KR100594519B1 - Non-Aqueous Protic Peptide Formulations - Google Patents

Abstract

The present invention relates to stable nonaqueous protic preparations of peptide compounds. These stable formulations contain peptides in nonaqueous protic solvents. They can be stored for long periods at elevated temperatures and are particularly useful in implantable delivery devices for long term administration of drugs.

Description

Non-Aqueous Protic Peptide Formulations

This application claims 35 U.S.C. Priority is claimed to US Patent Application No. 60 / 021,129 (1996.7.3) under 119 (e), which is hereby incorporated by reference.

The present invention relates to stable nonaqueous protic preparations of peptide compounds. In particular, formulations having high concentrations of peptide compounds are provided.

references

The following references are referred to numerically within square brackets ([]) in the relevant parts of the specification.

1.Zoladex (Goserelin Acetate Implant), Physician's Desk Reference, 50th edition, p 2858-2861 (1996).

2. US Pat. No. 3,914,412 (October 21, 1975).

3. U.S. Patent 4,547,370 (October 15, 1985).

4. US Pat. No. 4,661,472 (April 28, 1987).

5. U.S. Patent 4,689,396 (August 25, 1987).

6. US Pat. No. 4,851,385 (1989.7.25.).

7. US Pat. No. 5,198,533 (1993.3.30.).

8. US Pat. No. 5,480,868 (1996.1.2.).

9. International Patent Publication No. WO92 / 20711 (Nov. 26, 1992).

10. International Patent Publication No. WO95 / 00168 (1995.1.5.).

11. International Patent Publication No. WO 95/04540 (1995.2.16.).

12. "Stability of Gonadorelin and Triptorelin in Aqueous Solution", V.J. VJ Helm, B. Double You. BW Muller, Pharmaceutical Research , 7/12, p 1253-1256 (1990).

13. "New Degradation Product of Des-Gly ¹⁰ -NH ₂ -LH-RH-Ethylamide (Fertirelin) in Aqueous Solution", J. J. Okada, T. T. Seo, F. F. Kasahara, K. K. Takeda, S. S. Kondo, J. of Pharmaceutical Sciences , 80/2, p 167-170 (1991).

14. "Characterization of the Solution Degradation Product of Histrelin, a Gonadtropin Releasing Hormone (LHRH) Agonist", A. R. AR Oyler, R. E. RE Naldi, J. R. JR Lloyd, D.A. DA Graden, C.J. CJ Shaw, M. L. Ml Cotter, J. of Pharmaceutical Sciences , 80/3, p 271-275 (1991).

15. "Parenteral Peptide Formulations: Chemical and Physical Properties of Native Luteinizing Hormone-Releasing Hormone (LHRH) and Hydrophobic Analogues in Aqueous Solution", M.F. MF Powell, L.M. LM Sanders, A. A. Rogerson, V. V. Si, Pharmaceutical Research , 8/10, p 1258-1263 (1991).

16. "Degradation of the LHRH Analog Nafarelin Acetate in Aqueous Solution", D.M. DM Johnson, R. A. RA Pritchard, W.F. WF Taylor, D. D. Conley, G. G. Zuniga, K. G. KG McGreevy, Intl. J. of Pharmaceutics , 31, p 125-129 (1986).

17. "Percutaneous Absorption Enhancement of Leuprolide", M. Y. MY Fu Lu, D. D. Lee, G.S. GS Rao, Pharmaceutical Research , 9/12, p 1575-1576 (1992).

18. Lutrepulse (gonadorelin acetate for IV injections), Physician's Desk Reference, 50th edition, p 980-982 (1996).

19. Factrel (gonadorelin HCl for subcutaneous or IV injection), Physician's Desk Reference, 50th edition, p 2877-2878 (1996).

20. Lupron (leuprolide acetate for subcutaneous injection), Physician's Desk Reference, 50th edition, p 2555-2556 (1996).

21. Lupron depot (leuprolide acetate for depot suspending), Physician's Desk Reference, 50th edition, p 2556-2562 (1996).

22. "Pharmaceutical Manipulation of Leuprorelin Acetate to Improve Clinical Performance", H .. H. Toguchi, J. of Intl. Medical Research , 18, p 35-41 (1990).

23. "Long-Term Stability of Aqueous Solutions of Luteinizing Hormone-Releasing Hormone Assessed by an In-Vitro Bioassay and Lipid Chromatography", Y. F. YF Shi, R. J. RJ Sherins, D. D. Brightwell, J. F. JF Gallelli, D.C. Chatterji, J. of Pharmaceutical Sciences , 73/6, p 819-821 (1984).

24. "Peptide Liquid Crystals: Inverse Correlation of Kinetic Formation and Thermodynamic Stability in Aqueous Solution :, M.F. Powell, J. Fleitman, L.M. Sanders, V.C., Pharmaceutical Research, 11 / 9, p 1352-1354 (1994).

25. "Solution Behavior of Leuprolide Acetate, an LHRH Agonist, as Determined by Circular Dichroism Spectroscopy", M. E. ME Powers, A. A. Adejei, M. Y. Fu Lu, M. MC Manning, Intl. J. of Pharmaceutics , 108, p 49-55 (1994).

26. "Preparation of Three-Month Depot Injectable Microspheres of Leuprorelin Acetate Using Biodegradable Polymers", Pharmaceutical Research , 11/8, p 1143-1147 (1994).

The disclosures of each of these documents, patents or patent applications are hereby incorporated by reference in their entirety to the same extent as to specifically and individually include the language of each individual document, patent and patent application.

