Patent Application: US-51916405-A

Abstract:
the invention relates to the use in the preparation of medicines having activity of the antihypertensive type , useful for treating or preventing hypertension , of one or more peptides having inhibiting activity on ace with ic 50 values of the order of or less than 60 μm , selected from the group of peptides having the following amino acid sequences : thr - val - tyr asn - met - ala - ile - asn - pro - ser - lys phe - ala - leu - pro - gln - tyr phe - pro - gln - tyr - leu - gln - tyr phe - ala - leu - pro - gln - tyr - leu - lys asn - met - ala - ile - asn - pro phe - ala - leu - pro . the invention also relates to a pharmaceutical composition containing as its active principle an effective quantity of one or more of the above - cited peptides in combination with a pharmaceutically acceptable vehicle . the invention also relates to a food product , in particular suitable for supplementing the diet of people subject to hypertension or seeking to prevent the appearance thereof , containing an effective quantity of one or more of the above - cited peptides , in combination with food supports , in particular proteins , lipids , or carbohydrates . such a food product may also include at least one of the peptides having the following amino acid sequences : ala - leu - asn - glu - ile - asn - gln - phe - tyr - gln - lys ala - leu - asn - glu - ile - asn - gln - phe - tyr tyr - leu .

Description:
the invention is described below in greater detail by way of the following non - limiting example : five grams ( g ) of ammonium caseinate were dissolved in 200 milliliters ( ml ) of 20 mm acetate buffer having a ph of 6 . 6 , and containing 3 . 5 m of urea , 35 mm of ethylenediaminetetraacetic acid ( edta ), and 0 . 1 % of 2 - mercaptoethanol , and then 20 g of deae - cellulose de 23 balanced in 150 ml of the same buffer were added . the resulting mixture was stirred for 15 minutes ( min ) at 25 ° c . and then filtered on a no . 41 filter [ whatman ]. the retentate was eluted with twice 250 ml of acetate - urea - edta buffer in 2 - mercaptoethanol . the three filtrates were grouped together . this first stirring - filtering cycle served to eliminate a fraction f0 . the following casein fractions ( f1 and f2 ) were eluted using the same procedure , adding 30 mm and 70 mm of cacl 2 respectively to the buffer . edta was added to the fractions in amounts of 15 mm to the fraction f1 , 45 mm to the fraction f1 , and 85 mm to the fraction f2 . the filtrates f0 , f1 , and f2 , were dialyzed against ultrapure water and then lyophylized , after which they were subjected to electrophoresis using a polyacrylamide - urea gel in order to reveal the fractioning . the fraction f1 contained α s2 casein . the purification of the α s2 casein was finished off by hydrophobic interaction chromatography on a tskgel phenyl 5pw column [ tosohaas , stuttgart , germany ] having dimensions of 150 millimeters ( mm )× 32 . 5 mm . the fraction f1 ( 1 milligram per milliliter ( mg · ml − 1 )) was put into solution in a 0 . 48 m sodium phosphate buffer at ph 6 . 4 , containing 2 . 5 m of urea and in the presence of 0 . 1 % 2 - mercaptoethanol , and then filtered on a 0 . 45 micrometer ( μm ) pvdf filter [ pall corporation , ann arbor , mich ., united states ]. twenty mg of protein solution were injected . a non - linear gradient going from 0 . 48 m to 0 . 037 m of sodium phosphate heaving a ph of 6 . 4 and containing 2 . 5 m of urea was applied at a flow rate of 6 . 0 milliliters per minute ( ml · min − 1 ) as follows : from 480 mm to 126 mm ( 18 min ), 126 mm ( 3 min ), from 126 mm to 103 mm ( 3 min ), 103 mm ( 3 min ), from 103 mm to 72 mm ( 5 min ), 72 mm ( 5 min ), from 72 mm to 37 mm ( 4 min ), 37 mm ( 17 min ). the collected bovine α s2 casein was dialyzed , lyophylized , and stored under a vacuum at + 4 ° c . the α s2 casein was put into solution at a concentration of 0 . 05 % ( w / v ) in 100 ml of 67 mm sodium phosphate buffer at a ph of 8 . 1 containing 0 . 02 % sodium nitride . bovine pancreatic trypsin ( e . c . 3 . 4 . 21 . 4 ) immobilized on agarose beads and treated by tpck ( n - tosyl - l - phenylalanine chloromethylketone ) [ sigman , saint louis , miss ., united states ] was added , after washing in the preceding buffer and filtering several times , to the α s2 casein solution in order to obtain a concentration of 0 . 