AU690573B2 - Process for the preparation of colon specific reduction sensitive polymers - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: 0*

S

Name of Applicant: Danbiosyst UK Ltd Actual Irventor(s): Etienne Schacht Ian Wilding Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: PROCESS FOR THE PREPARATION OF COLON SPECIFIC REDUCTION SENSITIVE POLYMERS Our Ref 385705 POF Code: 1554/239077 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- PROCESS FOR THE PREPARATION OF COLON SPECIFIC REDUCTION SENSITIVE POLYMERS The present invention relates to a process for the preparation of specific reduction sensitive polymers selected from the group consisting of azo-containing polymers, (ii) disulfide-containing polymers, and (iii) azo- and disulfide-containing polymers, the process comprising the steps of copolymerizing an azo- and/or disulfide-containing a,w-difunctional reagent by polycondensation or polyaddition with a suitable a,w-difunctional comonomer.

The process according to the invention has for purpose the preparation of azo- and/or disulfide-contai- 15 ning polymers which can be completely degraded or decomposed into monomeric units by bacteria in the reductive medium of the human colon intestine after a short incubation period. Said polymers are useful for manufacturing tablets and capsules intented for a site specific drug 20 delivery in the lower part of the gastrointestinal tract.

From W091/11175, a process is already known for preparing colon specific reduction sensitive polymers selected from the group consisting of azo-containing polymers; 25 (ii) disulfide-containing polymers, and (iii) azo- and disulfide-containing polymers.

Said process comprises the step of copolymerizing an azo- and/or disulfide-containing a,w-difunctional comonomer according to the general reaction scheme illustrated below n Y-Ri-R-XX-R-RY n HX-R 3 -XH Y-Ri-R-XX-R-R 2

-Z-X-R

3

-XH

mmwith -XH -OH -Y -COOH, CO-Hal, COOAlkyl, -N=C=O, -CH-CH(R), -SOHal 0 -XX- -S-Sand R alkyl, aryl R R, alkyl, aryl, alkylaryl groups optionally substituted

R

3 alkylidene, arylidene, alkyla- rlidene optionally substituted 15 polyether, polyester hal halogen radical, e.g. Cl, Br with -Y H-X- -Z-Xand Z C=O, CH 2 -CH-OH, SO, and whereby X and Y are interchangeable in the above formulas.

25 Said process allows the preparation of a multitude of azo- and/or disulfide-containing polymers through a variation in the a,w-difunctional reagents. The final physicochemical and physical properties (hydrophilicity, permeability, thermal properties, rheoligical properties) can be widely varied.

The molecular weight of the resulting polymers is advantageously determined by gel permeation chromatography. It can be altered by adjusting the degree and type monomers used in the reaction mixture.

The molecular weight of the polymers prepared by the process of the invention is adjusted by modifying c. L. the molar ratios of the functional groups reacting with one another. For high molecular weight polymers equivalent amounts of reactive functional groups should be used.

It is well known to those skilled in the art that molecular weights for polymers must be above about 1000 to avoid adsorption in the gastrointestinal tract and that polymers for use in the gastrointestinal tract can have average molecular weights up to 10,000,000. (See U.S.Patent No. 4,298,595, Col. 25, lines 14-33).

The polymers are especially appropriated for preparing specific drug delivery systems. The latter can be matrix type systems or reservoir type systems. In the S: former case the azo- and/or disulfide-containing polymer is a major part of the drug containing compartment. In the latter part the azo- and/or disulfide-containing polymer is used to encapsulate a drug loaded core.

Owing to the property that azo- and/or disulfide polymers are stable in the fluids of the mouth, 20 the stomach and the upper intestine the polymers according to the invention can pass the mouth, stomach and upper intestine without being destructed. In the lower S. part of the gastrointestinal tract, these reduction sensitive polymers are too slowly cleaved and fragmented by S 25 the reductive medium, all or not enzym mediated, ,Go that enclosed active agent is not completely released at the site of degradation.