The luteinizing hormone releasing hormone (LHRH), also known as gonadotropin releasing hormone (GnRH), is a decapeptide with a structure of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ to be. It is secreted by the hypothalamus and binds to receptors on the pituitary gland, releasing luteinizing hormone (LH) and follicle stimulating hormone (FSH). LH and FSH stimulate the gonads to synthesize steroid hormones. Numerous analogues of LHRH are known, including LHRH related peptides acting as agonists and LHRH related peptides acting as antagonists [1-15]. LHRH analogs are known to be useful in the treatment of hormone-dependent diseases such as prostate cancer, benign prostatic hyperplasia, endometrial hyperplasia, uterine fibroids, uterine fibroids, precocious puberty or breast cancer, and are known to be useful as contraceptives [8]. Sustained release administration is preferred for both agonist LHRH related compounds that reduce the number of available receptors after repeated dosing to inhibit the production of steroid hormones and antagonist LHRH related compounds that must be administered continuously for continued inhibition of endogenous LHRH [ 8].

Ongoing parenteral delivery of drugs, particularly peptide drugs, offers many advantages. The use of implantable devices to continuously deliver a wide variety of drugs or other beneficial actives is well known in the art. Typical devices are described, for example, in US Pat. No. 5,034,229; 5,057,318 and 5,110,596. Each of these patents is hereby incorporated by reference in its entirety.

In general, oral bioavailability of peptides comprising LHRH related compounds is low [16-17].

Current commercial formulations of LHRH, analogs and related compounds used for parenteral infusion contain relatively low concentrations of LHRH related compounds (0.05-5 mg / ml) and may also contain excipients such as mannitol or lactose. Aqueous solution [18-20]. Formulations of such LHRH related compounds may be refrigerated or stored for a short time at room temperature.

Available depot formulations of LHRH related compounds administered for sustained release over one to three months include 15% LHRH related compounds dispersed in a matrix of D, L-lactic and glycolic acid copolymers provided in a cylindrical form to be injected subcutaneously. Formulations [1] and formulations comprising microparticles comprising a core of gelatin and an LHRH-related compound surrounded by an envelope of a D, L-lactic acid and glycolic acid copolymer. These microparticles are suspended in diluent for subcutaneous or intramuscular injection [21, 26]. These products should be stored at temperatures below room temperature. Aqueous formulations of LHRH related compounds are known to exhibit chemical and physical instability as well as to degrade after irradiation [12-16, 22-25].

The formulation shown to be stable (t ₉₀ approximately 5 years) was a very low concentration (25 μg / ml) buffered (10 mM, ionic strength 0.15) aqueous solution stored at a temperature not higher than room temperature (25 ° C.) [15].

There is a need for stable formulation of peptides.

<Overview of invention>

The present invention provides a stable non-aqueous formulation that is a solution of peptide compounds in a non-aqueous protic solvent. In particular, formulations having a peptide at a concentration of at least about 10% are provided. These stable formulations can be stored for extended periods at elevated temperatures (eg, 37 ° C.) and are particularly useful in implantable delivery devices for prolonged delivery of drugs (eg, 1-12 months or longer).

In one aspect, the present invention provides a stable non-aqueous formulation of a peptide compound containing at least one peptide compound in at least one non-aqueous protic solvent. Particularly preferred formulations contain at least about 10% (w / w) peptide compound.

In another aspect, the present invention provides a method for preparing a stable non-aqueous formulation of a peptide compound, comprising dissolving at least one peptide compound in at least one non-aqueous protic solvent. Preferred formulations contain at least about 10% (w / w) peptide compound.

In another aspect, the present invention relates to administering to a patient suffering from a disease suffering from a disease that can be alleviated by administration of a peptide compound an effective amount of a stable non-aqueous formulation containing at least one peptide compound in at least one non-aqueous protic solvent. It provides a method of treating a patient suffering from a disease that can be alleviated by the administration of said peptide compound.

1 shows the stability of a 40% leuprolide acetate solution in propylene glycol (PG) after 2 months at 80 ° C., measured by reverse phase HPLC (RP-HPLC).

FIG. 2 is the RP-HPLC of the same sample as FIG. 1 injected on size exclusion chromatography (SEC) showing that 3% dimers and trimers were formed and no aggregates were detected.

3 is an Arrhenius plot showing the loss of leuprolide from a 40% solution of leuprolide acetate in propylene glycol (PG).

4 shows the loss of leuprolide from a 40% leuprolide solution in PG over 4-6 months at 37 ° C., 50 ° C., 65 ° C. or 80 ° C. FIG.

FIG. 5 shows the chemical and physical stability of 40% leuprolide solution in PG after 4 months at 80 ° C. FIG.

6 shows the chemical stability of 40% leuprolide acetate solution in PG after 9 months at 37 ° C.

FIG. 7 shows the chemical stability of 40% leuprolide acetate solution in PG / acetate buffer (30:70) after one year at 37 ° C. FIG.

8 shows the physical stability of 40% leuprolide acetate solution in PG / acetate buffer (30:70) after one year at 37 ° C.

FIG. 9 shows the stability of a 40% leuprolide acetate solution in water (30:70) containing PG / preservative after 6 months at 60 ° C. after irradiation.

FIG. 10 shows the long term stability of 40% leuprolide acetate solution in PG / water (30:70) over 6 months at 37 ° C. after irradiation.

FIG. 11 shows the stability of 30% goserelin solution in PEG 600 / acetate buffer (30:70) after 14 days at 80 ° C. FIG.

