Hydrosoluble Polymeric Drug Carriers Derived from Citric Acid and

Chapter 26 Hydrosoluble Polymeric Drug Carriers Derived from Citric Acid and L-Lysine

Downloaded by NORTH CAROLINA STATE UNIV on May 4, 2013 | http://pubs.acs.org Publication Date: July 18, 1991 | doi: 10.1021/bk-1991-0467.ch026

J. Huguet, M. Boustta, and M. Vert L.S.M., U R A Centre Nationale de Recherche Scientifique 500, INSA de Rouen, B P 08, 76131 Mont-Saint-Aignan Cedex, France

New functional polyamides with carboxyl alcohol pendent groups were synthesized from two metabolites, namely c i t r i c acid and L - l y s i n e . These water-soluble macromolecules were obtained by step-growth polymerization using the interfacial method applied to protected L - l y s i n e and c i t r i c acid, the latter being in its diacyl chloride form. It is shown that a side reaction occured during the polycondensation with the formation of intramolecular cyclic imide groups. Chemical modification due to the cleavage of the protecting groups and the hydrolysis of the imide rings are discussed on the basis of IR and CNMR spectra. The imide rings were used advantageously to couple low molecular primary amine compounds. Resulting macromolecular conjugates were soluble in water at neutral pH regardless of the degree of substitution. 13

The interest of bioresorbable polymers is growing very fast in the field of therapy f o r both surgery (1) and drug delivery (2). Among the various systems which are presently studied to achieve controlled drug delivery such as implants, micro and nanoparticles, polymerized liposomes, hydrophobic microdomain-forming copolymers and macromolecular drugs or prodrugs, the latter basically require water-soluble bioresorbable macromolecules to act as drug c a r r i e r s (3). By bioresorbable polymers, we understand degradable synthetic polymeric systems which can yield low molecular weights degradation products eliminated from the body through natural pathways. One of our rationales f o r the synthesis of such polymers aiming at biomedical or pharmacological applications is to start from metabolites. At the moment, the most popular bioresorbable polymers are the aliphatic polyesters, especially those derived from lactic or glycol ic acids, two metabolites of the glycolic pathway. These polyesters are very attractive compounds to achieve diffusion-controlled and / or 0097-6156/91/0467-0405$06.00/0 © 1991 American Chemical Society

In Water-Soluble Polymers; Shalaby, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

406

WATER-SOLUBLE POLYMERS

degradation-controlled drug delivery because the physical, physico-chemical, mechanical and biological properties of resulting devices can be adjusted through chirality and enantiomeric distributions or by copolymerization to introduce achiral units as glycolic ones in polylactic chains (4). However, none of the members of this family is water-soluble. A few years ago, a route was found to make a water-soluble degradable polyester derived from a metabolite of the Krebs cycle, namely malic acid. This hydroxyacid metabolite is trifunctional and has to be monoprotected to allow the synthesis of linear macromolecules. Accordingly, benzyl malolactonate was synthesized and polymerized whereas Pd-charcoal catalyzed hydrogenolysis of protecting groups yielded p o l y ^ - m a l i c acid) -[-0-CH(C00H)-C0-] whose polymer chains have racemic or optically active


n

repeating units with pendent carboxylic acid groups (4-6). It has been shown that racemic poly(£-malic acid) is soluble in water regardless to pH, is non toxic when given i.m. or i.v. to mice (7) and does degrade in vitro to yield malic acid as ultimate degradation product (8). The coupling of low molecular weights amine or alcohol compounds was shown feasible through the pendent carboxylic acid group (9). However, a major drawback of such coupling is that water-insolubility comes up rapidly when the resulting new amide or ester pendent groups are hydrophobic. This occurs for no more than 20% substitution and then limit the possible load if water-solubility had to be preserved (10). The introduction of more than one hydrophilic ionic or non-ionic groups per repeating unit can reasonably provide a means to retain water-solubility even for systems with the attachment of one drug molecule per repeat unit. A few years ago, we became interested in developping linear water-soluble, polymeric drug c a r r i e r s not only degradable and derived f r o m metabolites but also allowing high degree of hydrophobic compound attachment with retention of water-solubility. One possibility was offered by multifunctional hydroxyacids of the Krebs cycle and we selected c i t r i c acid as a source of polymerizable monomers. In a f i r s t approach, we tried to make multifunctional aliphatic polyesters by ring opening polymerization of heterocyclic compounds derived from c i t r i c acid. Two derivatives were investigated : β-citrolactone dibenzyl ester, a β-lactone type monomer and citride tetrabenzyl ester, a dioxane dione type monomer (the term citride being used by reference to lactide and glycolide compounds). Unfortunately, these cyclic monomers appeared as non polymerizable so f a r and thus we have looked for other ways to make biodegradable polymers derived from c i t r i c acid (11). As an alternative, we selected the route of step-growth polymerization of the A A - B B type starting from citric acid as a diacid and from L - l y s i n e as a diamine comonomer. In this paper, we wish to report the synthesis of the resulting new family of functional polyamides bearing carboxyl and alcohol pendent groups (12).

