminal group, or a second terminal group; the diacyl linking group is selected from the group consisting of:

[0302]

[0303] and a residue of a camptothecin active substance, which is a residue of SN-38 linked to the diacyl linker via the C-10 or C-20 position.

[0304] In some embodiments, the dendrimer has: a core unit formed from a core unit precursor comprising two amino groups; building blocks, each of which is wherein the acyl group of each building block provides a covalent attachment point for linking to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linking to a subsequent generation building block, a first terminal group, or a second terminal group; the diacyl linking group is selected from the group consisting of:

[0305]

[0306] and the residues of the following camptothecin active substances

[0307]

[0308] In some embodiments, the dendrimer has: a core unit that is Building blocks, each of which is

[0309] wherein the acyl group of each building block provides a covalent attachment point for linking to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linking to a subsequent generation building block, a first terminal group, or a second terminal group; a diacyl linker group, which is

[0310] and the residues of the following camptothecin active substances

[0311]

[0312] In some embodiments, the dendrimer has a core unit that is

[0313] Building blocks, each of which is

[0314]

[0315] wherein the acyl group of each building block provides a covalent attachment point for linking to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linking to a subsequent generation building block, a first terminal group, or a second terminal group; a diacyl linker group, which is

[0316]

[0317] and the residues of the following camptothecin active substances

[0318]

[0319] In some embodiments, the dendrimer is:

[0320]

[0321] , wherein T1′ represents the first terminal group of:

[0322]

[0323] or T1′ represents H, wherein less than 5 T1′ are H; and T2′ represents the second terminal group A PEG group, wherein the PEG group is a methoxy-terminated PEG having a molecular weight in the range of about 1500 to 2500 Daltons, or T2' represents H, and wherein less than 5 T2' are H.

[0324] In some embodiments, the molecular weight of the dendrimer is 50 to 300 kDa. In some embodiments, the molecular weight of the dendrimer is 60 to 200 kDa. In one embodiment, the molecular weight of the dendrimer is 70 to 150 kDa. In one embodiment, the molecular weight of the dendrimer is 100 to 120 kDa.

[0325] In some embodiments, the % release of camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. after 6 hours of incubation ranges from 10 to 50%. In some embodiments, the % release of camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. after 6 hours of incubation ranges from 15 to 40%. In some embodiments, the % release of camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. after 6 hours of incubation ranges from 20 to 30%. In some embodiments, the 50% release of camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. after incubation ranges from 1 to 72 hours. In some embodiments, the 50% release of the camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. is in the range of 5 to 60 hours. In some embodiments, the 50% release of the camptothecin active substance released from the dendrimer in PBS buffer / DMA 9:1 (v / v) at pH 7.4 and 37° C. is in the range of 10 to 24 hours.

[0326] In some embodiments, after incubation, 50% release of the camptothecin active substance released from the dendrimer in rat plasma / PBS (5:1 v / v) at 37°C is 1 to 72 hours. In some embodiments, 50% release of the camptothecin active substance released from the dendrimer in rat plasma / PBS (5:1 v / v) at 37°C ranges from 1 to 24 hours. In some embodiments, 50% release of the camptothecin active substance released from the dendrimer in rat plasma / PBS (5:1 v / v) at 37°C ranges from 2 to 20 hours. In some embodiments, 50% release of the camptothecin active substance released from the dendrimer in rat plasma / PBS (5:1 v / v) at 37°C ranges from 3 to 10 hours.

[0327] In some embodiments, the dendrimer is any of the dendrimers described in the Examples below.

[0328] Composition

[0329] In some embodiments, the dendrimer is presented as a composition, preferably a pharmaceutical composition.

[0330] It should be understood that due to the nature of the synthetic process used to produce the dendrimers, there may be some variation in the molecular composition between the dendrimers present in a given composition. For example, as described above, one or more of the synthetic steps used to prepare the dendrimers may not be carried out completely to completion, which may result in the presence of dendrimers that do not all contain the same number of first or second end groups, or contain incomplete generations of building blocks.

[0331] Thus, provided are compositions comprising a plurality of dendrimers or pharmaceutically acceptable salts thereof, wherein the dendrimers are as defined herein,

[0332] The average number of first end groups per dendrimer in the composition is in the range of 24 to 32, and

[0333] The average number of second end groups per dendrimer in the composition is in the range of 24 to 32. In some embodiments, the average number of first end groups per dendrimer is in the range of 26 to 32, and wherein the average number of second end groups per dendrimer is in the range of 26 to 32. In some embodiments, the average number of first end groups per dendrimer is in the range of 28 to 32, and wherein the average number of second end groups per dendrimer is in the range of 28 to 32. In some embodiments, the average number of first end groups per dendrimer is in the range of 29 to 32, and wherein the average number of second end groups per dendrimer is in the range of 29 to 32. In some embodiments, the average number of first end groups per dendrimer is in the range of 30 to 32, and wherein the average number of second end groups per dendrimer is in the range of 30 to 32. In some embodiments, the composition is a pharmaceutical composition, and the composition comprises a pharmaceutically acceptable excipient.

[0334] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 26 first end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 28 first end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 30 first end groups.

[0335] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 26 second end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 28 second end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 30 second end groups.

[0336] In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 26 first end groups and at least 26 second end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 28 first end groups and at least 28 second end groups. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the dendrimers contain at least 30 first end groups and at least 30 second end groups.

[0337] The present invention also provides pharmaceutical formulations or compositions for both veterinary and human medical use, comprising a dendrimer of the invention or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, and optionally any other therapeutic ingredients, stabilizers, etc. The carrier must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly harmful to the recipient thereof. The compositions of the present invention may also include polymeric excipients / additives or carriers, such as polyvinylpyrrolidone, derivatized celluloses, such as hydroxymethylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose, Ficolls (a polymeric sugar), hydroxyethyl starch (HES), dextran (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin), polyethylene glycol, and pectin. The composition may further include diluents, buffers, citrate, trehalose, binders, disintegrants, thickeners, lubricants, preservatives (including antioxidants, inorganic salts (e.g., sodium chloride), antimicrobials (e.g., benzalkonium chloride), sweeteners, antistatic agents, sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty acid esters, steroids (e.g., cholesterol)), and chelating agents (e.g., EDTA, zinc and other such suitable cations). Other pharmaceutical excipients and / or additives suitable for use in the composition according to the present invention are listed in "Remington: The Science & Practice of Pharmacy", 19.sup.th ed., Williams & Williams, (1995)", and "Physician's Desk Reference", 52.sup.nd ed., Medical Economics, Montvale, NJ (1998), and "Handbook of Pharmaceutical Excipients", Third Ed., Ed. AHKibbe, Pharmaceutical Press, 2000".

[0338] The dendrimers of the present invention can be formulated into compositions, including compositions suitable for intranasal delivery, inhalation into the lungs, administration by aerosol or parenteral (including intraperitoneal, intravenous, subcutaneous or intramuscular injection). The composition can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. All methods include the step of associating the dendrimer with a carrier constituting one or more auxiliary components. Typically, the composition is prepared by associating the dendrimer with a liquid carrier to form a solution or suspension, or alternatively, by associating the dendrimer with a formulation component suitable for forming a solid, optionally granular product, and then, if warranted, the product is formed into the desired delivery form. When it is a granule, the solid formulation of the present invention will generally contain particles ranging in size from about 1 nanometer to about 500 microns. Typically, for solid formulations for intravenous administration, the diameter of the particles will generally be in the range of about 1 nm to about 10 microns. The composition may contain dendrimers of the invention, which are nanoparticles having a particle diameter below 1000 nm, for example between 5 and 1000 nm, in particular between 5 and 500 nm, more particularly between 5 and 400 nm, such as between 5 and 50 nm and in particular between 5 and 20 nm. In one embodiment, the composition contains dendrimers having an average size between 5 and 20 nm. In some embodiments, the dendrimers are polydispersed in the composition with a PDI between 1.01 and 1.8, in particular between 1.01 and 1.5, more particularly between 1.01 and 1.2. In some embodiments, the dendrimers are polydispersed in the composition with a PDI of about 1.1. In one example, the dendrimers are monodispersed in the composition.

[0339] In some preferred embodiments, the composition is formulated for parenteral delivery. For example, in one embodiment, the formulation can be a sterile lyophilized composition suitable for reconstitution in an aqueous vehicle prior to injection.

[0340] In one embodiment, formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the dendrimer, which may, for example, be formulated to be isotonic with the blood of the recipient.

[0341] In some embodiments, the composition is formulated for intraperitoneal delivery. Any suitable delivery method can be used. For example, in some embodiments, delivery can be performed by lavage or aerosol. In one embodiment, the composition is formulated for intraperitoneal delivery and is used to treat cancer in the peritoneal cavity, including malignant epithelial tumors (e.g., ovarian cancer) and peritoneal carcinomatosis (e.g., gastrointestinal cancer, particularly colorectal cancer, gastric cancer, gynecological cancer, and primary peritoneal tumors).

[0342] Also provided are pharmaceutical formulations suitable for administration by inhalation as an aerosol. These formulations contain a solution or suspension of the desired dendrimer or a salt thereof. The desired formulation can be placed in a chamber and atomized. Atomization can be accomplished by compressed air or by ultrasonic energy to form a plurality of droplets or solid particles containing the dendrimer or a salt thereof.

[0343] As described below, the dendrimers of the present invention can be administered, for example, in combination with one or more additional pharmaceutically active agents. In some embodiments, the dendrimers are provided in combination with additional active substances. In some embodiments, a composition is provided, comprising a dendrimer as defined herein or a pharmaceutically acceptable salt thereof, one or more pharmaceutically acceptable carriers and one or more additional pharmaceutically active agents, such as additional anticancer / oncology agents, such as small molecule cytotoxic agents, checkpoint inhibitors, or antibody therapies. Not only can the dendrimers of the present invention be administered with other chemotherapeutic drugs, but they can also be administered with other drugs, such as corticosteroids, antihistamines, analgesics, and drugs that help restore or prevent blood toxicity, such as cytokines.

[0344] In some embodiments, the composition is formulated for parenteral infusion as part of a chemotherapy regimen. In these embodiments, the composition may, for example, be substantially free of or completely free of solubilizing excipients, particularly solubilizing excipients such as polyethoxylated castor oil (e.g., such as those sold under the trade name Sold or sold under the trade name s sold under the trade name polysorbate A solubilizing excipient is an additive that helps the dendrimer dissolve in one or more solvents. In one embodiment, the composition is substantially free of or completely free of polyethoxylated castor oil (e.g., such as sold under the trade name Sold or sold under the trade name Products sold in the market) and polyethoxylated sorbitan monooleate (e.g., such as polysorbate In one embodiment, the composition is substantially or completely free of solubilizing excipients. By avoiding the use of certain solubilizing excipients, the dendrimer composition is less likely to cause side effects, such as acute or delayed hypersensitivity reactions, including life-threatening anaphylaxis and / or severe fluid retention, and / or eliminates the need for steroid pretreatment.

[0345] How to use

[0346] The dendrimers of the invention may be used to treat or prevent any disease, condition or symptom that an unmodified pharmaceutically active agent may be used to treat or prevent. Thus, there is also provided a dendrimer or pharmaceutical composition as described herein for use in therapy.

[0347] In some embodiments, the dendrimer is used in a method of treating or preventing cancer, for example, for inhibiting the growth of a tumor. In some embodiments, the dendrimer is used to treat cancer. Also provided is a method of treating cancer, comprising administering a therapeutically effective amount of a dendrimer to a subject in need thereof. Also provided is the use of a dendrimer as defined herein or a composition as defined herein in the preparation of a medicament for treating cancer.

[0348] In some embodiments, the cancer is a solid tumor. The cancer can be a primary or metastatic tumor. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor.

[0349] In some embodiments, the cancer is a cancer associated with a BRCA1 and / or BRCA2 mutation.

[0350] In some embodiments, the cancer is selected from the group consisting of colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, and cervical cancer. In some embodiments, the cancer is metastatic colon cancer or rectal cancer. In some embodiments, the cancer is metastatic pancreatic adenocarcinoma. In some embodiments, the cancer is metastatic ovarian cancer. In some embodiments, the cancer is non-small cell or small cell lung cancer. In some embodiments, the cancer is stage IV-B, recurrent or persistent cervical cancer. In some embodiments, the cancer is metastatic gastric cancer. In some embodiments, the cancer is metastatic esophageal cancer.

[0351] In some embodiments, a dendrimer for use in therapy (e.g., cancer therapy) has a core that is:

[0352]

[0353] In some embodiments, dendrimers for use in therapy (e.g., cancer therapy) have building blocks, each of which is:

[0354] More preferably

[0355] wherein the acyl group of each building block provides a covalent attachment point for linkage to the core or a previous generation building block; and wherein each nitrogen atom provides a covalent attachment point for linkage to a subsequent generation building block, a first terminal group, or a second terminal group.

[0356] In some embodiments, dendrimers for use in therapy (eg, cancer therapy) have first terminal groups (T1), each of which is:

[0357]

[0358] In some embodiments, the dendrimers used for therapy (e.g., cancer therapy) have second terminal groups, each of which is

[0359] PEG group

[0360] And wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of about 1500 to 2500 Daltons.

[0361] In some embodiments, dendrimers for use in therapy (eg, cancer therapy) have a first end group in the range of 26 to 32 and a second end group in the range of 26 to 32.

[0362] In some embodiments, the dendrimer used for therapy (e.g., cancer therapy) is

[0363]

[0364] Wherein T1′ represents the first terminal group of the following:

[0365]

[0366] or T1′ represents H, wherein less than 5 T1′ are H; and T2′ represents the first terminal group of:

[0367] PEG group

[0368] wherein the PEG group is a methoxy-terminated PEG having an average molecular weight in the range of about 1500 to 2500 Daltons, or T2' represents H, and wherein less than 5 T2' are H.

[0369] In some embodiments, the dendrimer used in therapy (eg, tumor therapy) is any of the dendrimers described in the following examples.

[0370] combination

[0371] In combination therapy, particularly during chemotherapy, drugs are often co-administered with other drugs. Thus, in some embodiments, the dendrimer is co-administered with one or more other pharmaceutically active agents, such as one or more other anticancer agents / drugs. The dendrimer and the one or more other pharmaceutically active agents may be administered simultaneously, subsequently, or separately. For example, they may be administered as part of the same composition, or by separate compositions. The one or more other pharmaceutically active agents may be, for example, anticancer agents used to treat colorectal cancer, gastric cancer, pancreatic cancer, prostate cancer, or breast cancer.

[0372] A specific example of a group of additional anti-cancer drugs is immunotherapeutic agents. As will be understood by those skilled in the art, the phrase 'immunotherapeutic agent' refers to an agent that directs an immune response to attack tumor cells, thereby enhancing the immune system's natural ability to combat disease. In some embodiments, the other pharmaceutically active agent is an immunotherapeutic agent. Thus, in some embodiments, the dendrimer is administered in combination with one or more immunotherapeutic agents. Cancer cells express tumor antigens, which are detected by antibody proteins in the immune system. When normal antibodies bind to external pathogens, the modified immunotherapeutic agent binds to the tumor antigens and labels them for the immune system to inhibit and / or kill. In some embodiments, the immunotherapeutic agent is an antibody.

[0373] One form of immunotherapy is checkpoint inhibitor therapy. These therapies target immune checkpoints, which are key regulators of the immune system that can attenuate the immune response to immune stimulation when stimulated. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint inhibitor therapy can block inhibitory checkpoints, thereby restoring immune system function. In some embodiments, the immunotherapy targets an immune checkpoint protein. Among the immune checkpoint proteins, the programmed cell death protein 1 (PD-1) receptor is expressed on the surface of activated T cells, and its ligands PD-L1 and PD-L2 are typically expressed on the surface of dendritic cells or macrophages. Immunotherapeutic agents that block or inhibit the binding of PD-L1 and / or PD-L2 on tumor cells to PD-1 on T cells prevent tumors from evading the immune response. In some embodiments, the immunotherapeutic agent is a reagent that binds to PD-1, PD-L1, CTL4, CD52, and / or CD20. In some embodiments, the immunotherapy is a PD-1 inhibitor and / or a PD-L1 inhibitor. In one example, the immunotherapeutic agent is a PD-1 inhibitor. In one example, the immunotherapeutic agent is a PD-L1 inhibitor. In one example, the immunotherapeutic agent is a CTL4 inhibitor. In one example, the immunotherapeutic agent is a CD52 inhibitor. In one example, the immunotherapeutic agent is a CD20 inhibitor.

[0374] Several PD-1 and PD-L1 inhibitors have been approved for the treatment of various cancers, including:

[0375] · Pembrolizumab Is approved for melanoma, including advanced melanoma, metastatic non-small cell lung cancer, and head and neck squamous cell carcinoma in patients with BRAF mutations;

[0376] · Nivolumab Is approved for the treatment of inoperable or metastatic melanoma, squamous cell lung cancer, renal cell carcinoma, and Hodgkin lymphoma;

[0377] Atezolizumab Approved for the treatment of lung cancer, bladder cancer, small cell lung cancer, and triple-negative breast cancer;

[0378] Avelumab Approved for the treatment of non-small cell lung cancer, gastric cancer, and Merkel cell carcinoma, and is currently in clinical trials for the treatment of bladder cancer, gastric cancer, head and neck cancer, mesothelioma, ovarian cancer, and kidney cancer;

[0379] Durvalumab Approved for the treatment of locally advanced or metastatic urothelial carcinoma and currently in clinical trials for the treatment of non-small cell lung cancer, advanced metastatic urothelial carcinoma, and recurrent head and neck cancer; and

[0380] Cemiliximab It is approved for the treatment of squamous cell skin cancer, myeloma, lung cancer, and metastatic cutaneous squamous cell carcinoma.

[0381] Several PD-1 and PD-L1 inhibitors are being tested for the treatment of various cancers, including spartalizumab, camrelizumab, sintilizumab, tislelizumab, and toripalimab. In addition, several PD-1 and PD-L1 inhibitors are in experimental development, including KN035, CK-301, AUNP12, CA-170, and BMS-986189.

[0382] Thus, in some embodiments, the immunotherapeutic is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and cemiplimab. In one example, the immunotherapeutic is pembrolizumab. In one example, the immunotherapeutic is nivolumab. In one example, the immunotherapeutic is atezolizumab. In one example, the immunotherapeutic is avelumab. In one example, the immunotherapeutic is durvalumab. In one example, the immunotherapeutic is cemiliximab. The immunotherapeutic may also be a combination of any two or more of the above immunotherapeutics. For example, the immunotherapeutic may be a combination of pembrolizumab and nivolumab.

[0383] Immunotherapeutic agents can be administered according to any suitable conventional method. Typically, immunotherapeutic agents are administered according to the product information of immunotherapeutic agents. When used in combination with the dendrimers of the present application, the dosage of the immunotherapeutic agent may be the same as the dosage of the immunotherapeutic agent monotherapy, or may be different (e.g., reduced dosage). In some embodiments, when used in combination with dendrimers, the dosage of the immunotherapeutic agent is the same as the dosage of the immunotherapeutic agent monotherapy. In some embodiments, when used in combination with dendrimers, the dosage of the immunotherapeutic agent is different from the dosage of the immunotherapeutic agent monotherapy (e.g., lower dosage). Those skilled in the art will appreciate that the dosage of the immunotherapeutic agent and the associated dosage regimen / timetable can be adjusted accordingly.

