PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`A61K 58/26, C07K 14/605
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
` WO 99/4336]
`(51) International Patent Classification 6 :
`
`
`(11) International Publication Number:
`
`
`
`(43) International Publication Date:
`2 September 1999 (02.09.99)
`
`
`(21) International Application Number:
`
`PCT/DK99/00080
`25 February 1999 (25.02.99)
`
`(22) International Filing Date:
`
`
`(30) Priority Data:
`
`DK
`27 February 1998 (27.02.98)
`0271/98
`
`
`
`
`
`(71) Applicant: NOVO NORDISK A/S [DK/DK]; Novo Allé,
`DK—2880 Bagsvaerd (DK).
`
`
`
`
`
`
`(72) Inventors: KNUDSEN, Liselotte, Bjerre; Valby Langgade
`49A, 1.
`tv., DK~2500 Valby (DK). HUUSFELDT, Per,
`
`
`Olaf; Applebys Plads 27,5. mf., DK—1411 Copenhagen K
`
`
`(DK). NIELSEN, Per, Franklin; Dalso Park 59, DK—3500
`erlose (DK). KAARSHOLM, Niels, C.; Clausholmvej 38,
`DK—2720 Vanlose (DK). OLSEN, Helle, Birk; Skolelodden
`
`
`23, DK—3450 Allergd (DK). THIM, Lars; Skiftevej 22,
`DK—2820 Gentofte (DK). BJORN, Soren, Erik; Marie
`Grubbes A116 47, DK—2800 Lyngby (DK).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TR, TI‘, UA, UG, UZ, VN, YU, ZW,
`ARIPO patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
`ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European patent (AT, BE, CH, CY, DE, DK, ES, FI,
`FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE,
`SN, TD, TG).
`
`Published
`
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`
`
`
`
`(54) Title: GLP—Z DERIVATIVES WITH HELIX—CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTURED MICEL-
`LAR—LIKE AGGREGATES
`
`(57) Abstract
`
`The present invention relates to a pharmaceutical composition comprising a GLP—2 derivative of improved solubility and/or stability,
`and to a method for improving the solubility and/or stability of GLP—2 or a fragment or an analogue thereof.
`
`
`
`CFAD Exhibit 1025
`CFAD Exhibit 1025
`
`1
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Zimbabwe
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`
`2
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`GLP—Z DERIVATIVES WITH HELIX—CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTURED MICEL-
`LAR—LIKE AGGREGATES
`
`Field of the invention
`
`The present invention relates to a pharmaceutical composition comprising a GLP-2 derivative
`
`of improved solubility and/or stability, and to a method for improving the solubility and/or stabi-
`
`lity of GLP-2 or a fragment or an analogue thereof.
`
`Background of the invention
`
`Peptides are widely used in medical practice, and since they can be produced by recombinant
`
`DNA technology it can be expected that their importance will increase also in the years to co-
`
`me. When native peptides or analogues thereof are used in therapy it is generally found that
`
`they have a high clearance. A high clearance of a therapeutic agent is inconvenient in cases
`
`where it is desired to maintain a high blood level thereof over a prolonged period of time since
`
`repeated administrations will then be necessary. Examples of peptides which have a high clea-
`
`rance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon, glu-
`
`cagon-like peptide-1, glucagon-like peptide-2, insulin-like growth factor-1, insulin-like growth
`
`factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase
`
`activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathy-
`
`roid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid
`
`stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods and analogues
`
`thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, aden-
`
`osine deaminase and ribonuclease. in some cases it is possible to influence the release profile
`
`of peptides by applying suitable pharmaceutical compositions, but this approach has various
`
`shortcomings and is not generally applicable.
`
`The amino acid sequence of GLP-2 and other preproglucagon fragments is given i.a. by
`
`Schmidt et al. (Diabetologia 28 704-707 (1985). Little is known about the physical chemical
`
`properties of GLP-2 but GLP-2 is expected, like GLP-t, to be a highly flexible and unstable
`
`molecule. GLP-2 and fragments thereof and analogues of GLP-2 and fragments thereof are
`
`potentially useful i.a. in regulation of appetite and in the treatment of small bowel syndrome.
`
`However, the high clearance limits the usefulness of these compounds, and thus there still is a
`
`need for improvements in this field.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`3
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`We recently found that derivatisation of this relatively small and very flexible molecule resul-
`
`ted in compounds whose plasma profile were highly protracted and still had retained activity
`(PCT application No. DK97/00360).
