Lyophilized products, pharmaceutical compositions, and kits comprising dye-peptide conjugates and derivatives

Abstract

Provided herein are lyophilized products comprising LS301 or derivatives or salts thereof, diluents for reconstitution, pharmaceutical composition formed from the lyophilized product, kits comprising vials containing the lyophilized product and diluent, kits comprising vials containing a solution of the lyophilized product in water and diluent, and related methods.

Description

LYOPHILIZED PRODUCTS, PHARMACEUTICAL COMPOSITIONS, AND KITS COMPRISING DYE-PEPTIDE CONJUGATESAND DERIVATIVES

[0001] This application claims the benefit of priority of the United States Provisional Patent Application Serial No. 63 / 500,629 filed on May 8, 2023, the disclosure of which is incorporated by reference in its entirety for all purposes.

[0002] The present disclosure generally relates to lyophilized products, pharmaceutical compositions, and kits comprising dye-peptide conjugates and derivatives, such as LS301.

[0003] Various cancers and other disorders are imaged by administering targeted fluorescence imaging probes. LS301 is an excellent near-infrared (NIR) imaging probe, especially because it is specific towards a target commonly found on several cancer types and some cell types associated with inflammatory disorders. The clinical formulation for an optical probe with hydrophobic properties ideally has one or more of the following features: adequate and accurate dosing, bioavailability, targeting efficacy, appropriate absorption, and fluorescence characteristics, and short-term stability (after reconstitution before dosing), and long-term storage stability (for example, as a packaged drug product). The hydrophobic property of LS301 affects tissue distribution and clearance, which can affect tumor tissue uptake and clearance from non-tumor tissue. In various embodiments, the lyophilized products and compositions described herein can accelerate tumor uptake, allowing for quick clinical intervention within as soon as an hour post-administration; and / or can provide good signal-to-background when administered a longer period of time before a procedure, even at low dose.

[0004] The present disclosure provides formulations of LS301 and derivatives and salts thereof for various biological applications, including lyophilized products, IV delivery, and topical application. Specifically, the present disclosure provides a lyophilized product comprising 2-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp- Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 2-20 wt.% calcium salt, 20-30 wt.% polysorbate, 10-60 wt.% £><?ta-cyclodextrin, 1-10 wt.% sodium acetate, optionally 40-60 wt.% dextrose, and optionally 1-10 wt.% histidine.

[0005] The present disclosure also provides a lyophilized product formed by a method comprising solubilizing a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg- Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration in a solution of 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% heto-cyclodextrin, and 0.2-2 wt.% sodium acetate, optionally 1-100 mM histidine in water at a ratio of about 1 gram of dye-peptide conjugate per about 10 liters of the solution; filtering the solution; and lyophilizing the filtered solution to form the lyophilized product.

[0006] The present disclosure further provides a pharmaceutical composition comprising a lyophilized product described herein and a diluent. In certain embodiments, the pharmaceutical composition comprises 1-4 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% heto-cyclodextrin, and 0.2-2 wt.% sodium acetate, 0.2-10 wt.% dextrose, 1-100 mM histidine; and 62-85.6 wt.% water.

[0007] The present disclosure provides a vial containing a lyophilized product disclosed herein.

[0008] The present disclosure provides a vial containing a pharmaceutical composition disclosed herein.

[0009] The present disclosure provides a kit comprising a first vial containing a lyophilized product disclosed herein and a second vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine to be reconstituted with 88-98.5 wt.% water.

[0010] The present disclosure provides a kit comprising a first vial containing a solution of a lyophilized product disclosed herein and a second vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine to be reconstituted with 88- 98.5 wt.% water.

[0011] The present disclosure provides a method of preparing a pharmaceutical composition, the method comprising mixing an amount of diluent into a vial comprising the lyophilized product disclosed herein sufficient to form a pharmaceutical composition comprising about 2 wt.% dye-peptide conjugate, wherein the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate 80, 1-100 mM histidine, and 88-98.5 wt.% water.

[0012] Other objects and features will be in part apparent and in part pointed out below.DETAILED DESCRIPTION

[0013] The present disclosure relates to formulations of a dye-peptide derivative (LS301) that selectively target tyrosine 23-phosphorylated annexin A2 (pANXA2) and accumulates in cancerous tissue, lymph nodes, and specific regions of inflammation, such as joint pathology in rheumatoid arthritis.LYOPHILIZED PRODUCT

[0014] The present disclosure provides a lyophilized product comprising 5-20 wt.% a dye- peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 5-20 wt.% calcium salt, 20-30 wt.% polysorbate, 40-60 wt.% hydroxypropyl-beta-cyclodextrin, and 1-10 wt.% sodium acetate.

[0015] In certain embodiments, the cypate is

[0016] In certain embodiments, at least one of the Cys amino acid residues is D-Cys.

[0017] In certain embodiments, the dye-peptide conjugate is LS301 and comprises the structural formula

[0018] In certain embodiments, the dye-peplide conjugate is LS301, comprising the structural formula

[0019] In certain embodiments, a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly- Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Tyr- OH (LS838) or pharmaceutically acceptable salts thereof.

[0020] In certain embodiments, the dye-peptide conjugate is LS838, a derivative of LS301. In certain embodiments, LS838 comprises the structural formula

[0021] In certain embodiments, the dye-peptide conjugate is LS838 and comprises the structural formula

[0022] In certain embodiments, LS838 may be substituted for LS301 in any of the lyophilized products or pharmaceutical compositions described herein.

[0023] In certain embodiments, LS301 is derivatized with a radionuclide chosen from, for example, fluorine-18, iodine-123, iodine-124, iodine-125, and iodine-131. This radiolabeling allows imaging of cancer in the human body noninvasively using nuclear imaging methods. The fluorescence allows optical methods to guide tissue biopsy, surgery, and assessment of surgical margins.

[0024] In certain embodiments, the lyophilized product comprises between 5 wt.% and 20 wt.% calcium salt, such as between 5 wt.% and 10 wt.%, between 10 wt.% and 15 wt.%, and between 15 wt.% and 20 wt.% calcium salt. Without wishing to be bound by theory, calcium ion enhances cellular uptake of the dye-peptide conjugate from the lyophilized product, enhancing, for example, accumulation and retention in tumors. In certain embodiments, the lyophilized product comprises at least 5 wt.% calcium salt. In certain embodiments, the lyophilized product comprises not more than 20 wt.% calcium salt. In certain embodiments, the lyophilized product comprises about 10 wt.% calcium salt. In certain embodiments, the calcium salt is chosen from calcium gluconate, calcium citrate, calcium phosphate, calcium lactate, calcium lactate gluconate, calcium acetate, and calcium carbonate. In certain embodiments, the calcium salt is calcium gluconate.

[0025] In certain embodiments, the lyophilized product comprises between 20 wt.% and 30 wt.% polysorbate, such as about 20 wt.%, about 21 wt.%, about 22 wt.%, about 23 wt.%, about 24 wt.%, about 25 wt.%, about 26 wt.%, about 27 wt.%, about 28 wt.%, about 29 wt.%, or about 30 wt.% polysorbate. Polysorbate is an emulsifier. In certain embodiments, the lyophilized product comprises at least about 20 wt.% polysorbate. In certain embodiments, the lyophilized product comprises not more than about 30 wt.% polysorbate. In certain embodiments, the lyophilized product comprises about 25 wt.% polysorbate. In certain embodiments, the polysorbate is chosen from polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). The number following the “polysorbate” part relates to the type of major fatty acid associated in the molecule. Monolaurate is indicated by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80. The number 20 following the “polyoxyethylene” part refers to the total number of oxyethylene (-CH2CH2O-) groups found in the molecule. In certain embodiments, the polysorbate is polysorbate 80.

[0026] In certain embodiments, the lyophilized product comprises between 40 wt.% and 60 wt.% of a bc / «-cyclodextrin, such as between 40 wt.% and 45 wt.%, between 45 wt.% and 50 wt.%, between 50 wt.% and 55 wt.%, or between 55 wt.% and 60 wt.% of a beta- cyclodextrin. In certain embodiments, the lyophilized product comprises about 50 wt.% beta- cyclodextrin. Beto-cyclodextrin is also referred to as “betadex” and is sold under the brand name Kleptose™. In certain embodiments, the bc / a-cyclodextrin is hydroxypropyl-beta- cyclodextrin, sold under the brand name Kleptose™ HBP, and typically comprising partial substitution with 2-hydroxypropyl ethers.

[0027] In certain embodiments, the lyophilized product comprises between 1 wt.% and 10 wt.% sodium acetate, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, or about 10 wt.% sodium acetate. In certain embodiments, the lyophilized product comprises at least 1 wt.% sodium acetate. In certain embodiments, the lyophilized product comprises no more than 10 wt.% sodium acetate. In certain embodiments, the lyophilized product comprises about 5 wt.% sodium acetate. In certain embodiments, the sodium acetate is sodium acetate trihydrate.

[0028] In certain embodiments, the lyophilized product has been adjusted to a target pH of about 3.0 with acetic acid.

