Formulations of manp and uses thereof

Abstract

The present disclosure provides for and includes pharmaceutical formulations comprising MANP peptides, methods of making such formulations, and uses of such formulations in the treatment of diseases and conditions for which the peptides contained in such formulations are suitable. The present disclosure further provides for and includes methods for increasing the stability of peptide formulations and for reducing aggregation during the production of peptide formulations.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 580,623, filed September 5, 2023, and U.S. Provisional Application No. 63 / 588,186, filed October 5, 2023, both of which are incorporated herein by reference in their entirety.

[0003] Merging of sequence lists

[0004] The sequence list, which was created on September 3, 2024 and contained in a file named “P35383WO00_SL.TXT” (1,996 bytes (measured in MS-Windows®)), is hereby submitted electronically and incorporated in its entirety by reference. Background Technology

[0005] Natriuretic peptides are peptides that can induce urinary sodium excretion—increasing sodium excretion in urine. Natriuretic peptides can be produced by brain, heart, kidney, and / or vascular tissues. The human natriuretic peptide family includes the cardiac hormones atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urinary dilatantin (URO). Natriuretic peptides function via two well-characterized guanylate cyclase receptors (NPR-A for ANP, BNP, and URO; NPR-B for CNP) and the second messenger cyclic guanosine monophosphate (cGMP) (Kuhn (2003) Circ. Res. 93:700-709; Tawaragai et al. (1991) Biochem. Biophys. Res. Commun. 175:645-651; and Komatsu et al. (1991) Endocrinol. 129:1104-1106). Numerous studies have confirmed that ANP and BNP exert therapeutic-related biological effects via GC-A and cGMP, such as urinary sodium excretion, vasodilation, inhibition of the renin-angiotensin-aldosterone system (RAAS), inhibition of cardiomyocyte hypertrophy and apoptosis, stimulation of angiogenesis, and inhibition of organ fibrosis.

[0006] Compared to ANP, mutant (or modified) atrial natriuretic peptide (MANP) is more effective in lowering blood pressure (BP), inducing urinary sodium excretion, and inhibiting aldosterone via pGC-A and its second messenger cGMP. International Patent Application No. PCT / US2017 / 060808 generally discloses MANP analogs exhibiting pGC-A functional gains and applicable to the treatment of cardiovascular, renal, and metabolic diseases. International Patent Application No. PCT / US2019 / 060401 generally discloses materials and methods for treating hypertension (including refractory hypertension) in combination with M-atrial natriuretic peptide (MANP) and a diuretic (e.g., furosemide). There is a need for MANP compositions and formulations that are stable in storage, transportation, and administration to patients. Summary of the Invention

[0007] The subject matter of this disclosure is based in part on the surprising finding that certain pharmaceutically acceptable MANP formulations containing sucrose, polysorbate-20, and acetate are stable in terms of long-term storage and transportation.

[0008] In one aspect, this disclosure provides a composition comprising MANP, acetate, sucrose, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is substantially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 10 mM. In one aspect, the concentration of sucrose is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of sucrose is about 275 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O.

[0009] In one aspect, this disclosure provides a composition comprising MANP, acetate, mannitol, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is substantially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 40 mM. The concentration of mannitol is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of mannitol is about 250 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O.

[0010] In one aspect, this disclosure provides a composition for treating hypertension, comprising MANP, acetate, sucrose, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is substantially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 10 mM. In one aspect, the concentration of sucrose is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of sucrose is about 275 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O.

[0011] In one aspect, this disclosure provides a composition for treating hypertension, comprising MANP, acetate, mannitol, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is substantially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 40 mM. In one aspect, the concentration of mannitol is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of mannitol is about 250 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O.

[0012] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20.

[0013] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 40 mM acetate, about 250 mM mannitol, and about 0.02% polysorbate 20.

[0014] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein less than 0.5% of the monomer aggregates after storage at 2-8°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0015] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 1% after storage at 5°C for 6 months, as measured by the height of the main peak obtained via reversed-phase HPLC. In another aspect, the composition experiences a purity loss of less than 0.5% after storage at 5°C for 6 months, as measured by the height of the main peak obtained via reversed-phase HPLC. In yet another aspect, the composition experiences a purity loss of less than 0.2% after storage at 5°C for 6 months, as measured by the height of the main peak obtained via reversed-phase HPLC.

[0016] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 10% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC. In another aspect, the composition experiences a purity loss of less than 5% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC. In yet another aspect, the composition experiences a purity loss of less than 3% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC.

[0017] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 10% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC. In another aspect, the composition experiences a purity loss of less than 5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC.

[0018] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition exhibits a purity loss of less than 0.2% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In another aspect, the composition exhibits a purity loss of less than 0.18% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography.

[0019] In one aspect, this disclosure provides a pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 0.5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In another aspect, the composition experiences a purity loss of less than 0.35% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography.

[0020] In one aspect, this disclosure provides a composition comprising MAMP, a buffer, a stabilizer / strainer, and a nonionic surfactant. In one aspect, the buffer is selected from the group consisting substantially of: acetate, acetic acid, alanine, arginine, aspartic acid, bicarbonate, N,N-bis(2-hydroxyethyl)glycine (bicine), carbonate, citrate, citric acid, glycine, glycylglycine, glutamic acid, histidine, lysine, malic acid, potassium phosphate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, sodium succinate, succinic acid, sulfate, nitrate, maleic acid, fumaric acid, tartaric acid, aspartic acid, N-tris(hydroxymethyl)methylglycine (tricine), tris(hydroxymethyl)aminomethane, and tromethamine. In one aspect, the buffer is an acetate. In one aspect, the concentration of the acetate is in the range of about 10 mM to 40 mM. In one aspect, the concentration of the acetate is about 10 mM. In one aspect, the concentration of the acetate is about 40 mM. In one aspect, the stabilizer / tension agent is selected from the group consisting essentially of: albumin, arginine, Brij 30, Brij 35, glucose, dimethyl sulfone, ethylenediaminetetraacetic acid, glycerol, glycerol, glycine, guanine, hydroxypropyl-β-cyclodextrin, lactose monohydrate, magnesium chloride, maltose, mannitol, methionine, 2-methylthioethanol, monothioglycerol, inositol, potassium chloride, poloxamer, polyethylene glycol, polysorbate 20, polysorbate 80, polyvinyl alcohol, polyvinylpyrrolidone, propylene glycol, protamine sulfate, sodium chloride, sorbitol, sucrose, thioglycolic acid, trehalose, and Triton. In one aspect, the stabilizer / tension agent is sucrose. In one aspect, the concentration of sucrose is approximately 275 mM. In one aspect, the stabilizer / tension agent is mannitol. In one aspect, the concentration of mannitol is approximately 250 mM. On one hand, the nonionic surfactant is selected from the group consisting essentially of: sorbitol polyoxyglyceride, polysorbate 20, polysorbate 40, sodium docusate, polysorbate 60, polysorbate 80, benzalkonium chloride, capryloyl hexanoyl polyoxyglyceride, hexadecyl pyridine chloride, lauroyl polyoxyglyceride, linoleyl polyoxyglyceride, caprylyl polyol 9, oleoyl polyoxyglyceride, poloxamer, polyoxyethylene 10 oleyl ether, polyoxyethylene 15 hydroxystearate. The following substances are present: nonyl alcohol ether 9, polyoxyethylene 20 cetearyl alcohol ether, polyoxyethylene 40 stearate, pullulan, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, sodium lauryl sulfate, sorbitol monolaurate, sorbitol monooleate, polyoxyethylene stearate, sorbitol monopalmitate, sorbitol monostearate, stearoyl polyoxyglyceride, sorbitol sesquioleate, sorbitol trioleate, and tyrosaliplatin. In one aspect, the nonionic surfactant is polysorbate 20. In another aspect, the concentration of polysorbate 20 is about 0.2%. In another aspect, the pH is about 5.5.On one hand, the osmotic pressure is approximately 300-420 mOsm / kgH2O. On the other hand, the osmotic pressure is approximately 310-390 mOsm / kgH2O.

[0021] In one aspect, this disclosure provides a vial containing a formulation comprising MANP, acetate, sucrose, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is substantially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 10 mM. In one aspect, the concentration of sucrose is about 275 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O.

[0022] In one aspect, this disclosure provides a lyophilized powder prepared according to the following steps: (a) combining MANP, acetate, sucrose, and polysorbate 20 in a liquid solution; and (b) lyophilizing the combination of step (a). In one aspect, the concentration of MANP in the liquid composition is about 2 mg / ml. In one aspect, MANP consists essentially of SEQ ID NO: 1. In one aspect, the concentration of acetate in the liquid composition is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate in the liquid composition is about 10 mM. In one aspect, the concentration of sucrose in the liquid composition is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of sucrose in the liquid composition is about 275 mM. In one aspect, the concentration of polysorbate 20 in the liquid composition is about 0.02%. In one aspect, the pH of the liquid composition is about 5.5. In one aspect, the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O. On the one hand, the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

[0023] In one aspect, this disclosure provides a lyophilized powder prepared according to the following steps: (a) combining MANP, acetate, mannitol, and polysorbate 20 in a liquid solution; and (b) lyophilizing the combination of step (a). In one aspect, the concentration of MANP in the liquid composition is about 2 mg / ml. In one aspect, MANP is essentially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate in the liquid composition is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate in the liquid composition is about 40 mM. In one aspect, the concentration of mannitol in the liquid composition is in the range of about 250 mM to about 275 mM. In one aspect, the concentration of mannitol in the liquid composition is about 250 mM. In one aspect, the concentration of polysorbate 20 in the liquid composition is about 0.02%. In one aspect, the pH of the liquid composition is about 5.5. In one aspect, the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O. On the one hand, the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

[0024] In one aspect, this disclosure provides a dry powder composition comprising MANP, acetate, sucrose, and polysorbate 20.

[0025] In one aspect, this disclosure provides a powder prepared by spray drying, wherein spray drying includes the following steps: (a) providing a liquid comprising MANP, acetate, sucrose and polysorbate 20; and (b) spray drying the liquid of step (a) using a spray drying apparatus.

[0026] In one aspect, this disclosure provides a freeze-dried powder prepared by freeze-drying, wherein freeze-drying includes the following steps: (a) providing a liquid comprising MANP, acetate, sucrose and polysorbate 20; and (b) freeze-drying the liquid of step (a) at a certain temperature for a sufficient time to convert the liquid composition into a solid.

[0027] In one aspect, this disclosure provides a lyophilized powder prepared by a method comprising the following steps: (a) providing a liquid comprising MANP, acetate, sucrose and polysorbate 20; and (b) lyophilizing the liquid of step (a).

[0028] In one aspect, this disclosure provides a pre-filled syringe containing MANP, acetate, sucrose, and polysorbate 20. In one aspect, the concentration of MANP is about 2 mg / ml. In one aspect, MANP is essentially composed of SEQ ID NO: 1. In one aspect, the concentration of acetate is in the range of about 10 mM to about 40 mM. In one aspect, the concentration of acetate is about 10 mM. In one aspect, the concentration of sucrose is about 275 mM. In one aspect, the concentration of polysorbate 20 is about 0.02%. In one aspect, the pH is about 5.5. In one aspect, the osmotic pressure is about 300-420 mOsm / kgH2O. In one aspect, the osmotic pressure is about 310-390 mOsm / kgH2O. Attached Figure Description

[0029] This document describes aspects of the present disclosure by way of example only, with reference to the accompanying drawings. Referring now to the drawings in detail, it should be emphasized that the details shown are by way of example and are used for illustrative discussion of aspects of the present disclosure. In this respect, the description and drawings, considered individually and together, will make it apparent to those skilled in the art how aspects of the present disclosure can be practiced.

[0030] Figure 1 An exemplary reversed-phase HPLC chromatogram is depicted.

[0031] Figure 2 The peptide concentrations measured for each formulation evaluated in Study 1 are depicted.

[0032] Figure 3 The pH values ​​measured for each formulation evaluated in Study 1 are depicted.

[0033] Figure 4 An exemplary C8 reversed-phase HPLC chromatogram of MANP is depicted.

[0034] Figure 5 The relative areas of the main peaks from the selected samples of Study 1, as measured by C8 RP-HPLC, are depicted.

[0035] Figure 6 An exemplary size exclusion chromatography (SEC) chromatogram of MANP is depicted.

[0036] Figure 7 The relative areas of the leading peaks from the sample in Study 1, as measured by SEC, are depicted.

[0037] Figure 8 The relative areas of the main peaks (monomers) from the sample in Study 1, as measured by SEC, are depicted.

[0038] Figure 9The relative areas of the back peaks from the sample in Study 1, as measured by SEC, are depicted.

[0039] Figure 10 The peptide concentrations measured for each formulation evaluated in Study 2 are depicted.

[0040] Figure 11 The differences in peptide concentrations for each formulation evaluated in Study 2 compared to T=0 are depicted.

[0041] Figure 12 The pH values ​​of each formulation evaluated in Study 2 are depicted compared to T=0.

[0042] Figure 13 An exemplary reversed-phase HPLC chromatogram from Study 2 using the primary reversed-phase method is depicted, showing the peak numbering scheme.

[0043] Figure 14A The relative areas of the leading peak 3 from the sample of Study 2, as measured by RP-HPLC, are depicted.

[0044] Figure 14B The relative areas of the leading peak 5 from the sample of Study 2, as measured by RP-HPLC, are depicted.

[0045] Figure 14C The relative areas of the main peaks from the sample in Study 2, as measured by RP-HPLC, are depicted.

[0046] Figure 15 The changes in the relative area of ​​the main peak in RP-HPLC for each formulation evaluated in Study 2 were depicted compared to T=0.

[0047] Figure 16 The estimated loss of the RP-HPLC main peak extrapolated to 12 months when the Study 2 sample was stored at 5 °C was depicted.

[0048] Figure 17A The relative areas of the back peak 1 from the sample of Study 2, as measured by RP-HPLC, are depicted.

[0049] Figure 17B The relative areas of the back peak 3 from the sample of Study 2, as measured by RP-HPLC, are depicted.

[0050] Figure 17C The relative areas of the back peak 5 from the sample of Study 2, as measured by RP-HPLC, are depicted.

[0051] Figure 18 The change in the relative area of ​​peak 1 after RP-HPLC for each formulation evaluated in Study 2 was depicted compared to T=0.

[0052] Figure 19A The relative area of ​​the front peak 1 from the sample of Study 2, as measured by SEC, is depicted.

[0053] Figure 19B The relative areas of the main peaks from the sample in Study 2, as measured by SEC, are depicted.

[0054] Figure 19C The relative areas of the back peak 1 from the sample of Study 2, as measured by SEC, are depicted.

[0055] Figure 20A The relative areas of the back peak 2 from the sample of Study 2, as measured by SEC, are depicted.

[0056] Figure 20B The relative areas of the back peak 3 from the sample of Study 2, as measured by SEC, are depicted.

[0057] Figure 21 The relative area variation of the SEC main peak (monomer) from the sample in Study 2 is depicted.

[0058] Figure 22 The pH values ​​measured for each formulation evaluated in Study 3 are depicted.

[0059] Figure 23 The measured peptide concentrations for each formulation evaluated in Study 3 are depicted.

[0060] Figure 24 The relative areas of the main peaks from the sample in Study 3, as measured by RP-HPLC with UV detection, are depicted.

[0061] Figure 25 The differences in the relative area of ​​the main peak for each formulation evaluated in Study 3 (at 5 °C, between T=0 and T=4 weeks) are depicted by RP-HPLC with UV detection.

[0062] Figure 26 The extrapolation loss of the relative area of ​​the main peak for each formulation evaluated in Study 3, measured by RP-HPLC with UV detection, is depicted (at 5 °C, between T=0 and T=4 weeks).

[0063] Figure 27 The relative areas of the main peaks from the sample in Study 3, as measured by RP-HPLC with fluorescence detection, are depicted.

[0064] Figure 28 The differences in the relative area of ​​the main peak for each formulation evaluated in Study 3 (at 5°C, between T=0 and T=4 weeks) are depicted by RP-HPLC with fluorescence detection.

[0065] Figure 29The extrapolation loss of the main peak relative area for each formulation evaluated in Study 3, measured by RP-HPLC with fluorescence detection, is depicted (between T=0 and T=4 weeks at 5 °C).

[0066] Figure 30 The relative areas of the main peaks from Study 3 at 5°C for T=0 and T=4 weeks, as measured by SEC, are depicted.

[0067] Figure 31 The study compared the predicted and measured values ​​of the PLS model using the RP-HPLC main peak purity of the samples from Studies 2 and 3 after 4 weeks at 5°C as the endpoint.

[0068] Figure 32 The regression coefficients of the PLS model were plotted using the purity of the RP-HPLC main peak after 4 weeks at 5°C for the samples from Studies 2 and 3 as the endpoint.

[0069] Figure 33 The effects of pH and peptide concentration on a PLS model were depicted, with the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint, based on samples from Studies 2 and 3.

[0070] Figure 34 The effects of pH and acetate on a PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. The peptide concentration was fixed at 2 mg / ml.

