Polymer derivatives of β-lactam antibiotics of the penicillin series

Polymer derivatives of β-lactam antibiotics of the penicillin series

119 Journal of Controlled Release, 10 (1989) 119-129 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands POLYMER DERIVATIVES ...

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Journal of Controlled Release, 10 (1989) 119-129 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

POLYMER

DERIVATIVES

OF /3-LACTAM

Evgenij F. Panarin and Mikhail institute of Macromolecular

Compounds

ANTIBIOTICS

OF THE PENICILLIN

SERIES

V. Solovskij

of the Academy

of Sciences of the U.S.S.R., Boishoipr.3

I, Leningrad

199004

(U.S.S.R.) (Received June 30, 1988; accepted in revised form March 1989) Key words: polymeric p-lactam antibiotics; vinyl pyrrolidone copolymers; antimicrobial activity; penicilinase degradation; soluble polymeric drug carriers

In order to elucidate the effect of the macromolecular nature of penicillins on their antimicrobial properties and to search for new @actam antibiotics resistant to inactivation by penicillinase, the homopolymerization and copolymerization with N-vinylpyrrolidone (VP) of o-vinyl-, p-acryloyl-, p-,m-,o-methacryloylaminophenoxymethylpenicillins and p-methacryloylaminooxacillin were carried out. Depending on the conditions of the process, water-soluble homopolymers of unsaturatedpenicillins and their copolymers with VP having intrinsic viscosity []gTM KCI= 0.1Z1.90 dl/gand iodometric activity of 160-520 g/rng were obtained. The VP copolymers containing up to 42% of units of non-inactivated antibiotic were prepared by the attachment of 6-aminopenicillanic acid to carboxyl-containing VP copolymers by using the method of mixed anhydrides. The physico-chemical properties of the polymer penicillins obtained and the kinetics of their inactivation by penicillinases of Bacillus licheniformis 749/c and Bacillus cereus were investigated. It was established thatpolymer antibioticsgreatly exceed monomer penicillins with respect to their resistance to the hydrolytic effect of penicillinases. It was established in the in vitro microbiological experiments that polymer penicillins exhibit high activity with respect to the bacteria sensitive to benzylpenicillin. This activity depends on the molecular weight of these penicillins and the character of distribution of penicillin units along the polymer chain. The contrast to benzylpenicillin, polypenicillins at low concentration (I-100 &ml) affect penicillinase-forming Staphylococcus.

INTRODUCTION Penicillins (I) are inhibitors of synthesis of the cell wall of bacteria. They interrupt the crosslinking stage of peptidoglycan macromolecules which form the cell wall of Gram-positive bacteria [l-3]. The crosslinking is carried out by peptide side chains terminated by Dalanyl-D-alanine dipeptide, penicillin being its structural analogue. The conformation of penicillin corresponds to that of the dipeptide in

the transition state of the moment of breaking of the peptide bond [ 41. Hence penicillin competes with n-alanyl-D-alanine dipeptide for the bonding to the active center of peptidases, which induce the crosslinking of peptidoglycan macromolecules and acylates peptidases, transforming them into the inactive form. A synthetic polymer containing penicillin units in the side chain may presumably also be regarded as a substrate for peptidases. It may be expected that polymer penicillin will interact with pep-

120

tidases localized in the external layer of the cytoplasmic membrane of bacteria and will thus exhibit its antimicrobial properties. On the basis of these assumptions we carried out an investigation on the insertion of polymer structures into the acyl side radical of penicillins, R (I), i.e., the synthesis of polymer penicillins. In this case the polymers obtained can maintain their j?-lactam ring intact and hence exhibit antimicrobial activity, in contrast to the polymerization of penicillins using their /3-lactam rings [El]. Polymer penicillins were obtained by two procedures: (1) by the polymerization of monomeric penicillins I (into the acyl-side radical of which a double bond had been introduced), and (2) by the bonding of 6-aminopenicillanic acid (6-APA), II, the chemical nucleus of penicillins, to carboxyl-containing copolymers of Nvinylpyrrolidone.

paring methacryloyl (acryloyl) aminophenoxyacetic acids (IIIa-d), o-vinylphenoxyacetic acid (IV) and 5-methyl-3- (p-methacryloylamino)phenylisoxazol-4-carboxylic (V) acid have been developed [ 7-91. CHZ=C-C-NH I

