Patent Application: US-67134003-A

Abstract:
the present invention relates to a glp - 1 peptide having the following formula , or a pharmaceutically acceptable salt thereof : x - his - ala - glu - gly - thr - phe - thr - ser - asp - val - ser - ser - tyr - leu - glu - gly - gln - ala - ala - lys - glu - phe - lle - ala - trp - leu - val - lys - gly - arg - y wherein x is a rigidifying hydrophobic moiety and wherein y is selected from the group consisting of oh , nh 2 and gly - oh . moreover , the present invention relates to pharmaceutical compositions comprising a therapeutically effective amount of a peptide of the present invention , or a pharmaceutically acceptable salt thereof , in association with at least one constituent selected from a pharmaceutically acceptable carrier , diluent , and excipient .

Description:
as used herein , the term “ subject ” is intended to mean a mammal selected from the group consisting of human , porcine , bovine , caprine , ovine , feline , canine and equine . as used herein , the term “ glp - 1 analogues ” is intended to mean analogues which are biologically - active glp - 1 peptides that include glp - 1 ( 7 – 37 ) oh , glp - 1 ( 7 – 36 ) nh 2 and their derivatives ; these derivatives include peptides that contain amino acid substitutions , made with the intention of improving solubility ( replacement of hydrophobic amino acids with hydrophilic amino acids , pegylation of terminal carboxyl groups or the ε - amino group of lysine ), conferring resistance to oxidation ( substitution of met , trp , gin , asn ), increasing biological potency in in vitro and in vivo assays ( one or more amino acid substitutions at positions 11 , 12 , 16 , 22 , 23 , 24 , 25 , 27 , 30 , 33 , 34 , 35 , 36 , or 37 , but not those at 8 ), or increasing half - life in circulation ( acyl ( c 12 – c 18 ) modifications of the ε - amino group of lysine ). as used herein , the term “ rigidifying hydrophobic moiety ” is intended to mean a conformationally rigid moiety , which has a limited number of spatial orientations due to the presence of one or more rigidifying elements in its backbone such as , a double bond , a triple bond , or a saturated or unsaturated ring , which have little or no conformational mobility . as a result , the number of conformers or rotational isomers is reduced when compared to the corresponding straight , or unsubstituted and saturated aliphatic chain . these rigidifying hydrophobic moieties are divided into the following groups : as used herein , the term “ unit dosage form ” refers to physically discrete units , suitable as unitary dosages for human subjects and other mammals ; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect , in association with one / or more suitable pharmaceutical excipients . as used herein , the term “ aryl ” refers to phenyl , 1 - naphthyl , and 2 - naphthyl . as used herein , the term “ substituted aryl ” refers to phenyl , 1 - naphthyl , and 2 - naphthyl having a substituent selected from lower alkyl , lower alkoxy , lower alkylthio , halo , hydroxy , trifluoromethyl , amino , — nh ( lower alkyl ), — n ( lower alkyl ) 2 , as well as di - and tri - substituted phenyl , 1 - naphthyl , or 2 - naphthyl , the di - and tri - substituted phenyl , 1 - naphthyl , and 2 - naphthyl comprising a substituent selected from the group consisting of methyl , methoxy , methylthio , halo , hydroxy , and amino . the present invention also includes salt forms of glp - 1 analogs . a glp - 1 analog of the present invention may be sufficiently acidic or sufficiently basic to react with any of a number of organic and inorganic bases , and organic and inorganic acids , to form a salt . acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid , hydrobromic acid , hydroibdic acid , sulfuric acid , phosphoric acid , and the like , and organic acids such as p - toluenesulfonic acid , methanesulfonic acid , oxalic acid , p - bromophenyl - sulfonic acid , carbonic acid , succinic acid , citric acid , benzoic acid , acetic acid , and the like . examples of such salts include sulfate , pyrosulfate , bisulfate , sulfite , bisulfite , phosphate , monohydrogenphosphate , dihydrogenphosphate , metaphosphate , pyrophosphate , chloride , bromide , iodide , acetate , propionate , decanoate , caprylate , acrylate , formate , isobutyrate , caproate , heptanoate , propiolate , oxalate , malonate , succinate , suberate , sebacate , fumarate , maleate , butyne - 1 , 4 - dioate , hexyne - 1 , 6 - dioate , benzoate , chlorobenzoate , methylbenzoate , dinitrobenzoate , hydroxybenzoate , methoxybenzoate , phthalate , sulfonate , xylenesulfonate , phenylacetate , phenylpropionate , phenylbutyrate , citrate , lactate , gamma - hydroxybutyrate , glycolate , tartrate , methanesulfonate , propanesulfonate , naphthalene - 1 - sulfonate , naphthalene - 2 - sulfonate , mandelate , and the like . preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid . a more preferred acid addition salt is formed with hydrochloric acid . base addition salts include those derived from inorganic bases , such as ammonium or alkali or alkaline earth metal hydroxides , carbonates , bicarbonates , and the like . such bases , useful in preparing the salts of this invention , thus include sodium hydroxide , potassium hydroxide , ammonium hydroxide , potassium carbonate , and the like . salt forms of glp - 1 analogs are particularly preferred . of course , when the compounds of the present invention are used for therapeutic purposes , the compounds may also be in the form of a salt , however , the salt must be a pharmaceutically acceptable salt . the present invention relates to a glp - 1 peptide , modified by the covalent attachment of a hydrophobic group to the n - terminus , thus resulting in protease resistance , more specifically dpp iv resistance . given the sequence information as disclosed herein , and the state of the art in solid phase protein synthesis , glp - 1 analogs can be obtained via chemical synthesis . the principles of solid phase chemical synthesis of polypeptides are well known in the art ( dugas and penney , 1981 ; merrifield , 1962 ; stewart and young , 1969 ). examples for synthesizing the peptides of the present invention are provided below . the present invention also relates to pharmaceutical compositions comprising a glp - 1 analog of the present invention , in combination with a pharmaceutically acceptable carrier , diluent , or excipient . such pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art , and are administered individually or in combination with other therapeutic agents , preferably via parenteral routes . especially preferred routes of administration include intramuscular and subcutaneous administration . parenteral daily dosages , preferably a single daily dose , are typically in the range of from about 1 mcg / kg to about 100 mcg / kg of body weight , although lower or higher dosages may be administered . the required dosage will depend upon the severity of the condition of the patient and upon such criteria as the patient &# 39 ; s height , weight , sex , age , and medical history . in making the compositions of the present invention , the active ingredient , which comprises at least one peptide of the present invention , is usually mixed or diluted with an excipient . when an excipient is used as a diluent , it may be a solid , semi - solid , or liquid material which acts as a vehicle , carrier , or medium for the active ingredient . examples of suitable excipients include , but are not limited to lactose , dextrose , sucrose , trehalose , sorbitol , mannitol , starches , gum acacia , calcium silicate , microcrystalline cellulose , polyvinylpyrrolidone , cellulose , water , syrup , and methyl cellulose . the formulations can additionally include lubricating agents such , but not limited to , talc , magnesium stearate and mineral oil . the formulations may further include wetting agents , emulsifying and suspending agents , preserving agents such as methyl - and propylhydroxybenzoates , sweetening agents or flavoring agents . the compositions of the invention can be formulated so as to provide quick , sustained or delayed release of the active ingredient following administration to the patient , by employing formulation procedures well known in the art . the compositions of the present invention are preferably formulated in unit dosage form , with each dosage normally containing from about 1 mcg to about 10 mg of the active ingredient . additional pharmaceutical methods may be employed to control the duration of action . controlled release preparations may be achieved by the use of polymers to complex or absorb a