[0029] The present disclosure also provides a lyophilized product formed by a method comprising solubilizing a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg- Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration in a solution of 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% heto-cyclodextrin, and 0.2-2 wt.% sodium acetate in water at a ratio of about 1 gram of dye-peptide conjugate per about 10 liters of the solution; filtering the solution; and lyophilizing the filtered solution to form the lyophilized product.

[0030] In certain embodiments, the solution comprises between 1 wt.% and 4 wt.% calcium salt in water, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, or about 4 wt.% calcium salt in water. In certain embodiments, the solution comprises at least 1 wt.% calcium salt in water. In certain embodiments, the solution comprises not more than about 4 wt.% calcium salt in water. In certain embodiments, the calcium salt for the solution is chosen from calcium gluconate, calcium citrate, calcium phosphate, calcium lactate, calcium lactate gluconate, calcium acetate, and calcium carbonate. In certain embodiments, the calcium salt in the solution is calcium gluconate.

[0031] The term “solubilize” or “solubilizing,” as defined herein, refers to a solute, such as LS301, dissolving and / or suspending into a solvent or medium to form a mixture.

[0032] In certain embodiments, the solution comprises between 4 wt.% and 6 wt.% polysorbate in water, such as about 4 wt.%, about 5 wt.%, or about 6 wt.% polysorbate in water. In certain embodiments, the solution comprises at least about 4 wt.% polysorbate in water. In certain embodiments, the solution comprises not more than about 6 wt.% polysorbate in water. In certain embodiments, the polysorbate for the solution is chosen from polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) in water. In certain embodiments, the polysorbate for the solution is polysorbate 80.

[0033] In certain embodiments, the solution comprises between 8 wt.% and 12 wt.% beta- cyclodextrin in water, such as about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, or about 12 wt.% 6<?ta-cyclodextrin in water. In certain embodiments, the solution comprises at least about 8 wt.% £>eta-cyclodextrin in water. In certain embodiments, the solution comprisesnot more than about 12 wt.% fteto-cyclodextrin in water. In certain embodiments, the beta- cyclodextrin in the solution is hydroxypropyl-beta-cyclodextrin.

[0034] In certain embodiments, the solution comprises between 0.2 wt.% and 2 wt.% sodium acetate in water, such as between 0.2 wt.% and 0.5 wt.%, between 0.5 wt.% and 1.0 wt.%, between 1.0 wt.% and 1.5 wt.%, or between 1.5 wt.% and 2.0 wt.% sodium acetate in water. In certain embodiments, the solution comprises at least about 0.2 wt.% sodium acetate in water. In certain embodiments, the solution comprises not more than about 2 wt.% sodium acetate in water.

[0035] In certain embodiments, the solution has a ratio of about 1 gram of dye -peptide conjugate per about 10 liters of the solution. A person of skill in the art can adjust the ratio of the dye-peptide conjugate and the solution based on the amounts of ingredients predetermined for the lyophilized product.PHARMACEUTICAL COMPOSITIONS

[0036] The present disclosure further provides a pharmaceutical composition comprising a lyophilized product described herein and a diluent. In certain embodiments, the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water. In other embodiments, the diluent is water, such as water for injection.

[0037] In certain embodiments, the diluent comprises 1-10 wt.% dextrose, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, or about 10 wt.% dextrose. In certain embodiments, the diluent comprises at least about 1 wt.% dextrose. In certain embodiments, the diluent comprises not more than about 10 wt.% dextrose. In certain embodiments, the diluent comprises about 4 wt.% dextrose.

[0038] In certain embodiments, the diluent comprises 0.5-2 wt.% polysorbate, such as between 0.5 wt.% and 1 wt.%, between 1 wt.% and 1.5 wt.%, or between 1.5 wt.% and 2 wt.% polysorbate. In certain embodiments, the diluent comprises at least about 0.5 wt.% diluent. In certain embodiments, the diluent comprises not more than about 2 wt.% polysorbate. In certain embodiments, the diluent comprises about 1 wt.% polysorbate. In certain embodiments, the polysorbate is polysorbate 80.

[0039] In certain embodiments, the diluent comprises 1-100 mM histidine, such as between 1 mM and 10 mM, between 10 mM and 20 mM, between 20 mM and 30 mM, between 30 mMand 40 mM, between 40 mM and 50 mM, between 50 mM and 60 mM, between 60 mM and 70 mM, between 70 mM and 80 mM, between 80 mM and 90 mM, or between 90 mM and 100 mM histidine. In certain embodiments, the diluent comprises at least about 1 mM histidine. In certain embodiments, the diluent comprises not more than about 100 mM histidine. In certain embodiments, the diluent comprises about 10 mM histidine.

[0040] In various embodiments, the balance of the diluent is water, such as 88-98.5 wt.% water, for example, about 88 wt.%, about 89 wt.%, about 90 wt.%, about 91 wt.%, about 92 wt.%, about 93 wt.%, about 94 wt.%, about 95 wt.%, about 96 wt.%, about 97 wt.%, about 98 wt.%, or about 98.5 wt.% water. In certain embodiments, the diluent comprises at least about 88 wt.% water. In certain embodiments, the diluent comprises not more than about 98.5 wt.% water. In certain embodiments, the diluent comprises about 75 wt.% water.

[0041] In certain embodiments, the diluent comprises about 4 wt.% dextrose, about 1 wt.% polysorbate, about 10 mM histidine, and about 95 wt.% water.

[0042] In certain embodiments, a sufficient quantity of the diluent is added to the lyophilized product to obtain a concentration of dye-peptide conjugate of about 2 wt.% of the total composition; that is, a pharmaceutical composition comprising the components of the lyophilized product and the diluent.

[0043] In certain embodiments, when lyophilized product is combined with the diluent, a mixture suitable for injection forms within two minutes. In these embodiments, the mixture is suitable for use for at least eight hours when stored at room temperature (20-24 °C) and at least 24 hours when stored between 2 and 8 °C.

[0044] In certain embodiments, the pharmaceutical composition comprises 1-4 wt.% a dye- peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% beto-cyclodextrin, 0.2-2 wt.% sodium acetate, 1-10 wt.% dextrose, 1-100 mM histidine, and 88-98.5 wt.% water.

[0045] In certain embodiments, the pharmaceutical composition comprises 1-4 wt.% a dye- peptide conjugate, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, or about 4 wt.% dye- peptide conjugate. In certain embodiments, the pharmaceutical composition comprises at least 1 wt.% dye-peptide conjugate. In certain embodiments, the pharmaceutical composition comprises LS301.

[0046] In certain embodiments, the solution comprises between 1 wt.% and 4 wt.% calcium salt, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, or about 4 wt.% calcium salt. In certain embodiments, the solution comprises at least about 1 wt.% calcium salt. In certain embodiments, the solution comprises not more than about 4 wt.% calcium salt. In certain embodiments, the calcium salt for the solution is chosen from calcium gluconate, calcium citrate, calcium phosphate, calcium lactate, calcium lactate gluconate, calcium acetate, and calcium carbonate. In certain embodiments, the calcium salt in the solution is calcium gluconate.

[0047] In certain embodiments, the pharmaceutical composition comprises between 4 wt.% and 6 wt.% polysorbate, such as about 4 wt.%, about 5 wt.%, or about 6 wt.% polysorbate. In certain embodiments, the pharmaceutical composition comprises at least about 4 wt.% polysorbate. In certain embodiments, the pharmaceutical composition comprises not more than about 6 wt.% polysorbate. In certain embodiments, the polysorbate for the pharmaceutical composition is chosen from polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). In certain embodiments, the polysorbate for the pharmaceutical composition is polysorbate 80.

[0048] In certain embodiments, the pharmaceutical composition comprises between 8 wt.% and 12 wt.% be / u-cyclodexlrin, such as about 8 wt.%, about 9 wt.%, about 10 wt.%, about 11 wt.%, or about 12 wt.% beta-cyclodextrin. In certain embodiments, the pharmaceutical composition comprises at least about 8 wt.% beta-cyclodextrin. In certain embodiments, the pharmaceutical composition comprises not more than about 12 wt.% beta-cyclodextrin. In certain embodiments, the beto-cyclodextrin in the pharmaceutical composition is hydroxypropyl- era-cyclodextrin.

[0049] In certain embodiments, the pharmaceutical composition comprises between 0.2 wt.% and 2 wt.% sodium acetate, such as between 0.2 wt.% and 0.5 wt.%, between 0.5 wt.% and 1.0 wt.%, between 1.0 wt.% and 1.5 wt.%, or between 1.5 wt.% and 2.0 wt.% sodium acetate. In certain embodiments, the pharmaceutical composition comprises at least about 0.2 wt.% sodium acetate. In certain embodiments, the pharmaceutical composition comprises not more than about 2 wt.% sodium acetate. In certain embodiments, the pharmaceutical compositioncomprises about 1 wt.% sodium acetate. In certain embodiments, the sodium acetate is sodium acetate trihydrate.