II

I

\

/I

Rl

9

0

6CH2C00H

III III

o , R,

=

H, p-isomer

III

b , R,

=

CHJ

III



, R,

=

CH,,

, R,

=

CH3,0-fsomer

1IId

, p-

tsorncr

m-tsomer

CH,=CH I 0CH2COOH

R-CC-NH-CH-CH C-N-CH 0

IV \

/

COOH

I IO,R

=

C6H50CH2-

Ib,R

=

C6H5-i-;-

CH3

-

CH2=‘-~-NH-$--&-t’“‘” N

i-CH3 N\

‘0’ Ic,R

H

2

=

C6H5CH2--

N_CH_C~S~C/CH3 I I I\OHo-N-cT

COOH II

The following monomer penicillins were synthesized: unsaturated derivatives of phenoxymethylpenicillin (PhOMP), Ia, and oxacillin (Ox) -6-methyl-3-phenylisoxazol-4-carbamido penicillanic acid lb. Phenoxymethylpenicillin and oxacillin are acid-stable antibiotics used in medicine [ 61. For the synthesis of unsaturated PhOMP and Ox derivatives, methods for pre-

,C-CH3 0

V

The monomeric penicillins p-acryloylamino (AA) PhOMP, p-,m-,o-methacryloylamino (MAP) PhOMP and p-MAOx were obtained by the acylation of 6-APA with mixed anhydrides (VI) of unsaturated acids IIIa-d, IV, V and ethoxyformic acid and isolated in the form of acids (VII) of potassium or sodium salts (VIII) (Scheme 1). When the second method of preparation of polymer penicillins was applied, the copolymers of VP with crotonic acid (CA), IXd, /?phenylacrylic acid ( P-PhAA), IXb, and o-vinylphenoxyacetic acid (o-VPhOAA), X, were used as the initial polymer carriers.

121 (C2H5)3N ____t C2H5COOCt

R-COOH

RCOOCOOC2H5

+

H2N-CH-CH

VI 04

AS\

I I

R-C-NH-CH-CH II

ij

t---N

-tH

/

CH3

ih

-COOH

/s\c

-

CH3COOM

Ofi

11

5

1

1

I’C+

k -

I; -

k~-c00~

04 VII

Scheme 1. Synthesis

of monomeric

100-m

IX IXa,R2

=

MATERIALS

CH3

;

IXb,R2=

VIII

, MI

K,Na

penicillins.

1

H2C--cCH2

t

,CH3

R-C-NH-CH-CH

C6H5

AND METHODS

Monomers

6-Aminopenicillanic acid was provided by the U.S.S.R. Research Institute of Antibiotics (RIA) in the form of a crystalline substance with m.p. 207 oC and iodometric activity of 960 pglmg. p-Acryloylamino phenoxyacetic acid IIIa and p- and m-methacryloylaminophenoxyacetic acids IIIb and 111~ were synthesized by the method described in Refs. [7,8] by the acylation of p- and m-aminophenoxyacetic acids with anhydrides of acrylic and methacrylic acid, re-

spectively, and o-methacryloylaminophenoxyacetic acid III was obtained by the alkylation of o-methacryloylaminophenol with monochloroacetic acid [ 71. o-Vinylphenoxyacetic acid IV was synthesized by the action of monochloroacetic acid on o-vinylphenol according to the method described in Ref. [lo]. 5-Methyl-3- (p-methacryloylamino)phenylisoxazol-4-carboxylic acid V was obtained by the acylation of 5-methyl-3- (p-amino)phenylisoxazol-4-carboxylic acid with methacrylic anhydride [ 91. Unsaturated phenoxymethylpenicillins and p-methacryloylaminooxacillin (VIII) were obtained by the method of mixed anhydrides on the basis of 6-APA and the corresponding unsaturated acids IIIa-d, IV and V [9,11]. Carboxyl-containing copolymers of VP (IXa,b,X) were obtained by the copolymerization of VP with crotonic, P-phenylacrylic and o-vinylphenoxyacetic acids in ethanol in the presence of AIBN [ 121. Polymer penicillins

The polymerization of potassium salts of unsaturated PhOMP andp-MAOx was carried out at 3” C in a 0.1 A4 phosphate buffer (pH 6.87.0) at an initial monomer concentration of O.l0.4 mol/l [ 91. The polymerization was initiated with the aid of the K.$$Os-NaHSO, redox system. The concentration of K=&Os was 0.3-1.1 wt.% and the weight ratio NaHSO, : K&O8 was

