PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`
`
`(22) International Filing Date:
`
`25 February 1999 (25.02.99)
`
`(30) Priority Data:
`0268/98
`0272/98
`
`27 February 1998 (27.02.98)
`27 February 1998 (27.02.98)
`
`DK
`DK
`
`(71) Applicant: NOVO NORDISK A/S [DK/DK]; Novo Allé,
`DK~2880 Bagsvaerd (DK).
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 6 :
`
`
` WO 99143341
`(11) International Publication Number:
`A61K 38/26, C07K 14/605
`
`(43) International Publication Date:
`2 September 1999 (02.09.99)
`
`
`PCT/DK99/00084
`(21) International Application Number:
`(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, 'I'I‘, 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).
`
`
`
`(72) Inventors: KNUDSEN, Liselotte, Bjerre', Valby Langgade
`49A, 1.
`tv., DK—2500 Valby (DK). HUUSFELDT, Per,
`Olaf; Applebys Plads 27, 5. mf., DK—14ll Copenhagen K
`(DK). NIELSEN, Per, Franklin; Dalso Park 59, DK—3500
`Veerlose (DK). KAARSHOLM, Niels, C.; Clausholmvej 38,
`DKfi2720 Vanlose (DK). OLSEN, Helle, Birk; Skolelodden
`23, DK—3450 Allemd (DK). BJORN, Soren, Erik; Marie
`Grubbes A116 47, DK—2800 Lyngby (DK).
`
`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—l 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—l derivative of improved solubility and/or stability,
`and to a method for improving the solubility and/or stability of GLP—1 or a fragment or an analogue thereof.
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`Zimbabwe
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`Lesotho
`LS
`SI
`Slovenia
`Armenia
`FI
`LT
`Lithuania
`SK
`Slovakia
`Austria
`FR
`LU
`SN
`Luxembourg
`Senegal
`Australia
`GA
`LV
`Latvia
`SZ
`Swaziland
`GB
`MC
`Monaco
`TD
`Chad
`Azerbaijan
`GE
`MD
`Bosnia and Herzegovina
`Republic of Moldova
`Togo
`Barbados
`GH
`MG
`Madagascar
`Tajikistan
`GN
`MK
`Belgium
`Turkmenistan
`The former Yugoslav
`Burkina Faso
`GR
`Republic of Macedonia
`Turkey
`HU
`Mali
`Bulgaria
`Trinidad and Tobago
`Benin
`IE
`Ukraine
`Mongolia
`Brazil
`IL
`Mauritania
`Uganda
`Belarus
`IS
`Malawi
`United States of America
`IT
`Canada
`Mexico
`Uzbekistan
`JP
`Viet Nam
`Central African Republic
`Niger
`KE
`Netherlands
`Congo
`Yugoslavia
`KG
`Switzerland
`Norway
`KP
`COte d'Ivoire
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`KR
`
`LC
`Ll
`LK
`LR
`
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`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
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
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`WO 99/43341
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`PCT/DK99/00084
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`GLP—I DERIVATIVES WITH HELIX—CONTENT EXCEEDING 25 %, FORMING PARTIALLY STRUCTURED MICEL-
`LAR—LIKE AGGREGATES
`
`Field of the invention
`
`5
`
`The present invention relates to a pharmaceutical composition comprising a GLP-1 derivative
`
`of improved solubility and/or stability, and to a method for improving the solubility and/or stabi-
`
`lity of GLP—1 or a fragment or an analogue thereof.
`
`Background of the invention
`
`10
`
`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.
`
`The hormones regulating insulin secretion belong to the so-called enteroinsular axis, desig-
`
`15
`
`nating a group of hormones, released from the gastrointestinal mucosa in response to the
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`presence and absorption of nutrients in the gut, which promote an early and potentiated re-
`
`lease of insulin. The enhancing effect on insulin secretion, the so-called incretin effect, is
`
`probably essential for a normal glucose tolerance. Many of the gastrointestinal hormones,
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`including gastrin and secretin (cholecystokinin is not insulinotropic in man), are insulinotro-
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`20
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`pic, but the only physiologically important ones, those that are responsible for the incretin
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`effect, are the glucose-dependent insulinotropic polypeptide, SIP, and glucagon-like peptide-
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`1 (GLP-1). Because of its insulinotropic effect, GlP, isolated in 1973 (1) immediately attrac—
`
`ted considerable interest among diabetologists. However, numerous investigations carried
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`out during the following years clearly indicated that a defective secretion of GlP was not in-
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`25
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`volved in the pathogenesis of insulin dependent diabetes mellitus (lDDM) or non insulin-
`
`dependent diabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropic hormone, GIP
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`was found to be almost ineffective in NIDDM (2). The other incretin hormone, GLP-1 is the
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`most potent insulinotropic substance known (3). Unlike GIP, it is surprisingly effective in sti-
`
`mulating insulin secretion in NIDDM patients. In addition, and in contrast to the other insuli-
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`30
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`notropic hormones (perhaps with the exception of secretin) it also potently inhibits glucagon
`secretion. Because of these actions it has pronounced blood glucose lowering effects parti-
`cularly in patients with NIDDM.