[0050] In certain embodiments, the pharmaceutical composition comprises 1-10 wt.% dextrose, such as about 1 wt.%, about 2 wt.%, about 3 wt.%, about 4 wt.%, about 5 wt.%, about 6 wt.%, about 7 wt.%, about 8 wt.%, about 9 wt.%, or about 10 wt.% dextrose. In certain embodiments, the pharmaceutical composition comprises at least about 1 wt.% dextrose. In certain embodiments, the pharmaceutical composition comprises not more than about 10 wt.% dextrose. In certain embodiments, the pharmaceutical composition comprises about 4 wt.% dextrose.

[0051] In certain embodiments, the pharmaceutical composition comprises 0.5-2 wt.% polysorbate, such as between 0.5 wt.% and 1 wt.%, between 1 wt.% and 1.5 wt.%, or between 1.5 wt.% and 2 wt.% polysorbate. In certain embodiments, the pharmaceutical composition comprises at least about 0.5 wt.% pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises not more than about 2 wt.% polysorbate. In certain embodiments, the pharmaceutical composition comprises about 1 wt.% polysorbate. In certain embodiments, the polysorbate is polysorbate 80.

[0052] In certain embodiments, the pharmaceutical composition comprises 1-100 mM histidine, such as between 1 mM and 10 mM, between 10 mM and 20 mM, between 20 mM and 30 mM, between 30 mM and 40 mM, between 40 mM and 50 mM, between 50 mM and 60 mM, between 60 mM and 70 mM, between 70 mM and 80 mM, between 80 mM and 90 mM, or between 90 mM and 100 mM histidine. In certain embodiments, the pharmaceutical composition comprises at least about 1 mM histidine. In certain embodiments, the pharmaceutical composition comprises not more than about 100 mM histidine. In certain embodiments, the pharmaceutical composition comprises about 10 mM histidine.

[0053] In various embodiments, the balance of the pharmaceutical composition is water, such as 88-98.5 wt.% water, for example about 88 wt.%, about 89 wt.%, about 90 wt.%, about 91 wt.%, about 92 wt.%, about 93 wt.%, about 94 wt.%, about 95 wt.%, about 96 wt.%, about 97 wt.%, about 98 wt.%, or about 98.5 wt.% water. In certain embodiments, the pharmaceutical composition comprises at least about 88 wt.% water. In certain embodiments, the pharmaceutical composition comprises not more than about 98.5 wt.% water. In certain embodiments, the pharmaceutical composition comprises about 75 wt.% water.

[0054] In various embodiments, the balance of the pharmaceutical composition is water, such as 62-86 wt.% water, for example, about 62 wt.%, about 63 wt.%, about 64 wt.%, about 65 wt.%, about 66 wt.%, about 67 wt.%, about 68 wt.%, about 69 wt.%, about 70 wt.%, about 71 wt.%, about 72 wt.%, about 73 wt.%, about 74 wt.%, about 75 wt.%, about 76 wt.%, about 77 wt.%, about 78 wt.%, about 79 wt.%, about 80 wt.%, about 81 wt.%, about 82 wt.%, about 83 wt.%, about 84 wt.%, about 85 wt.%, or about 86 wt.% water. In certain embodiments, the pharmaceutical composition comprises at least about 62 wt.% water. In certain embodiments, the pharmaceutical composition comprises not more than about 86 wt.% water.

[0055] In certain embodiments, the pharmaceutical composition comprises about 2 wt.% LS301, about 2 wt.% calcium salt, about 5 wt.% polysorbate, about 10 wt.% beta- cyclodextrin, and about 1.3 wt.% sodium acetate, about 4 wt.% dextrose, about 10 mM histidine, and about 75 wt.% water.

[0056] In certain embodiments, the pharmaceutical composition has a pH between 5.5 and 6.5.

[0057] In certain embodiments, the pharmaceutical composition has an osmolality of about 330 mOsm / kg.

[0058] The present disclosure further provides a pharmaceutical composition comprising a lyophilized product described herein and a diluent. In certain embodiments, the pharmaceutical composition comprises 1-4 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% heta-cyclodextrin, and 0.2-2 wt.% sodium acetate, 0.2-10 wt.% dextrose, 1-100 mM histidine; and 62-86 wt.% water.

[0059] The present disclosure provides a vial containing a lyophilized product disclosed herein.

[0060] The present disclosure provides a vial containing a pharmaceutical composition disclosed herein.

[0061] The present disclosure provides a method of preparing a pharmaceutical composition, the method comprising mixing an amount of diluent into a vial comprising a lyophilized product disclosed herein sufficient to form a pharmaceutical composition comprising about 2 wt.% dye-peptide conjugate, wherein the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.%polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water. Any diluent disclosed herein can be used with this method.DERIVATIVES OF LS301

[0062] The lyophilized products and pharmaceutical compositions described herein may use an LS301 derivative or a salt thereof. In certain embodiments, the derivative comprises at least one imaging agent or at least one treatment agent. In one embodiment, the derivative comprises an imaging agent. In certain embodiments, the derivative comprises an imaging agent and a treatment agent. Irrespective of the embodiment, the derivative may be conjugated to the cyclic peptide directly via a covalent bond or indirectly via a linker.

[0063] Several imaging agents are suitable for use to the extent that they can detect or monitor the localization of the derivative. In one embodiment, the imaging agent comprises an optical imaging agent. Suitable optical imaging agents include fluorophores, organic fluorescent dyes, luminescent imaging agents, fluorescent lanthanide complexes, and fluorescent semiconductor nanocrystals.

[0064] The compounds and derivatives described herein may operate along the electromagnetic spectrum, including visible and infrared wavelengths, such as near-infrared (NIR), short-wavelength infrared (SWIR), middle-wavelength infrared (MWIR), long wavelength infrared (LWIR), and far-infrared. SWIR has the advantage over NIR of deeper tissue penetration.

[0065] Examples of suitable visible (400-700 nm) fluorescent dyes include fluorescein, FITC, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647) and DyDelight Dyes. Suitable near-infrared (NIR) (700-1400 nm) fluorescent dyes include carbocyanine dyes, such as cypate and its derivatives.Luminescence imaging agents include luminescent lanthanide chelates and bioluminescence compounds (e.g., bacterial Lux, eukaryotic Luc, or Rue systems). In a specific embodiment, an imaging agent is a carbocyanine dye or a derivative thereof. Suitable carbocyanine dyes are known in the art. In certain embodiments, the dye-peptide conjugate comprises a carbocyanine dye chosen from Cypate (cypate 4), LS288, LS798, LS276, LS843, Cypate 3, and Cypate 2. Cypates are known in the art. In many embodiments, including LS301, the cypate is typically cypate 4. A dye-peptide conjugate comprising a carbocyanine dye maycomprise a nonionic group (i.e., polyethylene glycol) or a positively charged moiety (i.e.,+NMe3) conjugated to a free carboxylic acid group of a cypate.

[0066] Short-wave infrared (SWIR) (1400-3000 nm) operates at longer wavelengths than NIR. Many NIR dyes produce signals in the SWIR, albeit weaker than the signal in the NIR range. MWIR is between 3000 nm and 8000 nm (3-8 pm). LWIR is between 8000 and 15000 nm (8-15 pm. Far infrared is between 15 and 1,000 pm. To visualize in these ranges, one of skill in the art would select a dye active at those wavelengths. That is, the cypate portion of the peptide-dye conjugate would be selected to operate at the chosen wavelengths.

[0067] Alternatively, a derivative comprising a carbocyanine dye may comprise a functional group for conjugating a radioisotope, treatment agent, or another biologically active molecule. Non- limiting examples of biologically active molecules include nanoparticles, small organic molecules, peptides, proteins, organometallics, drugs, antibiotics, and carbohydrates. In certain embodiments, the biologically active molecule is <500 Da. In certain embodiments, a functional group is chosen from alkyne, azido (N3), and a chelating agent. As used herein, a “chelating agent” is a molecule that forms multiple chemical bonds with a single metal atom. Examples of chelating agents include, but are not limited to, iminodicarboxylic and polyaminopolycarboxylic reactive groups, diethylenetriaminepentaacetic acid (DTPA), l,4,7,10-tetraazacyclododecane-l,4,7,10- tetraacetic acid (DOTA), tetramethyl heptanedionate (TMHD), 2,4-pentanedione, ethylenediamine-tetraacetic acid disodium salt (EDTA), ethyleneglycol- O, O'- bis(2- aminoeihyl)-M A''W'-lelraacelic acid (EGTA), A-(2-hydroxyethyl)ethylenediamine-A,jV',A'- triacetic acid trisodium salt (HEDTA), nitrilotriacetic acid (NTA), and 1,4,8,11- tetraazacyclotetradecane-A,A',M",A'"-tetraacetic acid (TETA), deferoxamine (DFO), and derivatives thereof.