[0071] Figure 35 The effects of pH and histidine on a PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. The peptide concentration was fixed at 2 mg / ml.

[0072] Figure 36 The effects of pH and succinate on a PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. The peptide concentration was fixed at 2 mg / ml.

[0073] Figure 37 The effects of glycine and HP-b-CD on the main peak purity of the RP-HPLC sample after 4 weeks at 5°C were depicted using a PLS model with the endpoint of the peptide concentration. The peptide concentration was fixed at 2 mg / ml and the pH was fixed at 5.0.

[0074] Figure 38 The effects of mannitol and sucrose on the PLS model were depicted based on the purity of the main peak in RP-HPLC after 4 weeks at 5°C, using Study 2 samples as the endpoint. Peptide concentration was fixed at 2 mg / ml, and pH was fixed at 5.0.

[0075] Figure 39 The effects of ArgHCl and sucrose on the main peak purity of the PLS model after 4 weeks at 5°C were depicted based on RP-HPLC using Study 2 samples as the endpoint. The peptide concentration was fixed at 2 mg / ml, and the pH was fixed at 5.0.

[0076] Figure 40 The predicted and measured values ​​of the second PLS model were compared using the RP-HPLC main peak purity of the samples from Studies 2 and 3 after 4 weeks at 5°C as the endpoint.

[0077] Figure 41 The regression coefficients of the second PLS model were plotted using the RP-HPLC main peak purity of the samples from Studies 2 and 3 after 4 weeks at 5°C as the endpoint.

[0078] Figure 42 The effects of pH and peptide concentration on a second PLS model were depicted, with the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint, based on samples from Studies 2 and 3.

[0079] Figure 43 The effects of pH and acetate on the second PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. The peptide concentration was fixed at 2 mg / ml.

[0080] Figure 44 The effects of pH and histidine on the second PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. The peptide concentration was fixed at 2 mg / ml.

[0081] Figure 45 The effects of glycine and HP-b-CD on the main peak purity of the RP-HPLC sample after 4 weeks at 5°C were depicted using a second PLS model, with the peptide concentration fixed at 2 mg / ml and pH fixed at 6.0.

[0082] Figure 46 The effects of mannitol and sucrose on the main peak purity of the RP-HPLC sample after 4 weeks at 5°C were depicted using a second PLS model with this as the endpoint. Peptide concentration was fixed at 2 mg / ml, and pH was fixed at 6.0.

[0083] Figure 47 The effects of pH and acetate on a second PLS model were depicted, using the purity of the RP-HPLC main peak after 4 weeks at 5°C as the endpoint for samples from Studies 2 and 3. Peptide concentrations were fixed at 2 mg / ml, and sucrose concentrations were fixed at 250 mM.

[0084] Figure 48 The pH values ​​of the four samples in the study were described with and without stirring.

[0085] Figure 49 The pH values ​​of the four samples in the study were described under both freeze-thaw stress and no freeze-thaw stress.

[0086] Figure 50 The peptide concentrations of the four samples in the study were depicted with and without stirring.

[0087] Figure 51 The peptide concentrations of the four samples in the study were depicted under both freeze-thaw stress and no freeze-thaw stress.

[0088] Figure 52 The purity of the main peak of the four samples in Study 4, as measured by RP-HPLC, was described under both stirred and unstirred conditions.

[0089] Figure 53 The purity of the main peak of the four samples under freeze-thaw stress and without freeze-thaw stress was described, as measured by RP-HPLC.

[0090] Figure 54 The changes in the purity of the main peak of Study 4 samples under freeze-thaw stress and stirring stress (T=0 – after stress) were depicted as measured by RP-HPLC.

[0091] Figure 55 The monomer content of Study 4 samples under both stirring and non-stirring stresses was described, as measured by SEC.

[0092] Figure 56 The monomer content of Study 4 samples under freeze-thaw stress and without freeze-thaw stress, as measured by SEC, is depicted.

[0093] Figure 57 The changes in the main peak (monomer) of the Study 4 samples after experiencing stirring stress and freeze-thaw stress (T=0 – after stress) are depicted as measured by SEC.

[0094] Figure 58 The peptide concentrations of the five samples studied were depicted.

[0095] Figure 59 The pH values ​​of the five samples in the study were described.

[0096] Figure 60 An exemplary RP-HPLC chromatogram with labeled peaks is depicted.

[0097] Figure 61 The relative areas of the peaks (preliminary peak 3) representing methionine oxide species in Study 5 samples at different time points, as measured by RP-HPLC, are depicted.

[0098] Figure 62 The relative areas of the peaks (front peak 3) representing methionine oxide species in Study 5 samples at different free methionine concentrations, as measured by RP-HPLC, are depicted.

[0099] Figure 63 The relative area changes of the main peaks in Study 5 samples (without Met – with XmM Met) were depicted as measured by RP-HPLC.

[0100] Figure 64 The relative area of ​​the main peak, as measured by RP-HPLC, was plotted as a function of methionine concentration.

[0101] Figure 65 The relative areas of the leading peaks of the five samples in Study 5 at different time points, as measured by SEC, are depicted.

[0102] Figure 66 The relative areas of the main peaks of the five samples in the study at different time points are depicted, as measured by SEC. Figure 65 The relative areas of the leading peaks of the five samples in Study 5 at different time points, as measured by SEC, are depicted.

[0103] Figure 67 The relative areas of the back peaks of the five samples in Study 5 at different time points, as measured by SEC, are depicted.

[0104] Figure 68 The relative areas of the front peaks of the five samples from Study 5 at different methionine concentrations, as measured by SEC, are depicted.

[0105] Figure 69 The relative areas of the main peaks of the five samples in the study at different methionine concentrations, as measured by SEC, are depicted.

[0106] Figure 70 The relative areas of the peaks in the five samples from Study 5 at different methionine concentrations, as measured by SEC, are depicted.

[0107] Figure 71 The pH values ​​measured for validation samples (inverted and non-inverted) stored at 5°C are depicted.

[0108] Figure 72 The pH values ​​measured for validation samples stored at 25°C and 40°C are depicted.

[0109] Figure 73 The peptide concentrations measured in validation samples (inverted and non-inverted) stored at 5°C are depicted.

[0110] Figure 74 The peptide concentrations measured in validation samples stored at 25°C and 40°C are depicted.

[0111] Figure 75 The monthly peptide concentration loss of validation samples stored at 5°C, 25°C, and 40°C was depicted.

[0112] Figure 76 An exemplary RP-HPLC chromatogram with suggested peak identification is depicted.

[0113] Figure 77 The relative areas of the main peaks in a validation sample (non-inverted) stored at 5 °C as measured by RP-HPLC over time are depicted.

[0114] Figure 78 The relative areas of the main peaks in a validation sample (inverted) stored at 5°C, as measured by RP-HPLC, are depicted over time.

[0115] Figure 79 The relative areas of the main peaks in the validation sample (non-inverted) stored at 25°C as measured by RP-HPLC over time are depicted.

[0116] Figure 80 The relative areas of the main peaks in a validation sample (non-inverted) stored at 40 °C as measured by RP-HPLC over time are depicted.

[0117] Figure 81 The monthly variation of the relative area of ​​the main peak of the verification sample at different temperatures, as measured by RP-HPLC, is depicted.

[0118] Figure 82 The monthly predicted variation of the relative area of ​​the main peak in a validation sample (non-inverted) stored at 5 °C, as measured by RP-HPLC, is depicted.

[0119] Figure 83 The monthly predicted variation of the relative area of ​​the main peak in a validation sample (inverted) stored at 5 °C, as measured by RP-HPLC, is depicted.

[0120] Figure 84 The monthly predicted variation of the relative area of ​​the main peak in validation samples stored at 25°C, as measured by RP-HPLC, is depicted.

[0121] Figure 85 The monthly predicted changes in the relative area of ​​the main peak of the validation sample stored at 40 °C, as measured by RP-HPLC, are depicted.

[0122] Figure 86 The relative areas of the main peaks over time are depicted for validation samples (non-inverted) stored at 5°C, as measured by SEC.

[0123] Figure 87 The relative areas of the main peaks over time are depicted for a validation sample (inverted) stored at 5°C, as measured by SEC.

[0124] Figure 88 The relative areas of the main peaks over time are depicted for validation samples (non-inverted) stored at 25°C, as measured by SEC.

[0125] Figure 89 The relative areas of the main peaks over time are depicted for validation samples (non-inverted) stored at 40°C, as measured by SEC.

[0126] Figure 90 The monthly variation of the relative area of ​​the main peak of the verification sample at different temperatures, as measured by SEC, is depicted.

[0127] Figure 91 The monthly predicted variation of the relative area of ​​the main peak in a validation sample (non-inverted) stored at 5°C, as measured by SEC, is depicted.

[0128] Figure 92 The monthly predicted variation of the relative area of ​​the main peak of a validation sample (inverted) stored at 5°C, as measured by SEC, is depicted.

[0129] Figure 93 The monthly predicted variation of the relative area of ​​the main peak of the validation sample stored at 25°C, as measured by SEC, is depicted.

[0130] Figure 94 The monthly predicted variation of the relative area of ​​the main peak of the validation sample stored at 40°C, as measured by SEC, is depicted. Detailed Implementation

[0131] This disclosure describes and includes pharmaceutically acceptable formulations of MANP or its pharmaceutically acceptable salts, which contain sucrose, polysorbate-20, and acetate.

[0132] This description is not intended to be a detailed list of all different ways in which this disclosure may be implemented or all features that may be added to this disclosure. For example, a feature described with respect to one embodiment may be incorporated into other embodiments, and a feature shown with respect to a particular embodiment may be removed from that embodiment. Therefore, this disclosure contemplates that in some embodiments of this disclosure, any feature or combination of features set forth herein may be excluded or omitted. Furthermore, many variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from this disclosure. In other instances, well-known structures, interfaces, and processes have not been shown in detail so as not to unnecessarily obscure this disclosure. Nothing in this specification is intended to be construed as denying any part of the full scope of this disclosure. Therefore, the following description is intended to illustrate some specific embodiments of this disclosure and not to exhaustively specify all permutations, combinations, or variations thereof.

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used herein in describing this disclosure is for the purpose of describing particular aspects or embodiments only and is not intended to be limiting of this disclosure.

[0134] All publications, patent applications, patents, and other references cited herein are incorporated in their entirety for the purpose of teaching in relation to the sentences and / or paragraphs in which they are referenced. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions for equivalent techniques that would be obvious to a person skilled in the art.

[0135] Unless the context otherwise indicates, it is expressly intended that the various features of this disclosure described herein may be used in any combination. Furthermore, this disclosure also contemplates that in some embodiments of this disclosure, any features or combinations of features set forth herein may be excluded or omitted.

[0136] The methods disclosed herein include and encompass one or more steps or actions for implementing the described methods. Method steps and / or actions may be interchanged without departing from the scope of this disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiments, the order and / or use of specific steps and / or actions may be modified without departing from the scope of this disclosure.

[0137] As used in the specification and appended claims of this disclosure, unless the context clearly indicates otherwise, the singular forms “a”, “an” and “the” are intended to include the plural forms as well.

[0138] As used herein, “and / or” means and covers any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”) context.

[0139] As used herein, the terms “about” or “approximately” when referring to measurable values ​​(such as length, frequency, measurement, etc.) are intended to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified quantity.

[0140] As used herein, phrases such as “between X and Y” and “between approximately X and Y” should be interpreted as including both X and Y. As used herein, phrases such as “between approximately X and Y” mean “between approximately X and approximately Y” and phrases such as “from approximately X to Y” mean “from approximately X to approximately Y”.

[0141] As used herein, the term "exemplary" is used to mean serving as an example, illustration, or description. Any embodiment or aspect described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments or aspects, nor does it imply exclusion of equivalent structures and techniques known to those skilled in the art. Rather, the term "exemplary" is used to present concepts in a concrete manner, and the subject matter disclosed is not limited to such examples.

[0142] Composition

[0143] In one aspect, this disclosure provides and includes compositions comprising MAP, acetate, sucrose, and polysorbate 20. In another aspect, this disclosure provides and includes compositions comprising MAP, acetate, mannitol, and polysorbate 20. In yet another aspect, this disclosure provides and includes compositions comprising MAP, a buffer, a stabilizer or tension agent, and a nonionic surfactant.

[0144] In one aspect, this disclosure provides compositions comprising MAP. As used herein, “MANP” is an ANP-based peptide whose amino acid sequence comprises a mature human ANP sequence of 28 amino acids and a carboxyl terminus of an additional 12 amino acids. Without being bound by theory, MAP is a pGC-A / cGMP activator that can significantly reduce blood pressure and vascular resistance. In one aspect, MAP comprises the peptide SLRRSSCFGGRMDRIGAQSGLGCNSFRYRITAREDKQGWA having the sequence shown in SEQ ID NO: 1. In one aspect, MAP may be a variant of the sequence shown in SEQ ID NO: 1. In one aspect, MAP may contain the amino acid sequence shown in SEQ ID NO: 1, and one or more amino acid additions, subtractions, or substitutions. In one aspect, MAP may contain the amino acid sequence shown in SEQ ID NO: 1, and one or more amino acid additions, subtractions, and substitutions. In one aspect, MAP may contain the amino acid sequence shown in SEQ ID NO: 1, and one, two, three, four, five, six, seven, eight, nine, or ten single amino acid residues added, subtracted, or substituted. On one hand, MANP may contain the amino acid sequence shown in SEQ ID NO: 1, as well as one, two, three, four, five, six, seven, eight, nine or ten single amino acid residues added, subtracted and substituted.

[0145] In one aspect, any amino acid residue shown in SEQ ID NO: 1 may be subtracted, and any amino acid residue (e.g., any of the 20 conventional amino acid residues or any other type of amino acid, such as ornithine or citrulline) may be added to the sequence shown in SEQ ID NO: 1. In another aspect, MANP may contain one or more chemical structures, such as ε-aminocaproic acid; hydroxylated amino acids, such as 3-hydroxyproline, 4-hydroxyproline, (5R)-5-hydroxy-L-lysine, isohydroxylysine, and 5-hydroxy-L-valine; and / or glycosylated amino acids, such as amino acids containing monosaccharides (e.g., D-glucose, D-galactose, D-mannose, D-glucosamine, and D-galactosamine) or combinations of monosaccharides.

[0146] MAPs with one or more amino acid additions, subtractions, or substitutions relative to the representative MAP sequence shown in SEQ ID NO: 1, also referred to herein as “variant” MAPs, can be generated using any suitable method. In one aspect, amino acid substitutions can be performed by selecting substitutions that do not significantly differ in maintaining (a) the structure of the peptide backbone in the substituted region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect on the volume of the side chain. For example, naturally occurring residues can be grouped according to side chain characteristics into several groups: (1) hydrophobic amino acids (ortholeucine, methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids affecting chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions performed within these groups can be considered conserved substitutions. Non-limiting examples of useful conservative substitutions may include, but are not limited to, valine substituted with alanine, lysine substituted with arginine, glutamine substituted with asparagine, glutamic acid substituted with aspartic acid, serine substituted with cysteine, asparagine substituted with glutamine, aspartic acid substituted with glutamic acid, proline substituted with glycine, arginine substituted with histidine, leucine substituted with isoleucine, isoleucine substituted with leucine, arginine substituted with lysine, leucine substituted with methionine, leucine substituted with phenylalanine, glycine substituted with proline, threonine substituted with serine, serine substituted with threonine, tyrosine substituted with tryptophan, phenylalanine substituted with tyrosine, and / or leucine substituted with valine.

[0147] On one hand, MANPs may include one or more non-conservative substitutions. Non-conservative substitutions typically require exchanging a member from one of the aforementioned categories for a member from another category. Such production may be ideal for providing large quantities or alternative embodiments of such compounds. Whether an amino acid alteration results in a functional polypeptide can be readily determined by measuring the specific activity of the polypeptide variant using methods disclosed herein, for example.

[0148] On the one hand, MANP can have a length of, for example, 35 to 45 amino acid residues (e.g., 35 to 40, 40 to 45, 35 to 37, 36 to 38, 37 to 39, 38 to 40, 39 to 41, 40 to 42, 41 to 43, 42 to 44, or 43 to 45 amino acid residues).

[0149] In one aspect, the composition comprises a concentration of MANP effective in treating the disease. In another aspect, the composition comprises MANP at concentrations of about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1.0 mg / ml, about 2.0 mg / ml, about 3.0 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, or about 10.0 mg / ml. In one aspect, the composition comprises MANP at a concentration of at least 0.1 mg / ml, at least 0.2 mg / ml, at least 0.3 mg / ml, at least 0.4 mg / ml, at least 0.5 mg / ml, at least 0.6 mg / ml, at least 0.7 mg / ml, at least 0.8 mg / ml, at least 0.9 mg / ml, at least 1.0 mg / ml, at least 2.0 mg / ml, at least 3.0 mg / ml, at least 4.0 mg / ml, at least 5.0 mg / ml, at least 6.0 mg / ml, at least 7.0 mg / ml, at least 8.0 mg / ml, at least 9.0 mg / ml, or at least 10.0 mg / ml. In another aspect, the composition comprises MANP at a concentration of about 0.5 mg / ml to 5.0 mg / ml. In another aspect, the composition comprises MANP at a concentration of about 0.75 mg / ml to 4.0 mg / ml. In another aspect, the composition comprises MANP at a concentration of about 1.0 mg / ml to 3.0 mg / ml. In one respect, the composition contains MANP at a concentration of about 2.0 mg / ml.