`
`Hitherto little was known about the physico-chemical and solution structural properties of
`
`GLP—2 derivatives. Such knowledge is a prerequisite for rational handling during e.g. produc-
`
`tion, purification and formulation work and is eventually important for understanding of the
`
`structural basis for the protraction mechanism.
`
`Solubility limitations and the low stability against the actions of endogenous diaminopeptidyl
`
`peptidase limits the usefulness of GLP-2 derivatives, and thus there still is a need for improve-
`
`ments in this field. Accordingly, it is one object of the present invention to provide pharmaceuti-
`
`cal solutions comprising GLP-2 derivatives with improved solubility and stability.
`
`Summary of the invention
`
`Preproglucagon, from which GLP-2 originates, is synthesized la. in the L-cells in the distal ille-
`
`um, in the pancreas and in the brain. Processing of preproglucagon to give GLP-1 and GLP-2
`
`occurs mainly in the L-cells. GLP-2 is a 33 amino acid residue peptide possibly in some tissue
`
`expended to 34 amino acid residues. A simple system is used to describe fragments, ana-
`
`logues and derivatives of GLP-2. Thus, for example, Lys2°GLP-2(1-33) designates a fragment
`
`of GLP-2 formally derived from GLP—2 by deleting the amino acid residues No. 34 and substi-
`
`1O
`
`15
`
`20
`
`25
`
`tuting the naturally occurring amino acid residue in position 20 (Arg) by Lys. Similarly,
`ArgS°Ly535(N‘-tetradecanoyl)GLP-2(1-35) designates a derivative of a GLP—2 analogue formally
`
`derived from GLP—2 by C—terminal addition of a Lys residue, exchange of the naturally occur-
`
`ring amino acid residue in position 30 (Lys) with an Arg residue and tetradecanoylation of the 5-
`
`amino group of the Lys residue in position 35.
`
`PCT application No. DK97/00360 describes various GLP-2 derivatives that are found to be
`
`very protracted. Whereas GLP-2 and GLP-2 analogues are molecules to which no defined so-
`
`lution structure can be ascribed, we found that some of these protracted GLP-Z derivatives
`
`may exist in a partially structured micellar-like aggregated form which is stable over a wide
`
`concentration range.
`
`30
`
`35
`
`4
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`3
`
`Circular Dichroism (CD) can be used to show that the GLP-2 derivatives have a certain par—
`
`tially structured conformation independent of their concentration. In contrast, for normal GLP-2
`
`an increase in the helix content is seen with increasing concentration, from 10-15% to 30-35%
`(at 500 uM concentration) in parallel with peptide self-association. For the GLP-2 derivatives
`
`forming partially structured micellar—like aggregates in aqueous solution the helix content re-
`
`mains constant above 30% at concentrations of 10 uM. The aggregated structured conformati-
`
`on is an inherent property of the derivative present in water or dilute aqueous buffer without the
`
`need for any additional structure-inducing components. Note that the CD signal is proportional
`
`to the average content of a-helix in the peptides, Le, a CD value of -1 corresponds to 10% a-
`
`1O
`
`helix content under these conditions.
`
`Thus, in its broadest aspect, the present invention relates to a pharmaceutical composition
`
`comprising a GLP-2 derivative which has a helix content as measured by CD at 222 nm in H20
`
`at 22 i 2 °C exceeding 25%, preferably in the range of 25% to 50%, at a peptide concentration
`
`15
`
`of about 10 uM.
`
`20
`
`25
`
`30
`
`The size of the partially helical, micelle-like aggregates may be estimated by size-exclusion
`
`chromatography. Similarly, the apparent (critical micelle concentrations) CMC’s of the pepti-
`
`des may be estimated from the concentration dependent fluorescence in the presence of
`
`appropriate dyes (eg. Brito, R. & Vaz, W. (1986) Anal. Biochem. 152, 250-255).
`
`That the derivatives have a partially structured micellar—like aggregate conformation in aqueous
`
`solutions makes them more soluble and stable in solution as compared to the native peptide.
`
`The increased solubility and stability can be seen by comparing the solubility after 9 days of
`
`standing for a derivative and normal GLP-2(1-34) in a pharmaceutical formulation, eg. 5 mM
`
`phosphate buffer, pH 6.9 added 0.1 M NaCl.