[0068] An imaging agent emits a signal that a signal-transducing machine can detect. In some cases, an imaging agent can emit a signal spontaneously, such as when the detectable label is a radionuclide. In other cases, the imaging agent emits a signal due to being stimulated by an external field, such as when the imaging agent is a relaxivity metal. Examples of signals include, without limitation, gamma rays, X-rays, visible light, infrared energy, and radio waves. Non- limiting examples of modalities of imaging may include magnetic resonance imaging (MRI), ultrasound (US), computed tomography (CT), Positron EmissionTomography (PET), Single Photon Emission Computed Tomography (SPECT), Optical coherence tomography (OCT), and optical imaging (01, bioluminescence, and fluorescence).

[0069] In an alternative embodiment, the derivative comprises a radiological imaging agent. In certain embodiments, the derivative comprises two imaging agents, for example a carbocyanine dye or derivative thereof and a radioisotope. The radioisotope may be conjugated to the carbocyanine dye or conjugated to the peptide residue, such as Tyr. Many radioisotopes can be detected and are suitable for use herein. Examples of radiological imaging agents include, but are not limited to, antimony-124, antimony-125, arsenic-74, barium-103, barium-140, beryllium-7, bismuth- 206, bismuth-207, cadmium- 109, cadium- 115, calcium-45, cerium-139, cerium-141, cerium-144, cesium-137, chromium-51, gadolinium- 153, gold-195, gold-199, hafnium-175-181, indium-i l l, iridium-192, iron-55, iron-59, krypton-85, lead-210, manganese-54, mercury-197, mercury-203, molybdenum-99, neodymium- 147, neptunium-237, nickel-63, niobium-95, osmium-185, palladium- 103, platinum-195, praseodymium- 143, promethium- 147, protactinium-233, radium- 226, rhenium-186, rubidium-86, ruthenium- 103, ruthenium- 106, scandium-44, scandium-46, selenium-75, silver-110, silver-111, sodium-22, strontium-85, strontium-89, strontium-90, sulfur-35, tantalum-182, technetium-99, tellurium- 125, tellurium- 132, thallium-204, thorium- 228, thorium-232, thallium-170, tin-113, titanium-44, tungsten-185, vanadium-48, vanadium- 49, ytterbium- 169, yttrium- 88, yttrium-90, yttrium-91, zinc-65, and zirconium. In a further alternative embodiment, the radiological imaging agent is selected from the group consisting of technetium-99, indium-il l, strontium-90, iodine-125, thallium-201, fluorine-18, carbon- 11, carbon-13, nitrogen-13, oxygen-15, copper-64, lutetium-177, yttrium-90, and iodine-123, iodine-124, iodine-125, and iodine-131. In certain embodiments, a radioisotope images and treats. It is known in the art that radioisotopes function as both imaging and treatment agents. For example, since iodine-131 has both beta and gamma decay modes and can be used for radiotherapy or imaging. Thus, the derivative may be131I-LS301 orniI-LS838.

[0070] Many other imaging agents are suitable in the derivatives, for example, gadolinium, metalloporphyrin, ferric chloride, ferric ammonium citrate, and ferrioxamine methanesulfonate for magnetic resonance imaging.

[0071] In certain embodiments, the treatment agent in the derivative may be conjugated to the peptide via one or more linkers. In other embodiments having more than one linear peptide or one or more cyclic peptides, the individual peptides may optionally be conjugatedvia one or more linkers. Many linkers are suitable. Typically, the linker imparts flexibility to the derivative. Generally speaking, the chain of atoms defining the linker can and will vary depending upon the embodiment.

[0072] In certain embodiments, the linker comprises one or more amino acids. Amino acid residue linkers are usually at least one residue and can be 50 or more residues. In an embodiment, a linker may be about 1 to about 10 amino acids. In another embodiment, a linker may be about 10 to about 20 amino acids. In still another embodiment, a linker may be about 20 to about 30 amino acids. In another embodiment, a linker may be about 30 to about 40 amino acids. In different embodiments, a linker may be about 40 to about 50 amino acids. In other embodiments, a linker may be more than 50 amino acids. For instance, a linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. In a specific embodiment, a linker is 1 amino acid.

[0073] Any amino acid residue may be used for the linker. Typical amino acid residues used for linking are glycine, serine, alanine, leucine, lysine, glutamic and aspartic acid, or the like. For example, a linker may be (AAS)n, (AAAL)n, (GnS)n. or (G2S)n, wherein A is alanine, S is serine, L is leucine, and G is glycine, and wherein n is an integer from 1-20, or 1-10, or 3-10. Accordingly, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Thus, in certain embodiments, a linker includes, but is not limited to, (AAS)n, (AAAL)n, (GnS)n. or (G2S)n, wherein A is alanine, S is serine, L is leucine, and G is glycine and wherein n is an integer from 1-20, or 1-10, or 3-10. In a specific embodiment, a linker is one glycine.

[0074] In a further embodiment, the linker comprises hydrocarbyl or substituted hydrocarbyl groups. In certain embodiments, the linker is from about 1 to about 50 atoms in length. Alternatively, the linker is about 2 about 30 atoms in length. In an embodiment, the linker is from about 4 to about 20 atoms in length. The linker may comprise a variety of heteroatoms that may be saturated or unsaturated, substituted or unsubstituted, linear or cyclic, or straight or branched. The chain of atoms defining the linker will typically be selected from the group consisting of carbon, oxygen, nitrogen, sulfur, selenium, silicon, and phosphorus. In an alternative embodiment, the chain of atoms is selected from the group consisting of carbon, oxygen, nitrogen, sulfur, and selenium. In an embodiment, the linker comprises substantially carbon and oxygen atoms. In addition, the chain of atoms defining the linker may be substituted or unsubstituted with atoms other than hydrogen, including, but not limited to,hydroxy, keto (=0), or acyl, such as acetyl. Thus, the chain may optionally include one or more ether, thioether, selenoether, amide, or amine linkages between hydrocarbyl or substituted hydrocarbyl regions. Exemplary linkers include ethylene glycol and aminohexanoic acid. More specifically, a linker may be a polyethylene glycol linker. Such a linker may be referred to as a heterobifunctional PEG linker or a homobifunctional PEG linker.

[0075] In certain embodiments, a linker further comprises one or more spacers. Spacers are known in the art. Non-limiting examples of spacers include 2-aminoethoxy-2-ethoxy acetic acid (AEEA) linkers, AEEEA linkers, and AEA linkers. In a specific embodiment, a linker further comprises one or more 2-aminoethoxy-2-ethoxy acetic acid (AEEA) linkers.KITS

[0076] The present disclosure provides a kit comprising a first vial containing a lyophilized product disclosed herein and a second vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate 80, and about 10 mM histidine to be reconstituted with 88- 98.5 wt.% water.

[0077] In certain embodiments, the diluent, once reconstituted, comprises 4 wt.% dextrose, 1 wt.% polysorbate 80, 10 mM histidine, and 95 wt.% water. In certain embodiments, a sufficient quantity of the diluent is added to the lyophilized product to obtain a concentration of dye-peptide conjugate of 2 wt.% of the total composition in the vial.

[0078] In certain embodiments, the pharmaceutical composition is stored frozen or between 2 and 8 °C and mixed with a second vial of a diluent to prepare final pharmaceutical composition for dosing.

[0079] In certain embodiments, the lyophilized product is stored frozen or between 2 and 8 °C and be mixed with a commercially available buffer, such as normal saline, phosphate buffered saline, or the like, to prepare final pharmaceutical composition for dosing.

[0080] In certain embodiments, the kit comprises instructions for administration. Such kits can aid the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and mixed immediately before use. If desired, such packaging of the components can be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil, such as a blisterpack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0081] Ampules or vials may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other suitable containers include bottles fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a hottie having a stopper that a hypodermic injection needle can pierce. Other containers may have two compartments separated by a readily removable membrane that, upon removal, permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0082] In certain embodiments, kits can be supplied with instructional materials. For example, instructions may be printed on paper or another substrate and / or supplied as an electronic -readable medium or video. In addition, detailed instructions for the kit may not be physical; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0083] A control sample or a reference sample, as described herein, can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample previously obtained from a healthy subject, a group of healthy subjects, or a wild-type subject or sample. A control or reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.ADMINISTRATI N

[0084] The actual dosage amount of a compound of the present disclosure or composition comprising a compound of the present disclosure administered to a subject may be determined by physical and physiological factors such as type of animal treated, such as a human subject, age, sex, body weight, the severity of the condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the subject and on the route of administration. A skilled artisan may determine these factors. The practitioner responsible for administration will typically determine the concentration of activeingredient(s) in a composition and the appropriate dose(s) for the individual subject. The individual physician may adjust the dosage in the event of any complication.

[0085] Agents and compositions described herein can be administered according to methods described herein in various means known to the art. The agents and composition can be used therapeutically as exogenous or endogenous materials. Exogenous agents are those produced or manufactured outside the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some device (biological or other) for delivery within or to other organs in the body.