[0150] This disclosure provides compositions of MAP contained in a suitable buffer. In one aspect, the buffer is selected from the group consisting of: acetate, bicarbonate, carbonate, citrate, N,N-bis(2-hydroxyethyl)glycine, histidine, glycine, glutamate, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, tris(hydroxymethyl)aminomethane, diglycinate, N-tricine, malic acid, succinate, sulfate, nitrate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. In one aspect, the buffer is a citrate. In another aspect, the buffer is an acetate. In another aspect, the buffer is Tris-HCl. In another aspect, the buffer is a phosphate. In another aspect, the buffer is histidine. In one aspect, the composition comprises MAP in an acetate buffer.

[0151] The buffer can be present at a concentration suitable for adjusting the pH of the composition. In one aspect, the composition comprises a buffer at a concentration of about 1 mM, about 2 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 500 mM. In one aspect, the composition comprises a buffer at a concentration of at least 1 mM, at least 2 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, or at least 500 mM. In another aspect, the composition comprises a buffer at a concentration of about 1 mM to about 50 mM. In another aspect, the composition comprises a buffer at a concentration of about 5 mM to about 40 mM. In another aspect, the composition comprises a buffer at a concentration of about 10 mM to about 40 mM. In another aspect, the composition comprises an acetate at a concentration of about 10 mM. In another aspect, the composition comprises an acetate at a concentration of about 14 mM.

[0152] The pH of the composition is selected for the stability of the MANP peptide. In one aspect, the pH of the composition is in the range of about 4.0 to about 9.0. In another aspect, the pH of the composition is in the range of about 5.0 to about 7.0. In another aspect, the pH of the composition is in the range of about 5.0 to about 7.0. In another aspect, the pH of the composition is in the range of about 5.0 to about 6.0. In another aspect, the pH of the composition is about 5.0. In another aspect, the pH of the composition is about 5.5. In another aspect, the pH of the composition is about 6.0.

[0153] The tensioning agent may be present at a concentration suitable for adjusting the stability of the composition. In one aspect, the composition comprises any known tensioning agent, including but not limited to propylene glycol, sorbitol, sucrose, glycine, mannitol, lactose monohydrate, arginine, glucose, trehalose, sodium chloride, potassium chloride, glycerol, glycerol, inositol, and dimethyl sulfone.

[0154] In one aspect, the composition comprises sucrose. In another aspect, the composition comprises sucrose at a concentration ranging from about 200 mM to about 300 mM. In another aspect, the composition comprises sucrose at a concentration ranging from about 250 mM to about 275 mM. In another aspect, the composition comprises sucrose at a concentration of about 275 mM. In one aspect, the composition comprises mannitol. In another aspect, the composition comprises mannitol at a concentration ranging from about 200 mM to about 300 mM. In another aspect, the composition comprises mannitol at a concentration ranging from about 250 mM to about 275 mM. In another aspect, the composition comprises mannitol at a concentration of about 250 mM.

[0155] Stabilizers containing high molecular weight polymers may be present at concentrations suitable for adjusting the stability of the composition. In one aspect, the composition contains any known stabilizer, including but not limited to hydroxypropyl-β-cyclodextrin, polyethylene glycol (e.g., PEG3350), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), poloxamer (Pranic F68 and F127), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), carboxy- / hydroxycellulose or derivatives thereof (e.g., HPC, HPC-SL, HPC-L and HPCM), Triton X-100, Brij 30, Brij 35, sulfur-containing substances such as monothioglycerol, mercaptoacetic acid, and 2-methylthioethanol, and various salts (e.g., sodium chloride).

[0156] Additional stabilizers may be included, such as those that further enhance the therapeutic activity of the MANP peptide. Examples of such stabilizers include, but are not limited to, methionine and EDTA, which prevent methionine oxidation, and nonionic surfactants, which prevent aggregation associated with freeze-thaw cycles and mechanical shearing.

[0157] In one aspect, the composition comprises polysorbate 20. In another aspect, the composition comprises polysorbate 20 at a concentration ranging from about 0.005% to 0.5%. In another aspect, the composition comprises polysorbate 20 at a concentration ranging from about 0.01% to 0.1%. In another aspect, the composition comprises polysorbate 20 at a concentration ranging from about 0.01% to 0.02%. In another aspect, the composition comprises polysorbate 20 at a concentration of about 0.02%. In another aspect, the composition comprises polysorbate 80. In another aspect, the composition comprises polysorbate 80 at a concentration ranging from about 0.005% to 0.5%. In another aspect, the composition comprises polysorbate 80 at a concentration ranging from about 0.01% to 0.1%. In another aspect, the composition comprises polysorbate 80 at a concentration ranging from about 0.01% to 0.02%. In another aspect, the composition comprises polysorbate 80 at a concentration of about 0.02%. In one aspect, the composition comprises hydroxypropyl-β-cyclodextrin. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration ranging from about 1 mM to 200 mM. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration ranging from about 15 mM to 100 mM. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration of about 15 mM. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration of about 25 mM. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration of about 50 mM. In another aspect, the composition comprises hydroxypropyl-β-cyclodextrin at a concentration of about 100 mM. In one aspect, the composition comprises methionine. In another aspect, the composition comprises methionine at a concentration ranging from about 1 mM to about 50 mM. In another aspect, the composition comprises methionine at a concentration ranging from about 5 mM to about 25 mM. In another aspect, the composition comprises methionine at a concentration ranging from about 10 mM to about 20 mM. In one aspect, the composition contains methionine at a concentration of about 5 mM. In another aspect, the composition contains methionine at a concentration of about 10 mM. In yet another aspect, the composition contains methionine at a concentration of about 20 mM.

[0158] In one aspect, the osmotic pressure of the composition is in the range of about 250 mOsm / kgH2O to about 500 mOsm / kgH2O. In another aspect, the osmotic pressure of the composition is in the range of about 290 mOsm / kgH2O to about 400 mOsm / kgH2O. In another aspect, the osmotic pressure of the composition is in the range of about 300 mOsm / kgH2O to about 420 mOsm / kgH2O. In yet another aspect, the osmotic pressure of the composition is in the range of about 310 mOsm / kgH2O to about 390 mOsm / kgH2O.

[0159] As used herein, the term "surfactant" refers to any molecule or ion consisting of a water-soluble (hydrophilic) head and a lipophilic (lipophilic) segment. Surfactants preferentially accumulate at interfaces, with their hydrophilic portion facing the water (hydrophilic phase) and their lipophilic portion facing the oil or hydrophobic phase (i.e., glass, air, oil, etc.). The concentration at which a surfactant begins to form micelles is called the critical micelle concentration, or CMC. Furthermore, surfactants reduce the surface tension of liquids. Surfactants are also called amphiphilic compounds. The term "detergent" is a general synonym for surfactant. Surfactants can be anionic (e.g., chenodeoxycholic acid, cholic acid, tuftediocin, tuftediocin aglycone, N-lauroyl sarcosine, lithium dodecyl sulfate, sodium dodecyl sulfate, sodium hexanesulfonate, taurine chenodeoxycholic acid, sodium dodecyl sulfate, or sodium lauryl sulfate) or cationic (e.g., alkyltrimethylammonium bromide, benzalkonium chloride, dimethyl dioctadecyl ammonium bromide, dodecyl ethyl dimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, ethyl hexadecyl dimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, hexadecyl trimethyl ammonium bromide, polyoxyethylene (10)-N-tallow-1,3-Diaminopropane, tonzoamine bromide and / or trimethyl(tetradecyl)ammonium bromide) or nonionic (e.g., BigCHAP, bis(polyethylene glycol bis[imidazolylcarbonyl]), block copolymers such as polyethylene oxide / polypropylene oxide block copolymers such as poloxamer, poloxamer 188 and poloxamer 407, Brij 35, Brij 56, Brij 72, Brij 76, Brij 92V, Brij 97, Brij 58P, Polyoxyethylene Castor Oil EL, Decylene Glycol Monododecyl Ether, N-Decanyl-N-Methylglucosamine, n-Dodecyl-N-Methylglucamide, Alkyl Polyglucoside, Ethoxylated Castor Oil, Heptaethylene Glycol Monodecyl Ether, Heptaethylene Glycol Monododecyl Ether, Heptaethylene Glycol Monotetradecyl Ether, Hexaethylene Glycol Monododecyl Ether, Hexaethylene Glycol Monohexadecyl Ether, Hexaethylene Glycol Monooctadecyl Ether, Hexaethylene Glycol Monotetradecyl Ether, Igepal CA-630, Igepal CA-630, Methyl-6-O-(N-heptylcarbamoyl)-β-D-glucopyranoside, Non-ethylene glycol monododecyl ether, N-nonanoyl-N-methylglucosamine, N-nonanoyl-N-methylglucosamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-β-D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether, Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether, Polyethylene glycol diglycidyl ether, Polyethylene glycol ether W-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 2 0 isohexadecanyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolyl carbonyl), polyoxyethylene 25 propylene glycol stearate, bark saponins, Tergitol, tetradecyl-β-D-maltodextrin, tetraethylene glycol monodecyl ether, tetraethylene glycol monotetradecyl ether, tetraethylene glycol monotetradecyl ether, triethylene glycol monodecyl ether, triethylene glycol monododecyl ether, triethylene glycol monohexadecyl ether, triethylene glycol monooctyl ether, triethylene glycol monotetradecyl ether, triethylene glycol monooctyl ether, triethylene glycol monotetradecyl ether, Triton, Tween, tylosap, sphingomyelins (sphingomyelins) and glycosphingomyelins (ceramides, gangliosides), phospholipids and n-undecyl-β-D-glucopyranoside.

[0160] In one respect, the pharmaceutically acceptable formulations described herein are stable for long-term storage and transport. As used herein, “stability” can mean “chemical stability” or “physical stability.” As used herein, a substance is “chemically stable” if it is not particularly reactive in a given environment and retains its useful properties within a defined period of intended usability. As used herein, a substance is “physically stable” if its original physical properties (including, but not limited to, appearance, palatability, homogeneity, solubility, and suspendability) are retained within a defined time period. In one respect, the pharmaceutically acceptable formulations described herein are chemically stable for long-term storage and transport. In another respect, the pharmaceutically acceptable formulations described herein are physically stable for long-term storage and transport.

[0161] Composition for treating hypertension

[0162] In one aspect, this disclosure provides and includes compositions for treating hypertension, the compositions comprising MANP, acetate, sucrose, and polysorbate 20.

[0163] As used in this article, “treatment of a disease” refers to the management and care of a patient who has developed a disease, condition, or disorder. The aim of treatment is to combat the disease, condition, or disorder. Treatment includes the administration of active compounds to eliminate or control the disease, condition, or disorder, and to alleviate symptoms or complications associated with the disease, condition, or disorder.

[0164] As used in this article, "effective dose" means a dose that is sufficient to be effective for the patient compared to no treatment or placebo treatment.

[0165] The MANP-containing compositions described herein can be administered to patients requiring treatment of hypertension. These compositions can be administered to patients requiring such treatment at several sites, such as at local sites (e.g., skin and mucous membrane sites), at sites bypassing absorption (e.g., intra-arterial, intra-venous, intracardiac administration), and at sites involving absorption (e.g., intradermal, subcutaneous, intramuscular, or intraperitoneal administration).

[0166] The compositions described herein can be administered to patients requiring such treatment via several routes of administration, such as tongue, sublingual, buccal, intraoral, oral, gastric and enteral, nasal, lungs (e.g., via bronchioles and alveoli or combinations thereof), epidermis, dermis, transdermal, vagina, rectum, eye (e.g., via conjunctiva), urethra, and parenteral.

[0167] The compositions described herein can be administered in a number of dosage forms, including but not limited to solutions, suspensions, emulsions, microemulsions, multiple emulsions, foams, ointments, pastes, patches, creams, tablets, coated tablets, lotions, capsules (e.g., hard gelatin capsules and soft gelatin capsules), suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalers, eye drops, ophthalmic ointments, ophthalmic lotions, vaginal pessaries, vaginal rings, vaginal ointments, injectable solutions, in situ transformation solutions (e.g., in situ gelation, in situ coagulation, in situ precipitation, in situ crystallization), infusion solutions, and implants.

[0168] The compositions described herein can be further formulated into or attached (e.g., through covalent, hydrophobic, and electrostatic interactions) to drug carriers, drug delivery systems, and advanced drug delivery systems to further enhance the stability of the compounds of this embodiment, increase bioavailability, increase solubility, reduce adverse effects, achieve time-based therapies known to those skilled in the art, and increase patient compliance, or any combination thereof. Examples of carriers, drug delivery systems, and advanced drug delivery systems include, but are not limited to, polymers (e.g., cellulose and its derivatives), polysaccharides (e.g., dextran and its derivatives), starch and its derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic acid and polyglycolic acid and their block copolymers, polyethylene glycol, carrier proteins (e.g., albumin), gels (e.g., thermal gelation systems), block copolymer systems known to those skilled in the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and their dispersions, L2 phases and their dispersions (phase behavior in lipid-water systems is known to those skilled in the art), polymeric micelles, multiple emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and their derivatives, and dendritic polymers.

[0169] The compositions described herein can also be formulated as solids, semi-solids, powders, and solutions for pulmonary administration, using, for example, metered-dose inhalers, dry powder inhalers, and nebulizers, or other devices known to those skilled in the art.

[0170] The compositions described herein can also be used in the formulation of controlled-release, sustained-release, long-acting-release, delayed-release, and slow-release drug delivery systems. In one aspect, the compositions described herein can be used in the formulation of parenteral controlled-release and sustained-release systems known to those skilled in the art (both systems result in a reduction in the number of administrations). In another aspect, the compositions described herein are subcutaneous controlled-release and sustained-release systems. Examples of useful controlled-release systems and compositions, without limiting the scope of this disclosure, are hydrogels, oil gels, liquid crystals, polymer micelles, microspheres, and nanoparticles. Methods for producing controlled-release systems that can be used with the compositions of this disclosure include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure homogenization, encapsulation, spray drying, microencapsulation, coagulation, phase separation, solvent evaporation to produce microspheres, extrusion, and supercritical fluid processes. Generally refer to Handbook of Pharmaceutical Controlled Release (Wise, DL editor Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences, Volume 99: Protein Formulation and Delivery (MacNally, EJ editor Marcel Dekker, New York, 2000).

[0171] In one aspect, parenteral administration is performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe (optionally a pen syringe). In another aspect, parenteral administration can be performed using an infusion pump. In another aspect, the compositions described herein can be solutions, suspensions, or powders for the administration of MANP as a nasal or pulmonary liquid or powder spray. In another aspect, pharmaceutical compositions containing MANP can also be suitable for transdermal administration (e.g., by needle-free injection or from patches, iontophoresis patches, or via mucosal application).

[0172] In one respect, the compositions described herein can be formulated for administration via the pulmonary route in a carrier, as a solution, suspension, or dry powder, using any known type of device suitable for pulmonary drug delivery. Examples of these include, but are not limited to, the three general types of aerosol generation for pulmonary drug delivery, and may include jet or ultrasonic nebulizers, metered-dose inhalers, or dry powder inhalers (see Yu J, Chien Y W. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997) 395-453).

[0173] In one aspect, the compositions described herein can be formulated into a dry form by freeze-drying (e.g., lyophilization), spray drying, or air drying. In another aspect, the compositions described herein can be formulated into a dry form for storage. In another aspect, the compositions described herein can be formulated into a dry form for transportation. In another aspect, the compositions described herein can be formulated into a dry form to improve stability.

[0174] Pharmaceutical Composition

[0175] As used herein, "pharmaceutical composition" means a product comprising an active compound or a salt thereof, and pharmaceutical excipients such as buffers, preservatives, and optionally stress-relieving agents and / or stabilizers. Therefore, pharmaceutical compositions are also referred to in the art as pharmaceutical preparations. In one aspect, a pharmaceutical preparation is a lyophilized preparation to which a physician or patient adds a solvent and / or diluent before use. In another aspect, a pharmaceutical preparation is a dried preparation that can be used without any pre-dissolution (e.g., lyophilized or spray-dried). In yet another aspect, a pharmaceutical preparation is a lyophilized preparation to which a physician or patient adds a solvent and / or diluent before use.

[0176] As used in this article, "pharmaceutical acceptable" means suitable for normal pharmaceutical use, i.e., it has not caused adverse events in patients.

