`Pedersen et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,114,833 B2
`*Feb. 14, 2012
`
`US008114833B2
`
`(54) PROPYLENE GLYCOL-CONTAINING
`PEPTIDE FORMULATIONS WHICHARE
`OPTIMIAL FOR PRODUCTION AND FOR USE
`IN INJECTION DEVICES
`
`(75) Inventors: Tina Bjeldskov Pedersen, Smerum
`(DK); Claude Bonde, Lyngby (DK);
`Dorthe Kot Engelund, Holte (DK)
`(73) Assignee: Novo Nordisk A/S, Bagsvaerd (DK)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 663 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(21) Appl. No.: 11/435,977
`(22) Filed:
`May 17, 2006
`
`(65)
`
`Prior Publication Data
`US 2007/OO 10424 A1
`Jan. 11, 2007
`
`Related U.S. Application Data
`(63) Continuation
`of
`application
`PCT/DK2004/000792, filed on Nov. 18, 2004.
`(60) Provisional application No. 60/524,653, filed on Nov.
`24, 2003.
`Foreign Application Priority Data
`
`(30)
`
`No.
`
`Nov. 20, 2003 (DK) ................................. 2003 O1719
`
`(51) Int. Cl.
`(2006.01)
`A6 IK38/26
`(52) U.S. Cl. ............................................ 514/2:530/308
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
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`(Continued)
`Primary Examiner — Christina Bradley
`(74) Attorney, Agent, or Firm — Michael J. Brignati
`(57)
`ABSTRACT
`The present invention relates to pharmaceutical formulations
`comprising a peptide and propylene glycol, to methods of
`preparing Such formulations, and to uses of such formulations
`in the treatment of diseases and conditions for which use of
`the peptide contained in Such formulations is indicated. The
`present invention further relates to methods for reducing the
`clogging of injection devices by a peptide formulation and for
`reducing deposits on production equipment during produc
`tion of a peptide formulation.
`31 Claims, 7 Drawing Sheets
`
`MYLAN INST. EXHIBIT 1001 PAGE 1
`
`
`
`US 8,114,833 B2
`Page 2
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`
`MYLAN INST. EXHIBIT 1001 PAGE 2
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`
`
`US 8,114,833 B2
`Page 3
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`European Pharmacopoeia, 3rd Edition, 2.2.3, 1997, pp. 17-8, Council
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`Journal of Medicinal Chemistry, 2000, vol.43, pp. 1664-9.
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`Edition, 1980, PT. 79, p. 1406.
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`Martin A. et al., Physical Pharmacy; Physical Chemical Principles in
`the Pharmaceutical Sciences, 1983, 3rd Edition, p. 232.
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`the Pharmaceutical Sciences, 1983, 3rd Edition, p. 323.
`Sigma Product Information on Gly-Gly Buffer Dated Mar. 16, 2010.
`Stenesh, J. Biochemistry, 1998, pp. 67-9.
`United States Pharmacopoeia, 24th Edition, 1999, pp. 1977-8.
`Villanueva Penacarril M.L. Potent Glycognic Effect of Glp-1(7-36)
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`* cited by examiner
`
`MYLAN INST. EXHIBIT 1001 PAGE 3
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 1 of 7
`
`US 8,114,833 B2
`
`FIGURE 1
`
`
`
`MYLAN INST. EXHIBIT 1001 PAGE 4
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 2 of 7
`
`US 8,114,833 B2
`
`
`
`FIGURE 2
`
`Mannitol
`
`Glyce
`
`MYLAN INST. EXHIBIT 1001 PAGE 5
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 3 of 7
`
`US 8,114,833 B2
`
`FIGURE 3
`
`
`
`starteresa in
`
`stra a
`
`is
`
`tarter startlycaretailures
`
`Myo-inositol
`
`Matose
`
`Glycerol
`
`MYLAN INST. EXHIBIT 1001 PAGE 6
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 4 of 7
`
`US 8,114,833 B2
`
`FGURE 4
`
`
`
`Glycine
`
`LactoSe
`
`Mannito
`
`MYLAN INST. EXHIBIT 1001 PAGE 7
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 5 of 7
`
`US 8,114,833 B2
`
`FIGURE 5
`
`
`
`MYLAN INST. EXHIBIT 1001 PAGE 8
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 6 of 7
`
`US 8,114,833 B2
`
`FIGURE 6
`
`
`
`MYLAN INST. EXHIBIT 1001 PAGE 9
`
`
`
`U.S. Patent
`
`Feb. 14, 2012
`
`Sheet 7 Of 7
`
`US 8,114,833 B2
`
`FGURE 7
`
`
`
`Mento
`
`MYLAN INST. EXHIBIT 1001 PAGE 10
`
`
`
`US 8,114,833 B2
`
`1.
