Grafting amino drugs to poly(styrene-alt-maleic anhydride) as a potential method for drug release

Abstracts

Drug delivery systems based on polymer-drug conjugates give an improved treatment with lower toxicity or side effects and be used for the treatment of different diseases. Conjugates of biodegradable poly(styrene-alt-maleic anhydride) (PSMA), with a therapeutic agents such as amantadine hydrochloride, amlodipine, gabapentin, zonisamide and mesalamine, were afforded by the formation of the amide bonds of the amino drugs that reacted with the PSMA anhydride groups. The amounts of covalently conjugated drugs were determined by a¹ H NMR spectroscopic method, and the in vitro release rate in buffer solution (pH 1.3) was studied at body temperature 37 ºC. In kinetic studies, different dissolution models were examined to obtain drug release data and the collected data were well-fitted to the Korsmeyer-Peppas equation, revealing a dominant Fickian diffusion mechanism for drug release under the in vitro conditions.

drug release; polymer-drug; poly(styrene-alt-maleic anhydride); PSMA; kinetic

Sistemas de liberação de fármacos baseados em conjugados polímero-fármaco fornecem um tratamento melhorado com menor toxidade ou efeitos colaterais e são usados no tratamento de diferentes doenças. Conjugados de poli(estireno-alt-anidrido maleico) (PSMA) biodegradável, com agentes terapêuticos tais como cloridrato de amantadina, amlopidina, gabapentina, zonisamida e mesalamina, foram gerados pela formação de ligações de amidas dos fármacos amino que reagiram com os grupos anidridos do PSMA. As quantidades de fármacos covalentemente conjugados foram determinadas por ressonância magnética nuclear (NMR) de¹ H, e a taxa de liberação in vitro em solução tampão (pH 1.3) foi estudada à temperatura do corpo (37 ºC). Em estudos de cinética, modelos de dissolução diferentes foram examinados a fim de se obter dados de liberação do fármaco e os dados coletados se ajustaram bem a equação de Korsmeyer-Peppas, revelando um mecanismo de difusão Fickaniano dominante na liberação do fármaco em condições in vitro.

ARTICLE

Grafting amino drugs to poly(styrene-alt-maleic anhydride) as a potential method for drug release

Ardeshir Khazaei^I,*; Shahnaz Saednia^I,*; Javad Saien^I; Masoud Kazem-Rostami^II; Mahdieh Sadeghpour^III; Maryam Kiani Borazjani^IV; Fatemeh Abbasi^I

^IFaculty of Chemistry, Bu-Ali Sina University, PO Box 651783868, Hamedan, Iran

^IIYoung Researchers Club and Elite, Takestan Branch, Islamic Azad University, Takestan, Iran

^IIIDepartment of Chemistry, Takestan Branch, Islamic Azad University, Takestan, Iran

^IVFaculty of Science, Department of Chemistry, Bushehr Payame Noor University (PNU), PO Box 1698, Bushehr, Iran

ABSTRACT

Drug delivery systems based on polymer-drug conjugates give an improved treatment with lower toxicity or side effects and be used for the treatment of different diseases. Conjugates of biodegradable poly(styrene-alt-maleic anhydride) (PSMA), with a therapeutic agents such as amantadine hydrochloride, amlodipine, gabapentin, zonisamide and mesalamine, were afforded by the formation of the amide bonds of the amino drugs that reacted with the PSMA anhydride groups. The amounts of covalently conjugated drugs were determined by a¹ H NMR spectroscopic method, and the in vitro release rate in buffer solution (pH 1.3) was studied at body temperature 37 ºC. In kinetic studies, different dissolution models were examined to obtain drug release data and the collected data were well-fitted to the Korsmeyer-Peppas equation, revealing a dominant Fickian diffusion mechanism for drug release under the in vitro conditions.

Keywords: drug release, polymer-drug, poly(styrene-alt-maleic anhydride), PSMA, kinetic

RESUMO

Sistemas de liberação de fármacos baseados em conjugados polímero-fármaco fornecem um tratamento melhorado com menor toxidade ou efeitos colaterais e são usados no tratamento de diferentes doenças. Conjugados de poli(estireno-alt-anidrido maleico) (PSMA) biodegradável, com agentes terapêuticos tais como cloridrato de amantadina, amlopidina, gabapentina, zonisamida e mesalamina, foram gerados pela formação de ligações de amidas dos fármacos amino que reagiram com os grupos anidridos do PSMA. As quantidades de fármacos covalentemente conjugados foram determinadas por ressonância magnética nuclear (NMR) de¹ H, e a taxa de liberação in vitro em solução tampão (pH 1.3) foi estudada à temperatura do corpo (37 ºC). Em estudos de cinética, modelos de dissolução diferentes foram examinados a fim de se obter dados de liberação do fármaco e os dados coletados se ajustaram bem a equação de Korsmeyer-Peppas, revelando um mecanismo de difusão Fickaniano dominante na liberação do fármaco em condições in vitro.