[0384] For example, when administered as an immunotherapeutic monotherapy, pembrolizumab is administered every three weeks at 200 mg intravenously for 30 minutes. When administered in combination with a dendrimer, the dose of pembrolizumab can be the same as the dose of pembrolizumab monotherapy, or the dose of pembrolizumab can be different from the dose of pembrolizumab monotherapy.

[0385] Thus, in some embodiments, the dendrimer is administered in combination with any one or more of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and cemiplimab. In some embodiments, the dendrimer is administered in combination with nivolumab. In some embodiments, the dendrimer is administered in combination with atezolizumab. In some embodiments, the dendrimer is administered in combination with avelumab. In some embodiments, the dendrimer is administered in combination with durvalumab. In some embodiments, the dendrimer is administered in combination with cemiplimab.

[0386] In the treatment of cancer, immunotherapeutics may be administered in combination with radiation therapy. Thus, in some embodiments, dendrimers or compositions comprising dendrimers are administered in combination with immunotherapeutics (such as pembrolizumab) and in combination with radiation therapy. Radiation therapy may be administered to a patient before, simultaneously with, or after administration of a dendrimer according to the invention.

[0387] Another example of an additional group of anticancer drugs is poly ADP ribose polymerase (PARP) inhibitors. PARP enzymes are a family of proteins involved in many cellular processes, including but not limited to DNA repair, genome stability, and programmed cell death. As a result, inhibition of PARP enzymes presents an attractive anticancer target. In some embodiments, the additional anticancer drug is a PARP inhibitor.

[0388] Several PARP inhibitors are currently known, and a selection of them has been approved for sale. For example, olaparib, rucapabrib, niraparib, and talazoparib have all been approved by the USFDA. Therefore, in some embodiments, the additional anticancer drug is olaparib. In some embodiments, the additional anticancer drug is rucapabrib. In some embodiments, the additional anticancer drug is niraparib. In some embodiments, the additional anticancer drug is talazoparib.

[0389] When used alone as a chemotherapeutic agent, Olaparib is administered orally as a tablet or capsule containing 100mg or 150mg of Olaparib. The recommended daily dose of Olaparib is 300mg, taken twice a day, resulting in an equivalent total daily dose of 600mg. When used in combination with the dendrimer of the present application, the daily dose of Olaparib can be the same as the daily dose of Olaparib monotherapy, or can be different (e.g., a reduced daily dose). In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is the same as the daily dose of Olaparib monotherapy. In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is different from the daily dose (e.g., a lower dose) for Olaparib monotherapy. In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is 600mg. In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is about 600mg. In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is less than 600 mg. In some embodiments, the daily dose of Olaparib is less than about 600, 500, 400, 300, 200, 100, or 50 mg. In some embodiments, the daily dose of Olaparib is between about 50 and 600 mg, 100 and 600 mg, 200 and 600 mg, or 300 and 600 mg. In some embodiments, the daily dose of Olaparib is between about 300 and 600 mg. In some embodiments, the daily dose of Olaparib is in the range of 300 to 500 mg, 300 to 400 mg, 200 to 500 mg, 200 to 400 mg, 200 to 300 mg, 100 to 500 mg, 100 to 400 mg, 100 to 300 mg, or 100 to 200 mg.

[0390] In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is about 8 mg / kg. In some embodiments, when used in combination with a dendrimer, the daily dose of Olaparib is less than 8 mg / kg. In some embodiments, the daily dose of Olaparib is less than about 8, 7, 6, 5, 4, 3, or 2 mg / kg. In some embodiments, the daily dose of Olaparib is between about 2 and 8 mg / kg, 3 and 8 mg / kg, 4 and 8 mg / kg, 5 and 8 mg / kg, 6 and 8 mg / kg, 2 and 7 mg / kg, 3 and 7 mg / kg, 4 and 7 mg / kg, 5 and 7 mg / kg, 2 and 6 mg / kg, 3 and 6 mg / kg, 4 and 6 mg / kg, 2 and 5 mg / kg, 3 and 5 mg / kg, or 2 and 4 mg / kg. One skilled in the art will appreciate that the daily dose of Olaparib can be adjusted accordingly.

[0391] The agents of the combination therapy may involve separate dosing regimens. For example, the dendrimer may need to be administered at a time interval different from the time interval at which the other anticancer agent needs to be administered, i.e., the PARP inhibitor (e.g., olaparib). In some embodiments, a therapeutically effective amount of the dendrimer is administered to a subject in need thereof at a predetermined frequency. In some embodiments, the dendrimer is administered to a subject in need thereof according to a dosing regimen in which the dendrimer is administered once every one to four weeks. In some embodiments, the dendrimer is administered to a subject in need thereof according to a dosing regimen in which the dendrimer is administered once every three to four weeks. In some embodiments, the PARP inhibitor is administered to a subject in need thereof according to a dosing regimen in which the dendrimer is administered once every three to four weeks. In some embodiments, the PARP inhibitor is administered to a subject in need thereof according to a dosing regimen in which the PARP inhibitor is administered once, twice, three times, four times, or five times a day. In some embodiments, the PARP inhibitor is administered once a day. In some embodiments, the PARP inhibitor is administered twice a day. In some embodiments, the PARP inhibitor is administered three times a day. In some embodiments, the PARP inhibitor is administered in a weekly regimen consisting of 5 days of administration and 2 days of non-administration. In some embodiments, the 2 days are consecutive. In some embodiments, Olaparib is administered to a subject in need thereof according to a dosage regimen of administering Olaparib once, twice, or three times a day. In some embodiments, Olaparib is administered once a day. In some embodiments, Olaparib is administered twice a day. In some embodiments, Olaparib is administered three times a day. In some embodiments, Olaparib is administered with a weekly regimen consisting of 5 days of administration and 2 days of non-administration. In some embodiments, the 2 days are continuous.

[0392] The PARP inhibitor may be administered according to any conventional means. In addition, the PARP inhibitor need not necessarily be administered via the same route as the dendrimer. In some embodiments, the PARP inhibitor is administered orally. In some embodiments, the dendrimer is administered intravenously. In some embodiments, the dendrimer is administered intravenously and the PARP inhibitor is administered orally. In some embodiments, olaparib is administered orally. In some embodiments, the dendrimer is administered intravenously and olaparib is administered orally.

[0393] Other PARP inhibitors besides olaparib, rucapabrib, niraparib and talazoparib are known, and the use of such other PARP inhibitors in combination with dendrimers also forms part of the present invention. For example, veliparib, CEP9722, E7016, iniparib and 3-aminobenzamide are known PARP inhibitors. In some embodiments, the additional anticancer drug is veliparib. In some embodiments, the additional anticancer drug is CEP9722. In some embodiments, the additional anticancer drug is E7016. In some embodiments, the additional anticancer drug is iniparib. In some embodiments, the additional anticancer drug is 3-aminobenzamide.

[0394] Olaparib, also known as AZD-2281, MK-7339, and It is an FDA-approved targeted therapy for cancer. It is particularly useful for treating cancers in patients with hereditary BRCA1 or BRCA2 mutations, including but not limited to some ovarian cancers, breast cancers, and prostate cancers. In some embodiments, the combination of dendrimers and PARP inhibitors (e.g., olaparib) is used to treat cancers associated with BRCA1 mutations. In some embodiments, the combination is used to treat cancers associated with BRCA2 mutations. In some embodiments, the combination is used to treat cancers associated with homologous recombination repair mutations (HRRm) or homologous recombination defects (HRDlower). HRR genes include BRCA1 / 2, BRIP1, ATM, RAD54L, and CDK12. In some embodiments, the combination is used to treat solid tumors. In some embodiments, the combination is used to treat homologous recombination repair mutations (HRRm) or homologous recombination defects (HRD) positive (e.g., HRD score ≥42, Myriad MyChoice HRD) solid cancers. In some embodiments, the combination is used to treat cancers associated with ovarian cancer. In some embodiments, the combination is used to treat cancers associated with breast cancer. In some embodiments, the combination is used to treat prostate cancer. In some embodiments, the combination is used to treat pancreatic cancer. In some embodiments, the combination is used to treat colorectal cancer. In some embodiments, the combination is used to treat gastric cancer. In some embodiments, the combination is used to treat small cell lung cancer or non-small cell lung cancer. In some embodiments, the additional anticancer drug is olaparib, and the combination is used to treat ovarian cancer. In some embodiments, the additional anticancer drug is olaparib, and the combination is used to treat breast cancer. In some embodiments, the additional anticancer drug is olaparib, and the combination is used to treat prostate cancer.

[0395] In the treatment of cancer, PARP inhibitors may be administered in combination with radiation therapy. Thus, in some embodiments, a dendrimer or a composition comprising a dendrimer is administered in combination with a PARP inhibitor such as olaparib and in combination with radiation therapy. Radiation therapy may be administered to a patient prior to, concurrently with, or after administration of a dendrimer according to the present disclosure.

[0396] Examples of other pharmaceutically active agents include, but are not limited to, chemotherapeutic and cytotoxic agents, tyrosine kinase inhibitors, checkpoint inhibitors, EGFR inhibitors, and monoclonal antibody therapies. In some embodiments, the EGFR inhibitor is an EGFR antibody. An example of an EFGR antibody is cetuximab. Cetuximab is an anti-epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER-1) antibody. It is suitable for treating patients with EGFR expression, RAS wild-type metastatic colorectal cancer together with irinotecan and for head and neck squamous cell carcinoma. Other EGFR antibodies include necitumumab (squamous nSCLC combined with gemcitabine and cisplatin) and panitumumab. Cetuximab is administered at 400 mg / m 2 Initial dose followed by weekly doses of 250 mg / m 1 hour before irinotecan, platinum-based therapy with fluorouracil, or FOLFIRI 2 . A Cochrane review (2016) reported that patients administered cetuximab in combination with chemotherapy did not experience further delays in the spread of lung cancer and did not prolong life compared with chemotherapy alone. In the first-line setting, the phase III CRYSTAL trial (cetuximab combined with irinotecan as first-line treatment for metastatic colorectal cancer) demonstrated that cetuximab improved upon standard chemotherapy, specifically reducing the risk of progression (8.9 vs. 8 months, HR 0.85; p = 0.048, enhancing tumor response (46.9% vs. 38.7%, OR 1.40; p = 0.004) and radical resection (R0) of metastases with curative intent (p = 0.002). However, OS analysis did not show a statistically significant difference between treatment groups (19.9 vs. 18.6; HR 0.93, p=0.31). However, the inventors have demonstrated that the combination of cetuximab and dendrimers provides improved properties in the HT-29 cell mouse xenograft colorectal cancer model in mice when administered in combination with the camptothecin-containing dendrimers of the present invention. Examples of other EGFR inhibitors include tyrosine kinase inhibitors such as neranitinib (HER2+ breast cancer), osimertinib (T790M+ NSCLC), erlotinib (mPC or NSCLC), gefitinib (NSCLC or metastatic cancer) and lapatinib (HER2+ breast cancer), vandetanib (thyroid cancer) and dacomitinib (NSCLC). In some embodiments, the dendrimer is administered in combination with an EGFR inhibitor (e.g., an EGFR antibody such as cetuximab), and the combination is used to treat, for example, colorectal cancer.

[0397] For the treatment of colorectal cancer, the dendrimers can be administered with, for example, 5-fluorouracil and / or folinic acid, cetuximab, bevacizumab, capecitabine or a platinate (including cisplatin). Examples of other pharmaceutically active agents suitable for various indications include:

[0398] Temozolomide, anlotinib, cyclophosphamide, and niraparib for Ewing sarcoma;

[0399] Anlotinib, ramucirumab, apatinib, cisplatin, and rucapabrib for esophageal cancer;

[0400] Veliparib, DS-8201a, and trastuzumab for breast cancer;

[0401] alitinib, cyclophosphamide, cisplatin, bevacizumab, temozolomide for neuroblastoma;

[0402] Enzalutamide, for prostate cancer;

[0403] Erlotinib, for pancreatic cancer.

[0404] dose

[0405] It should be understood that a therapeutically effective amount refers to a dendrimer administered in an amount sufficient to alleviate or prevent to some extent one or more symptoms of the condition or disorder being treated. The therapeutically effective amount of a dendrimer can be based on, for example, the amount of dendrimer administered. Alternatively, it can be determined based on the amount of camptothecin active substance (e.g., SN-38) that the dendrimer can theoretically deliver, for example, based on the loading of the camptothecin active substance on the dendrimer.

[0406] Dendrimers can be administered by any suitable route, including, for example, dendrimers can be administered intravenously. Irinotecan is typically administered as an IV infusion for 30-90 minutes. In some embodiments, dendrimers are delivered as IV push injections. In some embodiments, dendrimers are administered IV over a period of 0.5 minutes to 90 minutes, or 0.5 minutes to 60 minutes, or 0.5 minutes to 15 minutes, or 0.5 minutes to 5 minutes. In another example, dendrimers can be administered intraperitoneally. The route of administration can be, for example, for a disease or condition that a subject has. For example, in some embodiments, the disease or condition can be an intraperitoneal malignancy, such as gynecological or gastrointestinal cancer, and the dendrimer can be administered intraperitoneally. In some embodiments, dendrimers can be used to treat cancers of the peritoneal cavity, such as malignant epithelial tumors (e.g., ovarian cancer) or metastatic peritoneal cancer (e.g., gastrointestinal cancer, particularly colorectal cancer, gastric cancer, gynecological cancer, and primary peritoneal tumors), and the dendrimers are administered intraperitoneally.

[0407] In some embodiments, the amount of dendrimer administered is sufficient to deliver between 2 and 100 mg of active agent / m 2 Between 2 and 50 mg active agent / m 2 Between 2 and 40 mg active agent / m 2 Between 2 and 30 mg active agent / m 2 Between 2 and 25 mg active agent / m 2 Between 2 and 20 mg active agent / m 2 Between 5 and 50 mg active agent / m 2 Between 10 and 40 mg active agent / m 2 Between 15 and 35 mg active agent / m 2 Between 10 and 20 mg / m 2 Between 20 and 30 mg / m 2 Between, or between 25 and 35 mg active agent / m 2 A dose of 10 mg / kg of active agent in mice should be approximately equivalent to 30 mg / m 2 Human dose (FDA Guidance 2005). (To convert human mg / kg doses to mg / m 2 , this number can be multiplied by 37, FDA Guidance 2005).

[0408] In some embodiments, a therapeutically effective amount of a dendrimer is administered to a subject in need thereof at a predetermined frequency. In some embodiments, a dendrimer is administered to a subject in need thereof according to a dosing regimen in which the dendrimer is administered once every one to four weeks. In some embodiments, a dendrimer is administered to a subject in need thereof according to a dosing regimen in which the dendrimer is administered once every three to four weeks.

[0409] It has been surprisingly found that the dendrimers of the present invention contain residues of a camptothecin active substance, i.e., SN-38, which has increased potency compared to direct administration of irinotecan, the clinically approved SN-38 prodrug. Examples of irinotecan therapy include and As used herein, the terms "unconjugated" and "released" refer to the drug, i.e., the camptothecin active substance, which has dissociated or cleaved from the dendrimer. Such dissociation or cleavage may occur in vivo after administration of the drug-dendrimer conjugate.

[0410] As described above, the dendrimers can be administered in combination with other active agents, such as EGFR inhibitors, such as cetuximab, PARP inhibitors, such as olaparib, or immunotherapeutic agents, such as pembrolizumab. Typically, for such combinations, the dose of each agent administered is lower than the dose of the monotherapy required to achieve a therapeutic or prophylactic effect. In some embodiments, when administered in combination with cetuximab, the dose of the dendrimer is sufficient to deliver between about 2 and about 100 mg of the active agent / m 2 Between about 2 and about 50 mg active agent / m 2 Between about 2 and about 40 mg active agent / m 2 Between about 2 and about 30 mg active agent / m 2 Between about 2 and about 25 mg active agent / m 2 Between about 2 and about 20 mg active agent / m 2 Between about 5 and about 50 mg active agent / m 2 Between about 10 and about 40 mg active agent / m 2 Between about 15 and about 35 mg active agent / m 2 Between about 10 and about 20 mg / m 2 Between about 20 and about 30 mg / m 2 Between, or between about 25 and about 35 mg active agent / m 2 and the dose of cetuximab is about 50 mg / m 2 About 400 mg / m 2 In the range of about 50 mg / m 2 About 250 mg / m 2 In the range of about 50 mg / m2 About 150 mg / m 2 In some embodiments, when olaparib is administered in combination with a dendrimer, the dose of the dendrimer is sufficient to deliver between about 2 and about 100 mg of active agent / m 2 Between about 2 and about 50 mg active agent / m 2 Between about 2 and about 40 mg active agent / m 2 Between about 2 and about 30 mg active agent / m 2 Between about 2 and about 25 mg active agent / m 2 Between about 2 and about 20 mg active agent / m 2 Between about 5 and about 50 mg active agent / m 2 Between about 10 and about 40 mg active agent / m 2 Between about 15 and about 35 mg active agent / m 2 Between about 10 and about 20 mg / m 2 Between about 20 and about 30 mg / m 2 Between, or between about 25 and about 35 mg active agent / m 2 In some embodiments, when pembrolizumab is administered in combination with a dendrimer, the dose of the dendrimer is sufficient to deliver between about 2 and about 100 mg of the active agent / m 2 Between about 2 and about 50 mg active agent / m 2 Between about 2 and about 40 mg active agent / m 2 Between about 2 and about 30 mg active agent / m 2 Between about 2 and about 25 mg active agent / m 2 Between about 2 and about 20 mg active agent / m 2 Between about 5 and about 50 mg active agent / m 2 Between about 10 and about 40 mg active agent / m 2 Between about 15 and about 35 mg active agent / m 2 Between about 10 and about 20 mg / m 2 Between about 20 and about 30 mg / m 2 Between, or between about 25 and about 35 mg active agent / m 2 and / or the dose of pembrolizumab is between about 100 mg / day to about 400 mg / day, or about 100 mg / day to about 200 mg / day.

[0411] When the dendrimer is administered in combination with an EGFR inhibitor such as cetuximab, a PARP inhibitor such as olaparib, or an immunotherapeutic such as pembrolizumab, they can be administered, for example, sequentially. In some embodiments, the other therapeutic agent (e.g., an EGFR inhibitor such as cetuximab; a PARP inhibitor such as olaparib; or an immunotherapeutic such as pembrolizumab) is administered prior to administration of the dendrimer. For example, it can be administered at least 15 minutes, at least 30 minutes, at least 45 minutes in advance. For example, it can be administered no more than 24 hours, no more than 12 hours, no more than 6 hours, no more than 3 hours, no more than 2 hours, or no more than 90 minutes in advance. In some embodiments, the other therapeutic agent (e.g., an EGFR inhibitor such as cetuximab; a PARP inhibitor such as olaparib; or an immunotherapeutic such as pembrolizumab) is administered about 1 hour prior to administration of the dendrimer.