Several segmented disulfide-containing polyamides and hydrophilic groups suitable for reductive cleavage were prepared by polycondensation of 3,3'dithiobissuccinimidylpropionate and a, w-amino-terminated poly(tetramethyleneoxide) or tetraethyleneglycol diamine (JEFFAMINE EDR-192). Polymer films were incubated in a ml sample taken from a SHIME reactor, t.m. Simultating Human Intestinal Microbial Ecosystem, which is simulating the microbial contents of human colon.

Also, segmented polyurethanes comprising disulfide and hydrophilic groups intended for the preparation of capsules which specific dry delivery were synthetized by reaction of 4,4'-diphenylmethane diisocyanate (MDI), an a,w-hydroxy-terminated prepolymer and a disulfidecontaining chain extender. "s hydroxy-terminated prepolymers were used poly(tetramethyleneoxide), poly(propyleneoxide), polycaprolactone, poly(ethyleneoxide), poly- (ethyleneoxide/polypropyleneoxide).

In a same manner, segmented polyurethanes comprising azo-aromatic and hydrophilic groups were synthesized by reaction of m-xylylene diisocyanate with a mix- S; ture of m,m'-dihydroxyazobenzene, poly(ethylene glycol) S 15 and 1,2-propanediol. A hydrophilic drug (FOY-305) was enclosed in a capsule made of these polymers or coated with these polymers. The capsules or coated pellets were incubated in a SHIME-reactor reproducing a culture of human intestinal flora. Samples of reduction sensitive S 20 polymers are taken over time at measured intervals. The degradation in molecular weight is observed by performing gel permeation chromatography on the samples. Although the drug was released from these pellets, the molecular weight did not decrease substantially. Hence there was no S 25 degradation of the main chain azo groups to amine groups.

A series of azo-containing polyamides were prepared by polycondensation of an azo-containing diacid chloride with an c.igomeric a,w-diamine. The polymers were incubated in an in-vitro reductive medium. A plurality of tests have indicated that numerous azo-containing polymers become stickly and colourless during incubation in a SHIME-reactor simulating a culture of human intestinal flora. Changes in structure were monitored by 3C, Raman, UV and GPC analysis. The observed discolouring of these polymers is not necessarily due to degradation, but RA4/ No o ~p-nrP- -a-r I can be due to partial reduction of the azo to the hydrazo form.

The thermodynamic conditions for the reductive cleavage of the azo polymers were determined by voltametric measurements.

It was shown that for the hydrophilic azo-containing polyamide, incubation in the reductive medium led to degradation of the azo bond with formation of amines.

The molecular weight of the azo-polymers does not decrease substantively. The partial reduction of azoto hydrazo-group occurs slowly and there is no degradation of the main claim azo groups to amine groups.

Duration incubation, the polymers become stickly and colourless. During reoxidation, the original 15 colours reappears and the molecular weight raises to the initial value.

These experiments indicate that for this type of polyamide no degradation of the azo-bond to aminogroups occurs. Changes in colour and mechanical proper- 20 ties are due to a conversion of the azo- to the hydrazogroups. These finding are in good agreement with those of Kimura et al. in EP-A-0 398 472. They have shown in an article published in Polymer 33(1992)5294-5299 that by incubation of azo-containing polyurethanes in a culture S. 25 of human intestinal flora, no molecular weight decrease occured. As from these results, they concluded that there was no degradation of azo groups to amines, only to hydrazo groups.

The present invention has for subject matter a process whereby the thermodynamic conditions for the reductive cleavage of azo- or disulfide-containing polymers is improved by incubation of said polymers in a in-vitro reductive medium.