122

1: 1.5. The copolymerization of VP with unsaturated penicillin acids VII was carried out in bulk or in an organic solvent (DMF or acetone ) at 60” C in the presence of 0.5 wt.% of AIBN [ 131. The binding of 6-APA to carboxyl-containing VP copolymers was performed according to the method of Ref. [ 141. In all cases polymer penicillins were isolated by precipitation into dry acetone and vacuum-dried. Penicillinases of Bacillus licheniformis c and Bacillus cereus

749/

These penicillinases were provided by RIA in the form of dry preparations in vessels with an activity of lo6 units. The enzymes were dissolved in 11 of phosphate buffer at pH 6.8. The solutions obtained were diluted to the desired penicillinase concentration and stored at 3°C. Methods

of polypenicillin

investigation

The intrinsic viscosity of polypenicillins was determined at 25°C in a 0.5 M solution of KC1 in an Ubbelohde viscometer. The UV spectra of aqueous solutions of mono- and polypenicillins were recorded with an SF-26 spectrophotometer in a cell with a layer thickness of 1 cm. The IR spectra were recorded with a Nippon Bunko spectrometer using KBr pellets. The iodometric activity of polypenicillins was determined by a published method [ 151. The kinetics of enzymatic hydrolysis of mono- and polypenicillins were studied by the method of Ref. [ 161, the initial substrate concentration was 1.5 x 10e3 mol/l and the enzyme concentration [E] varied from 4.7 x 10m7 to 2.0 x 10W5 mol/l.

RESULTS AND DISCUSSION Monomer

penicillins

The main characteristics of the resulting unsaturated phenoxymethylpenicillins and pmethacryloylaminooxacillin are given in Table

1. The level of iodometric activity of monomer PhOMPs indicates that they were isolated at a high degree of purity, whereas the values of minimum inhibiting concentrations (MIC ) show their high antimicrobial activity with respect to Staphylococcus aureus 209 P. The introduction of the unsaturated acylamino group into the acyl-side radical of oxacillin and phenoxymethylpenicillin resulted in a certain decrease in activity. In this case the o-isomer is less active than the p- and m-derivatives. The change in the PhOMP structure caused by the insertion of the double bond also had a certain effect on its sensitivity to penicillinase. All monomer penicillins with the exception of oMAPhOMP are hydrolyzed by penicillinase at a lower rate than PhOMP. Polymerization

of monomer

penicillins

Since the p-la&am ring of antibiotics of the penicillin series is very labile, the homopolymerization of salts of unsaturated PhOMP and p-MAOx was carried out in aqueous buffer solutions at pH 6.8-7.0 at a temperature of 3 ‘C using the K2S208-NaHS03 systems as redox initiators. As can be seen from data given in Table 2, under these conditions unsaturated penicillins are readily polymerized forming high molecular weight products. The highest polyobserved for mmerization rate was MAPhOMP: for 3.5 h, 80% of this monomer was transformed into the polymer, and the lowest rate was found for o-VPhOMP. Depending on the initiator and monomer concentrations used, water-soluble poly-PhOMP and poly-p-MAOx with intrinsic viscosities [v] ranging from 0.3 to 1.5 dl/g were obtained. The reduced viscosity of aqueous solutions of the polymers increases with dilution (Fig. 1). This increase shows that polymer penicillins are typical polyelectrolytes. For a tentative evaluation of the molecular weight of the polypenicillins obtained, GPC on Sephadex and Acrylex gels was used. The polymers were usually eluted in the form of narrow peaks (Fig. 2) with low retention volume val-

123 TABLE I Properties of salts of unsaturated penicillinsVII1 Penicillin

M

T melt. ("C)

Iodometric activitya k/mg)

Bate of inactivation by penicillinaseb, VEX lo7(mol/l min)

MIC St. aur. 209P k/ml)

o-VPhOMP p-AAPhOMP p-MAPhOMP m-MAPhOMP o-MAPhOMP p-MAOx PhOMP

K K K K K Na K

232-234 229-231 215-218 211-212 220-221 180-182 230-232

990 1000 1000 1000 1000 940 1000

5.6 5.1 5.6 4.0 8.1 resistant 7.2

0.03 0.12 0.25 0.05 0.50 6.2 0.03

“Calculated activity - 1000 pg/mg. bAt [S,] =1.5x10A3 mol/l, [E] =4.7X10-7mol/l. TABLE 2 Polymerization of potassium salts of unsaturated phenoxymethylpenicillins in a buffer solution (pH 7.0) at T= 3 ‘C Monomer Designation