[0412] Pharmacokinetics, Efficacy, and Side Effects

[0413] In some embodiments, the dendrimers of the invention provide one or more of the following: increased therapeutic drug exposure (AUC), increased half-life (t 1 / 2 ), increased T max , reduced C max , and / or reduced toxicity. For example, topotecan is a camptothecin active substance having the following structure:

[0414]

[0415] It has been approved for the treatment of ovarian and lung cancer under the trade name This can be done, for example, after administration of a dendrimer comprising topotecan as the camptothecin active substance and after administration of an equal amount of unconjugated drug (e.g. ) and then compared. In this context, the term "equivalent" means that a dose of dendrimer administered would provide the same number of moles of camptothecin active substance as in a dose of unconjugated drug administered if all camptothecin active substance present as part of the dendrimer were released.

[0416] In some embodiments, the dendrimers of the invention provide one or more of the following: increased therapeutic drug exposure (AUC), increased half-life (t 1 / 2 ), increased T max , reduced C max, and / or reduced toxicity. For example, as described above, irinotecan is a prodrug of the camptothecin active substance SN-38. For example, after administering a dendrimer comprising SN-38 as a camptothecin active substance, and after administering an equal amount of irinotecan (e.g., ) and then compared. As used herein, the term “equivalent” means that administration of a dose of dendrimer, if all SN-38 present as part of the dendrimer is released, will provide the same number of moles of free SN-38 as provided by a dose of irinotecan, assuming that all irinotecan prodrug is cleaved into SN-38.

[0417] In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide one or more of the following: increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased T max , reduced C max , and / or reduced toxicity.

[0418] In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide one or more of the following compared to an equivalent dose of SN-38 (e.g., free SN-38): increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased T max , reduced C max In some embodiments, the dendrimer comprises residues of SN-38 and is administered in an amount of the dendrimer containing an equal or greater amount of SN-38 than the amount of SN-38 provided by administration of the maximum tolerated dose of irinotecan, and provides one or more of the following compared to administration of the maximum tolerated dose of irinotecan: increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased T max , reduced C max , and / or reduced toxicity.

[0419] In some embodiments, the dendrimer comprises residues of SN-38 and is administered in an amount of the dendrimer containing an equal or less amount of SN-38 than the amount of SN-38 provided by administration of the maximum tolerated dose of irinotecan, and provides one or more of the following as compared to administration of the maximum tolerated dose of irinotecan: increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased T max , reduced C max , and / or reduced toxicity.

[0420] In some embodiments, the dendrimer comprises residues of SN-38 and is administered in an amount of dendrimer containing an equal or greater amount of SN-38 as compared to the maximum tolerated dose of SN-38, and provides one or more of the following as compared to administration of the maximum tolerated dose of SN-38: increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased Tmax, decreased Cmax, and / or reduced toxicity.

[0421] In some embodiments, the dendrimer comprises residues of SN-38 and is administered in an amount of the dendrimer containing an equal amount or less amount of SN-38 as compared to the maximum tolerated dose of SN-38, and provides one or more of the following as compared to administration of the maximum tolerated dose of SN-38: increased therapeutic drug exposure (AUC) of free SN-38, increased half-life (t 1 / 2 ), increased Tmax, decreased Cmax, and / or reduced toxicity.

[0422] In some embodiments, administration of the dendrimer provides at least twice the therapeutic drug exposure (AUC) of the free camptothecin active agent compared to direct administration of an equivalent dose of the camptothecin active agent (eg, free camptothecin active agent).

[0423] In some embodiments, the dendrimer comprises residues of SN-38, and administration of the dendrimer provides at least twice the therapeutic drug exposure (AUC) of free SN-38 compared to direct administration of an equivalent dose of irinotecan.

[0424] In some embodiments, the dendrimer comprises SN-38 and provides at least twice the therapeutic drug exposure (AUC) of free SN-38 compared to administration of an equivalent amount of irinotecan administered at its maximum tolerated dose.

[0425] In some embodiments, the dendrimer comprises SN-38 and provides at least twice the therapeutic drug exposure (AUC) of free SN-38 compared to administration of an equivalent amount of SN-38 administered at its maximum tolerated dose.

[0426] The beneficial property of dendrimers that gradually release free SN-38 over time allows therapeutically effective concentrations of the active substance to be sustained over a long period of time without the high C max This means that dendrimers generally have an improved side effect / toxicity profile compared to administration of the same amount of SN-38 or irinotecan by itself, and may allow administration of dendrimers in amounts containing greater amounts of camptothecin active material (e.g., SN-38) bound to the dendrimer than would be provided by administration of the maximum tolerated dose of SN-38 or irinotecan.

[0427] Oncology drugs often have significant side effects, which are attributed to systemic toxicity, such as hematotoxicity, neurotoxicity, cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity, and gastrointestinal reactions. For example, camptothecin such as irinotecan can cause side effects such as: diarrhea, myelosuppression, neutropenia, neutropenic fever, neutropenic infection, leukopenia, thrombocytopenia, lymphopenia, hypersensitivity, kidney damage, renal failure, pulmonary toxicity (dyspnea, cough, pneumonia, interstitial lung disease), teratogenicity, nausea, vomiting, dehydration, abdominal pain, septic shock, constipation, anorexia, mucositis, anemia, fatigue, pain, fever, infection, dizziness, drowsiness, confusion, vasodilation, hypotension, thromboembolic events, abnormal bilirubin, rash, hair loss, or weight loss.

[0428] In some embodiments, the administration of dendrimers reduces the occurrence of side effects and / or toxicity compared to the administration of the same dose of free camptothecin active substance. In some embodiments, the side effects are diarrhea, and / or vomiting / nausea, and / or myelosuppression.

[0429] In some embodiments, the camptothecin active substance is a residue of SN-38, and the administration of dendrimers reduces the incidence of side effects and / or toxicity compared to the administration of an equal dose of irinotecan. In some embodiments, the side effects are diarrhea and / or myelosuppression.

[0430] The toxicity of a drug refers to the degree of damage caused to an organism and is measured by its effect on a target. In oncology, one such measurement of toxicity in animal models is weight loss, which determines the maximum tolerated dose (MTD). In humans, toxicity is usually determined by specific adverse events (AE events), which usually identify dose-limiting toxicity. It should be understood that, generally in oncology, there is a narrow therapeutic window and off-target toxicity is considered a normal side effect of killing tumor cells.

[0431] In some embodiments, when used in a method of treating cancer, such as in colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, or cervical cancer, the administration of dendrimers provides reduced toxicity compared to the administration of an equal dose of free camptothecin active substance. In some embodiments, the camptothecin active substance is SN-38, and when used in a method of treating cancer, such as in colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, or cervical cancer, the administration of dendrimers provides reduced toxicity compared to the administration of an equal dose of irinotecan.

[0432] In some embodiments, the incidence of adverse reactions is comparable to that of the direct administration of the drug, as measured by the number of patients with a particular AE (e.g., diarrhea, myelosuppression, neutropenia, neutropenic fever, neutropenic infection, leukopenia, thrombocytopenia, lymphopenia, hypersensitivity reaction, renal impairment, renal failure, pulmonary toxicity (dyspnea, cough, pneumonia, interstitial lung disease), teratogenicity, nausea, vomiting, dehydration, abdominal pain, septic shock, constipation, anorexia, mucositis, anemia, fatigue, pain, fever, infection, dizziness, somnolence, confusion, vasodilation, hypotension, thromboembolic events, abnormal bilirubin, rash, alopecia, or weight loss). The dendrimers provide at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in toxicity compared to an equivalent dose of the free camptothecin active, or a reduction in the severity of the AE as measured by a reduction in the grade of the AE (Common Terminology Criteria for Adverse Events, "CTCAE") compared to direct administration of an equivalent dose of the free camptothecin active in at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the patient population compared to direct administration of an equivalent dose of the free camptothecin active.

[0433] In some embodiments, administration of the dendrimer provides less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% toxicity compared to direct administration of an equivalent dose of the free camptothecin active agent, or the severity of the toxicity is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to direct administration of an equivalent dose of the free camptothecin active agent.

[0434] In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and is selected by having a specific AE (e.g., diarrhea, myelosuppression, neutropenia, neutropenic fever, neutropenic infection, leukopenia, thrombocytopenia, lymphopenia, hypersensitivity reaction, renal impairment, renal failure, pulmonary toxicity (dyspnea, cough, pneumonia, interstitial lung disease), teratogenicity, nausea, vomiting, dehydration, abdominal pain, septic shock, constipation, anorexia, mucositis, anemia, fatigue, pain, fever, infection, dizziness, somnolence, confusion, vasodilation, hypotension, thromboembolic events, abnormal bilirubin, skin The dendrimers provide at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% reduction in toxicity as measured by the number of patients experiencing an AE (e.g., rash, alopecia, or weight loss) compared to directly administered an equivalent dose of irinotecan, or a reduction in severity of the AE as measured by a reduction in the grade of the AE compared to directly administered an equivalent dose of irinotecan in a patient population that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than directly administered an equivalent dose of the free camptothecin active substance.

[0435] In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer provides less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the incidence of toxicity (e.g., diarrhea, bone marrow suppression, neutropenia, neutropenic fever, neutropenic infection, leukopenia, thrombocytopenia, lymphopenia, hypersensitivity reaction, renal impairment, renal failure, pulmonary toxicity (dyspnea, cough, pneumonia, interstitial lung disease), teratogenicity, nausea, vomiting, dehydration, abdominal pain, septic shock, constipation, anorexia, mucositis, anemia, fatigue, pain, fever, infection, dizziness, somnolence, confusion, vasodilation, hypotension, thromboembolic events, abnormal bilirubin, rash, hair loss, or weight loss) compared to direct administration of an equivalent dose of irinotecan.

[0436] A significant problem associated with the clinical use of irinotecan is its tendency to cause gastrointestinal toxicity. Frequent and often severe gastrointestinal toxicity, especially diarrhea, limits its wider application. Early symptoms of gastrointestinal toxicity include diarrhea, vomiting, sweating and abdominal cramps. In fact, such symptoms have been reported in up to 80% of patients taking irinotecan. The complex pharmacology and metabolism of irinotecan are considered to be the cause of the associated gastrointestinal toxicity. It is believed that irinotecan, a prodrug, is converted to its active form SN-38 by carboxyl-esterase, which is mainly found in the liver in humans. Subsequently, SN-38 is glucuronidated to SN-38-glucuronide (SN-38G) by hepatic uridine diphosphate glucuronosyltransferase-1A1 (UDP-GT 1A1). Both SN-38 and SN-38G are excreted via urine and bile. Once in the intestinal cavity, SN-38G is decoupled back to SN-38 by bacterial β-glucuronidase. Free luminal SN-38, either bile or SN-38G unconjugated, is believed to be the cause of irinotecan-induced diarrhea.

[0437] Compared to direct administration of irinotecan, the use of dendrimers (SN-38) containing camptothecin active substances, such as described herein, can avoid gastrointestinal toxicity, or reduce the incidence of gastrointestinal toxicity. This may be the result of ester cleavage to release the free drug (i.e., SN-38), which may occur more predominantly outside the liver, thus avoiding or reducing the tendency for biliary excretion.

[0438] Thus, in some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces gastrointestinal toxicity and / or related side effects compared to direct administration of an equivalent dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces gastrointestinal toxicity and / or related side effects as measured by the number of patients with gastrointestinal toxicity and / or related side effects compared to direct administration of an equivalent dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces gastrointestinal toxicity and / or related side effects in at least 5%, or at least 10%, or at least 20%, or at least 30%, at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of patients compared to direct administration of an equivalent dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and the administration of the dendrimer reduces gastrointestinal toxicity symptoms and / or related side effects selected from the group consisting of acute diarrhea, delayed diarrhea, vomiting, sweating, and abdominal cramps. In one example, the residue of the camptothecin active substance is a residue of SN-38, and the administration of the dendrimer reduces gastrointestinal toxicity symptoms and / or related side effects selected from the group consisting of acute diarrhea, delayed diarrhea, vomiting, sweating, and abdominal cramps in at least 5%, or at least 10%, or at least 20%, or at least 30%, at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of patients. In one example, the residue of the camptothecin active substance is a residue of SN-38, and the administration of the dendrimer reduces acute diarrhea compared to the direct administration of an equivalent dose of free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces delayed diarrhea compared to direct administration of an equal dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces vomiting compared to direct administration of an equal dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces sweating compared to direct administration of an equal dose of a free camptothecin active substance (e.g., irinotecan). In one example, the residue of the camptothecin active substance is a residue of SN-38, and administration of the dendrimer reduces abdominal cramping compared to direct administration of an equal dose of a free camptothecin active substance (e.g., irinotecan).

[0439] The dendrimers of the present invention surprisingly achieve a sustained pharmacokinetic profile for the unconjugated or released drug, resulting in a significant increase in AUC compared to the equivalent or normalized amount of free drug. The sustained pharmacokinetic profile and the associated increased release / unconjugated camptothecin active substance AUC indicate that the drug will be present in the body for a longer time at a therapeutically effective level. It should be understood that exposure to the drug for a longer time is desirable because it can prolong the therapeutic effect of the drug and allow for a reduced dosing frequency. In some embodiments, the dendrimer provides an increased therapeutic drug exposure / area under the curve (AUC) of the camptothecin active substance compared to the direct administration of an equal dose of free camptothecin active substance. In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and the dendrimer provides an increased therapeutic drug exposure / area under the curve (AUC) of SN-38 compared to the direct administration of an equal dose of irinotecan. AUC is the area under the curve of drug concentration in plasma versus time. AUC represents total drug exposure over time. It should be understood that AUC is generally proportional to the total amount of drug delivered to the body. In some embodiments, the dendrimer achieves a more sustained in vivo pharmacokinetic profile for concentration levels of released SN-38 compared to the pharmacokinetic profile for SN-38 concentration levels achieved by administration of an equal dose of free irinotecan.

[0440] In some embodiments, the dendrimer provides an increased therapeutic drug exposure / area under the curve (AUC) of the camptothecin active substance compared to direct administration of an equivalent dose of the free camptothecin active substance. In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and the dendrimer provides an increased therapeutic drug exposure / area under the curve (AUC) of SN-38 compared to direct administration of an equivalent dose of irinotecan. In some embodiments, administration of the dendrimer provides at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 10 times, or at least 15 times greater therapeutic drug exposure (AUC) of the camptothecin active substance compared to direct administration of an equivalent dose of the free camptothecin active substance. In some embodiments, the residue of camptothecin is the residue of SN-38, and administration of the dendrimer provides at least 100, at least 200 ng / h / mL, at least 300 ng / h / mL, at least 400 ng / h / mL, at least 500 ng / h / mL, at least 600 ng / h / mL, at least 700 ng / h / mL, at least 800 ng / h / mL, at least 900 ng / h / mL, at least 1000 ng / h / mL, at least 2000 ng / h / mL, at least 3000 ng / h / mL, at least 5000 ng / h / mL, or at least 10000 ng / h / mL of free SN-38. In some embodiments, the residue of camptothecin is the residue of SN-38, and administration of the dendrimer provides between about 100 ng / h / mL and about 10000 ng / h / mL, or between about 1000 ng / h / mL and about 10000 ng / h / mL, or between about 2000 ng / h / mL and about 5000 ng / h / mL of free SN-38.

[0441] Embodiments of the present invention dendrimers can release camptothecin active substances so as to achieve therapeutically effective concentrations of the active substances over a sustained period of time without achieving high C max level, thereby reducing the possibility of AE.

[0442] The maximum drug concentration (C max ) is the maximum (or peak) serum concentration of a drug achieved in a specified compartment or test area of ​​the body after the drug has been administered and before a second dose is administered. It should be understood that while it is important to be able to administer a pharmaceutical agent at a level sufficient to achieve therapeutic concentration levels, if the maximum concentration level achieved is high, there is an increased risk of encountering off-target effects, side effects, and increased toxicity. This is particularly a problem for compounds with short half-lives, because in these cases, in order to provide therapeutically effective levels of the active substance over an extended period of time, it may be necessary to increase the dose and therefore increase the C max, resulting in an increased likelihood of side effects. Therefore, it is highly desirable to be able to deliver pharmaceutically active agents in a form that provides therapeutically effective levels for a sustained period of time while avoiding the need to achieve very high maximum concentrations (C max ) level.

[0443] In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide a reduced C of free SN-38 compared to administration of an equivalent dose of SN-38. max In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide a reduced C of free SN-38 compared to administration of an equivalent dose of irinotecan. max In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide a C of free SN-38 that is less than 0.8 times, less than 0.75 times, less than 0.5 times, compared to administration of an equivalent dose of SN-38. max In some embodiments, the residue of camptothecin is the residue of SN-38, and administration of the dendrimer provides less than about 50 ng / mL, less than about 40 ng / mL, or less than about 30 ng / mL of free SN-38.

[0444] As described above, dendrimers according to the present invention exhibit sustained exposure when administered in vivo. In some embodiments, the pharmacokinetic profile of the camptothecin active substance released from the dendrimer is observed to have an increased terminal half-life (t 1 / 2 In some embodiments, the residue of the camptothecin active substance is SN-38, and the SN-38 released from the dendrimer has an increased terminal half-life (t 1 / 2 In some embodiments, the dendrimers of the invention comprise residues of SN-38 and provide at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 15-fold higher t of free SN-38 than administration of an equivalent dose of SN-38. 1 / 2 . The half-life of a drug is the time it takes for the plasma concentration of the drug to be halved. It will be appreciated that an increased (i.e., longer) half-life may be desirable because it results in a longer period of exposure to therapeutically effective concentrations of the drug. This also results in the need for less frequent dosing. In some embodiments, the residue of camptothecin is a residue of SN-38, and administration of the dendrimer results in a pharmacokinetic profile of free SN-38 (i.e., SN-38 that has been released from the dendrimer) having a t of at least about 5 hours, at least about 10 hours, at least about 20 hours, at least about 25 hours, or at least about 30 hours. 1 / 2In some embodiments, the camptothecin residue is a residue of SN-38, and administration of the dendrimer provides a pharmacokinetic profile of free SN-38 having a T of at least about 0.5 hours, at least about 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours. max .

[0445] In some embodiments, when used in a method of treating cancer, such as colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, or cervical cancer, the camptothecin active agent released from the dendrimer has an increased terminal half-life (t t ) compared to directly administering an equivalent dose of the free camptothecin active agent. 1 / 2 ).

[0446] In some embodiments, the camptothecin active substance residue is SN-38, and when used in a method of treating cancer, such as colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer, or cervical cancer, the SN-38 released from the dendrimer has an increased terminal half-life (t ) compared to directly administering an equivalent dose of irinotecan. 1 / 2 ).