Results and Discussion Monomers

synthesis

In order to achieve proper polycondensation, we have used partial protection of the α-hydroxy part of the tetrafunctional c i t r i c acid and of the carboxylic acid group of the trifunctional L - l y s i n e to make both compounds bifunctional.


26.

Drug

HUGUET ET AL.

Carriers

Derived

from

Citric

Acid

and L-Lysine

407

The diacyl chloride type-AA monomer was obtained in two steps f r o m acid or 2-hydroxy 1,2,3-propanetricarboxylic acid according to following reactions : 1

CH -C0 H 21 2

2

0

>

H0-Ç-C0 H 2

I

ru _rn u CH -C0 H

D_rw-_n R-f*-

RCHO

2

2

s o c

,

>

W A

J

R-ÇH—0 Τ \

2

Vu - c o H

CH -C0C1 2

r\

I 0

citric the

—S

C H

2-coci

0

R = -CCI 3 c i t r o c h l o r a l (13) or R = - C H citrobenzal (14)


B

S

R = -CC1 R = -C H B

S

3

I 2

T r i f unctional L-lysine was protected by esterification of the carboxylic group with benzylalcohol tobecome bifunctional. with p. toluene sulfonic acid to yield the L - l y s i n e benzylester ditosylate 3 (15). H N-(CH )^-CH-NH 2

2

C0 H 2

2

+ C H - 0 H + 2 TosH 2

C H B

> T o s , N H f - ( C H ) - C H - N H f , Tos® G

2

u

C0 -CH -C H

S

2

2

B

s

3 L - l y s i n e benzylester

ditosylate

Interfacial polycondensation Interfacial polycondensation of 1 or 2 with 3 were carried out in non miscible water/organic solvent mixtures under vigorous stirring. The reaction time was never more than 10 minutes. The use of a dichloromethane / dichloro-1,2 ethane mixture as the solvent medium instead of benzene was ineffective to increase molecular weights. The highest values of GPC molecular weights ( M ^ 20000 in G p c

dioxane with respect to polystyrene standards) were obtained with an excess of 10 to 20 % of diacyl chlorides. By reacting the protected diacyl chlorid with the protected diamine, the corresponding polyamide-type polymer was expected, i.e. a polyamide with the initial protecting groups, namely the oxolactonic group f o r c i t r i c units and the benzyl ester one f o r L - l y s i n e units according to reaction I.

PLCAIp


408



Actually, we have found that interfacial polycondensation yielded a largely imidified hydroxylated polyamide imide polymer with a small content of residual oxolactonic groups. As the polycondensation reaction was carried out in a non miscible alkaline water / organic solvents mixture, we concluded that oxolactonic ring was cleavable in this rather alkaline medium. The liberated hydroxy acid group was then available to react with newly formed amide bond to yield intramolecular imide group (reaction II). The crude copolymers namely PLCAIp appeared as yellowish solids and were soluble in many usual organic solvents such as CHCl^, acetone, dioxane, T H F , DMF and insoluble in water, alcohols, benzene and diethylether. The presence of imide rings was indentified by IR spectrometry. FTIR spectra of these crude polymers exhibited 6 bands in the 1500-1900 cm zone (figure 1). The 3 bands located at 1550, 1660 and 1740 cm were assigned to the >NH (amid II), >C=0 (amid I) vibrations of amide groups and to the >C=0 of benzyl ester group respectively. The band located at 1820 cm characteristic of the υ >C=0 vibration of oxolactonic ring largely disappeared whereas two unexpected bands were detected at 1710 and 1780 cm . These bands were assigned to the >C=0 vibration of cyclic imide ring as refered to polysuccinimide (16,17). In the case of the crude polymer derived from citrochloral dichloride, the_ two bands corresponding to - C C l ^ group usually located at 820, 860 cm were found very much decreased too. Hydrogenolysis of protecting groups In order to liberate the carboxyl protected groups of L - l y s i n e units, we have used successfully the Pd-charcoal hydrogenolysis method. Hydrogenolysis carried out in N-methyl-pyrrolidone at 60°C, yielded the polyamide imide copolymers (PCLAI,H), bearing carboxyl and hydroxyl hydrophilic pendent groups as shown in reaction III.