The present invention proposes a process for the preparation of colon specific reduction sensitive polymers selected from the group consisting of qL- PCI~-~ azo-containing polymers; (ii) disulfide-containing polymners, and (iii) azo- and disuif ide-containing polymers, said process comprising the following successive steps A] copolymerizing an azo- and/or disuif ide-containing c~a,w±-difunctional reagent by polycondensation or polyaddition with a suitable a,w-difunctional comonomer according to the general reaction scheme illustrated -below OV.n Y-RI-R-XX-R-R,Y n HX-R 3

-XH--

Y-R-R-:<X-R-R

2

(-Z-X-R

3

Z-R

1

-R-XX-R-R

2 3 3

-XH

20 WITH HXR-H.sahyrpilc.mooe *with -X O COC.Hl 4~kl CHC() Sa *9

-NH

1

-HS

and R alkyl, aryl

R

1 R2 alkyl, aryl, alkylaryl groups optionally substituted by methoxy-groups alkylidene, arylidene, alkylarylidene all or not substituted /i R iA4 polyether, polyester hal =halogen radical, e.g. Cl, Br with -Y H-X- -Z-Xand Z C=0, CH,-CH-OH, SO, X and Y being interchangeable in the above formulas, B] Reduction of the copolymer formed in step A in an invitro reductive medium, and C] Partial reoxidation of the copolymer reduced in step

B.

According to a particularity of the invention, the process comprises the 15 copolymerization of dithio bis-(succinimidyl propionate) with a hydrophilic a, o- .difunctional comonomer.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is Snot intended to exclude other additives or components or integers or steps.

The reduction sensitive polymers are prepared according to the methods described hereunder, Description of the starting materials Jeffamine ED-600 (Mn=600) is a commercial product purchased from Texaco Chemical Company (Austin, USA). The polymer is dried by azeotropic

S

removal of water from toluene solution.

The amine content is determined by titration Jeffamine ED-600 2.96 meq/g.

CeOIERVAEISSAWUIV4OELEESPECI%7MO4-4.lOC Azobenzene-4,4'-diacid chloride was prepared from the corresponding azobenzene-4,4'-dicarboxylicacid as described in the literature All other reagents were obtained from Janssen Chimica (Beerse, Belgium).

The chloroform was washed with sulfuric acid and water and subsequently dried over calciumhydride.

Tridtylamine was purified by reacting with tosylchloride and ninhydrin, respectively.

It was finally dried over calciumhydride.

Methods IR spectra were recorded on a Beckmann IR 4230' apparatus. UV spectra were measured on a Kontron Uvikon 810 double beam spectrophotometer.

'H-NMR spectra were recorded using a 360 MHz Bruker WH-360 apparatus.

13 C-NMR spectra were obtained at 100 MHz on a Varian Unity-400 spectrometer using a 5 mm broadband probe.

The molecular weight of the polymers was determined by gel permeation chromatography. A PL gel mixed D column was used with chloroform as eluent. For calibration, polystyrene standards were used.

FT-Raman spectra were recorded using a Bruker IFS 66FT IR spectrometer equipped with a FRA 106 FT Raman module.

Voltametric measurements were carried out on a EG&G Princeton Applied Research Potentistat/Galvanostat Model 273 using a Pd electrode as working electrode and a saturated calomel electrode (SCE) as reference electrode.

Viscosity was measured using a Ubbelohde viscosimeter (Haake).

The preparation of a polyamide starting from Jeffamine ED-600 and azobenzene-4,4'-diacid chloride L, td~l is achieved according to a method described by M. BALASUBRAMANIAN et al., in Makromol. Chem. 180 (1979) 2517-2519. The 10 g (14.80 mmol) of Jeffamine ED-600, dissolved in 100 ml dry, alcohol free chloroform, was placed in a three necked flask and stirred under nitrogen atmosphere. To this solution 7 ml (50 mmol) tridthylamine was added. The mixture was stirred for 15 min at -10 0

C.