[0177] As used herein, “excipient” means a chemical compound commonly added to a pharmaceutical composition, such as a buffer, tensile agent, preservative, etc. In another sense, the term “excipient” broadly refers to any component other than the active therapeutic ingredient. The excipient can be an inert, inactive, and / or non-pharmacologically active substance. Formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th edition (1995) and any subsequent editions). Non-limiting examples of excipients include: solvents, diluents, buffers, preservatives, tensile agents (e.g., isotonic agents), chelating agents, and stabilizers (e.g., oxidation inhibitors, aggregation inhibitors, surfactants, and / or protease inhibitors).

[0178] This disclosure provides a pharmaceutical composition comprising MANP, acetate, sucrose, and polysorbate 20. In one aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20. In another aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 40 mM acetate, about 250 mM mannitol, and about 0.02% polysorbate 20.

[0179] This disclosure also provides a pharmaceutical composition comprising MANP, acetate, sucrose, and polysorbate 20, which exhibits minimal aggregation during long-term storage. As used herein, “aggregation” or “aggregate formation” describes the physical interactions between the polypeptide molecules described herein (e.g., MANP) resulting in the formation of oligomers that may remain soluble or produce large, visible aggregates that precipitate from solution. As used herein, “storage” or “stored” describes the liquid pharmaceutical compositions or formulations described herein that, once prepared, are not immediately administered to a patient but are packaged for storage, in liquid form, in a frozen state, or in a dry state, for later reconstitution into a liquid form or other forms suitable for administration to a patient. Without being bound by theory, aggregates formed by polypeptides during storage of liquid pharmaceutical compositions may adversely affect the biological activity of the polypeptide, resulting in a loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, when administering polypeptide-containing pharmaceutical compositions using infusion systems, aggregate formation may cause other problems, such as clogging of tubing, membranes, or pumps.

[0180] In one aspect, the pharmaceutical compositions described herein are stored at about 1-10°C, about 2-5°C, or about 3-5°C. In another aspect, the pharmaceutical compositions described herein are stored at about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C, about 2°C, or about 1°C.

[0181] On one hand, the amount of peptides can be assessed using UV spectroscopy. Without being bound by theory, peptides absorb in the UV region of the electromagnetic spectrum, where aromatic amino acids such as tryptophan absorb at approximately 280 nm, disulfide bonds absorb at approximately 250–320 nm, and peptide bonds absorb at approximately 190 nm and 210–220 nm. On one hand, the concentration of a peptide can be determined by dividing the absorbance by the path length of light through the sample and the extinction coefficient of the peptide. On one hand, the amount of a peptide (e.g., MANP as described herein) can be determined by measuring the absorbance of UV light through the peptide sample. On one hand, the amount of a peptide (e.g., MANP as described herein) can be determined by measuring the absorbance of UV light at 190–250 nm. On one hand, the presence or degree of monomer aggregation can be assessed using UV spectroscopy. The precipitation of aggregates reduces the amount of monomers in solution, which can be detected by a decrease in peptide concentration.

[0182] On the one hand, the presence or degree of monomer aggregation can be assessed visually. Without being bound by theory, large aggregates that scatter light result in a cloudy appearance and, in some cases, gel formation. On the other hand, monomer aggregation can be assessed visually.

[0183] On one hand, the presence or degree of monomer aggregation can be assessed using size exclusion chromatography (SEC). Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated according to their size, molecular weight, or hydrodynamic volume. Without being bound by theory, an aqueous solution is used to transport the molecules of interest through a column packed with tiny porous beads, typically made of dextran, agarose, or polyacrylamide polymers. As the solution travels down the column, the molecules enter the pores. Larger particles cannot enter as many pores and therefore elute from the column more quickly. Smaller molecules travel through more pores and elute from the column more slowly. The eluent is collected at a constant volume or fraction, and the collected fraction is analyzed by spectroscopic techniques to determine the concentration of the eluted particles. On one hand, UV-Vis spectroscopy is used to determine the concentration of the eluted peptide. On another hand, UV absorption at 220 nm is used to determine the concentration of the eluted peptide. On another hand, multi-angle laser light scattering (MALS) is used to determine the concentration of the eluted peptide. On yet another hand, refractive index measurements are used to determine the concentration of the eluted peptide. On one hand, viscosity measurements are used to determine the concentration of the eluted peptides.

[0184] SEC elution spectra of a sample provide information about the sample's quality and conformational heterogeneity. An elution spectrum is a graph of the amount of sample eluted over time, where molecules of similar size elute at approximately the same time. Without being bound by theory, the presence of peaks in an elution spectrum indicates the presence of molecules of different sizes, with the main peak typically containing monomers (when there is little or no aggregation), higher molecular weight peaks typically containing aggregates (e.g., dimers, trimers, and more advanced aggregates), and lower molecular weight peaks containing degradation products. Without being bound by theory, the peak height indicates the concentration of a specific size of peptide in the eluent, and the area under the peak indicates the total amount of a specific size of peptide. Therefore, the relative area of ​​a specific peak indicates the amount of a specific size of peptide. That is, the higher the relative area of ​​the main peak, the higher the amount of monomers and the lower the amount of aggregates. For example, a loss in the relative area of ​​the main peak may indicate monomer aggregation, precipitation from solution, or loss of purity.

[0185] In one aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein less than 0.5% of monomer aggregates are observed after storage at 2-8°C for 24 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In another aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the purity loss of the composition is less than 0.2% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In yet another aspect, the purity loss of the composition is less than 0.18% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In one aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition exhibits a purity loss of less than 0.5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography. In another aspect, the composition exhibits a purity loss of less than 0.35% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via size exclusion chromatography.

[0186] On one hand, the chemical stability of a sample can be assessed using high-performance liquid chromatography (HPLC). HPLC is a widely used analytical chemistry technique for separating compounds in chemical mixtures. These separations utilize the pressure-driven flow of a mobile phase through a column packed with a stationary phase. The mobile phase carries the liquid sample through the column to the detector, and compounds or analytes are separated due to varying degrees of interaction with the stationary phase. In reversed-phase HPLC (RP-HPLC), the stationary phase is typically a chemically bonded inorganic oxide, while the mobile phase is typically a water-organic solvent mixture (e.g., a mixture of acetonitrile, water, and trifluoroacetic acid). In RP-HPLC, the sample passes through the column, and different compound groups interact differently with the stationary phase, resulting in different retention times depending on their chemical properties. Therefore, different analytes or components in the sample are separated. Without being bound by theory, the presence of peaks in the elution spectrum indicates the presence of molecules with different chemical compositions. For samples with a single analyte, the main peak typically contains the analyte (when there is little or no degradation), while other peaks indicate different degradation products, the amount of which is affected by temperature, analyte concentration, and formulation composition. On the one hand, the relative height of the main peak indicates the amount of undegraded peptides (e.g., MANP as described herein) in the sample. For example, a loss in main peak height may indicate that some peptides have degraded, precipitated from solution, or suffered a loss of purity. On the other hand, the relative area of ​​the main peak indicates the amount of undegraded peptides (e.g., MANP as described herein) in the sample. That is, the higher the relative area of ​​the main peak, the higher the amount of undegraded peptides and the lower the amount of degradation products. For example, a loss in the relative area of ​​the main peak may indicate that some peptides have degraded, precipitated from solution, or suffered a loss of purity.

[0187] In one aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 1% after storage at 5°C for 6 months, as measured by the height of the main peak obtained via reversed-phase HPLC. In another aspect, the composition experiences a purity loss of less than 0.5% after storage at 5°C for 6 months, as measured by the height of the main peak in reversed-phase HPLC. In another aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the composition experiences a purity loss of less than 10% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via reversed-phase HPLC. In another aspect, the composition experiences a purity loss of less than 5% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak in reversed-phase HPLC. In one aspect, the purity loss of the composition after storage at 5°C for 12 months is less than 3%, as measured by the relative area of ​​the main peak in reversed-phase HPLC. In another aspect, the pharmaceutical composition described herein comprises about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose, and about 0.02% polysorbate 20, wherein the purity loss of the composition after storage at 5°C for 24 months is less than 10%, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC. In yet another aspect, the purity loss of the composition after storage at 5°C for 24 months is less than 5%, as measured by the relative area of ​​the main peak in reversed-phase HPLC.

[0188] Reagent kits, vials, and pre-filled syringes

[0189] This disclosure provides a kit for treating hypertension in patients in need, comprising the composition described herein. In one aspect, the kit for treating hypertension in patients in need comprises the composition described herein and an injection device for dispensing a dose of the composition. In one aspect, the injection device is a syringe. In another aspect, the injection device is a dose-limiting syringe. In another aspect, the injection device is a pen syringe. In another aspect, the injection device is a catheter. In another aspect, the injection device is a syringe with an auto-injector.

[0190] This disclosure provides vials containing the compositions described herein. The long-term stability of the composition is determined by the vial being made of a material that does not interact with or degrade the composition stored therein. In one aspect, the vial is a glass container. In another aspect, the vial includes a glass body and a cap. In another aspect, the vial is an ampoule. In one aspect, the vial contains a formulation comprising MANP, acetate, mannitol, and polysorbate 20. In another aspect, the vial contains a formulation comprising MANP, acetate, sucrose, and polysorbate 20. In one aspect, the composition described herein stored in the vial experiences a purity loss of less than 1% after storage at 5°C for 6 months, as measured by the height of the main peak obtained via reversed-phase HPLC. In one aspect, the composition described herein stored in the vial experiences monomer aggregation of less than 0.5% after storage at 2-8°C for 24 months, as measured by the relative area of ​​the main peak obtained via SEC. In one aspect, the composition described herein stored in the vial experiences a purity loss of less than 10% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via RP-HPLC. In one aspect, the compositions described herein, stored in vials, exhibit a purity loss of less than 10% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via RP-HPLC. In another aspect, the compositions described herein, stored in vials, exhibit a purity loss of less than 0.2% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained via SEC. In yet another aspect, the compositions described herein, stored in vials, exhibit a purity loss of less than 0.5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained via SEC.

[0191] This disclosure also provides pre-filled syringes containing the compositions described herein. In one aspect, a pre-filled syringe is a syringe pre-filled with medication before being dispensed to an end user who will administer the medication to a patient. In another aspect, the pre-filled syringe contains the compositions described herein in a medication container (e.g., a syringe barrel), a resilient plunger for dispensing the medication, and an attached hypodermic needle or a feature allowing the user to attach a needle before administering the medication so that the medication can be delivered directly from the syringe to the patient through the needle. The user of the syringe will typically be required to receive training in injection administration skills and may be the patient, a physician, a nurse, or other caregiver such as a family member. In one aspect, the compositions disclosed herein are present in the pre-filled syringe at concentrations of about 0.1 mg / ml, about 0.2 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 5 mg / ml, about 10 mg / ml, about 20 mg / ml, about 50 mg / ml, about 100 mg / ml, about 200 mg / ml, and about 500 mg / ml. On the one hand, pre-filled syringes can be used to administer approximately 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml, 2.6 ml, 2.7 ml, 2.8 ml, 2.9 ml, 3.0 ml, 3.1 ml, 3.2 ml, 3.3 ml, 3.4 ml, 3.5 ml, 3.6 ml, 3.7 ml, 3.8 ml, and 3.9 ml per dose to each patient. The compositions described herein in ml, about 4.0 ml, about 4.1 ml, about 4.2 ml, about 4.3 ml, about 4.4 ml, about 4.5 ml, about 4.6 ml, about 4.7 ml, about 4.8 ml, about 4.9 ml, about 5.0 ml, about 5.1 ml, about 5.2 ml, about 5.3 ml, about 5.4 ml, about 5.5 ml, about 5.6 ml, about 5.7 ml, about 5.8 ml, about 5.9 ml, about 6.0 ml, about 7.0 ml, about 8.0 ml, about 9.0 ml, about 10.0 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, or about 100 ml.

[0192] Example

[0193] The following examples illustrate this disclosure. The examples listed herein illustrate several aspects of this disclosure, but should not be construed as limiting the scope of this disclosure in any way.

[0194] Example 1: Formulation Development Research

[0195] The main objective of this study is to develop a liquid formulation containing MANP that is stable when stored at 2–8°C, with a target concentration of approximately 2 mg / ml, a pH of 7.4, and an osmotic pressure of 290–310 mOsm / L.

[0196] The following equipment was used in this study, as shown in Table 1.

[0197] Table 1: Materials used in the study

[0198]

[0199] Methods: UV analysis

[0200] To prevent sample loss and errors caused by UV sample preparation, the concentration of each formulation was measured using a SoloVPE. Sample concentration was measured by adding 100 μL of material to a small, disposable UV container of the SoloVPE. A new fiber optic probe was installed, and the instrument measured the sample absorbance using an extinction coefficient of 1.58 mL*mg⁻¹ cm⁻¹, correcting for background scattering. The extinction coefficient was calculated based on the primary amino acid sequence of the peptide. After analysis, the sample was removed from the disposable container using a pipette. Both the disposable container and the fiber optic probe were then discarded. This procedure was repeated for each sample.

[0201] Method: pH analysis

[0202] After sample preparation, the pH of each formulation was checked, and the measured sample pH was within ±0.1 of the target pH. Before starting the analysis, the pH probe was calibrated using three pH standards ordered from Fisher. The pH of the formulation was measured by inserting the pH probe into the sample and waiting for the measurement to stabilize, which may take 1 to 2 minutes. After analysis, the pH probe was rinsed with 18 MΩ water for one minute and stored in pH storage solution.

[0203] Method: Osmotic pressure measurement

[0204] Osmometry analysis was performed using an Advanced Instruments Osmo 1. At the start of the analysis, a reference standard of 290 mOsm was analyzed to ensure the instrument was functioning correctly. After the reference standard was passed, the sample was analyzed. 20 μL of the material was removed and analyzed using the Osmo 1. After analysis, the chamber was cleaned using a chamber cleaner. This procedure was repeated for each sample.

[0205] Method: Size exclusion chromatography (SEC)

[0206] The SEC method is used to measure and track changes in the physical stability of peptide stability samples. The parameters of the SEC analysis are described below:

[0207] Column: Sepax Zenix-C Sec-80

[0208] Manufacturer: Sepax

[0209] Specifications: 7.8 x 300 mm

[0210] Part number: 233080-7830

[0211] Mobile phase: 50% acetonitrile and 50% water, 0.1% TFA.

[0212] Sample concentration (mg / mL): 2 mg / mL

[0213] Autosampler temperature: 5℃ ± 3℃

[0214] Column temperature: 25℃

[0215] Flow rate: 0.8 mL / min

[0216] Injection volume: 7 μg (3.75 μL for 2 mg / mL).

[0217] UV settings: 220 nm, and

[0218] Data acquisition time: 10 Hz.

[0219] Method: Reversed-phase chromatography (RP)

[0220] This method is used to measure and track changes in the chemical stability of peptides. Several reversed-phase methods were used to study the chemical stability of peptides. The first study used the Original method and the C8 method. The Primary reversed-phase method was used in all subsequent studies. An exemplary chromatogram of the C8 method is shown in [image / image / etc.]. Figure 1 The parameters of the Original inversion method are described as follows:

[0221] Column: Waters XBridge C18, 4.6 x 150 mm, 3.5 μm,

[0222] Manufacturer: Waters

[0223] Part number: 186003034

[0224] Mobile phase A: 0.2% methanesulfonic acid (MSA) aqueous solution.

[0225] Mobile phase B: 100% acetonitrile

[0226] Sample concentration (mg / mL): 2 mg / mL

[0227] Autosampler temperature: 5.0 ± 3.0℃

[0228] Column temperature: 40℃

[0229] Flow rate: 1.2 mL / min

[0230] Injection volume: 5 μL, and

[0231] UV setting: 215 nm.

[0232] The parameters of the C8 inversion method are described as follows:

[0233] Column: Aeris Widepore, 3.6 μm XB-C8, 250 x 2.1 mm,

[0234] Manufacturer: Phenomenex

[0235] Part number: 00G-4481-AN

[0236] Mobile phase A: Water, 0.1% TFA

[0237] Mobile phase B: Acetonitrile, 0.1% TFA

[0238] Sample concentration (mg / mL): 2.5 μg on column.

[0239] Autosampler temperature: 5 ± 3℃

[0240] Column temperature: 40℃

[0241] Flow rate: 0.4 mL / min

[0242] Injection volume: 2.5 μL, and

[0243] UV setting: 220 nm.

[0244] The parameters of the Primary inversion method are described in Table 2, and are as follows:

[0245] Column: XSelect CSH, C18, 3.5 μm

[0246] Manufacturer: Waters

[0247] Specifications: 4.6 x 150 mm

[0248] Part number: 186005270

[0249] Mobile phase A: 15% acetonitrile, 85% water, 0.1% TFA.

[0250] Mobile phase B: 30% acetonitrile, 70% water, 0.1% TFA.

[0251] Mobile phase C: 80% acetonitrile, 20% water, 0.1% TFA.

[0252] Sample concentration (mg / mL): 2 mg / mL stock solution injection.

[0253] Autosampler temperature: 8 ± 3℃

[0254] Column temperature: 40℃

[0255] Flow rate: 0.4 mL / min

[0256] Injection volume: 1.25 μL

[0257] UV settings: 220 nm, and

[0258] Data acquisition time: 10 Hz.