PLCAl,

Resulting PLCAI,H was found soluble in methanol, DMF, DMSO and insoluble in acetone, dioxane, ethers, chlorinated and aromatic organic solvents. In water, the solubility of PLCAI,H was found to depend on the proportion of imide groups whereas its Na salt form obtained by neutralization with sodium hydroxide was totally soluble. The FTIR spectra of the copolymers PLCAI,H and PLCAI,Na in the 1500-1900 cm zone are shown in figure 2. The benzyl ester band located at 1740 cm is no longer present. We also found that the typical 1810 cm



26.

0

Drug

HUGUET ET AL.

Carriers

Derived

I

ι

ι

ι

ι

4000

3500

3000

2500

2000

from

ι 1750 cm -1

Citric

ι 1500

Acid

and L-Lysine

1 1250

Figure 1. FTIR spectrum of PLCAIp corresponding polycondensation of citrochloral dichloride and protected

409

1

1

1000

670

to interfacial L-lysine.


410



Figure 2.

FTIR spectra of PLCAI.H (-

- - -)

and P L C A . N a (


).

26.

HUGUET ET AL.

Drug

Carriers

Derived

from

Citric

Acid

and L-Lysine

411

band due to oxolactonic ring is no longer detectable in the IR spectrum whereas the two imide bands located at 1780 and 1710 cm are still present. Therefore, we concluded that hydrogenolysis cleaved not only the benzyl ester protecting groups but also the residual oxolactonic ones. The IR bands of remaining imide groups were particularly visible in PLCAI,Na because of the shifting of carboxylic >C=0 vibration f r o m 1715 cm to 1600 cm by neutralization with sodium hydroxide.


Hydrolysis of imide functions It is well known that cyclic imides are stable in acidic medium but react in basic medium (18). The 100 % alkaline hydrolysis of cyclic imide groups present in PLCAI,H copolymers yielded the sodium salt of poly (L-lysinecitramide) (PLCA,Na), the initially desired multifunctional polyamide. However, because of the detour through the imidified copolymers and because of the two possible ways to open the non-symmetric imide cycle, namely a and b, the actual PLCA chains include two types of L-lysine / citric acid enchainments, namely succinic and glutaric types corresponding respectively to (a) and (b) openings in reaction IV, with two different side chains, respectively carboxy and carboxymethylene.

The acidic derivative of PLCA,Na (PLCA,H) was obtained by ion-exchange on a cationic resin in the H form. PLCA, H appeared as a white solid which was soluble in organic solvents such as alcohols, DMF, DMSO and also in water regardless of pH. Compounds with molecular weights in the 20000-60000 daltons range (as determined by S A L L S ) can be easily achieved. In figure 3, comparison is made of the IR spectra of the initially imidified copolymer (H form) and of a functional polyamide obtained after complete hydrolysis. The IR spectrum of the latter clearly shows 3 main bands in the 1500-1800 cm region assigned to >C=0 carboxylic groups at 1730 cm and to amide groups at 1650 cm (amide I) and 1550 cm (amide II), the two imide bands visible in the imidified copolymer spectrum having totally disappeared. The C NMR spectrum of the sodium salt form of the poly (lysine citramide) was recorded in D^O (fig. 4). Seven signals were observed 1

between 20 and 80 ppm and assigned to the various main chain carbon atoms. Five singulets were correlated to the five carbon atoms of lysine unit : C^ for the asymétrie methine at 55.3 ppm and C , C , C 7

f i

q

and C}C)

f o r the four


412



Figure 3.

FTIR spectra of PLCAI,H (

- -)

and P L C A , H (


).

26.

HUGUET ET AL.

Drug

Carriers

Derived

from

Citric

Acid

and

413

L-Lysine


Ci.

Ci •

c

j m

n

11 M

Il j

j 1111

160

M n

ι j

» j n

11 M 1 1 1 j n

60

Figure 4.

U L

n

11111

j 111 M ι fi ι j ι η

S 0 1 3

3

i

ι i l 111 j 1 1 1 1 1 1 M ι j 1111

40

il » il j II

20

C NMR spectrum of P L C A , N a in D 0 . o


414

WATER-SOLUBLE P O L Y M E R S

methylene carbon atoms at 28.1, 22.6, 31.3 and 39.2 ppm. The last two signals located at 44.5 and 74.7-75.2 ppm were respectively assigned to the quaternary and C ^ - C ^ methylene carbon atoms of the c i t r i c unit. Structured resonances were also detected and assigned respectively to carbonyl atoms C^, C of amide groups at 172-172.8 ppm and to side chains 5

carbonyl atoms C

, C

at 176-177 ppm.