The present invention relates to the unexpected finding that dissolving peptide compounds in non-aqueous protic solvents results in the formation of stable formulations. Known aqueous formulations of peptide compounds, which are dilution buffered aqueous solutions containing excipients such as EDTA or ascorbic acid, which must be stored at low temperatures (4-25 ° C.), are decomposed such as acid / base catalysis, deamidation, racemization and oxidation reactions. Pathways form decomposition products. In contrast, the formulations claimed in the present invention stabilize the peptide compound at elevated temperatures (eg, 37 ° C. to 80 ° C.) or at high concentrations (ie, at least about 10%).

Standard peptide and protein preparations consist of dilute aqueous solutions. Two important aspects of peptide preparations include solubilization and stabilization of drug molecules.

Solubilization of peptides under aqueous conditions is standard since they are similar to natural state. However, solubilization under nonaqueous conditions is not known. We have found that peptide preparations in nonaqueous protic solvents are possible.

Stability of peptides is usually achieved by changing one or more of pH, buffer type, ionic strength and excipients (EDTA, ascorbic acid, etc.). In these formulations, the degradation pathways that require water (hydrolysis, deamidation, racemization) cannot be sufficiently stable. In contrast, in the present invention, high concentrations of peptides formulated with non-aqueous solutions such as propylene glycol and polyethylene glycol have been shown to be chemically and physically stable. Such solvents are considered to be nonaqueous protic solvents. Some nonaqueous protic solvents do not have the large dipole moments required for stabilization of the rate determining step and thus may serve to reduce the rate of degradation.

The present invention consists in the use of nonaqueous protic solvents such as propylene glycol and polyethylene glycol to stabilize high concentrations of peptide and protein preparations against both chemical and physical degradation. The findings of the present invention provide the fact that the use of propylene glycol or polyethylene glycol improves the total solubility and stability of the peptide at various formulation conditions, including high concentrations and elevated temperatures, enabling the delivery of the peptide to implantable delivery devices that would otherwise not be possible. Is done.

A. Definition :

The terms used herein have the following meanings:

The term "chemical stability" means that the decomposition products produced by chemical pathways, such as oxidation or hydrolysis, are formed in an acceptable proportion. Specifically, formulations are considered chemically stable if up to about 20% of degradation products are formed after two months at 37 ° C.

The term "physical stability" means that agglomerates (eg, dimers, trimers and higher forms) are formed in an acceptable proportion. Specifically, the formulation is considered physically stable if less than about 15% aggregates form after 2 months at 37 ° C.

The term "stable formulation" means that at least about 65% of the chemically and physically stable compounds remain after 2 months (or equivalent conditions at elevated temperature) at 37 ° C. Particularly preferred formulations are those which retain at least about 80% of chemically and physically stable compounds under these conditions. Particularly preferred stable formulations are those which do not exhibit degradation after sterile irradiation (eg gamma, beta or electron beam).

The term “peptide” and / or “peptide compound” means a polymer of up to about 50 amino acid residues bonded together by an amide (CONH) bond. Analogs, derivatives, agonists, antagonists and pharmaceutically acceptable salts of any one of these are included in this term. The term also refers to peptides and / or peptide compounds having peptomimetic units as part of their structure, and / or amino acids or natural amino acids modified or derived from D-amino acids, D- or L-configuration. Include.

The term “LHRH-related compound” means luteinizing hormone releasing hormone (LHRH) and its analogs and pharmaceutically acceptable salts. LHRH agonists and antagonists of octa-, nona- and decapeptides are included in the term LHRH related compounds, including natural LHRH. Particularly preferred LHRH related compounds include LHRH, leuprolide, goserelin, naparelin and other known active agonists and antagonists [1-21].

The term "high concentration" means at least about 10% (w / w) up to the maximum solubility of a particular compound.

The term "excipient" means more or less inert material in a formulation which is added as a diluent or vehicle or added to provide form or roughness. Excipients include solvents such as EtOH (which are used to dissolve the drug in the formulation), nonionic surfactants such as Tween 20 (which are used to solubilize the drug in the formulation) and benzyl alcohol and methyl or propyl parabens. And preservatives, which are used to prevent or inhibit microbial growth.

The term "non-aqueous protic solvent" means a non-aqueous solvent capable of forming hydrogen bonds or providing protons, including hydrogen bonded to oxygen or nitrogen. Examples of non-aqueous protic solvents are polyethylene glycols (PEGs), propylene glycol (PG), polyvinylpyrrolidone (PVP), methoxypropylene glycol (MPEG), glycerol and glycofurol.

The term "polar aprotic solvent" means a polar solvent that does not contain acidic hydrogen and does not act as a hydrogen bond donor. Examples of polar aprotic solvents are dimethyl sulfoxide (DMSO), dimethylformamide (DMF), hexamethylphosphorotriamide (HMPT) and n-methyl pyrrolidone.

B. Preparation of the formulation :

The present invention relates to nonaqueous preparations of peptide compounds in nonaqueous protic solvents that are stable for extended periods at elevated temperatures. Standard dilute aqueous formulations of peptides and proteins require adjustment of buffer type, ionic strength, pH, and excipients (eg, EDTA and ascorbic acid). In contrast, the formulations claimed in the present invention obtain stabilization of the peptide compound by using a nonaqueous protic solvent. In particular, the formulations of the present invention provide stability of compounds at high concentrations (at least about 10% (w / w)).