[0447] It is understood that any one or more of improved therapeutic drug exposure (AUC), increased half-life (t 1 / 2 ) and reduced drug toxicity can provide better clinical efficacy. In some embodiments, the administration of the dendrimer provides enhanced clinical efficacy compared to the administration of an equal dose of a free camptothecin active substance. In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and the administration of the dendrimer provides enhanced clinical efficacy compared to the administration of an equal dose of irinotecan. In some embodiments, compared to the direct administration of an equal dose of a free camptothecin active substance, the dendrimer provides an improvement in efficacy properties selected from the group consisting of: progression-free survival, progression time, objective response rate (PR+CR), total response rate, overall survival, and duration of response. In some embodiments, the residue of the camptothecin active substance is a residue of SN-38, and compared to the direct administration of an equal dose of irinotecan, the dendrimer provides an improvement in efficacy properties selected from the group consisting of: progression-free survival, progression time, objective response rate (PR+CR), total response rate, overall survival, and duration of response.

[0448] Dendrimer Synthesis

[0449] The dendrimers of the invention may be prepared by any suitable method, for example by reacting a camptothecin active intermediate with a dendrimer intermediate already containing a PEG or PEOX group to introduce the pharmaceutically active agent, or by reacting an intermediate comprising a lysine group, a camptothecin active residue and a residue of a PEG or PEOX group with a dendrimer intermediate. Thus, in a fifth aspect, there is provided a process for producing a dendrimer as defined herein, comprising:

[0450] a)

[0451] a1) reacting a camptothecin active substance intermediate with a dendrimer intermediate or a salt thereof under amide coupling conditions, wherein the camptothecin active substance intermediate is

[0452]

[0453] wherein X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group;

[0454] The dendrimer intermediate comprises:

[0455] i) a core unit (C); and

[0456] ii) building units (BU), each building unit being a lysine residue or an analog thereof;

[0457] wherein the core unit is covalently linked to two building blocks via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in a building block;

[0458] The dendrimer is a five-generation building block dendrimer;

[0459] wherein the building blocks of different generations are covalently linked to each other via an amide bond formed between a nitrogen atom present in one building block and a carbon atom of an acyl group present in another building block;

[0460] The dendrimer further comprises:

[0461] iv) a plurality of second end groups (T2), each second end group comprising a PEG or PEOX group;

[0462] wherein at least half of the outer building blocks have a nitrogen atom covalently linked to a second terminal group and have an unsubstituted nitrogen atom available for reaction with the first intermediate; and

[0463] a2) subjecting the product of step a1) to deprotection conditions to remove the protecting group PG,

[0464] or

[0465] b)

[0466] b1) Under amide coupling conditions, react the surface unit intermediate with the dendrimer intermediate or its salt, wherein the surface unit intermediate is:

[0467] PEG / PEOX group

[0468] wherein the PEG group is a PEG-containing group and the PEOX group is a PEOX-containing group;

[0469] X is -OH or a leaving group, or X together with the C(O) group to which it is attached forms a carboxylate salt; and wherein PG is a protecting group;

[0470] The dendrimer intermediate comprises:

[0471] i) a core unit (C); and

[0472] ii) a building unit (BU), each building unit being a lysine residue or an analogue thereof;

[0473] wherein the core unit is covalently linked to two building units via amide bonds, each amide bond being formed between a nitrogen atom present in the core unit and a carbon atom of an acyl group present in the building unit;

[0474] The dendrimer intermediate is a four-generation building unit dendrimer intermediate;

[0475] wherein the building units of different generations are covalently linked to each other via amide bonds formed between a nitrogen atom present in one building unit and a carbon atom of an acyl group present in another building unit;

[0476] and wherein the nitrogen atoms present in the outer building units of the dendrimer intermediate are unsubstituted; and

[0477] b2) In the case where the surface unit intermediate contains the protecting group PG, subject the product of step b1) to deprotection conditions to remove PG.

[0478] Process variants a) and b) involve the formation of an amide bond by reaction of a -C(O)X group with an amine group present in the dendritic intermediate. Any suitable amide forming conditions may be used. Examples of typical conditions include the use of a suitable solvent (e.g. dimethylformamide), optionally a suitable base, and at a suitable temperature (e.g. ambient temperature, e.g. in the range of 15°C to 30°C). Where X is a leaving group, any suitable leaving group may be used, such as an activated ester. Where X is a -OH group or where X forms a carboxylate together with the C(O) group to which it is attached, this group is typically converted to a suitable leaving group prior to reaction with the dendritic intermediate, for example by using a suitable amide coupling agent such as PyBOP.

[0479] Where a protecting group PG is used, any suitable group may be used which is inert or substantially inert to the reaction conditions following step a1) or b1), but which can subsequently be removed under mild reaction conditions which do not result in a significant level of side reactions to the dendritic structure, i.e., such that a good yield of the final dendrimer can be obtained. Protecting groups are known in the art, and for hydroxyl groups, examples include: silyl ethers, such as TBDMS or TBDPS; acid labile ether protecting groups, such as THP or MOM groups; or reducible ether protecting groups, such as benzyl groups.

[0480] Any suitable separation and / or purification technique may be used, for example the dendrimer may be obtained by dissolution in a suitable solvent (eg THF) and precipitation by addition of an anti-solvent (eg MTBE).

[0481] The camptothecin active substance intermediate used in variant a) itself is obtainable, for example, by reaction of the camptothecin active substance (for example in protected form) with diglycolic anhydride.

[0482] The surface unit intermediate used in variant b) can itself be obtained, for example, by the following steps:

[0483] i) making a PEG or PEOX intermediate, wherein the PEG or PEOX intermediate is:

[0484] PEG / PEOX Group

[0485] wherein the PEG group is a PEG-containing group and the PEOX group is a PEOX-containing group, and

[0486] X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate;

[0487] With the following reaction,

[0488]

[0489] wherein PG1 is an amine protecting group (such as a Boc or Cbz group), and PG2 is an acid protecting group (such as a methyl ester or a benzyl ester);

[0490] ii) deprotecting PG1;

[0491] iii) reacting the product of step ii) with a camptothecin active intermediate, wherein the camptothecin active intermediate is:

[0492]

[0493] wherein X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and

[0494] iv) Deprotecting PG2.

[0495] The dendrimer intermediate used in variant a) can itself be obtained, for example, by a continuous process comprising the following steps:

[0496] i) reacting a core unit (C) containing an amino group with a building unit, which is a protected lysine or an analog thereof, containing a -C(O)X group, wherein X is -OH or a leaving group or -CO(X) to form a carboxylate, and wherein the amino group present in the lysine or an analog thereof is protected, to form an amide bond between the core unit and the building unit;

[0497] ii) deprotecting the protecting groups present on the building blocks;

[0498] iii) reacting a free amino group present on a building block with another building block, said other building block being a protected lysine or an analog thereof, which contains a -C(O)X group, wherein X is -OH or a leaving group or -C(O)X forms a carboxylate, and wherein the amino group present in the lysine or an analog thereof is protected, to form an amide bond between building blocks of different generations;

[0499] iv) deprotecting the protecting groups present on the building blocks;

[0500] v) repeating steps iii) and iv) until four generations of building units are generated;

[0501] vi) reacting the free amino groups present on the building blocks with

[0502] PEG / PEOX Group

[0503] wherein PG is a protecting group, and wherein X is -OH or a leaving group, or wherein X forms a carboxylate together with the C(O) group to which it is attached to form an amide bond therebetween; and

[0504] vii) Deprotecting the protecting group PG.

[0505] Alternatively, the dendrimer intermediate used in variant a) may be obtained, for example, by carrying out steps i) to v) as described above, and:

[0506] vi) reacting a free amino group present on a building block with another building block, said other building block being a protected lysine or an analog thereof, which contains a -C(O)X group, wherein X is -OH or a leaving group or -C(O)X forms a carboxylate, and wherein the amino group present in the lysine or an analog thereof is orthogonally protected, to form an amide bond between building blocks of different generations;

[0507] vii) deprotecting the first set of amino protecting groups;

[0508] viii) reacting the free amino groups present on the building blocks with

[0509] PEG / PEOX Group

[0510] wherein the PEG group is a PEG-containing group, and X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate;

[0511] vii) deprotecting the second set of amino protecting groups.

[0512] The dendrimer intermediate used in variant c) can itself be obtained, for example, by carrying out steps i) to v) as described above for variant a).

[0513] The present invention also provides a synthetic intermediate for preparing dendrimers. Therefore, the present invention also provides an intermediate for preparing dendrimers, which is

[0514]

[0515] wherein X is -OH or a leaving group, or wherein X together with the C(O) group to which it is attached forms a carboxylate; and wherein PG is a protecting group.

[0516] Also provided are intermediates for producing dendrimers, which are

[0517] PEG / PEOX Group

[0518] wherein the PEG group is a PEG-containing group, the PEOX group is a PEOX-containing group; X is -OH or a leaving group, or wherein X forms a carboxylate together with the C(O) group to which it is attached; and wherein PG is a protecting group. Such intermediates can be prepared, for example, as described above.

[0519] The present invention will now be described with reference to the following examples that illustrate some specific aspects of the invention. However, it should be understood that the particularity of the following description of the invention does not supersede the generality of the foregoing description of the invention.

[0520] Example

[0521] Example 1: Synthesis and Characterization

[0522] The dendrimers represented in the following examples include the core and the building units in the outermost generation of the dendrimers. The underground generations are not described. The dendrimer BHALys[Lys] 32 represents a 5-generation dendrimer having the formula BHALys[Lys] 2 [Lys] 4 [Lys] 8 [Lys] 16 [Lys] 32

[0523] Relates to the theoretical number of ε-surface amino groups on the dendrimer that can be replaced by PEG ～2100 The actual average number of PEG 1 groups attached to BHALys[Lys] 32 was determined by ～2300 H NMR experiments (see the following part of this example, entitled Characterization).

[0524] BHALys[Boc] 2

[0525] Solid α,ε-(Boc) 2 -(L)-lysine p-nitrophenyl ester (2.787 kg, 5.96 mol) was added to a solution of aminodiphenylmethane (benzylamine) (0.99 kg, 5.4 mol) in anhydrous acetonitrile (4.0 L), DMF (1.0 L) and triethylamine (1.09 kg) over 15 minutes. The reaction mixture was stirred overnight at 20 °C. Then the reaction mixture was heated to 35 °C and an aqueous sodium hydroxide solution (0.5 N, 10 L) was added slowly over 30 min. The mixture was stirred for another 30 min and then filtered. The solid cake was washed with water and dried to constant weight (2.76 kg, 5.4 mol), 100% yield. 1 H NMR (CD 3 OD) δ 7.3 (m, 10H, Ph calcd 10H); 6.2 (s, 1H, CH-Ph 2 ​Calculate 1H); 4.08(m,α-CH,1H),3.18(br,ε-CH 2 ) and 2.99(m,ε-CH 2 2H); for β,γ,δ-CH 2 , a total of 1.7-1.2(br,β,γ,δ-CH 2 ) and 1.43 (s, tBu), and tBu 25H calculated 24H. MS (ESI+ve) found 534.2 [M+Na] + Calculated as C 29 H 41 N 3 O 5 Na[M+Na] + 534.7.

[0526] BHALys[HCl] 2

[0527] A solution of concentrated HCl (1.5 L) in methanol (1.5 L) was slowly added to the BHALys[Boc] in three portions at a rate that minimized excessive foaming. 2 (780.5 g, 1.52 mol) in a stirred suspension of methanol (1.5 L). The reaction mixture was stirred for an additional 30 min and then concentrated under vacuum at 35 ° C. The residue was dissolved in water (3.4 L) and concentrated twice under vacuum at 35 ° C and then stored under vacuum overnight. Acetonitrile (3.4 L) was then added and the residue was concentrated again under vacuum at 35 ° C to give BHALys[HCl] 2 , as a white solid (586 g, 1.52 mol), 100% yield. 1 H NMR (D 2 O) δ7.23 (br m, 10H, Ph calculated 10H); 5.99 (s, 1H, CH-Ph 2 calculated for 1H); 3.92 (t, J = 6.5 Hz, α-CH, 1H, calculated for 1H); 2.71 (t, J = 7.8 Hz, ε-CH 2 , 2H, calculated for 2H); 1.78 (m, β, γ, δ-CH 2 ,2H),1.47(m,β,γ,δ-CH 2 , 2H), and 1.17 (m, β, γ, δ-CH 2 , 2H, total 6H calculated 6H). MS (ESI+ve) found 312 [M+H] + calculated as C 19 H 26 N 3 O[M+H]+312.

[0528] BHALys[Lys] 2 [Boc] 4

[0529] To BHALys[HCl] 2 To a suspension of (586 g, 1.52 mmol) in anhydrous DMF (3.8 L) triethylamine (1.08 kg) was slowly added to maintain the reaction temperature below 30 °C. Solid α,ε-(Boc) was added in three portions. 2 -(L)-Lysine p-nitrophenolate (1.49 kg) was added and stirred slowly for 2 hours between additions. The reaction was stirred overnight. Aqueous sodium hydroxide solution (0.5 M, 17 L) was slowly added to the well-stirred mixture and stirred until the solid precipitate moved freely. The precipitate was collected by filtration and the solid filter cake was washed with water (2×4 L) and then with acetone / water (1:4, 2×4 L). The solid was slurried again with water, then filtered and dried under vacuum overnight to obtain BHALys[Lys] 2 [Boc] 4 (1.51 kg), 100% yield. 1 H NMR (CD 3 OD)δ7.3(m,10H,Ph calculated 10H);6.2(s,1H,CH-Ph 2 calculated for 1H); 4.21 (m, α-CH), 4.02 (m, α-CH) and 3.93 (m, α-CH, total 3H, calculated for 3H); 3.15 (m, ε-CH 2 ) and 3.00(m,ε-CH 2 Total 6H, calculated 6H); for β, γ, δ-CH 2 , a total of 1.7-1.3(br,β,γ,δ-CH 2 ) and 1.43 (s, tBu), and tBu 57H calculated 54H. MS (ESI+ve) found 868.6 [M-Boc] + ; 990.7[M+Na] + Calculated as C 51 H 81 N 7 O 11 Na[M+Na]+991.1.

[0530] BHALys[Lys] 2 [HCl] 4

[0531] BHALys[Lys] 2 [Boc] 4(1.41 kg, 1.46 mol) was suspended in methanol (1.7 L). Hydrochloric acid (1.7 L) was mixed with methanol (1.7 L) and the resulting solution was added to the dendrimer suspension in four portions and stirred for 30 minutes. The solvent was removed under reduced pressure and treated with two consecutive strips of water (3.5 L) followed by two consecutive strips of acetonitrile (4 L) to give BHALys[Lys] 2 [HCl] 4 (1.05 Kg, 1.46 mmol), quantitative yield. 1 H NMR (D 2 O) δ7.4 (br m, 10H, Ph calculated 10H); 6.14 (s, 1H, CH-Ph 2 calculated for 1H); 4.47 (t, J = 7.5 Hz, α-CH, 1H), 4.04 (t, J = 6.5 Hz, α-CH, 1H), 3.91 (t, J = 6.8 Hz, α-CH, 1H, total 3H, calculated for 3H); 3.21 (t, J = 7.4 Hz, ε-CH 2 ,2H),3.01(t,J=7.8Hz,ε-CH 2 ,2H) and 2.74 (t,J=7.8Hz,ε-CH 2 , 2H, total 6H, calculated 6H); 1.88 (m, β, γ, δ-CH 2 ),1.71(m,β,γ,δ-CH 2 ),1.57(m,β,γ,δ-CH 2 ) and 1.35(m,β,γ,δ-CH 2 Total 19H, calculate 18H).

[0532] BHALys[Lys] 4 [Boc] 8

[0533] BHALys[Lys] 2 [HCl] 4 (1.05 Kg, 1.47 mol) was dissolved in DMF (5.6 L) and triethylamine (2.19 L). α,ε-(Boc) was added in three portions. 2 -(L)-lysine p-nitrophenolate (2.35 kg, 5.03 mol) was added and the reaction was stirred at 25°C overnight. A solution of NaOH (0.5 M, 22 L) was added and the resulting mixture was filtered, washed with water (42 L) and then air dried. The solid was dried under vacuum at 45°C to give BHALys[Lys] 4 [Boc] 8 (2.09 Kg, 1.11 mol), 76% yield.1 HNMR (CD 3 OD)δ7.3(m,10H,Ph calculated 10H);6.2(s,1H,CH-Ph 2 calculated for 1H); 4.43 (m, α-CH), 4.34 (m, α-CH), 4.25 (m, α-CH) and 3.98 (br, α-CH, total 7H, calculated for 7H); 3.15 (br, ε-CH 2 ) and 3.02(br,ε-CH 2 Total 14H, calculated 14H); for β, γ, δ-CH 2 , a total of 1.9-1.2(br,β,γ,δ-CH 2 ) and 1.44(br,s,tBu), and tBu122H calculates 144H.

[0534] BHALys[Lys] 4 [TFA] 8

[0535] To BHALys[Lys] at 0°C 4 [Boc] 8 To a stirred suspension of (4 g, 2.13 mmol) in DCM (18 mL) was added TFA (13 mL). The solid was dissolved and the solution was stirred overnight under an argon atmosphere. The solvent was removed in vacuo and residual TFA was removed by trituration with ether (100 mL). The product was redissolved in water and then freeze-dried to give BHALys[Lys] 4 [TFA] 8 , as an off-white solid (4.27 g, 2.14 mmol), quantitative yield. 1 H NMR (D 2 O) δ7.21 (br m, 10H, Ph calculated 10H); 5.91 (s, 1H, CH-Ph 2 Calculated for 1H); 4.17 (t, J = 7.4 Hz, α-CH, 1H), 4.09 (t, J = 7.1 Hz, α-CH, 1H), 4.02 (t, J = 7.2 Hz, α-CH, 1H, 3.84 (t, J = 6.5 Hz, α-CH, 2H), 3.73 (t, J = 6.7 Hz, α-CH, 1H), 3.67 (t, J = 6.7 Hz, α-CH, 1H, total 7H, calculated for 7H); 3.0 (m, ε-CH 2 ),2.93(m,ε-CH 2 ) and 2.79(b,ε-CH 2 , total 15H, calculated 14H); 1.7 (br, β, γ, δ-CH 2),1.5(br,β,γ,δ-CH2),1.57(m,β,γ,δ-CH 2 ) and 1.25 (br, β, γ, δ-CH2 total 45H, calculated 42H). MS (ESI+ve) found 541.4 [M+2H] 2+ ; Calculated as C 55 H 99 N 15 O 7 [M+2H] 2+ 541.2.