[0259] Table 2: Primary Reverse Phase Gradient

[0260]

[0261] Methods: General sample preparation, aseptic filtration, and sample filling

[0262] The 10 mg / mL peptide solution was prepared on the same day of preparation, and the 2X buffer was prepared the day before. The 10 mg / mL peptide solution was prepared by weighing the peptide into a sterile container and adding Milli-Q water to achieve the target peptide concentration. The volume of the 10 mg / mL peptide solution was calculated based on the target sample volume. The calculated volume of peptide was then added to a new sterile container. The volume of 2X buffer added to the peptide solution was half the target sample volume. The pH of the peptide and 2X buffer was then measured using a pH probe. If the pH value exceeded ± 0.1 pH units, the sample pH was adjusted with 0.1 M NaCl. The sample was then brought to the target sample volume with Milli-Q water, and the pH value and peptide concentration were measured again.

[0263] The samples were aseptically filtered through a clean hood wiped with 70% ethanol. Each formulation was loaded into a sterile syringe equipped with a sterile filter. The sample was then slowly pushed through the filter into a sterile container. After sterile filtration, the samples were placed into vials.

[0264] Methods: Stirring study

[0265] According to the study, the samples were stirred at 25°C for one day. For each temperature, two different samples were prepared: a stirred sample, 0.5 mL of which was placed in a 2 mL vial; and a QS sample, 0.2 mL of which was placed in a 2 mL vial and placed next to the shaking plate as a control. The stirred sample was placed horizontally in the sample box and shaken at 590 rpm (this speed is based on the orbital radius of the vibrator). After 24 hours, both the stirred sample and the control were analyzed.

[0266] Method: Freeze-thaw procedure

[0267] The fourth round of sample preparation was frozen at -80°C for 10 minutes and thawed at 5°C for 15 minutes. These studies used 2 mL vials, with 0.25 mL of the raw material added to each vial and subjected to 5 freeze-thaw cycles. During the fifth freeze-thaw cycle, the sample was placed at -80°C overnight and thawed at 5°C the following day. The samples were analyzed after undergoing five freeze-thaw cycles.

[0268] Methods: Multivariate statistical modeling using partial least squares regression (PLS)

[0269] Partial Least Squares Regression (PLS) is used to further elucidate the impact of various formulation parameters on the stability of Phase 1 and Phase 2 samples. For any large numerical matrix, when a reasonable number of samples exist (collectively forming the so-called X matrix), a mathematical model can be constructed to explain the largest amount of variance in the dependent variable (Y matrix) of interest. The single best description of the relationship between the variation in the X matrix and the endpoint (Y matrix) is called the first principal component, PC1. The next important component (in terms of describing the variance in the Y matrix) is called the second principal component, PC2, and so on. Typically, only one or two PCs are needed to explain most of the variance in the Y matrix. Each of these PCs contains some contribution from the individual variables in the X matrix. If a variable in the X matrix contributes significantly to the construction of a given PC, that variable is considered significant. In fact, for a given model, regression coefficients can be calculated for each variable in the X matrix, where the model is a combination of a certain number of PCs to adequately describe the Y matrix. In summary, PLS takes information from the X matrix, calculates the required number of PCs, and constructs a suitable model. The model that includes all samples is called the calibrated model. The total coefficient of determination (r²) indicates the quality of the model. All PLS calculations were performed using Unscrambler software (CAMO, Corvallis, OR). A PLS analysis using a single variable in the Y matrix is ​​called a PLS1 analysis. A model that fits multiple variables to the Y matrix is ​​called a PLS2 analysis.

[0270] Full cross-validation of all calibration models was performed using standard techniques. In short, one sample was removed at a time, the dataset was recalibrated, and a new model was built. This process was repeated until all calibration samples had been removed and quantized into validation models. Therefore, the first set containing all samples is called the calibration set, and the set after cross-validation is called the validation set. The folding knife algorithm was used to determine the statistical significance of any factors used in constructing the PLS models described above.

[0271] Results of Study 1: Determination of the optimal pH and buffer concentration for MANP through visual inspection, pH, and UV assays.

[0272] Study 1 aimed to investigate the effects of different pH values, buffer types, and different stabilizers / strain modifiers on the stability of MANP. The test conditions were as follows:

[0273] Table 3: Formulation Design in Study 1

[0274]

[0275] Stability of samples was characterized by visual inspection, pH, and UV testing. During Study 1, all samples except formulations 2, 4, and 6 showed signs of physical instability (Tables 4 and 5). After T=0, the signs of physical instability observed for most samples were turbidity and gel formation.

[0276] The changes in peptide concentration in the samples were consistent with the observations from visual inspection (Table 6). Samples showing signs of physical instability also exhibited significant decreases in peptide concentration, with losses reaching up to 100%. Figure 2 Throughout the study, the peptide concentrations of formulations 2, 4, and 6 varied by less than 5% compared to T=0. The pH of the stability samples remained constant throughout the study. Figure 3 ).

[0277] Table 4: Visual characterization of the stability samples from Study 1 at T=0 and T=1 week at 40℃.

[0278]

[0279] Table 5: Visual characterization of the stability samples from Study 1 at 25°C for 2 weeks and at 5°C for 3 weeks.

[0280]

[0281] Table 6: Peptide concentration and pH of the stability samples from Study 1

[0282]

[0283] Results of Study 1: Determination of the optimal pH and buffer concentration for MANP by RP-HPLC

[0284] Stability samples were characterized by reversed-phase chromatography. A typical chromatogram measured using the C8 reversed-phase method is shown below. Figure 4 The study describes the increase in intensity of multiple peaks before and after the main peak when the peptide is subjected to stress at 25°C. Each peak represents a different degradation product, the amount of which is affected by temperature, peptide concentration, and formulation composition.

[0285] Only three formulations (formulations 2, 4, and 6) in Study 1 were feasible in terms of stability as determined by RP-HPLC (Table 7). All other formulations showed signs of physical instability (concentration loss, precipitation, turbidity, or gelation). These degraded samples were not further analyzed. The main peak of formulation 1 began with a highest T0 relative area (%) of 97.28, while formulations 4 and 6 were both approximately 96.4 (%). When the samples were stored at 5°C for 3 weeks, the main peak of formulation 1 changed by less than 0.1%, while formulations 2 and 3 showed an increase ( Figure 5 At 25°C for 2 weeks, all formulations showed some loss in the relative area (%) of the main peak, with formulation 6 experiencing the largest loss of 2.7%, while formulation 4 showed virtually no loss. The RP-HPLC data collected in Study 1 indicate that the C8 method is sensitive to chemical changes but still requires further improvement.

[0286] Table 7: Reversed-phase data of stability samples from formulations 2, 4, and 6 of Study 1

[0287]

[0288] Table 7 (continued): Reversed-phase data of stability samples from formulations 2, 4, and 6 of Study 1

[0289]

[0290] Results of Study 1: Determination of the optimal pH and buffer concentration for MANP using SEC

[0291] Size exclusion chromatography (SEC) is used to monitor changes in the physical stability of peptides and also provides some chemical information. The peaks eluting before the main peak are high molecular weight (HMW) substances, such as dimers or larger polymers. The peaks eluting after the main peak are low molecular weight (LMW) substances, such as peptide fragments resulting from chemical degradation. Examples of peptide analysis by SEC are shown below. Figure 6 middle.

[0292] Sample 1 was analyzed by SEC, where the initial monomer (main peak) content was approximately 98%, and closer to 95% except for formulation 10 (Tables 8-11). Samples exhibiting signs of macroscopic physical instability at later time points were not analyzed, partly to prevent contamination of the SEC column. In samples with lower pH stability (between 4.5 and 6.5), monomer loss was caused by an approximately 1% increase in HMW and LMW substances. Figures 6 to 9 For samples with higher pH stability (between 7.5 and 8), monomer loss was associated with a significant increase (approximately 10% to 14%) in both high molecular weight wheat (HMW) and low molecular weight wheat (LMW) substances. Formulations 2, 4, and 6 were the best performing formulations, with monomer losses of less than 1%.

[0293] Table 8: SEC results of the T=0 stability sample in Study 1

[0294]

[0295] Table 9: SEC results of the stability samples at 40℃ for Study 1 (T=1 week)

[0296]

[0297] Table 10: SEC results of the stability samples from Study 1 at 25℃ for T=2 weeks

[0298]

[0299] Table 11: SEC results of the stability samples from Study 1 at 5℃ for 3 weeks.

[0300]

[0301] These results indicate greater instability above pH 6.5. The use of citrate buffers leads to precipitation and reduced peptide solubility. Mannitol was found to be more beneficial than NaCl in maintaining solubility. The SEC results largely reflect the solubility conclusions, with a preferred pH range of approximately 5 to 6.

[0302] Study 2 Results: The optimal pH and buffer concentration for MANP were determined through visual inspection, peptide concentration, and pH.

[0303] Based on the results of Study 1, Study 2 focused on using buffers such as acetate and histidine (His) in a weakly acidic pH range, and included non-electrolytes as tension modifiers. The formulations tested are shown in Table 12 below.

[0304] Table 12: Formulation Design in Study 2

[0305]

[0306] Visual examination of the Study 2 samples revealed that they remained clear throughout the four-week timeframe (Table 13). A few formulations showed significant losses of peptide concentration (up to 92%), such as formulations 7, 9, 13, and 16 (Table 14). Many of these compositions contained ArgHCl. Actual peptide concentrations are plotted on... Figure 10 The relative loss of peptide concentration was plotted in [the following text is incomplete and requires further context]. Figure 11 The actual initial pH value is shown in the figure. Figure 12 middle.

[0307] Table 13: Visual characterization of stability samples from Study 2 at 5°C from T=0 to T=4 weeks

[0308]

[0309] Table 13 (continued): Visual characterization of stability samples from Study 2 at 5°C from T=0 to T=4 weeks

[0310]

[0311] Table 14: Peptide concentration and pH of stability samples in Study 2

[0312]

[0313] Results of Study 2: Determination of the optimal pH and buffer concentration for MANP by RP-HPLC

[0314] The stability of the sample was characterized by reversed-phase chromatography using the Primary reversed-phase method described in the above-described method section. Figure 13Examples of RP-HPLC chromatograms for Study 2 samples using this method are provided. RP-HPLC results for all time points in Study 2 are listed in Tables 15 to 18. Those formulations showing peptide content loss also exhibited significant increases in the first peaks 3 and 5. Figure 14A and Figure 14B ), and the loss of main peak intensity ( Figure 14C ).

[0315] The relative area changes of the two leading peaks and the main peak in RP-HPLC are shown in Figures 14A to 14C The results showed that most MANP formulations exhibited only minor differences, except for formulations 7, 9, 13, and 16. The loss of the main peak was considerable at high temperatures, but the loss was minimal for samples stored at 5°C. Figure 15 Linear extrapolation of the main peak purity using RP-HPLC indicated that multiple formulations from Study 3 maintained over 95% of the main peak relative intensity after one year at 5°C. Figure 16 In the most unstable formulations (7, 13, and 16), the post-peak also showed a significant increase. Figures 17A to 17C The relative change of peak 1 is shown in... Figure 18 middle.

[0316] Table 15: Inverse phase data of the T=0 stability sample in Study 2

[0317]

[0318] Table 15 (continued): Inverse phase data of the T=0 stability sample in Study 2

[0319]

[0320] Table 16: Reverse phase data of the stability samples at 40℃ for 1 week in Study 2

[0321]

[0322] Table 16 (continued): Reversed-phase data of the stability samples at 40℃ for 1 week in Study 2

[0323]

[0324] Table 17: Reverse-phase data of samples from the study of stability at 25℃ for 2 weeks (T=2 weeks)

[0325]

[0326] Table 17 (continued): Reversed-phase data of samples with stability at 25℃ for 2 weeks (T=2 weeks)

[0327]

[0328] Table 18: Reverse phase data of the stability samples at 5℃ for 4 weeks in the study 2

[0329]

[0330] Table 18 (continued): Reversed-phase data of the stability samples at 5℃ for 4 weeks in the study 2

[0331]

[0332] Study 2 Results: Determination of the optimal pH and buffer concentration for MANP using SEC

[0333] Stability samples from Study 2 were also analyzed using SEC, as summarized in Tables 19 to 22. The largest monomer losses appeared to occur in formulations 7, 13, and 16. Figure 19B ), which is accompanied by an increase in the latter peak 1 ( Figure 19C Other increases in subsequent peaks were also observed. Figures 20A to 20B This indicates that these samples were degraded to some extent through fragmentation or protein hydrolysis. Therefore, the monomer content reduction was more pronounced in these samples, although some loss was also observed in most formulations. Figure 21 However, many formulations showed monomer loss of <0.5% over a four-week period at 5°C.

[0334] Table 19: SEC results of the two samples with T=0 stability.

[0335]

[0336] Table 20: SEC results of the samples with stability at 40℃ for 1 week in Study 2

[0337]

[0338] Table 21: SEC results of samples with stability at 25℃ for 2 weeks (T=2 weeks)

[0339]

[0340] Table 22: SEC results of the samples with stability at 5℃ for 4 weeks in the study 2

[0341]

[0342] Study 3 Results: Optimal pH and buffer concentration of MANP were optimized through visual inspection, peptide concentration, and pH analysis.

[0343] A third study was initiated to optimize the optimal pH and buffer conditions for MANP. Furthermore, this study investigated combinations of sucrose, mannitol, HP-b-CD, and Gly as potential stabilizers. The twelve formulations evaluated in Study 3 are shown in Table 23.

[0344] Table 23: Formulation Design in Study 3

[0345]

[0346] Note: The out-of-phase data at T0 and T=4 weeks 5℃ were collected in triplicate and were not compared with those at T=2 weeks 5℃.

[0347] Similar to Study 2, visual inspection of these stability samples showed no evidence of particle formation, precipitation, or discoloration (Table 24). pH values ​​measured at each time point are summarized in Table 25. Figure 22 These values ​​were very close to the target values ​​and did not change significantly throughout the study. Similarly, peptide concentrations were close to the target values ​​and did not change throughout the study (Table 26). Figure 23 The pH range selected for this study provides adequate physical stability.

[0348] Table 24: Visual characterization of stability samples from Study 3 at T=0, T=2 weeks at 5℃, and T=4 weeks at 5℃.

[0349]

[0350] Table 25: pH of stability samples in Study 3

[0351]

[0352] Table 26: Peptide concentrations in stability samples from Study 3

[0353]

[0354] Results of Study 3: Optimal pH and buffer concentration of MANP were optimized using RP-HPLC.

[0355] The stability of formulation 3 at 5°C for up to four weeks was monitored using the primary RP-HPLC method. The RP-HPLC results measured by UV are summarized in Tables 27 and 28. During the four-week period at 5°C, the loss of chemical purity as determined by RP-HPLC was quite small (<0.5%) (Table 29). Formulations 3, 9, and 10 showed the least loss of purity during storage. These differences are illustrated graphically. Figure 24 and Figure 25 The extrapolation loss of the relative area of ​​the main peak in Study 3 samples using RP-HPLC with UV detection is shown in the figure. Figure 26 middle.

[0356] Table 27: Reversed-phase data of stability samples (formulations 1-6) at 5°C and T=0 weeks and T=4 weeks as determined by UV in Study 3

[0357]

[0358] Table 27 (continued): Reversed-phase data of stability at 5°C and T=0 and T=4 weeks by UV measurement for samples (formulations 1-6) in Study 3.

[0359]

[0360] Table 28: Reversed-phase data of stability samples (formulations 7-12) at 5°C and T=0 weeks (T=4 weeks) determined by UV in Study 3.

[0361]

[0362] Table 28 (continued): Reversed-phase data of stability samples (formulations 7-12) at 5°C and T=0 weeks (T=4 weeks) determined by UV in Study 3.

[0363]

[0364] Table 29: Purity of the reversed-phase main peak (mean and standard deviation) determined by UV in Study 3

[0365]

[0366] The RP-HPLC measurements discussed above were performed using UV detection. The RP-HPLC method also uses fluorescence detection runs in an effort to provide greater selectivity for peptides relative to other non-protein substances. Tables 30 and 31 provide a summary of RP-HPLC results using fluorescence detection. A comparison of the initial peak purity with the peak purity after storage at 5°C is shown in Table 32. Generally, the trends observed with UV detection are similar to those observed with RP-HPLC using fluorescence detection. Losses are <0.5%, and the ranking of optimal formulations is the same.

[0367] Table 30: Reversed-phase data of stability samples (formulations 1-6) at 5°C and T=0 weeks by fluorescence determination in Study 3.

[0368]

[0369] Table 30 (continued): Reversed-phase data of stability samples (formulations 1-6) at 5°C and T=0 weeks by fluorescence determination in Study 3.

[0370]

[0371] Table 31: Reversed-phase data of stability samples (formulation 7-12) at 5°C and T=0 weeks (T=4 weeks) determined by fluorescence assay in Study 3.

