European Journal of Pharmaceutical Sciences 17 (2002) 207216 www.elsevier.

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Gembrozil encapsulation and release from microspheres and macromolecular conjugates

a , *, Monika Barbaric a , Branka Zorc a , Dario Voinovich b , Anita Martinac a , Jelena Filipovic-Grcic a Ivan Jalsenjak
b a Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica 1, 10 000 Zagreb, Croatia Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Trieste, P. le Europa 1, 34127 Trieste, Italy

Received 28 February 2002; received in revised form 5 September 2002; accepted 11 September 2002

Abstract The purpose of this study was to evaluate and compare the ability of the macromolecular conjugates and microspheres to modify the release rate of gembrozil (Gem). Gem was covalently linked to two similar polymers: poly[a,b-(N -2-hydroxyethyl-DL-aspartamide)] (PHEA) and poly[a,b-(N -3-hydroxypropyl-DL-aspartamide)] (PHPA) by an ester linkage. The polymerdrug conjugates obtained (PHEAG(13) and PHPAG) differ in weight-average molecular weight, length of spacer and Gem content. Microspheres, composed of chitosans of different molecular weight alone or as a mixture with (2-hydroxypropyl)methylcellulose (HPMC), PHEA or PHPA and with different theoretical polymer / drug ratio (2:1 and 3:1, w / w) were prepared by spray drying. The microparticulate systems were morphologically characterised by scanning electron microscopy, particle size analysis and Gem content was determined. In vitro dissolution tests were performed to evaluate the feasibility of conjugates and microspheres in modulating Gem release. The results obtained show that microspheres are always suitable to modulate Gem release and that the best conditions are achieved by microspheres composed of the low molecular weight chitosan (CL) combined with PHPA or HPMC with either 2:1 or 3:1 (w / w) polymer / drug ratio. The PHEAG conjugates exhibited rapid Gem release within less than 2 h, while the PHPAG conjugate showed sustained Gem release proles over a 10-h period. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Chitosan microspheres; Macromolecular conjugates; Gembrozil; Sustained release; Poly[a,b-(N -2-hydroxyethyl-DL-aspartamide)]; Poly[a,b(N -3-hydroxypropyl-DL-aspartamide)]

1. Introduction Gembrozil is the lipid-regulating agent, which is generically classied as a bric acid derivative. It appears to be most useful in the treatment of lipoprotein disorders characterised by elevation of very-low-density (VLD) lipoproteins and plasma triglycerides, since it lowers triglycerides and both total and VLD-cholesterol, while increasing high-density-lipoprotein (HDL)-cholesterol levels (Todd and Ward, 1988). It is rapidly absorbed after
Abbreviations : C, chitosan; C80, chitosan food grade 80; C90, chitosan food grade 90; CH, chitosan of high molecular weight; CL, chitosan of low molecular weight; CM, chitosan of medium molecular weight; Gem, gembrozil; HPMC, (2-hydroxypropyl)methylcellulose; PHEA, poly[a,b(N -2-hydroxyethyl-DL-aspartamide)]; PHPA, poly[a,b-(N-3-hydroxypropyl-DL-aspartamide)] * Corresponding author. Tel.: 1 385-1-461-2608; fax: 1 385-1-4612691. E-mail address: jelena.lipovic-grcic@fbf.tel.hr (J. Filipovic-Grcic).

oral administration and its short plasma half-life requires relatively frequent dosing. Some gastrointestinal symptoms and rash were observed as side effects of gembrozil treatment. In order to improve its pharmacokinetics and bioavailability, aliphatic and aromatic gembrozil esters (Piccoli et al., 1994), benzamides (Sircar and Holmes, 1983), nicotinic acid (Hoee, 1981) and 3-ethoxy derivatives (Wang et al., 1996) were synthesised. The aim of this work was to investigate the feasibility of controlled delivery systems in order to optimise the therapeutic properties of gembrozil and to lower its side effects. For this purpose two different types of delivery systems were evaluated: macromolecular drug conjugates and microspheres. Macromolecular drug carrier systems in which drugs are covalently linked to polymers have been largely studied and suggested as an effective way to prolong the pharmacological activity, minimize unfavourable side effects and toxicity, decrease the required dose and increase the

0928-0987 / 02 / $ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 02 )00190-2