`PROPYLENE GLYCOL-CONTAINING
`PEPTIDE FORMULATIONS WHICHARE
`OPTIMIAL FOR PRODUCTION AND FOR USE
`IN INJECTION DEVICES
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This Application is a continuation of International Appli
`cation serial no. PCT/DK2004/000792 filed Nov. 18, 2004
`and claims priority from U.S. application Ser. No. 60/524,653
`filed Nov. 24, 2003 and from Danish Application serial no. PA
`2003 01719 filed Nov. 20, 2003.
`
`FIELD OF THE INVENTION
`
`10
`
`15
`
`The present invention relates to pharmaceutical formula
`tions comprising a peptide and propylene glycol, to methods
`of preparing Such formulations, and to uses of such formula
`tions in the treatment of diseases and conditions for which use
`of the peptide contained in Such formulations is indicated.
`The present invention further relates to methods for reducing
`the clogging of injection devices by a peptide formulation and
`for reducing deposits on production equipment during pro
`duction of a peptide formulation.
`
`25
`
`BACKGROUND OF THE INVENTION
`
`2
`preferred embodiment, the pharmaceutical formulations of
`the invention further contain a buffer and a preservative.
`The present invention also relates to methods for producing
`the pharmaceutical formulations of the invention.
`In one embodiment, the method for preparing a peptide
`formulation comprises:
`a) preparing a first Solution by dissolving preservative,
`propylene glycol and buffer in water,
`b) preparing a second solution by dissolving the peptide in
`water;
`c) mixing the first and second solutions; and
`d) adjusting the pH of the mixture in c) to the desired pH.
`In another embodiment, the method for preparing a peptide
`formulation comprises:
`a) preparing a first solution by dissolving preservative and
`buffer in water;
`b) adding propylene glycol to the first Solution;
`c) mixing the first Solution with a second solution contain
`ing peptide dissolved in water, and
`d) adjusting the pH of the mixture in c) to the desired pH.
`In yet another embodiment, the method for preparing a
`peptide formulation comprises:
`a) preparing a solution by dissolving preservative, buffer
`and propylene glycol in water;
`b) adding the peptide to the solution of step a); and
`c) adjusting the pH of the solution of step b) to the desired
`pH.
`The present invention further relates to methods of treat
`ment using the pharmaceutical formulations of the invention
`where the compositions are administered in an amount effec
`tive to combat the disease, condition, or disorder for which
`administration of the peptide contained in the formulation is
`indicated.
`In addition the present invention also relates to a method
`for reducing deposits on production equipment during pro
`duction of a peptide formulation, where the method com
`prises replacing the isotonicity agent previously utilized in
`said formulation with propylene glycol at a concentration of
`between 1-100 mg/ml.
`In one embodiment, the reduction in deposits on the pro
`duction equipment during production by the propylene gly
`col-containing formulation relative to that observed for the
`formulation containing the previously utilized isotonicity
`agent is measured by a simulated filling experiment.
`The present invention also relates to a method for reducing
`deposits in the final product during production of a peptide
`formulation, where the method comprises replacing the iso
`tonicity agent previously utilized in said formulation with
`propylene glycolata concentration of between 1-100 mg/ml.