Introduction

Nowadays, in order to improve the curative effect of drugs, drug delivery systems are studied and developed. Two general procedures are used to supply the polymers containing pendant bioactive substituents. One method consists of the polymerization of drug containing monomers.¹ In this method, bioactive compounds, which have reactive functional groups, such as carboxyl,^2-6 hydroxyl,^7-11 sulfhydryl^12-14 and amino,^15-19 can be converted to their derivatives with the capability of polymerization, and then the drug containing monomers becomes polymerized by currently polymerization methods. Another one uses drugs attached to the prefabricated polymers.²⁰ In these systems, drugs or their derivatives are chemically linked to the synthesized^21,22 or natural^23,24 polymeric chains by chemical reaction.

Poly(styrene-alt-maleic anhydride) (PSMA) is a commercially available copolymer, including reactive anhydride groups. Anhydride groups of PSMA can easily react with hydroxyl, sulfhydryl or amine groups.^25-27 PSMA copolymers were applied to clinically deliver neocarzinostatine,^28-31 which is an antitumor protein. Henry et al. ³¹ showed that showed that alkyl amine derivatives of PSMA are capable of destabilizing biological membranes at acidic pH values and showed how this activity can be modulated for use in intracellular drug delivery applications.

In this work, our group reported novel types of polymer-drug conjugates based on PSMA and amino medicinal compounds, such as amantadine (antiviral and anti-parkinson),^32-34 amlodipine (anti-hypertensive),^35,36 gabapentin (treatment of epilepsy),^37-39 zonisamide (anti-convulsant)^40,41 and mesalamine (anti-inflammatory). ⁴² Amino drugs containing -NH₂ were linked up with PSMA by amide bond formation, and then, hydrolysis reactions were carried out in buffer solution (pH 1.3) at 37 ºC, as the human gastric simulated conditions. Drug release kinetics and release mechanisms are afforded by acquiring experimental data in different ranges of times.

Experimental

Materials and apparatus

Styrene, maleic anhydride, methanol, hydrogen chloride, benzoyl peroxide, potassium chloride and organic solvents were purchased from Merck Chemicals branch in Iran, and used without further purification. Dimethylformamide (DMF) was dried over MgSO₄ for 72 h, and then distilled once, and tetrahydrofuran (THF) was refluxed on Na/benzophenone for 8 h and distilled twice before use. Amantadine, gabapentin, amlodipine, zonisamide and mesalamine were received from Alborz-Daro Antibiotic Manufacturer, Iran. Buffer solution (pH 1.3) was prepared by addition of KCl aqueous solution (25 mL, 0.2 mol L^-1) to HCl (33.6 mL, 0.2 mol L^-1) in a volumetric flask.

All the UV-Vis spectrophotometric data were recorded by using a Shimadzu UV-265 spectrophotometer. A Shimadzu 435-U-04 FTIR spectrophotometer was used to obtain the FTIR (Fourier infrared spectroscopy) spectra of powder pressed KBr pellet samples. ¹ H nuclear magnetic resonance (NMR) spectra were performed in DMSO-d₆ sample solutions by a Bruker 300 MHz spectrometer. Molecular weight of PSMA was determined by a Waters-150 GPC analysis instrument (mobile phase: THF, flow rate: 1.0 mL min^-1, and column temperature: 30 ºC).

Preparation of poly(styrene-alt-maleic anhydride)

Poly(styrene-alt-maleic anhydride) (PSMA) was prepared through a thermally initiated free-radical polymerization of styrene and maleic anhydride³¹ (Scheme 1a): a mixture of equimolar amounts (0.0432 mol) of styrene (4.99 mL) and maleic anhydride (4.236 g) was added to THF (25 mL) solution with benzoyl peroxide (0.0043 mol, 1.042 g) as polymerization initiator agent, and then, poured into a flask equipped with a reflux condenser and a magnetic stirrer under nitrogen atmosphere. Polymerization was carried out at 80 ºC for 7 h. The viscous liquid, which resulted from the polymerization reaction, was washed in cold methanol to precipitate pure PSMA polymer. The purified PSMA was filtered off and dried under vacuum at room temperature.