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solubility of the drug, as well as alter the body distribution and ensure adequate drug delivery to target cells or tissues (Duncan and Spreaco, 1994). Microparticulate systems have great potential, being able to convert poorly soluble, poorly absorbable and labile biologically active substances into promising drugs. Polymers used for conjugation of gembrozil in this work were PHEA and PHPA. PHEA is an especially interesting and promising drug carrier since it is water soluble, non-toxic, no antigenic, and biodegradable when exposed to a complex set of enzymes (Neri et al., 1973; et al., 1979). Many pharmacologically active Drobnk agents, bearing carboxylic, amino or hydroxyl groups have been covalently linked to PHEA (see, for example, Zorc et al., 1993; Giammona et al., 1994, 1995, 1998). Also, we attempted to develop drug-loaded microspheres based on chitosans and mixtures of chitosans and HPMC, PHEA or PHPA in order to investigate the inuence of the polymeric composition of the microspheres on drug content and on drug release. Chitosan is a natural, non-toxic, biodegradable, biocompatible and mucoadhesive polysaccharide. Its ability to be made into solutions, lms, bres, beads as well as microspheres has lead to many pharmaceutical applications (Kas, 1997; Kotze et al., 1999). Chitosan itself reduces blood cholesterol levels and thus it could be expected that entrapment of gembrozil into chitosan microspheres could potentiate that effect (Furda, 2000). Although many polymers are used in the pharmaceutical formulations, the most widely utilised are the cellulose derivatives. The use of hydrophilic cellulose ether such as HPMC, has played an important role in the development of sustained release drug delivery systems. HPMC can take up and retain large amounts of water, which inuences the physical and chemical properties of polymer and drug release prole (Nokhodchi and Rubinstein, 2001). Microspheres were produced by spray drying, which is a rapid high-yield technique that is applicable at industrial scale. The ability of macromolecular conjugates and microspheres to modify the release rate of gembrozil were evaluated and compared.

(Japan), and gembrozil and L-aspartic acid were from Aldrich (USA). All other chemicals used were of analytical grade and purchased from Kemika (Croatia).

2.2. Preparation of polymers and polymer Gem conjugates

The outline of the procedure for the preparation of the polymerGem conjugates (Scheme 1) was the same as the method described previously (Lovrek et al., 2000). The synthesis of the PHEAG ( 5 ) and PHPAG ( 6 ) was essentially esterication of the polyhydroxyl polymers PHEA ( 2 ) and PHPA ( 3 ) by the azole activated Gem, Gem-Bt ( 4 ). In short, a solution of the PHEA ( 2 ) or PHPA ( 3 ), Gem-Bt ( 4 ), and triethylamine (TEA) in DMF was stirred at room temperature (3 days). The reaction mixture was evaporated under reduced pressure. The sticky residue was then triturated with cyclohexane, acetone and ether in order to remove benzotriazole, amine and eventually unbound 4, since these compounds were soluble in the used solvents and conjugates 5 or 6 were not. The nal loose products PHEAG ( 5 ) or PHPAG ( 6 ) were ltered off. The PHEA ( 2 ) was prepared by thermal polycondensation of L-aspartic acid in the presence of phosphoric acid and subsequent aminolysis of polysuccinimide (PSI ( 1 )) et al., with ethanolamine (Neri et al., 1973; Jakopovic 1996; Lovrek et al., 2000). PHEA weight-average molecular masses were 31 000, 56 000 and 61 000 for PHEA(1), PHEA(3) and PHEA(2), respectively (Table 1), and they were determined by the viscosimetric method (Antoni et al., 1974). In order to prolong the distance between main chain and hydroxyl side groups, aminolysis of PSI ( 1 ) was carried out with 3-hydroxypropylamine as well. In this way a new hydroxy-functionalised polyaspartamide polymer PHPA ( 3 ) was prepared. It could be considered that its Mw was very close to 61 000, Mw of PHEA(2), since both polymers were derived from the same PSI fraction ( 1 ) and PSI was aminolysed under analogous conditions. The average molecular masses of PSI and PHEA, determined according to the MarkHouwink equation for PSI [h ] 5 1.32 3 10 2 2 3 M 0.76 (Vlasak et al., 1979) and for PHEA w 23 0.87 [h ] 5 2.32 3 10 3 M w (Neri et al., 1973), respectively, revealed that both polymers had practically the same polymerisation degree. The drug content in polymerGem conjugates was estimated by UV spectroscopy using the molar absorption coefcient for Gem e 276 5 1866 l / mol / cm (in 96% EtOH, c 5 4.99 3 10 2 4 M). The weight percentage of Gem in PHEAG(13) was in the range from 29 to 53%, and in PHPAG, 20% (Table 1). The drug content depended on the molar ratio of the reactant 4 and monomer units of the corresponding polymer 1, 2 or 3. The proof that Gem was covalently bound in the synthesised polymerdrug conjugates could be found in