[0372]

[0373] Table 31 (continued): Reversed-phase data of stability samples (formulations 7-12) at 5°C and T=0 weeks by fluorescence determination in Study 3.

[0374]

[0375] Table 32: Purity of the reversed-phase main peak (mean and standard deviation) determined by fluorescence in Study 3

[0376]

[0377] Even after being stored at 5°C for four weeks, the relative area of ​​the RP-HPLC main peak remained close to 97%. Figure 27 and Figure 28 When extrapolated to 24 months, the purity of most main peaks remained above 93%. Figure 29 ).

[0378] Study 3 Results: Optimal pH and buffer concentration of MAP were optimized using SEC.

[0379] The same stability samples from Study 3 were analyzed using SEC. During four weeks of storage at 5°C, the monomer content showed almost no change (Table 33). Figure 30 As determined by SEC, there was almost no measurable loss of peptides due to aggregate formation. The main degradation pathway appears to be chemical in nature, therefore subsequent analysis focused on the chemical stability of MANP as determined by RP-HPLC.

[0380] Table 33: SEC data of samples from the study of stability at 5℃ for 3 weeks at T=0 and T=4.

[0381]

[0382] Since Studies 2 and 3 appeared to provide many promising formulations, further mathematical analysis of the data was performed. Various models were constructed using the PLS method, as shown below. The first PLS model used the peak purity (relative area) after four weeks at 5°C as the endpoint, drawing on the results of Studies 2 and 3 (a total of 28 formulations). No formulations were identified as outliers, with succinate being calculated as a significant factor. Predicted values ​​versus measured values ​​are shown below. Figure 31 In the calibration and validation sets, both exhibited very high r-values ​​(>0.92), indicating that the PLS model has fairly high quality. The regression coefficients are as follows: Figure 32 The values ​​provided are large and negative for succinate. The negative regression coefficient indicates that lower concentrations will produce higher levels of RPHPLC purity. In other words, succinate is considered a significant destabilizing factor in this model.

[0383] The response surface to the effects of pH and peptide concentration is shown in Figure 33Within the pH range used in these studies, the model predicted better chemical stability at higher pH values ​​(e.g., close to 6.0) compared to lower pH values ​​(close to 4.5 to 5.0). The effect of peptide concentration was relatively small, but the model indicated that stability at 2 mg / ml was comparable to that at lower peptide concentrations.

[0384] This response surface was generated in the absence of any buffer. If we investigate the effect of pH in the presence of acetate (…), Figure 34 The response is quite complex. Note that these are quadratic models, which allow for nonlinear responses as a function of concentration. Additionally, a pH buffer interaction term is specifically included. Therefore, the effect of acetate as a function of pH can and does vary. At lower pH values, acetate is predicted to have a stabilizing effect, but at higher pH values, it offers little (if any) benefit, while stability is higher. His buffers show a similar trend to acetate (…). Figure 35 However, the impact is relatively small. One buffer that is clearly and significantly harmful is succinate (…). Figure 36 ).

[0385] The model indicates that HP-b-CD tends to have lower stability, while the effect of Gly is almost zero. Figure 37 The prediction of the effects of mannitol and sucrose is non-linear. Figure 38 Sucrose reaches its maximum stability at around 100-200 mM. In contrast, ArgHCl has a greater effect and tends towards lower stability. Figure 39 Given the significant negative effects of ArgHCl and succinate, a new model was constructed to extract those formulations in order to identify the factors / excipients most favorable to stability.

[0386] The next PLS model used the same endpoint as the previous model (main peak purity determined by RP-HPLC after four weeks at 5°C), but excluded formulations containing succinate and ArgHCl. Two of the remaining formulations (Studies 3, F11 and Studies 2, F12) were identified as outliers. In this new model, many factors (peptide concentration, Gly, HP-b-CD, and mannitol) were found to be significant. The model's predicted values ​​versus the measured values ​​are shown in [Figure / Table / Insert Table ... Figure 40 The model is slightly noisier than previous models, partly due to the smaller number of formulations included. Regression coefficients are summarized in a bar chart. Figure 41 Significant factors are shown in blue shaded bars.

[0387] The effects of pH and peptide concentration in the second PLS model are shown in... Figure 42The model indicates that a slightly higher pH is beneficial, at least in the absence of a buffer. Simultaneously, higher stability is observed at higher peptide concentrations. Therefore, future research tends to focus on formulations at 2 mg / ml. If the peptide concentration is fixed at 2 mg / ml, the effects of acetate and pH can be investigated. Figure 43 At the upper end of the pH range (near pH 6.0), acetate is predicted to help stabilize MANP. Conversely, the model indicates that His would be harmful under the same conditions. Figure 44 Therefore, based on this analysis, acetate appears to be the preferred option.

[0388] The model predicts that both HP-b-CD and Gly will reduce chemical stability during storage. Figure 45 Conversely, both mannitol and sucrose showed some degree of stabilizing effect. Figure 46 To provide some degree of quantification for the model predictions, the chemical purity in the absence of excipients is shown as 97.3% (as indicated by the circle in the lower left corner). In the presence of 250 mM sucrose, the purity at storage increases to 97.6%. However, the addition of mannitol appears to be slightly more helpful, thus improving storage stability to 97.8% at the same concentration. Given that mannitol and sucrose appear to have some stabilizing effect, the effects of acetate and pH incorporating 250 mM sucrose were re-plotted. Figure 47 Response surface methodology indicates that acetate will improve stability even in the presence of stabilizers such as sucrose, if used at pH 6.0 and the maximum concentration (40 mM).

[0389] Results of Study 3: Optimal pH and buffer concentration of MANP were optimized using RP-HPLC.

[0390] The fourth study investigated the effect of surfactants on the stability of MANP using two different optimized base formulations (40 mM acetate, 270 mM sucrose, pH 5.5 and pH 6.0). The design of Study 4 is shown in Table 4.

[0391] Table 34: Formulation Design in Study 4

[0392]

[0393] Study 4 Results: The surfactants and base formulation of MANP were determined through visual inspection, peptide concentration, and pH.

[0394] Visual examination of the samples revealed no evidence of particulate matter or turbidity, except for some samples after stirring (Table 35). This included two samples without added surfactant (1 and 7) and a pH 5.5 formulation containing HP-b-CD instead of a surfactant. Apart from this, all samples remained clear even after stress. The pH of the samples remained almost unchanged even after stirring or repeated freeze-thaw (F / T) cycles (Table 36). Figure 48 and Figure 49 Peptide concentrations were also unaffected by these two stresses (Table 37). Figure 50 and Figure 51 ).

[0395] Table 35: Visual characterization of the freeze-thaw and stirring stability of the four samples studied.

[0396]

[0397] Table 36: pH of samples from Study 4 after stirring and freeze-thaw cycles

[0398]

[0399] Table 37: Peptide concentrations in the stirred and freeze-thawed samples from Study 4

[0400]

[0401] Results of Study 4: The surfactants and basic formulation of MANP were determined by reversed-phase chromatography.

[0402] In Study 4, the effects of F / T and stirring on the MANP formulation were evaluated using the RP-HPLC Primary method. These results are listed in Tables 38 and 39. Some minor purity loss was observed in the stirred samples (…). Figure 52 However, samples exposed to the F / T cycle suffered almost no loss (if any). Figure 53 The loss plot of relative main peak area determined by RP-HPLC indicates that there is little difference between formulations based on surfactant composition. Figure 54 This is to be expected when assessing chemical stability under short-term interfacial stress.

[0403] Table 38: Reversed-phase data of the stirred samples in Study 4

[0404]

[0405] Table 38 (continued): Reversed-phase data of the stirred samples in Study 4

[0406]

[0407] Table 39: Reversed-phase data of freeze-thawed samples from Study 4

[0408]

[0409] Table 39 (continued): Reversed-phase data of the stirred samples from Study 4

[0410]

[0411] Study 4 Results: Determination of MANP's surfactants and base formulation via SEC

[0412] These same Study 4 samples were also tested using SEC to determine whether any aggregation occurred under interfacial stress. These SEC results are shown in Tables 40 and 41. Stirred samples showing turbidity were not analyzed. For the remaining stirred samples, there were some minor variations in monomer content (…). Figure 55 The same applies to samples exposed to F / T cycles. Figure 56 The monomer loss of the four samples before and after stress is illustrated in the figure below. Figure 57 Overall, PS 20 appears to perform slightly better than PS 80. Furthermore, the lowest concentration of PS 20 appears to provide adequate stability. Additionally, stability at pH 6.0 appears to be slightly better than at pH 5.5.

[0413] Table 40: SEC results for Study 4 samples with and without stirring stress (AG) and QS

[0414]

[0415] Table 41: SEC results for Study 4 samples with and without stirring stress (AG) and QS

[0416]

[0417] Study 5: Effect of methionine oxidation on MANP stability

[0418] A final study was conducted to determine the formulation of MANP. Due to the oxidative potential of MANP to Met, four formulations were prepared, as listed in Table 42. This study will help determine whether the addition of a certain amount of free Met helps reduce peptide oxidation.

[0419] Table 42: Study on the design of 5-methionine formulations

[0420]

[0421] Study 5 Results: Methionine oxidation of MANP was determined by visual characterization, peptide concentration, and pH.

[0422] Each sample was evaluated by visual inspection (Table 46). All samples remained clear and colorless throughout Study 5. No significant changes in pH or peptide concentration occurred during the study (Table 47). Peptide concentrations of individual stored samples ( Figure 58 ) and pH value ( Figure 59 The figure illustrates that these values ​​changed very little during the storage stability study.

[0423] Table 43: Stability of MET Samples from Study 5: Visual Characterization, pH, Osmolarity, and Peptide Concentration of Samples

[0424]

[0425] Results of Study 5: Determination of methionine oxidation in MANPs by reversed-phase chromatography

[0426] The analysis of the five samples by RP-HPLC focused on maintaining the overall purity of the main peak, but also on minimizing the elution of Met oxides at approximately 19 minutes. Figure 60 RP-HPLC data are summarized in Table 44. If the relative areas of the major Met oxidation products are plotted as a function of free Met concentration (…), Figure 61 As the Met content increases, oxidation shows a moderate but discernible decrease. Studies on the effect of free Met concentration indicate that adding Met at 5 mM provides some protection, but higher concentrations do not significantly improve the situation. Figure 62 If we study the purity of the main peak, we can see a similar trend (). Figure 63 and Figure 64 ).

[0427] Table 44: Inverted Data from Study 5

[0428]

[0429] Table 44 (continued): Study 5 Inverted Data

[0430]

[0431] Results of Study 5: Determination of Methionine Oxidation in MANPs via SEC

[0432] Samples from Study 5 were also analyzed using SEC because interfacial stress typically produces aggregates and high molecular weight (HMW) substances. HMW substances showed a certain increase, particularly at 25°C and 40°C (Table 45). The amounts and changes of the leading, main, and trailing peaks are plotted on... Figures 65 to 70 In the middle. If we see the effect of Met at a given point in time ( Figures 68 to 70 If ), then Met has almost no effect on the degree of aggregation.

[0433] Table 45: SEC Data from Study 5

[0434]

[0435] Study 5 demonstrates that even the addition of only 5 mM Met can protect MANP from Met oxidation to some extent. Meanwhile, Met appears to have no detrimental effect on the physical stability of these formulations (free Met also provides no protection against moderate aggregation that occurs during interfacial stress).

[0436] in conclusion

[0437] Five rounds of formulation screening were conducted on MANP. Early in the project, higher pH values ​​and the use of succinate buffers were found to be detrimental to physical stability, with precipitation and turbidity observed. Overall, the primary degradation pathway was chemical instability. Aggregation occurred minimally under storage conditions. Even under interfacial stress (stirring, F / T cycling), aggregation was minimal. Therefore, shelf-life limitation will be related to the ability to control chemical degradation, as measured by RP-HPLC.

[0438] The optimal pH appears to be close to 6.0, where acetate is the preferred buffer. At this pH, a 40 mM acetate concentration appears to favor maximizing the purity of the RP-HPLC main peak over time at 5 °C. His is a potential alternative buffer. Both mannitol and sucrose appear to be good stabilizers and can also act as stress modifiers. The use of electrolytes (ArgHCl, NaCl) leads to greater chemical instability. Adding a small amount of PS 20 (0.01 or 0.02%) appears to protect against interfacial stress. Furthermore, adding a small amount of free Met (5 mM) reduces the degree of Met oxidation in MANP.

[0439] Example 2: Stability Verification Study

[0440] Four formulations were prepared with peptide concentrations set at 2 mg / mL, PS 20 at 0.02%, and pH ranges from 5 to 6 (Table 46). The formulations were buffered with histidine and acetate at concentrations ranging from 10 mM to 40 mM. Stabilizers / strain modifiers were sucrose and mannitol at concentrations ranging from 200 mM to 250 mM. Previous studies have shown that HP-β-CD improves peptide stability and was included in validation studies at 25 mM.

[0441] Formulation stability tests were conducted for up to 6 months at 5°C, 25°C, and 40°C (Table 47). The 5°C samples had two different placement orientations: non-inverted and inverted, with the formulation in contact with the cap when inverted.

[0442] Table 46: Validation Formulations

[0443]

[0444] Table 47: Temperature Study

[0445]

[0446] General Sample Preparation

[0447] The 10 mg / mL peptide solution was prepared on the same day of preparation, and the 2X buffer was prepared the day before. The 10 mg / mL peptide solution was prepared by weighing the peptide into a sterile container and adding Milli-Q water to achieve the target peptide concentration. The volume of the 10 mg / mL peptide solution was calculated based on the target sample volume. The calculated volume of peptide was then added to a new sterile container. The volume of 2X buffer added to the peptide solution was half the target sample volume. The pH of the peptide and 2X buffer was then measured using a pH probe. If the pH value exceeded ± 0.1 pH units, the sample pH was adjusted with 0.1 M NaCl. The sample was then brought to the target sample volume with Milli-Q water, and the pH value and peptide concentration were measured again.

[0448] The samples were aseptically filtered through a clean hood wiped with 70% ethanol. Each formulation was loaded into a sterile syringe equipped with a sterile filter. The sample was then slowly pushed through the filter into a sterile container. After sterile filtration, the samples were placed into vials.

[0449] pH value, osmotic pressure, and peptide concentration

[0450] The validation samples were characterized by visual inspection, pH value, and peptide concentration, as shown in Table 48.

[0451] The pH of each formulation was measured, and the measured sample pH was found to be within ±0.1 of the target pH. Before starting the analysis, the pH probe was calibrated using three pH standards ordered from Fisher. The pH of the formulation was measured by inserting the pH probe into the sample and waiting for the measurement to stabilize, which may take 1 to 2 minutes. After the analysis, the pH probe was rinsed with 18 MΩ water for one minute and stored in pH storage solution.

[0452] The peptide concentration of each formulation was measured using SoloVPE. The operator measured the sample concentration by adding 100 μL of material to a small, disposable UV container of SoloVPE. A new fiber optic probe was installed, and the instrument used a 1.58 mL*mg... -1 cm -1 The extinction coefficient is used to measure the sample absorbance and correct for background scattering. After analysis, the sample is removed from the disposable container using a pipette. Both the disposable container and the fiber optic probe are then discarded. This procedure is repeated for each sample.

[0453] During the 6-month period, formulations 2, 3, and 4 showed no signs of physical instability; the solutions remained unchanged in color, were visually clear, and no signs of particle formation were observed. Formulation 1 showed no signs of physical instability for up to 3 months, but a gel formed at the bottom of the vial at 6 months.

[0454] Table 48: Visual Characterization of Validation Samples

[0455]

[0456]

[0457] *Sample inverted

[0458] The pH value, peptide concentration, and osmotic pressure of the verification samples were measured and are shown in Table 49.

[0459] Osmometry analysis was performed using an Advanced Instruments Osmo 1. At the start of the analysis, a reference standard of 290 mOsm was analyzed to ensure the instrument was functioning correctly. After the reference standard was passed, the sample was analyzed. 20 μL of material was removed and analyzed using the Osmo 1. After analysis, the chamber was cleaned using a chamber cleaner. This procedure was repeated for each sample.

[0460] Osmolarity was measured only at T=0 and ranged from 315 to 392 mOsm / kgH2O. Throughout the study, the pH change for all samples was less than 0.05 compared to the T=0 value, meaning no significant changes were observed during the study. Figure 71 and Figure 72 ).

[0461] Table 49: pH, osmotic pressure, and peptide concentration of the validation samples

[0462]

[0463]

[0464] *Sample inverted

[0465] The peptide concentrations of formulations 2, 3, and 4 changed by less than 10% after 6 months at 5°C. Figure 73 and Figure 74 When stored at 40°C for more than 3 months, the peptide concentrations of all formulations decreased, while this phenomenon was not observed at 5°C and 25°C. Figure 74 Unlike other formulations, Formulation 1 did experience a significant decrease in peptide concentration after 6 months of stable storage at 5°C, from approximately 2 mg / mL to approximately 0.8 mg / mL. Figure 73 The loss of peptide concentration in Formulation 1 at 5°C appears to be due to the formation of a gel by the peptides at the 6-month time point.