[0086] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0087] Agents and compositions described herein can be administered in various methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled- release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0088] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition like that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled time at a selected site. Polymeric materials include poly anhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0089] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes. Carrier-based systems for molecular or biomolecular agent delivery can provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo,' prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific deliver}' of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve the taste of the product; or improve the shelf life of the product.

[0090] Compositions of the compounds disclosed herein suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be a bolus, electuary, or paste.

[0091] To a large extent, the choice of excipient depends on factors such as the particular mode of administration, the effect of the excipient on the solubility and stability of the active ingredient, and the nature of the dosage form.

[0092] The pharmaceutical compositions provided herein may be provided in unit dosage forms or multiple-dosage forms. Unit-dosage forms, as used herein, refer to physically discrete units suitable for administration to human and animal subjects and packaged individually as is known in the art. Each unit dose contains a predetermined quantity of the active ingredient(s) sufficient to produce the desired therapeutic effect associated with the pharmaceutical carriers or excipients. Unit-dosage forms include ampoules, syringes, and individually packaged tablets and capsules. Unit dosage forms may be administered in fractions or multiples thereof. A multiple-dosage form is a plurality of identical unit dosage forms packaged in a single container to be administered in a segregated unit-dosage form. Examples of multiple-dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons.

[0093] The pharmaceutical compositions provided herein may be administered alone or in combination with one or more other compounds provided herein, one or more other active ingredients. The pharmaceutical compositions herein may be formulated in various dosage forms for oral, parenteral, and topical administration. The pharmaceutical compositions may also be formulated as a modified release dosage form, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmedrelease, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art). The pharmaceutical compositions provided herein may be administered at once or multiple times at time intervals. It is understood that the precise dosage and duration of treatment may vary with the patient’s age, weight, and condition and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro tests or diagnostic test data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the pharmaceutical compositions.DEFINITIONS

[0094] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0095] The terms “comprise,” “have,” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open-ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has,” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0096] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain 1embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” indicates that a value includes the standard deviation of the mean for the device or method employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending on the desired properties obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0097] When ranges of values are disclosed, and the notation “from m ... to n2” or “between m . . . and n 2” or “m-nz” is used, where m and m are the numbers, then unless otherwise specified, this notation is intended to include the numbers themselves and the range between them. This range may be integral or continuous between and including the end values. By way of example, the range “from 2 to 6 carbons” is intended to include two, three, four, five, and six carbons, since carbons are counted in integer units. Compare, by way of example, the range “from 1 to 3 pM (micromolar),” which is intended to include 1 pM, 3 pM, and everything in between to any number of significant figures (e.g., 1.255 pM, 2.1 pM, 2.9999 pM, etc.).

[0098] As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” refers to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met therequired toxicological and manufacturing testing standards or is included in the Inactive Ingredient Guide prepared by the US Food and Drug Administration. “Pharmacologically active” (or “active”) as in a “pharmacologically active” (or “active”) derivative or analog refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. The term “pharmaceutically acceptable salts” includes acid addition salts which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, tosylic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0099] The term “pharmaceutically-acceptable carrier” is art-recognized. It refers to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in compatibility with the subject composition and its components and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as com starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buflfenng agents, such as magnesium hydroxide and aluminum hydroxide; algimc acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in lyophilized products and pharmaceutical compositions.

[0100] In some embodiments, “pharmaceutically acceptable salt” refers to acid addition salts with an inorganic or organic acid. Lists of suitable salts are found in WO 87 / 05297, Johnston e / al., published September 11, 1987; Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418; and J. Pharm. Sci., 66, 2 (1977), eachincorporated herein by reference in its entirety. A reference for the preparation and selection of pharmaceutical salts of the present disclosure is P. H. Stahl & C. G. Wermuth “Handbook of Pharmaceutical Salts,” Verlag Helvetica Chimica Acta, Zurich, 2002, which is incorporated herein by reference in its entirety. The organic or inorganic acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxyhenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1 ,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic, glucoheptonic, 3 -phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid, and the like. In some embodiments, “pharmaceutically acceptable salt” refers to base addition salts with an inorganic or an organic base. Inorganic bases which may be used to prepare salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, manganese, aluminum hydroxides, carbonates, bicarbonates, phosphates, and the like; particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium hydroxides, carbonates, bicarbonates, or phosphates. Organic bases from which may be used to prepare salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0101] As used herein, “treating,” “treatment,” and the like means ameliorating a disease, so as to reduce, ameliorate, or eliminate its cause, its progression, its severity, or one or more of its symptoms, or otherwise beneficially alter the disease in a subject. In certain embodiments, “treating” or “treatment” of a subject at risk refer to reducing the risk of developing a disease, or of disease progression to a worse state. Prevention of a disease may involve complete protection from disease, for example as in the case of prevention of infection with a pathogen, or may involve prevention of disease progression, for example from prediabetes to diabetes. For example, prevention of a disease may not mean complete foreclosure of any effect related to the diseases at any level, but instead may mean prevention of the symptomsof a disease to a clinically significant or detectable level. Prevention of diseases may also mean prevention of the progression of a disease to a later stage of the disease.

[0102] The term “therapeutic agent” is art-recognized and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance acting locally or systemically in a subject. Examples of therapeutic agents, also referred to as “drugs,” are described in well-known literature references such as the Merck Index (14th edition), the Physicians’ Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition). These therapeutic agents include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment.

[0103] As used herein, “formulation” can refer to a lyophilized product or a pharmaceutical composition or a reconstitution diluent. The agents and compositions described herein can be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier to provide the form for proper administration to the subject. As used herein, “lyophilized product” refers to a product that has undergone freeze-drying, known as lyophilization, to remove water and preserve its structure and activity. In these embodiments, the lyophilized product is a dry and stable form of the drug, providing, for example, increased shelf-life and reduced storage costs.

[0104] A “stable” formulation, lyophilized product, or pharmaceutical composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about -80 °C and about 60 °C, for a commercially reasonable time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.“Weight percent” or “wt%” or “% (w / w)” refers to the ratio of the weight specified component in relation to the weight of the total composition, unless specified otherwise.

[0105] “Water for injection” or “WFI” refers to a type of purified water used for pharmaceutical applications, particularly in the preparation of drugs and medical devices. It is a highly purified form of water that meets the standards of the United States Pharmacopeia(USP), Chapter <1231>. WFI is substantially free of any substances that could potentially harm patients. WFI is produced via distillation or reverse osmosis, which removes impurities and other contaminants from the water, resulting in water substantially free of dissolved solids, organic matter, and microorganisms. In certain embodiments, when a diluent or pharmaceutical composition comprises water, the water is water for injection. The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes, which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered with one or more additional agents or other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0106] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce the dosage frequency. Controlled-release preparations can also be used to affect the onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations may initially release an amount of an agent(s) that produces the desired therapeutic effect and gradually and continually release other amounts of the agent to maintain the therapeutic effect over an extended period. To maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of the agent being metabolized or excreted from the body. Various inducers may stimulate the controlled release of an agent, e.g., a change in pH, temperature, enzymes, water, or other physiological conditions or molecules.

[0107] Agents or compositions described herein can also be used with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treating the disease, disorder, or condition.

[0108] “Effective amount” and “therapeutically effective amount” of an agent, compound, drug, composition, or combination is an amount that is nontoxic and effective for producingsome desired therapeutic effect upon administration to a subject or patient (e.g., a human subject or patient). The precise therapeutically effective amount for a subject may depend upon, e.g., the subject’s size and health, the nature and extent of the condition, the therapeutics or combination of therapeutics selected for administration, and other variables known to those of skill in the art. The effective amount for a given situation is determined by routine experimentation and is within the clinician’s judgment.

[0109] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of all examples or exemplary language (e.g., “such as”) provided concerning certain embodiments herein is intended merely to better illuminate the present disclosure and does not limit the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0110] Groupings of alternative elements or embodiments of the present disclosure are not construed as limitations. Each group member can be referred to and claimed individually or combined with other group members or elements found herein. One or more group members can be included or deleted from a group for convenience or patentability reasons. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

[0111] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0112] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.ENUMER TED EMBODIMENTS

[0113] Also disclosed herein are the following embodiments.

[0114] Embodiment 1. A lyophilized product comprising:2-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,2-20 wt.% calcium salt,20-30 wt.% polysorbate,10-60 wt.% of a beta-cyclodextrin,1-10 wt.% sodium acetate, optionally 40-60 wt.% dextrose, and optionally 1-10 wt.% histidine.

[0115] Embodiment 2. The lyophilized product of Embodiment 1, wherein the cypate is chosen from chosen from LS288, LS798, LS276, LS843, Cypate 2, Cypate 3, Cypate 4, Cy5, Cy5.5, Cy7, and Cy9.

[0116] Embodiment 3. The lyophilized product of Embodiment 2, wherein the cypate is Cy5.5.

[0117] Embodiment 4. The lyophilized product of Embodiment 2, wherein the cypate is Cy7.