Then 4.4 g (13.03 mmol) azobenzene-4,4'-diacid chloride, dissolved in 100 ml, dry ethanol free chloroform, was added at once at the reaction mixture. This mixture was stirred for 15 minutes at -10 0 C and then allowed to reach room temperature. Stirring was continued for 24 hours.

The reaction mixture was extracted with 0.1 M HC1 (3 x 150 ml), water, 0.1 M NaOH (3 x 150 ml). The chloroform layer was dried over MgS0 4 filtered and evaporated to dryness. The resulting polymer was obtained as en orange coloured waxy solid.

iH NMR spectroscopy (CDCl 3 360 MHz) 1,1 ppm CH 3 7 of PPG ether); 6 1,35 ppm CH 3 3 of PPG ether); 20 6 3,65 ppm CH, a of PEG ether); 6 3,8 ppm CH f of PPG ether); 8 4,4 ppm (broadened, N-H amide); 6 7,95 ppm aromatic protons).

IR-spectroscopy (film on KBr discs) u 3300 cm N-H stretching vibration, u 1650 cm' C=O stretching vibration, u 1540 cm" N-H bending vibration, u 1600 cm C-H aromatic stretching vibration, u 1100 cm"' C-O stretching vibration.

Casting of films A concentrated solution (ca. 30%) of the azo polyamide in chloroform was casted on a siliconized glass plate. After evaporation of the solvent in a hot air ventillated oven, the film was removed from the glass plate.

Preparation of in-vitro reductive medium A solution of sodium phosphate buffer (0.25 M, pH 6.5) was boiled and further cooled under an atmosphere of Argon. While stirring, sodium sulphide and cystein are added to give concentrations of 50 mM for each.

The redox potential of this medium, determined with a Pt redox-electrode, was -430 mV 10 mV) and remained constant for at least 4 days. The reductivity of this medium was assayed by incubation of sulfalazine. The formed 5-ASA was determined qualitatively and quantitatively by HPLC analysis.

Incubation of films in chemical reductive medium An azo polyamide ca. 0,1 g (tickness 100 Am) was incubated in a solution of 30 ml reductive medium for periods from 3 hours varying to 2 days. At regular time intervals, films were removed from the medium, washed with water, dried in vacuo and analysed by GPC. FT-Raman and 3 C-NMR measurements.

Incubation of films in simulatin human intestinal medium

SHIME)

An azo polyamide ca. 0.05 g (thickness 100 mm) was incubated in 15 ml batch medium of a SHIME- 20 reactor for periods from 12 hours varying to 3 days. The SHIME-reactor consists of 5 vessels which represent the ileum (2 vessels) and the colon (3 vessels). The redox potential -250 mV 50 mV) is daily measured with a Pt redox-electrode. The incubation flasks were kept at 37 0

C.

25 At regular time intervals, films were removed from the medium, washed with water, dried in vacuo and analysed by S. GPC, Raman and '1C-NMR measurements.

Results and Discussion I) Synthesis of co-polyamides containing disulfide The disulfide-containing polymers were prepared by polycondensation of 3,3'-dithiobissuccinimidyl propionate and a,w-amino terminated prepolymer.

3,3'-dithiobissuccinimidyl propionate (DDSP) was synthetized according to the following reaction schema

I,,

HOOC- (CHW) 2

,-COOH

0 dicyclohexylcarbonate N-OH dioxane

O

00 0 0 II II OCO-(CH ),-S-S-(CH,)2-OCON C C I I O (DDSP) 0 To a solution of 5 gm (23.778 mmol) 3,3'-dithiobissuccinimidyl propionate (DDSP) in 125 ml dry dioxane in 250 ml two neck flask under nitrogen ntmosphere was added a solution of 6.25 gm (54.3 mmol) Nhydroxysuccinimide (NHS) The reaction mixture was cooled to 06C in an ice-acetone bath followed by addition of 11.25 gm (54.52 mmol) dicyclohexylcarbonate (DCC). The 30 reaction mixture was stirred for 1 hour at 0 C and the stirring was continued overnight at room temperature. The precipitated dicyclohexylurea (DCU) was filtered off, washed with dry dioxane, the filtrate was concentrated to Sone/third and was l:.ft overnight in refrigerator.