2. Materials and methods

2.1. Reagents and chemicals

Different molecular weight chitosans (CH, CM and CL) were purchased from Fluka (Switzerland): CH (Mw 600 000; deacetylation degree 83%), CM (Mw 400 000; deacetylation degree 83.5%), and CL (Mw 150 000; deacetylation degree 87.4%). Chitosan food grade 90 (C90) and 80 (C80) were obtained from Syntapharm (Germany) and were used without further purication: C90 (deacetylation degree 91%), C80 (deacetylation degree 84%). HPMC was purchased from Shin-Etsu Chemicals

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Scheme 1. Procedure for the preparation of the polymerGem conjugates.

Table 1 Preparation and characteristics of PHEAG and PHPAG conjugates Conjugate PHEA(1)G PHEA(2)G PHEA(3)G PHPAG Polymer PHEA(1) PHEA(2) PHEA(3) PHPA Mw of polymer 31 000 61 000 56 000 61 000 Gem content (wt%) 53 34 29 20

PolymerGem conjugates produced and their main characteristics are listed in Table 1.

2.3. Preparation of microspheres

Microspheres were prepared by spray drying (Buchi 190 mini spray drier, Switzerland). The drying conditions were as follows: ow rate of 0.25 l / h, inlet air temperature of 120 8C and outlet air temperature of 75 8C. Microspheres containing Gem with different polymeric composition were prepared (Tables 24).

IR- and UV-spectra. The IR-spectra of 5 and 6 showed an ester carbonyl band at 1725 cm 2 1 . All prepared conjugates absorbed UV-light in the same absorption ranges as Gem, whereas PHEA and PHPA themselves had no UV-absorption at these wavelengths.

2.3.1. Chitosan microspheres with Gem Different types of chitosans or mixtures of chitosans (Table 2) at xed concentration (1%, w / v) were solubil-

Table 2 Main characteristics of chitosan microspheres with Gem Chitosan Viscosity of chitosan solution (mPa / s)a Theoretical polymer / drug ratio (w / w) 2:1 Mean diameter ( mm) CL CM CH 1 CL CH C90 C80
a b

3:1 Drug content (wt%) 2263 2463 2764 2562 2564 3362 Entrapment efciency (wt%)b 67610 73610 80612 7567 76612 9866 Mean diameter ( mm) 2.661.1 2.661.2 2.461.1 2.661.0 2.360.9 2.461.0 Drug content (wt%) 1863 1662 2263 1562 1862 1564 Entrapment efciency (wt%)b 73611 6369 89612 6267 7166 61615

100 200 400 70 190

2.360.9 2.660.9 2.260.8 2.661.1 2.360.9 2.661.2

1% solution in 1% acetic acid; according to Certicate of Analysis provided from the producer. Entrapment efciency 5 theoretical drug content / actual drug content 3 100. Values are mean6S.D. (n 5 3).

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Table 3 Main characteristics of HPMC and C / HPMC microspheres with Gem Polymers Theoretical polymer / drug ratio (w / w) 2:1 Mean diameter ( mm) HPMC C80 / HPMC C90 / HPMC CL / HPMC
a

3:1 Drug content (wt%) 3264 3364 3064 3362 Entrapment efciency (wt%)a 96611 99612 90611 9866 Mean diameter ( mm) 3.861.4 3.061.5 3.161.6 3.261.3 Drug content (wt%) 2561 1964 2163 2562 Entrapment efciency (wt%)a 10064 74615 84612 99.567

3.661.3 2.861.7 2.961.5 3.361.5

Entrapment efciency 5 theoretical drug content / actual drug content 3 100. Values are mean6S.D. (n 5 3).

ized in 0.5% acetic acid solution. In order to obtain the microspheres with different theoretical polymer / drug ratio Gem was dissolved at different concentrations (2 and 3%, w / v) in ethanol and added to chitosan solution in a 1:6 (v / v) ratio. Table 2 lists all the batches of Gem-loaded chitosan microspheres produced.

