United States Patent [19J
`Stevenson et al.
`
`[54] NON-AQUEOUS POLAR APROTIC PEPTIDE
`FORMULATIONS
`
`[75]
`
`Inventors: Cynthia L. Stevenson; Steven J.
`Prestrelski, both of Mountain View,
`Calif.
`
`[73] Assignee: ALZA Corporation, Palo Alto, Calif.
`
`[21] Appl. No.: 08/874,233
`
`[22] Filed:
`
`Jun. 13, 1997
`
`[51]
`
`Related U.S. Application Data
`[60] Provisional application No. 60/022,699, Jul. 3, 1996.
`Int. Cl.6
`
`............................. A61K 38/00; C07K 5/00;
`C07K 7/00
`[52] U.S. Cl. ............................. 514/15; 530/313; 530/328
`[58] Field of Search ..................................... 530/328, 313;
`514/15
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3,914,412 10/1975 Gendrich et al. ....................... 424/177
`4,547,370 10/1985 Roeske ...................................... 514/15
`4,661,472
`4/1987 Rivier et al. .............................. 514/15
`4,689,396
`8/1987 Roeske et al. .......................... 530/313
`4,851,385
`7/1989 Roeske ...................................... 514/15
`5,034,229
`7/1991 Magruder et al. ...................... 424/422
`5,057,318 10/1991 Magruder et al. ...................... 424/438
`5,110,596
`5/1992 Magruder et al. ...................... 424/438
`5,198,533
`3/1993 Schally et al. .......................... 530/313
`5,480,868
`1/1996 Kamei et al. ............................. 514/15
`
`FOREIGN PATENT DOCUMENTS
`
`1098151
`2008403
`2119248
`2119248
`WO92/20711
`WO94/19020
`WO95/00168
`WO95/04540
`WO97/27840
`
`12/1964
`11/1978
`4/1983
`11/1983
`11/1992
`9/1994
`1/1995
`2/1995
`8/1997
`
`United Kingdom ............. A61K 3/00
`United Kingdom .
`United Kingdom .
`United Kingdom ........... A61K 37/26
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`
`OIBER PUBLICATIONS
`
`Fu Lu, et al., "Percutaneous Absorption Enhancement of
`9/12,
`pp.
`Leuprolide", Pharmaceutical Research,
`1575-1576 (1992).
`Helm, et al., "Stability of Gonadorelin and Triptorelin in
`Aqueous Solution", Pharmaceutical Research, 7/12, pp.
`1253-1256 (1990).
`Johnson, et al., "Degradation of the LH-RH Analog Nafare(cid:173)
`lin Acetate in Aqueous Solution", Intl. J. of Pharmaceutics,
`31, pp. 125-129 (1986).
`Okada, et al., "Preparation of Three-Month Depot Injectable
`Microspheres of Leuprorelin Acetate Using Biodegradable
`Polymers", Pharmaceutical Research, 11/8, pp. 1143-1147
`(1994).
`
`I 1111111111111111 11111 111111111111111 IIIII IIIII 1111111111111111 Ill lllll llll
`US005932547A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,932,547
`Aug. 3, 1999
`
`of
`Product
`"New Degradation
`al.,
`et
`Okada,
`Des-Gly1°-NH2-LH-RH-Ethylamide (Fertirelin) in Aque(cid:173)
`ous Solution", J. of Pharmaceutical Sciences, 80/2, pp.
`167-170 (1991).
`Oyler, et al., "Characterization of the Solution Degradation
`Products of Histrelin, a Gonadotropin Releasing Hormone
`(LH/RH) Agonist'', J. Pharmaceutical Sciences, 80/3, pp.
`271-275 (1991).
`Powell, et al., "Peptide Liquid Crystals: Inverse Correlation
`of Kinetic Formation and Thermodynamic Stability in Aque(cid:173)
`ous Solution", Pharmaceutical Research, 11/9, pp.
`1352-1354 (1994).
`Powell, et al., "Parenteral Peptide Formulations: Chemical
`and Physical Properties of Native Luteinizing Hormone-R(cid:173)
`eleasing Hormone (LHRH) and Hydrophobic Analogues in
`Aqueous Solution", Pharmaceutical Research, 8/10, pp.