The precipitated traces of DCU was filtered off and the filtrate was evaporated on rotary evaporator. The white crude product was recrystallized by refluxing it in a mixture (r50 ml) of dry acetone and diethyl ether (1:1) for l,12 hrs. The DCU impurities was found to be more soluble in the refluxing mixture and the pure DDSP was less soluble, the insoluble product was filtered and dried in vacuum. The last step was repeated 2-3 times ar~nuuar~ mrr~a~rmar~ ll ~lliaarr~~l- 11, till ro DCU was evidented in the NMR of tho product.

B) Copolymerization of DDSP with Jeffamine EDR-192 and PTHF-diamine 750 B-i) 1:1 PTHF-750 EDR-192 2.

0 0 9 N--C-(CH4 2 HI) N-R-NRh lr i- 0 0 (PTHF-750) (JEFFAMINE EDR 192) CHCl 3

TEA

00 Ut Ii: )I

-C(CH

2

S-(CH

2 )-C-NH-PTHF 750-NH) 05

C-(CH

2

)-S-S-(CH

2 C-NH-EDR 192-NH] 0 5 :ti::with EDR-192 1{ 2 N(CH2CH 2 0) 3

CH

2

CH

2

NH

2 *1JHF 750 H 2 N- (CR 2 )4 4-0 [CH 2 CH2CH 2

CH

2

(CR

2 4

NH

2 Procedure To a cooled solution (OOC) of 2.51 gm *o a so (24.75 meq) EDR-192 in 15 ml dry chloroform, 10.55 gm (24.75 meq) PTHF-750 in 15 ml dry CHC1 3 and 13.69 ml triethylapine was added a solution of DDSP (49.5 meq) in ml CHC1 3 The reaction mixture was stirred at OOC for 1 hour and the stirring was continued for 36 hrs at room temperature.

The chloroform layer was extracted with C1 0.1 M H 2 O NaOH 0.1 M (3x) and finally with H 2 0 The CHC1 3 layer was dried over MgSO 4 overnight and U i 1 III then was filtered and the filtrate was evaporated in rotary evaporator. The product was further dried on vacuum.

Results of the co-polyamides not treated in a in-vitro reductive medium.

1) Good film forming 2) GPC (NMP) Weight average molecular weight 21791 Dispersity 2.3 3) Colonic degradation see Table 1.

II) Degradation of disulfide-cointaining co-polyamides in isolated colonic medium Polymer films were incubated in a 15 ml sample taken from a SHIME reactor. The SHIME reactor (Simulating 15 Human Intestinal Microbial Ecosystem) is simulating the microbial contents of human colon. The flasks were degase S o sed by flushing it with nitrogen and then was incubated at 37 0 C. At regular times samples were taken and analyzed by GPC (eluent NMP). Undissolved polymer samples were 20 washed, dried under vacuum and the analyzed by GPC. The soluble polymers were removed by freeze-drying the medium and the residue was dissolved ir filtered to avoid inorganic salts and the analyzed by GPC.

OxOT 4

V

6 0 0 )V

I

44

S

4

*SSS

4 5

*SS*

9** 4*5

S.

S

*5 TABLE 1 Colonic degradation of copolymer B-1) Incuba- Insoluble part Soluble part tion time Mw Mn Polydisp. Mw Mn Polydisp.

(hrs) 0.0 21884 6104 3.58 15162 2594 5.84 14492 2621 5.52 2.0 13046 2318 5.62 3557 789 11022 2090 5.27 12398 2047 6.057 2893 731 3.958 26.5 14101 2550 5.53 2835 718 3.948 47.0 1341 2220 6.188 1921 717 2.679 Comment Slow degradation.