2.3.2. HPMC microspheres with Gem HPMC was dissolved in a mixture of ethanol and water (2:3, v / v). The polymer concentration was 1% (w / v). In order to obtain the microspheres with different theoretical polymer / drug ratio Gem was dissolved at different concentrations (2 and 3%, w / v) in ethanol and added to HPMC solution in a 1:6 (v / v) ratio. The mixtures were spray-dried under the conditions described above. The characteristics of microspheres prepared are given in Table 3. 2.3.3. C / HPMC microspheres with Gem For the preparation of the C / HPMC microspheres chitosan and the HPMC solutions were prepared as described above at 1% (w / v) concentration. The type of chitosan varied between the preparations while the C / HPMC ratio was kept constant (1:1, w / w). The ethanolic solution of Gem (2 or 3%, w / v) was mixed with solution of polymers in 1:6 (v / v) ratio. Mixtures were spray-dried under the conditions described in Section 2.3. The microspheres obtained and their characteristics are given in Table 3. 2.3.4. CL / PHEA and CL / PHPA microspheres with Gem The chitosan of low molecular weight (CL) at xed

concentration of 1% (w / v) was solubilized in 0.5% acetic acid solution. Polymers, PHEA(13) or PHPA, were dissolved in puried water at different concentrations (Table 4). Gem was dissolved in ethanol (2 or 3%, w / v). Different mixtures were prepared by varying PHEA used, while the CL / G ratio was kept constant (2:1, w / w). For the preparation of the CL / PHPA microspheres two mixtures were prepared by varying CL / G ratio (2:1 and 3:1, w / w) (Table 4). As for the PHEA / G and PHPA / G ratios the same ratios were used as present in the PHEAG and PHEAG conjugates (Table 1). The mixtures were spraydried under the conditions described in Section 2.3. Properties of the microspheres prepared are given in Table 4.

2.4. Encapsulation efciency determination

The drug content of the microspheres was determined spectrophotometrically ( l 5 276 nm; Ultrospec Plus, Pharmacia LKB) after digestion of microspheres with 0.1 M HCl. The microspheres prepared using HPMC were digested with the mixture of 0.1 M HCl and ethanol. Preliminary studies showed that the presence of dissolved polymer did not interfere with the Gem absorbance at 276 nm. Each determination was carried out in triplicate.

2.5. Particle size distribution

The microscopical image analysis technique for determination of particle size distribution was used. Microsphere sizes and distribution were determined with

Table 4 Preparation and the main characteristics of CL / PHEA and CL / PHPA microspheres with Gem Microsphere sample CL / PHEA(1) CL / PHEA(2) CL / PHEA(3) CL / PHPA(2:1) CL / PHPA(3:1)
a

Polymer PHEA(1) PHEA(2) PHEA(3) PHPA PHPA

Concentration of polymer solution (%) 4.36 9.54 12.20 20.38 20.38

CL / polymer ratio (w / w) 2.3:1 1.04:1 1:1.22 1:2 1:1.36

CL / G (w / w) 2:1 2:1 2:1 2:1 3:1

Mean diameter ( mm) 2.461.0 2.360.9 2.561.1 2.761.2 2.661.1

Drug content (wt%) 2362 1463 1562 962 762

Entrapment efciency (wt%)a 87611 71611 83611 66615 58612

Entrapment efciency 5 theoretical drug content / actual drug content 3 100. Values are mean6S.D. (n 5 3).

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Olympus BH-2 microscope, equipped with a computercontrolled image analysis system (Optomax V, Cambridge).

2.6. Scanning electron microscopy ( SEM)

The shape and surface characteristics of the microparticles were observed by scanning electron microscopy. The microspheres were sputter-coated with Au / Pd using a vacuum evaporator (Edwards) and examined using a scanning electron microscope (Philips 500, Eindhoven) at 10 kV accelerating voltage.