`1258-1263 (1991).
`Powers, et al., "Solution Behavior of Leuprolide Acetate, an
`LHRH Agonist, as Determined by Circular Dichroism Spec(cid:173)
`troscopy", Intl. J. of Pharmaceutics, 108, pp. 49-55 (1994).
`Shi, et al., "Long-Term Stability of Aqueous Solutions of
`Luteinizing Hormone-Releasing Hormone Assessed by an
`In-Vitro Bioassay and Liquid Chromatography", J. of Phar(cid:173)
`maceutical Sciences, 73/6, pp. 819-821 (1984).
`Toguchi, "Pharmaceutical Manipulation of Leuprorelin
`Acetate to Improve Clinical Performance", J. of Intl. Medi(cid:173)
`cal Research, 18, pp. 35-41 (1990).
`Factrel (gonadorelin HCI for subcutaneous of IV injection),
`Physician's Desk Reference, 50th Edition, pp. 2877-2878
`(1996).
`Lupron (leuprolide acetate for subcutaneous injection), Phy(cid:173)
`sician's Desk Reference, 50th Edition, pp. 2555-2556
`(1996).
`Lupron Depot (leuprolide acetate for depot suspension),
`Physician's Desk Reference, 50th Edition, pp. 2556-2562
`(1996).
`Lutrepulse (gonadorelin acetate for IV injection), Physi(cid:173)
`cian's Desk Reference, 50th Edition, pp. 980-982 (1996).
`Zoladex (goserelin acetate implant), Physician's Desk Ref(cid:173)
`erence, 50th Edition, pp. 2858-2861 (1996).
`
`Primary Examiner-Avis M. Davenport
`Attorney, Agent, or Firm-Pauline Ann Clarke; Stephen F.
`Stone; Mary Ann Dillahunty
`
`[57]
`
`ABSTRACT
`
`This invention relates to stable non-aqueous polar aprotic
`formulations of peptide compounds. These stable formula(cid:173)
`tions comprise peptide in non- aqueous polar aprotic sol(cid:173)
`vent. They may be stored at elevated temperatures for long
`periods of time and are especially useful in implantable
`delivery devices for long term delivery of drug.
`
`34 Claims, 8 Drawing Sheets
`
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`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
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`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 1 of 8
`
`5,932,547
`
`Peptide
`
`~
`
`I
`
`I
`
`0.0
`
`~
`I
`10.0
`
`I
`
`...J\...J-.
`I
`
`I
`20.0
`Time (min)
`
`FIG. 1
`
`I
`30.0
`
`I
`
`I
`40.0
`
`Monomer
`
`0.0
`
`10.0
`
`Dimer
`
`\
`
`~
`
`t
`
`'
`30.0
`
`I
`
`40.0
`
`20.0
`Time (min)
`
`FIG. 2
`
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`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 2 of 8
`
`5,932,547
`
`-2 -r-~--------------------,
`
`l:l
`
`In Kobs DMSO
`
`0
`(/) ::
`C
`"'
`.c
`0
`~
`
`C
`
`-3
`
`-4
`
`-5
`
`-6 ~------,----r----r---r---r----r---,r--r-----,
`0.0033
`0.0032
`0.0031
`0.0030
`0.0029
`0.0028
`
`1/T
`
`FIG. 3
`
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`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 3 of 8
`
`5,932,547
`
`120 - - - - - - - - - - - - - - - - - - - - - ,
`
`cu
`"C
`
`J:) .. C.
`
`:l cu
`..J
`::e
`
`0
`
`80
`
`60
`
`40
`
`20
`
`[:] % Leuprolide
`
`• % Chemical
`
`C % Di/Trimer
`0 Mass Balance
`
`oJ!ll=.::_.!;-_-r--r---.---r---.--r----1
`a
`o
`6
`4
`2
`(months)
`
`Time
`
`FIG. 4
`
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`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 4 of 8
`
`5,932,547
`
`4.4
`
`cu
`
`"C ·--0 ...
