CA1059694A - Method for manufacturing of substances with -nh2 group which are released from polymers by enzymes - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The present invention relates to a method of manufactur-ing a macromolecular form of active substances with -NH2 groups which is useful for protracted release of the active component by an enzymatic cleavage, wherein an N-substituted amide or ester of acrylic or methacrylic acid, which contains at least three chained atoms in its side chain terminated by the carboxylic car-bon atom, is condensed with the.alpha.-amine group of an aminoacid of L-configuration, as .beta.-phenylalanine, tyrosine, tryptophan, lysine, arginine, glycine, alanine, leucine, citrulline or ornithine, and then an active component is bonded by its -NH2 groups to the car-boxylic group of this amino acid and the resulting monomer is copolymerised with a hydrophilic monomeric component based on N-alkymethyacrylamide, N-alkyacrylamide, N,N-dialkylacrylamide where alkyl has 1 to 6 carbon atoms and may contain 1 to 3 -OH
groups, or glycol methacrylate or glycol acrylate, where glycol is ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, triethylene glycol and tetraethylene glycol, alone or in a mixture, and if desired with further monomeric components which may act as crosslinking agent. The polymeric form prepared by the method according to the present invention enables to control the physical and biological fate of the active substance by the nature and molecular weight of the carrier-polymer.

Description

~5'~69~

~ he invention relate~ to a method of manufaoturing apolymeric form of active sub~tances. An ob~ect o~ the present in~ention is ~ method for producing a macromolecular preparation which liberates an acti~e component by enzymatic hydrolysi~O
~ he e~fect o~ many active ~3ubstances introduced into the biological en~ironment is limited by their short resiaental time or inacti~ation which fact nece33itates increa~ed doses ~or treatment.
To pre~ent thi~ dra~back, some sub~tance~ are ~pplied in a form o~ low-~oluble derivatives or complexes w~ich ~hould create a local depot which releases the e~ective compound only "~
Yery 810~1y.
Substance~ bonded to polymers were te~ted in other cases.
If the polymeric form of the ~ubstance it~elf is not acti~e, the low-molecular-weight active form ha~ to be cleaved of~, This cleavage i9 hydrolytical in some cases and proceed~ by a ~uitable ~elocity which i9~ however~ di~ficult to control. Other author~
proposed the en~ymatic hydrolysis, but neither a chemical bond specific ~or enzyme~ ~a~ preparedJ nor the enzymatic cleavage wae directly proved. Other studies described analy~es of the enzyma tic cleavability~ however) natural polymers or in~oluble synthetic polymers were used.
~ ow, it ha~ been found that a chemical bond ~pecific for enzym~ can be prepared in the ~ide chain of synthetic polym~r~ by econo~ic~lly and advantageous way and that an aotive compound oon taining -NH2 group~ may be bonded through this bond providing th 810~ cleavage of thi~ compound in its active form through the ;,-acti~ity of en~yme~ present in biological en~ironment~
A~ object of the invention is a method for preparation 30 of the polymeric form of a biological active ~ub~tance containing ~ X2 groups, ~herein an active component i~ bonded by it~ -NH2 group to a ~ide chain with the terminal -~-aminoacid specific for ,; , , , :

16~S9~i;94 he selected system. This side chain is a part of the polymer unit contained in the amount of 1 - 50 mol.~, especially 1 - 15 mol.%, in a crosslinked or non-crosslinked copolymer containing a hydrophilic component based on N-aIkylmethacrylamide, N,N~
dialkylacrylamide, N-alkylacrylamide, where alkyl has 1 to 6 carbon atoms and may contain 1 to 3 -OH groups, or glycol methacrylate or glycol acrylate, where glycol is ethylene glycol, propylene glycol, butylene glycol, diethylene glycol r triethylene glycol, or tetraethylene glycol, alone or in a mixture, while the side chain between the polyvinylic chain and the active component has the minimum length of 6 atoms.
A method for preparation of thè polymeric form according ;
to the invention consists in condensing an N-substituted amide of acrylic or methacrylic acid, which contains at least three chained atoms in its side chain terminated by the carboxylic carbon atom, with the ~-amino group of an aminoacid having L-configuration, followed by bonding of the aminoacid carboxylic groups with the -NH~ group of an active component and the subse~uent copolymeri-zation ofthe resulting monomer with a hydrophilic monomeric component based on N-alkylmethacrylamide, N-alkylacrylamide, N,N-dialkylacrylamide, where alkyl has 1 to 6 carbon atoms and may contain 1 to 3 -OH groups, or glycol methacrylate or glycol acrylate, where gIycol is ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, triethylene glycol, and tetraethylene glycol, alone or in a mixture.
The aminoacid of L-configuration is preferably selected from the group consisting of ~-phenylalanine, tyrosine, tryptophan, ~;
lysine, arginine, glycine, alanine, leucine, citrulline and ornithine.
The procedure for manufacturlng the polymeric form of an active substance may also consist in bonding of a component, which forms an active ester, to the carboxylic group of the side chain or the terminal aminoacid by the known methods, instead of bonding the active component, so that an activated polymer is