Self-coupling of amine-tvpe compounds to PLCAI polymers The coupling of amine-type compound can be basically achieved via two different routes : by using coupling reagents which can react with the pendent carboxylic acid groups of citric o r / a n d L - l y s i n e units or by self-coupling on the cyclic imide groups present in the polymeric intermediates. We have tried to directly couple some primary amines such as 2-aminobutane, 1-phenylethylamine and amphetamine to PLCAI,H under the same conditions reported by Saudek et a l . f o r poly succinimide (18). The coupling of amphetamine (Amp) to a 100 % imidified copolymer was carried out in hot DMF solutions with an excess of amine. Because of the two possibilities of ring opening, two isomeric amid-acid conjugates were actually obtained (reaction V).

H N-CH-CH 2

CH I C H

-NH-ÇH-(CH )-I

3

2

C0 H

2

B

2

S

V (a) „

ÇH -C I

(b)

2

B

C0-NH-CH-CH ..OH.

b

2|

- f - N H - Ç H - (CH ) - N H - C - C H - Ç - C H - C 2

C0

2

Amp

2

2

Ï

0

1 X ο)

NH-CH-CH. I C H - C 6H n,s 2

ÇH

ι Θ co 2

Amp

3

2

-Ç-CH 2

OH

e

G

The final amount of the covalently bound amino derivative was deduced f r o m the C NMR spectrum (fig. 5) of the sodium salt of P L C A Amp. From the two peaks corresponding respectively to the asymétrie carton atoms of L - l y s i n e unit (C^ at 57 ppm) and of amphetamine {C^ at 49 ppm), we have found that 100 % amphetamine was covalently bound to polymer chains. In spite of this high degree of substitution which corresponds to one attached amphetamine per each citric unit, the resulting polymer chains were still soluble in water because of the remaining hydroxyl and carboxylate hydrophilic groups. Conclusion We have shown that it is possible to make highly hydrophilic water soluble drug c a r r i e r s by polycondensation of metabolites such as c i t r i c acid and L - l y s i n e after protection of some of the functional groups present in these molecules. In the course of polycondensation reaction, intramolecular imid groups are formed which can be used to couple directly primary amine compounds by avoiding the use of coupling reagents. High degree of coupling


H U G U E T E T AL.

Drug

Carriers

Derived

from Citric

Acid

and

L-Lysine


26.

Figure 5. C NMR spectrum in the 45-80 ppm range of amphetamine coupled to PLCAI in D O .


415

416


can be achieved without losing the water solubility. Now, it is attempted to know whether such polylysinecitramide which carries breakable bonds in the main chain can be biodegradable. Experimental part Typical interfacial polycondensation L-lysine

benzylic

ester

ditosylate

(7.6 g),

CO Na

(7.45 g)

and

dodecyl


3 sodium sulfate (0.5 g) were dissolved in water-benzene mixture (60 cm , V/V, 66/33) in a Waring type mixer and s t i r r e d at 12-15000 t / m n . Citrobenzal dichloride (4.54 g) dissolved in benzene (40 cm ) and CO^Na^ (8.2 g)dissolved in water (20 c m ) were added simultaneously. F i r s t , 90% of these two solutions were introduced within 6 minutes. The reaction was allowed to stir f o r 2 minutes and then, the remaining 10% were added within two m i n . . The polymer precipitated as a yellow solid during the reaction. The medium was then acidified with dilute HCI. The polymer was washed with water and dried under vacuum at 50°C. 5.1 g of PLCAIp were recovered (yield « 83%). 3

Catalytic hydrogenolysis of PLCAIp PLCAIp (5 g) dissolved in N-methyl pyrrolidone and 10% Pd-charcoal catalyst (1 g) was allowed to s t i r under hydrogen atmosphere at 60°C until no more H ^ was absorbed. After 24 h . , the catalyst was f i l t r a t e d and the solvent evaporated to yield a slightly colored paste. The crude compound was dissolved in methanol, precipitated by ether and dried. 4.1 g of PLCAI,H (white solid) were recovered (yield » 84%). Hydrolysis of imide functions