`C. = 4.2
`
`cu
`-I
`
`~
`
`C -
`
`4.0
`
`l:J
`
`37°c
`
`• so 0 c
`
`a
`ss 0 c
`~ 80°C
`
`3.8 ..1.-----.,.----r---r----i----,---~-~8
`6
`4
`2
`O
`(months)
`
`Time
`
`FIG. 5
`
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`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 5 of 8
`
`5,932,547
`
`),( . . ·, . .
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`Novo Nordisk A/S Ex. 2010, P. 6
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 6 of 8
`
`5,932,547
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`Novo Nordisk A/S Ex. 2010, P. 7
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 7 of 8
`
`5,932,547
`
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`
`Novo Nordisk A/S Ex. 2010, P. 8
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`U.S. Patent
`
`Aug. 3, 1999
`
`Sheet 8 of 8
`
`5,932,547
`
`(cid:127)
`
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`
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`
`Novo Nordisk A/S Ex. 2010, P. 9
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`1
`NON-AQUEOUS POLAR APROTIC PEPTIDE
`FORMULATIONS
`
`5,932,547
`
`2
`23. "Long-Term Stability of Aqueous Solutions of Lutein(cid:173)
`izing Hormone-Releasing Hormone Assessed by an
`In-Vitro Bioassay and Liquid Chromatography", Y. F. Shi,
`R. J. Sherins, D. Brightwell, J. F. Gallelli, D. C. Chatterji,
`J. of Pharmaceutical Sciences, 73/6, pages 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.
`Si, Pharmaceutical Research, 11/9, pages 1352-1354
`(1994).
`25. "Solution Behavior of Leuprolide Acetate, an LHRH
`Agonist, as Determined by Circular Dichroism
`Spectroscopy", M. E. Powers, A Adejei, M. Y. Fu Lu, M.
`C. Manning, Intl. J. of Pharmaceutics, 108, pages 49-55
`(1994).
`26. "Preparation of Three-Month Depot Injectable Micro-
`spheres of Leuprorelin Acetate Using Biodegradable
`Polymers", Pharmaceutical Research, 11/8, pages
`1143-1147 (1994).
`The disclosure of each of the above publications, patents
`or patent applications is hereby incorporated by reference in
`its entirety to the same extent as if the language of each
`individual publication, patent and patent application were
`25 specifically and individually incorporated by reference.
`Luteinizing hormone-releasing hormone (LHRH), also
`known as gonadotropin releasing hormone (GnRH), is a
`decapeptide with the structure:
`pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 . It is
`30 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 ana(cid:173)
`logs of LHRH are known, including peptides related to
`35 LHRH which act as agonists and those which act as antago(cid:173)
`nists. [1-15] LHRH analogs are known to be useful for
`treating hormone-dependent diseases such as prostate
`cancer, benign prostatomegaly, endometriosis,
`hysteromyoma, metrofibroma, precocious puberty, or mam-
`40 mary cancer and as contraceptives. [8] Sustained release
`administration is preferred for both agonist LHRH-related
`compounds, which reduce the number of available receptors
`after repeated administration so that the production of ste(cid:173)
`roid hormones is suppressed, and antagonist LHRH-related
`45 compounds, which must be continually administered for
`persistent inhibition of endogenous LHRH. [8]
`The sustained parenteral delivery of drugs, especially
`peptide drugs, provides many advantages. The use of
`implantable devices for sustained delivery of a wide variety
`50 of drugs or other beneficial agents is well known in the art.
`Typical devices are described, for example, in 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.
`In general, oral bioavailability of peptides, including
`55 LHRH-related compounds, is low. [16-17]
`Currently marketed formulations of LHRH, its analogs
`and related compounds which are used for parenteral injec(cid:173)
`tion are aqueous solutions which contain relatively low
`concentrations of LHRH-related compounds (0.05 to 5
`60 mg/ml) and may also contain excipients such as mannitol or
`lactose.[18-20] Such formulations of LHRH-related com(cid:173)
`pounds must either be stored refrigerated or may be stored
`at room temperature for short periods of time.
