HK1020677B - Aqueous formulations of lhrh-related peptide compounds, methods for their preparation and uses thereof - Google Patents

Description

Aqueous preparation of LHRH related peptide compound, preparation method and application

Technical Field

The present invention relates to high concentration stable aqueous peptide compound formulations.

Background

Reference documents:

the following references are cited as numerals in brackets ([ ]) in relevant parts of the specification.

Zoladex (acetate ruin implant), Physician's Desk Reference, 50 th edition, p2858-2861 (1996).

Us patent 3,914,412 published on 21/10/2.1975.

Us patent 4,547,370 published 10/15/3.1985.

Us patent 4,661,472 published on 28.4. 4.1987.

Us patent 4,689,396 published 25.8. 5.1987.

Us patent 4,851,385 published 25/7/6.1989.

Us patent 5,198,533 published 3.30. 7.1993.

Us patent 5,480,868 published on 2.1. 8.1996.

WO 92/20711 published on 11/26/9.1992.

WO 95/00168 published on 5.1. 10.1995.

WO 95/04540 published on 2/16/11.1995.

"stability of Gonadorelin (Gonadorelin) and Triptorelin (Triptorelin) in aqueous solution", v.j.helm, b.w.muller, pharmaceutical research, 7/12, p1253-1256 (1990).

13.“Des-Gly¹⁰-NH₂-new degradation products of LH-RH-acetamide (Fertirelin) in aqueous solution ", j.okada, t.seo, f.kasahara, k.takeda, s.kondo, journal of pharmaceutical science 80/2, p167-170 (1991).

"characterization of the degradation products of the solution of the gonadotropin releasing hormone (LHRH) agonist Histrelin (Histrelin)" A.R.OYER, R.E.Naldi, J.R.Lloyd, D.A.Graden, C.J.Shaw, M.L.Cotter, J.Pharmacology, 80/3, 271-275 (1991).

"parenteral peptide preparation: chemical and physical properties of natural luteinizing hormone-releasing hormone (LHRH) and hydrophobic analogues in aqueous solution ", m.f. powell, l.m. sanders, a.rogerson, v.si, pharmaceutical research, 8/10, p1258-1263 (1991).

"degradation of the LHRH analogue Nafarelin Acetate (Nafarelin Acetate) in aqueous solution", d.m. johnson, r.a. pritchard, w.f. taylor, d.conley, g.zuniga, k.g. mcgreeny, international journal of pharmacy, 31, p125-129 (1986).

"enhancement of percutaneous absorption of Leuprolide", m.y.fu Lu, d.lee, g.s.rao, pharmaceutical research, 9/12, p1575-1576(1992).

Lutresulse (gonadorelin acetate for Intravenous (IV) injection), Physician's desk reference, 50 th edition, p980-982 (1996).

Factrel (gonadorelin hydrochloride for subcutaneous or IV injection), Physician's desk reference, 50 th edition, p2877-2878 (1996).

Lupron (leuprolide acetate for subcutaneous injection), Physician's Desk Reference, 50 th edition, p2555-2556(1996).

Lupron stock (leuprolide acetate for storage of suspensions), Physician's desk reference, 50 th edition, p2556-2562 (1996).

"pharmaceutical procedures for the improvement of clinical performance of leuprolide acetate", h.toguchi, journal of international medical research, 18, p35-41 (1990).

"evaluation of the Long-term stability of aqueous solutions of luteinizing hormone-releasing hormone by in vitro biological tests and liquid chromatography", Y.F.Shi, R.J.Sherins, D.Brightwell, J.F.Gallelli, D.C.Chatterji, J.of Pharmaceutical sciences, 73/6, p819-821 (1984).

"peptide liquid crystals: in aqueous solution, kinetic composition and thermodynamic stability are inversely related ", m.f. powell, j.fleitman, l.m. sanders, v.c. si, pharmaceutical research, 11/9, p1352-1354 (1994).

"solution behavior of LHRH agonist leuprorelin acetate as determined by circular dichroism", m.e. powers, a.adejei, m.y.fu Lu, m.c. manning, international journal of medicine, 108, p49-55 (1994).

"preparation of injectable leuprolide acetate microspheres storable for 3 months using biodegradable polymers", pharmaceutical research, 11/8, p1143-1147 (1994).

The disclosures of each of the foregoing publications, patents or patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Luteinizing Hormone Releasing Hormone (LHRH), also known as gonadotropin releasing hormone (GnRH), is a decapeptide having the following structure:

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. This hormone 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 analogs of LHRH are known, including those LHRH-related peptides that act as agonists and as antagonists. [1-15]LHRH analogues are known to be useful in the treatment of hormone dependent diseases such as prostate cancer, benign prostatic hypertrophy, endometriosis, uterine fibroids, precocious puberty or breast cancer and as contraceptives. [8]Sustained release administration is preferred for both LHRH-related compounds, which are agonists that reduce the number of receptors available to inhibit steroid hormone production after repeated administration, and LHRH-related compounds, which are antagonists that must be administered continuously to continuously inhibit endogenous LHRH. [8]

Sustained delivery of drugs, particularly peptide drugs, via the parenteral tract has many benefits. The use of implant devices to provide sustained delivery of a wide variety of different drugs or other useful agents is well known to those skilled in the art, and typical devices are described in, for example, U.S. Pat. nos. 5,034,229; 5,057,318; and 5,110,596. The disclosure of each of these patents is incorporated herein by reference.