K&Cs (mass % )

Polymerization time (h)

Iodometric activity

Polymer yield (%)

Polymer penicillin ]?I?52 (dl/g)

990 990 990 1000 1000 1000 1000 980 940

Iodometric activity (pg/mg)

(pg/mg) o-VPhOMP o-VPhOMP o-VPhOMP p-AAPhOMP p-AAPhOMP p-MAPhOMP p-MAPhOMP m-MAPhOMP p-MAOx

KCI

0.30 0.30 0.20 0.45 0.30 0.75 1.10 1.60 0.25

90 22 90 60 40 24 24 3.5 24

ues. These values show that the polymers were of high molecular weight. In contrast, monomer penicillins were characterized by much higher retention volume values. The GPC method made it also possible to evaluate the homogeneity of these polypenicillins. Polymer fractions I (Fig. 2) or samples of poly-PhOMP free of monomer admixtures were subjected to further investigation. The structure of polymer penicillins was confirmed by quantitative iodometric analysis and by UV and IR spectroscopy. The iodometric activity of all polymer PhOMP was relatively high:

67 41 69 79 66 80 79 80 58

0.60 0.85 1.14 0.29 0.65 1.48 0.98 0.61 0.74

580 650 580 730 490 600 620 520 490

490-650 pg/mg (Table 2) but lower than that of the initial monomers (Table 1).This fact indicates that when polymerization proceeds in an aqueous buffer solution (pH 7.0) at 3°C it is not possible to avoid completely the cleavage of the /?-lactam ring of the antibiotic. The UV spectra of poly-PhOMP revealed a shift in the maximum of absorption bands towards the short wavelength range as compared to their position in the spectra of the initial monomers (Fig. 3) as a result of polymerization. The IR spectra of poly-PhOMP exhibit the following bands: the vc=o band of the /I-lactam ring in the

124

9 48

0.7

5

4

3

02

L 0.6

1.0

as a5 a4 43

02.

L4

c

if/&

Fig. 1. Reduced viscosity of poly-AAPhOMP concentration.

solutions

vs.

Fig. 3. UV spectra in water: 1, potassium salt of pMAPhOMP; 2, potassium salt of m-MAPhOMP; 3, potassium salt of poly-p-MAPhOMP; 4, potassium salt of polym-MAPhOMP.

bulk and in an organic solvent in the presence of AIBN. Monomer penicillins were used in the form of acids. It was found (Table 3) that in the copolymerization under these conditions it is possible to obtain low molecular weight copolymers with [q] = 0.08-0.40 dl/g. However, just as in the case of homopolymerization, marked inactivation of the antibiotic is observed even when the copolymer was isolated at low conversion. The resulting VP copolymers contained not more than 25-30 wt.% of noninactivated penicillin units. Fig. 2. Chromatography of poly-PhOMP on Sephadex G75, column 2x20 cm, eluent 0.1 N NaCI, elution rate 20 ml/h: 1, potassium salt of poly-o-VPhOMP, [q] =0.85 dl/ g; 2, potassium salt of poly-p-AAPhOMP, [q ] =0.65 dl/g; 3, potassium salt of o-VPhOMP.

1780 cm-’ range, the vcso band of the amide group in the 1650-1680 cm-’ range and that of the carboxylate group in the 1390 cm-’ range. In addition to homopolymers, copolymers of monomer penicillins and VP were also prepared. Our purpose was to obtain polymers with less frequent unit arrangement along the chain as compared to that in homopolymers and to establish the effect of the microstructure of polypenicillins on their antimicrobial activity. In this connection, the copolymerization of VP with unsaturated PhOMP was investigated in

Binding of 6-APA to carboxyl-containing copolymers of vinylpyrrolidone

The reaction with 6-APA of VP copolymers IXa,b and X, the carboxyl groups of which had been activated by the transformation into mixed anhydrides with ethoxyacetic acid, resulted in the formation of terpolymers: VP-CA-6crotonoylaminopenicillanic acid (6-CrAPA), VP-P-PhAA-6- cinnamoylaminopenicilXI, lanic acid (6-CAPA), XII, and VP-o-VPhOAAo-VPhOMP, XIII (Scheme 2). Some characteristics of the terpolymers obtained are given in Table 4. The data in Table 4 show that the degree of completion of 6-APA