- 2 -~~
1~59694 obtained after the above mentioned copolymerization, which i~
further used for bonding of the active component by its -~2 group or for bonding o~ an aminoacyl derivative of this active component with the afore~aid I~ ~ -aminoacids by the known method~.
~ he choice of the manufacturing method depends on the character o~ the copolymer u~ed and~ e~pecially, on the character of the ~ctive comp~nent. ~owever~ it i9 advantageou~ to prepare first the monomer which has the terminal carboxylic group ~or bonding the active component esteri~ied with p~nitrophenol9 2,3, 5-trichlorophenol, 8-hydroxyquinoline~ 5-chloro-8-hydroxyquinoline, N_hyaroxysuccinimide~ or NOhydro~yphthalimide. This monomer i9 copolymerized ~ith a ~ubstituted acrylamide or methacrylamide according to the Czechoslovak Patent Nr. ....... 0. (PV 2879-74)o ~he re~ulting copolymer i~ ~table if ~tored in a cool place and under an i~ert atmo~phere, It react~ in a suitable solvent with -~H2 groups of the active component forming the abo~e described cleavable bond.
~he procedure via reacti~e e~ters (see Example 2) i~ ;
economical with re~pect to utilizakion of the active component and enables to prepsre the sctivated poIymeric carrier ~or bonding o~
v~rious active compounds.
~ he polymeric form prepared by the method according to the present in~ention has numercu~ advantage~. It enables to con trol the physical and biological fate of the acti~e ~ubstance by the nature and molecular weight of the carrier-polymer. ~he rate o~ cleavage can be controlled by the length of the side ohain (c~. Example ~)~ l`he active component i~ protected by it~ bonding ~ot only again~t the liberation but also against it~ in~ctivation (e.g. the hi~tamine bond in Example 4 make~ the attack o~ histamin-ase, i.e. monoaminooxidase 9 impo~sible).
~he ob~ect of the present invention is further illustra-

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1~5~6~4 ted in examples of performance~ without, however, limiting the ~cope of the invention, A copolymer I containing 97.6 mol~% of N(2-hydroxypropyl) methacrylamide (HPMA) and 2.4 mol,% of N_methacryloylglycylpheny-lalanylnitranilide (MGPN) and a copolymer II containing 98.18 -mol.% of HPMA and 1,82 mol,~ o~ nitrophenyl N-(methacryloyl)-glycinatewerepreparedbythedirectcopolymerization accordingto the C~echoslovak PatentNo. 173,8460f October'7,1976. Thecopolymer lQ II was dissolved in dimethylsulfoxide to the 10~ solution and the ~ame volume of a 0,5% solution of ~-phenylalanyl p-nitranilide (a) or glycyl-p-nitranilide (b) in the same solvent wa added, ~he mixture was allowed to stand overnight at 37 C, l'he copolymer ~as precipitated in a tenfold excess of the acetone~ether mixture (1 1)7 washed, di~solved in dimethylformamide to the lO~o solution and lyophili~ed giving the KoIIa and KoIIb products~ respectively, ~he product~ were used as substrates for the enzymatic cleavage in the procedure according to B,~, Erlanger~ ~, Edel and A,G, Cooper (Arch. ~iochem, ~iophys. 1966, 115, 206), N_(Succinyl)_~_pheny_ lalanyl-p-nitranilide (SPNA) served as a reference.
The polymer with bonded glycyl-p-ni-tranilide did not exhibit any cleavage even after 24 hours of incubation, The clea-vage rate of the polymer-bonded I_phenylalanylni-tranilide reference (SPNA = 100%) i9 given in the following table:
Initial content of nitranilide Cleavage rate (SPN~ = 100%) in the copolymer Chymotrypsin Pronase _________ _ ______ ____________ ________________~_____--__________ ~
KoI 2.4 mol,% 23 % - `
KoIIa 1.25 mol~% 15 % 82%
KoIIb 1.56 mol.% O % 0%

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59~34 The re~ults prove that the formed bond betwee-n the side chain and p-nitranilide as the model compound i9 accessible for enzyme already if the side-chain length is 6 atoms and that bo-th pro-ducts obtained by the direct copolymerization and by the poly-mer analogous reaction of the activated polymer are equally suit-able.
EXAMPIE_2 A copolymer IV containing 96 mol.% of HPMA and 4 mol.%
of N_methacryloylglycylglycyl(~-phenylalanyl)nitranilide was prepared by the direct copolymerization according.to ths Czechoslovak Patent NOa 173,846 similarly as in Example l.
This polymer and the polymer KoI with bonded I~phenylalanyl_p-nitranilide described in the Example I (KoI) were u~ed in various concentrations (5xlO 5M, lO 4M~ 2xlO 4M, 7xlO 4M~ lO 3M, 5xlO 3M
calculated on nitranilide) as substrates for cleavage by chymo- . :
trypsin according to the method in Example la The incubation ~or .~
24 hours was allowed at -the concentration 7xlO 4~. The percentage ;.-.
of cleavage was then determined and if it did not reach 100%, fresh enzyme wa~ added and incubation was carried out again for 2 hours. As far a~ the cleavage did not increase~ the achieved percentage of cleavage was consldered the limit value, Constant~ Km (Michaelis~ constant showing the enzyme affinity to the substrate) and Vmax (the maximum rate of cleavage showing the decomposition of an enzyme-sub~trate complex to enzyme and product) were calculated from the results. ~he results in the ., following table show that the cleavage -rate as well as the limit cleavage can be advantageously controlled by the leng-th of the - side chain.