`
`In the present text, 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 pepti-
`de. Such addition can take place either at the N-terminal end or at the C-terminal end of the
`
`parent peptide or both.
`
`5
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`4
`
`The term “derivative” is used in the present text to designate a peptide in which one or more of
`
`the amino acid residues of the parent peptide have been chemically modified, e.g. by alkylati-
`
`on, acylation, ester formation or amide formation.
`
`The term “a GLP-2 derivative” is used in the present text to designate a derivative of GLP-2 or
`
`an analogue thereof. In the present text, the parent peptide from which such a derivative is
`
`formally derived is in some places referred to as the “GLP-2 moiety” of the derivative.
`
`In a preferred embodiment, the present invention relates to pharmaceutical composition accor-
`
`ding to claim 1, wherein the concentration of GLP-2 derivative is not less than 0.5 mg/_m|, prefe-
`rably not less than about 5 mg/ml, more preferred not less than about 10 mg/ml and, prefe-
`
`rably, not more than about 100 mg/ml.
`
`The pharmaceutical composition of the invention preferably comprises a GLP-2 derivative whe-
`
`rein at least one amino acid residue of the parent peptide has a lipophilic substituent attached.
`
`More preferred are compositions comprising a GLP-2 derivative having a lipophilic substituent
`
`which is attached to any one of the amino acid residues in 20-34, preferably 30—34, most pre-
`ferred 30.
`
`1O
`
`15
`
`The pharmaceutical composition according to the invention, preferably further comprises one
`
`20
`
`or more of the following substances:
`
`. a pharmaceutically acceptable vehicle or carrier;
`
`0 an isotonic agent, preferably selected from the group consisting of sodium chloride, mannitol
`
`and glycerol;
`
`0 a preservative, preferably selected from the group consisting of phenol, m-cresol, methyl p-
`
`25
`
`hydroxybenzoate, butyl p—hydroxybenzoate and benzyl alcohol;
`
`0
`
`a buffer, preferably selected from the group consisting of sodium acetate, citrate, glycylgly-
`
`cine, histidine, 2-phenylethanol and sodium phosphate; and
`
`o
`
`a surfactant capable of improving the solubility and/or the stability of the GLP-Z derivative,
`
`preferable selected from poloxymer 188, tween 20 and tween 80.
`
`6
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`5
`
`In a preferred embodiment, the pharmaceutical composition of the invention comprises a GLP-
`
`2 derivative wherein the lipophilic substituent comprises from 4 to 40 carbon atoms, preferably
`
`from 8 to 25 carbon atoms.
`
`The lipophilic substituent is preferably attached to an amino acid residue in such a way that a
`
`carboxyl group of the lipophilic substituent forms an amide bond with an amino group of the
`
`amino acid residue, or, the lipophilic substituent is attached to an amino acid residue in such a
`
`way that an amino group of the lipophilic substituent forms an amide bond with a carboxyl
`
`group of the amino acid residue.
`
`in a preferred embodiment the pharmaceutical composition according to the invention compri-
`
`10
`
`ses a GLP-2 derivative wherein the lipophilic substituent is attached to the parent peptide by
`
`means of a spacer.
`
`The spacer is preferably, in one embodiment, an unbranched alkane a,co~dicarboxylic acid
`
`group having from 1 to 7 methylene groups, preferably two methylene groups, which form a
`
`bridge between an amino group of the parent peptide and an amino group of the lipophilic sub-
`stituent.
`
`15
`
`The spacer is preferably, in another embodiment, an amino acid residue except Cys, or a di-
`
`peptide such as Gly-Lys or any unbranched alkane a,m-aminoacid having from 1 to 7 methyle-
`
`ne groups, preferably 2-4 methylene groups, which form a bridge between an amino group of
`
`the parent peptide and an amino group of the lipophilic substituent.
`
`20
`
`In a preferred embodiment, the lipophilic substituent comprises a partially or completely hydro-
`
`genated cyclopentanophenathrene skeleton.
`
`In another preferred embodiment, the lipophilic substituent is a straight-chain or branched alkyl
`group.
`
`25
`
`The lipophilic substituent is preferably the acyl group of a straight-chain or branched fatty acid,
`the acyl group more preferably being:
`
`0
`
`selected from the group comprising CH3(CH2)nCO-, wherein n is 4 to 38, preferably
`
`CH3(CH2)6CO-, CH3(CH2)BCO—, CH3(CH2)1OCO-, CH3(CH2),?_CO-, CH3(CH2)14CO-,
`
`CH3(CH2)1SCO—, CH3(CH2)1BCO-, CH3(CH2)ZOCO- and CH3(CH2)ZZCO—; or
`
`. an acyl group of a straight-chain or branched alkane a,oa-dicarboxylic acid; or
`
`7
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`6
`
`.