125 TABLE 3 Copolymerization of VP (M,) and unsaturated penicillins (M,) at 65°C in the presence of 0.5 wt.% of AIBN

in

Ml

Solvent

Copolymer yield ( % )

Copolymerization time (h)

the monomer mixture

Copolymer [~K.GKKCI (dl/g)

Iodometric activity

[WI

(mol%)

(pg/mg)

(mol %) p-MAPhOMP p-MAPhOMP p-MAPhOMP p-MAPhOMP p-MAOx

acetone in bulk DMFA acetone acetone

10 15 20 25 6

0.25 0.10 0.08 0.34 0.40

22 15 36 36 20

5 3 10 8 0.75

-

-CH

19 25 34 41 8.4

-CHr

CH-

CICOOC2H?

I R

found

calcd.

160 150 270 250 300

470 560 640 730 270

CH -CH-

I

I b

4

H2CNN\C=0

(C2H33

X

cl

I I Ii& -CHz

?

I

0-T2H5 -Cl+--CH

-CH-CH

CH -CH

CHz

I H2C/N’C=0 I H2C-cH2

-CH-

I “j& I I HN-CH-CH I ,C0’

A

H2C /N’C=O 1 I H2C CH2

c’+

/ c -CH3

“AJ

b-~~C*H5~I

/d-CO’ \

H

-+ COON(C2H5J3 I

c! XI -XIII XI,

R:CHx,

X=

-C-

,

XIII,RrH,

OCH2-C-

X;

II 0 XII

R=C,5H5,

II

,

0

X=-C-

, II 0

Scheme 2. Binding of 6-APA to carboxyl-containing

copolymers

of VP.

TABLE 4 Terpolymers of N-vinylpyrrolidone Initial copolymer Designation

rll122i KC, (dUg)

WI

(mol%)

Terpolymer yield (%)

Terpolymer Iodometric activity (pg/mg)

S(%)

found

found

calcd.

degree of conversion

calcd.

(%) VP-6CrAPA VP-6-CrAPA VP-6-CAPA VP-o-VPhOAA VP-o-VPhOAA

0.18 0.12 0.23 0.55 0.40

9 20 24 22 35

81 79 77 68 72

180 250 320 310 420

250 450 560 550 690

73.5 56.4 57.0 56.4 60.1

degree of conversion (%)

1.54 2.23

2.12 3.84

72.6 58.2

2.12 2.91

3.68 4.63

57.5 62.8

126

condensation determined from both the data of iodometric analysis of reaction products and the sulfur content in these products was the same. This indicates that in the course of the reaction and isolation of final terpolymers, no hydrolytic cleavage of the /?-lactam ring of penicillin units occurs. The IR spectra of terpolymers exhibit the bands characteristic of carbonyl groups in the /?-lactam (1780 cm-l) and pyrrolidone (1680 cm-l) rings, the band of the COO- group (1390 cm-l ) and the stretching vibrations band of the trialkyl substituted quaternary ammonium ion (2900 cm- ’ ) . Hence, the results of the analysis of the terpolymers show that the method of mixed anhydrides makes it possible to insert 6-APA into the structure of carboxyl-containing VP copolymers. In this case water-soluble terpolymers containing up to 42% of non-inactivated penicillin units may be obtained in 7080% yield. The advantages of this method are, first, its simplicity, because it is not necessary to synthesize unsaturated penicillin derivatives and to polymerize them. Secondly, it is possible to control the molecular weight of polypenicillin in the stage of the preparation of the initial copolymers.

paths of synthesis of these preparations is the insertion of sterically hindered structures into the acyl-side radical of penicillins [ 181. In this connection it was of great interest to evaluate the resistance to penicillinase of polymer penicillins obtained. Figure 4 shows the results of the investigation of the kinetics of inactivation of poly-pMAPhOMP and poly-o-VPhOMP and initial monomer antibiotics by penicillinase of Bacillus licheniformis 749/c at [E] = 4.7~ low7 mol/ 1. It is clear that poly-p-MAPhOMP and polyo-VPhOMP are inactivated by penicillinase much more slowly than monomer penicillins. It should be noted that the low degree of inactivation is not related to the inhibition of the enzyme by high molecular weight penicillins. This was proven by a control experiment (Fig. 4)) which showed that benzylpenicillin (BP) added to the poly-p-MAPhOMP-penicillinase system 50 min after the start of the reaction was inactivated at a rate proportional to the initial concentration of the enzyme. Hence, the complete retention of penicillinase activity in the presence of polypenicillin was confirmed and it was established that the polymer PhOMP is not a penicillinase inhibitor. Its stability to this en-