3 PLCAI,H (4 g) were dissolved in aqueous 0.7 Ν NaOH (180 cm ). The mixture was allowed to stir f o r 45 min. at room temperature. The basic solution was then diluted with water and dialyzed against neutral water through semipermeable dialysis tube (Wiskase type - cut off 6-8000). The final solution was concentrated, f i l t r a t e d through a 0.45 μπι Millipore f i l t e r and finally freeze-dried. 3.1 g of P L C A , N a were recovered (yield ^ 60%). Coupling of amphetamine to PLCAI.H PLCAI,H (1.5 g) (100% imide rings with respect to c i t r i c acid repeating units), amphetamine (19 cm ) and DMF (2 cm ) were heated f o r 3 days at 60°C. The amine excess was evaporated under _vacuum. After solubilizing the resulting polymer in aqueous NaCI M (100 cm ), the solution was dialyzed against neutral water through a Viskase tube (cut off 3500). The polymeric solution was then concentrated and percolated through an exchange cationic resin (Na form), and then, freeze-dried. A brownish solid (1.6 g) containing 100% of coupled amine (as evaluated by C NMR) was recovered (yield * 65%).


26.

Drug

HUGUET ET AL.

Carriers

Derived

from

Citric

Acid

and

L-Lysine

417

IR spectra IR spectra were recorded with a PERKIN-ELMER 1760 Molecular weights

FTIR.

determination


Molecular weights of PLCAIp in dioxane were evaluated by GPC using a Waters apparatus equipped with μ-styragel columns and refractive index detection. Data are expressed with respect to GPC peaks in reference to polystyrene standards. For P L C A , H examples, molecular weights were evaluated by laser light scattering by using a Malvern SALLS diffusiometer. NMR spectra 62.8 Mhz C NMR spectra were recorded at 30°C by using a BRUCKER WH250 (ESSO Research Center-Mont-Saint-Aignan - FRANCE). 1 3

Acknowledgements Authors are indebted to Mr. Plaindoux and the 13 contribution to the recording of C NMR spectra.

Esso

Co.

for

their

helpful

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Vert, M.; Christel, P.; Chabot, F.; Leray, J. In Macromolecular Biomaterials; Hastings, G.W.; Ducheyne, P., Eds.; CRC Press: Boca Raton, 1984; p 117. Vert, M. In Polymers in Controlled Drug Delivery; Illum, L.; Davis, S.S., Eds.; Wright: Bristol, 1987; p 117. Vert, M. In Therapeutic Drug carrier Systems; Bruck, S.D.,Ed.; CRC Press: Boca Raton, 1986; p 291. Vert, M.; Lenz, R.W. Polym. Prep. 1979, 20, 608. Guerin, P.; Vert, M.; Braud, C.; Lenz, R.W. Polym. Bull.1985, 14, 187. Arnold, S.C.; Lenz, C.W. Makromol. Chem., Macromol.Symp. 1986, 6, 285. Braud, C . ; Bunel,C.; Vert, M . ; Bouffard, Ph.; Clabaut, M.; Delpech,B. Proc. IUPAC 24th Int. Symp. Macromolecules, Amherst, 1982, p 384. Braud, C.; Bunel, C.; Vert, M. Polym. Bull. 1985, 13, 293. Bunel, C.; Vert, M. IUPAC 24th Microsymp., Prague, 1983, 52. Braud, C.; Vert, M. In Polymers as Biomaterials; Shalaby, S.W., Hoffman, A . S . ; Ratner, B.D.; Horbett, T . A . , Eds.; Plenum Press: New-York, 1984; Ρ 1 . Boustta, M. Ph. D; Thesis, Rouen University, France, 1988. Boustta, M.; Huguet, J.; Vert, M. Br. Fr. PV 88-02956. Boesenken, J. Versl. Akad. Wetenshap. 1926, 35, 1084. Eggerer, H., Liebigs Ann. Chem. 1963, 666, 192. Izumiya, N.; Makisumi, S. J. Chem. Soc. Japan, 1957, 78, 662. Adler, A.J.; Fasman, G.D.; Blout, E.R. J. Am. Chem. Soc. 1963, 85, 90. Rodriguez-Galan, Α . ; Munoz-Guerra, S.; Subirana-Buichong, J.A.; Sekiguchi, H. Makromol. Chem., Macrom. Symp., 1986, 6, 277. Kovacs, J.; Kovacs, H.N.; Konyves, I.; Czaszar, J.; Vajda, T.; Mix, H. J. Org. Chem., 1961, 26, 84. Vlasak, J . ; Rypacek, F.; Drobnik, J.; Saudek, V. J. Polym. Sci., Pol. Symp. 1979, 66, 59.

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Hydrosoluble Polymeric Drug Carriers Derived from Citric Acid and

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