[0536] BHALys[Lys] 8 [Boc] 16

[0537] α,ε-(Boc) 2 -(L)-lysine p-nitrophenolate (1.89 g, 4.05 mmol) in DMF (25 mL) was added to BHALys[Lys] 4 [NH 2 TFA] 8 (644mg, 0.32mmol) and triethylamine (0.72mL, 5.2mmol) in DMF (25mL), and the reaction was stirred overnight under argon atmosphere. The reaction mixture was poured on ice / water (500mL), then filtered and the collected solid was dried overnight under vacuum. The dried solid was fully washed with acetonitrile to obtain BHALys[Lys] 8 [Boc] 16 , as an off-white solid (0.82 g, 0.22 mmol), 68% yield. 1 H NMR (CD 3 OD) δ7.3 (m, 10H, Ph calculated 10H); 6.2 (br s, 1H, CH-Ph 2 calculated for 1H); 4.48 (br, α-CH), 4.30 (br, α-CH) and 4.05 (br, α-CH, total 16H calculated for 15H); 3.18 (br, ε-CH 2 ) and 3.02(m,ε-CH 2 Total 31H, calculated 30H); for β, γ, δ-CH 2 , a total of 1.9-1.4(br,β,γ,δ-CH 2 ) and 1.47 (br, s, tBu), and tBu 240H calculated 234H. MS (ESI+ve) found 3509 [M+H-(Boc) 2 ] + Calculated as C 173 H306 N 31 O 43 [M+H-(Boc) 2 + 3508.5; 3408 [M+H-(Boc) 3 + Calculated as C 168 H 298 N 31 O 41 [M+H-(Boc) 3 + 3408.4。

[0538] BHALys[Lys] 8 [TFA] 16

[0539] A solution of TFA / DCM (1:1, 19 mL) was slowly added to a stirred suspension of BHALys[Lys] 8 [Boc] 16 (800 mg, 0.22 mmol) in DCM (25 mL). The solid dissolved and the solution was stirred overnight under an argon atmosphere. The solvent was removed in vacuo and residual TFA was removed by repeated freeze-drying of the residue to afford BHALys[Lys] 8 [TFA] 16 , as an off-white lyophilizate (848 mg, 0.22 mmol), in quantitative yield. 1 H NMR (D 2 O) δ 7.3 (br m, 10H, Ph calculated 10H); 6.08 (s, 1H, CH-Ph 2 calculated 1H); 4.3 (m, α-CH), 4.18 (m, α-CH), 4.0 (m, α-CH) and 3.89 (m, α-CH, total 16H, calculated 15H); 3.18 (br, ε-CH 2 ) and 2.94 (m, ε-CH 2 total 32H, calculated 30H); 1.9 (m, β, γ, δ-CH 2 ), 1.68 (m, β, γ, δ-CH 2 ) and 1.4 (m, β, γ, δ-CH 2 total 99H, calculated 90H). MS (ESI+ve) found 2106 [M+H] + Calculated as C 103 H 194 N 31 O 15 [M+H] + 2106.9。 ​​​

[0540] BHALys[Lys] 16 [Boc] 32

[0541] α,ε-(Boc) 2 -(L)-Lysine p-nitrophenolate (1.89 g, 4.05 mmol) in DMF (25 mL) was added to BHALys[Lys] 8 [TFA] 16 (644 mg, 0.32 mmol) and triethylamine (0.72 mL, 5.2 mmol) in DMF (25 mL) and the reaction was stirred overnight under an argon atmosphere. The reaction was poured onto ice / water (500 mL) and then filtered and the collected solid was dried under vacuum overnight. The dried solid was washed thoroughly with acetonitrile to give BHALys[Lys] 16 [Boc] 32 , as an off-white solid (0.82 g, 0.22 mmol), 68% yield. 1 H NMR (CD 3 OD) δ7.28 (m, 9H, Ph calculated 10H); 6.2 (br s, 1H, CH-Ph 2 Calculated 1H); 4.53(br,α-CH),4.32(br,α-CH)and 4.05(br,α-CH,total 35H, calculated 31H); 3.18(br,ε-CH 2 ) and 3.04(m,ε-CH 2 Total 67H, calculated 62H); for β,γ,δ-CH 2 , a total of 1.9-1.5(br,β,γ,δ-CH 2 ) and 1.47 (br, s, tBu), and tBu 474H calculated 474H. MS (ESI+ve) found 6963 [M+H-(Boc) 4 ] + Calculated as C 339 H 610 N 63 O 87 [M+H-(Boc) 4 ] + 6960.9; 6862[M+H-(Boc) 5 ] + Calculated as C 334 H 604 N 63 O 85 [M+H-(Boc) 5 ] + 6860.8.

[0542] BHALys[Lys] 16 [TFA] 32

[0543] A solution of TFA / DCM (1:1, 19 mL) was slowly added to BHALys[Lys] 16 [Boc] 32 (800 mg, 0.11 mmol) was added to a stirred suspension in DCM (25 mL). The solid was dissolved and the solution was stirred overnight under an argon atmosphere. The solvent was removed under vacuum and the residual TFA was removed by repeated freeze drying of the residue to give BHALys[Lys] 16 [TFA] 32 , as off-white lyophilizate (847 mg, 0.11 mmol), quantitative yield. 1 H NMR (D 2 O) δ7.3 (br m, 11H, Ph calculated 10H); 6.06 (s, 1H, CH-Ph 2 calculated for 1H); 4.3 (m, α-CH), 4.19 (m, α-CH), 4.0 (m, α-CH) and 3.88 (m, α-CH, total 35H, calculated for 31H); 3.15 (br, ε-CH 2 ) and 2.98(m,ε-CH 2 Total 69H, calculated 62H); 1.88 (m, β, γ, δ-CH 2 ),1.7(m,β,γ,δ-CH 2 ) and 1.42(m,β,γ,δ-CH 2 Total 215H, calculated 186H). MS (ESI+ve) found 4158 [M+H] + Calculated as C 199 H 386 N 63 O 31 [M+H]+4157.6

[0544] HO-Lys(α-BOC)(ε-PEG ～2300 )

[0545] DIPEA (0.37 mL, 2.10 mmol) was added to NHS-PEG ～2300 (2.29 g, 1.05 mmol) (PEG ～2100 represents a methoxy-terminated PEG group with an average molecular weight of about 2300 Da, and wherein NHS represents NHS-C(O)CH 2) and N-α-BOC-L-lysine (0.26 g, 1.05 mmol) in an ice-cooled mixture of DMF (20 mL). The stirred mixture was allowed to warm to room temperature overnight, then any remaining solids were filtered (0.45 μm PALL acrodisc), and the solvent was removed in vacuo. The residue was taken up in ACN / H 2 O (1:3, 54 mL) and purified by PREP HPLC (Waters Xbridge C18, 5 μm, 19×150 mm, 25 to 32% ACN (5-15 min), 32 to 60% ACN (15 to 20 min), no buffer, 8 mL / min, RT=17 min) to give 1.41 g (56%) HO-Lys(BOC)(PEG 2100 ). 1 H NMR (CD 3 OD)δ3.96-4.09(m,1H),3.34-3.87(m,188H);3.32(s,3H),3.15(q,J=6.0Hz,2H),2.40(t,J=6.2Hz,2H),1.28-1.88(m,6H),1.41(s,9H).

[0546]

[0547] To BHA Lys [Lys] 16 [TFA] 32 To a stirred mixture of PEG (0.19 g, 24 μmol) in DMF (20 mL) was added DIPEA (0.86 mL, 4.86 mmol). The mixture was then added dropwise to PyBOP (0.62 g, 1.20 mmol) and HO-Lys(BOC-Lys)(PEG ～2300 ) (2.94 g, 1.20 mmol) in a stirred mixture of DMF (20 mL). The reaction mixture was stirred overnight and then diluted with water (200 mL). The aqueous mixture was centrifuged (5K membrane, 20 L water). The retentate was freeze-dried to provide 1.27 g (73%) of the desired dendrimer. HPLC (C8 XBridge, 3×100 mm, gradient: 5% ACN (0-1 min), 5-80% ACN / H 2 O) (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm.0.1%TFA) Rf (min) = 8.52. 1H NMR (300MHz, D 2O)δ(ppm):1.10-2.10(m,Lys CH 2 (β,χ,δ) and BOC,666H),3.02-3.36(m,Lys CH 2 (ε),110H),3.40(s,PEG-OMe,98H),3.40-4.20(m,PEG-OCH 2 ,5750H+Lys CH surface,32H),4.20-4.50(m,Lys,CH internal 32H),7.20-7.54(m,BHA,8H). 1 H NMR showed about 29 PEGs.

[0548]

[0549] Add 1.27g (17.4μmol) BHALys[Lys] 32 [α-BOC] 32 [ε-PEG ～2300 ] 32 The mixture was stirred overnight at room temperature in TFA / DCM (1:1, 20 mL). The volatiles were removed in vacuo and the residue was dissolved in water (30 mL). The mixture was then concentrated. The process was repeated two more times and then freeze-dried to give 1.35 g (106%) of the desired product as a viscous colorless oil. HPLC (C8 XBridge, 3×100 mm, gradient: 5% ACN (0-1 min), 5-80% ACN / H 2 O) (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm. 0.1% TFA) Rf (min) = 8.51. 1 HNMR (300MHz, D 2 O)δ(ppm):1.22-2.08(Lys CH 2 ((β,χ,δ),378H),3.00-3.26(Lys CH2(ε),129H),3.40(PEG-OMe,96H),3.45-4.18(PEG-OCH 2 ,5610H+Lys CH surface,32H),4.20-4.46(Lys, CH interior,33H),7.24-7.48(8H,BHA). 1 H NMR showed about 29 PEGs.

[0550] Characterization

[0551] Table 1 shows the different batches synthesized The actual number of PEG chains on the dendrimer is also determined by 1 H NMR calculations.

[0552] Table 1. Different batches of

[0553]

[0554] Calculation from proton NMR spectra For batch 1: PEG number = number of protons in the PEG region of the NMR spectrum (integrated) (3.4-4.2 ppm) / number of protons in a single PEG chain (NMR spectrum)

[0555] =5594H / 202H

[0556] =27.69 (about 28 PEG units)

[0557] PEG stands for –C(O)CH 2 -PEG ～2300 , where PEG ～2300 It represents a methoxy-terminated PEG group with an average molecular weight of about 2300 Daltons.

[0558] The theoretical number of α surface amino groups on the dendrimer that can be substituted with linker-SN-38 is related to the number of α surface amino groups on the dendrimer that can be substituted with linker-SN-38. By using the integrals comparing the PEG region to the SN-38 region, 1 H NMR spectroscopy experiments determined the connection to BHALys[Lys] 32 The actual average number of linker-SN-38 groups.

[0559] Example 2: Synthesis of Example Dendrimers

[0560] Dendrimer constructs were prepared in which SN-38 was covalently linked via the C-10 or C-20 position to a diacid linker that was attached to the α-amino position on the outermost lysine layer of the dendrimer.

[0561] Table 2.

[0562]

[0563] Shown below are the Glu (glutaric acid), TDA (thiodiacetic acid), DGA (diglycolic acid), and 2,5-THF (tetrahydrofuran-2,5-dicarboxylic acid) linkers used, along with SN-38, a representative of a dendrimer with PEG and SN-38 residues attached via a DGA diacyl linker, and surface building blocks for compounds 2e / 2f.

[0564]

[0565]

[0566] PEG stands for –C(O)CH 2 -PEG ～2300 , where PEG ～2300 represents a methoxy terminated PEG group with an average molecular weight of about 2300 Daltons. In the above examples, the DGA linker was attached to the α-amino group on the outer lysine layer (Example 2e).

[0567] ● indicates the residues of SN-38.

[0568]

[0569] Example 2a: Synthesis

[0570] (i) Glu-C10-SN-38

[0571]

[0572] To a magnetically stirred suspension of SN-38 (600 mg, 1.53 mmol) and glutaric anhydride (349 mg, 3.06 mmol) in DCM (19 mL) at room temperature was added triethylamine (426 μL, 3.06 mmol). The mixture was stirred at room temperature overnight, after which the suspension had dissolved. A portion (7.5 mL) of the reaction mixture (0.45 μL) was filtered, diluted with DCM (10 mL), and washed with phosphate buffer (5% NaCl and 1% NaHCO). 2 PO 4 The solution was washed with 5% HCl (adjusted to pH 3) in water until the washings remained at pH 3 (4×9 mL). The pH of the combined aqueous phases was adjusted to 3 with 1 M HCl and the aqueous layer was extracted with EtOAc (3×20 mL). The combined organic phases (ca. 70 mL) were washed with brine (30 mL) and MgSO 4 The volatiles were then removed in vacuo and the residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 20-60% ACN / H 2 O (5-50 min), 0.1% TFA, RT = 33 min) to give 106 mg (35%) of the product as a yellow solid.

[0573] LCMS (C8, gradient: 15% ACN / H 2O (0-1min), 15-25% ACN (1-2min), 25% ACN (2-8min), 25-80% ACN (8-10min), 80% ACN (10-11min), 80-15% ACN (11-13min), 15% ACN (13-15min), 10mM ammonium formate (pH 6.8), 0.4mL / min, Rf (min) = 6.04. ESI+ (ve) observation [M] + =507. Calculated as C 27 H 26 N 2 O 8 =507Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.88(dd,J=7.4,7.2Hz,3H),1.29(dd,J=7.6,7.4Hz,3H),1.81-1.97(m,4H),2.40(dd, J=7.4,7.2Hz,2H),2.73(t,J=7.4Hz,2H),3.15-3.23(m,2H),5.34(s,2H),5.44(s,2H),6.51(br s,1H),7.33(s,1H),7.68(dd,J=9.1,2.5Hz,1H),8.02(d,J=2.5Hz,1H),8.21(d,J=9.1Hz,1H),12.18(brs,1H).

[0574] (ii)

[0575]

[0576] To a magnetically stirred mixture of Glu-C10-SN-38 (215 mg, 424 μmol) and PyBOP (221 mg, 424 μmol) in DMF (9 mL) was added 1% d-Hydroxypropene (20% d-Hydroxypropene) ... (757 mg, 10.2 μmol) and NMM (179 μL, 1.63 mmol). After 16 h at room temperature, the reaction mixture was added to a chilled (0 °C) solution of 1% AcOH in 3:7 ACN / water (200 mL), filtered and filtered through ultrafiltration (0.005 m 2, 10kDa, regenerated cellulose filter) was concentrated to 30mL. The concentrate was further ultrafiltered with 13×30mL diafiltration (1% AcOH in 3:7ACN / MQ water). The retentate was freeze-dried to give 931mg of product. The substance was dissolved in THF (4.5mL) and added to cooled MTBE (20mL) at 0°C. The mixture was stirred at 0°C (1h) and the resulting precipitate was separated by filtration. The product was vacuum dried (16h) to give 783mg (89%) of the desired substance as a yellow solid.

[0577] HPLC (C8 Xbridge, 3×100 mm) gradient: 5% sACN / H 2 O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 243nm, 0.1% TFA, 0.4mL / min, Rf (min) = 8.69. 1 H NMR (300MHz, CD 3 OD) δ (ppm): 0.65-3.04 (m, 706H), 3.04-3.27 (m, 79H), 3.36 (s, 93H), 3.37-4.14 (m, 5,615H), 4.14-4.63 (m, 76H), 5.12-5.89 (m, 50H), 6.97-7.80 (m, 114H). The SN-38 loading was determined by comparing the integrated area of ​​the aromatic compounds of SN-38 (4H) with the integrated area of ​​PEG. In the above example, the aromatic region is 104H (114H-10H) from the BHA part of the dendrimer. 104H / 4H=26SN-38 molecules / dendrimer. The theoretical molecular weight of the conjugate with 28 PEG2300 chains and 26 DGA-SN-38 is 83,374 Da. The drug loading (% w / w) can then be calculated by multiplying the molecular weight of SN-38 (392) by the number of SN-38 molecules on the dendrimer divided by the total molecular weight of the construct, i.e. (392 x 26) / 83,374 = 12.2%.

[0578] Example 2b: Synthesis

[0579] (i) OtBu-Glu-C20-SN-38-O(Boc)

[0580]

[0581] To a magnetically stirred suspension of Boc-C10-SN-38 (Zhao, H. et al, Bioconjugate Chem., 2008, 19, 849-859) (48 mg, 0.097 mmol) in DCM (1.8 mL) was added (0.2 mL) of mono-tert-butyl glutarate (25 mg, 0.14 mmol), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 27 μL, 0.16 mmol) and DMAP (4-dimethylaminopyridine, 4 mg, 0.031 mmol) in DCM at room temperature. The mixture was stirred at room temperature for 3 h until the reaction was judged to be >80% complete by HPLC. The reaction mixture was diluted with 8 mL of DCM and washed with NaHCO successively. 3 Aqueous solution (1%, 2×2.5 mL), water (2.5 mL) and HCl (0.1 M, 2×2.5 mL) were used for washing. 4 Dried and filtered. The volatiles were then removed in vacuo and the residue was dried in vacuo (2 h) to give 63 mg (98%) of the product as a yellow solid.

[0582] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 8.38. ESI + (ve) observation [M] + =663. Calculated as C 36 H 42 N 2 O 10 =663Da. 1 H NMR (300 MHz, CDCl 3 )δ(ppm):0.97(dd,J=7.4,7.3Hz,3H),1.37-1.48(m,3H),1.41(s,9H),1.61( s,9H),1.83-1.97(m,2H),2.06-2.37(m,4H),2.46-2.67(m,2H),3.12-3.20(m ,2H),5.25(s,2H),5.41(d,J=17.3Hz,1H),5.68(d,J=17.3Hz,1H),7.17(s,1 H), 7.67 (dd, J = 9.2, 2.3Hz, 1H), 7.90 (d, J = 2.3Hz, 1H), 8.22 (d, J = 9.2Hz, 1H).

[0583] (ii) Glu-C20-SN-38

[0584]

[0585] A solution of OtBu-Glu-C20-SN-38-O(Boc) (59 mg, 0.089 mmol) in TFA (4 mL) was stirred magnetically at room temperature overnight. The volatiles were then removed in vacuo and the residue was azeotroped with DCM, mixed with water and lyophilized to give 59 mg (>95%) of the product as a white-yellow solid.

[0586] LCMS (C18, gradient: 5% ACN / H 2 O (0-1min), 5-60% ACN (1-10min), 60% ACN (10-11min), 60-5% ACN (11-13min), 5% ACN (13-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 9.41. ESI + (ve) observation [M] + =507. Calculated as C 27 H 26 N 2 O 8 =507Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.91(dd,J=7.4,7.3Hz,3H),1.29(dd,J=7.6,7.4Hz,3H),1.71-1.81(m,2H),2.10-2.17(m,2H),2.30(t,J=7.4Hz,2H),2.56 (t,J=7.4Hz,2H),3.08(dd,J=14.9,7.4,2H),5.28(s,2H),5.48(s,2H),6.93(s,1H),7.39-7.43(m,2H),8.01-8.04(m,1H),10.33(br s,1H).

[0587] (iii)

[0588]

[0589] To a magnetically stirred mixture of Glu-C20-SN-38 (13 mg, 26 μmol) and PyBOP (14 mg, 26 μmol) in DMF (0.5 mL) was added A mixture of 4-(4-(4-(4-piperidin-2-yl)-2-nitropropene) (50 mg, 0.60 μmol) and DIPEA (16 μL, 97 μmol) also in DMF (1 mL) was added. After 16 hours at room temperature, the volatiles were removed and the residue was purified by size exclusion chromatography (Sephadex, LH-20, ACN). Appropriate fractions were combined and concentrated as judged by HPLC. The residue was then dissolved in water, filtered (0.45 μm) and lyophilized to give 45 mg (79%) of the desired material as a yellow solid.