`
`GLP-1, a product of the proglucagon (4), is one of the youngest members of the secretin-VIP
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`35
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`family of peptides, but is already established as an important gut hormone with regulatory
`function in glucose metabolism and gastrointestinal secretion and metabolism (5). The glu-
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`cagon gene is processed differently in the pancreas and in the intestine. in the pancreas (9),
`
`the processing leads to the formation and parallel secretion of 1) glucagon itself, occupying
`
`positions 33-61 of proglucagon (PG); 2) an N—terminal peptide of 30 amino acids (PG (1-30))
`
`often called glicentin-related pancreatic peptide, GRPP (10, 11); 3) a hexapeptide corre-
`
`5
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`sponding to PG (64-69); 4) and, finally, the so—called major proglucagon fragment (PG (72-
`
`158)), in which the two glucagon-like sequences are buried (9). Glucagon seems to be the
`
`only biologically active product. In contrast, in the intestinal mucosa, it is glucagon that is bu-
`
`ried in a larger molecule, while the two glucagon—like peptides are formed separately (8). The
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`following products are formed and secreted in parallel: 1) glicentin, corresponding to PG (1-
`
`10
`
`69), with the glucagon sequence occupying residues Nos. 33—61 (12); 2) GLP-1(7-36)amide
`
`(PG (78-107))amide (13), not as originally believed PG (72-107)amide or 108, which is inac-
`
`tive). Small amounts of C-terminally glycine-extended but equally bioactive GLP-1(7-37),
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`(PG (78—108)) are also formed (14); 3) intervening peptide-2 (PG (111-122)amide) (15); and
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`4) GLP-2 (PG (126-158)) (15, 16). A fraction of glicentin is cleaved further into GRPP (PG
`
`15
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`(1730)) and oxyntomodulin (PG (33—69)) (17, 18). Of these peptides, GLP-1, has the most
`
`conspicuous biological activities.
`
`The amino acid sequence of GLP-1 is given i.a. by Schmidt et al. (Diabetologia 28 704—707
`
`(1985). Although the interesting pharmacological properties of GLP-1(7-37) and analogues
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`20
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`thereof have attracted much attention in recent years only little is known about the structure
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`of these molecules. The secondary structure of GLP-1 in micelles has been described by
`
`Thorton et al. (Biochemistry 33 3532-3539 (1994)), but in normal solution, GLP-1 is conside-
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`red a very flexible molecule. Surprisingly, we found that derivatisation of this relatively small
`
`and very flexible molecule resulted in compounds whose plasma profile were highly protrac-
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`25
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`ted and still had retained activity (PCT application No. DK97/OO340).
`
`While much attention has been focused on the pharmacological properties of acylated GLP-
`
`1 derivatives, hitherto little is known about their physico-chemical and solution structural pro-
`
`perties. Such knowledge is a prerequisite for rational handling during e.g. production, purifi—
`
`30
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`cation and formulation work and is eventually important for understanding of the structural
`basis for the protraction mechanism.
`
`GLP-1 and analogues of GLP-1 and fragments thereof are potentially useful i.a. in the treat-
`
`ment of type 1 and type 2 diabetes. However, solubility limitations and the low stability against
`
`35
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`the actions of endogenous diaminopeptidyl peptidase limits the usefulness of these com-
`
`pounds, and thus there still is a need for improvements in this field. Accordingly, it is one object
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`WO 99/43341
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`PCT/DK99/00084
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`3
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`of the present invention to provide pharmaceutical solutions comprising GLP-1 derivatives with
`
`improved solubility and stability.
`
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`bolite in vivo. J Clin Endocrinol Metab 1995; 80: 952—957.