2 units of nα - benzoyl - l - arginine ethyl ester ( baee ) per ml . hydrolysis took place at 37 ° c . for 24 hours . the reaction was stopped by diluting the mixture twice using 4 % acetonitrile containing 0 . 2 % trifluoroacetic acid ( tfa ), and then filtering on a 0 . 45 μm polyvinylidene fluoride ( pvdf ) filter . the hydrolysate was conserved at − 30 ° c . c — fractioning the hydrolysate by reverse - phase hplc in a gradient of acetonitrile the hydrolysate was fractioned on a c18 xterra ™ column [ waters , milford , mass ., united states ] having dimensions of 250 mm × 4 . 6 mm thermostated to 37 ° c . 500 μl of sample ( 0 . 25 mg · ml − 1 ) were injected . the elution profile had an isocratic phase of 3 min at 1 . 6 % acetonitrile in water ( in the presence of 0 . 1 % tfa ) followed by a linear gradient serving to reach 40 % acetonitrile in 87 min at a rate of 1 ml · min − 1 . the peptide profile is shown in fig1 where the absorbance at 215 nanometers ( nm ) is plotted up the ordinate and elution time along the abscissa . five of the seven peptides of the group selected in the context of the present invention correspond to the peptide peaks referenced 1 to 4 in fig1 . these peptides were selected and lyophylized twice . they were identified by determining their amino acid composition by the hamilton ( 23 ) ninhydrine method and by mass spectrometry coupled to the hplc , esi - lc / ms (“ electrospray source ionization ”), or by ms / ms , mass spectrometry in tandem . the peak 1 collected at 25 min contains the peptide tvy ( seq id no . : 1 ). the peak 2 collected at 29 min contains the peptide nmainpsk ( seq id no . : 2 ). the peak 3 collected at 57 min contains the peptide falpqy id no . : 3 ). the peak 4 collected at 60 min contains the peptides fpqylqy ( seq id no . 4 ) and falpqylk ( seq id no . : 5 ). the other two peptides , nmainp ( seq id no . : 6 ) and falp ( seq id no . : 7 ) can be obtained by chemical synthesis using conventional methods . the same applies to the five peptides that are preferably obtained by fractioning the total trypsic hydrolysate of α s2 casein . d — in vitro test of the peptides on the enzyme for the angiotensin i converting enzyme ( ace ) the main experiment relies on measuring the residual activity of ace on a synthesized substrate of hippurhyl - his - leu - oh in the presence of a potentially inhibiting peptide [ cushman and cheung ( 24 )]. the hippuric acid that was released was assayed by hplc and its quantity compared with a reference having no inhibitor . incubation was performed in a 50 mm ches buffer with a ph of 8 . 3 containing 5 mm of hippuryl - his - leu - oh , 350 mm of nacl , 3 . 33 u · l − 1 ace , and 5 % ethanol . the mixture ( final volume : 150 μl ), after 10 min of pre - incubation without the enzyme , was incubated for 60 min at 37 ° c . the reaction was stopped with captopril ( 5 μm ), edta ( 1 mm ), and tfa ( 0 . 067 %). the hippuric acid that was released was quantified by hplc using a c18 symmetry ® column [ waters , milford , mass ., united states ] with dimensions of 150 mm × 2 . 1 mm and thermostated at 37 ° c . the samples were filtered on a 0 . 45 μm pvdf filter and 40 μl were injected . an acetonitrile gradient in water ( in the presence of 0 . 1 % tfa ) was applied at a rate of 0 . 25 ml · min − 1 . the elution gradient went from 13 % to 50 % acetonitrile in 7 min , and then reached 99 % in 0 . 5 min , and was maintained at that value for 1 . 5 min . the method of determining the ic 50 was validated by comparing the value found for captopril ( 0 . 022 μm ), a known ace inhibitor , with biological values ( 0 . 023 μm [ cushman et al . ( 25 )], 0 . 018 μm [ duncan et al . ( 26 )], 0 . 007 μm [ pihlanto - leppälä et al . ( 27 )]. the four chromatographic peaks ( 1 to 4 ) collected from the trypsic hydrolysate of α s2 casein and corresponding to the five peptides of the group selected in the context of the present invention were tested twice at a concentration of 50 μm of primary amines . the chromatographic peaks numbered 5 to 7 were tested under the same conditions . the results obtained are given in fig2 where the inhibition percentage is plotted up the ordinate and the chromatographic peak number along the abscissa . it can be seen that the peaks 1 to 4 containing the peptides of the group selected in the context of the present invention inhibits ace at more than 40 %, and of those peaks , peak no . 