Examples of peptides and peptide compounds that can be formulated using the present invention include peptides having biological activity, or peptides that can be used to treat diseases or other pathological conditions. Examples include adrenal cortical stimulating hormones, angiotensin I and II, atrial sodium excretory peptides, bombesin, bradykinin, calcitonin, cesulin, dynophine A, alpha and beta endorphins, endothelin, enkephalins, endothelial growth factor, Fertirelin, Follicle gonadotropin releasing peptide, Galanine, Glucagon, Gonadorrelin, Gonadotropin, Goserelin, Growth hormone releasing peptide, Hysterlin, Insulin, Leuprolide, LHRH, Motilin, Na Parelin, neurotensin, oxytocin, somatostatin, substance P, tumor necrosis factor, tryptorelin and vasopressin. Analogs, derivatives, antagonists, agonists and pharmaceutically acceptable salts of these substances can also be used.

Peptide compounds useful in the formulations and methods of the invention may be used in the form of salts, preferably pharmaceutically acceptable salts. Useful salts are known to those skilled in the art and include salts with inorganic acids, organic acids, inorganic bases or organic bases. Preferred salts are acetate salts.

Peptides and peptide compounds that are readily soluble in nonaqueous protic solvents are preferred for use in the present invention. One skilled in the art will readily be able to determine which compound will be useful based on solubility, i.e., the compound should be dissolved in an acceptable amount that may be a pharmaceutically effective amount in a particular nonaqueous protic solvent. Preferred solubility is at least about 10% (w / w). Particularly preferred peptide compounds are LHRH related compounds, including leuprolide and leuprolide acetate.

The proportion of peptides can vary depending on the compound, the disease to be treated, the solubility of the compound, the expected dose and the duration of administration. ( E.g. , The Pharmacological Basis of Therapeutics , Gilman et al., 7th edition (1985)) and Pharmaceutical Sciences, Remington, 18th edition (1990) The concentration of peptides in high concentration formulations may range from about 10% (w / w) or more up to the maximum solubility of the compound. The preferred range is about 20 to about 60% (w / w). Currently more preferred ranges are from about 30 to about 50% (w / w) and the most preferred range is from about 35 to about 45% (w / w).

In general, stable formulations of the invention can be prepared by simply dissolving the desired amount of the desired peptide compound in the selected non-aqueous protic solvent. We have found that in polymeric solvents such as PEG, the solubility tends to be inversely proportional to the molecular weight of the solvent. Preferred nonaqueous protic solvents include propylene glycol (PG), polyethylene glycol (PEG), methoxypropylene glycol (MPEG), glycerol and polyvinylpyrrolidone (PVP).

It is known to those skilled in the art that it may be beneficial to add water, buffers, solubilizers such as nonionic surfactants, excipients such as EDTA, and preservatives such as benzyl alcohol, methyl or propyl parabens to the pharmaceutical formulation of the peptide. . (See, eg, Pharmaceutical Sciences , Remington, 18th Edition (1990)) Such active agents can optionally be added to the formulations claimed in the present invention.

C. Method :

We have found that stable non-aqueous preparations of peptide compounds can be prepared by dissolving the peptide compounds to be formulated in a non-aqueous protic solvent.

We tested these peptide compound formulations, especially those of LHRH related compounds, leuprolide, by accelerated aging at elevated temperatures and measuring the chemical and physical stability of the formulations. These findings (eg, Table III and shown in FIGS. 1, 2, 6 and 7) demonstrate that these formulations were stable at 37 ° C. for about one year or longer storage conditions.

We also tested the stability of peptide compound formulations prepared as described herein after 2.5 megarad gamma irradiation. The results shown in Table IV show that these formulations remained chemically and physically stable after such irradiation. Electron beam irradiated formulations have also been found to be stable.

As shown in Table I below, we dissolve (or attempt to dissolve) various peptide preparations, specifically leuprolide, goserelin, LHRH, bradykinin, insulin and trypsinogen, and then at elevated temperatures. The stability was tested by accelerated aging. The stability of the formulations was measured. The results are presented in Table I as half-lives at 37 ° C. assuming E _a = 22.2 mm 3 / mol. The various peptides tested were soluble in the nonaqueous protic solvents tested and remained stable under the test conditions. The solubility of certain peptides in water and the stability of the resulting solutions are readily determined using routine procedures known to those skilled in the art.

Stability of Peptides in Non-aqueous Protic Solvents Formulation Half-life ^* (temperature) 40% leuprolide in PG 5.2 years (37 ℃) 40% goserelin in PG 6.2 months (80 ℃) 20% LHRH in PG 1.2 years (65 ℃) 20% Bradykinin in PG 3.2 months (65 ℃) 20% insulin in PG Decomposes within 24 hours (65 ℃) 40% Trypsinogen in PG Insoluble 40% Trypsinogen in PEG Insoluble 20% Trypsinogen in PEG 7.7 months (65 ℃) ^* Half-life at 37 ° C assuming E _a = 22.2 μs / mole

The 40% leuprolide formulation in propylene glycol stored for 6 months at 37 ° C. showed linear degradation as measured by the total loss of peptide from solution. Analyzing these data, the activation energy (E _a ) was 16.6 kW / mol and t ₉₀ was 9.6 months, which shows the stability of these formulations at elevated temperatures.

We also unexpectedly find that certain peptide formulations of the invention are bacteriostatic (ie, inhibit bacterial growth), bactericidal (ie, kill bacteria) and sparger (ie, kill spores). . In particular, 50-400 mg / ml leuprolide formulations showed bacteriostatic, bactericidal and sparse activity. The stability of the sample was not affected by the spiking of bacteria, indicating that enzymes released from killed or lysed bacteria did not adversely affect the stability of the product. This proves that these agents did not help with enzyme activity.