[0118] Embodiment 5. The lyophilized product of Embodiment 1 , wherein the dye-peptide conjugate is LS301 comprising the structural formula

[0119] Embodiment 6. The lyophilized product of Embodiment 2, wherein the dye-peptide conjugate is LS301 comprising the structural formula

[0120] Embodiment 7. The lyophilized product of any one of Embodiments 1 to 6, comprising about 10 wt.% calcium salt.

[0121] Embodiment 8. The lyophilized product of Embodiment 7, wherein the calcium salt is chosen from calcium gluconate, calcium citrate, calcium phosphate, calcium lactate, calcium lactate gluconate, calcium acetate, and calcium carbonate.

[0122] Embodiment 9. The lyophilized product of Embodiment 8, wherein the calcium salt is calcium gluconate.

[0123] Embodiment 10. The lyophilized product of any one of Embodiments 1 to 9, comprising about 25 wt.% polysorbate.

[0124] Embodiment 11. The lyophilized product of any one of Embodiments 1 to 10, wherein the polysorbate is polysorbate 80.

[0125] Embodiment 12. The lyophilized product of any one of Embodiments 1 to 11 , comprising about 50 wt.% beta-cyclodextrin.

[0126] Embodiment 13. The lyophilized product of any one of Embodiments 1 to 12, wherein the beta-cyclodextrin is hydroxypropyl-beta-cyclodextrin.

[0127] Embodiment 14. The lyophilized product of any one of Embodiments 1 to 13, comprising 5-7 wt.% sodium acetate.

[0128] Embodiment 15. The lyophilized product of any one of Embodiments 1 to 14, wherein the sodium acetate is sodium acetate trihydrate.

[0129] Embodiment 16. The lyophilized product of any one of Embodiments 1 to 15, comprising about 40-60 wt% dextrose.

[0130] Embodiment 17. The lyophilized product of Embodiment 16, comprising about 56 wt% dextrose.

[0131] Embodiment 18. The lyophilized product of any one of Embodiments 1 to 17, comprising about 1-10 wt% histidine.

[0132] Embodiment 19. The lyophilized product of Embodiment 18, comprising about 2 wt% histidine.

[0133] Embodiment 20. The lyophilized product of any one of Embodiments 1 to 19, adjusted to a target pH 3.0 with acetic acid.

[0134] Embodiment 21. A lyophilized product formed by a method comprising: solubilizing a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acidresidue is independently in a D or L configuration in a solution of 1-4 wt.% calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% beta-cyclodextrin, and 0.2-2 wt.% sodium acetate, optionally 0.2-10 wt% dextrose, and optionally 1 to 100 mM histidine in water at a ratio of 1 gram of dye-peptide conjugate per 10 liters of the solution; filtering the solution; and lyophilizing the filtered solution to form the lyophilized product.

[0135] Embodiment 22. The lyophilized product of any one of Embodiments 1 to 21, comprising not more than 5%, not more than 3%, not more than 2%, not more than 1 .5%, not more than 1.3%, or not more than 1.2% of an oxidative dimer of the LS301 or the derivative or the salt thereof having a relative retention time of about 0.88 during reverse-phase high- performance liquid chromatography (HPLC).

[0136] Embodiment 23. A lyophilized product comprising:2-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, and not more than 5% of an oxidative dimer of the LS301 or the derivative or the salt thereof having a relative retention time of about 0.88 during reverse-phase high-performance liquid chromatography (HPLC).

[0137] Embodiment 24. The lyophilized product of Embodiment 23, comprising not more than 3%, not more than 2%, not more than 1.5%, not more than 1.3%, or not more than 1.2% of the oxidative dimer of LS301.

[0138] Embodiment 25. A pharmaceutical composition comprising the lyophilized product of any one of Embodiments 1 to 23 and a diluent, wherein the diluent optionally comprises 0.2-10 wt% dextrose and 1 to 100 mM histidine.

[0139] Embodiment 26. The pharmaceutical composition of Embodiment 25, wherein the lyophilized product comprises:2-5 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,2-5 wt.% calcium salt,20-25 wt.% polysorbate,10-15 wt.% of a 6<? / u-cyclodextrin,1-5 wt.% sodium acetate,40-60 wt.% dextrose, and1-5 wt.% histidine; and the diluent is water for injection.

[0140] Embodiment 27. The pharmaceutical composition of Embodiment 25, wherein the lyophilized product comprises:5-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,5-20 wt.% calcium salt,20-30 wt.% polysorbate,40-60 wt.% of a 6<? / < -cyclodexlrin, and1-10 wt.% sodium acetate; and wherein the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water.

[0141] Embodiment 28. The pharmaceutical composition of Embodiment 27, wherein the diluent comprises 4 wt.% dextrose, 1 wt.% polysorbate, about 10 mM histidine, and 95 wt.% water.

[0142] Embodiment 29. The pharmaceutical composition of any one of Embodiments 25 to 28, wherein a sufficient quantity of the diluent is added to the lyophilized product to obtain a concentration of dye-peptide conjugate of 2 wt.% of the pharmaceutical composition.

[0143] Embodiment 30. The pharmaceutical composition of any one of Embodiments 25 to 28, comprising:1-4 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,1-4 wt.% calcium salt,4-6 wt.% polysorbate,8-12 wt.% ie / a-cyclodextrin,0.2-2 wt.% sodium acetate,0.2-10 wt.% dextrose, about 10 mM histidine, and62-86 wt.% water.

[0144] Embodiment 31. The pharmaceutical composition of Embodiment 30, comprising: about 2 wt.% LS301, about 2 wt.% calcium salt, about 5 wt.% polysorbate, about 10 wt.% feeto-cyclodextrin, about 1.3 wt.% sodium acetate, about 4 wt.% dextrose, about 10 mM histidine, and about 75 wt.% water.

[0145] Embodiment 32. The pharmaceutical composition of any one of Embodiments 25 to 31 having a pH between 5.5 and 6.5.

[0146] Embodiment 33. The pharmaceutical composition of any one of Embodiments 25 to 32, having an osmolality of about 330 mOsm / kg.

[0147] Embodiment 34. A vial containing the lyophilized product of any one of Embodiments 1 to 24.

[0148] Embodiment 35. A vial containing the pharmaceutical composition of any one of Embodiments 25 to 33.

[0149] Embodiment 36. A kit comprising a vial containing the lyophilized product of any one of Embodiments 1 to 24 and a vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine, to be reconstituted with 88-98.5 wt.% water.

[0150] Embodiment 37. A kit comprising a vial containing a solution of the lyophilized product of any one of Embodiments 1 to 24 in water, and a vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine, to be reconstituted with 88-98.5 wt.% water.

[0151] Embodiment 38. The kit of Embodiment 36 or 37, wherein the diluent, once reconstituted, comprises 4 wt.% dextrose, 1 wt.% polysorbate, about 10 mM histidine, and 95 wt.% water.

[0152] Embodiment 39. of a pharmaceutical composition formed in the vial.

[0153] Embodiment 40. A method of preparing a pharmaceutical composition, the method comprising mixing an amount of diluent into a vial comprising the lyophilized product of anyone of Embodiments 1 to 24 sufficient to form a pharmaceutical composition comprising about 2 wt.% dye-peptide conjugate.

[0154] Embodiment 41. The method of Embodiment 40, wherein the diluent comprises 1- 10 wt.% dextrose, 0.5-2 wt.% polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water.EXAMPLES

[0155] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure and thus can constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 - Dye-peptide conjugate LS301

[0156] All the fluorenylmethyloxycarbonyl (Fmoc) amino acids and Fmoc-Lys (Boc)-Wang Resin were purchased from AAPPTec (Louisville, KY, USA). Dichloromethane (DCM), acetic acid, acetic anhydride, thioanisole, phenol, hydroxybenzotriazole (HOBt), N,N- diisopropylethylamine (DIEA), A-trityl-1 ,2-ethanediamine, phenol, thioanisole, dimethylformamide (DMF), A,A'-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), iodine, methyl tert-butyl ether (MTBE), O-(7- azabenzotriazol- l -yl)- / V,Y,Y',Y'- tetramethyluronium hexafluorophosphate (HATU), and dexamethasone (DEX) were purchased from Sigma-Aldrich (St Louis, MO). Hematoxylin and eosin (H&E) stains were purchased from MilliporeSigma (St Louis, MO). Rabbit antipANXA2 (phospho-Tyr24) antibody was purchased from Signalway Antibody (College Park, MD). AlexaFluor 594- conjugated donkey antirabbit antibody was purchased from Thermo Fisher Scientific (Waltham, MA).