Peptides, including LHRH-related compounds, are generally less bioavailable orally. [16-17]

Currently marketed aqueous formulations of LHRH for parenteral injection, their analogues and related compounds usually contain relatively low concentrations of LHRH-related compounds (0.05-5mg/ml), and may also contain excipients such as mannitol or lactose. [18-20] this LHRH-related compound preparation must be stored in a refrigerator or left at room temperature for a short period of time.

Depot formulations of LHRH-related compounds which upon administration can be released continuously over a period of 1-3 months include a preparation for subcutaneous injection containing 15% LHRH-related compounds [1] dispersed in a cylindrical matrix of a copolymer of D, L-lactic acid and glycolic acid and a preparation containing microparticles comprising a core containing LHRH-related compounds and gelatin and a shell of a copolymer of D, L-lactic acid and glycolic acid. These microparticles are suspended in a diluent for subcutaneous or intramuscular injection. [21, 26] these products must be stored at room temperature or lower. Aqueous formulations of LHRH-related compounds are known to exhibit chemical and physical instability and to degrade upon exposure to light. [12-16, 22-25]

Shows stability (t)₉₀About 5 years) is a very low concentration (25. mu.g/ml) aqueous buffered (10mM buffer, 0.15 ionic strength) solution stored at a temperature not exceeding room temperature (25 ℃). [15]

There is a need for stable, high concentration aqueous peptide formulations.

Brief description of the invention

The present invention provides stable aqueous formulations of peptide compounds in solution in water at a concentration of at least about 10%. These stable, high concentration formulations can be stored at higher temperatures (e.g., 37 ℃) for longer periods of time, and are particularly useful for implantable delivery devices that deliver drugs over long periods of time (e.g., 1-12 months or longer). The aqueous formulation may optionally include buffers, excipients, ethanol (EtOH), surfactants or preservatives.

In one aspect, the invention provides a stable aqueous peptide compound formulation comprising at least about 10% (w/w) peptide compound and water.

In another aspect, the invention provides a method of preparing a stable aqueous peptide compound formulation, the method comprising dissolving at least about 10% (w/w) of a peptide compound in water.

In another aspect, the invention provides a method of treating a subject having a disease state ameliorated by the administration of a peptide compound, the method comprising administering to the subject an effective amount of a stable aqueous formulation comprising at least about 10% (w/w) peptide compound and water.

Brief Description of Drawings

FIG. 1 shows the stability of a 40% leuprolide acetate solution in water, after standing at 80 ℃ for 2 months, as determined by reverse phase HPLC (RP-HPLC).

FIG. 2 shows the results of the same sample as in FIG. 1 injected into Size Exclusion Chromatography (SEC). This figure shows very few aggregates, with aggregates consisting of dimer and trimer products, and no higher aggregates detected.

FIG. 3 is an Arrhenius plot (Arrhenius plot) showing the loss of leuprolide in a 40% solution of leuprolide acetate in water.

Figure 4 shows the chemical and physical stability of a 40% leuprolide acetate solution in water after standing at 80 ℃ for about 3 months.

Figure 5 shows that the loss of leuprolide acetate from a 40% solution of leuprolide acetate in water during 3 to 6 months at 37 ℃, 50 ℃, 65 ℃ or 80 ℃ follows quasi-first order kinetics.

Figure 6 shows the chemical and physical stability of a 40% leuprolide acetate solution in water after 9 months at 37 ℃.

FIG. 7 shows the stability of a 30% solution of triptyline in acetate buffer and mannitol after 14 days at 80 ℃.

FIG. 8 shows that gelled and ungelled aqueous formulations of leuprolide (370mg/ml) remain stable during 6 months at 37 ℃.

Detailed Description

The present invention describes the unexpected discovery that dissolving a high concentration (i.e., at least about 10%) of a peptide compound in water results in a stable formulation. Previously known formulations of peptidal compounds are dilute buffered aqueous solutions containing excipients such as EDTA or ascorbic acid, which must be stored at low temperatures (4-25 ℃) and which can form degradation products using degradation pathways such as acid/base catalyzed hydrolysis, deamidation, racemization and oxidation. In contrast, the formulations claimed in the present invention stabilize high concentrations of peptide compounds at higher temperatures (e.g., 37 ℃ to 80 ℃), thereby enabling delivery of peptides in implantable delivery devices, which has not previously been feasible.