III) Mechanical tests Tensile testing 20 The tensile strength of the polymer samples was measured using a Hounsfield type H1OKM tensile machine.

The samples was tested at 25 0 C at a crosshead speed (tensile rate) of 20 mm/min using 100 N cell.

Film preparation Polymer solutions containing 10% w/w of the polymer in chloroform were cast on a T&flon plate. The plate was covered with a watch glass to slow down solvent evaporation, solidified by atmospheric air drying. The films were carefully removed from the plate and dried to constant weight in vacuum at 30 0 C. The thickness of the film was measured using micrometer Forster-isometer S 2.320 (Belgium). The film was cutted for tensile strength measurements using hand press cutting knife from Berg Schmid model HK500.

1.3 Disulfide-containinq polyurethanes The disulfide-containing polyurethanes are pre-

I--

14 pared by polycondensation of 4, 4 1-diphenylrnethane diisocyanate (MDI), an a,wi-hydroxy-terminated prepolymer and a disuif ide-containing chain extender, according to the following reaction scheme

HO-R-OH

DIMAc 0 0 4HO-CHCH-S-S-CH.CH,-OH 05 r 0 0 0 I- I :-0-CHCH,-S-S-CHCH-0-C-NH-MDI-NH-c-OR--C-N-MDI-NH As hydroxy-terminated prepolymers were used poly (tetramethyleneoxide), poly (propyleneoxide), poly (caprolactone), poly(ethyleneoxide), poly(ethyleneoxide/proo pyleneoxide).

Synthesis of azo-containing Volvamides The azo-containing polymers were synthesized by *0 polycondensation of azobenzene-4,41-diacid chloride and as 25 oligomeric a,ca-diamines in chloroform as solvent and tridthylamine as proton acceptor. The azobenzene-4, 4 1-diacid chloride was prepare from the corresponding azobenzene- 4,4'-dicarboxylic acid by reaction with thionylchloride in toluene as solvent. The oligomeric diamines selected was hydrophylic (Jef famine ED-EGO) which resulted in water solubae polymer: HOOC0 Q N=N- Q -COOK I (4) 0 0 C Ii -C HN -R-NH, TEAk I CHCI; 0 0 it 11 C NC-NI-.-R-NH4 (9) 0 *0*S

S

S

S

*SOS

*5

S

*5 0*SS

SS

S.

00 0 5 S S 0 R=HI C- (CC CH, CH- 3 CH- 3 a Characterisation of the polymers was done by IH-NMR and IR spectroscopy. Table 2 shows the molecular 2..eight of both azo polymers as determined by GPC using polystyrene as standards.

~c Ri 7Ar a TABLE 2.

Molecular weiaht of the azo-containing polvamides Polymer Diamine Molecular weight Hydrophylic azo Jeffamine ED-600 Mw 16500 polymer Mn 7900 Reduction of azo-containin polvamides in chemical reductive medium The degradation of the azo polymers was investigated in an in-vitro reductive medium. During incubation, the films become sticky and colourless. Upon drying of the incubated polymer at 50 0 C in air, the samples showed a rapid colour recovery from colourless to orange.

Table 3 shows molecular weight as determined by GPC using polystyrene as standards and the results of viscosity measurements for said hydrophylic azo polymer before and after incubation in a chemical reductive medium.

TABLE 3 Changes in molecular weight of after incubation in the chemical in-vitro reductive medium Incubation time Mw Mn 0 16380 7280 6 4410 2120 24 4230 1880 3790 1730 48 2860 1215 72 2060 940 0 9 *9 9 9. 9 9 GPC analysis incubation times (table of samples taken at different 3) show a remarkable decrease in

~I

anaRIRbu*rramrmrmn~3 mrrarrr~ lar~ col 17 molecular weight. After 3 hours incubation, the molecular weight is apparently reduced of a factor 10 faible original value and remains so for the further 48 hours of incubation (see table However, during reoxidation, the original colour appears and the molecular weight as determined by GPC raises to the initial value. The films dissolved completely in the reductive medium after 6 hours of incubation.