`
`selected from the group comprising HOOC(CH2)mCO-, wherein m is from 4 to 38, preferably
`
`from 4 to 24, more preferred selected from the group comprising HOOC(CH2),4CO-,
`
`HOOC(CH2)1GCO-, HOOC(CH2)1BCO-, HOOC(CH2)2(,CO— and HOOC(CH2)ZZCO-.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`CH3(CH2)p((CH2)qCOOH)CHNH-CO(CH2)ZCO-, wherein p and q are integers and p+q is an in-
`
`teger of from 8 to 33, preferably from 12 to 28.
`
`In another preferred embddiment, the lipophilic substituent is a group of the formula
`
`CH3(CH2),CO-NHCH(COOH)(CH2)ZCO-, wherein r is an integer of from 10 to 24.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`1O
`
`CH3(CH2)SCO-NHCH((CH2)ZCOOH)CO-, wherein s is an integer of from 8 to 24.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`-NHCH(COOH)(CH2)4NH-CO(CH2),,CH3, wherein u is an integer of from 8 to 18.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`-NHCH(COOH)(CH2)4NH-COCH((CHZ)ZCOOH)NH-CO(CH2)WCH3, wherein w is an integer of
`from 10 to 16.
`
`15
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`-NHCH(COOH)(CH2)4NH-CO(CH2)ZCH(COOH)NH-CO(CH2)XCH3, wherein x is an integer of
`from 10 to 16.
`
`In another preferred embodiment, the lipophilic substituent is a group of the formula
`
`-NHCH(COOH)(CH2)4NH-CO(CH2)2CH(COOH)NH-CO(CH2)yCH3, wherein y is zero or an inte-
`ger of from 1 to 22.
`
`In a preferred embodiment the pharmaceutical composition according to the invention, compri-
`
`ses a GLP-2 derivative wherein the parent peptide is selected from the group comprising GLP-
`
`2(1-30); GLP-2(1-31); GLP—2(1-32); GLP-2(1-33); GLP-2(1-34) and GLP-2(1-35).
`
`20
`
`25
`
`In a further preferred embodiment the pharmaceutical composition according to the invention,
`
`comprises a GLP-2 derivative wherein the parent peptide has the following amino acid sequ-
`ence:
`
`8
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`X1HXZDGSFSDEMNTXaLDX“LAX5X“DFlNWLX7X8TK|TDX9
`
`7
`
`wherein X1 is NHZ, DFPEEVAIVEELGRR, DFPEEVTlVEELGRR, DFPEEVNIVEELRRR, or a
`
`fragment thereof,
`
`X2 is Ala or Gly,
`
`X3 is He or Val,
`
`X4 is Asn, Ser or His,
`
`X5 is Ala or Thr,
`
`X‘3 is Arg or Lys,
`
`10
`
`X7 is He or Leu,
`
`X8 is Gln or His, or
`
`X9 is OH, Lys, Arg, Arg-Lys, Lys-Arg, Arg-Arg or Lys-Lys.
`
`The pharmaceutical composition according to the invention preferably comprises a GLP-2 deri-
`
`15
`
`vative wherein a total of up to fifteen, preferably up to ten, more preferably up to six, amino a-
`
`cid residues have been exchanged with any a-amino acid residue which can be coded for by
`
`the genetic code.
`
`The parent peptide is most preferably selected from the group comprising:
`
`LySZOGLP-2(1-33);
`
`20
`
`LysZOArga°GLP-2(1 -33);
`
`Arga°Lyss4GLP-2(1 —34);
`
`Arg3°Ly835GLP-2(1-35);
`
`Arga0'35Lysz°GLP—2(1—35);
`
`ArgssGLP-2(1-35).