Standard peptide and protein formulations consist of dilute aqueous solutions. Two important aspects of peptide formulations include the dissolution and stabilization of drug molecules. In general, peptides can be stabilized by varying one or more of the following factors, pH, buffer type, ionic strength, excipients (EDTA, ascorbic acid, etc.). In contrast, the highly concentrated peptides of the present invention formulated in water provide a stable solution.

The present invention includes the use of high concentrations of peptides dissolved in aqueous solutions to stabilize peptide formulations against chemical and physical degradation.

A. Defining:

the following terms used herein have the following meanings:

the term "chemical stability" refers to the formation of an acceptable percentage of degradation products resulting from chemical pathways such as oxidation or hydrolysis. In particular, the formulation is considered to be chemically stable if no more than about 20% of decomposition products are formed after 2 months at 37 ℃.

The term "physical stability" refers to the formation of an acceptable percentage of aggregates (e.g., dimers, trimers and larger structures). In particular, the formulation is considered to be physically stable if no more than about 15% of the aggregates are formed after 2 months at 37 ℃.

The term "stable formulation" refers to a peptide compound that retains at least about 65% of its chemical and physical stability after being placed at 37 ℃ for 2 months (or under the same conditions at higher temperatures). Particularly preferred are formulations that retain at least about 80% of the chemically and physically stable peptide under these conditions. Particularly preferred stable formulations are those that do not exhibit degradation upon sterilizing irradiation (e.g., gamma, beta or electron beam).

The terms "peptide" and/or "peptidic compound" refer to polymers of up to about 50 amino acid residues linked together by amide (CONH) bonds, also including analogs, derivatives, agonists, antagonists and pharmaceutically acceptable salts of any of them, and also including peptides and/or peptidic compounds having D-amino acids, modified, derivatized or non-naturally occurring amino acids in the D-or L-configuration and/or peptone mimetic units that are part of their structure.

The term "LHRH-related compounds" refers to Luteinizing Hormone Releasing Hormone (LHRH) and analogs and pharmaceutically acceptable salts thereof, and the term LHRH-related compounds includes, in addition to native LHRH, eight-, nine-and decapeptide LHRH agonists and antagonists, with particularly preferred LHRH-related compounds including LHRH, leuprolide, goserelin, nafarelin and other known active agonists and antagonists. [1-21]

The term "high concentration" refers to a concentration of a particular LHRH-related compound of at least about 10% (w/w) up to maximum solubility.

The term "excipient" refers to an inert or high or low material added to a formulation as a diluent or carrier to shape or thicken. The excipients are different from solvents used to dissolve drugs in the formulation, such as EtOH, non-ionic surfactants used to solubilize drugs in the formulation, such as tween 20, and preservatives used to prevent or inhibit microbial growth, such as benzyl alcohol and methyl or propyl parabens.

The term "buffering capacity" refers to the ability to reduce any pH change that would otherwise occur by the addition of an acid or base to a solution due to the presence of an acid/base pair mixture in the solution.

The term "polar aprotic solvent" refers to 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), hexamethylphosphoric triamide (HMPT) and N-methylpyrrolidone.

B. Preparation of the preparation:

highly concentrated aqueous liquid formulations of peptide compounds are described which are stable when left at elevated temperatures for extended periods of time. Standard dilute aqueous peptide and protein formulations require adjustment of buffer type, ionic strength, pH and excipients (such as EDTA and ascorbic acid) to achieve stability. In contrast, the formulations claimed in the present invention achieve stability of the peptide compounds by using high concentrations (at least about 10%, w/w) of the compounds dissolved in water.

Examples of peptides and peptide compounds that can be formulated using the present invention include those that are biologically active or can be used to treat disease or other pathological conditions. They include, but are not limited to: corticotropin, angiotensin I and II, atrial natriuretic peptide, bombesin, bradykinin, calcitonin, cerebellin, dynorphin a, alpha and beta endorphin, endothelin, enkephalin, epidermal growth factor, fertirelin, follicular gonadotropin releasing peptide, galanin, glucagon, gonadorelin, gonadotropin, ghrelin, histrelin, insulin, leuprorelin, LHRH, motilin, nafarelin, neurotensin, oxytocin, somatostatin, substance P, tumor necrosis factor, triptorelin and vasopressin. Analogs, derivatives, antagonists, agonists and pharmaceutically acceptable salts of the foregoing may also be used.

Depending on the particular peptide compound to be formulated, ionic strength and pH are factors that are of considerable concern. For example, we have found that preferred aqueous formulations of leuprolide acetate have a low ionic strength and a pH of about 4 to 6.

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

Hydrophilic and water-soluble peptidic compounds are preferred for use in the present invention, and one skilled in the art can readily determine which compound is useful depending on the water solubility of the compound, i.e., the compound must be soluble in water in an amount of at least about 10% (w/w), preferably the amount is a pharmaceutically effective amount, and particularly preferred peptidic compounds are LHRH-related compounds including leuprolide and leuprolide acetate, etc.