`Available depot formulations of LHRH-related com-
`65 pounds administered for sustained release over a period of
`1-3 months include a formulation comprised of 15%
`LHRH-related compound dispersed in a matrix ofD,L-lactic
`
`20
`
`15
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`This application claims priority under 35 U.S.C. 119(e) 5
`to U.S. application Ser. No. 60/022,699 filed Jul. 3, 1996, the
`disclosure of which is incorporated herein by reference.
`FIELD OF THE INVENTION
`This invention relates to stable non-aqueous polar aprotic
`formulations of peptide compounds and more particularly to 10
`formulations of peptide compounds at high concentrations.
`BACKGROUND OF THE INVENTION
`References:
`The following references are referred to by numbers in
`brackets ([]) at the relevant portion of the specification.
`1. Zoladex (goserelin acetate implant), Physician's Desk
`Reference, 50th Edition, pages 2858-2861 (1996).
`2. U.S. Pat. No. 3,914,412, issued Oct. 21, 1975.
`3. U.S. Pat. No. 4,547,370, issued Oct. 15, 1985.
`4. U.S. Pat. No. 4,661,472, issued Apr. 28, 1987.
`5. U.S. Pat. No. 4,689,396, issued Aug. 25, 1987.
`6. U.S. Pat. No. 4,851,385, issued Jul. 25, 1989.
`7. U.S. Pat. No. 5,198,533, issued Mar. 30, 1993.
`8. U.S. Pat. No. 5,480,868, issued Jan. 2, 1996.
`9. WO92/20711, published 26 Nov. 1992.
`10. WO95/00168, published 5 Jan. 1995.
`11. WO95/04540, published 16 Feb. 1995.
`12. "Stability of Gonadorelin and Triptorelin in Aqueous
`Solution", V. J. Helm, B. W. Muller, Pharmaceutical
`Research, 7/12, pages 1253-1256 (1990).
`13. "New Degradation Product of Des-Gly1°-NH2-LH-RH(cid:173)
`Ethylamide (Fertirelin) in Aqueous Solution", J. Okada,
`T. Seo, F. Kasahara, K. Takeda, S. Kondo, J. of Pharma(cid:173)
`ceutical Sciences, 80/2, pages 167-170 (1991).
`14. "Characterization of the Solution Degradation Product
`of Histrelin, a Gonadotropin Releasing Hormone (LHRH)
`Agonist'', A R. Oyler, R. E. Naldi, J. R. Lloyd, D. A
`Graden, C. J. Shaw, M. L. Cotter, J. of Pharmaceutical
`Sciences, 80/3, pages 271-275 (1991).
`15. "Parenteral Peptide Formulations: Chemical and Physi(cid:173)
`cal Properties of Native Luteinizing Hormone-Releasing
`Hormone (LHRH) and Hydrophobic Analogues in Aque(cid:173)
`ous Solution", M. F. Powell, L. M. Sanders, A. Rogerson,
`V. Si, Pharmaceutical Research, 8/10, pages 1258-1263
`(1991).
`16. "Degradation of the LHRH Analog Nafarelin Acetate in
`Aqueous Solution", D. M. Johnson, R. A Pritchard, W. F.
`Taylor, D. Conley, G. Zuniga, K. G. McGreevy, Intl. J. of
`Pharmaceutics, 31, pages 125-129 (1986).
`17. "Percutaneous Absorption Enhancement of Leuprolide",
`M. Y. Fu Lu, D. Lee, G. S. Rao, Pharmaceutical
`Research, 9/12, pages 1575-1576 (1992).
`18. Lutrepulse (gonadorelin acetate for IV injection), Phy(cid:173)
`sician's Desk Reference, 50th Edition, pages 980---982
`(1996).
`19. Factrel (gonadorelin HCI for subcutaneous or IV
`injection), Physician's Desk Reference, 50th Edition,
`pages 2877-2878 (1996).
`20. Lupron (leuprolide acetate for subcutaneous injection),
`Physician's Desk Reference, 50th Edition, pages
`2555-2556 (1996).
`21. Lupron depot (leuprolide acetate for depot suspension),
`Physician's Desk Reference, 50th Edition, pages
`2556-2562 (1996).
`22. "Pharmaceutical Manipulation of Leuprorelin Acetate to
`Improve Clinical Performance", H. Toguchi, J. of Intl.