Enzymatic hydrolysis of polymer penicillins An essential defect of biosynthetic benzyl(1~) and phenoxymethylpenicillins, decreasing their therapeutic effect, is the low efficiency of these antibiotics with respect to resistant strains of microorganisms, the number of which is continuously increasing. At present, 80% of clinical strains of pathogenic staphylococci are resistant to the action of biosynthetic penicillins [ 171. One of the determining factors of the resistance of microorganisms to penicillins is known to be [ 181 the fact that these organisms produce and excrete into the environment specific enzymes, penicillinases (/Llactamases) which inactivate penicillin. Hence, the problem arises of a search for antibiotics of the penicillin series resistant to penicillinase. One of the

20

Fig. 4. Hydrolysis licheniformis [E]=4.7~10-~ ( [q] = 1.48 dl/g), 0.56~ 10e3 mol/l VPhOMP; 3, ( n

40

60

80

400

fW

of penicillins by penicillinase of Bacillus 749/c, [so]=1.5x10-3 mol/l, mol/l at 37°C: 1, poly-p-MAPhOMP 50 min after the start of the reaction of benzylpenicillin was added; 2, poly-o) p-MAPhOMP, (0 ) o-VPhOMP.

127

zyme is due to specific properties of the macromolecule. The rate of hydrolysis of the polymer substrate by penicillinase of Bacillus licheniformis 749/c did not vary greatly when the enzyme concentration was increased. Thus, the extent of hydrolysis of poly-o-VPhOMP with [~]=085dl/ginsolutionat [E]=2~10-~ mol/l was only 28% for 48 h. It is significant that the polymer subsequently isolated from the solution exhibited biological activity. Figure 5 illustrates the kinetics of hydrolysis of the monomer p-MAPhOMP, its copolymer with VP at a composition ratio of 1:4 and [q] = 0.24 dl/g, poly-p-MAPhOMP with [q] = 1.05 dl/g and the oxacillin antibiotic by cereus at penicillinase of Bacillus [E] = 4.8~ lop6 mol/l. Just as in the case of penicillinase of Bacillus licheniformis 749/c, a linear time dependence of the decrease in the concentration of the substrate (both monomer and polymer substrates) is observed. This confirms the zero order of the enzymatic reactions under investigation and makes it possible to calculate their rates (V,). It is clear from Fig. 5 that at [E] = 4.8 x 10e6 mol/l the antibiotic

oxacillin, resistant to penicillinase, is already rate considerable destroyed at a (V,= 2.8~ 10m3 mol/l min). Under the same conditions, the inactivation rate of poly-pMAPhOMP is less by a factor of 3 ( V, = 0.9 x 10m3mol/l min) than that of oxacillin hydrolysis and by a factor of 80 than that of inactivation of the monomer p-MAPhOMP for which V,= 74 x 10e3 mol/l min. Not only the homopolymers of unsaturated penicillins but also their copolymers with VP of much lower molecular weight were found to be resistant to penicillinase. In the above example, the V, of the copolymer of VP andp-MAPhOMP is close to that of the p-MAPhOMP homopolymer and is 1.4 X 1O-3 mol/l min. Consequently, the results of the study on the inactivation of polymer PhOMP by the preparations of pure isolated penicillinases show that the transformation of the monomer PhOMP into the polymer permits a drastic increase in its resistance to penicillinase. This protective effect is also manifested with respect to penicillinases of various types and is due to steric hindrances induced by the main polymer chain and penicillin side fragments. These steric effects hinder the formation of the enzyme-substrate complex and limit the accessibility of the P-lactam ring of polypenicillins to the active centre of the enzyme. Antimicrobial

Fig. 5. Hydrolysis of penicillins by penicillinase of Bacillus cereus, [S,,] =1.5X 10e3 mol/l, [E] =3.2X 1Om6mol/l at 37°C: l,p-MAPhOMP, 2, oxacillin; 3, copolymer of VP-pMAPhOMP (composition 4: 1, [q] =0.24 dl/g); 4, poly-pMAPhOMP ( [q] = 1.05 dl/g).