The proportion of peptide varies with the compound, the disease to be treated, the solubility of the compound, the intended dose and the duration of administration (see, for example, the pharmacological basis of therapy, Gilman et al, 7 th edition (1985) and the pharmaceutical sciences, Remington, 18 th edition (1990), the contents of which are incorporated herein by reference). The concentration of the peptide compound ranges from at least about 10% (w/w) to the maximum solubility of the compound, preferably ranges from about 20 to about 60% (w/w), more preferably ranges from about 30 to about 50% (w/w), and most preferably ranges from about 35 to about 45% (w/w).

In general, stable formulations of the invention can be prepared by simply dissolving a therapeutically effective amount of the desired peptide compound in water, although the pH can be adjusted.

It is known to those skilled in the art that it is advantageous to add buffers, excipients, solvents such as EtOH, solubilizers such as nonionic surfactants and preservatives to pharmaceutical peptide formulations (see, e.g., pharmaceutical sciences, Remington, 18 th edition (1990)). Such agents may optionally be added to the formulations claimed in the present invention.

C. The methodology is as follows:

we have found that stable aqueous peptide compound formulations can be prepared by dissolving a high concentration (at least about 10%) of the peptide compound formulated in water.

We examined the stability of formulations of these peptide compounds, in particular the LHRH-related compound leuprolide, by subjecting the formulations to elevated temperatures to accelerate their aging and measuring the chemical and physical stability of the formulations. The results of these studies (as shown in table III and figures 1, 2 and 6) indicate that these formulations are stable upon storage at 37 ℃ for nearly 1 year or more.

We also tested the stability of peptide compound formulations prepared as described herein after 2.5 mrad gamma irradiation. The results shown in Table IV indicate that these formulations remain chemically and physically stable after such irradiation. The formulations were also found to be stable after electron beam irradiation.

As shown in table I, we examined the stability of a number of different peptide formulations, in particular leuprolide, goserelin, LHRH, angiotensin I, bradykinin, calcitonin, insulin, trypsinogen and vasopressin, by dissolving (or trying to dissolve) the formulations in water and then subjecting the formulations to accelerated aging at higher temperatures. Stability of the formulations was determined and the results shown in Table I are for hypothetical E_aHalf life at 37 ℃ at 22.2 kcal/mole. The large amount of peptide to be detected is soluble in water and can be placed on a test stripThe part is kept stable. The solubility of a particular peptide in water and the stability of the resulting solution can be readily determined using conventional methods well known to those skilled in the art.

Table I: stability of peptides formulated in Water

Preparation	Half life*(temperature)
		40% leuprorelin	9.7 years (37 deg.C)
40% of norrelin	19.3 months (80 deg.C)
		20％LHRH	2.5 years (65 ℃ C.)
20% angiotensin I	Insoluble gel (65 ℃ C.)
		20% bradykinin	8.5 months (65 deg.C)
40% calcitonin	Not dissolved (80 ℃ C.)
		20% calcitonin	9.6 months (80 deg.C)
5% calcitonin	23.5 months (50 ℃ C.)
		20% insulin	Insoluble gel (65 ℃ C.)
40% trypsinogen	Insoluble gel (65 ℃/80 ℃ C.)
		20% trypsinogen	Insoluble gel (65 ℃ C.)
40% vasopressin	Degradation (80 ℃ C.)
		20% vasopressin	14.3 days (65 ℃ C.)
Suppose EaHalf life at 37 ℃ of 22.2kcal/mole*

From the total loss of peptide in solution: the 40% leuprolide formulation dissolved in water showed linear degradation upon storage at 37 ℃ for 6 months. These data were analyzed to obtain activation energy (E)_a) Is 22.2kcal/mole, t₉₀At 13.8 months, these formulations were shown to be stable at higher temperatures.

We have also unexpectedly found that certain peptide formulations of the invention are bacteriostatic (i.e. inhibit the growth of bacteria), bacteriocidal (i.e. cause the death of bacteria) and sporicidal (i.e. kill spores). In particular, the leuprorelin preparation of 50-400mg/ml exhibits bacteriostatic, bactericidal and sporicidal activity. The stability of the samples was not affected by the addition of bacteria, indicating that the enzymes released by the killed and lysed bacteria did not adversely affect the stability of the product and that these preparations had no benefit on the enzyme activity.

It is known that some peptides, such as calcitonin and leuprolide, are physically less stable when formulated in aqueous solutions, they exhibit aggregation, gelation and fibrillation. For example, leuprolide can be induced to gel by increasing peptide concentration, introducing salt or gentle agitation. Improving physical stability may facilitate parenteral administration, including administration using implantable drug delivery systems, and the like.