`Medical Research, 18, pages 35-41 (1990).
`
`Novo Nordisk A/S Ex. 2010, P. 10
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`

`

`5,932,547
`
`3
`and glycolic acids copolymer presented as a cylinder to be
`injected subcutaneously [1] and a formulation comprised of
`microparticles comprising a core of LHRH-related com(cid:173)
`pound and gelatin surrounded by a shell of D,L-lactic and
`glycolic acids copolymer. These microparticles are sus- s
`pended in a diluent for injection either subcutaneously or
`intramuscularly. [21, 26] These products must be stored at
`room temperature or lower. Aqueous formulations of
`LHRH-related compounds are known to exhibit both chemi-
`cal and physical instability, as well as degradation after 10
`irradiation. [12-16, 22-25]
`Formulations which have been shown to be stable (t90
`about five years) have been very low concentration (25
`µg/ml) aqueous, buffered (10 mM, ionic strength of 0.15)
`solutions stored at temperatures no higher than room tern- 15
`perature (25° C.). [15]
`There is a need for stable formulations of peptides.
`
`4
`FIG. 7 illustrates that increasing the concentration of the
`peptide leuprolide in DMSO solution increased stability at
`80° C.
`FIG. 8 illustrates that increasing the moisture content of
`40% leuprolide-DMSO formulations resulted in decreased
`stability at 80° C.
`FIG. 9 illustrates that, in the formulations shown in FIG.
`8, chemical degradation products increased with increasing
`moisture.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The present invention is drawn to the unexpected discov(cid:173)
`ery that dissolving peptide compounds in non-aqueous polar
`aprotic solvents results in stable formulations. Previously
`known formulations of peptide compounds, which are dilute
`buffered aqueous solutions containing excipients such as
`EDTA or ascorbic acid which must be stored at low tem-
`20 peratures ( 4--25° C.), form degradation products using deg(cid:173)
`radation pathways such as acid/base catalyzed hydrolysis,
`deamidation, racemization and oxidation. In contrast, the
`presently claimed formulations stabilize peptide compounds
`at elevated temperatures (e.g., 37° C. to 80° C.) and at high
`25 concentrations (i.e., at least about 10%).
`Standard peptide and protein formulations consist of
`dilute aqueous solutions. Drug stability is usually achieved
`by varying one or more of the following: pH, buffer type,
`ionic strength, excipients (EDTA, ascorbic acid, etc). For
`30 these formulations, degradation pathways requiring water
`(hydrolysis, deamidation, racemization) cannot be fully sta(cid:173)
`bilized. In contrast, in the present invention, peptides for(cid:173)
`mulated in non-aqueous solutions, such as dimethyl sulfox(cid:173)
`ide (DMSO) and dimethyl formamide (DMF), were shown
`3s to be chemically and physically more stable than those
`formulated in water. DMSO and DMF are considered polar
`aprotic solvents. Aprotic solvents would be expected to
`decrease the rate of degradation since they lack the ability to
`contribute protons to degradation reactions. Conversely,
`40 solvents that are more polar than water (for example, the
`dipole moment of water is 1.85, for DMF is 3.82, and for
`DMSO is 3.96) would be expected to increase the rate of
`degradation since they can assist in stabilizing the rate
`determining step and increasing the rate of degradation.
`4s However, we discovered that the overall effect of polar
`aprotic solvents was generally to stabilize solutions of
`peptides.
`The invention consists of using non-aqueous, aprotic
`solvents such as DMSO or DMF to stabilize peptide for-
`so mulations against both chemical and physical degradation.
`The discovery consists of the realization that use of DMSO
`or DMF improves the overall stability of peptides in a wide
`range of formulation conditions, including high concentra(cid:173)
`tions and elevated temperatures, thus making possible the
`ss delivery of peptides in long term implantable devices that
`would not otherwise be feasible.
`A Definitions:
`As used herein, the following terms have the following
`meanings:
`The term "chemical stability" means that an acceptable
`percentage of degradation products produced by chemical
`pathways such as oxidation or hydrolysis is formed. In
`particular, a formulation is considered chemically stable if
`no more than about 20% breakdown products are formed
`after two months at 37° C.