Some peptides, such as calcitonin and leuprolide, are known to be physically unstable, exhibiting aggregation, gelling, and fibrosis when formulated in solutions in aqueous solutions as well as non-aqueous protic solvents. For example, leuprolide can be induced into the gel by increasing peptide concentration, introducing a salt, or gently stirring. Improving physical stability may make parenteral administration easier, including administration using an implantable drug delivery system.

Unexpectedly, it has been found that the addition of a polar aprotic solvent, such as DMSO, to the nonaqueous protic formulations of some peptides such as leuprolide, goserelin and calcitonin prevents the gelation of the formulation. This is clearly because the non-aqueous polar aprotic solvent forms the peptide in a random coil / alpha helix configuration that does not fold back into the beta sheet structure. Therefore, these solvents have an antigelling effect.

In addition, the stability of liquid formulations of leuprolide (370 mg / ml) and gelling (by stirring) in non-aqueous protic solvent PG was studied in vivo and in rats at 37 ° C., respectively. The results are shown in Table II below, which shows that both the gelling and liquid formulations remained stable over 12 weeks.

Study on the stability of liquid and gelling formulations of leuprolide in PG Research Time (Note) Liquid (residual%) Gelation (% remaining) Long-term stability 6 98.50 Long-term stability 12 98.00 Rat 6 97.40 Rat 12 95.90

The main aspect of the present invention is that the non-aqueous solution containing the peptide compound in the non-aqueous protic solvent is stable for a long time at high temperature. Such formulations are stable even when used at high concentrations. Therefore, these formulations are advantageous in that they can be stored for a long time at temperatures above room temperature. They are also suitable for use in implantable delivery devices.

The study in the following examples was carried out using the following methods.

1. Preparation of leuprolide acetate solution

Weigh leuprolide acetate (e.g., purchased from Mallinckrodt, St. Louis, MO) and, if necessary, vehicle (using 80 ° C., vortexing, stirring, and / or centrifugation). PG, PEG, MPEG, PG / H ₂ O, PG / H ₂ O, PEG / PG, MPEG / H ₂ O, PG containing EDTA) and dissolved in the appropriate concentration (w / w). The term "PEG" refers to PEG 300 unless otherwise indicated. The term “anhydrous PG” refers to a PG formulation prepared in a low humidity environment (ie, dry N ₂ atmosphere).

Unless otherwise indicated, the leuprolide free base content was calculated from a certificate of analytical potency value of 37% free base. This was 40% leuprolide acetate except as indicated.

2. Manufacture of reservoir

Appropriate leuprolide acetate solution was charged to the reservoir of an implantable drug delivery device (eg, as disclosed in US Patent Application No. 08 / 595,761, incorporated herein by reference). The charged device was then tested for stability. The formulations were filled with titanium plugs or polymer reservoirs plugged at each end with a polymer stopper. The filled reservoir was then sealed in a polyfoil bag and placed in a stability test oven.

It should be noted that the formulation inside the reservoir of these devices must be completely isolated from the external environment.

3. Reversed-HPLC (RP-HPLC)

All stability samples were analyzed for leuprolide concentration and peak area percent using gradient elution reverse phase HPLC analysis with refrigerated autosampler (4 ° C.) to minimize sample degradation. The chromatographic conditions used are listed below.

RP-HPLC Chromatography Conditions

Explanation parameter column HaiSil C18, 4.6 × 250mm, S / N 5103051 Flow rate 0.8 ml / min Injection volume 20 μl detection 210 nm Lyuprolide Retention Time 25 to 30 minutes Mobile phase A = 100 mM sodium phosphate, pH 3.0B = 90% acetonitrile / water gradient minute 0 5 25 40 41 46 46.1 50 B% 15 26.5 26.5 65 85 85 15 15

Typically between 4 and 6 different concentration levels of leuprolide standard (in water) between 0.1 and 1.2 mg / ml were tested with stability samples. Stability samples were handled with the standard set so that there were no more than 40 samples between the standard sets. All peaks between the empty volume and 45 minutes of run were integrated. The integrated peak area for the leuprolide standard was plotted as a function of concentration. Regression analysis was then used to calculate leuprolide concentrations for the stability samples. % Peak area for leuprolide peaks, sum of all peaks eluted before leuprolide (labeled "others"), and sum of all peaks eluted after leuprolide (labeled "aggregates") ) Was also recorded and plotted as a function of sample time point.

4. Size Exclusion Chromatography (SEC)

Selected stability samples were analyzed for peak area percent and molecular weight using an isocratic solution SEC analysis with refrigerated autosampler (4 ° C.). The chromatographic conditions used are listed below.

<SEC Chromatography Conditions>

Explanation parameter column Pharmacia Peptide, HR 10/30, 10 × 300 mm Flow rate 0.5 ml / min Injection volume 20 μl detection 210 nm Lyuprolide Retention Time About 25 minutes Mobile phase 100 mM ammonium phosphate, pH 2.0, 200 mM sodium chloride, 30% acetonitrile

Empty volume and total volume for the size exclusion column were needed to calculate the molecular weight. Empty volume and total volume were measured using a Bio-Rad high molecular weight standard and 0.1% acetone, respectively. Retention times for the first and acetone peaks in Biorad standards were recorded and converted to volume units using the following equation. Since these values are constant for a particular SEC column and HPLC system, the empty volume and total volume were measured again each time the SEC column or HPLC system was changed. The stability sample was then run after running the standard. The standard mixture contains about 0.2 mg / ml of the following peptides: Bursin (Mur = MW = 449), WLFR peptide (MW = 619), Angiotensin (MW = 1181), GRF (MW = 5108) and Cytochrome C (MW = 12394). ). These standards were chosen because they encompass the leuprolide molecular weight and all have a basic pH (9.8-11.0) similar to leuprolide.