We have unexpectedly found that the addition of a polar aprotic solvent such as DMSO to an aqueous formulation of certain peptides such as leuprolide, goserelin and calcitonin prevents gelling of the formulation. This is apparently because the non-aqueous polar aprotic solvents form the peptide into a random coil/alpha helix configuration that does not refold into a beta sheet structure and thus does not gel, and therefore, these solvents have an anti-gelling agent effect.

In addition, studies of gelled and ungelled aqueous formulations of leuprolide (370mg/ml) stored at 37 ℃ for 6 weeks showed a chemical stability profile similar to that of the RP-HPLC assay, which is shown in FIG. 8. The stability of aqueous liquid and gelled (by stirring) leuprolide formulations (370mg/ml) was similarly studied in vitro and in vivo in rats at 37 ℃ respectively, and the results are shown in table II, which shows that the gelled and liquid formulations remain stable during 18 weeks of storage.

Table II: study of the stability of liquid and gelled aqueous leuprolide formulations

Study of	Time (week)	Of liquid (residual%)	Gelled (% residual)
				Long term stabilityProperty of (2)	6	98.00
Long term stability	12	91.50
				Long term stability	18	93.50
Rat	4		94.80
				Rat	6		93.50
Rat	12		92.30
				Rat	18		92.60

The main aspect of the present invention is that the aqueous solution containing a high concentration of the peptide compound is stable after being left at high temperature for a long period of time. Thus, these formulations are advantageous in that they can be shipped and/or stored at room temperature or at temperatures above room temperature for extended periods of time, and they are also suitable for use in implantable delivery devices.

Disclosure of embodiments of the invention

The following methods were used in the following examples.

1. Preparation of leuprorelin acetate solution

Leuprolide acetate (from e.g. Mallinckrodt, st. louis, Missouri) is weighed out and added to the weighed carrier (sterile distilled water, ethanol/water or water with non-ionic surfactant) in the appropriate concentration (w/w) and then stirred gently until dissolved.

Unless otherwise stated, the leuprolide free base content was calculated to be 37% free base as demonstrated by the analytical potency values, which unless otherwise stated was 40% leuprolide acetate.

2. Preparation of the reservoir

The reservoir of an implantable drug delivery device (disclosed in U.S. patent application serial No. 08/595,761, incorporated herein by reference) is filled with a suitable leuprolide acetate solution and the filled device is then tested for stability. A titanium or polymer reservoir closed at each end by a polymer plug is filled with the formulation, and the filled reservoir is then sealed in a multi-foil pouch and placed in an oven for testing stability.

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

3. Reverse phase-HPLC (RP-HPLC)

All stability samples were analyzed for leuprolide concentration and% peak area using a gradient elution reverse phase HPLC assay in which a refrigerated autosampler (4 ℃) was used to minimize sample degradation. The chromatography conditions used were as follows:

RP-HPLC chromatographic conditions

Description of the invention	Parameter(s)
		Column	HaiSil C18，4.6×250mm，S/N 5103051
Flow rate of flow	0.8ml/min
		Injection amount	20μl
Detection of	210nm
		Leuprorelin retention time	Between 25 and 30 minutes
Mobile phase	100mM sodium phosphate, pH3.0B 90% acetonitrile/water
		Gradient of gradient	Min 052540414646.150% B1526.526.56585851515

Leuprolide standards (dissolved in water) at 4 to 6 different concentration levels (typically 0.1-1.2mg/ml) were run through the column with the stability samples sandwiched between the standard sample series, with no more than 40 samples between the standard sample series. The peak areas integrated for the empty column volume and for the run 45 minutes were integrated, the integrated peak areas for the leuprolide standard were plotted as a function of concentration, and then the leuprolide concentration for the stability samples was calculated using linear regression. The% peak area of the leuprolide peak, the sum of all peaks eluted before leuprolide (labeled "other"), the sum of all peaks eluted after leuprolide (labeled "aggregate") were also recorded and plotted as a function of the time point of sampling.

4. Size Exclusion Chromatography (SEC)

Selected stability samples were analyzed for% peak area and molecular weight using a non-gradient solution SEC test using a freeze sampler (4 ℃). The chromatography conditions used were as follows:

SEC chromatographic conditions

Description of the invention	Parameter(s)
		Column	Pharmacia Peptide，HR10/30，10×300mm
Flow rate of flow	0.5ml/min
		Injection amount	20μl
Detection of	210nm
		Leuprorelin retention time	About 25 minutes
Mobile phase	100mM ammonium phosphate, pH2.0, 200mM sodium chloride, 30% acetonitrile

The column volume and total volume of the size exclusion column required to calculate molecular weight can be determined using Bio-Rad high molecular weight standards and 0.1% acetone, respectively. The retention times of the first peak and acetone peak of the Bio-Rad standard 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 column volume and total volume should be re-determined each time there is a change in the SEC column and HPLC system. Then the standard was run, followed by the stability sample. The standard mixture contained about 0.2mg/mL of the following peptides: fadrocyte peptide (MW 449), WLFR peptide (MW 619), angiotensin peptide (MW 1181), GRF (MW 5108), and cytochrome C (MW 12394), which were chosen because they sandwich leuprorelin molecular weight and they all have basic pis similar to leuprorelin (9.8-11.0).