`In one embodiment, the reduction in deposits in the final
`product is measured by a reduction in the number of vials
`and/or cartridges of the propylene glycol-containing formu
`lation that must be discarded due to deposits relative to num
`ber of vials and/or cartridges of the formulation containing
`the previously utilized isotonicity agent that must be dis
`carded due to deposits.
`The present invention further relates to a method for reduc
`ing the clogging of injection devices by a peptide formula
`tion, where the method comprises replacing the isotonicity
`agent previously utilized in said formulation with propylene
`glycol at a concentration of between 1-100 mg/ml.
`In one embodiment, the reduction in clogging of the injec
`tion device by the propylene glycol-containing formulation
`relative to that observed for the formulation containing the
`previously utilized isotonicity agent is measured in a simu
`lated in use study.
`
`30
`
`35
`
`The inclusion of isotonicity agents in peptide-containing
`pharmaceutical formulations is widely known and one of the
`more common isotonic agents used in such formulations is
`mannitol. However, the present inventors have observed that
`mannitol causes problems during the production of peptide
`formulations as it crystallizes resulting in deposits in the
`production equipment and in the final product. Such deposits
`increase the need to clean the filling equipment during pro
`duction of the formulation and this results in reduced produc
`tion capability. In addition, Such deposits may also result in
`reduced yield of the final product since vials/cartridges con
`taining the peptide formulation may need to be discarded if
`particles are present. Finally, the present inventors have
`observed that in peptide formulations to be administered by
`injection, the presence of mannitol results in clogging of
`injection devices.
`45
`Accordingly, it is desirable to identify an alternative iso
`tonic agent to mannitol for inclusion in peptide-containing
`formulations and in particular, for inclusion in peptide for
`mulations which are administered by injection.
`
`40
`
`50
`
`SUMMARY OF THE INVENTION
`
`The present inventors have discovered that peptide formu
`lations containing propylene glycol at certain concentrations
`exhibit reduced deposits in production equipment and in the
`final product and also exhibit reduced clogging of injection
`devices. The present compositions may be formulated with
`any peptide and are also physically and chemically stable thus
`rendering them shelf-stable and Suitable for invasive (e.g.
`injection, Subcutaneous injection, intramuscular, intravenous
`or infusion) as well as non-invasive (e.g. nasal, oral, pulmo
`nary, transdermal or transmucosal e.g. buccal) means of
`administration.
`The present invention therefore relates to a pharmaceutical
`formulation comprising a peptide and propylene glycol,
`where the propylene glycol is present in a concentration of
`1-100 mg/ml and the pH of the formulation is from 7-10. In a
`
`55
`
`60
`
`65
`
`MYLAN INST. EXHIBIT 1001 PAGE 11
`
`
`
`3
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 shows a photograph of dried droplets on microscope
`slides of from left to right, placebo (no peptide) formulations
`containing no isotonic agent (e only water, preservative and
`buffer), mannitol, Sorbitol. Xylitol. Sucrose or glycerol as the
`isotonic agent with the far right slide containing mannitol
`with peptide Arg, Lys’(N-(y-GlucN'-hexadecanoyl)))-
`GLP-1(7-37).
`FIG. 2 shows light microscopy pictures of from left to
`right, some of the dried droplets of placebo formulations
`containing mannitol, arginin, inositol or glycerol as the iso
`tonic agent.
`FIG.3 shows light microscopy pictures of clogged needles
`dosed with placebo formulations containing myoinositol,
`maltose or glycerol as the isotonic agent.
`FIG. 4 shows light microscopy pictures of deposits on
`needles dosed with placebo formulations containing glycine,
`lactose or mannitol as the isotonic agent.
`FIG. 5 shows filling equipment after 24 hours simulated
`filling with Arg, Lys’(N-(y-Glu(N'-hexadecanoyl)))-
`GLP-1 (7-37) medium containing myo-inositol.
`FIG. 6 shows deposits on filling equipment after 24 hours
`simulated filling with a mannitol-containing placebo formu
`lation.
`FIG. 7 shows deposits on needles dosed with mannitol (top
`panel) and propylene glycol (bottom panel)-containing
`Arg, Lys'(N-(y-Glu(N'-hexadecanoyl)))-GLP-1 (7-37)
`formulations.