Spectroscopic evidence for the formation of the hydrazo bond during reduction of the azo bond Evidence for the formation of hydrazo groups during reduction of the hydrophobic azo polymer was gained by 1 3 C-NMR analysis.

Table 4 gives different chemical shifts for the oxidized and reduced form of the hydrophobic azo polymer.

The aromatic carbon resonances were assigned based on additivity rules [25] using a para X-Ph-Y structure with Y -N=N-Ph and, X -CON(CH 3 )2 for the oxidized polymer and with Y -NH-NH and X -CON(CH 3 2 for the reduced 0 20 one. The calculated values are in good agreement with those determined experimentally for the oxidized and the reduced form.

rdIn conclusion it can be stated that both poly- S 0* mer forms, having a few characteristic carbon resonance S. 25 lines C3 and C5), can easily be discriminated from each other by "C-NMR.

The "C-NMR sp,;ctra of the reoxidized polymers, after incubation in the chemical reductive medium and in the SHIME reactor, are clearly mixtures of the reduced and oxidized forms. The polymer which was incubated in the chemical reductive medium, reoxidized for three days in air atmosphere, still contains mainly (about 90%) of the reduced form. The polymer which was incubated in the SHIME-reactor, reoxidized for about two weeks in air atmosphere, contains mainly (about 55%) of the oxidized 18 form.

0 45 o -N-C-A Qy-N=N- Q -C-NH 0 4 5 0 HN-C NH -C-NH FT-Raman spectroscopy is an attractive tool to :o study the reduction of the azo groups. The aromatic ring vibrations in both spectra appear at 1613 cm 1 and 1607 The typical Raman shift at 1460 cm 1 can be assigned to the N=N functional group and is present in S 20 the oxidized form. This Raman signal can be used to differentiate between the oxidized and reduced compounds.

FT-Raman can thus be applied as a complementary nondestructive technique for this purpose. For the reduced s.o' polymer the N=N stretching band at 1460 cm' is missing S 25 and replaced by a broad band at 1298 This band is due to the symmetrical NH-NH stretching. The reoxidized polymers contain a shift at 1460 cm L 1 characteristic for the N=N group. However, the higher relative intensity of the Raman line at 1460 cm"' of the in SHIME- reactor incubated polymer can be correlated with a higher amount of the oxidfzed polymeric form. These results are totally in agreement with the 13C-NMR data.

Voltametric experiments Literature data [28-29) show that the electrochemical reduction of an azo bond proceeds via a two- 1 electron step mechanism towards the hydrazo group. Reduction surpassing the hydrazo-derivatives generally requires more energy than the azo-hydrazo reaction. If the difference in energy is great enough, the reduction may be represented by two waves (figure 2e 2e" S--NH-NH-- 2 -NH, 2

-NH-NH

2I 2W- In some cases, however, azo compounds are known to be reduced to the amines, by a four-electron step reaction (figure 6).

4e- 4 e 2 -NH 2 4o H+ 0 0 Voltametric measurements have been carried out 20 in water, in which tetrabutylammoniumbromide was used as oelectrolyte. By using water as a solvent, only the water soluble, hydrophylic polymer could be investigated.

From table 5 we can notice that the redox potential of .o is -230 mV vs. SCE 6 mV vs. SHE). This explains S 25 why the polymer is reduced in the chemical reductive medium.

Groups such as hydroxy, amino, methoxy, which increase the electron density on the azo linkage, show a shift towards higher (less negative) redox potentials and proceed by a four-electron step reduction towards amines [30,31). Voltametric measurement of the water solubl azobenzene-4,4'-dicarboxylic acid showed a lower half wave potential than the methoxy substituted analogu, OCH3 HOOC N=N

COOH

There is a difference of 140 mV in redox potential between both products. This implies that the reduction of the methoxy substituted diacid is more favourable.