2.7. In vitro release of Gem from conjugates and microspheres

In vitro release proles of Gem from conjugates and microspheres were examined in phosphate buffer, pH 7.4. The drug loaded microspheres containing 10 mg of Gem were put into rotating basket (50 rpm) and placed in 250 ml of the dissolution medium, thermostated at 37 8C. The release from conjugates was assessed through dialysis membranes (Spectr / Por membranes Mwco 1214 000), which were placed in continuously-stirred 250 ml volumes of phosphate buffer at 37 8C. At scheduled time intervals, agitation was stopped, the samples (2 ml) were withdrawn and replaced with fresh medium. The samples were ltered and assayed spectrophotometrically at 276 nm. All experiments were carried out in triplicate and average values were plotted.

conjugates with the polyaspartamide polymers PHEA and PHPA in phosphate buffer. The initial rapid release was common to all PHEAG samples. Drug release progressed in extent of more than 80% within 2 h. The dissolution rate of Gem from the PHEAG(1) conjugate with the lowest drug content (29%) was the fastest. However, it could be concluded that molecular mass of polymers used for conjugation and drug content did not signicantly affect Gem release prole from the PHEAG conjugates. In the case of the PHPAG conjugate the release was slow and linear. On the rst 50 min, the release of Gem was about 20%, and it took 12 h to release 96% of Gem. This reduction in release rate could be explained by longer distance between the main chain and hydroxyl side groups and differences in polymer / drug ratio and lipophilicity.

3.2. Characterisation of chitosan microspheres with gem and drug release

Five samples of chitosan of different molecular weight were used for the preparation of microspheres. The main characteristics of chitosans used and microspheres prepared are shown in Table 2. The amount of chitosan varied among the preparations while the amount of Gem was kept constant. The preparation method produced well-formed microspheres with good morphological characteristics for all batches prepared as shown in Fig. 2. Particle size analyses revealed that the microspheres were characterised by not so narrow size distributions with mean diameter ranging between 2.360.9 and 2.661.2 mm. Chitosan molecular weight, polymeric composition and polymer / drug ratio in the microspheres did not inuence particle size characteristics. The encapsulation efciencies were always very high, between 61 and 98% (Table 2). In the case of theoretical

3. Results and discussion

3.1. In vitro release of Gem from PHEA and PHPA conjugates

Fig. 1 shows the dissolution proles of Gem from its

Fig. 1. The release proles of gembrozil from PHEAG and PHPAG conjugates: ( s ) PHEA(1)G; ( h ) PHEA(2)G; ( ^ ) PHEA(3)G; ( d ) PHPAG.

Fig. 2. SEM micrograph of CL microspheres with polymer / drug ratio 3:1 (w / w).

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polymer / drug ratio (3:1, w / w), the highest entrapment, 89%, was obtained when the mixture of CL and CH chitosans was used (1:1, w / w) for encapsulation, followed by the CL chitosan with 73% encapsulation efciency. The microspheres prepared with theoretical polymer / drug ratio 2:1 (w / w) showed higher entrapment of Gem than the microspheres prepared with theoretical polymer / drug ratio 3:1 for the same type of chitosan used. Exception was, again, the microspheres prepared with the CL chitosan alone and the mixture of CL and CH chitosans, which entrapped less Gem as polymer / drug ratio decreased. These results indicated that no direct correlation could be drawn between the viscosities of chitosans used for microencapsulation and entrapment efciency obtained, and suggested that the CL probably differed in cationic

density (having degree of deacetylation 87%) from other types of chitosan used. Consequently, it differed in Gem afnity. The release proles of Gem from the chitosan microspheres with different polymeric composition in vitro are shown in Fig. 3. All batches of Gem loaded microspheres showed the most signicant differences in drug release in the rst 4 h and completely released the drug in 12 h. A better control of drug release was obtained with the microspheres made of the polymer / drug ratio 3:1 (w / w) than with the microspheres made of the polymer / drug ratio 2:1 (w / w), except for microspheres made of the mixture of CH and CL chitosans (Fig. 3d). For CH microspheres, Gem release proles did not seem critically dependent on

Fig. 3. The release proles of Gem from chitosan microspheres made of theoretical polymer / drug ratio 2:1 (w / w) ( s ) and 3:1 (w / w) ( j ), using different types of chitosan: (a) CL, (b) CM, (c) CH, (d) CH 1 CL, (e) C80, and (f) C90.