Peak area% was recorded for all peaks. The molecular weight for the isolated species was calculated using the following equations.

V _s = flow rate (ml / min) × sample peak retention time (minutes)

V _o = flow rate (ml / min) × empty volume retention time (minutes)

V _t = flow rate (ml / min) × total volume retention time (minutes)

 In the above formulas,

Vs = standard or sample volume

Vo = empty volume

Vt = total volume

Vs was calculated for the peak of each peptide standard. The Kd for each peptide standard was then calculated using the values for Vt and Vo measured earlier. The regression curve from the plot of logMW for Kd ⁻¹ was used to determine the molecular weight for each peak in the stability sample. Peak area% for stability samples was also recorded.

5. Device and materials

The equipment and materials used for RP-HPLC and SEC are as follows:

Waters Millennium HPLC system (Waters Chromatography, Milford, Mass.), Consisting of 717 automated samplers, 626 pumps, 6000S controllers, 900 photodiode array detectors, and 414 refractive index detectors.

HPLC vials for 48-position and 96-position (Waters Chromatography, Milford, Mass.).

Hisil C18, 120 A, 5 μm 4.6 × 250 mm HPLC column (Higgins Analytical, Mountain View, CA).

Pharmacia peptide, HR 10/30 SEC column (Pharmacia Biotech, Peacecatter, NJ).

6. Purity

Stability samples were analyzed using RP-HPLC. The area under the curve for the leuprolide peaks ÷ the sum of the area under the curve for all peaks is% purity. It should be noted that the data for the concentration percentages presented with the purity% data (Examples 6, 8, 9 and 10) are not definite. The analytical method used to measure% concentration in these experiments is not reliable.]

The following examples are provided to illustrate the invention and should not be construed as limiting the scope of the invention in any way.

Example 1

Accelerated Stability Study of Leproprolide Acetate Formulations

A formulation of 40% (w / w) leuprolide acetate (corresponding to 37% leuprolide free base) in the vehicle is prepared as described above, and also filling the reservoir of the implantable drug delivery device as described above. It was used for. Some reservoirs are made of polymeric material, while some are made of titanium.

The charged device was accelerated aging by storing at room temperature (80-88 ° C.) for 7 days in an incubator (Precision Scientific or Thelco). This corresponds to about 6 months at 37 ° C. or about 1 year at room temperature (25 ° C.) assuming an activation energy (E _a ) of 16.6 kW / mol.

Samples were analyzed using RP-HPLC and SEC as described above to determine the chemical and physical stability of the aged formulations.

The results presented in Table III below demonstrate that these formulations were able to maintain the stability of leuprolide, an LHRH related compound. In each case, at least 65% of leuprolide was maintained.

Stability of the Leuprolide Acetate Non-Aqueous Protic Formulation After 7 Days at an Elevated Temperature Temperature (℃) Reservoir material Formulation % Of leuprolide on day 7 88 polymer 40% of PG 70 88 polymer 40% of PG / H ₂ O (70/30) 73 88 polymer 40% in PEG / H ₂ O (90/10) 77 88 titanium 40% of PG 87 88 polymer 20% in PEG / PG (50/50) 74 88 polymer 20% in PEG / H ₂ O (88/12) 68 80 polymer 40% of PG 74 80 titanium 40% of PG 80 80 titanium 40% in PEG / H ₂ O (90/10) 86 80 titanium 40% of PG 87 80 titanium 40% of PG 80 80 titanium 1% in PG 40% in EDTA 80 80 polymer 40% of MPEG 350 / H ₂ O (50/50) 83 80 titanium 40% in anhydrous PG 76

Example 2

Stability Study of Irradiated Leprolide Acetate Formulations

A formulation of 40% (w / w) received leuprolide acetate (equivalent to 37% leuprolide free base) in PG was prepared as described above and also stored in a drug delivery device as described above. Used to fill the group. All reservoirs were made of polymeric material.

The filled device was irradiated with 2.5 megarad gamma radiation. Samples were shipped to Sterigenics, Tustin, Calif. And gamma irradiated in a batch mode (cobalt 60). Samples labeled "cold" were shipped and irradiated on dry ice. The sample was then accelerated aging as in Example 1. Samples were taken on days 0 and 7 and analyzed by RP-HPLC and SEC as described above to determine the chemical and physical stability of the irradiated formulation.

The results presented in Table IV below demonstrate that these leuprolide acetate formulations are stable after irradiation. In all cases, at least 65% of leuprolide was maintained and the level of aggregate formation was low.

Stability of 40% (w / w) leuprolide acetate protic formulations after 2.5 megarad gamma irradiation in a polymer reservoir Formulation Irradiation Day 7 leuprolide% (RP-HPLC) SEC 0 days 7 days Monomer% Di / trimeric% Monomer% Di / trimeric% 40% of PG has exist 88 97.4 0.9 94.5 3.7 40% of PG none 75 98.8 0.03 91.9 5.9 40% of PG cold 75 98.3 0.2 92.2 5.6

Example 3

Solubility Study of Leuprolide Acetate in PG

A leuprolide acetate formulation in PG was prepared as described above. The formulation was heated at 80 ° C. to facilitate dissolution of leuprolide in PG. The data is presented in Table V below.