peptide of the present invention . the controlled delivery of the active ingredient ( peptide ) may be exercised by selecting appropriate macromolecules ( for example , polyesters , polyamino acids , polyvinylpyrrolidone , ethylene vinylacetate copolymers , methylcellulose , carboxymethylcellulose , and protamine sulfate ), the concentration of the macromolecules as well as the methods of incorporation . such teachings are disclosed in remington &# 39 ; s pharmaceutical sciences ( 1980 ). another possible method to control the duration of action by controlled release preparations , is to incorporate a peptide of the present invention into particles of a polymeric material such as polyesters , polyamino acids , hydrogels , poly ( lactic acid ) or ethylene vinylacetate copolymers . the present invention is illustrated in further detail by the following non - limiting examples . solid phase synthesis of glp - 1 ( 7 – 37 ) cooh or glp - 1 ( 7 – 37 ) conh 2 and coupling of rigid hydrophobic pharmacophores the analogues of the present invention are made by solid phase peptide synthesis , using fluorenylmethoxycarbonyl - protected l - amino acids ( peptide synthesis protocols , 1994 ). completion of coupling was monitored by the kaiser color test . the organic acids disclosed in table 1 were coupled by the same method as used for coupling amino acids . the crude peptides were further purified by preparative hplc on vydac c 18 - columns using an acetonitrile gradient in 0 . 1 % tfa . the peptides were vacuum - dried to remove acetonitrile , and lyophilized from 0 . 1 % tfa . purity was assessed by analytical hplc and masses were determined by maldi - tof mass spectroscopy using a voyager biospectrometry workstation ( perspective systems ). the peptides were prepared as tfa salts and dissolved in saline for administration to animals . rinm5f cells ( atcc # crl - 2058 ) were grown to confluence and used 4 days following confluency . aqueous stock solutions ( 1 mm ) of glp - 1 , 5 , 8 , 16 , and 17 , also containing 0 . 1 % bsa , were made prior to the assay . the numbers refer to compounds listed in table 2 . cells were pre - incubated with 100 μl rpmi containing 0 . 5 mm ibmx for 10 minutes at 37 ° c . diluted ( 10 − 5 to 10 − 11 m ) peptides ( 100 μl ) were added to the wells and incubated for 40 minutes . at the end of the incubation period , the supernatant was collected and assayed for camp using a radioimmunoassay kit ( diagnostic product corporation ). maximal responses ( pmol / mg protein ) and ic 50 ( nm ) values were evaluated from the dose - response curves and data presented in table 3 . glp - 1 peptides ( 1 , 5 and 10 μg per mouse ) were injected i . p . into overnight fasted cd - 1 normal female mice ( 6 wk . charles river , montreal , canada ) 5 minutes prior to an oral glucose challenge ( 40 % glucose solution ); glucose ( 1 g / kg body weight ) was administered by oral gavage at t = 0 minutes , and blood samples were drawn by tail vein excision ; blood glucose levels ( mmol / l ) were determined at t = 0 , 10 , 20 , 30 , 60 , 90 and 120 minutes using a one - touch glucometer ( lifescan canada , burnaby , bc , canada ). the area under the curve ( auc glucose [ mmol / l * 120 min ]) was calculated using the trapezoidal method ( n = 3 – 4 animals per group ). the results are shown in fig1 . the horizontal line represents vehicle control ( n = 50 ). there was a dose dependent improvement in glucose clearance in all cases . at the highest dose tested , compounds 5 , 7 , 8 , 14 , and 15 were clearly superior in efficacy to an identical same dose of glp - 1 ( 7 – 37 ) cooh . dose - effect of compound 7 on glucose and insulin levels in normal cd - 1 female mice an ogtt was done as described above . plasma insulin levels were determined using a commercial ria kit ( linco research ). the results are shown in fig2 . compound 7 was administered ip and blood glucose ( mmol / l ) and plasma insulin levels ( ng / ml ) by ria ( linco research ) were measured . it was found that there was no effect on glucose disposal using 1 μg doses . however , 10 μg of compound 7 significantly improved glucose clearance in cd - 1 mice . correspondingly , the plasma insulin levels were higher at 15 and 60 minutes following treatment with 10 μg of compound 7 . a genetic mutation at the leptin receptor