[0466] When predicting monthly peptide concentration loss at three temperatures, formulation 1 showed the greatest loss compared to other formulations. Figure 75 The peptide concentration of formulation 4 is predicted to remain unchanged when stored at 5°C and 25°C.

[0467] Reversed-phase high-performance liquid chromatography (RP-HPLC)

[0468] A reversed-phase (RP) HPLC method was developed for formulation work because it can detect and quantify a variety of degradation products. Figure 76 Therefore, this method provides a reliable indication of the chemical purity of MANP. In short, the parameters used for RP-HPLC analysis are as follows:

[0469] Column: XSelect CSH, C18, 3.5 µm, 4.6 mm x 150 mm, from Waters (part number 186005270).

[0470] Mobile phase A: 15% acetonitrile, 85% water, 0.1% TFA.

[0471] Mobile phase B: 30% acetonitrile, 70% water, 0.1% TFA.

[0472] Mobile phase C: 80% acetonitrile, 20% water, 0.1% TFA.

[0473] Sample concentration (mg / mL): 2 mg / mL stock solution injection.

[0474] Autosampler temperature: 8 ± 3℃

[0475] Column temperature: 40℃

[0476] Flow rate: 0.4 mL / min

[0477] Injection volume: 1.25 μL

[0478] UV settings: 220 nm, and

[0479] Data acquisition time: 10 Hz.

[0480] Table 50: Gradient of Reverse-Phase Flow Phase

[0481]

[0482] Figure 76 Exemplary reversed-phase chromatograms with suggested peak identification are shown. Results of RP-HPLC analysis of the validation study samples over a 6-month period are listed in Tables 51 to 54.

[0483] Table 51: RP-HPLC data at T=0 and T=1 months

[0484]

[0485] Table 52: RP-HPLC data for T=1 month

[0486]

[0487] Table 53: RP-HPLC data at T=3 months

[0488]

[0489] Table 54: RP-HPLC data at T=6 months

[0490]

[0491] *Sample inverted

[0492] The results showed that, after 6 months at 5°C, formulation 4 showed the best performance with a monthly loss of 0.18%, compared to formulation 1 which had the worst stability (0.74% monthly loss). Figure 77 Similar results were observed when the sample was inverted. Figure 78 When the sample is stored at 25°C, the loss of the relative area of ​​the main peak increases to 3.5% to 4%. Figure 79 When the sample is stored at 40°C, the loss of the relative area of ​​the main peak increases to 18-22%. Figure 80 ).

[0493] Figure 81 The predicted loss of the main peak is shown monthly. When the relative area of ​​the main peak at 5°C for 12 months was predicted by RP-HPLC, Formulation 1 had lost 8.93% ( Figure 82 In contrast, formulation 4 showed a loss of only 2.21% after 12 months. Based on RP-HPLC data at 5°C, the stability of formulation 3 was only slightly lower. Inverting the vials for storage did not significantly alter the estimated degradation rate. Figure 83 At 25°C, the degradation of these most stable formulations increased significantly (more than 20-fold). Figure 84 At 40°C, the degradation of even the most stable formulations also increased significantly. Figure 85 ).

[0494] Size exclusion chromatography

[0495] Stability was also monitored using SEC, although formulation screening clearly indicated that the main degradation process was associated with chemical instability. High molecular weight (HMW) substances marked as leading peaks in the SEC were ≤0.50% at T0 (Table 55). After six months at 5°C, no formulations showed HMW levels ≥0.50% (Table 57). A graphical representation of monomer loss as determined by SEC under different storage conditions is shown in... Figures 86 to 89 Based on these results, the monthly losses at different storage temperatures were estimated. Figure 90Based on these data, formulation 1 exhibits the worst stability, while formulation 4 demonstrates the best stability, particularly when stored at 5°C. Figures 91 to 94 ).

[0496] Table 55: Size Exclusion Chromatography Data at T=0 and T=1 Months

[0497]

[0498] Table 56: Size Exclusion Chromatography Data at T=3 Months

[0499]

[0500] Table 57: Size Exclusion Chromatography Data at T=6 Months

[0501]

[0502] *Sample inverted

[0503] Subvisible particle analysis (Flow Cam)

[0504] The subvisible particle (SVP) levels of these validation samples were measured and are summarized in Table 58. While some samples arrived at GLB thawed, the six-month-old samples remained intact due to dry ice transport. The data clearly show that Formulation 1 exhibits a significantly increased SVP level compared to the other samples. The other three compositions have moderate SVP levels over a larger size range and are all within typical USP limits. Formulation 3 may have a slightly lower SVP level, but all formulations demonstrate significantly better stability than Formulation 1.

[0505] Table 58: Flow Cam (particle analysis) data from T=0 to T=6 months

[0506]

[0507] *Sample inverted

[0508] in conclusion

[0509] Four different MANP formulations were subjected to a six-month stability study at 5°C, 25°C, and 40°C. During the study, appearance, peptide content, and pH remained virtually unchanged (if any). Stability was monitored using two different HPLC methods: RP-HPLC and SEC. MANPs are more sensitive to chemical instability, therefore RP-HPLC provided the most sensitive and detailed stability assessment. These results showed that formulation 4 was the most stable, with formulation 3 providing almost the same good stability profile. Both were formulated at pH 5.5 using acetate buffer. Formulation 1 was significantly less stable than the other formulations, as can be seen from the SEC results. Formulation 1 also showed the highest level of SVP. Therefore, formulation 4, containing sucrose as a stabilizer at pH 5.5, is the preferred candidate, with formulation 3 being the best alternative candidate.

[0510] Example

[0511] As will be apparent from the foregoing, aspects of this disclosure can be embodied in various ways, including but not limited to the following:

[0512] Example 1: A composition comprising MANP, acetate, sucrose and polysorbate 20.

[0513] Example 2: The composition according to Example 1, wherein the concentration of MANP is about 2 mg / ml.

[0514] Example 3: The composition according to Example 1, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0515] Example 4: The composition according to Example 1, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0516] Example 5: The composition according to Example 1, wherein the concentration of the acetate is about 10 mM.

[0517] Example 6: The composition according to Example 1, wherein the concentration of sucrose is in the range of about 250 mM to about 275 mM.

[0518] Example 7: The composition according to Example 1, wherein the concentration of sucrose is about 275 mM.

[0519] Example 8: The composition according to Example 1, wherein the concentration of polysorbate 20 is about 0.02%.

[0520] Example 9: The composition according to Example 1, wherein the pH is about 5.5.

[0521] Example 10: The composition according to Example 1, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0522] Example 11: The composition according to Example 1, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0523] Example 12: A composition comprising MANP, acetate, mannitol and polysorbate 20.

[0524] Example 13: The composition according to Example 12, wherein the concentration of MANP is about 2 mg / ml.

[0525] Example 14: The composition according to Example 12, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0526] Example 15: The composition according to Example 12, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0527] Example 16: The composition according to Example 12, wherein the concentration of the acetate is about 40 mM.

[0528] Example 17: The composition according to Example 12, wherein the concentration of mannitol is in the range of about 250 mM to about 275 mM.

[0529] Example 18: The composition according to Example 12, wherein the concentration of mannitol is about 250 mM.

[0530] Example 19: The composition according to Example 12, wherein the concentration of polysorbate 20 is about 0.02%.

[0531] Example 20: The composition according to Example 12, wherein the pH is about 5.5.

[0532] Example 21: The composition according to Example 12, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0533] Example 22: The composition according to Example 12, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0534] Example 23: A composition for treating hypertension comprising MANP, acetate, sucrose and polysorbate 20.

[0535] Example 24: The composition according to Example 23, wherein the concentration of MANP is about 2 mg / ml.

[0536] Example 25: The composition according to Example 23, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0537] Example 26: The composition according to Example 23, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0538] Example 27: The composition according to Example 23, wherein the concentration of the acetate is about 10 mM.

[0539] Example 28: The composition according to Example 23, wherein the concentration of sucrose is in the range of about 250 mM to about 275 mM.

[0540] Example 29: The composition according to Example 23, wherein the concentration of sucrose is about 275 mM.

[0541] Example 30: The composition according to Example 23, wherein the concentration of polysorbate 20 is about 0.02%.

[0542] Example 31: The composition according to Example 23, wherein the pH is about 5.5.

[0543] Example 32: The composition according to Example 23, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0544] Example 33: The composition according to Example 23, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0545] Example 34: A composition for treating hypertension comprising MANP, acetate, mannitol and polysorbate 20.

[0546] Example 35: The composition according to Example 34, wherein the concentration of MANP is about 2 mg / ml.

[0547] Example 36: The composition according to Example 34, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0548] Example 37: The composition according to Example 34, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0549] Example 38: The composition according to Example 34, wherein the concentration of the acetate is about 40 mM.

[0550] Example 39: The composition according to Example 34, wherein the concentration of mannitol is in the range of about 250 mM to about 275 mM.

[0551] Example 40: The composition according to Example 34, wherein the concentration of mannitol is about 250 mM.

[0552] Example 41: The composition according to Example 34, wherein the concentration of polysorbate 20 is about 0.02%.

[0553] Example 42: The composition according to Example 34, wherein the pH is about 5.5.

[0554] Example 43: The composition according to Example 34, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0555] Example 44: The composition according to Example 34, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0556] Example 45: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20.

[0557] Example 46: A pharmaceutical composition comprising about 2 mg / ml MANP, about 40 mM acetate, about 250 mM mannitol and about 0.02% polysorbate 20.

[0558] Example 47: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein less than 0.5% of monomer aggregates are present after storage at 2-8°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0559] Example 48: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 1% after being stored at 5°C for 6 months, as measured by the height of the main peak obtained by reversed-phase HPLC.

[0560] Example 49: The pharmaceutical composition according to Example 48, wherein the purity loss of the composition is less than 0.5% after storage at 5°C for 6 months, as measured by the height of the main peak obtained by reversed-phase HPLC.

[0561] Example 50: The pharmaceutical composition according to Example 48, wherein the purity loss after storage at 5°C for 6 months is less than 0.2%, as measured by the height of the main peak obtained by reversed-phase HPLC.

[0562] Example 51: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 10% after being stored at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

[0563] Example 52: The pharmaceutical composition according to Example 51, wherein the purity loss of the composition is less than 5% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

[0564] Example 53: The pharmaceutical composition according to Example 51, wherein the purity loss of the composition is less than 3% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

[0565] Example 54: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 10% after being stored at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

[0566] Example 55: The pharmaceutical composition according to Example 54, wherein the purity loss of the composition is less than 5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

[0567] Example 56: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 0.2% after being stored at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0568] Example 57: The pharmaceutical composition according to Example 56, wherein the purity loss after storage at 5°C for 12 months is less than 0.18%, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0569] Example 58: A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 0.5% after being stored at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0570] Example 59: The pharmaceutical composition according to Example 58, wherein the purity loss of the composition is less than 0.35% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

[0571] Example 60: A composition comprising MANP, a buffer, a stabilizer / strainer, and a nonionic surfactant.

[0572] Example 61: The composition according to Example 60, wherein the buffer is selected from the group consisting substantially of: acetate, acetic acid, alanine, arginine, aspartic acid, bicarbonate, N,N-bis(2-hydroxyethyl)glycine (bicine), carbonate, citrate, citric acid, glycine, glycylglycine, glutamic acid, histidine, lysine, malic acid, potassium phosphate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, sodium succinate, succinic acid, sulfate, nitrate, maleic acid, fumaric acid, tartaric acid, aspartic acid, N-tris(hydroxymethyl)methylglycine (tricine), tris(hydroxymethyl)aminomethane, and tromethamine.

[0573] Example 62: The composition according to Example 61, wherein the buffer is an acetate.

[0574] Example 63: The composition according to Example 62, wherein the concentration of the acetate is in the range of about 10 mM to 40 mM.

[0575] Example 64: The composition according to Example 62, wherein the concentration of the acetate is about 10 mM.

[0576] Example 65: The composition according to Example 62, wherein the concentration of the acetate is about 40 mM.

[0577] Example 66: The composition according to Example 60, wherein the stabilizer / tension agent is selected from the group consisting essentially of: albumin, arginine, Brij 30, Brij 35, glucose, dimethyl sulfone, ethylenediaminetetraacetic acid, glycerol, glycerol, glycine, guanine, hydroxypropyl-β-cyclodextrin, lactose monohydrate, magnesium chloride, maltose, mannitol, methionine, 2-methylthioethanol, monothioglycerol, inositol, potassium chloride, poloxamer, polyethylene glycol, polysorbate 20, polysorbate 80, polyvinyl alcohol, polyvinylpyrrolidone, propylene glycol, protamine sulfate, sodium chloride, sorbitol, sucrose, thioglycolic acid, trehalose, and Triton.

[0578] Example 67: The composition according to Example 60, wherein the stabilizer / tension agent is sucrose.

[0579] Example 68: The composition according to Example 67, wherein the concentration of sucrose is about 275 mM.

[0580] Example 69: The composition according to Example 60, wherein the stabilizer / tension agent is mannitol.

[0581] Example 70: The composition according to Example 69, wherein the concentration of mannitol is about 250 mM.

[0582] Example 71: The composition according to Example 60, wherein the nonionic surfactant is selected from the group consisting substantially of: sorbitol polyoxyglyceride, polysorbate 20, polysorbate 40, sodium docusate, polysorbate 60, polysorbate 80, benzalkonium chloride, capryloyl hexanoyl polyoxyglyceride, hexadecyl pyridine chloride, lauroyl polyoxyglyceride, linoleyl polyoxyglyceride, octylphenyl polyol 9, oleoyl polyoxyglyceride, poloxamer, polyoxyethylene 10 oleyl ether, polyoxyethylene Ethylene 15-hydroxystearate, nonyl alcohol ether 9, polyoxyethylene 20-cetearyl alcohol ether, polyoxyethylene 40-stearate, pullulan, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, sodium lauryl sulfate, sorbitol monolaurate, sorbitol monooleate, polyoxyethylene stearate, sorbitol monopalmitate, sorbitol monostearate, stearoyl polyoxyglyceride, sorbitol sesquioleate, sorbitol trioleate, and teroxaprol.

[0583] Example 72: The composition according to Example 60, wherein the nonionic surfactant is polysorbate 20.

[0584] Example 73: The composition according to Example 71, wherein the concentration of polysorbate 20 is about 0.2%.

[0585] Example 74: The composition according to Example 60, wherein the pH is about 5.5.

[0586] Example 75: The composition according to Example 60, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0587] Example 76: The composition according to Example 60, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0588] Example 77: A vial containing a formulation comprising MANP, acetate, sucrose and polysorbate 20.

[0589] Example 78: The vial according to Example 77, wherein the concentration of MANP is about 2 mg / ml.

[0590] Example 79: The vial according to Example 77, wherein the MAP consists essentially of SEQ ID NO: 1.

[0591] Example 80: The vial according to Example 77, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0592] Example 81: The vial according to Example 77, wherein the concentration of the acetate is about 10 mM.

[0593] Example 82: The vial according to Example 77, wherein the concentration of sucrose is about 275 mM.

[0594] Example 83: The vial according to Example 77, wherein the concentration of polysorbate 20 is about 0.02%.

[0595] Example 84: A vial according to Example 77, wherein the pH is about 5.5.

[0596] Example 85: The vial according to Example 77, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0597] Example 86: The vial according to Example 77, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0598] Example 87: A lyophilized powder prepared according to the following steps:

[0599] (a) Combination in liquid solution: MANP, acetate, sucrose, and polysorbate 20; and

[0600] (b) The combination described in freeze-drying step (a)

[0601] Example 88: The lyophilized powder according to Example 87, wherein the concentration of MANP in the liquid composition is about 2 mg / ml.

[0602] Example 89: The freeze-dried powder according to Example 87, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0603] Example 90: The lyophilized powder according to Example 87, wherein the concentration of the acetate in the liquid composition is in the range of about 10 mM to about 40 mM.

[0604] Example 91: The lyophilized powder according to Example 87, wherein the concentration of the acetate in the liquid composition is about 10 mM.

[0605] Example 92: The freeze-dried powder according to Example 87, wherein the concentration of sucrose in the liquid composition is in the range of about 250 mM to about 275 mM.

[0606] Example 93: The freeze-dried powder according to Example 87, wherein the concentration of sucrose in the liquid composition is about 275 mM.

[0607] Example 94: The freeze-dried powder according to Example 87, wherein the concentration of polysorbate 20 in the liquid composition is about 0.02%.

[0608] Example 95: The lyophilized powder according to Example 87, wherein the pH of the liquid composition is about 5.5.

[0609] Example 96: The freeze-dried powder according to Example 87, wherein the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O.

[0610] Example 97: The freeze-dried powder according to Example 87, wherein the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

[0611] Example 98: A lyophilized powder prepared according to the following steps:

[0612] a. Combination in liquid solution: MANP, acetate, mannitol, and polysorbate 20; and

[0613] b. The combination described in freeze-drying step (a).