[0590] HPLC (C8 Xbridge, 3×100 mm) gradient: 5% sACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 0.1% formic acid, 0.4 mL / min, Rf (min) = 8.13. 1 H NMR (300MHz, CD 3 OD) δ (ppm): 0.62-3.26 (m, 1, 010H), 3.36 (s, 103H), 3.37-4.10 (m, 6, 100H), 4.11-4.50 (m, 72H), 4.57 (s, 38H), 5.17-5.88 (m, 50H), 6.92-8.11 (m, 144H). According to Example 2a, the number of SN-38 molecules was calculated to be 32, and the drug loading was 13.6% w / w.

[0591] Example 2c: Synthesis

[0592] (i) OtBu-TDA-C10-SN-38

[0593]

[0594] To a magnetically stirred suspension of SN-38 (100 mg, 0.26 mmol) in DCM (2 mL) was added a solution of mono-tert-butyl thiodiglycolate (53 mg, 0.33 mmol / L), EDC (67 μL, 0.43 mmol) and DMAP (31 mg, 0.26 mmol) in DCM (0.5 mL) at room temperature. The mixture was stirred overnight at room temperature. The volatiles were removed in vacuo and the residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 35-70% ACN / water (5-40 min), 0.1% TFA) to give 10 mg (7%) of OtBu-TDA-C20-SN-38 (RT=35.8 min) and 10 mg (7%) of OtBu-TDA-C10-SN-38 (RT=37.5 min) as a yellow solid.

[0595] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 5.97. ESI + (ve) observation [M] + =581. Calculated as C 30 H 32 N 2 O 8 S = 581 Da.

[0596] (ii) TDA-C10-SN-38

[0597]

[0598] Method A:

[0599] A solution of OtBu-TDA-C10-SN-38 (10 mg, 17.2 μmol) in TFA (2 mL) was magnetically stirred at room temperature overnight. The volatiles were then removed in vacuo and the residue was azeotroped with DCM, mixed with water and lyophilized to give 9 mg (>95%) of the product as a yellow solid.

[0600] Method B:

[0601] Triethylamine (71 μL, 0.51 mmol) was added to a magnetically stirred suspension of SN-38 (100 mg, 0.25 mmol) and thiodiglycolic anhydride (85%, 79 mg, 0.51 mmol) in DCM (3 mL) at room temperature. The suspension dissolved rapidly and the mixture was stirred at room temperature overnight. After 16 hours, the reaction was judged to be >80% complete by HPLC. The reaction mixture was diluted with 3 mL of DCM and eluted with phosphate buffer (5% NaCl and 1% NaH 2 PO 4 The precipitate was washed in water (adjusted to pH 3 with 5% HCl) until the washings remained at pH 3 (3×6 mL). The pH of the combined aqueous phases was adjusted to 3 with 1 M HCl and the aqueous layer was extracted with more DCM (2×20 mL). The combined organic phases were washed with MgSO 4 Dry, filter and remove volatiles in vacuo.Dry the residue in vacuo to give 81 mg (60%) of product as a yellow solid.

[0602] LCMS (C8, gradient: 15% ACN / H 2 O (0-1min), 15-25% ACN (1-2min), 25% ACN (2-8min), 25-80% ACN (8-10min), 80% ACN (10-11min), 80-15% ACN (11-13min), 15% ACN (13-15min), 10mM ammonium formate (pH 6.8), 0.4mL / min, Rf (min) = 5.44. ESI+ (ve) observation [M] + =526. Calculated as C 26 H 24 N 2 O 8 S = 525 Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.88(dd,J=7.4,7.2Hz,3H),1.30(dd,J=7.7,7.4Hz,3H),1.81-1.94(m,2H),3.16-3.23(m,2H),3.53(s,2H),3.83(s,2H),5. 34(s,2H),5.44(s,2H),6.52(s,1H),7.34(s,1H),7.70(dd,J=9.1,2.5Hz,1H),8.04(d,J=2.5Hz,1H),8.25(d,J=9.1Hz,1H),12.73(br s,1H).

[0603] (iii)

[0604]

[0605] At room temperature, (28 mg, 0.37 μmol) and NMM (6.5 μL, 59 μmol) in DMF (0.7 mL) were added to a magnetically stirred mixture of TDA-C10-SN-38 (8 mg, 15 μmol) and PyBOP (8 mg, 15 μmol) in DMF (0.3 mL). After 16 h at room temperature, the volatiles were removed and the residue was purified by size exclusion chromatography (Sephadex, LH-20, MeOH). Appropriate fractions were combined and concentrated as judged by HPLC, dissolved in water, filtered (0.45 μm) and lyophilized to give 29 mg (89%) of the desired substance as a yellow-orange solid. HPLC (C8 Aeris WIDEPORE, 100×2.1 mm), gradient: 5% ACN / H

[0606] O (0 - 1 min), 5 - 80% ACN (1 - 7 min), 80% ACN (7 - 12 min), 80 - 5% ACN (12 - 13 min), 5% ACN (13 - 15 min), 214 nm, 10 mM ammonium formate, 0.4 mL / min, Rf(min) = 7.94. 2 1H NMR (300 MHz, CD 1 OD) δ (ppm): 0.16 - 3.27 (m, 696H), 3.36 (s, 108H), 3.37 - 4.14 (m, 6,135H), 4.14 - 4.67 (m, 78H), 4.97 - 5.88 (m, 56H), 6.82 - 8.56 (m, 138H). Based on Example 2a, the number of SN-38 molecules was calculated to be 32 and the drug loading was 13.8%. 3 3

[0607] Example 2d: Synthesis of

[0608] (i) OtBu-TDA-C20-SN-38-O(Boc)

[0609]

[0610] To a magnetically stirred suspension of Boc-C10-SN-38 (306 mg, 0.62 mmol) and thiodiglycol mono-tert-butyl ester (192 mg, 0.93 mmol) in DCM (8 mL) was added EDC (187 μL, 1.05 mmol) and DMAP (23 mg, 0.19 mmol) at 0 ° C. The mixture was stirred at 0 ° C for 15 minutes and then at room temperature for 3 h until the reaction was judged to be >70% complete by HPLC. The reaction mixture was filtered (0.45 μm) and diluted with 7 mL DCM. 15 mL phosphate buffer (5% sodium chloride and 1% NaHCO 3) was added. 2 PO 4 The pH was adjusted to 3 with 5% HCl in water, and then 15 mL of EtOAc was added. After phase separation, the organic layer was washed with MgSO 4 Dry, filter (0.7 μm) and concentrate. The residue is purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 30-90% ACN / H 2 O (5-40 min), 0.1% TFA, RT = 44 min) to give 230 mg (54%) of the product as a yellow solid.

[0611] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 8.28. ESI + (ve) observation [M] + =681. Calculated as C 35 H 40 N 2 O 10 S = 681 Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.94(dd,J=7.4,7.3Hz,3H),1.28(dd,J=7.6,7.5Hz,3H),1.38(s,9H),1.54(s,9H),2.10-2.23(m,2H),3.16-3.24(m,2H),3.36-3.51(m,2H, partially hidden in DMSO-d 6peak), 3.70 (dd, J = 19.6, 15.2 Hz, 2H), 5.35 (s, 2H), 5.52 (s, 2H), 7.16 (s, 1H), 7.74 (dd, J = 9.2, 2.5 Hz, 1H), 8.10 (d, J = 2.5 Hz, 1H), 8.18 (d, J = 9.2 Hz, 1H).

[0612] (ii)TDA-C20-SN-38

[0613]

[0614] A solution of OtBu-TDA-C20-SN-38-O(Boc) (333 mg, 0.49 mmol) in TFA (20 mL) was magnetically stirred at room temperature for 4 h. The volatiles were removed in vacuo, and the residue was azeotroped with DCM, cooled to 0°C, mixed with water and lyophilized to give 337 mg (>95%) of the product as a yellow-orange solid.

[0615] LCMS (C8, gradient: 15% ACN / H 2 O (0-1min), 15-25% ACN (1-2min), 25% ACN (2-8min), 25-80% ACN (8-10min), 80% ACN (10-11min), 80-15% ACN (11-13min), 15% ACN (13-15min), 10mM ammonium formate (pH 6.8), 0.4mL / min, Rf (min) = 5.93. ESI+ (ve) observation [M] + =525. Calculated as C 26 H 24 N 2 O 8 S = 525 Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.94(dd,J=7.3,7.4Hz,3H),1.29(dd,J=7.6,7.4Hz,3H),2.08-2.21(m,2H),3.09(dd,J=14.9,7.3Hz,2H),3.42(dd,J=17.6 ,15.6Hz,2H),3.68(dd,J=21.5,15.0Hz,2H),5.29(s,2H),5.50(s,2H),7.07(s,1H),7.38-7.42(m,2H),8.00-8.03(m,1H),10.31(br s,1H).

[0616] (iii)

[0617]

[0618] To a magnetically stirred mixture of TDA-C20-SN-38 (250 mg, 476 μmol) and PyBOP (248 mg, 476 μmol) in DMF (10 mL) was added A mixture of 1% AcOH (851 mg, 11.5 μmol) and NMM (201 μL, 1.83 mmol) in DMF (12 mL). After 16 h at room temperature, the reaction mixture was added to a chilled (0 °C) solution of 1% AcOH in 3:7 ACN / water (220 mL), filtered and filtered through ultrafiltration (0.005 m 2 , 10kDa, regenerated cellulose filter) was concentrated to 35mL. The concentrate was further ultrafiltered with 17×35mL diafiltration (1% AcOH in 3:7ACN / water). The retentate was lyophilized to give 983mg of product. The substance was dissolved in THF (4.5mL) and added to cooled (0°C) MTBE (20mL). The mixture was stirred at 0°C (1h) and the resulting precipitate was separated by filtration. The product was vacuum dried (72h) to provide 816mg (82%) of the desired substance as a yellow solid.

[0619] HPLC (C8 Xbridge, 3×100 mm), gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 243 nm, 10 mM ammonium formate, 0.4 mL / min, Rf (min) = 8.70. 1 H NMR (300MHz, CD 3 OD) δ (ppm): 0.65-2.48 (m, 532H), 2.52-3.26 (m, 122H), 3.36 (s, 102H), 3.37-4.13 (m, 5, 803H), 4.13-4.66 (m, 99H), 5.15-5.99 (m, 60H), 6.84-7.99 (m, 183H). According to Example 2a, the number of SN-38 molecules was calculated to be 31, and the drug loading was 14.1%.

[0620] Embodiment 2e: Synthesis

[0621] (i)DGA-C10-SN-38

[0622]

[0623] Triethylamine (106 μL, 0.77 mmol) was added to a magnetically stirred suspension of SN-38 (100 mg, 0.26 mmol) and diglycolic anhydride (89 mg, 0.77 mmol) in DCM (3 mL) at room temperature. The mixture was stirred at room temperature. After 20 minutes, the reaction was diluted with DMF / acetonitrile (1:5 v / v, containing 0.1% TFA) and purified by preparative HPLC (BEH300 Waters XBridge C18, 5 μM, 30×150 mm, 10-50% ACN / H 2 O (5-40 min, 0.1% TFA, RT = 36.3 min) to afford 33 mg (25%) of the product as a yellow solid.

[0624] LCMS (C8, gradient: 15% ACN / H 2 O (0-1min), 15-25% ACN (1-2min), 25% ACN (2-8min), 25-80% ACN (8-10min), 80% ACN (10-11min), 80-15% ACN (11-13min), 15% ACN (13-15min), 10mM ammonium formate (pH 6.8), 0.4mL / min, Rf (min) = 5.11. ESI+ (ve) observation [M] + =509. Calculated as C 26 H 24 N 2 O 9 =508Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.88(dd,J=7.4,7.2Hz,3H),1.30(dd,J=7.7,7.5Hz,3H),1.81-1.94(m,2 H),3.15-3.22(m,2H),4.27(s,2H),4.60(s,2H),5.34(s,2H),5.44(s,2H),6.51(br s,1H),7.33(s,1H),7.72(dd,J=9.1,2.5Hz,1H),8.08(d,J=2.5Hz,1H),8.23(d,J=9.1Hz,1H),12.79(br s,1H).

[0625] (ii)

[0626]

[0627] To a magnetically stirred mixture of DGA-C10-SN-38 (63 mg, 124 μmol) and PyBOP (64 mg, 124 μmol) in DMF (2 mL) at room temperature was added (213 mg, 2.87 μmol) and NMM (50 μL, 0.46 mmol) in DMF (3 mL) likewise. After 16 h at room temperature, the volatiles were removed and the residue was purified by size-exclusion chromatography (Sephadex, LH-20, ACN). Appropriate fractions were combined and concentrated as judged by HPLC. The residue was then dissolved in water, filtered (0.45 μm) and lyophilized to give 211 mg (85%) of the desired substance as a yellow solid.

[0628] HPLC (C8 Xbridge, 3×100 mm) gradient: 5% sACN / H 2 O (0 - 1 min), 5 - 80% ACN (1 - 7 min), 80% ACN (7 - 12 min), 80 - 5% ACN (12 - 13 min), 5% ACN (13 - 15 min), 243 nm, 0.1% TFA, 0.4 mL / min, Rf (min) = 8.35. 1 H NMR (300 MHz, CD 3 OD) δ (ppm): 0.68 - 2.37 (m, 506H), 2.53 - 3.27 (m, 148H), 3.36 (s, 102H), 3.37 - 4.03 (m, 5,940H), 4.03 - 4.74 (m, 169H), 4.93 - 5.77 (m, 60H), 6.87 - 8.58 (m, 136H). The number of SN-38 molecules was calculated to be 31 and the drug loading was 14.1% according to Example 2a.

[0629] Example 2f: Synthesis of

[0630] (i) OtBu-DGA-C20-SN-38

[0631]

[0632] To a magnetically stirred suspension of SN-38 (100 mg, 0.26 mmol) and diethylene glycol mono-tert-butyl ester (63 mg, 0.33 mmol) in DCM (1.5 mL) was added EDC (76 μL, 0.43 mmol) and DMAP (31 mg, 0.26 mmol) in DCM (1 mL) at room temperature. The mixture was stirred at room temperature for 48 h. The reaction mixture was then filtered (0.22 μm) and the volatiles were removed in vacuo. The residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 30-65% ACN / H 2 O (5-40 min, 0.1% TFA, RT = 37 min) to give 29 mg (20%) of the product as a yellow solid.

[0633] LCMS (C18, gradient: 5% ACN / H 2 O (0-1min), 5-60% ACN (1-10min), 60% ACN (10-11min), 60-5% ACN (11-13min), 5% ACN (13-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 11.83. ESI + (ve) observation [M] + =565. Calculated as C 30 H 32 N 2 O 9 =565Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.92(dd,J=7.4,7.3Hz,3H),1.29(dd,J=7.6,7.5Hz,3H),1.40(s,9H ),2.08-2.21(m,2H),3.09(dd,J=15.0,7.4Hz,2H),4.08(s,2H),4.39(d,J=17. 0Hz,1H),4.56(d,J=17Hz,1H),5.29(dd,J=20.5,18.9Hz,2H),5.51(s,2H),7.0 0(s,1H),7.40(s,1H),7.41(d,J=7.4,2.6Hz,1H),8.01-8.04(m,1H),10.31(br s,1H).

[0634] (ii)DGA-C20-SN-38

[0635]

[0636] A solution of OtBu-DGA-C20-SN-38 (29 mg, 0.051 mmol) in TFA (5 mL) was magnetically stirred at room temperature overnight. The volatiles were then removed in vacuo and the residue was azeotroped with DCM, mixed with water and lyophilized to give 23 mg (88%) of the product as a yellow solid.

[0637] LCMS (C18, gradient: 5% ACN / H 2 O (0-1min), 5-60% ACN (1-10min), 60% ACN (10-11min), 60-5% ACN (11-13min), 5% ACN (13-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 9.12. ESI+ (ve) observation [M+H] + =509. Calculated as C 26 H 24 N 2 O 9 =508Da. 1 H NMR (300 MHz, DMSO-d 6 )δ(ppm):0.92(dd,J=7.4,7.3Hz,3H),1.29(dd,J=7.6,7.4Hz,3H),2.08-2 .21(m,2H),3.09(dd,J=15.0,7.4Hz,2H),4.12(s,2H),4.40(d,J=17.0Hz,1 H),4.57(d,J=17Hz,1H),5.29(s,2H),5.51(s,2H),7.00(s,1H),7.40(s,1 H),7.41(dd,J=7.0,2.5Hz,1H),8.02-8.05(m,1H),10.31(s,1H),12.75(br s,1H).

[0638] (iii)

[0639]

[0640] To a magnetically stirred mixture of DGA-C20-SN-38 (20 mg, 39 μmol) and PyBOP (20 mg, 39 μmol) in DMF (0.6 mL) was added 1% dHO in DMF (1 mL) at room temperature. A mixture of 4-nitropropene (70 mg, 0.91 μmol) and NMM (16 μL, 146 μmol) was prepared. After 16 hours at room temperature, the volatiles were removed and the residue was purified by size exclusion chromatography (Sephadex, LH-20, MeOH). Appropriate fractions were combined and concentrated as judged by HPLC, then dissolved in water, filtered (0.45 μm) and lyophilized to give 72 mg (89%) of the desired material as a yellow-orange solid.

[0641] HPLC (C8 Aeris WIDEPORE, 100×2.1 mm), gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 10 mM ammonium formate, 0.4 mL / min, Rf (min) = 7.96. 1 H NMR (300MHz, CD 3 OD) δ (ppm): 0.53-3.24 (m, 813H), 3.36 (s, 106H), 3.37-4.04 (m, 6,070H), 4.04-4.65 (m, 155H), 5.18-5.91 (m, 53H), 6.75-8.37 (m, 137H). According to Example 2a, the number of SN-38 molecules was calculated to be 32, and the drug loading was 13.9%.

[0642] 2g

[0643] (i) OtBu-modified-Glu-C10-SN-38

[0644]

[0645] To a magnetically stirred suspension of SN-38 (100 mg, 0.26 mmol) and modified mono-tert-butyl glutarate (72 mg) in DCM (2 mL) was added EDC (76 μL, 0.43 mmol) and DMAP (31 mg, 0.26 mmol) in DCM (0.5 mL) at room temperature. The mixture was stirred overnight at room temperature, after which the reaction was judged to be 70% complete by HPLC. The mixture was filtered (0.2 μm), concentrated to about 3 mL in vacuo and diluted with 3 mL of acetonitrile containing 0.1% TFA. The solvent was removed in vacuo again and the residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 35% ACN / water (0-5 min), 35-70% ACN (5-40 min), 70% ACN (40-43 min), 70-80% ACN (43-44 min), 80% ACN (44-49 min), 80-35% ACN (49-50 min), 35% ACN (50-60 min), 0.1% TFA) to give 101 mg (67%) of OtBu-modified-Glu-C10-SN-38 (RT=48.9 min) as a yellow solid after lyophilization.

[0646] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 7.46. ESI + (ve) observation [M] + = 591. Calculated value of compound = 591 Da.

[0647] (ii) Modified-Glu-C10-SN-38

[0648]

[0649] A solution of OtBu-modified-Glu-C10-SN-38 (95 mg) in TFA (10 mL) was magnetically stirred at room temperature overnight. The volatiles were removed in vacuo, and the residue was azeotroped with DCM, mixed with water and lyophilized to give 90 mg (>95%) of modified-Glu-C10-SN-38 as a yellow-orange solid.