4 containing the peptides fpqylqy ( seq id no . 4 ) and falpqylk ( seq id no . 5 ), peak no . 3 containing the peptide falpqy ( seq id no . 3 ), and peak no . 1 containing the peptide tvy ( seq id no . 1 ) inhibit ace at more than 70 %. synthetic peptides were used to determine the ic 50 values of these five peptides precisely . the peptides were initially tested twice at concentrations lying in the range 0 . 1 μm and 250 μm to 500 μm in order to obtain an estimate of their ic 50 value , and then tested in triplicate on an appropriate range of concentrations . the results obtained are given by the graphs of fig3 where the logarithm of the activity / inhibition ratio is plotted up the ordinate and the logarithm of peptide concentration along the abscissa . this enables the inhibition curve to be liberalized and enables the ic 50 values to be deduced therefrom using straight line equations . the ic 50 values are summarized in table 1 . they are all of the order of or less than 60 μm , it being observed that the peptides falpqy ( seq id no . 3 ) and falpqylk ( seq id no . 5 ) have the best performance with an ic 50 value of 4 . 3 μm . the seven peptides in the group selected in the context of the present invention have amino acid sequences that are different from those of the eight inhibitor peptides described in the past [ fitzgerald and meisel ( 28 ), yamamoto and takano ( 29 ), pihlanto - leppälä ( 30 ), nurminen ( 31 ), takano ( 32 )], including those reported by maeno et al . ( 13 ) obtained using α s2 casein : cnα s2 -( f198 - 202 ), cnα s2 -( f190 - 197 ), and cnα s2 -( f189 - 193 ). as mentioned above , two peptides of the group selected in the context of the present invention , obtained by fractioning the trypsic hydrolysate of α s2 casein gave values of ic 50 of less than 5 μm , and two others gave values for ic 50 less than 20 μm , thereby classifying them amongst the most active inhibitors of ace amongst natural peptides obtained by a mono - enzymatic process on milk proteins . the two peptides nmainp ( seq id no . 6 ) and falp ( seq id no . 7 ) which are not obtained directly by fractioning the trypsic hydrolysate of α s2 casein are remarkable firstly in that they possess a prolyl residue at their c - terminal end , which is common with certain other ace - inhibiting peptides [ maruyama , et al . ( 33 ), kohmura et al . ( 34 , 35 , 36 ), nakamura et al . ( 37 )], and secondly in that their amino acid sequence is completely contained in the other two peptides nmainpsk ( seq id no . 2 ) and falpqy ( seq id no . 3 ) which are obtained directly by such fractioning . as a result , it is possible to envisage that the use of the second two peptides ( seq id nos . 2 and 3 ) as medicine , or as a food supplement , could lead to in vivo formation of the first two peptides ( seq id nos . 6 and 7 ) by breaking the appropriate peptide bonds . it should be observed that using at least one of the seven peptides of the group selected in the context of the present invention for preparing medicines , food products , or food supplements may be performed in combination with one or more other peptides , having ace - inhibiting activity but having an ic 50 value greater than 60 μm . this would occur when implementing total trypsic hydrolysate of α s2 casein or a fraction thereof containing at least one peptide of the group . such a combination could be advantageous for in vivo inhibiting activity on ace . ( seq id no . 8 ), cnα s2 -( f81 - 91 ), alneinqfyqk , ala - leu - asn - glu - ile - asn - gln - phe - try - gln - lys , peak 5 eluted at 52 min ; ( seq id no . 9 ), cnα s2 -( f81 - 89 ), alneinqfy , ala - leu - asn - glu - ile - asn - gln - phe - tyr , peak 6 eluted at 59 min ; ( seq id no . 10 ), cnα s2 -( f206 - 207 ), yl , tyr - leu , peak 7 eluted at 31 min ; which may also be obtained by fractioning the trypsic hydrolysate of α s2 casein and which inhibits ace in the range 25 % to 35 % at a concentration of 50 μm of primary amines ( fig2 ). ( 1 ) grosclaude , f ., 1988 , le polymorphisme des principales lactoprotéines bovines , inra prod . anim ., 1 , 5 - 17 . ( 2 ) swaisgood , h . e ., 1992 , chemistry of the caseins in p . f . fox : advanced dairy chemistry , volume 1 , proteins , blackie academic & amp ; professional , london , united kingdom , 63 - 109 . 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