[0157] LS301 (cypate-cyclic (DCys-Gly-Arg-Asp-Ser-Pro-Cys)- Lys-OH) was synthesized as previously reported. Briefly, the linear GRD peptide, H-DCys (Acm)-Gly-Arg (Pbf)-Asp (tBu)-Ser (tBu)-Pro-Cys (Acm)-Lys (Boc)-OH, was prepared via a CEM Liberty Blue microwave peptide synthesizer (Matthews, NC, USA) on the Fmoc-Lys (Boc)- wang resin. The resin (0.1 mmol) was swelled in DCM for 1 h before use. Fmoc-amino acids (0.5 mmol,5 eq), coupling reagent (HBTU, 0.5 mmol, 5 eq), and DIEA (1 mmol, 10 eq) were added to the resin. The mixture was reacted for 15 min under microwave irradiation (100W, 90 °C). The resin was washed three times with DME

[0158] Deprotection of Fmoc group was performed by treatment of 20% piperidine / DMF for 5 min under microwave irradiation (100 W, 90 °C). The peptidyl resin was washed. The peptide was cyclized through the disulfide bridge with iodine (1.2 eq) in DMF for 90 min. Subsequently, cypate (3 eq) was conjugated to the cyclic peptide on a solid support in the presence of DTC (5 eq) in DMF to afford the LS301 peptidyl resin. The resin was then treated with a cleavage cocktail of TFA: thioanisole: phenol: water (85:5:5:5, v / v / v / v) for 90 min at room temperature. The cleaved peptide product was concentrated in vacuo before purification by reverse-phase high-performance liquid chromatography (HPLC; Gilson, Middleton, WI, USA). Analytical HPLC determined product purity (>95%). The compound identity was confirmed by electrospray ionization mass spectrometry on a Shimadzu LCMS-2020 Mass Spectrometer (Columbia, MD) with peaks observed at 1470 (M+l) and 735 (M+2 / 2).

[0159] Table 1 shows the specifications for LS301, including test items, test methods, and acceptance criteria. Table 2 shows the results of two batches of LS301 examined under the standards from Table 1. Data from these studies support storage of LS301 at -20°C for at least 24 months.

[0160] Table 1 - Specifications for LS301

[0161] Table 2: Analysis result of a reference standardExample 2 - Lyophilized product (LS30LIT)

[0162] A lyophilized product was formed by solubilizing LS301 from Example 1 in a solution of calcium gluconate, polysorbate 80, hydroxypropyl-Z?eta-cyclodextrin, and sodium acetate in water at a ratio of 1 gram of dye-peptide conjugate per 10 liters of the solution, mixing the solution, filtering the mixed solution, and lyophilizing the filtered solution to form the lyophilized product.

[0163] The amounts of each component in the lyophilized product are shown in Table 3. Table 4 compares the prior lyophilized product LS301-HSA (disclosed in copending US patent application no. 17 / 119,305) and the lyophilized product LS301-IT shown in Table 1. Table 5 shows the lyophilized product release specifications for LS301, including test items, test methods, and acceptance criteria.

[0164] Table 3: Composition of LS301 Lyophilized Product (LS301-IT)API = active pharmaceutical ingredient; JP = Japanese Pharmacopoeia; NF = National Formulary; Ph Eur = European Pharmacopoeia; qs = quantum sufficit (sufficient quantity);USP = United States Pharmacopeia.a% w / w was calculated based on the density value of 1.0099 g / mL at 5 °C.bNot listed in FDA’s Inactive Ingredient Database. Approved as a prescription and over-the- counter (OTC) drug as a sterile injection at 100 mg / mL (10%) and in tablet dosage forms.

[0165] Table 4: Comparison of Lyophilized Products

[0166] API = active pharmaceutical ingredient, HS A = human serum albumin; NA = not applicable; qs = quantum sufficif, WFI = water for injection.

[0167] Table 5: LS301 Release SpecificationEU = endotoxin unit; ID = identification; NMT = not more than; USP = United States Pharmacopeia; UV = ultraviolet.

[0168] From these results, the recommended long-term storage conditions for LS301-IT and LS301-HSA are -20°C to 8 °C in a lyophilized powder form with protection from light. Under these conditions, both LS301 lyophilized products have been shown to be stable. However, many preclinical and nonclinical formulation studies require the reconstituted pharmaceutical compositions to be prepared and maintained in solution for extended periods.

[0169] Under these conditions, the LS301-IT lyophilized product was stable for at least 18 months.

[0170] In certain embodiments, the lyophilized products and pharmaceutical compositions were stored at about 5 °C. The lyophilized products and pharmaceutical compositions may also be stored under a low-oxygen atmosphere or an inert gas, such as nitrogen or argon. Evidence suggests that the primary degradation pathway is an oxidative impurity, as shown in Table 5 in the not more than 5.0% impurities. A major impurity was observed at a relativeretention time (RRT) of 0.88 during high-performance liquid chromatography (HPLC). This impurity was identified as an oxidative dimer of LS301. In certain embodiments, this impurity can be controlled to not more than 3.0%, not more than 2.0%, not more than 1.5%, not more than 1.3%, or not more than 1.2%. Typically, a person of skill in the art would wish to minimize the concentration of impurities and degradation products to avoid unwanted side effects and / or reduced efficacy of the active.Example 3 - Reconstitution Diluent and Storage Stability of Solutions

[0171] While developing LS301-IT, it was observed that the reconstitution using water for injection or saline did not maintain in solution suitable for injection for the desired time of at least four hours. In particular, the LS301-HSA lyophilized product did not solubilize readily when introduced to saline or water for injection but required sonication, which increased the temperature and potentially the impurities after fully reconstituted. The aggregation and solubility issues were apparent visibly.

[0172] Therefore, a reconstitution diluent was developed to achieve a timing of reconstitution of less than two minutes, such as between about 57 seconds and about 29 seconds. The diluent was manufactured by compounding, sterile filtration, and aseptic filling into vials, for example, containing at least 3 mL. The LS301-IT lyophilized product, when reconstituted with the diluent, did not present any issues for the reconstitution to become a dose ready for injection. The LS301-IT preparation (once reconstituted with diluent) meets the acceptance criteria for injections as defined in the USP for particulate matter. The components of the reconstitution diluent are shown below in Table 6.

[0173] Table 6: Composition of Reconstitution DiluentACS = American Chemical Society; ChP = Chinese Phannacopoeia; JP = Japanese Pharmacopoeia; Ph Eur = European Pharmacopoeia; qs = quantum sufficit; USP = United States Pharmacopeia.

[0174] In certain embodiments, the lyophilized product is formulated with the solid ingredient of the diluent so that it can be reconstituted with water for injection, as shown in Table 7.

[0175] Table 7: LS301-IT formulated for reconstitution with water for injection

[0176] After reconstitution in the diluent, serial dilutions for LS301-IT (the lyophilized product from Table 3) and LS301-HSA were prepared from 2000 nmol to about 1 nmol. Replica sets of the LS301-IT and the LS301-HSA dilutions were stored with light protection at -20 °C, 4 °C, and room temperature (20-24 °C). Following reconstitution in diluent and at each time point before spectral analysis, each sample was inspected visually to assess sample homogeneity. Precipitation and flocculation were recorded.

[0177] For the spectroscopic study, the aliquots were also prepared in low-binding tubes before the solutions were transferred to a disposable cuvette (Brandtech Cat#759150) for steady-state measurements. All mixtures were vortexed for 30 seconds before thespectroscopy and kept in the dark except during analysis. The absorption and steady-state fluorescence spectra were recorded on a Beckman DU640 Spectrophotometer and a Horiba Fluorolog-3 spectrofluorometer. All measurements were conducted in triplicate at room temperature on Day 0 (preparation), Day 1, Day 3, and weekly through 6 weeks. Samples were excited at 760 nm to obtain most of the emission peak feature.

[0178] Both of the LS301 lyophilized products were stored at -20 °C and were thawed by placing them at room temperature for 10 minutes. No precipitate was observed in either sample. No noticeable precipitation or flocculation was observed for the LS301-TT dilutions stored at 4 °C or room temperature. The LS301-HSA samples stored at 4 °C and room temperature were stable through Week 1 as no flocculant was observed. From Week 2 through the end of the study (Week 6), some floc appeared in the solution. The floc increased throughout the study. However, after gentle swirling of the sample tubes, the floc redissolved.

[0179] The absorption spectrum of each sample was recorded before each fluorescence measurement. The peak absorbance of all the samples did not noticeably change during the 6- week testing period, indicating that the fluorescence loss of the samples was not from photobleaching but rather aggregation or dimerization.

[0180] The peak intensity of the fluorescence of each sample was recorded, calculated, and converted to a percent change compared to the fluorescence intensity of the freshly prepared sample (Day 0). Table 8 summarizes the cumulated loss in fluorescence signal for each pharmaceutical composition of LS301 at each of the three temperatures.

[0181] Table 8: Stability After Six Weeks of Storage.

[0182] LS301-IT dilutions stored at -20 °C had the best stability as they lost only about 3.5% of the fluorescence signal over 6 weeks. The LS301-IT dilutions stored at 4 °C lost about 4.5% of the fluorescence signal, while the LS301-IT samples stored at RT lost ~8% of the fluorescence signal by Week 6. The resulting solution suitable for injection was stablewhen stored for at least eight hours between 20 and 24 °C and 24 hours when stored between 2 and 8 °C.