Leuprolide% in PG Leproprolide Acetate Weight (mg) PG weight (mg) Total weight Leuprolide acetate% 148.6 225.7 374.3 39.70 154 183.7 337.7 45.60 146.8 147.2 294 49.93

Example 4

Long-term accelerated stability study of leuprolide acetate in PG

A 40% (w / w) leuprolide acetate solution in PG was prepared as described above, stored in a reservoir, stored at 80 ° C. for 2 months, and analyzed. The results shown in FIG. 1 (RP-HPLC) and FIG. 2 (SEC) show that after 2 months 55.9% of leuprolide was recovered with only 37.2% chemical degradation and 15.2% physical aggregate. These formulations were stable (as described above) after 7 days at 80 ° C. (corresponding to 2 months at 37 ° C.).

A 40% (w / w) leuprolide acetate solution in PG was prepared as described above, stored in a reservoir, stored at 80 ° C. for 4 months, and analyzed using RP-HPLC. 5 is a plot of leuprolide, its chemical and physical degradation products, recovered over four months. In addition, the sum of these three elements is presented as mass balance. The results show that all peptide materials can be described as intact leuprolides or as degraded species, indicating that no degradation processes or degradation products were unknown in stability studies.

A 40% (w / w) leuprolide acetate solution in PG was prepared as described above and placed in a reservoir, stored at 37 ° C., 50 ° C., 65 ° C. or 80 ° C. for 4-6 months, and then RP-HPLC The analysis was carried out. The results presented in FIG. 4 show that leuprolide degradation is consistent with pseudo first order motion. In addition, as discussed below, FIG. 3 shows that leuprolide degradation in PG is consistent with linear Areneus motion. Therefore, accelerated stability studies are a valid technique for evaluating the stability of leuprolide and extrapolating it back to 37 ° C.

A 40% (w / w) leuprolide acetate solution in PG was prepared as described above, stored in a reservoir, stored at 37 ° C., 50 ° C., 65 ° C. or 80 ° C. and analyzed using RP-HPLC. The results were calculated as described in Physical Pharmacy: Physical Chemical Principles in the Pharmaceutical Sciences , 3rd edition, Martin et al., Chapter 14 (1983), which had an E _a of 16.6 dl / mol, t ₉₀ showed 9.6 months at 37 ° C. The data is shown below and an Arrhenius plot of the data is shown in FIG. 3.

PG ℃ Kobs (month ^-1 ) t _1/2 (month) 37 1.12 × 10 ^-2 61.6 50 3.13 × 10 ^-2 22.2 65 8.64 × 10 ^-2 8.0 80 0.322 2.4 E _a = 16.6 dl / mol

Example 5

Long-term stability studies of leuprolide acetate in PG

The chemical stability of the 40% leuprolide acetate solution prepared and analyzed as described above is shown in FIG. 6. After 9 months at 37 ° C., less than 5% (3.1%) of chemical degradation products (represented as “early”) and less than 10% (5.6%) based on RP-HPLC data but closely match the SEC data. There was at least 90% (90.1%) of leuprolide, in which physical aggregates (represented as “late”) were formed.

Example 6

Long-term stability studies of leuprolide acetate in PG / acetate buffer

A 30% (w / w) leuprolide acetate solution in PG / acetate buffer (pH 5.0, 0.0282M) (30:70) was prepared as described above, then placed in a glass ampoule and irradiated as described above. And stored at 37 ° C. for 1 year. Analysis (as described above) by RP-HPLC (FIG. 7) and SEC (FIG. 8) show that these agents are stable. After 9 months, RP-HPLC showed that more than 70% chemically active leuprolide was present in the formulation. SEC results showed that 90% of the physically stable leuprolides were present after 9 months at 37 ° C.

Example 7

Long-term accelerated stability study of leuprolide acetate in PG / water

40% (w / w) leuprolide acetate formulation in water containing PG / preservative (30:70) is mixed with 0.18% methyl paraben and 0.025% propylparaben with water and contains 30:70 PG / preservative solution Water was prepared, and then leuprolide acetate was dissolved in this solution as described above. This formulation was placed in a glass ampoule as described above, then irradiated and stored at 60 ° C.

Purity was analyzed over 6 months as described above. The results are shown in FIG. These data show that the purity of these formulations was at least 90% at 45 days and about 65% at 6 months. Very high standard deviations were seen at the 90 day data points.

Example 8

Long-term stability studies of leuprolide acetate in PG / water

40% (w / w) leuprolide acetate formulations in PG / water (30:70) were prepared as described above, placed in glass ampoules, irradiated and stored at 37 ° C. for 6 months as described above. Then it was analyzed using HPLC.

The results shown in FIG. 10 show that more than 70% of leuprolide remains after 6 months.

Example 9

Accelerated Stability Study of Goserelin in PEG 600 / Acetate Buffer

30% (w / w) goserelin preparation in PEG 600 / acetate buffer (30:70), prepared as described above for leuprolide acetate, was stored for 14 days at 80 ° C. in a glass ampoule, as described above. Purity was analyzed as well.

The results shown in FIG. 11 show that more than 65% goserelin remains after 9 days.