[0614] Example 99: The lyophilized powder according to Example 98, wherein the concentration of MANP in the liquid composition is about 2 mg / ml.

[0615] Example 100: The freeze-dried powder according to Example 98, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0616] Example 101: The lyophilized powder according to Example 98, wherein the concentration of the acetate in the liquid composition is in the range of about 10 mM to about 40 mM.

[0617] Example 102: The lyophilized powder according to Example 98, wherein the concentration of the acetate in the liquid composition is about 40 mM.

[0618] Example 103: The lyophilized powder according to Example 98, wherein the concentration of mannitol in the liquid composition is in the range of about 250 mM to about 275 mM.

[0619] Example 104: The freeze-dried powder according to Example 98, wherein the concentration of mannitol in the liquid composition is about 250 mM.

[0620] Example 105: The freeze-dried powder according to Example 98, wherein the concentration of polysorbate 20 in the liquid composition is about 0.02%.

[0621] Example 106: The lyophilized powder according to Example 98, wherein the pH of the liquid composition is about 5.5.

[0622] Example 107: The freeze-dried powder according to Example 98, wherein the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O.

[0623] Example 108: The freeze-dried powder according to Example 98, wherein the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

[0624] Example 109: A dry powder composition comprising MANP, acetate, sucrose and polysorbate 20.

[0625] Example 110: A powder prepared by spray drying, wherein the spray drying includes the following steps:

[0626] a. Provide a liquid containing MAMP, acetate, sucrose, and polysorbate 20; and

[0627] b. Spray dry the liquid from step (a) using a spray drying apparatus.

[0628] Example 111: A freeze-dried powder prepared by freeze-drying, wherein the freeze-drying includes the following steps:

[0629] a. Provide a liquid containing MAMP, acetate, sucrose, and polysorbate 20; and

[0630] b. Freeze-dry the liquid from step (a) at a certain temperature for a sufficient time to convert the liquid composition into a solid.

[0631] Example 112: A lyophilized powder prepared by a method comprising the following steps:

[0632] a. Provide a liquid containing MAMP, acetate, sucrose, and polysorbate 20; and

[0633] b. The liquid from step (a) is freeze-dried.

[0634] Example 113: A prefilled syringe containing MANP, acetate, sucrose and polysorbate 20.

[0635] Example 114: A pre-filled syringe according to Example 113, wherein the concentration of MANP is about 2 mg / ml.

[0636] Example 115: The pre-filled syringe according to Example 113, wherein the MANP is substantially composed of SEQ ID NO: 1.

[0637] Example 116: The pre-filled syringe according to Example 113, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

[0638] Example 117: The pre-filled syringe according to Example 113, wherein the concentration of the acetate is about 10 mM.

[0639] Example 118: The pre-filled syringe according to Example 113, wherein the concentration of sucrose is about 275 mM.

[0640] Example 119: The pre-filled syringe according to Example 113, wherein the concentration of polysorbate 20 is about 0.02%.

[0641] Example 120: A pre-filled syringe according to Example 113, wherein the pH is about 5.5.

[0642] Example 121: A pre-filled syringe according to Example 113, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

[0643] Example 122: A pre-filled syringe according to Example 113, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

[0644] Although the invention has been described with reference to specific aspects, those skilled in the art will understand that various changes can be made and elements of the invention can be substituted with equivalents without departing from the scope of the invention. Furthermore, many modifications can be made to specific aspects or materials taught in the invention without departing from the scope of the invention. Therefore, the invention is not intended to be limited to the specific aspects disclosed, but rather to encompass all aspects falling within the scope and spirit of the appended claims.

Claims

1. A composition comprising MAMP, acetate, sucrose, and polysorbate 20.

2. The composition according to claim 1, wherein the concentration of MANP is about 2 mg / ml.

3. The composition according to claim 1, wherein the MAP is substantially composed of SEQ ID NO:

1.

4. The composition according to claim 1, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

5. The composition according to claim 1, wherein the concentration of the acetate is about 10 mM.

6. The composition according to claim 1, wherein the concentration of sucrose is in the range of about 250 mM to about 275 mM.

7. The composition according to claim 1, wherein the concentration of sucrose is about 275 mM.

8. The composition according to claim 1, wherein the concentration of the polysorbate 20 is about 0.02%.

9. The composition according to claim 1, wherein the pH is about 5.

5.

10. The composition according to claim 1, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

11. The composition according to claim 1, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

12. A composition comprising MANP, acetate, mannitol and polysorbate 20.

13. The composition according to claim 12, wherein the concentration of MANP is about 2 mg / ml.

14. The composition of claim 12, wherein the MANP is substantially composed of SEQ ID NO:

1.

15. The composition of claim 12, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

16. The composition according to claim 12, wherein the concentration of the acetate is about 40 mM.

17. The composition of claim 12, wherein the concentration of mannitol is in the range of about 250 mM to about 275 mM.

18. The composition according to claim 12, wherein the concentration of mannitol is about 250 mM.

19. The composition according to claim 12, wherein the concentration of the polysorbate 20 is about 0.02%.

20. The composition according to claim 12, wherein the pH is about 5.

5.

21. The composition according to claim 12, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

22. The composition according to claim 12, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

23. A composition for treating hypertension, the composition comprising MANP, acetate, sucrose and polysorbate 20.

24. The composition according to claim 23, wherein the concentration of MANP is about 2 mg / ml.

25. The composition of claim 23, wherein the MANP is substantially composed of SEQ ID NO:

1.

26. The composition of claim 23, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

27. The composition according to claim 23, wherein the concentration of the acetate is about 10 mM.

28. The composition of claim 23, wherein the concentration of said sucrose is in the range of about 250 mM to about 275 mM.

29. The composition according to claim 23, wherein the concentration of sucrose is about 275 mM.

30. The composition of claim 23, wherein the concentration of the polysorbate 20 is about 0.02%.

31. The composition according to claim 23, wherein the pH is about 5.

5.

32. The composition according to claim 23, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

33. The composition according to claim 23, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

34. A composition for treating hypertension, the composition comprising MANP, acetate, mannitol and polysorbate 20.

35. The composition of claim 34, wherein the concentration of MANP is about 2 mg / ml.

36. The composition of claim 34, wherein the MANP is substantially composed of SEQ ID NO:

1.

37. The composition of claim 34, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

38. The composition according to claim 34, wherein the concentration of the acetate is about 40 mM.

39. The composition of claim 34, wherein the concentration of mannitol is in the range of about 250 mM to about 275 mM.

40. The composition according to claim 34, wherein the concentration of mannitol is about 250 mM.

41. The composition according to claim 34, wherein the concentration of polysorbate 20 is about 0.02%.

42. The composition according to claim 34, wherein the pH is about 5.

5.

43. The composition according to claim 34, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

44. The composition according to claim 34, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

45. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20.

46. ​​A pharmaceutical composition comprising about 2 mg / ml MANP, about 40 mM acetate, about 250 mM mannitol and about 0.02% polysorbate 20.

47. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein less than 0.5% of monomer aggregates are present after storage at 2-8°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

48. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition loses less than 1% of purity after being stored at 5°C for 6 months, as measured by the height of the main peak obtained by reversed-phase HPLC.

49. The pharmaceutical composition of claim 48, wherein the purity loss of the composition is less than 0.5% after storage at 5°C for 6 months, as measured by the height of the main peak obtained by reversed-phase HPLC.

50. The pharmaceutical composition of claim 48, wherein the purity loss of the composition is less than 0.2% after storage at 5°C for 6 months, as measured by the height of the main peak obtained by reversed-phase HPLC.

51. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition loses less than 10% of purity after being stored at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

52. The pharmaceutical composition of claim 51, wherein the purity loss of the composition is less than 5% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

53. The pharmaceutical composition of claim 51, wherein the purity loss of the composition is less than 3% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

54. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition loses less than 10% of purity after being stored at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

55. The pharmaceutical composition of claim 54, wherein the purity loss of the composition is less than 5% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by reversed-phase HPLC.

56. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 0.2% after being stored at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

57. The pharmaceutical composition of claim 56, wherein the purity loss of the composition is less than 0.18% after storage at 5°C for 12 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

58. A pharmaceutical composition comprising about 2 mg / ml MANP, about 10 mM acetate, about 275 mM sucrose and about 0.02% polysorbate 20, wherein the composition has a purity loss of less than 0.5% after being stored at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

59. The pharmaceutical composition of claim 58, wherein the purity loss of the composition is less than 0.35% after storage at 5°C for 24 months, as measured by the relative area of ​​the main peak obtained by size exclusion chromatography.

60. A composition comprising MAMP, a buffer, a stabilizer / strainer, and a nonionic surfactant.

61. The composition of claim 60, wherein the buffer is selected from the group consisting substantially of: acetate, acetic acid, alanine, arginine, aspartic acid, bicarbonate, N,N-bis(2-hydroxyethyl)glycine (bicine), carbonate, citrate, citric acid, glycine, glycylglycine, glutamic acid, histidine, lysine, malic acid, potassium phosphate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, sodium succinate, succinic acid, sulfate, nitrate, maleic acid, fumaric acid, tartaric acid, aspartic acid, N-tris(hydroxymethyl)methylglycine (tricine), tris(hydroxymethyl)aminomethane, and tromethamine.

62. The composition according to claim 60, wherein the buffer is an acetate.

63. The composition according to claim 62, wherein the concentration of the acetate is in the range of about 10 mM to 40 mM.

64. The composition according to claim 62, wherein the concentration of the acetate is about 10 mM.

65. The composition according to claim 62, wherein the concentration of the acetate is about 40 mM.

66. The composition of claim 60, wherein the stabilizer / tension agent is selected from the group consisting substantially of: albumin, arginine, Brij 30, Brij 35, glucose, dimethyl sulfone, ethylenediaminetetraacetic acid, glycerol, glycerol, glycine, guanine, hydroxypropyl-β-cyclodextrin, lactose monohydrate, magnesium chloride, maltose, mannitol, methionine, 2-methylthioethanol, monothioglycerol, inositol, potassium chloride, poloxamer, polyethylene glycol, polysorbate 20, polysorbate 80, polyvinyl alcohol, polyvinylpyrrolidone, propylene glycol, protamine sulfate, sodium chloride, sorbitol, sucrose, thioglycolic acid, trehalose, and Triton.

67. The composition of claim 60, wherein the stabilizer / tension agent is sucrose.

68. The composition according to claim 67, wherein the concentration of sucrose is about 275 mM.

69. The composition according to claim 60, wherein the stabilizer / tension agent is mannitol.

70. The composition according to claim 69, wherein the concentration of mannitol is about 250 mM.

71. The composition according to claim 60, wherein the nonionic surfactant is selected from the group consisting substantially of: sorbitol polyoxyglyceride, polysorbate 20, polysorbate 40, sodium docusate, polysorbate 60, polysorbate 80, benzalkonium chloride, capryloyl hexanoyl polyoxyglyceride, hexadecyl pyridine chloride, lauroyl polyoxyglyceride, linoleyl polyoxyglyceride, octylphenyl polyol 9, oleoyl polyoxyglyceride, poloxamer, polyoxyethylene 10 oleyl ether, polyoxyethylene 15% hydroxystearate, nonyl alcohol ether 9, polyoxyethylene 20 cetearyl alcohol ether, polyoxyethylene 40 stearate, pullulan, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, sodium dodecyl sulfate, sorbitol monolaurate, sorbitol monooleate, polyoxyethylene stearate, sorbitol monopalmitate, sorbitol monostearate, stearoyl polyoxyglyceride, sorbitol sesquioleate, sorbitol trioleate, and teroxaprol.

72. The composition according to claim 60, wherein the nonionic surfactant is polysorbate 20.

73. The composition according to claim 72, wherein the concentration of the polysorbate 20 is about 0.2%.

74. The composition according to claim 60, wherein the pH is about 5.

5.

75. The composition according to claim 60, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

76. The composition according to claim 60, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

77. A vial containing a formulation comprising MANP, acetate, sucrose and polysorbate 20.

78. The vial according to claim 77, wherein the concentration of MANP is about 2 mg / ml.

79. The vial of claim 77, wherein the MANP is substantially composed of SEQ ID NO:

1.

80. The vial according to claim 77, wherein the concentration of said acetate is in the range of about 10 mM to about 40 mM.

81. The vial according to claim 77, wherein the concentration of the acetate is about 10 mM.

82. The vial according to claim 77, wherein the concentration of sucrose is about 275 mM.

83. The vial according to claim 77, wherein the concentration of the polysorbate 20 is about 0.02%.

84. The vial according to claim 77, wherein the pH is about 5.

5.

85. The vial according to claim 77, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

86. The vial according to claim 77, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

87. A lyophilized powder prepared according to the following steps: a. A combination in liquid solution of: MANP, acetate, sucrose, and polysorbate 20; and b. The combination of the freeze-drying step (a).

88. The lyophilized powder according to claim 87, wherein the concentration of MANP in the liquid composition is about 2 mg / ml.

89. The freeze-dried powder according to claim 87, wherein the MANP is substantially composed of SEQ ID NO:

1.

90. The lyophilized powder according to claim 87, wherein the concentration of the acetate in the liquid composition is in the range of about 10 mM to about 40 mM.

91. The lyophilized powder according to claim 87, wherein the concentration of the acetate in the liquid composition is about 10 mM.

92. The freeze-dried powder according to claim 87, wherein the concentration of sucrose in the liquid composition is in the range of about 250 mM to about 275 mM.

93. The freeze-dried powder according to claim 87, wherein the concentration of sucrose in the liquid composition is about 275 mM.

94. The freeze-dried powder according to claim 87, wherein the concentration of polysorbate 20 in the liquid composition is about 0.02%.

95. The lyophilized powder according to claim 87, wherein the pH of the liquid composition is about 5.

5.

96. The freeze-dried powder according to claim 87, wherein the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O.

97. The freeze-dried powder according to claim 87, wherein the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

98. A lyophilized powder prepared according to the following steps: a. A combination in liquid solution of: MANP, acetate, mannitol, and polysorbate 20; and b. The combination of the freeze-drying step (a).

99. The lyophilized powder according to claim 98, wherein the concentration of MANP in the liquid composition is about 2 mg / ml.

100. The freeze-dried powder according to claim 98, wherein the MANP is substantially composed of SEQ ID NO:

1.

101. The lyophilized powder according to claim 98, wherein the concentration of the acetate in the liquid composition is in the range of about 10 mM to about 40 mM.

102. The lyophilized powder according to claim 98, wherein the concentration of the acetate in the liquid composition is about 40 mM.

103. The lyophilized powder according to claim 98, wherein the concentration of mannitol in the liquid composition is in the range of about 250 mM to about 275 mM.

104. The freeze-dried powder according to claim 98, wherein the concentration of mannitol in the liquid composition is about 250 mM.

105. The freeze-dried powder according to claim 98, wherein the concentration of polysorbate 20 in the liquid composition is about 0.02%.

106. The lyophilized powder according to claim 98, wherein the pH of the liquid composition is about 5.

5.

107. The freeze-dried powder according to claim 98, wherein the osmotic pressure of the liquid composition is about 300-420 mOsm / kgH2O.

108. The freeze-dried powder according to claim 98, wherein the osmotic pressure of the liquid composition is about 310-390 mOsm / kgH2O.

109. A dry powder composition comprising MANP, acetate, sucrose and polysorbate 20.

110. A powder prepared by spray drying, wherein the spray drying comprises the following steps: a. Provide a liquid containing MANP, acetate, sucrose, and polysorbate 20; and b. Spray dry the liquid from step (a) using a spray drying apparatus.

111. A freeze-dried powder prepared by freeze-drying, wherein the freeze-drying comprises the following steps: a. Provide a liquid containing MANP, acetate, sucrose, and polysorbate 20; and b. Freeze-dry the liquid from step (a) at a certain temperature for a sufficient time to convert the liquid composition into a solid.

112. A lyophilized powder prepared by a method comprising the following steps: a. Provide a liquid containing MANP, acetate, sucrose, and polysorbate 20; and b. The liquid from step (a) is freeze-dried.

113. A prefilled syringe containing MANP, acetate, sucrose and polysorbate 20.

114. The pre-filled syringe of claim 113, wherein the concentration of MANP is about 2 mg / ml.

115. The pre-filled syringe of claim 113, wherein the MAP consists substantially of SEQ ID NO:

1.

116. The pre-filled syringe of claim 113, wherein the concentration of the acetate is in the range of about 10 mM to about 40 mM.

117. The pre-filled syringe of claim 113, wherein the concentration of the acetate is about 10 mM.

118. The pre-filled syringe of claim 113, wherein the concentration of sucrose is about 275 mM.

119. The pre-filled syringe of claim 113, wherein the concentration of polysorbate 20 is about 0.02%.

120. The pre-filled syringe of claim 113, wherein the pH is about 5.

5.

121. The pre-filled syringe according to claim 113, wherein the osmotic pressure is about 300-420 mOsm / kgH2O.

122. The pre-filled syringe according to claim 113, wherein the osmotic pressure is about 310-390 mOsm / kgH2O.

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