General procedure for preparation of drug-loaded polymer

The grafting of selected drugs onto PSMA was carried out by reacting the drug amino group with the anhydride groups of PSMA to form amide bonds (Scheme 1b). Each drug was added with an equimolar ratio of monomers. For this purpose, PSMA (0.005 mol, 1.00 g) was dissolved in dry DMF (25 mL) by magnetic stirrer, and then, 0.005 mol of a selected drug, amantadine (0.939 g), gabapentin (0.856 g), zonisamide (1.061 g), mesalamine (0.766 g) and except for amlodipine (0.0025 mol, 1.202 g), due to its larger molecules and lower reactivity, were added to the solutions. The reaction was carried out at room temperature under nitrogen atmosphere for 12 h. Drug loaded polymer was purified by washing with distilled water and dried under vacuum at room temperature. The crude product of PSMA-MSE was gummy and sticky, so it was washed with cold diethyl ether to reach its powder form.

In vitro drug release study

In vitro drug release studies were performed by placing drug loaded polymer samples in a predefined volume of buffer (pH 1.3) at 37 ºC. The quantity of released drugs was measured by UV-Vis spectrophotometer; the absorbance was recorded in the maximum wavelength value (λ_max) related to each drug (Table 1). Standard calibration curves (drug concentration vs. absorbance at appropriate λ_max) for suitable dilutions of drugs in buffer solution (pH 1.3) were first obtained. Sampling to determine the amount of released drugs (in ppm) was performed at different times (ranges as shown in Figure 1).

Results and Discussion

PSMA copolymerization

The free radical copolymerization of PSMA is facile and the copolymer readily affordable. As long as the polymerization was going on, the vinylic peaks disappeared in the¹ H NMR spectrum of PSMA, and aliphatic peaks such as methylene (CH₂) and methyne (CH) replaced them. Overlapped broad peaks between d 1.1-2.8 and 6-7.9 ppm are due to methylene/methane and aromatic ring protons of styrene, respectively. Methyne protons of maleic anhydride appear between d 3.3-3.5 ppm. Average molecular weight of PSMA was determined by gel permeation chromatography (GPC) (Table 2). In the FTIR spectrum for PSMA, anhydride group peaks appear at 1856, 1799 (cyclic anhydride C=O) and 1224 cm^-1 (cyclic C-O-C).

Grafted drugs to PSMA

Spectroscopic characterizations of the functional groups proved that drugs were grafted with covalent bonds to PSMA. The FTIR spectra show that the carboxylic acid and amide groups are formed after the drug amine group attacked the anhydride groups of PSMA. It is noticeable that the corresponding peaks of anhydride groups of PSMA are not entirely removed due to the unreacted remaining parts of the polymeric chains. Table 3 summarizes the FTIR spectra of drug loaded PSMA samples.

Using¹ H NMR data, the molar ratio of attached drug per monomer unit was calculated separately for each PSMA-drug copolymer sample. The percentages of attached drugs per monomer unit were about 17.8, 23.0, 39.3, 33.6 and 25.7 for amantadine, amlodipine, gabapentin, zonisamide and mesalamine, respectively.

In vitro drug release investigations of drug-loaded PSMA

All the drugs used in this work are oral, hence, in vitro hydrolysis investigations of drug-loaded PSMAs were done according to the simulated gastric juice condition of human body⁴³ at acidic pH values (range of 1.1-1.5). In such acidic medium, drug-loaded PSMA hydrolyses were completed gradually as shown in Figure 1, in this way, the drug release is more controlled and permanent. In another study, which was done in simulated intestine media (at higher pHs such as 5, 6 and 7.4), the drug release was carried out by cleavage of the polymeric backbone and were more rapidly accomplished.

The drug release percentage and the cumulative release profiles of amantadine, amlodipine, gabapentin, zonisamide and mesalamine from their polymeric backbones PSMA-AAE, PSMA-ALE, PSMA-GBN, PSMA-ZNE and polymer matrix. Higuchi⁴⁵ described the release of drugs PSMA-MSE are presented in Figure 1. Drugs are released from porous and insoluble matrices as a square root of rather fast at the beginning of the process. However, a mild time dependent process based on Fickian diffusion, as variation appeared as the process proceeds.