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Substrate Km (mM) Vmax ~imit cleavage ___ ~__ ____________________________ _____________ ___ _______ :SPN~ 0,0~6 1.00 100 %
KoI 2.0 0.7 28.9 %
KoIV 2.22 8.7 100 %

EX~
~ -(Phenylalan,yl)histamine was prepared by -the method described by Ho Arold and ~. Rietschel (Z. Chem~ 1969, 9? 144) and used in the polymer analogous reaction with the copolymer ''~
KoII de~cribed in Example 1. The copolymer (KoV~ was obtained `
which contained 1.1~ of N_LNL(methacryloyl)glycyl~ phenylalanyl-histamine unit~. It was incubated with chymotrypsin by the method described in ~xample 1~ The released histamine was determined according to P.A, Shore, A. Burkhalter and V,H. Cohn, Jr.
(J, Pharmacol, exp, theor~ 127, 182, 1959). The initial Goncentra- `
tion of the bonded histamine was lxlO 3M and the chymotryp~in con-centration 0.5 mg/ml. The initial cleavage ,rate was constant for ' 80 min with the slope K= 1,2xlO 6 mole min lmg 1. ~he limit cleavage was 28%.

, EXAMPIE 4 ,~
An activated copolymer of N-(2-hydroxypropyl)methacryla_ ',' mide with nitrophenyl methacryloylglycylglycinate cro~slinked with trimethylolpropane trimethacr~late, prepared according to the Czechoslovak Patent No. 173,846 was dispersed in '~
dimethylformamide to the 5% w/w ~uspension~ Dimethyl~ormamide solutions of N_phenylalanyl,N'-i~onicotinylhydrazide hydroohloride (2 ml of 0.5% solution) and triethylamine (0.1 ml of 3% ~olution) were added to 2 ml of the suspension under continuous stirring. ', ~he mixture wa~ allooe d to stand at 37 C overnight and the polymer i, was isolated by centrifugation and washed with water as long as the washings gave reaction on chlorides, ~he ~uspension of the pro-duct was incubated with chymotrypsin under the same conditions as ,' ~ . ~' .. ..

~59~9~
in Example 1. Hydrazide oE isonicotinic acid cleaved after 2 hours was determined in the supernatant after centrifugation spec-trophotometrically. The amount cleaved after this -time recalculated to the whole amount of the polymer was 1.8 mg.
E~AMPLE 5 The following copolymers were prepared, reprecipitated and refined according to the Czechoslovak Patent No. 173,846 93.2 mol.% of N-ethylacrylamide with 6.8 mol.% of MGPN (see Example 2), 97.3 mol.% of N-acryloyImorpholine with 2.7 mol.% of MGPN, and 92 mol.% of triethylane glycolmonomethacrylate with 8 mol.% of MGPN. The enzymatic cleavability of p-nitranilide was tested analogously as in Example 1. The cleavage was followed at ,~
37C for 15 min and expressed in percent of the reference SPNA
cleavage amounting 15.2, 23.5 and 18.7%, respectively.

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Claims (3)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Method of manufacturing a macromolecular form of active substances with -NH2 groups which is useful for protracted release of the active component by an enzymatic cleavage, wherein an N-substituted amide or ester of acrylic or methacrylic acid, which contains at least three chained atoms in its side chain terminated by the carboxylic carbon atom, is condensed with the a-amino group of an aminoacid of L-configuration, and then an active component is bonded by its -NH2 group to the carboxylic group of this amino acid and the resulting monomer is copolymerized with a hydrophilic monomeric component based on N-alkylmethacryl-amide, N-alkylacrylamide, N,N-dialkylacrylamide, where alkyl has 1 to 6 carbon atoms and may contain 1 to 3 -OH groups, or glycol methacrylate or glycol acrylate, where glycol is ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, triethylene glycol and tetraethylene glycol, alone or in a mixture, and if desired with further monomeric components which may act as a crosslinking agent.

2. A method according to claim 1, wherein the terminal carboxylic group of the .alpha.-aminoacid is converted in a reactive ester, then the copolymerization is carried out and the active component is bonded to the prepared reactive polymer.

3. A method according to claims 1 or 2, wherein the aminoacid is selected from the group consisting of .beta.-phenylalanine, tyrosine, tryptophan, lysine, arginine, glycine, alanine, leucine, citrulline and ornithine.