activity

of polymer penicillins

In order to evaluate the biological activity of polymer penicillins, their antimicrobial properties and acute toxicity were investigated. The values of minimum polypenicillin concentrations inhibiting the growth of Staphylococcus aureus 209 P sensitive to BP are given in Table 5. It is clear that all the polymers synthesized, regardless of their structure and method of preparation, exhibit relatively high antimicrobial activity. The values of MIC found for them are 0.25-16.0 pg/ml. Hence, it was established that the macromolecular nature of the new penicillins does not prevent the maintenance of

128

TABLE 5 Antimicrobial activity of polymer penicillins Preparation

Iodometric activity

[ q]gy,$KCI

MIC

(dl/g)

St. cur. 209P (fig/mg)

hdmd Poly-o-VPhOMP Poly-o-VPhOMP Poly-p-MAPhOMP Poly-p-MAPhOMP Poly-p-MAPhOMP Poly-o-MAPhOMP Poly-p-AAPhOMP Poly-p-MAOx VP-p-MAPhOMP (I)” VP-CrAPA (II)” VP-CAPA

(II)”

Benzylpenicillin

0.25

580 580 590 560 600 550 730 300 180 250

0.60 1.14 0.47 0.80 1.48 1.90 0.20 0.40 0.12 0.10

320

0.23

(0.05)b 0.24

_

(0.08jb 0.03

1000

1.6 4.0 16.0 16.0 16.0 2.0 10.0 8 (1.3)b 0.18

“Method of preparation. bMIC value referred to the antibiotics content of the polymer. TABLE 6 Activity of the VP-p-MAPhOMP copolymer (25 mol.% of PMAPhOMP, [q]iys KCIco.34 dl/g) with respect to staphylococci strains 102, 132, 1235 and 1582 resistant to penicillinase Preparation

VP-pMAPhOMP Benzylpenicillin

MIC (pg/mg)

for staphylococci strains

103

132

1235

1582

1.6 (0.6)” 125

25 (10)” 160

100 (40)” 2500

100 (40)” 2500

“MIC values referred to the antibiotics

content of the polymer.

antimicrobial properties. This suggests that the mechanism of antimicrobial action of polymer penicillins is the same as that of their low molecular weight analogues. It should be noted that although molecular weight virtually does not influence the sensitivity to p-lactamases, it profoundly affects the antimicrobial activity. The antimicrobial activity of polypenicillin decreases with increasing molecular weight. This decrease is due to the decreasing possibility of transport of macromolecules through the peptidoglycan shell with increasing size, because only macromolecules with a molecular weight of lO,OOO-100,000 can

pass through the cell walls of bacteria [ 191. The optimum molecular weight of polypenicillins should probably be (10-70) x 103, [ ~1 =O.l-0.3 dl/g. In this case polymer penicillins can rapidly diffuse through the cell walls of bacteria. Copolymers of unsaturated PhOMP showed a slightly higher antimicrobial activity than did homopolymers. Hence, it was established that the type of distribution of units along the macromolecule chain profoundly affects the antimicrobial activity of polypenicillins. This phenomenon is presumably due to the higher mobility of penicillin units in the copolymer chain, their higher accessibility to the place of action in the bacterial cell and to a decrease in the negative charge of the macromolecule, which facilitates the interaction with the negatively charged cell. Polymer penicillins at low concentrations (1.6-100 pug/ml) affect the staphylococcus resistant to penicillinase and considerably exceed BP in this respect (Table 6 ) . They are effective with respect to both penicillinase, forming strains of moderate resistance (MI& = 125 staphylococcus ,ug/ml ) and h’ig hl y resistant strains ( MICsp = 2500 pg/ml) isolated clinically. Hence, the experiments in which staphylococcus strains resistant to BP were used confirmed the resistance of polymer PhOMP to hydrolytic action of penicillinases detected in experiments in which purified enzymes were used. The acute toxicity of poly-PhOMP was low. For polymers with [r/l = 0.3-0.5 dl/g, the maximum tolerated dose (in experiments on mice with intravenous administration) is 500 mg of live weight.

CONCLUSIONS

This investigation showed that the insertion of p-lactam antibiotics into the structure of a synthetic polymer, either by the polymerization of monomer penicillins or by the bonding

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of 6-APA to carboxyl-containing polymers, makes it possible to obtain polymer penicillins characterized by resistance to P-lactamases and by high antimicrobial activity. This result indicates that it is possible to use synthetic polymers for overcoming the resistance of bacteria to antibiotics.