In order to analyze the kinetics of drug release from PSMA matrix, it was clear that, in vitro release results were fitted by first order equations, according to Higuchi⁴⁵ and Korsmeyer-Peppas. A first order model (equation 1) describes the systems in which the rate of drug release is linearly proportional to the drug concentration on the polymer matrix. Higuchi⁴⁵ described the release of drugs from porous and insoluble matrices as a square root of time dependent process based on Fickian diffusion, aspresented in equation 2. ^46-48 Korsmeyer et al. ⁴⁹ derived a simple relationship which described drug release from a polymeric system (equation 3).^49-51

where C_t is the amount of drug released at time t, C_∞ is the maximum amount of released drug experimentally measured after long times (more than 200 h), k₁ is the first order drug release constant, k_H is the Higuchi rate constant, k_KP is the Korsmeyer-Peppas rate constant and n is the kinetic order. To study the release kinetics, data obtained from in vitro drug release studies were plotted as -ln[1 - (C_t/C_∞)] vs. time in the first order model, as amount of drug released vs. square root of time in the Higuchi model and as log (C_t/C_∞) vs. log t in Korsmeyer-Peppas equation.

The obtained kinetic data of drugs release from drug-loaded PSMA, together with the coefficient of determination (r²), are listed in Table 4. Fitting data to the three kinetic models and investigation of the r² values show that the Kormeyer-Peppas model agrees with the release curves most accurately.

Water uptake

The water diffusion into PSMA-drug copolymers is based on Fickian diffusion that supposes proportionality between the flow and concentration gradient. The water uptake (S_w) of the conjugated polymer was determined gravimetrically in distilled water at room temperature overnight (W_s). Before each measurement, the dry weight of polymer (W_d) which was dried under vacuum overnight was also determined. The excess of water was gradually removed with filter paper. The water uptake was determined with the following equation:⁵²

Swelling percentages were obtained as 30, 75, 66, 55 and 70% for PSMA, PSMA-AAE, PSMA-ALE, PSMA-GBN and PSMA-ZNE, respectively. It is worth mentioning that PSMA-MSE is entirely soluble in water.

These results indicate that, drug-loaded PSMA systems have more water uptake potential in comparison with the original polymer. This is expected due to the possible hydrogen bonding of the carboxylic acid groups, in the backbone of the drug loaded polymeric chains, increasing its swelling.

Mechanism of drug release

To explain the nature of the drug release mechanism, Korsmeyer et al. ⁴⁹ developed a simple empirical model, which described drug release from a polymeric system. Some processes may be classified as either purely diffusion or purely erosion controlled; many others can only be interpreted as being governed by both. The analysis of experimental data in the light of equation 3, as well as the interpretation of the corresponding release exponent values, leads to a better understanding of the balance between these mechanisms. ^53-55 The equation can be written as:

where (C_t/C_∞) in equation 5, is the mass fraction of drug released at time t and n is a characteristic of the drug-polymer system.

By determining the release exponent, it is possible to obtain information about the physical mechanism controlling the drug release from a particular device. ^50,56 Based on the value of this exponent n, the drug transport was classified as given in Table 5. ⁵⁷

The exponent n in this work varies from 0.256 to 0.441 for all the formulations (Table 4), i.e., less than 0.45; therefore, this suggests that Fickian diffusion is the main release mechanism under the conditions used. Drug mass transfer flow in this mechanism is related to the product of a molecular diffusivity and a concentration gradient. In order to further characterize the drug release process, the mean dissolution time (t_m) was calculated for each drug according to the following equation. ⁵⁴

This parameter reflects the level of drug release retarding by the polymer matrix and the in vitro media. The t_m obtained values are presented in Figure 2.

Mean dissolution times are within 40.4 to 99.9 h. Among the used drugs, zonisamide and gabapentin exhibit the highest and lowest dissolution times, respectively. This can be attributed to the resonance between S=O and NH bonds (between PSMA and zonisamide) which in turn causes the bond cleavage to occur in longer times.

Conclusions

The covalent grafting of five well-known drugs to PSMA was successfully done by the formation of amides from the reaction of amino group drugs with the anhydride groups of PSMA. In vitro release of amantadine, amlodipine, gabapentin, zonisamide and mesalamine from their corresponding drug-loaded PSMA was also studied, and indicated that the drug release can occur smoothly during long times, and this can be considered as an useful method for drug delivery. The kinetic analysis and the mean dissolution time indicate that Fickian diffusion controls the release of the drugs under study, and also zonisamide and gabapentin exhibit the slowest and the fastest release rates, respectively.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as a PDF file.

Acknowledgements

The authors gratefully acknowledge partial support of this work by the Research Affairs Office of Bu-Ali Sina University Center of Excellence in Development of Chemical Method (CEDCM) and Prof. Abbas Afkhami for his help in this research.

Submitted: March 19, 2013

Published online: June 11, 2013

Supplementary Information

The supplementary material is available in pdf: [Supplementary material]

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Publication Dates

• Publication in this collection
  31 July 2013
• Date of issue
  July 2013

History

• Received
  19 Mar 2013
• Accepted
  11 June 2013