The% peak area of all peaks was recorded and the molecular weight of each isolated peak was calculated using the following equation:

V_sflow rate (mL/min) × retention time of sample peak (min)

V_oFlow rate (mL/min) × retention time of empty column volume peak (min)

V_tFlow rate (mL/min) × retention time of total volume peak (min)

Wherein: v_sStandard or sample volume

V_oEmpty column volume

V_tTotal volume

Calculate V for each peptide Standard Peak_sThen using the previously determined V_tAnd V_oValues were calculated for Kd of each peptide standard. Using logMW vs Kd^-1The linear regression line in the plot of (a) determines the molecular weight of each peak in the stability sample and the% peak area of the stability sample is also recorded.

5. Instruments and materials

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

a Waters Millennium HPLC system consisting of a 717 autosampler, 626 pump, 6000S controller, 900 photodiode matrix detector and 414 refractive index detector (Waters Chromatography, Milford, MA).

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

HaiSil C18, 120A, 5 μm 4.6X 250mm HPLC column (Higgins analytical, Mountain View, CA).

Pharmacia Peptide, HR 10/30 SEC column (Pharmacia Biotech, Piscataway, NJ).

6. Purity of

Stability samples were analysed using RP-HPLC and the area under the leuprolide peak curve divided by the sum of the areas under all peak curves equals% purity [ it should be noted that the% concentration data expressed as% purity data (examples 5, 6 and 7) are not conclusive and the analytical method used to determine% concentration in these experiments is unreliable ].

The following examples are provided to illustrate the invention and are not meant to limit the invention in any way

The scope of the invention.

Example 1

Accelerated stability study of leuprorelin acetate reagent

A 40% (w/w) leuprolide acetate formulation (equivalent to 37% leuprolide free base) dissolved in sterile distilled water, ethanol/water (70/30) or water containing 10% tween 20 was prepared as described above and used to fill the reservoir of the implantable drug delivery device described above. Some reservoirs are made of polymeric materials and some are made of titanium.

The aging was accelerated by placing the filled devices in an incubator (Precision scientific or Therco) and storing at higher temperature (80-88 ℃) for 7 days. Assuming activation energy (E)_a) 22.2kcal/mole, the above conditions correspond to about 1.5 years at 37 ℃ or about 4 years at room temperature (25 ℃).

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

The results shown in Table III indicate that these aqueous formulations are capable of maintaining the stability of the LHRH related compound leuprolide. At least 65% of leuprolide was retained in each case. However, during the course of the study, a large amount of the formulation was evaporated from the reservoir along with EtOH, indicating that long term storage of formulations containing high concentrations of volatile solvents of the EtOH type at higher temperatures may be problematic. It was also found that the formulation containing the non-ionic surfactant 10% tween 20 was not more stable than the aqueous solution without this solubilising agent.

TABLE III

Stability of 40% (w/w) leuprolide acetate aqueous formulation after standing at elevated temperature for 7 days

Temperature (. degree.C.)	Reservoir material	Preparation	Percent leuprorelin on day 7
				88	Polymer and method of making same	40% in water	68
88	Titanium (IV)	40% in water	71
				88	Polymer and method of making same	40% in water	66*
88	Polymer and method of making same	40% in ethanol/water (70/30)	85**
				88	Polymer and method of making same	40% in 10% Tween 20	65
80	Polymer and method of making same	40% in water	83
				80	Polymer and method of making same	40% in water	80
80	Polymer and method of making same	40% in water	78
				80	Polymer and method of making same	40% in water	79
80	Polymer and method of making same	40% in water	83
				80	Polymer and method of making same	40% in water	77
80	Polymer and method of making same	40% in water	79
				80	Polymer and method of making same	40% in water	74
80	Polymer and method of making same	40% in water	88

^*10% is evaporated

^**60% is evaporated

Example 2

Stability studies of irradiated leuprolide acetate formulations

A 40% (w/w) leuprolide acetate formulation (equivalent to 37% leuprolide free base) dissolved in water was prepared as described above, and used to fill the reservoirs of the above implantable drug delivery devices, some of which were made of polymeric material and some of which were made of titanium.

The filled device was subjected to 2.5 mrad gamma irradiation, the sample was mounted to Sterigenics (Tustin, California) and gamma irradiated in batch mode (cobalt 60). The samples were then accelerated aged as in example 1. Samples labeled "cold" were mounted on dry ice and irradiated, sampled on days 0 and 7, and analyzed using RP-HPLC and SEC as described above to determine the chemical and physical stability of the irradiated formulations.