`
`47. Deacon CF, Nauck MA, Toft-Nielsen M, Pridal L, Willms B, Holst JJ. 1995. Both subcu—
`
`10
`
`taneous and intravenously administered glucagon-iike peptide-1 are rapidly degraded
`
`from the amino terminus in type ll diabetic patients and in healthy subjects. Diabetes 44:
`1126-1131.
`
`Summary of the invention
`
`15
`
`Human GLP-1 is a 37 amino acid residue peptide originating from preprogiucagon which is
`
`synthesised i.a. in the L-cells in the distal ileum, in the pancreas and in the brain. Processing of
`
`preprogiucagon to give GLP—1(7—36)amide, GLP-1(7-37) and GLP-2 occurs mainly in the L-
`
`cells. A simple system is used to describe fragments and analogues of this peptide. Thus, for
`
`20
`
`example, GIy”-GLP-1(7-37) designates a fragment of GLP—1 formally derived from GLP-1 by
`
`deleting the amino acid residues Nos. 1 to 6 and substituting the naturally occurring amino acid
`
`residue in position 8 (Ala) by Gly. Similarly, Lys3“(N5-tetradecanoyl)-GLP-1(7-37) designates
`
`GLP—1 (7-37) wherein the s-amino group of the Lys residue in position 34 has been tetradeca-
`
`noyiated. Where reference in this text is made to C-terminally extended GLP-1 analogues, the
`
`25
`
`amino acid residue in position 38 is Arg unless othenivise indicated, the optional amino acid
`
`residue in position 39 is also Arg unless othenNise indicated and the optional amino acid resi-
`
`due in position 40 is Asp unless othenivise indicated. Also, if a C-terminaliy extended analogue
`
`extends to position 41, 42, 43, 44 or 45, the amino acid sequence of this extension is as in the
`
`corresponding sequence in human preprogiucagon unless othenNise indicated.
`
`30
`
`PCT application No. DK97l00340 describes various GLP-1 derivatives that are found to be
`
`very protracted. Whereas GLP-1 and GLP-1 analogues are molecules to which no defined so—
`
`lution structure can be ascribed, we found that some of these protracted GLP-1 derivatives
`
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`9
`
`may exist in a partially structured micellar—like aggregated form which is stable over a wide
`
`concentration range.
`
`Circular Dichroism (CD) can be used to show that the GLP—1 derivatives have a certain par-
`
`5
`
`tially structured conformation independent of their concentration. in contrast, for normal GLP-
`
`1(7—37) an increase in the helix content is seen with increasing concentration, from 10-15% to
`
`30-35% (at 500 pM concentration) in parallel with peptide self-association. For the GLP-1 deri-
`
`vatives forming partially structured micellar-like aggregates in aqueous solution the helix con—
`
`tent remains constant above 30% at concentrations of 10 pM. The aggregated structured con-
`
`10
`
`formation is an inherent property of the derivative present in water or dilute aqueous buffer wit—
`
`hout the need for any additional structure-inducing components.
`
`Thus, in its broadest aspect, the present invention relates to a pharmaceutical composition
`
`comprising a GLP—1 derivative which has a helix content as measured by CD at 222 nm in H20
`
`15
`
`at 22 i 2 °C exceeding 25%, preferably in the range of 25% to 50%, at a peptide concentration
`
`of about 10 pM.
`
`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-
`
`20
`
`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.
`
`25
`
`The increased solubility and stability can be seen by comparing the solubility after 9 days of
`
`standing for a derivative and normal GLP-1(7-37) 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
`
`30
`
`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.
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`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 alkyiati-
`
`on, acylation, ester formation or amide formation.
`
`10
`
`15
`
`The term “a GLP-1 derivative" is used in the present text to designate a derivative of GLP-1 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-1 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-1 derivative is not less than 0.5 mg/ml, 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-1 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-1 derivative having a lipophiliesubstituent
`
`which is attached to any one of the amino acid residues in position 18-38, preferably 26-34.
`
`The pharmaceutical composition according to the invention, preferably further comprises one
`
`20
`
`or more of the following substances:
`
`- a pharmaceutically acceptable vehicle or carrier;
`
`. 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;
`
`. a buffer, preferably selected from the group consisting of sodium acetate, citrate, glycylgly-
`
`cine, histidine, 2-phenylethanol and sodium phosphate; and
`
`. a surfactant capable of improving the solubility and/or the stability of the GLP-1 derivative,
`
`preferable selected from poloxymer 188, tween 20 and tween 80.