`The term "physical stability" means that an acceptable
`percentage of aggregates (e.g., dimers, trimers and larger
`
`SUMMARY OF THE INVENTION
`The present invention provides stable non-aqueous for(cid:173)
`mulations which are solutions of peptide compounds in
`polar aprotic solvents. In particular, the peptide compounds
`are formulated at concentrations of at least about 10%.
`These stable formulations may be stored at elevated tem(cid:173)
`peratures (e.g., 37° C.) for long periods of time and are
`especially useful in implantable delivery devices for long
`term delivery (e.g., 1-12 months or longer) of drug.
`In one aspect, the invention provides stable non-aqueous
`formulations of peptide compounds, said formulations com(cid:173)
`prising at least one peptide compound in at least one polar
`aprotic solvent. In a preferred embodiment, the formulation
`comprises at least about 10% (w/w) peptide compound.
`In another aspect, the invention provides methods for
`preparing a stable non-aqueous formulation of a peptide
`compound, said methods comprising dissolving at least one
`peptide compound in at least one polar aprotic solvent.
`Preferred formulations comprise at least about 10% (w/w)
`peptide compound.
`In yet a further aspect, the invention provides methods for
`treating a subject suffering from a condition which may be
`alleviated by administration of a peptide compound, said
`methods comprising administering to said subject an effec(cid:173)
`tive amount of a stable non-aqueous formulation comprising
`at least one peptide compound in at least one polar aprotic
`solvent.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 illustrates the stability of 40% leuprolide acetate
`solution (w/w) in dimethylsulfoxide (methylsulfoxide or
`DMSO) after two months at 80° C. as measured by reverse
`phase HPLC (RP-HPLC).
`FIG. 2 shows the same sample as FIG. 1 injected by size
`exclusion chromatography (SEC). This figure shows that
`there is very little aggregation, and what aggregation there
`is is comprised of dimer and trimer products, with no higher
`order aggregation.
`FIG. 3 presents the Arrhenius plot showing the loss of
`leuprolide from 40% solutions of leuprolide acetate in
`dimethylsulfoxide (DMSO).
`FIG. 4 illustrates the chemical and physical stability of a 60
`40% leuprolide solution in DMSO after six months at 80° C.
`FIG. 5 illustrates the loss of leuprolide from a 40%
`leuprolide acetate solution in DMSO over a period of six
`months at 37° C., 50° C., 65° C. or 80° C.
`FIG. 6 illustrates the chemical stability of a 40% leupro- 65
`lide acetate solution in DMSO over a period of nine months
`at 37° C.
`
`Novo Nordisk A/S Ex. 2010, P. 11
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`5,932,547
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`5
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`10
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`5
`forms) is formed. In particular, a formulation is considered
`physically stable if no more that about 15% aggregates are
`formed after two months at 37° C.
`The term "stable formulation" means that at least about
`65% chemically and physically stable peptide compound
`remains after two months at 37° C. ( or equivalent conditions
`at an elevated temperature). Particularly preferred formula(cid:173)
`tions are those which retain at least about 80% chemically
`and physically stable peptide under these conditions. Espe(cid:173)
`cially preferred stable formulations are those which do not
`exhibit degradation after sterilizing irradiation ( e.g., gamma,
`beta or electron beam).
`The terms "peptide" and/or "peptide compound" mean
`polymers of up to about 50 amino acid residues bound
`together by amide (CONH) linkages. Analogs, derivatives,
`agonists, antagonists and pharmaceutically acceptable salts
`of any of these are included in these terms. The terms also
`include peptides and/or peptide compounds which have
`D-amino acids, modified, derivatized or non-naturally
`occurring amino acids in the D- or L- configuration and/or 20
`peptomimetic units as part of their structure.
`The term "LHRH-related compound" means luteinizing
`hormone releasing hormone (LHRH) and its analogs and
`pharmaceutically acceptable salts. Octa-, nona- and
`decapeptide LHRH agonists and antagonists are included in 25
`the term LHRH-related compounds, as is native LHRH.