The results shown in table IV indicate that these leuprolide acetate formulations are stable after irradiation. At least 65% of the leuprolide was retained in each case, while only low levels of aggregates were produced.

TABLE IV

Stability of 40% (w/w) leuprolide acetate aqueous formulation after 2.5 mrad gamma irradiation

Reservoir material	Preparation	Irradiation	Leuprorelin% on day 7 (RP-HPLC)	SEC
								Day 0		Day 7
				Monomer%	Dimer/trimer%	Monomer%	Dimer/trimer%
								Polymer and method of making same	40% in water	Is that	75	90.4	1.2	80.9	3.9
Polymer and method of making same	40% in water	Whether or not	75	99.8	0.2	82.4	3.1
								Polymer and method of making same	40% in water	Cold	79	89.4	0.2	80.3	3.1
Titanium (IV)	40% in water	Is that	83	98.5	1.1	84.9	2
								Titanium (IV)	40% in water	Whether or not	Not testing	99.6	0	96.6	0
Titanium (IV)	40% in water	Is that	81	98.8	0.9	94.7	2.4
								Titanium (IV)	40% in water	Whether or not	82	99.9	0	95	1.9
Titanium (IV)	40% in water	Is that	73	99.1	0.9	88.3	3
								Titanium (IV)	40% in water	Is that	79	99	0.8	94.3	3.4
Titanium (IV)	40% in water	Is that	74	98.6	0.5	90.9	3.6

Example 3

Long-term accelerated stability study of water-soluble leuprolide acetate

A40% (w/w) leuprolide acetate solution dissolved in water was prepared, filled into a reservoir, stored at 80 ℃ for 2 months and analyzed as described above. The results shown in fig. 1(RP-HPLC) and 2(SEC) indicate that 81.1% leuprolide can be recovered after 2 months with only 14.6% chemical degradation and 5.1% physical aggregation.

Leuprolide acetate solution (dissolved in water, 40% (w/w)) was prepared, loaded, stored and analyzed as described above. Figure 4 is a graph of leuprolide and its chemical and physical degradation products recovered over a 3 month period. The sum of these three factors is also expressed as mass balance. The results show that we can attribute all peptide material to either intact leuprolide or its degradation products, indicating that stability studies have not missed unknown degradation processes or products.

Leuprolide acetate solution (40% (w/w) in water) was prepared as described above, filled into a reservoir, stored at 37 ℃, 50 ℃, 65 ℃ or 80 ℃ and analyzed using RP-HPLC. The results shown in fig. 5 show the loss of leuprolide from these solutions over a period of 3 to 6 months, indicating that leuprolide degradation follows quasi-first order kinetics. In addition, as discussed below, fig. 3 demonstrates that degradation of leuprolide dissolved in water follows linear arrhenius kinetics, and therefore, accelerated stability studies are an effective technique to assess the stability of leuprolide and push back to 37 ℃.

A40% (w/w) leuprolide acetate solution dissolved in water was prepared as described above, filled into a reservoir, stored at 37 deg.C, 50 deg.C, 65 deg.C or 80 deg.C, and analyzed using RP-HPLC. According to physical medicine: physical chemistry principle in pharmaceutical sciences (physical pharmacy): the results calculated as described by Physical Chemical Principles in the pharmaceutical sciences), 3 rd edition, Martin et al, Chapter 14 (1983) indicate the E of these solutions_aIs 22.2kcal/mole, t₉₀It was 13.8 months.

Data are shown below and an arrhenius plot of data is shown in figure 3.

Water (W)
			℃	Kobs (moon)-1)	t1/2(moon)
37	7.24×10-3	95.7
			50	3.21×10-2	21.6
65	0.111	6.3
			80	0.655	1.1

E_a＝22.2kcal/mole

Example 4

Long-term stability study of water-soluble leuprolide acetate

The chemical stability of the 40% leuprolide acetate solution prepared and analyzed as described above is shown in figure 6. After 9 months at 37 ℃ over 85% (88.3%) leuprolide was still present, with less than 10% (8.4%) of chemical degradation products formed (indicated as "early" in the figure according to the RP-HPLC profile) and less than 5% (3.5%) of physical aggregation (indicated as "late" in the figure according to the RP-HPLC data, but in good agreement with the SEC data).

Example 5

Accelerated stability study of goserelin

A30% (w/w) performance relin preparation dissolved in an acetate buffer (pH5.0, 0.0282M) containing 3% mannitol was prepared as described above, and then the preparation was stored in an ampoule at 80 ℃ for 14 days and analyzed for purity.

The results shown in figure 7 indicate that about 65% of sexual relin is retained after 9 days.

Example 6

Stability Studies of sex relin formulations

40-45% (w/w) goserelin formulation dissolved in 3% mannitol-containing acetate buffer or salt (0.9% NaCl) -containing acetate buffer was prepared as described above and placed in a polymer container.