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the polymer / drug ratio employed (Fig. 3c). When microspheres of hydrophilic polymers are immersed in water, they swell and form a gel diffusion layer that hinders the outward transport of the drug within the matrix, hence producing a controlled release effect (Lim et al., 2000). As the amount of polymer increases, the thickness of the hydrogel layer increases as well and the drug diffusion is more retarded. That can explain the slower release of lipophilic Gem from microspheres with higher theoretical polymer / drug ratio. In addition, chitosan can bind lipophilic compounds, which could also affect the Gem release prole (Furda, 2000). When comparing the Gem release proles from microspheres composed of different molecular weight chitosans and with the same theoretical polymer / drug ratio it could be seen that CL and CM microspheres with theoretical polymer / drug ratio 2:1 and 3:1 (w / w) are characterised by insignicantly different Gem dissolution proles (Fig. 3a and b). The fastest Gem release was achieved with the microspheres prepared using CH for both polymer / drug ratios employed. The C80 and C90 microspheres showed drug dissolution proles ranging between the release proles of the CH and CL microspheres (Fig. 3e and f). Fig. 3 also shows an initial burst (ranging between 10 and 50% in 10 min) of Gem release from all batches of microspheres. This is most likely due to the presence of Gem on the surface of the microsphere and a certain amount of Gem that was not entrapped at all. Its reduction in crystallinity, caused by spray drying, enhanced its dissolution rate (Moyano et al., 1995). The initial rapid release may have a functional use in providing an initial dose during the drug delivery, minimising any lag period. The CL (3:1, w / w) microspheres present the lowest burst effect and the most regular Gem dissolution prole.

Fig. 4. The release proles of Gem from HPMC microspheres made of theoretical polymer / drug ratio: ( s ) 2:1, ( j ) 3:1 (w / w).

Due to the polymer swelling described previously, the release of Gem was slower as polymer / drug ratio increased. The microspheres with higher drug content were expected to be more porous than those with low drug content, which might facilitate the release of residual drug from microspheres (Wan et al., 1994). This could explain the signicant difference in the percentage of the Gem released from the HPMC microspheres differing in polymer / drug ratio.

3.4. Characterisation of C / HPMC microspheres with Gem and drug release

The main characteristics of microspheres prepared are given in Table 3. The highest entrapment efciency (99.5%) was obtained for the CL / HPMC microspheres with the theoretical polymer / drug ratio 3:1 (w / w), although all microspheres exhibited high Gem entrapment efciency (7499%). The C / HPMC microspheres obtained were spherical in shape and could easily be resuspended in water, like chitosan and the HPMC microspheres. As expected, the average sizes of the C / HPMC microspheres were between the sizes of C and HPMC microspheres, ranging between 2.861.7 and 3.361.5 mm. The C / HPMC microsphere dissolution proles are shown in Fig. 5. About 80% of Gem was released from all C / HPMC microspheres within 4 h. The CL / HPMC microspheres (drug contents of 33 and 25%) show similar dissolution proles (Fig. 5a). The Gem release was slower from the C80 / HPMC and C90 / HPMC microspheres made of the polymer / drug ratio 3:1 (w / w) than from the C80 / HPMC and C90 / HPMC microspheres made of the polymer / drug ratio 2:1 (w / w) (Fig. 5b,c). This could be explained by polymer swelling, but at the same time by higher drug content of the microspheres made of polymer / drug ratio 2:1 (w / w) and consequently higher porosity.

3.3. Characterisation of HPMC microspheres with Gem and drug release

The main characteristics of microspheres obtained are given in Table 3. The entrapment efciency was very high (96 and 100%), giving the microspheres with 32 and 25% Gem content. The HPMC microspheres were larger than the chitosan microspheres, characterised by size distributions with mean diameters of 3.661.3 and 3.861.4 mm for the microspheres made of the polymer / drug ratio 2:1 and 3:1 (w / w), respectively. Polymer / drug ratio did not inuence particle size characteristics and Gem entrapment efciency. The HPMC microsphere dissolution proles are shown in Fig. 4. Gem release from the HPMC microspheres with polymer / drug ratio 2:1 (w / w) was completed within 5 h while only 65% of Gem from the HPMC microspheres with the polymer / drug ratio 3:1 (w / w) was released in that period.