`Particularly preferred LHRH-related compounds include
`LHRH, leuprolide, goserelin, nafarelin, and other known
`active agonists and antagonists. [1-21]
`The term "high concentration" means at least about 10% 30
`(w/w) and up to the maximum solubility of the particular
`peptide.
`The term "excipient" means a more or less inert substance
`in a formulation which is added as a diluent or vehicle or to
`give form or consistency. Excipients are distinguished from
`solvents such as EtOH, which are used to dissolve drugs in
`formulations, and from non-ionic surfactants such as Tween
`20, which are used to solubilize drugs in formulations, and
`from preservatives such as benzyl alcohols or methyl or
`propyl parabens, which are used to prevent or inhibit micro(cid:173)
`bial growth.
`The term "polar aprotic solvent" means a polar solvent
`which does not contain acidic hydrogen and does not act as
`a hydrogen bond donor. Examples of polar aprotic solvents
`are dimethylsulfoxide (DMSO), dimethylformamide
`(DMF), hexamethylphosphorotriamide (HMPT), and
`n-methyl pyrrolidone.
`The term "non-aqueous protic solvent" means a non-polar
`solvent which contains hydrogen attached to oxygen or
`nitrogen so that it is able to form hydrogen bonds or donate
`a proton. Examples of apolar protic solvents are polyethyl(cid:173)
`ene glycols (PEGs), propylene glycol (PG), polyvinylpyr(cid:173)
`rolidone (PVP), methoxypropylene glycol (MPEG), glyc(cid:173)
`erol and glycofurol.
`B. Preparation of Formulations:
`The present invention is drawn to non-aqueous formula(cid:173)
`tions of peptide compounds in polar aprotic solvent which
`are stable for prolonged periods of time at elevated tem(cid:173)
`peratures. Standard dilute aqueous peptide and protein for(cid:173)
`mulations require manipulation of buffer type, ionic 60
`strength, pH and excipients (e.g., EDTA and ascorbic acid)
`to achieve stability. In contrast, the claimed formulations
`achieve stabilization of peptide compounds by the use of
`non-aqueous polar aprotic solvents. In particular, stability of
`high concentrations (at least about 10%, w/w) of compound 65
`has been provided by the formulations of the present inven-
`tion.
`
`6
`Examples of peptides and peptide compounds which may
`be formulated using the present invention include those
`peptides which have biological activity or which may be
`used to treat a disease or other pathological condition. They
`include, but are not limited to adrenocorticotropic hormone,
`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, goserelin,
`growth hormone releasing peptide, histrelin, insulin,
`leuprolide, LHRH, motilin, nafarelin, neurotensin, oxytocin,
`somatostatin, substance P, tumor necrosis factor, triptorelin,
`and vasopressin. Analogs, derivatives, antagonists, agonists
`and pharmaceutically acceptable salts of the above may also
`15 be used.
`The peptide compounds useful in the formulations and
`methods of the present invention can be used in the form of
`a salt, preferably a pharmaceutically acceptable salt. Useful
`salts are known to those of skill 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 which are readily
`soluble in non-aqueous polar aprotic solvents are preferred
`for use in the present invention. One of skill in the art can
`easily determine which compounds will be useful on the
`basis of their solubility, i.e., the compound must be soluble
`in the particular non-aqueous polar aprotic solvent to at least
`an acceptable amount. Preferred solubilities are at least
`about 10% (w/w). Particularly preferred peptide compounds
`are LHRH-related compounds, including leuprolide and
`leuprolide acetate.
`The proportion of peptide may vary depending on the
`compound, the condition to be treated, the solubility of the
`compound, the expected dose and the duration of adminis(cid:173)
`tration. (See, for example, The Pharmacological Basis of
`35 Therapeutics, Gilman et al., 7th ed. (1985) and Pharmaceu(cid:173)
`tical Sciences, Remington, 18th ed. (1990), the disclosures
`of which are incorporated herein by reference.) The con(cid:173)
`centration of peptide in high concentration formulations may
`range from at least about 10% (w/w) to the maximum
`40 solubility of the compound. A preferred range is from about
`20 to about 60% (w/w). The currently more preferred range
`is from about 30 to about 50% (w/w) and a most preferred
`range is about 35 to about 45% (wlw).