The containers were stored in an oven at 37 ℃ for 1 month.

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

The results shown below show that these aqueous formulations maintain the stability of the LHRH related compound, relin. In each case, at least 98% of the goserelin was retained.

Medicine	Carrier	Purity%	Concentration%
				Sexual ruilin	Acetate buffer/mannitol acetate buffer/salt	98.198.0	54.250.1

Example 7

Stability studies of nafarelin formulations

A 15% (w/w) nafarelin formulation dissolved in acetate buffer containing 3% mannitol was prepared as described above and placed in a polymeric container.

The containers were stored in an oven at 37 ℃ for 1 month.

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

The results shown below demonstrate that these aqueous formulations maintain the LHRH related compound nafarexStability of the forest, as at least 98% of nafarelin is retained.

Medicine	Carrier	Purity%	Concentration%
				Nafarelin	Acetate buffer/mannitol	98.8	18.3

Modifications of the above-described modes for carrying out various embodiments of the present invention will be readily apparent to those skilled in the art after receiving the above-listed teachings of the present invention. The foregoing examples are intended to illustrate and not to limit the invention, the scope of which is defined by the following claims.

Claims (23)

1. A stable aqueous therapeutic formulation of an LHRH-related peptide compound selected from the group consisting of leuprolide, LHRH, nafarelin and goserelin, wherein said formulation contains at least 10% (w/w) of at least one of said LHRH-related peptide compounds and water, formulated to be stable upon standing at 37 ℃ for at least 2 months.

2. The formulation of claim 1, comprising at least 20-60% (w/w) LHRH-related peptide compounds.

3. The formulation of claim 1, comprising at least 30% (w/w) LHRH-related peptide compounds.

4. The formulation of claim 1, which is formulated to remain stable upon irradiation.

5. The formulation of claim 1, which is formulated to be stable upon standing at 80 ℃ for at least 2 months.

6. The formulation of claim 1 which is stable when left at 37 ℃ for at least 1 year.

7. The formulation of claim 1, which is suitable for use in an implantable drug delivery device.

8. The formulation of claim 1, further comprising at least one additive selected from the group consisting of buffers, excipients, additional solvents, solubilizers, and preservatives.

9. The formulation of claim 8, wherein the peptidic compound is leuprolide or goserelin and the additional solvent added is a polar aprotic solvent.

10. The formulation of claim 1 consisting of leuprolide acetate dissolved in sterile distilled water at 30% to 50% (w/w).

11. The formulation of claim 1, which is formulated as a gel.

12. The formulation of claim 8, said additional solvent being at least one non-aqueous polar aprotic solvent.

13. The formulation of claim 12, wherein the non-aqueous polar aprotic solvent is DMSO or DMF.

14. The formulation of claim 1, wherein the peptide compound is 50-400mg/ml leuprolide, said formulation also having bacteriostatic, bactericidal or sporicidal activity without the use of conventional bacteriostatic, bactericidal or sporicidal agents.

15. A method for preparing a stable aqueous therapeutic formulation of an LHRH-related peptide compound, the method comprising:

preparing an aqueous medium;

selecting at least one LHRH-related peptide compound selected from the group consisting of leuprolide, LHRH, nafarelin and goserelin, capable of forming a stable formulation in the aqueous medium at a concentration of at least 10% (w/w) such that at least 65% of the chemically and physically stable LHRH-related peptide compound remains after 2 months at 37 ℃; and

dissolving the at least one LHRH-related peptide compound in an aqueous medium to a concentration of at least 10% (w/w).

16. The method of claim 15, wherein the step of dissolving the at least one peptide compound in the aqueous medium comprises dissolving the at least one peptide compound in the aqueous medium to a concentration of 20-60% (w/w).

17. The method of claim 15, further comprising the step of adding one or more additives selected from the group consisting of buffers, excipients, additional solvents, solubilizers, and preservatives.

18. The method of claim 15, wherein the preparation of the aqueous medium comprises providing sterile distilled water, the step of selecting at least one peptide compound comprises selecting leuprolide acetate, and the step of dissolving the at least one peptide compound in the aqueous medium comprises dissolving leuprolide acetate in distilled water to a concentration of 30-50% (w/w).

19. The method of claim 17, wherein the step of adding one or more additives comprises adding an additional solvent comprising at least one non-aqueous polar aprotic solvent.

20. The method of claim 19, wherein the at least one non-aqueous polar aprotic solvent is DMSO or DMF.

21. Use of a formulation according to claim 1 in the manufacture of a composition or an implantable drug delivery device useful in the treatment of a condition in which relief can occur following administration of a peptide compound.

22. The use of claim 21 wherein the condition is prostate cancer and the peptide compound is leuprolide or an LHRH antagonist.

23. The use of claim 21, wherein the peptide compound is leuprolide, which is present in an amount of 50-400mg/ml, and the condition requires bacteriostatic, bactericidal or sporicidal activity.