[0183] In the same time frame, the LS301-HSA samples stored at -20 °C lost about 8% of the fluorescence over 6 weeks, the 4 °C samples lost about 11.5% of the fluorescence signal, and the room temperature samples lost about 14% of the fluorescence signal after 6 weeks. Thus, LS301-HSA was less stable than LS301-IT under these conditions.

[0184] All concentrations for each composition followed the same pattern of lost fluorescence signal over 6 weeks. Thus, the lost fluorescence was independent of concentration.

[0185] Generally, fresh preparation of LS301 lyophilized product in diluent ensures the highest stability and fluorescence signal. However, based on these results, serial dilutions of either pharmaceutical composition can be prepared with the diluent, stored, and shipped frozen (e.g., -20 °C) with minimal loss of fluorescent signal. Pharmaceutical compositions containing LS301-IT are more stable than those of LS301-HSA when using the diluent for reconstitution, serial dilution, or long-term storage.

[0186] All references, patents or applications, U.S. or foreign, cited in the application are hereby incorporated by reference as if written herein in their entireties. Where any inconsistencies arise, material literally disclosed herein controls.

[0187] From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

CLAIMSWhat is claimed is:

1. A lyophilized product comprising:2-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo cypate-cyclo(Cys-Gly-Arg- Asp-Ser-Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,2-20 wt.% calcium salt,20-30 wt.% polysorbate,10-60 wt.% of a feeta-cyclodextrin,1-10 wt.% sodium acetate, optionally 40-60 wt.% dextrose, and optionally 1-10 wt.% histidine.

2. The lyophilized product of claim 1, wherein the cypate is chosen from chosen from LS288, LS798, LS276, LS843, Cypate 2, Cypate 3, Cypate 4, Cy5, Cy5.5, Cy7, and Cy9.

3. The lyophilized product of claim 2, wherein the cypate is Cy5.5.

4. The lyophilized product of claim 2, wherein the cypate is Cy7.

5. The lyophilized product of claim 1, wherein the dye-peptide conjugate is LS301 comprising the structural formulaSUBSTITUTE SHEET (RULE 26)6. The lyophilized product of claim 2, wherein the dye-peptide conjugate is LS3017. The lyophilized product of any one of claims 1 to 6, comprising about 10 wt.% calcium salt.48SUBSTITUTE SHEET (RULE 26)8. The lyophilized product of claim 7, wherein the calcium salt is chosen from calcium gluconate, calcium citrate, calcium phosphate, calcium lactate, calcium lactate gluconate, calcium acetate, and calcium carbonate.

9. The lyophilized product of claim 8, wherein the calcium salt is calcium gluconate.

10. The lyophilized product of any one of claims 1 to 9, comprising about 25 wt.% polysorbate.

11. The lyophilized product of any one of claims 1 to 10, wherein the polysorbate is polysorbate 80.

12. The lyophilized product of any one of claims 1 to 11, comprising about 50 wt.% beta- cyclodextrin.

13. The lyophilized product of any one of claims 1 to 12, wherein the % / c / -cyclodexlrin is hydroxypropyl-% / ( / -cyclodexlrin.

14. The lyophilized product of any one of claims 1 to 13, comprising 5-7 wt.% sodium acetate.

15. The lyophilized product of any one of claims 1 to 14, wherein the sodium acetate is sodium acetate trihydrate.

16. The lyophilized product of any one of claims 1 to 15, comprising about 40-60 wt% dextrose.

17. The lyophilized product of claim 16, comprising about 56 wt% dextrose.

18. The lyophilized product of any one of claims 1 to 17, comprising about 1-10 wt% histidine.

19. The lyophilized product of claim 18, comprising about 2 wt% histidine.

20. The lyophilized product of any one of claims 1 to 19, adjusted to a target pH 3.0 with acetic acid.

21. A lyophilized product formed by a method comprising: solubilizing a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser- Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration in a solution of 1-4 wt.%49SUBSTITUTE SHEET (RULE 26)calcium salt, 4-6 wt.% polysorbate, 8-12 wt.% Z?eta-cyclodextrin, and 0.2-4 wt.% sodium acetate, optionally 0.2-10 wt% dextrose, and optionally 1 to 100 mM histidine in water at a ratio of 1 gram of dye-peptide conjugate per 10 liters of the solution; filtering the solution; and lyophilizing the filtered solution to form the lyophilized product.

22. The lyophilized product of any one of claims 1 to 21, comprising not more than 5%, not more than 3%, not more than 2%, not more than 1.5%, not more than 1.3%, or not more than 1.2% of an oxidative dimer of the LS301 or the derivative or the salt thereof having a relative retention time of about 0.88 during reverse-phase high-performance liquid chromatography (HPLC).

23. A lyophilized product comprising:2-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser- Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration, and not more than 5% of an oxidative dimer of the LS301 or the derivative or the salt thereof having a relative retention time of about 0.88 during reverse-phase high-performance liquid chromatography (HPLC).

24. The lyophilized product of claim 23, comprising not more than 3%, not more than 2%, not more than 1.5%, not more than 1.3%, or not more than 1.2% of the oxidative dimer of LS301.

25. A pharmaceutical composition comprising the lyophilized product of any one of claims 1 to 23 and a diluent, wherein the diluent optionally comprises 0.2-10 wt% dextrose and 1 to 100 mM histidine.

26. The pharmaceutical composition of claim 25, wherein the lyophilized product comprises:2-5 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser- Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,2-5 wt.% calcium salt, 20-25 wt.% polysorbate,50SUBSTITUTE SHEET (RULE 26)10-15 wt.% of a feeto-cyclodextrin,1-5 wt.% sodium acetate,40-60 wt.% dextrose, and1-5 wt.% histidine; and the diluent is water for injection.

27. The pharmaceutical composition of claim 25, wherein the lyophilized product comprises:5-20 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser- Pro-Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,5-20 wt.% calcium salt,20-30 wt.% polysorbate,40-60 wt.% of a heta-cyclodextrin, and1-10 wt.% sodium acetate; and wherein the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water.

28. The pharmaceutical composition of claim 27, wherein the diluent comprises 4 wt.% dextrose, 1 wt.% polysorbate, about 10 mM histidine, and 95 wt.% water.

29. The pharmaceutical composition of any one of claims 25 to 28, wherein a sufficient quantity of the diluent is added to the lyophilized product to obtain a concentration of dye-peptide conjugate of 2 wt.% of the pharmaceutical composition.

30. The pharmaceutical composition of any one of claims 25 to 28, comprising:1-4 wt.% a dye-peptide conjugate chosen from cypate-cyclo(Cys-Gly-Arg-Asp-Ser-Pro- Cys)-Lys-OH (LS301), a derivative thereof, or a salt thereof, wherein each amino acid residue is independently in a D or L configuration,1-4 wt.% calcium salt,4-6 wt.% polysorbate,8-12 wt.% hc / cz-cyclodexlrin,0.2-2 wt.% sodium acetate,0.2-10 wt.% dextrose,51SUBSTITUTE SHEET (RULE 26)about 10 mM histidine, and62-86 wt.% water.

31. The pharmaceutical composition of claim 30, comprising: about 2 wt.% LS301, about 2 wt.% calcium salt, about 5 wt.% polysorbate, about 10 wt.% feeto-cyclodextrin, about 1.3 wt.% sodium acetate, about 4 wt.% dextrose, about 10 mM histidine, and about 75 wt.% water.

32. The pharmaceutical composition of any one of claims 25 to 31 having a pH between 5.5 and 6.5.

33. The pharmaceutical composition of any one of claims 25 to 32, having an osmolality of about 330 mOsm / kg.

34. A vial containing the lyophilized product of any one of claims 1 to 24.

35. A vial containing the pharmaceutical composition of any one of claims 25 to 33.

36. A kit comprising a vial containing the lyophilized product of any one of claims 1 to 24 and a vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine, to be reconstituted with 88-98.5 wt.% water.

37. A kit comprising a vial containing a solution of the lyophilized product of any one of claims 1 to 24 in water, and a vial containing a diluent comprising 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, and 1-100 mM histidine, to be reconstituted with 88-98.5 wt.% water.

38. The kit of claim 36 or 37, wherein the diluent, once reconstituted, comprises 4 wt.% dextrose, 1 wt.% polysorbate, about 10 mM histidine, and 95 wt.% water.

39. The kit of any of claims 36 to 38, wherein a sufficient quantity of the diluent is added to the lyophilized product to obtain a concentration of dye-peptide conjugate of 2 wt.% of a pharmaceutical composition formed in the vial.52SUBSTITUTE SHEET (RULE 26)40. A method of preparing a pharmaceutical composition, the method comprising mixing an amount of diluent into a vial comprising the lyophilized product of any one of claims 1 to 24 sufficient to form a pharmaceutical composition comprising about 2 wt.% dye-peptide conjugate.

41. The method of claim 40, wherein the diluent comprises 1-10 wt.% dextrose, 0.5-2 wt.% polysorbate, 1-100 mM histidine, and 88-98.5 wt.% water.53SUBSTITUTE SHEET (RULE 26)

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