`
`25
`
`30
`
`In a further preferred embodiment, the pharmaceutical composition of the present invention
`
`comprises a GLP-2 derivative which is selected from the group comprising
`
`Lysz°(NE-tetradecanoyl)GLP-2(1-33);
`
`Lysz°'3°-bis(NS-tetradecanoyl)GLP-2(1~33);
`
`LysZ°(NE—tetradecanoyl)Args°GLP-2(1-33);
`
`Arg3°Ly335(N‘-tetradecanoyl)GLP-2(1-35);
`
`Arg3°'35Lysz°(NE-tetradecanoyl)GLP-2(1—35);
`
`9
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`ArgasLy33°(NS-tetradecanoyl)GLP-2(1-35);
`
`ArgSOLys3“(NS-tetradecanoyl)GLP-2(1—34);
`
`Ly52°(Nf-(oa-carboxynonadecanoyl))GLP-2(1—33);
`
`Lys2°'3°-bis(NS-(oa-carboxynonadecanoyl))GLP-2(1~33);
`
`LysZ°(Ne-(oo-carboxynonadecanoyl))Arga°GLP-2(1-33);
`
`Arga°Ly535(Ne-(w—carboxynonadecanoyl))GLP-2(1-35);
`
`ArgmsLysz‘KNE—(co-carboxynonadecanoyl))GLP-2(1 —35);
`
`ArgasLys3°(NE-(m-carboxynonadecanoyl))GLP-2(1-35); and
`
`Args°Lys34(NE-(w-carboxynonadecanoyl))GLP—2(1-34).
`
`1O
`
`15
`
`20
`
`25
`
`30
`
`The present invention furthermore relates to a method for improving the solubility and/or stabi-
`
`lity of GLP—2 or a fragment or an analogue thereof, characterised in that a lipophilic substituent
`
`is introduced on any one of the amino acid residues of the parent peptide.
`
`By this method the lipophilic substituent is preferably introduced on any one of the amino acid
`
`residues in position 20—34, preferably‘30-34, most preferred 30.
`
`The lipophilic substituent preferably comprises from 4 to 40 carbon atoms, more preferably
`
`from 8 to 25 carbon atoms.
`
`In a preferred embodiment the lipophilic substituent is the acyl group of a straight-chain or
`
`branched fatty acid; preferably selected from the group comprising CH3(CH2)nCO-, wherein n is
`
`4 to 38, preferably CH3(CH2)SCO-, CH3(CH2)SCO-, CH3(CH2)1OCO-, CH3(CH2),2CO-,
`
`CH3(CH2)14CO—, CH3(CH2)1GCO-, CH3(CH2)1BCO-, CH3(CH2)ZOCO- and CH3(CH2)2ZCO-.
`
`The GLP-2 parent peptide is preferably GLP-2(1-30); GLP-2(1-31); GLP-2(1-32); GLP-2(1-33);
`
`GLP-2(1-34) and GLP-2(1-35).
`
`Detailed description of the invention
`
`To obtain a satisfactory protracted profile of action of the GLP-2 derivative, the lipophilic sub-
`
`stituent attached to the GLP-2 moiety preferably comprises 4-40 carbon atoms, in particular 8-
`
`25 carbon atoms. The lipophilic substituent may be attached to an amino group of the GLP-2
`
`moiety by means of a carboxyl group of the lipophilic substituent which forms an amide bond
`
`with an amino group of the amino acid to which it is attached. As an alternative, the lipophilic
`
`10
`
`10
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`9
`
`substituent may be attached to said amino acid in such a way that an amino group of the lipop-
`
`hilic substituent forms an amide bond with a carboxyl group of the amino acid. As a further op-
`tion, the lipophililic substituent may be linked to the GLP—2 moiety via an ester bond. Formally,
`the ester can be formed either by reaction between a carboxyl group of the GLP-2 moiety and
`a hydroxyl group of the substituent-to-be or by reaction between a hydroxyl group of the GLP-Z
`
`moiety and a carboxyl group of the substituent-to-be. As a further alternative, the lipophilic
`
`substituent can be an alkyl group which is introduced into a primary amino group of the GLP-2
`
`moiety.
`
`In one preferred embodiment of the invention, the lipophilic substituent is attached to the GLP-
`
`2 moiety by means of a spacer in such a way that a carboxyl group of the spacer forms an
`
`amide bond with an amino group of the GLP-2 moiety. Examples of suitable spacers are suc-
`
`cinic acid, Lys, Glu or Asp, or a dipeptide such as Gly-Lys. When the spacer is succinic acid,
`
`one carboxyl group thereof may form an amide bond with an amino group of the amino acid
`
`residue, and the other carboxyl group thereof may form an amide bond with an amino group of
`
`the lipophilic substituent. 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 the-
`
`reof 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 preferred embodiment, such a further spacer
`
`is succinic acid which forms an amide bond with the s-amino group of Lys and with an amino
`
`group present in the lipophilic substituent. In another preferred embodiment such a further spa-
`
`cer is Glu or Asp which forms an amide bond with the a-amino group of Lys and another amide
`
`bond with a carboxyl group present in the lipophilic substituent, that is, the lipophilic substituent
`
`is a Nf-acylated lysine residue.