`
`DESCRIPTION OF THE INVENTION
`
`The present invention relates to a pharmaceutical formula
`tion comprising a peptide or a mixture of peptides and pro
`pylene glycol where the final concentration of propylene
`glycol in the formulation is 1-100 mg/ml and the pH of the
`formulation is in the range of from 7-10.
`The pharmaceutical formulations of the invention are
`found to be optimal for production because they exhibit
`reduced deposits in production equipment relative to formu
`lations containing other isotonicity agents as measured by the
`simulated filling studies described in the Examples. In addi
`tion, the pharmaceutical formulations of the invention are
`found to be optimal for use in injection devices because they
`exhibit reduced clogging of the injection devices relative to
`formulations containing other isotonicity agents as measured
`by the simulated in use studies described in the Examples.
`The formulations of the present invention may be formu
`lated with any peptide where examples of Such peptides
`include, but are not limited to, glucagon, human growth hor
`mone (hGH), insulin, aprotinin, FactorVII, tissue plasmino
`gen activator (TPA), FactorVIIa, FFR-FactorVIIa, hepari
`nase, ACTH, Heparin Binding Protein, corticotropin
`releasing factor, angio-tensin, calcitonin, glucagon-like
`peptide-1, glucagon-like peptide-2, insulin-like growth fac
`tor-1, insulin-like growth factor-2, fibroblast growth factors,
`gastric inhibitory peptide, growth hormone-releasing factor,
`pituitary adenylate cyclase activating peptide, Secretin,
`enterogastrin, Somatostatin, Somatomedin, parathyroid hor
`mone, thrombopoietin, erythropoietin, hypothalamic releas
`ing factors, prolactin, thyroid stimulating hormones, endor
`phins, enkephalins, vasopressin, oxytocin, opiods, DPP IV.
`interleukins, immunoglobulins, complement inhibitors,
`serine protease inhibitors, cytokines, cytokine receptors,
`PDGF, tumor necrosis factors, tumor necrosis factors recep
`tors, growth factors and analogues as well as derivatives
`
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`US 8,114,833 B2
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`4
`thereof where each of these peptides constitutes an alternative
`embodiment of the present invention.
`In the present application, the designation “an analogue' is
`used to designate a peptide wherein one or more amino acid
`residues of the parent peptide have been substituted by
`another amino acid residue and/or wherein one or more
`amino acid residues of the parent peptide have been deleted
`and/or wherein one or more amino acid residues have been
`added to the parent peptide. Such addition can take place
`either at the N-terminal end or at the C-terminal end of the
`parent peptide or both. Typically “an analogue' is a peptide
`wherein 6 or less amino acids have been substituted and/or
`added and/or deleted from the parent peptide, more prefer
`ably a peptide wherein 3 or less amino acids have been sub
`stituted and/or added and/or deleted from the parent peptide,
`and most preferably, a peptide wherein one amino acid has
`been substituted and/or added and/or deleted from the parent
`peptide.
`In the present application, “a derivative' is used to desig
`nate a peptide or analogue thereof which is chemically modi
`fied by introducing an organic Substituent e.g. ester, alkyl or
`lipophilic functionalities, on one or more amino acid residues
`of the peptide or analogue thereof.
`In one embodiment, the peptide to be included in the for
`mulation of the invention is a GLP-1 agonist where “a GLP-1
`agonist' is understood to refer to any peptide which fully or
`partially activates the human GLP-1 receptor. In a preferred
`embodiment, the “GLP-1 agonist' is any peptide that binds to
`a GLP-1 receptor, preferably with an affinity constant(K) or
`a potency (ECs) of below 1 uM, e.g. below 100 nM as
`measured by methods known in the art (see e.g. WO
`98/08871) and exhibits insulinotropic activity, where insuli
`notropic activity may be measured in vivo or in vitro assays
`known to those of ordinary skill in the art. For example, the
`GLP-1 agonist may be administered to an animal and the
`insulin concentration measured over time.