TABLE 4 Half wave (E, 2 potentials 0@ .p PP p p p 20 25 Product E 112 (mV) E 1 r (mV) vs. SCE vs. SHE 9 -230 +6 HOOC N=N COOH +130 +366 CHO

OCH

3 +270 +506 HOOC N=N Q COOH When the hydrophylic azo polymer is incubated in a similar reductive medium, degradation proceeds with reduction of azo groups to amines. The solubility of the azo polymers in the reductive medium is the determining factor. Voltametric measurements have shown that all water-soluble azo compounds have a redox potential, more

I

positive than the reductive medium. These results indicate that the thermodynamic conditions for the reductive cleavage of azo bonds are fulfilled.

o 0 0 0 0 0.00 00 00 0 0 0 0 8000 0000 0*0* 0**0 *0* 0 0 4 0 *4 **00 4* 0* 00 00 0 9 00 40 0 0 4 0 0

Claims (7)

1. A process for the preparation of colon specific reduction sensitive polymers selected from the group consisting of azo-containing polymers; (ii) disulfide-containing polymers, and (iii) azo- and disulfide-containiag polymers, said process comprising the following successive steps A] copolymerizing an azo- and/or disulfide-containih9' a, w-difunctional reagent by polycondensation or polyaddition with a suitable a, w-difurctional cononomer according to the general reaction scheme illustrated below n Y-R,-R-XX-R-RY n HX-R 1 -XH Y-R-R-XX-R-R.- C -Z-X-R 1 -X-Z-R 1 I,.-Z-X-R 3 -XH WITH HX-R 3 -XH is a hydrophilic comononer with -XH -OH -Y -COOk, CO-Hal, COOAlkyl, -CH-CH(R), -SOHal 0 XX_ and R =alkyl, aryl R 1 R, alkyl, aryl, alkylaryl groups optionally sub- stituted by methoxy-groups =alkylidene, arylidene, alkylarylidene all or not substituted polyether, polyester hal =halogen radical, e.g. Cl, Br i _I with and Z -Y H-X- -Z-X- C=O, CHI-CH-OH, SO? X and Y being interchangeable in the above formulas, B] vitro Reduction of the copolymer formed in step A in an in- reductive medium, and Partial reoxidation of the copolymer reduced in step

2. The process according to claim 1, it comprises the copolymerization (succinimidyl propionate) with a hydrophylic comonomer. wherein of dithio bis a, -difunctional I *e I I I t I I I Iae. Ia Ic

3. The process according to claim 1 or 2, wherein 20 it comprises the copolymerization of dithio bis (succinimidyl propionate) with a hydrophylic a, -diamine functional comonomer.

4. The process of claim 1, in which the reoxidation is carried out by air drying.

The process of claim 1, in which the reduction is carried out in a reductive medium having a redox potential of 430 mv.

6. The process of claim 1, in which the reduction is carried out in a reductive medium containing sodium sulfide and cystein.

7. A process according to claim 1~ substantially as hereinbefore described. DATED: 27 February 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys for: DANBIOSYST UK LTD 0* S S *OS@ S SS S S S *5 S S S S .5 SS *S 5 S o 5 55 S S S c:.%wwNoRDwuwu~AAmNoDETEPEcr,744i-4DoO 1 0 A BS T RA CT PROCESS FOR THE PREPARATION OF COLON SPECIFIC REDUCTION SENSITIVE POLYMERS Specific reduction sensitive polymers se~lected :.*from the group consisting of azo-containing polymers, disulfide-containing polymers, and (iii) azo- and disulfide-containing polymers, are prepared by a process comprising the steps of copolymerizing an azo- and/or disulfide-containing 0!,wi-difunctional reagent by polycondengation or polyaddition with a a,w-difunctional comonomer. '0. 0* 0