[0650] LCMS (C8, gradient: 40% ACN / H 2O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 3.72. ESI + (ve) observation [M] + = 535. Calculated value of compound = 535 Da.

[0651] (iii)

[0652]

[0653] To a magnetically stirred mixture of modified-Glu-C10-SN-38 (33 mg) and PyBOP (32 mg, 61 μmol) in DMF (1 mL) was added A mixture of 4-(110 mg, 1.42 μmol) and NMM (25 μL, 227 μmol) in DMF (2 mL) was added. After 16 hours at room temperature, the volatiles were removed in vacuo and the residue was purified by size exclusion chromatography (Sephadex, LH-20, MeOH). Appropriate fractions were combined and concentrated as judged by HPLC. The residue was dissolved in water, filtered (0.45 μm) and lyophilized to give 120 mg (94%) of the desired material as a yellow solid.

[0654] HPLC (Aeris WIDEPORE, 100×2.1 mm), gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 214 nm, 10 mM ammonium formate, 0.4 mL / min, Rf (min) = 8.04. According to Example 2a, the number of SN-38 molecules was calculated to be 31 and the drug loading was 13.4%.

[0655] Embodiment 2h: Synthesis

[0656] (i) OtBu-modified-TDA-C10-SN-38

[0657]

[0658] To a magnetically stirred suspension of SN-38 (35 mg, 0.089 mmol) and modified thiodiglycolic acid mono-tert-butyl ester (24 mg) in DCM (1 mL) was added EDC (24 μL, 0.13 mmol) and DMAP (10 mg, 0.079 mmol) in DCM (0.5 mL) at room temperature. The mixture was stirred at room temperature overnight. The volatiles were removed in vacuo and the residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 35% ACN / water (0-5 min), 35-70% ACN (5-40 min), 70% ACN (40-43 min), 70-80% ACN (43-44 min), 80% ACN (44-49 min), 80-35% ACN (49-50 min), 35% ACN (50-60 min), 0.1% TFA) to afford 13 mg. (24%) of OtBu-modified-TDA-C10-SN-38 (RT=44.3 min) as a yellow solid.

[0659] (ii) Modified TDA-C10-SN-38

[0660]

[0661] A solution of OtBu-modified-TDA-C10-SN-38 (13 mg, 0.021 mmol) in TFA (2 mL) was magnetically stirred at room temperature overnight. The volatiles were then removed in vacuo, and the residue was azeotroped with DCM, mixed with water and lyophilized to give 14 mg (>95%) of modified-TDA-C10-SN-38 as a yellow-orange solid.

[0662] LCMS (C8, gradient: 15% ACN / H 2 O (0-1min), 15-25% ACN (1-2min), 25% ACN (2-8min), 25-80% ACN (8-10min), 80% ACN (10-11min), 80-15% ACN (11-13min), 15% ACN (13-15min), 10mM ammonium formate, 0.4mL / min, Rf (min) = 6.47 and 6.57 (two isomer peaks). ESI+ (ve) observation [M] + = 553. Calculated value of compound = 553 Da.

[0663] (iii)

[0664]

[0665] To a magnetically stirred mixture of modified-TDA-C10-SN-38 (64 mg, 116 μmol) and PyBOP (60 mg, 116 μmol) in DMF (2.5 mL) was added 1% ethanol in DMF (2.5 mL) at room temperature. A mixture of 4-(4-(2-nitro-1-yl)-2-nitropropene (199 mg, 2.68 μmol) and NMM (47 μL, 428 μmol) was added. After 16 hours at room temperature, the volatiles were removed in vacuo and the residue was purified by size exclusion chromatography (Sephadex, LH-20, MeOH). Appropriate fractions were combined and concentrated as judged by HPLC. The residue was dissolved in water, filtered (0.45 μm) and lyophilized to give 225 mg (96%) of the desired material as a yellow solid.

[0666] HPLC (C8 Xbridge, 3×100 mm) gradient: 5% sACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 243 nm, 0.1% TFA, 0.4 mL / min, Rf (min) = 8.54. According to Example 2a, the number of SN-38 molecules was calculated to be 32 and the drug loading was 14.3%.

[0667] Embodiment 2i: Synthesis

[0668] (i) 2,5-THF-C20-SN-38-O(Boc)

[0669]

[0670] To a magnetically stirred suspension of Boc-C10-SN-38 (Zhao, H. et al, Bioconjugate Chem., 2008, 19, 849-859) (188 mg, 0.38 mmol) and 3,8-dioxabicyclo[3.2.1]octane-2,4-dione (217 mg, 1.53 mmol) in DCM (7.5 mL) at 0°C was added triethylamine (265 μL, 1.91 mmol) and DMAP (12 mg, 95 μmol). The mixture was stirred at 0°C for 1 h until the reaction was >80% complete as judged by HPLC. The volatiles were removed in vacuo and the residue was purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 35-75% ACN / H 2O (5-50 min), 0.1% TFA) to give 151 mg (62%) of 2,5-THF-C20-SN-38 (RT = 34.5 min) as a yellow solid after lyophilization.

[0671] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 6.25. ESI + (ve) observation [M] + =635. Calculated as C 33 H 34 N 2 O 11 =635Da.

[0672] (ii) 2,5 / THF-C20-SN-38

[0673]

[0674] To a magnetically stirred solution of 2,5-THF-C20-SN-38-O(Boc) (151 mg, 0.24 mmol) in dichloromethane (1.4 mL) was added TFA (0.7 mL) at 0°C. The mixture was stirred at 0°C for 5 min and then at room temperature. After 2.5 h, the reaction was diluted with dichloromethane (20 mL). The volatiles were removed in vacuo, and the residue was azeotroped with DCM, cooled to 0°C, mixed with water and lyophilized to give 151 mg (>95%) of 2,5-THF-C20-SN-38 as a yellow-orange solid.

[0675] HPLC (C8 Xbridge, 3×100 mm), gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 243 nm, 10 mM ammonium formate, 0.4 mL / min, Rf (min) = 6.54. 1 H NMR (300 MHz, DMSO-d 6)δ(ppm):0.92(dd,J=7.4,7.3Hz,3H),1.29(dd,J=7.6,7.5Hz,3H),2.04-2.43(m,6H),3.09(dd,J=15.0,7.3Hz,2H),4.51 -4.58(m,1H),4.61-4.68(m,1H),5.29(s,2H),5.49(s,2H),6.96(s,1H),7.38-7.43(m,2H),8.03-8.08(m,1H),10.31(br s,1H).

[0676] (iii)

[0677]

[0678] To a magnetically stirred mixture of 2,5-THF-C20-SN-38 (125 mg, 233 μmol) and PyBOP (121 mg, 234 μmol) in DMF (8 mL) was added (417 mg, 5.6 μmol) and NMM (99 μL, 899 μmol) in DMF (7 mL). After 2 h at room temperature, the reaction mixture was added to a chilled (0 °C) solution of 1% AcOH in 3:7 ACN / water (150 mL), filtered (P3 sinter) and filtered through ultrafiltration (0.005 m 2 , 10kDa, regenerated cellulose filter) was concentrated to about 15mL. The concentrate was ultrafiltered with 40×15mL diafiltration (1% AcOH in 3:7ACN / MQ water). The retentate was lyophilized to give 486mg of product. The material was dissolved in THF (3.8mL) and added to cooled (0°C) MTBE (16mL). The mixture was stirred at 0°C (1h) and the resulting precipitate was separated by filtration. The product was vacuum dried (16h) to give 388mg (79%) of the desired material as a yellow solid.

[0679] HPLC (C8 Xbridge, 3×100 mm) gradient: 5% sACN / H 2 O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 243nm, 0.1% TFA, 0.4mL / min, Rf (min) = 8.29. 1 H NMR (300MHz, CD 3OD) δ (ppm): 0.27-3.27 (m, 837H), 3.36 (s, 100H), 3.37-3.90 (m, 5, 784H), 3.37-4.65 (m, 167H), 4.88-6.02 (m, 94H), 6.83-8.18 (m, 140H). According to Example 2a, the number of SN-38 molecules was calculated to be 32, and the drug loading was 14.4%.

[0680] Embodiment 2j: Synthesis

[0681] (i) DGA-C20-irinotecan

[0682]

[0683] To a magnetically stirred suspension of irinotecan hydrochloride (110 mg, 0.18 mmol) in DCM (5 mL) was added dimethylaminopyridine (82 mg, 0.67 mmol) at room temperature. After 10 minutes, the solid dissolved and diglycolic anhydride (51 mg, 0.44 mmol) was added. After 6 hours at room temperature, additional portions of dimethylaminopyridine (82 mg, 0.67 mmol) and diglycolic anhydride (51 mg, 0.44 mmol) were added. After stirring overnight at room temperature, the solvent was removed and the residue was dissolved in acetonitrile and purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 5-80% ACN / H 2 O (5-40 min, 0.1% ammonium formate, RT = 37 min) to give 48 mg (39%) of the product as a yellow solid.

[0684] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 7.77. ESI + (ve) observation [M] + =703. Calculated as C 37 H 42 N 4 O 10 =703Da.

[0685] (ii)

[0686]

[0687] To a magnetically stirred solution of DGA-irinotecan (30 mg, 43 μmol) and PyBOP (22 mg, 43 μmol) in DMF (1 mL) was added (92 mg, 1.1 μmol) in DMF (2 mL) solution, followed by NMM (19 μL, 173 μmol). After 16 hours at room temperature, the reaction mixture was concentrated in vacuo, then dissolved in MeOH (1.5 mL), and purified by SEC (Sephadex, LH-20, MeOH). The fractions containing the product were combined and concentrated in vacuo, and the resulting residue was dissolved in MQ water, filtered (0.2 μm acrodisc) and lyophilized to give the desired product as a light yellow solid (85 mg, 76% yield).

[0688] HPLC (C8 Xbridge, 3×100 mm), gradient: 5% ACN / H 2 O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 243nm, 0.1% TFA, 0.4mL / min, Rf (min) = 8.37min. 1 H NMR (300MHz, CD 3 OD-d 4 )(ppm): 0.83-2.43 (m, 1073), 2.84-3.24 (m, 324H), 3.36 (s, 113H), 3.44-3.99 (m, 5900H), 4.03-4.67 (m, 224H), 5.04-6.13 (m, 100H), 7.23-7.94 (m, 139H). According to Example 2a, the number of irinotecan molecules was calculated to be 32, and the drug loading was 19.1% w / w.

[0689] Embodiment 2k: Synthesis

[0690] (i) TDA-C20-irinotecan

[0691]

[0692] To a magnetically stirred suspension of irinotecan hydrochloride (112 mg, 0.18 mmol) in DCM (5 mL) was added dimethylaminopyridine (89 mg, 0.73 mmol) at room temperature. After 10 minutes, the solid dissolved and thiodiglycolic anhydride (60 mg, 0.45 mmol) was added. After 16 hours at room temperature, additional portions of dimethylaminopyridine (83 mg, 0.68 mmol) and thiodiglycolic anhydride (63 mg, 0.48 mmol) were added. After stirring for an additional 3 hours at room temperature, the solvent was removed and the residue was taken up in acetonitrile and purified by preparative HPLC (BEH 300 Waters Xbridge C18, 5 μM, 30×150 mm, 5-80% ACN / H 2 O (5-40 min, 0.1% ammonium formate, RT = 33 min) to give 94 mg (73%) of the product as a yellow solid.

[0693] LCMS (C8, gradient: 40% ACN / H 2 O (0-1min), 40-90% ACN (1-7min), 90% ACN (7-9min), 90-40% ACN (9-11min), 40% ACN (11-15min), 0.1% formic acid, 0.4mL / min, Rf (min) = 6.94. ESI + (ve) observation [M] + =719. Calculated as C 37 H 42 N 4 O 9 S = 719 Da.

[0694] (ii)

[0695]

[0696] To a magnetically stirred solution of TDA-irinotecan (38 mg, 52 μmol) and PyBOP (27 mg, 52 μmol) in DMF (1 mL) was added (105 mg, 1.4 μmol) in DMF (2 mL) solution, followed by NMM (23 μL, 210 μmol). After 16 hours at room temperature, the reaction mixture was concentrated in vacuo, then dissolved in MeOH (1.5 mL), and purified by SEC (Sephadex, LH-20, MeOH). The fractions containing the product were combined and concentrated in vacuo, and the resulting residue was dissolved in MQ water, filtered (0.2 μm acrodisc) and lyophilized to give the desired product as a yellow solid (110 mg, 85% yield).

[0697] HPLC (C8 Xbridge, 3×100 mm), gradient: 5% ACN / H 2 O (0-1min), 5-80% ACN (1-7min), 80% ACN (7-12min), 80-5% ACN (12-13min), 5% ACN (13-15min), 214nm, 0.1% TFA, 0.4mL / min. Rt=8.54min. 1 H NMR (300MHz, CD 3 OD-d 4 )(ppm): 0.65-2.72 (m, 816H), 2.98-3.32 (m, 231H), 3.36 (s, 137H), 3.38-4.10 (m, 6112H), 4.12-4.71 (m, 144H), 5.04-5.99 (m, 63H), 6.80-8.46 (m, 104H). According to Example 2a, the number of irinotecan molecules was calculated to be 24, and the drug loading was 15.4% w / w.

[0698] Example 3: Comparison of the release rate of linkers in PBS and plasma at 37°C and pH 7.4

[0699] A study was conducted to determine the rate of release of SN-38 from certain dendrimer compounds in 9:1 PBS (phosphate buffered saline):DMA at 37°C and pH 7.4. The results show that the % SN-38 released at 6 and 18 hours is shown in the table below.

[0700] Table 3.

[0701]

[0702] PBS release studies were performed by preparing a 1 mg / mL solution of the dendrimer in 9:1 v / v (PBS / DMA) immediately prior to incubation at 37°C. Samples were analyzed by HPLC at 6 h and 18 h, as well as other time points, comparing the % peak area of ​​SN-38 to that of the dendrimer construct. HPLC (C8 Xbridge, 3×100 mm), gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 243 nm, 10 mM ammonium formate buffer, 0.4 mL / min.

[0703] Release at other time points is shown in Fig.13 and 14 middle.

[0704] Table 4.

[0705]

[0706] Plasma release studies were performed by adding 0.8 mL of rat plasma (centrifuged and filtered) to 0.16 mL of a dendrimer solution (approximately 2 mg / mL SN-38 equivalent / saline). The mixture was vortexed (30 s) and then incubated at 37°C. Aliquots (0.1 mL) were removed at different time points and added to ACN (0.2 mL, 5% formic acid). The resulting mixture was vortexed (30 s), centrifuged (10 min, 4°C), filtered and analyzed by HPLC (C8 Xbridge, 3×100 mm, 0.4 mL / min, gradient: 5% ACN / H 2 O (0-1 min), 5-80% ACN (1-7 min), 80% ACN (7-12 min), 80-5% ACN (12-13 min), 5% ACN (13-15 min), 243 nm, 0.1% TFA, RT(SN-38)=7.0 min, RT(conjugate)=8.5 min.

[0707] Release data such as Fig.12 shown.

[0708] Example 4: In vitro anticancer drug screening assay

[0709] Studies were conducted using a cell counting (SRB) assay to determine the ability of the example dendrimers to inhibit the growth of HT-29 cells (a human colon cancer cell line). 50 is the concentration required to inhibit 50% of net cell growth.

[0710] Table 5.

[0711] Dendrimer number / compound <![CDATA[GI 50 μM]]> 2a 0.13,0.13 2e <0.01,<0.01 2f 0.05,0.04 2d 0.14 2c 0.03 2h 0.02 2g 0.8 2j 12 2k 46 Irinotecan 7 SN-38 <0.01

[0712] Example 5: Tolerance of Example Dendrimers in Balb / C Nude Mice

[0713] The tolerability of four example dendrimers (2a, 2f, 2i and 2h) was studied in Balb / C nude mice.

[0714] Sample preparation

[0715] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan was diluted in saline for daily dosing.

[0716] Experimental methods

[0717] Female Balb / c nude mice (6-8 weeks old) were injected intravenously with dendrimers (0.1 ml / 10 g body weight) once a week for 3 weeks (days 1, 8 and 15). The mice were weighed daily and observed for any signs of toxicity (weight loss, poor general health). The animals were monitored for 3-4 weeks after the last drug administration. Any mouse that exceeded the ethical endpoint (≥20% weight loss, poor general health) was immediately euthanized. The animals were fed a small dish of feed supplement every day (make sure to mix with food powder). The animals were evaluated for changes in body weight every 1-2 days after weekly dosing for three weeks. The maximum tolerated drug dose (MTD) was defined as the dose that causes no more than 10% body weight loss and whereby the animals return to baseline body weight within 7-10 days.

[0718] Results and discussion

[0719] The tolerability of the four SN-38 dendrimers and irinotecan was tested in 1-2 Balb / c mice. The maximum weight loss associated with each drug and tested dose is summarized in Table 6. Based on the weight loss observed in the SN-38 group, the final doses selected for the efficacy study were 2f and 2i, 15 mg / kg, 2h, 25 mg / kg, and 2a, 30 mg / kg. The 90 mg / kg dose was selected when mice treated with 100 mg / kg irinotecan showed limb pallor and lethargy.

[0720] Table 6: Tolerance of drug doses tested in Balb / c nude mice

[0721] Compound Tested dose (mg / kg) Maximum weight loss (%) Irinotecan 75,90,100 4,3,10 2a 10,30 0,10 2h 0.5,5,15,30 0,1,0,14 2f 15,20,30 4,10,22 2i 15,20,30 8,15,15

[0722] Weight loss was measured as a percentage of baseline body weight.

[0723] in conclusion:

[0724] Dendrimers 2f (15 mg / kg), 2i (15 mg / kg), 2h (25 mg / kg), and 2a (30 mg / kg) were injected intravenously into Balb / c nude mice once a week for three weeks and were well tolerated.

[0725] Example 6: Efficacy of Example Dendrimers in the SW620 Xenograft Model in Mice

[0726] The in vivo antitumor activity of four example dendrimers (2f, 2i, 2h & 2a) was studied in the SW620 (human colon cancer cell line) xenograft model.

[0727] Sample preparation

[0728] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan was diluted in saline for daily dosing.

[0729] Experimental methods

[0730] Female Balb / c nude mice (7-8 weeks old) were subcutaneously inoculated with 4×10 6 SW620 cells. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the twelfth day after transplantation, tumors of similar size (average tumor volume 135 mm 3 ) were randomly divided into 6 groups of 6 animals each (day 1). The treatment groups were normal saline, irinotecan (90 mg / kg), 2f (15 mg / kg), 2i (15 mg / kg), 2h (25 mg / kg) and 2a (25 mg / kg). All compounds were administered by tail vein injection at 0.1 ml / 10 g body weight on days 1, 8 and 15. Mice were fed a small dish of feed supplement every day (make sure to mix with food powder). The experiment ended on day 119, or earlier if ethical endpoints were met.