Example 10

Stability Study of Goserelin Formulations

40-45% (w / w) goserelin formulation in PEG 600 or PG / acetate buffer (30:70) was prepared as described above and placed in a polymer container. The vessel was stored for 1 month at 37 ° C. in an incubator. Samples were analyzed using RP-HPLC to determine the chemical stability of the aged formulation.

The results shown below demonstrate that these agents were able to maintain the stability of Goserelin, an LHRH related compound. In each case, at least 98% goserelin was maintained as indicated by purity data.

drug Vehicle Purity% Concentration% Goserelin PEG 600 99.3 23.6 Goserelin PG / Acetate Buffer (30:70) 98.2 49.7

Example 11

Stability Study of Naparelin Preparation

A 15% (w / w) naparelin preparation in PEG 600 or propylene glycol was prepared as described above for leuprolide and placed in a polymer container.

The vessel was stored for 1 month at 37 ° C. in an incubator.

Samples were analyzed using RP-HPLC to determine the chemical stability of the aged formulation.

The results shown below demonstrate that these formulations were able to maintain the stability of naparelin, an LHRH related compound. In each case, at least 99% of naparelin was maintained as indicated by purity data.

drug Vehicle Purity% Concentration% Nafarelin PEG 600 99.4 15.8 Nafarelin PG 99.4 12.9

Modifications of the foregoing manner of carrying out various embodiments of the invention will be apparent to those skilled in the art in accordance with the teachings of the invention described herein. The foregoing examples do not limit the invention, but are merely illustrative of the invention and the scope of the invention is defined by the following claims.

Claims (34)

a) 20-40% (w / w) of at least one peptide compound, wherein the peptide compound is an LHRH-related compound; And b) at least one non-aqueous protic solvent A stable non-aqueous preparation of peptide compounds containing, and stable at 37 ° C. for at least 1 month. A formulation according to claim 1 which contains 30 to 40% (w / w) peptide compound. The agent according to claim 1, wherein the peptide compound is selected from the group consisting of leuprolide, LHRH, naparelin and goserelin. The formulation of claim 1, which is stable after irradiation. The formulation according to claim 1, which is stable for at least 1 year at 37 ° C. The formulation of claim 1 suitable for use in an implantable drug delivery device. The formulation of claim 1, wherein said at least one non-aqueous protic solvent is selected from the group consisting of PG, PEG and glycerol. The formulation of claim 1, which forms a gel. The formulation of claim 1 further comprising at least one non-aqueous polar aprotic solvent. The formulation of claim 9, wherein said polar aprotic solvent is DMSO or DMF. The formulation of claim 1 further comprising water. The formulation of claim 1 further comprising at least one selected from the group consisting of excipients, surfactants, solubilizers and preservatives. The formulation according to claim 1, consisting essentially of leuprolide acetate which is 30% to 40% (w / w) LHRH-related compound in PG or PEG or mixtures thereof. Stable non-aqueous according to claim 1, comprising dissolving 20-40% (w / w) of at least one peptide compound, wherein the peptide compound is an LHRH-related compound, in at least one non-aqueous protic solvent. Method of preparation of the formulation. The method of claim 14, wherein at least 30-40% (w / w) of the peptide compound is dissolved. 15. The method of claim 14, wherein said peptide compound is selected from the group consisting of leuprolide, LHRH, naparelin and goserelin. The method of claim 14, further comprising adding at least one selected from the group consisting of excipients, surfactants, solubilizers and preservatives. 15. The method of claim 14, further comprising adding at least one non-aqueous polar aprotic solvent. 19. The method of claim 18, wherein said polar aprotic solvent is DMSO or DMF. The method of claim 14, further comprising adding water. 15. The method of claim 14, wherein said at least one non-aqueous protic solvent is selected from the group consisting of PG, PEG and glycerol. 15. The method of claim 14, wherein leuprolide acetate, which is 30% to 40% (w / w) of LHRH-related compound, is dissolved in PG or PEG or mixtures thereof. A method of using the formulation of claim 1 for the manufacture of a pharmaceutical composition for treating a patient suffering from a disease that can be alleviated by administration of an LHRH-related peptide compound. The method of claim 23, wherein the composition is for parenteral administration. The method of claim 23, wherein the composition is for long term continuous administration. The method of claim 23, wherein the composition is administered using an implantable drug delivery device. The method of claim 23, wherein said disease is prostate cancer and said peptide compound is leuprolide. The method of claim 23, wherein the composition is administered using an implantable drug delivery system. The method of claim 23, wherein said disease is prostate cancer and said peptide compound is an LHRH antagonist. The method of claim 14, wherein the process is performed under an atmosphere of inert gas. 31. The method of claim 30, wherein said atmosphere is dry nitrogen. a) 20-40% (w / w) of at least one peptide compound, wherein the peptide compound is an LHRH-related compound; b) at least one non-aqueous protic solvent; And c) contains at least one polar aprotic solvent, Stable non-aqueous formulations of LHRH-related peptide compounds that exhibit bacteriostatic, bactericidal or sparger activity without the use of conventional bacteriostatic, bactericidal or sparse agents. 20 to 40% (w / w) of at least one peptide compound, wherein the peptide compound is an LHRH-related compound, comprises dissolving in at least one non-aqueous protic solvent and at least one polar aprotic solvent The method for producing a stable non-aqueous preparation according to claim 32, wherein a conventional bacteriostatic agent, bactericide or spray agent is not added. Use of the agent according to claim 32 for the preparation of a pharmaceutical composition for treating a patient suffering from a disease that can be alleviated by the administration of an LHRH-related peptide compound.