`
`In another preferred embodiment of the present invention, the lipophilic substituent has a group
`
`which can be negatively charged. One preferred group which can be negatively charged is a
`
`carboxylic acid group.
`
`The parent peptide can be produced by a method which comprises culturing a host cell contai-
`
`ning a DNA sequence encoding the peptide and capable of expressing the peptide in a suitable
`
`nutrient medium under conditions permitting the expression of the peptide, after which the re-
`
`sulting peptide is recovered from the culture.
`
`1O
`
`15
`
`20
`
`25
`
`30
`
`11
`
`11
`
`

`

`W0 99/4336]
`
`PCT/DK99/00080
`
`10
`
`The medium used to culture the cells may be any conventional medium suitable for growing
`
`the host cells, such as minimal or complex media containing appropriate supplements. Suitable
`
`media are available from commercial suppliers or may be prepared according to published re-
`
`cipes (9.9. in catalogues of the American Type Culture Collection). The peptide produced by
`
`the cells may then be recovered from the culture medium by conventional procedures including
`
`separating the host cells from the medium by centrifugation or filtration, precipitating the prote-
`
`inaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulp-
`
`hate, purification by a variety of chromatographic procedures, 9.9. ion exchange chromato-
`
`graphy, gelfiltration chromatography, affinity chromatography, or the like, dependent onthe ty-
`
`pe of peptide in question.
`
`The DNA sequence encoding the parent peptide may suitably be of genomic or cDNA origin,
`
`for instance obtained by preparing a genomic or cDNA library and screening for DNA sequen-
`
`ces coding for all or part of the peptide by hybridisation using synthetic oligonucleotide probes
`
`in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Ma-
`
`niatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New
`
`York, 1989). The DNA sequence encoding the peptide may also be prepared synthetically by
`
`established standard methods, eg. the phosphoamidite method described by Beaucage and
`
`Caruthers, Tetrahedron Letters 22 (1981), 1859 — 1869, or the method described by Matthes et
`
`al., EMBO Journal 3 (1984), 801 - 805. The DNA sequence may also be prepared by polyme-
`
`rase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki
`
`et al., Science 239 (1988), 487 - 491.
`
`The DNA sequence may be inserted into any vector which may conveniently be subjected to
`
`recombinant DNA procedures, and the choice of vector will often depend on the host cell into
`
`which it is to be introduced. Thus, the vector may be an autonomously replicating vector, la. a
`
`vector which exists as an extrachromosomal entity, the replication of which is independent of
`
`chromosomal replication, 9.9. a plasmid. Alternatively, the vector may be one which, when in-
`
`troduced into a host cell, is integrated into the host cell genome and replicated together with
`
`the chromosome(s) into which it has been integrated.
`
`The vector is preferably an expression vector in which the DNA sequence encoding the peptide
`
`is operably linked to additional segments required for transcription of the DNA, such as a pro-
`
`1O
`
`15
`
`20
`
`'25
`
`30
`
`12
`
`12
`
`

`

`WO 99/43361
`
`'
`
`1 1
`
`PCT/DK99/00080
`
`moter. The promoter may be any DNA sequence which shows transcriptional activity in the
`
`host cell of choice and may be derived from genes encoding proteins either homologous or
`heterologous to the host cell. Examples of suitable promoters for directing the transcription of
`the DNA encoding the peptide of the invention in a variety of host cells are well known in the
`art, of. for instance Sambrook et al., supra.
`
`The DNA sequence encoding the peptide may also, if necessary, be operably connected to a
`
`suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translati-
`
`onal enhancer sequences. The recombinant vector of the invention may further comprise a
`DNA sequence enabling the vector to replicate in the host cell in question.
`
`1O
`
`The vector may also comprise a selectable marker, eg. a gene the product of which comple-
`
`ments a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kana-
`
`mycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.