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Fig. 5. The release proles of Gem from C / HPMC microspheres made of theoretical polymer / drug ratio 2:1 (w / w) ( s ) and 3:1 (w / w) ( j ), and varying the type of chitosan used: (a) CL / HPMC; (b) C80 / HPMC; (c) C90 / HPMC.

3.5. Characterisation of CL / PHEA and CL / PHPA microspheres with Gem and drug release
The main characteristics of the CL / PHEA and CL /

PHPA microspheres with Gem prepared by spray drying are given in Table 4. While the CL / G ratio was kept constant (2:1, w / w), different CL / PHEA microspheres were obtained by varying the type of PHEA used (PHEA(13)) and combined with CL in that way that the PHEA / G ratios were the same as in corresponding PHEA G conjugates. Two batches of the CL / PHPA microspheres were prepared with the CL / G ratios of 2:1 and 3:1 (w / w). In both batches the PHPA / G ratio was the same as in the corresponding PHPAG conjugate. The entrapment efciencies of the microspheres were between 58 and 87% w / w. The highest encapsulation efciency was obtained for the microspheres made of CL and PHEA(1) giving the microspheres with highest Gem content (23%, w / w). The CL / PHEA(2) and CL / PHEA(3) microspheres have the similar Gem content of 14 and 15%, respectively, while the entrapment efciency was lowest for the CL / PHPA microspheres (66 and 58% for CL / G ratio 2:1 and 3:1, respectively) resulting with low Gem content (9 and 7% w / w) as shown in Table 4. This could be attributed to the relatively high ratio of PHPA when compared to CL in the preparation. Fig. 6 shows release proles of Gem from the CL / PHEA and CL / PHPA microspheres in comparison with release proles of Gem from its PHEAG and PHPAG conjugates. The drug content inuenced the Gem release rates from the CL / PHEA microspheres in such a way that the release of Gem was faster as the drug content increased. From the CL / PHPA microspheres made of the CL / G ratio of 2:1 and 3:1 (w / w), the drug was released after an initial burst (about 20% in 10 min), with an almost constant rate for 8 h and the total release of about 60%. As shown in Fig. 6ac release of Gem from the PHEA G conjugates is faster than from the CL / PHEA microspheres containing both, Gem and PHEA. In case of the PHPAG conjugates and the CL / PHPA microspheres (Fig. 6d), the release proles were similar with slightly slower Gem release from the conjugates than from the microspheres. This could be attributed to the difference in drug content of the conjugates and the microspheres as well as to the physico-chemical nature of these two delivery systems. The Gem content of the PHEAG and PHPAG conjugates was signicantly (about two times) higher compared with the CL / PHEA and CL / PHPA microspheres (Tables 1 and 4). In the PHEAG and PHPAG conjugates, Gem is chemically bound to the polymer by an ester bond while it is physically entrapped into the matrix of the CL / PHEA and CL / PHPA microspheres. It appeared that the ester bond in the PHEAG conjugates was hydrolytically very labile and subsequent Gem release faster than its diffusion from the microspheres polymer matrix. PHPA has longer distance (propyl group) between main polymer chain and hydroxyl side groups than PHEA, which seemed to diminish hydrolysis of Gem from the corresponding conjugate. Also, relatively high ratio of this hydrophilic polymer in the CL / PHPA microspheres produced a more

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Fig. 6. The comparison of Gem release from CL / PHEA or CL / PHPA microspheres and PHEAG or PHPAG conjugates: ( s ) microspheres with: (a) CL / PHEA(1), (b) CL / PHEA(2), (c) CL / PHEA(3), and (d) CL / PHPA polymers; ( j ) conjugates with: (a) PHEA(1), (b) PHEA(2), (c) PHEA(3), and (d) PHPA.

hydrophilic matrix decreasing drugmatrix permeability and slowing the release of drug.

References
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4. Conclusion It may be concluded that both, the Gem conjugates and microspheres can modulate drug release, although microencapsulation is a more effective method in obtaining prolonged release of Gem than its conjugation with PHEA. The use of the appropriate mixtures of the CL chitosan and HPMC or PHPA produces Gem-loaded microspheres characterised by acceptable drug content and drug encapsulation efciency independent of the polymer / drug ratio. Furthermore, this polymeric composition induces the most regular drug release prole.

Acknowledgements This work was supported by grants 006250 and 006243 of the Ministry of Science and Technology of the Republic of Croatia.

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