`It has unexpectedly been found that increasing the con-
`45 centration of peptide that is dissolved in the non-aqueous
`polar aprotic solvent may increase the stability of the peptide
`formulation. For example, as seen in FIG. 7, when solutions
`of 5, 10, 20 and 40% leuprolide in DMSO were stored for
`8 weeks at 80° C. with samples taken periodically and
`50 analyzed to determine the percentage of leuprolide
`remaining, formulations containing higher concentrations of
`leuprolide were more stable than formulations with lower
`concentrations of leuprolide.
`Generally, the stable formulations of the present invention
`55 may be prepared by simply dissolving the desired amount,
`which may be a therapeutically effective amount, of the
`desired peptide compound in the selected non-aqueous polar
`aprotic solvent. Preferred polar aprotic solvents include
`DMSO and DMF.
`Increasing the water contained in the peptide formulations
`of the present invention increased peptide degradation as
`shown in FIG. 8. It appears that this increase may be due
`mainly to increasing chemical degradation products, with
`aggregation remaining relatively constant (FIG. 9).
`It has also been found that non-aqueous protic solvents
`such as PEG, PG and PVP may optionally be added to the
`claimed formulations.
`
`Novo Nordisk A/S Ex. 2010, P. 12
`Mylan Institutional v. Novo Nordisk
`IPR2020-00324
`
`

`

`7
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`10
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`8
`of the present invention. Studies of leuprolide-DMSO for(cid:173)
`mulations have shown that at 65° C. and 80° C. oxidation
`appears to be the major chemical degradation pathway.
`Conversely, at 37° C. and 50° C. hydrolysis and isomeriza-
`tion appear to be the predominant degradation routes for
`these formulations.
`We have also unexpectedly found that certain peptide
`formulations of the present invention are bacteriostatic (i.e.,
`inhibit bacterial growth), bactericidal (i.e., cause the death of
`bacteria), and sporicidal (i.e., kill spores). In particular,
`leuprolide formulations of 50-400 mg/ml exhibited
`bacteriostatic, bactericidal and sporicidal activity. The sta(cid:173)
`bility of the samples was unaffected by spiking with
`bacteria, indicating that the enzymes released from the killed
`15 and lysed bacteria did not adversely affect the stability of the
`product. This demonstrates that these formulations were not
`conducive to enzymatic activity.
`Some peptides, for example calcitonin and leuprolide, are
`known to be physically unstable, exhibiting aggregation,
`20 gelation and fibrillation when formulated in aqueous solu(cid:173)
`tion. Improving physical stability can increase
`bioavailability, alleviate sensitization and immune response,
`and allow for easier parenteral administration, including
`administration using implantable drug delivery systems.
`It has unexpectedly been found that certain peptides, such
`as leuprolide, goserelin and calcitonin, formulated in the
`non-aqueous polar aprotic solvents of the present invention
`do not gel. No gelation was found even after 12 months at
`37° C. This is apparently because non-aqueous polar aprotic
`30 solvents cause peptides to form a random coil/alpha helix
`conformation that does not refold into a beta sheet structure
`and, therefore, does not gel. Thus, these solvents have an
`anti-gellant effect.
`A major aspect of the invention is that non-aqueous
`35 solutions containing peptide compounds in polar aprotic
`solvents are chemically and physically stable at high tem(cid:173)
`peratures for long periods of time. Such formulations are
`stable even when high concentrations are used. Thus, these
`formulations are advantageous in that they may be shipped
`40 and stored at temperatures at or above room temperature for
`long periods of time. They are also suitable for use m
`implantable delivery devices.
`
`5
`
`C. Methodology:
`We have found that stable non-aqueous formulations of
`peptide compounds may be prepared by dissolving the
`peptide compound to be formulated in non-aqueous polar
`aprotic solvents.
`We have tested these peptide compound formulations,
`specifically formulations of the LHRH-related compound
`leuprolide, for stability by subjecting them to accelerated
`aging at elevated temperature and measuring the chemical
`and physical stability of the formulations. Results of these
`studies (shown, for example, in Table II and FIGS. 1, 2, 4
`and 6) demonstra