`Methods for identifying GLP-1 agonists are described in
`WO 93/19175 (Novo Nordisk A/S) and examples of suitable
`GLP-1 analogues and derivatives which can be used accord
`ing to the present invention includes those referred to in WO
`99/43705 (Novo Nordisk A/S), WO 99/43706 (Novo Nordisk
`A/S), WO 99/43707 (Novo Nordisk A/S), WO 98/08871
`(analogues with lipophilic substituent) and in WO 02/46227
`(analogues fused to serum albumin or to Fc portion of an
`Ig). (Novo Nordisk A/S), WO 99/43708 (Novo Nordisk A/S),
`WO99/43341 (Novo Nordisk A/S), WO 87/06941 (The Gen
`eral Hospital Corporation), WO 90/11296 (The General Hos
`pital Corporation), WO 91/11457 (Buckley et al.), WO
`98/43658 (Eli Lilly & Co.), EP0708179-A2 (Eli Lilly & Co.),
`EP 0699686-A2 (Eli Lilly & Co.), WO 01/98331 (Eli Lilly &
`Co).
`In one embodiment, the GLP-1 agonist is selected from the
`group consisting of GLP-1 (7-36)-amide, GLP-1 (7-37), a
`GLP-1 (7-36)-amide analogue, a GLP-1 (7-37) analogue, or a
`derivative of any of these.
`In one embodiment, the GLP-1 agonist is a derivative of
`GLP-1 (7-36)-amide, GLP-1 (7-37), a GLP-1 (7-36)-amide
`analogue or a GLP-1 (7-37) analogue, which comprises a
`lipophilic substituent.
`In this embodiment of the invention, the GLP-1 derivative
`preferably has three lipophilic substituents, more preferably
`two lipophilic Substituents, and most preferably one lipo
`philic substituent attached to the parent peptide (ie GLP-1 (7-
`36)-amide, GLP-1 (7-37), a GLP-1 (7-36)-amide analogue or
`a GLP-1 (7-37) analogue), where each lipophilic substituent
`(s) preferably has 4-40 carbon atoms, more preferably 8-30
`
`MYLAN INST. EXHIBIT 1001 PAGE 12
`
`
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`US 8,114,833 B2
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`carbonatoms, even more preferably 8-25 carbon atoms, even
`more preferably 12-25 carbon atoms, and most preferably
`14-18 carbon atoms.
`In one embodiment, the lipophilic Substituent comprises a
`partially or completely hydrogenated cyclopentanophenath
`rene skeleton.
`In another embodiment, the lipophilic substituent is a
`straight-chain or branched alkyl group.
`In yet another embodiment, the lipophilic substituent is an
`acyl group of a straight-chain or branched fatty acid. Prefer
`ably, the lipophilic Substituent is an acyl group having the
`formula CH-(CH2)CO—, wherein n is an integer from 4 to
`38, preferably an integer from 12 to 38, and most preferably
`is CH (CH2)CO—, CH (CH2)CO .CH (CH2)CO—,
`CH(CH2)CO
`CH-(CH2)CO
`and CH (CH)
`CO—. In a more preferred embodiment, the lipophilic Sub
`stituent is tetradecanoyl. In a most preferred embodiment, the
`lipophilic Substituent is hexadecanoyl.
`In a further embodiment of the present invention, the lipo
`philic Substituent has a group which is negatively charged
`Such as a carboxylic acid group. For example, the lipophilic
`Substituent may be an acyl group of a straight-chain or
`branched alkane C,c)-dicarboxylic acid of the formula HOOC
`(CH2)CO , wherein m is an integer from 4 to 38, prefer
`ably an integer from 12 to 38, and most preferably is HOOC
`25
`(CH2)CO HOOC(CH2)CO , HOOC(CH2)CO ,
`HOOC(CH2)CO or HOOC(CH2)CO-.