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ACKNOWLEDGEMENTS

The authors express their gratitude to Prof. N.A. Zaikina, Dr.Sc., G.E. Afinogenov, Dr.Sc,, and J.P. Fomina, Dr.Sc., who investigated the antimicrobial properties of monoand polypenicillins.

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REFERENCES 14 P.M. Blumberg and J.L. Strominger, Interaction of penicillin with the bacterial cell: Penicillin binding proteins and penicillin-sensitive enzymes, Bacterial. Rev., 38 (1974) 291-335. I.M. Frere, M. Leyh-Bouille, I.M. Ghuysen and H.R. Pernine, Interaction between p-la&am antibiotics and exocellular r&D-carboxypeptidase-transpeptidase of Streptomyces R-61, Eur. J. Biochem., 50 (1974) 203214. IL. Strominger, E. Willonghby, T. Kamiryo, P.M. Blumberg and R.R. Jocum, Penicillin-sensitive enzymes and penicillin binding components in bacterial cells, Ann. N.Y. Acad. Sci., 235 (1974) 210-224. B. Lee, Conformation of penicillin as transition stage analog of the substrate of peptidoglycan-transpeptidase, J. Mol. Biol., 61 (1971) 463-469. H. Smith and AC. Marshall, Polymers formed by some p-lactames antibiotics, Nature, 232 (1971) 45-46. SM. Navashin and J.P. Fomina, Manual on Antibiotics, Medistina, Moscow, 1974, pp. 80-97. E.F. Panarin and M.B. Berov, Synthesis of methacryloylamidophenols and methacryloylamidoacetic acids, Zh. Org. Chim., 4 (1968) 824-825.

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E.F. Panarin, E.A. Koltsova and M.B. Berov, Synthesis of acryloyl- and crotonoylaminophenoxyacetic acids, Zh. Org. Khim., 9 (1973) 73-74. E.F. Panarin and G.E. Afinogenov, Macromolecular antimicrobial substances and drugs, Zh. Vses. Khim. Ova. im D.I. Mendeleeva, 30 (1985) 378-386. B.B. Corson, W.J. Heintzelman, Z.H. Shwartzman, H.E. Tiefenthal, R.J. Lokken, J.E. Nickels, Y.R. Atwood and F.J. Pavlik, Preparation of vinylphenols and isopropenylphenols, J. Org. Chem., 23 (1958) 544-549. E.F. Panarin, M.V. Solovskij, M.B. Berov and M.V. Zhukova, Synthesis of unsaturated analogues of phenoxymethylpenicillin, Izv. Akad. Nauk SSSR, Ser. Khim., 10 (1974) 2300-2303. V.A. Kropachev, M.V. Solovskij and G.P. Akulov, Synthesis of N-vinylpyrrolidone copolymers labelled with tritium, Vysokomol. Soedin., 20B (1978) 715717. E.F. Panarin, M.V. Solovskij, 1.1. Gavrilova and M.V. Zhukova, Synthesis and investigation of polymer penicillin derivatives, in: Sinteticheskie Polimery v Meditsine, Fan, Tashkent, 1973, p. 25. E.M. Kleiner, L.B. Seniavina and AS. Khokhlov, Preparation and properties of phenylacetamidoand 6-phenoxyacetylaminopenicillane-3-acetic acids, Khim. Geterotsikl. Soedin., 5 (1966) 701-705. I.F. Alicino, Iodometric assay of natural and synthetic penicillins, 6-aminopenicillanic acid and cephalosporin C, Anal. Chem., 33 (1961) 648-649. E.F. Panarin and M.V. Solovskij, Relationship between the structure and resistance to penicillinase in the series of phenoxymethylpenicillins, Antibiotics, 10 (1971) 882-885. V.V. Smirnov, I.A. Vasilevskaya and S.R. Reznik, Antibiotics (in Russian) Vischa Shkola, Kiev, 1985, p. 181. F.P. Doyle and J.H.C. Nayler, Penicillins and related structures, in: N.Y. Hasper, A.B. Simmonds (Eds.), Advances in Drug Research, London, New York, Vol. 1,1964. R. Scherrer and P. Gerhardt, Molecular sieving by the Bacillus megaterium cell wall and protoplast, J. Bacteriol., 107 (1971) 718-735.