locus renders this strain of mice diabetic , and has been used as a useful model for diabetes . an ogtt was conducted as described in example 3 . peptides were injected intra - parentally ( ip ) at 5 μg / mouse doses . the results are shown in fig3 ( peptide administration , ogtt and glucose measurements were performed as described in the legend for fig1 ). three peptides , 7 , 8 and 15 performed better than glp - 1 in accelerating glucose disposal in this model . effect of glp - 1 analogues on plasma insulin levels in the intraperitoneal glucose tolerance test ( ipgtt ) using sprague - dawley rats sprague - dawley rats ( 300 – 350 g ) were fasted overnight , and injected with 1 g / kg glucose ( 2 ml ) ( over 15 – 20 seconds ), and blood glucose levels were determined at different times during a period of 3 hours using a portable glucometer . the drugs ( 10 μg / rat ) were dissolved in saline and injected into the femoral vein 5 minutes before the injection of glucose . thus 0 time represents blood glucose and insulin levels after drug administration but before glucose injection . plasma insulin levels were determined using a radioimmunoassay kit ( linco ). glucose and insulin levels are shown in mmol / l and ng / ml respectively . the insulinogenic index was calculated as delta insulin ( pm )/ delta glucose ( mm ). the results are shown in fig4 . in all groups of rats , the basal insulin levels were not affected by glp - 1 analogues alone . glucose injections produced rapid responses in plasma insulin levels and the values obtained at 0 , 30 , 60 , 90 minutes following glucose administration are shown in the bar graph . cross bars are shown to indicate the relative levels compared to saline ( n = 8 ) and glp - 1 ( n = 4 ) injections . based on these results , 5 ( n = 4 ) and 15 ( n = 4 ) produced higher average insulin levels than glp - 1 , whereas 7 ( n = 4 ) was similar to glp - 1 in response . the order of efficacy in these experiments was found to be 15 = 5 & gt ; 7 = glp - 1 & gt ; 8 = vehicle . c57bl / ks db / db mice were fasted overnight and then allowed free access to food for a period of 30 minutes . immediately following feeding , sc injections of vehicle , glp - 1 , 5 , 8 , or 17 ( 500 μg / kg ) were given , and glucose levels measured for 2 hours in blood drawn from a tail cut . blood glucose levels ( mmol / l ) were determined using an accucheck compact glucometer ( roche , germany ). the obtained data are presented in fig6 as mean ± sem . rats were fasted overnight , anesthetized ( isoflurane 2 %) and catheterized to receive intravenous glucose at a rate and concentration such as to maintain a glycemia between 16 and 18 mmol / l blood glucose . after reaching a stable glucose level and stabilizing it for a period of 1 hour , subcutaneous injections of vehicle , glp - 1 , 16 , or 17 ( 300 pg / kg ) were administered and glucose levels measured for an additional 2 hours . blood samples were drawn from interdigital punctures and blood glucose levels ( mmol / l ) monitored by an accucheck compact glucometer ( roche , germany ). the obtained data are presented in fig7 . aliquots ( 100 μg ) of glp - 1 ( 7 – 37 ) or 5 were incubated at room temperature ( 22 – 24 ° c .) in duplicates with 50 mu of dpp iv ( sigma - aldrich ), in 20 mm tris - hcl buffer , ph 8 . 0 . the reaction mixture was sampled at different times and directly analysed by rp - hplc using vydac c 18 columns . the peak areas were determined by chemstation rev . a . 05 . 01 data analysis software ( agilent technologies ). the observed data are expressed as the % remaining area in comparison to the undigested controls and are shown in fig8 . although the present invention has been described hereinabove by way of preferred embodiments thereof , it can be modified without departing from the spirit and nature of the subject invention as defined in the appended claims . the following references , to the extent that they provide exemplary procedural or other details supplementary to those set forth herein , are specifically incorporated herein by reference . u . s . pat . no . 5 , 512 , 549 u . s . pat . no . 5 , 545 , 618 adelhorst et al ., j . biol . chem ., 269 ( 9 ): 6275 – 6278 , 1994 . burcelin et al ., metabolism , 48 ( 2 ): 252 – 258 , 1999 . d &# 39 ; 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