[0731] Results and discussion

[0732] Drug Toxicity: All compounds were well tolerated and mean body weight loss did not exceed 10% in any treatment group. One animal from Group 2a was euthanized during the study due to weight loss on Day 99. The mean animal weight changes for each group are summarized below. Figure 1 Data representation

[0733] Mean percent weight change from baseline (Day 1) for each group; bars SEM. Graphs for each group are displayed until no fewer than 4 animals remain in the group.

[0734] Anti-tumor efficacy: Figure 2 and 3 The effects of drug treatment on SW620 tumor growth and survival are summarized separately. When the experiment ended, at day 119, no regrowth was observed in any group, and each tested drug induced complete tumor regression. In contrast, irinotecan treatment caused a delay in tumor growth, except for one tumor, which completely regressed after day 25. Graphs are shown until no fewer than 4 animals remain in the group. Figure 3 Shows from Figure 2 Kaplan-Meier survival curves of selected data from the data shown. The endpoint of the analysis was a tumor volume of 1200 mm3 Mice euthanized due to illness were examined.

[0735] in conclusion:

[0736] Intravenous injection of dendrimers 2f (15 mg / kg), 2i (15 mg / kg), 2h (25 mg / kg), and 2a (25 mg / kg) into Balb / c nude mice once a week for three weeks was well tolerated and induced regression of SW620 tumors in Balb / c nude mice.

[0737] Example 7: Efficacy of Example Dendrimers in the HT-29 Xenograft Model in Mice

[0738] The in vivo antitumor activity of three example dendrimers (2f, 2i and 2h) was studied in the HT-29 (human colon cancer cell line) xenograft model.

[0739] Sample preparation

[0740] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan was diluted in saline for daily dosing.

[0741] Experimental methods

[0742] Female Balb / c nude mice (7-8 weeks old) were subcutaneously inoculated with 5×10 6 HT-29 cells. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the fifteenth day after transplantation, tumors of similar size (average tumor volume 100 mm 3 ) were randomly divided into 5 groups of 10 animals each (day 1). Treatment groups were administered saline, irinotecan (90 mg / kg), 2f (15 mg / kg), 2i (15 mg / kg), and 2h (25 mg / kg). All compounds were administered via tail vein injection at 0.1 ml / 10 g body weight on days 1, 8, and 15. Mice were fed a small dish containing food supplement (mixed with food dust) every day. The experiment was terminated on day 130, or earlier if ethical endpoints were met.

[0743] ANOVA of tumor growth data was analyzed in GraphPad Prism followed by Dunnett's post hoc test. Survival curves were analyzed using the Mantel Cox rank test.

[0744] Intestinal Toxicity Analysis

[0745] A single iv dose of saline, 90 mg / kg irinotecan, or 15 mg / kg 2f was administered to tumor-bearing mice (n = 1). After 24 hours, the mice were euthanized, the intestines were removed and rinsed, rolled in a cassette, then fixed in formalin and paraffin-embedded. Tumors from each mouse were harvested, cut into two pieces, semi-fixed in formalin, and semi-frozen in OCT freezing medium. Sections of μm thickness were cut and stained with H&E. The slides were imaged on a BX-61 microscope and images were taken.

[0746] Tissue samples for immunohistochemistry and fluorescence analysis

[0747] Tumor-bearing mice (n = 2) were treated once weekly for three weeks with 2f (15 mg / kg), 2i (15 mg / kg), 2h (25 mg / kg), and irinotecan (90 mg / kg). Three days after the last dose, the mice were killed, tumors were harvested (half fixed in formalin, half fixed in OCT), intestines were prepared as above, and a portion of muscle was fixed in OCT. One untreated animal was harvested as described above.

[0748] Results and Discussion

[0749] Drug Toxicity

[0750] All compounds were well tolerated and the mean body weight loss did not exceed 7% in any treatment group. No animals were euthanized due to toxicity. The mean animal body weight changes for each group are summarized in Figure 4 . The data represent the mean percentage change in weight from baseline (day 1) for each group; bars are SEM. Graphs are shown for each group until there are no fewer than 6 animals remaining in the group.

[0751] Antitumor Efficacy

[0752] Figure 5 and 6 summarize the effects of each drug on HT-29 tumor growth and survival, respectively. Figure 6 The endpoint analyzed in 3 was a tumor volume of 1200 mm

[0753] Conclusion:

[0754] Example dendrimers of Examples 2f (15 mg / kg), 2i (15 mg / kg) and 2h (25 mg / kg) induced transient regression of HT-29 tumors in Balb / c nude mice.

[0755] Example 8: Efficacy of Example Dendrimers in Mouse MDA-MB-231 Xenograft Model

[0756] The in vivo antitumor activity of two example dendrimers (2f and 2h) was investigated in the MDA-MB-231 (human breast cancer cell line) xenograft model.

[0757] Sample preparation

[0758] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan was diluted in saline for daily dosing.

[0759] Experimental methods

[0760] Female Balb / c nude mice (7 weeks old) were subcutaneously inoculated with 3.5×10 6 MDA-MB-231 cells were cultured in PBS:Matrigel (1:1). Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the 14th day after transplantation, tumors of similar size (average tumor volume 110 mm 3 ) were randomly divided into 3 groups of 4 animals each (day 1). The treatment groups were normal saline, irinotecan (90 mg / kg), 2f (15 mg / kg), and 2h (25 mg / kg). All compounds were administered by tail vein injection at 0.1 ml / 10 g body weight on days 1, 8, and 15. Mice were fed a small dish of food supplement (mixed with food dust) every day. The experiment was terminated on day 70, or earlier if ethical endpoints were met.

[0761] Results and discussion

[0762] Drug toxicity

[0763] All compounds were well tolerated and mean body weight loss did not exceed 6% in any treatment group. The mean animal body weight changes for each group are summarized in Figure 7 Data represent mean percent weight change from baseline (Day 1) for each group; bars SEM. Graphs for each group are displayed until no fewer than 2 animals remain in the group.

[0764] Antitumor efficacy

[0765] Figure 8 and 9 The effects of each drug on MDA-MB-231 tumor growth and survival are summarized separately. Tumor volume is expressed as mean tumor volume (± SEM). Graphs are displayed until no less than 2 animals remain in the group. Figure 8 The end point of the analysis is 1200mm 3 of tumor volume.

[0766] Both SN-38 dendrimers exerted the same effect on tumors, initially inducing tumor regression until day 24, followed by tumor regrowth. Tumors in the irinotecan group continued to grow during drug treatment.

[0767] in conclusion:

[0768] Example dendrimers of Examples 2f (15 mg / kg) and 2h (25 mg / kg) induced regression of MDA-MB-231 tumors in Balb / c nude mice when administered IV once weekly for three weeks.

[0769] Example 9: Efficacy of Example Dendrimers in Mouse CAPAN-1 Xenograft Model

[0770] The in vivo antitumor activity of the example dendrimer (2f) administered intravenously and intraperitoneally in the CAPAN-1 (pancreatic cancer cell line) xenograft model was investigated.

[0771] Sample preparation

[0772] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before administration. Dendrimers were administered in mg / kg SN-38 equivalents.

[0773] Experimental methods

[0774] CAPAN-1 cells were inoculated subcutaneously in the buttocks of mice. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the fourteenth day after implantation, mice with tumors of similar size were randomly divided into 3 groups of 3 animals each (Day 1). The treatment groups were saline, 2f intravenous administration, and 2f intraperitoneal administration. The mice were fed a small dish of food supplement (mixed with food dust) every day.

[0775] Results and discussion

[0776] Drug toxicity

[0777] The mean animal weight changes for each group are summarized in Fig.10Data represent mean percent weight change from baseline (Day 1) for each group; bars SEM.

[0778] Antitumor efficacy

[0779] Fig.11 The effects of IV (intravenous) and IP (intraperitoneal) administration for 2f on CAPAN-1 tumor growth and survival are summarized.

[0780] in conclusion:

[0781] Example dendrimers of Example 2f induced CAPAN-1 tumor regression in mice when administered once weekly for three weeks either intravenously or intraperitoneally.

[0782] Example 10: Efficacy of Examples of Dendrimers Administered Alone or in Combination with Cetuximab in the HT-29 Xenograft Model in Mice

[0783] The efficacy of cetuximab at lower doses and with the EGFR agent cetuximab was explored in the HT-29 (human colon cancer cell line) xenograft model. In vivo antitumor activity of the combined dendrimers (2f).

[0784] Sample preparation

[0785] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan was diluted in saline for daily dosing.

[0786] Experimental methods

[0787] Female Balb / c nude mice (7-8 weeks old) were subcutaneously inoculated with 5×10 6 HT-29 cells. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the fifteenth day after transplantation, tumors of similar size (average tumor volume 100 mm 3) were randomly divided into 7 groups of 8 animals each (day 1). The treatment groups were saline, irinotecan (35 mg / kg), cetuximab (25 mg / kg), dendrimer 2f alone (10 mg / kg and 5 mg / kg), and dendrimer 2f (10 mg / kg and 5 mg / kg) in combination with cetuximab (25 mg / kg). All compounds were administered by tail vein injection at 0.1 ml / 10 g body weight on days 1, 8, 15 and 22. Mice were fed a small dish of food supplement (mixed with food dust) every day. Mice were culled if ethical endpoints were met. The endpoint of the analysis was 1200 mm 3 Tumor volume of 55 days or earlier appeared.

[0788] ANOVA of tumor growth data was analyzed in GraphPad Prism followed by Dunnett's post hoc test. Survival curves were analyzed using the Mantel Cox rank test.

[0789] Results and discussion

[0790] Drug toxicity

[0791] All compounds were well tolerated and mean body weight loss did not exceed 9% in any treatment group. No animals were euthanized due to toxicity.

[0792] Antitumor efficacy

[0793] Fig.15 and 16 The effects of each drug on HT-29 tumor growth and survival when administered alone are summarized. Irinotecan had little effect on this cell line (irinotecan-resistant cell line), while cetuximab had only a moderate effect. In contrast, dendrimer 2f showed a clear dose response with statistically significant tumor regression. Example 2f significantly prolonged survival compared to irinotecan (P<0.0001), as shown previously in Example 7. Example 2f also prolonged survival relative to cetuximab.

[0794] The higher dose of 10 mg / kg showed Figures 1 to 10 The almost identical effects shown indicate a wide therapeutic window between efficacy and safety.

[0795] Fig.17 and 18 The effects of irinotecan or dendrimers in combination with cetuximab, respectively, on HT-29 tumor growth and survival are summarized. Fig.18 Display from Fig.17Kaplan-Meier survival curves for selected data and vehicle are shown. The combination of irinotecan and cetuximab showed only very modest effects compared to each drug administered alone, whereas the combination of cetuximab and low-dose (5 mg / kg) 2f provided unexpected benefits on efficacy and survival, clearly indicating the potentiation effect of the dendrimer on the activity of cetuximab.

[0796] Example 11: Efficacy of Examples of Dendrimers Administered Alone or in Combination with Olaparib in the HT-29 Xenograft Model in Mice

[0797] The in vivo antitumor activity of dendrimers (2f) at lower doses and in combination with the PARP inhibitor olaparib was explored in the HT-29 (human colon cancer cell line) xenograft model.

[0798] Sample preparation

[0799] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately prior to administration. All dendrimers were administered in mg / kg SN-38 equivalents. Irinotecan clinical formulation (DBLTM-irinotecan, Hospira Australia Pty) was diluted in saline for daily dosing. Olaparib solid powder obtained from MedChemExpress was dissolved in DMSO and an oral formulation with a final concentration of 5 mg / ml was prepared in 10% DMSO: 15% (2-hydroxypropyl)-β-cyclodextrin in sterile water.

[0800] Experimental methods

[0801] Female Balb / c nude mice (7-8 weeks old) were subcutaneously inoculated with 5×10 6 HT-29 cells. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the thirteenth day after transplantation, tumors with similar sizes (average tumor volume 100 mm 3) were randomly divided into 8 groups of 10 animals each (day 1). Treatment groups were saline, irinotecan (80 mg / kg) or in combination with olaparib (50 mg / kg), dendrimer 2f alone (5 mg / kg and 8 mg / kg), and dendrimer 2f (5 mg / kg and 8 mg / kg) in combination with olaparib (50 mg / kg). All compounds were administered by tail vein injection at 0.1 ml / 10 g body weight on days 1, 8, and 15, except that (i) olaparib was administered orally for 5 days / 2 days off for 3 weeks, (ii) the group receiving dendrimer 8 mg / kg was administered the third dose on day 16, and (iii) in the dendrimer 8 mg / kg + olaparib group, -3 / 10 mice missed 1-2 olaparib doses in weeks 2 and / or 3. Mice were fed a small dish of food supplement (mixed with food dust) every day. If ethical endpoints were met, mice were culled. The endpoint for analysis was a tumor volume of 1200 mm 3 .

[0802] Statistical analysis of tumor growth was performed on day 28 when the first study animal reached the endpoint criteria for tumor burden.

[0803] Table 7: Analysis of percent tumor growth inhibition.

[0804] deal with Percent TGI P(vs vehicle) Olaparib 0 0.9999 Irinotecan 33 0.0446 Irinotecan + olaparib 37 0.0171 5mg / kg 2f 62 <0.0001 5mg / kg 2f+olaparib 100 <0.0001 8mg / kg 2f 97 <0.0001 8mg / kg 2f+olaparib 107 <0.0001

[0805] Results and discussion

[0806] Drug toxicity

[0807] All compounds were well tolerated and mean body weight loss did not exceed 8% in any treatment group. No animals were euthanized due to toxicity and all animals remained on the study until day 28.

[0808] Antitumor efficacy

[0809] Fig.19 The effects of each drug on HT-29 tumor growth when administered alone are summarized. Olaparib alone had no effect on tumor growth in this model. Irinotecan had a moderate effect on tumor growth (33% TGI at day 28). In contrast, dendrimer 2f showed a clear dose-dependent response with statistically significant tumor growth inhibition (62% and 94% TGI at day 28 for 5 mg / kg and 8 mg / kg, respectively). As shown in Examples 7 and 10 above, Example 2f significantly inhibited tumor growth and prolonged tumor regression compared to irinotecan (P<0.0001). Example 2f also prolonged tumor regression compared to Olaparib.

[0810] Fig.19The effects of irinotecan or dendrimers combined with olaparib on HT-29 tumor growth are also summarized. The combination of irinotecan and olaparib was superior to single-agent therapy, while the combination of olaparib and low-dose (5 mg / kg) 2f provided an unexpected benefit to efficacy with complete tumor growth inhibition, (100% TGI on day 28 clearly indicated that olaparib enhanced the anti-tumor activity of 2f. Similarly, the combination of olaparib and 8 mg / kg 2f also enhanced the activity with significant tumor regression on day 28 (% TGI>100%).

[0811] Example 12: Efficacy of Examples of Dendrimers Administered Alone or in Combination with PD-1 Inhibitors in the CT26 Xenograft Model in Mice

[0812] The in vivo antitumor activity of dendrimers (2f) in combination with PD-1 inhibitors will be explored in the CT26.WT (ATCC) (colon cancer cell line) syngeneic model.

[0813] Sample preparation

[0814] Dendrimer constructs were stored at -20°C until use. Constructs were dissolved in saline immediately before dosing. All dendrimers were administered in mg / kg SN-38 equivalents. In daily dosing, PD-1 antibody (InVivoMAb anti-mouse PD-1 (CD279) (Clone: ​​RMP1-14)) and isotype control (InVivoMAb rat IgG2a isotype control, anti-trinitrophenol) were diluted in saline.

[0815] Experimental methods

[0816] Female Balb / c mice (7-8 weeks old) were subcutaneously inoculated with 5×10 6 CT26 cells. Mice were weighed and tumors were measured 2-3 times per week using electronic calipers. Tumor volume (mm 3 ) is calculated as length (mm) / 2×width (mm) 2 On the thirteenth day after transplantation, tumors with similar sizes (average tumor volume 100 mm 3 Mice with 200 μg / kg of leukemia virus (CEV) were randomly divided into groups of 6 animals per group (day 1). Treatment groups were saline and compound 2f (15 mg / kg) alone or in combination with antibodies (200 μg ip on day 1 and 100 μg on days 4, 8, and 12). All dendrimer treatments were administered by tail vein injection at 0.1 ml / 10 g body weight on days 1, 8, and 15. Mice were culled if ethical endpoints were met. The endpoint for analysis was a tumor volume of 1200 mm 3 .

[0817] When the first study animal reaches the endpoint criteria for tumor burden, tumor growth analysis is performed.

[0818] Table 8: Expected tumor growth inhibition percentage (TGI) analysis.

[0819] deal with Percent TGI Vehicle + isotype control 0 Anti-PD-1 About 20% 15mg / kg 2f+isotype control About 60% (as mentioned above) 15mg / kg 2f+anti-PD-1 About 100%

[0820] Example 13: Pharmacokinetic Study

[0821] A single-dose toxicity study of 2f was conducted in rats for comparison with irinotecan. Five groups of Sprague-Dawley rats received a single slow bolus injection of vehicle, low (approximately 1.3 mg / kg SN-38 equivalents), medium (approximately 7.5 mg / kg SN-38 equivalents), or high dose (approximately 16.5 mg / kg SN-38 equivalents) of 2f or irinotecan 70 mg / kg (approximately 40 mg / kg SN-38 equivalents). (n=18 per group except vehicle n=6)

[0822] Blood samples were collected from alternating subgroups of 6 animals at 5m, 30m, 2h, 4h, 8h, 24h, 72h and 120h after administration and analyzed by LC-MS / MS for total and free SN-38) and pharmacokinetic properties. The results are summarized in the table below.

[0823] Table 9: Pharmacokinetic parameters of free SN-38.

[0824]

[0825] Table 10: Pharmacokinetic parameters of total SN-38.

[0826]

Claims

1. A dendrimer or a pharmaceutically acceptable salt thereof, wherein the dendrimer is in, T1′ represents the first terminal group of the following: or T1′ represents H, wherein less than 5 T1′ are H; and T2′ represents the second terminal group of: wherein the PEG group is a methoxy-terminated PEG having an average molecular weight of 2000 to 2200 Daltons, or T2' represents H, and wherein less than 5 T2' are H.

2. A pharmaceutical composition comprising the dendrimer of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

3. Use of the dendrimer according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a drug for treating cancer.

4. The use according to claim 3, wherein the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, lung cancer and cervical cancer.

5. The use according to claim 3, wherein the dendrimer is administered in combination with another anticancer drug.

6. Use according to claim 5, wherein the additional anti-cancer drug is an immunotherapeutic agent.

7. The use according to claim 6, wherein the immunotherapeutic agent is a PD-1 or PD-L1 inhibitor.

8. The use of claim 6, wherein the immunotherapeutic agent is selected from the group consisting of pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, and cemiplizumab.

9. The use of claim 8, wherein the immunotherapeutic agent is pembrolizumab.

10. The use according to claim 7, wherein the additional anticancer drug is a PARP inhibitor.

11. The use according to claim 10, wherein the additional anticancer drug is olaparib.

12. The use of claim 5, wherein the additional anticancer drug is an EGFR inhibitor.

13. The use according to claim 12, wherein the EGFR inhibitor is an EGFR antibody.

14. The use according to claim 13, wherein the EGFR antibody is cetuximab.

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