`In the GLP-1 derivatives of the invention, the lipophilic
`Substituent(s) contain a functional group which can be
`attached to one of the following functional groups of an
`amino acid of the parent GLP-1 peptide:
`(a) the amino group attached to the alpha-carbon of the
`N-terminal amino acid,
`(b) the carboxy group attached to the alpha-carbon of the
`C-terminal amino acid,
`(c) the epsilon-amino group of any Lys residue,
`(d) the carboxy group of the R group of any Asp and Glu
`residue,
`(e) the hydroxy group of the R group of any Tyr, Ser and
`Thr residue,
`(f) the amino group of the R group of any Trp, ASn, Gln,
`Arg, and His residue, or
`(g) the thiol group of the R group of any Cys residue.
`In one embodiment, a lipophilic Substituent is attached to
`the carboxy group of the R group of any Asp and Glu residue.
`In another embodiment, a lipophilic Substituent is attached
`to the carboxy group attached to the alpha-carbon of the
`C-terminal amino acid.
`In a most preferred embodiment, a lipophilic substituent is
`attached to the epsilon-amino group of any Lys residue.
`In a preferred embodiment of the invention, the lipophilic
`substituent is attached to the parent GLP-1 peptide by means
`of a spacer. A spacer must contain at least two functional
`groups, one to attach to a functional group of the lipophilic
`Substituent and the other to a functional group of the parent
`GLP-1 peptide.
`In one embodiment, the spacer is an amino acid residue
`except Cys or Met, or a dipeptide such as Gly-Lys. For pur
`poses of the present invention, the phrase “a dipeptide such as
`Gly-Lys' means any combination of two amino acids except
`Cys or Met, preferably a dipeptide wherein the C-terminal
`amino acid residue is Lys, His or Trp, preferably Lys, and the
`N-terminal amino acid residue is Ala, Arg, Asp, ASn, Gly,
`Glu, Gln, Ile, Leu, Val, Phe, Pro, Ser, Tyr, Thr, Lys, His and
`Trp. Preferably, an amino group of the parent peptide forms
`an amide bond with a carboxylic group of the amino acid
`residue or dipeptide spacer, and an amino group of the amino
`
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`acid residue or dipeptide spacer forms an amide bond with a
`carboxyl group of the lipophilic Substituent.
`Preferred spacers are lysyl, glutamyl, asparagyl, glycyl,
`beta-alanyl and gamma-aminobutanoyl, each of which con
`stitutes an individual embodiment. Most preferred spacers are
`glutamyland beta-alanyl. When the spacer is LyS, Glu or Asp,
`the carboxyl group thereof may form an amide bond with an
`amino group of the amino acid residue, and the amino group
`thereof may form an amide bond with a carboxyl group of the
`lipophilic Substituent. When Lys is used as the spacer, a
`further spacer may in Some instances be inserted between the
`e-amino group of Lys and the lipophilic Substituent. In one
`embodiment, such a further spacer is succinic acid which
`forms an amide bond with the e-amino group of Lys and with
`an amino group present in the lipophilic Substituent. In
`another embodiment such a further spacer is Glu or Asp
`which forms an amide bond with the e-amino group of Lys
`and anotheramide bond with a carboxyl group present in the
`lipophilic substituent, that is, the lipophilic substituent is a
`N-acylated lysine residue.
`In another embodiment, the spacer is an unbranched alkane
`C.()-dicarboxylic acid group having from 1 to 7 methylene
`groups, which spacer forms a bridge between an amino group
`of the parent peptide and an amino group of the lipophilic
`substituent. Preferably, the spacer is succinic acid.
`Inafurther embodiment, the lipophilic substituent with the
`attached spacer is a group of the formula CH3(CH2)NH
`CO(CH),CO , wherein p is an integer from 8 to 33, pref
`erably from 12 to 28 and q is an integer from 1 to 6, preferably
`2.
`Inafurther embodiment, the lipophilic substituent with the
`attached spacer is a group of the formula CH (CH),CO—
`NHCH(COOH)(CH),CO. , wherein r is an integer from 4
`to 24, preferably from 10 to 24.
`Inafurther embodiment, the lipophilic substituent with the
`attached spacer is a group of the formula CH3(CH2)CO—
`NHCH((CH2)COOH)CO