`
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`11
`
`In a preferred embodiment, the pharmaceutical composition of the invention comprises a GLP-
`
`1 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
`
`5
`
`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
`
`see a GLP-1 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 0i,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 GIy-Lys or any unbranched alkane a,w-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.
`
`The lipophilic substituent is preferably the acyl group of a straight-chain or branched fatty acid
`
`1
`
`25
`
`the acyl group more preferably being:
`
`.
`
`selected from the group comprising CH3(CH2),,CO-, wherein n is 4 to 38, preferably
`
`CHa(CH2)eCO-. CH3(CH2)sCO-, CH3(CH2)10CO'r CH3(CH2)1200-. CH3(CH2)14CO-,
`
`CH3(CH2)1GCO‘, CH3(CH2)1BCO', CH3(CH2)ZQCO‘ and CH3(CH2)2200'; or
`
`0 an acyl group of a straight-chain or branched alkane a,m-dicarboxylic acid; or
`
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`
`.
`
`selected from the group comprising HOOC(CH2),,,CO—, wherein m is from 4 to 38, preferably
`
`from 4 to 24, more preferred selected from the group comprising HOOC(CH2),4CO-,
`
`HOOC(CH2)16CO', HOOC<CH2)18CO-, HOOC(CH2)20CO' and HOOC(CH2)22CO'.
`
`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.
`
`ln another preferred embodiment, the lipophilic substituent is a group of the formula
`
`CH3(CH2),CO-NHCH(COOH)(CH2)2CO-, wherein r is an integer of from 10 to 24.
`
`in another preferred embodiment, the lipophilic substituent is a group of the formula
`
`10
`
`CH3(CH2)SCO-NHCH((CH2)2COOH)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((CH2)2COOH)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)2CH(COOH)NH-CO(CH2)XCH3, wherein x is an integer of
`from 10 to 16.
`
`20
`
`25
`
`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-1 derivative wherein the parent peptide is GLP-1(A-B) wherein A is an integer from
`
`1 to 7 and B is an integer from 38 to 45, or an analogue thereof.
`
`The parent peptide is preferably, in one embodiment, selected from the group comprising GLP-
`
`1(7-35); GLP—1(7—36); GLP-1(7-36)amide; GLP-1(7-37); GLP-1(7-33); GLP-1(7—39); GLP-1(7-
`
`40) and GLP-1(7-41); and analogues thereof.
`
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`13
`
`The parent peptide is preferably, in another embodiment, selected from the group comprising
`
`GLP-1(1—35); GLP-1(1-36); GLP-1(1—36)amide; GLP-1(1-37); GLP-1(1-38); GLP—1(1-39); GLP-
`
`1(1-40); GLP-1(1-41); and an analogues thereof.
`
`In yet another embodiment, the parent peptide is a GLP-1 analogue of formula I:
`
`5
`
`7
`
`8
`
`9
`
`10
`
`ll
`
`12
`
`l3
`
`14
`
`15
`
`16
`
`17
`
`His—Xaa—Xaa-Gly—Xaa—Phe—Thr—Xaa—Asp-Xaa—Xaa—
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`10
`
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa~Xaa—Xaa—Xaa—Phe—
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`I1e—Xaa-Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa
`
`15
`
`39
`
`40
`
`41
`
`42
`
`43
`
`44
`
`45
`
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa
`
`(I)
`
`wherein
`
`20
`
`Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 9 is Glu, Asp, or Lys,
`
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys,
`
`Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, or Lys,
`
`25
`
`Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys,
`
`Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,
`
`Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys
`
`Xaa at position 21 is Glu, Asp, or Lys,
`
`30
`
`Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 23 is Gln, Asn, Arg, Glu, Asp, or Lys,
`
`Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, lle, Val, Arg, Glu, Asp, or Lys,
`
`Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 26 is Lys, Arg, Gln, Glu, Asp, or His,
`
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`
`Xaa at position 27 is Glu, Asp, or Lys,
`
`Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys,
`
`Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,
`
`Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, lle, Val, Glu, Asp, or Lys,
`
`5
`
`Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Met, Leu, lle, Glu, Asp, or Lys,
`
`Xaa at position 34 is Lys, Arg, Glu, Asp, or His,
`
`Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys,
`
`Xaa at position 36 is Arg, Lys, Glu, Asp, or His, or
`
`Xaa at position 37 is Gly, Ala, Ser, Thr, Leu, lle, Val, Glu, Asp, or Lys, or is deleted,
`
`10
`
`Xaa at position 38 is Arg, Lys, Glu, Asp