`
`To direct a parent peptide of the present invention into the secretory pathway of the host cells,
`
`a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequ-
`
`ence) may be provided in the recombinant vector. The secretory signal sequence is joined to
`
`the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequ-
`
`ences are commonly positioned 5' to the DNA sequence encoding the peptide. The secretory
`
`signal sequence may be that normally associated with the peptide or may be from a gene en-
`
`coding another secreted protein.
`
`The procedures used to ligate the DNA sequences coding for the present peptide, the promo—
`
`ter and optionally the terminator and/or secretory signal sequence, respectively, and to insert
`
`them into suitable vectors containing the information necessary for replication, are well known
`
`to persons skilled in the art (of, for instance, Sambrook et al.,, supra).
`
`The host cell into which the DNA sequence or the recombinant vector is introduced may be any
`
`cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and
`
`higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, wit-
`
`hout limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.
`
`15
`
`20
`
`25
`
`30
`
`13
`
`13
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`12
`
`The GLP-2 derivatives of the invention can be prepared by introducing the lipophilic substituent
`
`into the parent GLP-2 or GLP-2 analogue using methods known per 39, see for example WO
`95/07931, the contents of which is hereby incorporated in its entirety by reference.
`
`Nf-acylation of a Lys residue can be carried out by using an activated amide of the acyl group
`
`to be introduced as the acylating agent, e.g. the amide with benzotriazole. The acylation is car-
`
`ried out in a polar solvent in the presence of a base.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Preparation and administration of the compositions
`
`Pharmaceutical compositions containing a GLP-2 derivative according to the present invention
`
`may be administered parenterally or orally to patients in need of such a treatment. Parenteral
`
`administration may be performed by subcutaneous, intramuscular or intravenous injection by
`
`means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can
`
`be performed by means of an infusion pump. A further option is a composition which may be a
`
`powder or a liquid for the administration of the GLP-2 derivative in the form of a nasal or pul-
`
`monal spray. As a still further option, the pharmaceutical compositions containing a the GLP-2
`
`derivatives of the invention can also be adapted to transdermal administration, e.g. from a
`
`patch, optionally a iontophoretic patch, ortransmucosal, e.g. bucal, administration.
`
`Pharmaceutical compositions containing a GLP-2 derivative of the present invention may be
`
`prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Scien-
`
`ces, 1985 or in Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
`
`Thus, the injectable compositions of the GLP-Z derivative of the invention can be prepared
`
`using the conventional techniques of the pharmaceutical industry which involves dissolving and
`
`mixing the ingredients as appropriate to give the desired end product.
`
`According to one procedure, the GLP-2 derivative is dissolved in an amount of water which is
`
`somewhat less than the final volume of the composition to be prepared. An isotonic agent, a
`
`preservative and a buffer is added as required and the pH value of the solution is adjusted - if
`
`necessary - using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as
`
`14
`
`14
`
`

`

`WO 99/43361
`
`PCT/DK99/00080
`
`13
`
`needed. Finally, the volume of the solution is adjusted with water to give the desired concen-
`
`tration of the ingredients.
`
`A composition for nasal administration of certain peptides may, for example, be prepared as
`
`described in European Patent No. 272097 (to Novo Nordisk AIS) or in WO 93/18785.
`
`According to one preferred embodiment of the present invention, the pharmaceutical composi-
`
`tions containing a GLP—2 derivative is provided in the form of a composition suitable for admini-
`
`stration by injection. Such a composition can either be an injectable solution ready for use or it
`
`can be an amount of a solid composition, e. g. a lyophilised product, which has to be dissolved
`in a solvent before it can be injected. The injectable solution preferably contains not less than
`
`about 0.5 mg/ml, preferably not less than about 5 mg/ml, more preferred not less than about 10
`
`mg/ml of the GLP-2 derivative and, preferably, not more than about 100 mg/ml of the GLP-2
`derivative.
`
`The pharmaceutical compositions containing a GLP-2 derivative of this invention can be used
`
`in the treatment of various diseases, including obesity, small bowel syndrome, Crohn's disea-
`
`se, ileitis, intestinal inflammation, gastric and duodenal ulceration, inflammatory bowel disease
`
`(IBD) and intestinal cancer damage therapy. The particular GLP—2 derivative to be used and
`
`the optimal dose level for any patient will depend on the disease to be treated and on a variety
`
`of factors including the efficacy of the specific peptide derivative employed, the age, body
`
`weight, physical activity, and diet of the patient, on a possible combination with other drugs,
`
`and on the severity of the case. it is reco