Departments of 1 Biochemistry and 2 Pharmacology, Postgraduate Institute of Medical Education & Research, Chandigarh 160 012, India
Received 20 August 2002; returned 27 October 2002; revised 17 December 2002; accepted 20 January 2003
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Abstract |
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Keywords: tuberculosis, pharmacokinetics, alginate, drug delivery, antitubercular drugs
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Introduction |
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The present study was planned to entrap antitubercular drugs isoniazid, rifampicin and pyrazinamide alone and in combination in alginate hydrogel microparticles and to evaluate their in vivo release profiles in guinea pigs. The formulation was also evaluated for chemotherapeutic potential against experimental tuberculosis.
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Materials and methods |
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Sodium alginate of medium viscosity (3500 cps), isoniazid, rifampicin and pyrazinamide were obtained from Sigma. All other reagents were of analytical grade and deionized water was used throughout the study.
Animals
Dunkin Hartley guinea pigs weighing 300400 g obtained from the Central Animal House, PGIMER, Chandigarh or Haryana Agricultural University, Hissar, India were used in this study. The animals were housed in an appropriate animal house facility under natural light conditions. They were fed standard pellet diet and water ad libitum. The work was approved by the Institute Animal Ethics Committee.
Culture
The culture of Mycobacterium tuberculosis H37Rv was originally obtained from the National Collection of Type Cultures (NCTC-7416), London, UK and was maintained on Youmans modified medium and Lowenstein Jensen medium.
Preparation and characterization of alginate gel microparticles
Two millilitres of isoniazid, rifampicin or pyrazinamide solution (25 g/L) was added to 2 mL of 2% alginate aqueous solution. For entrapment of isoniazid, rifampicin and pyrazinamide in combination, 2 mL of each drug (at a concentration of 25 g/L) was added to 6 mL of 2% alginate. Solutions were mixed and vortexed thoroughly for 510 min at 1000 rpm. This mixture was dropped through a 26G needle into 0.15 M CaCl2 solution, which was being continuously stirred. These droplets formed gel beads instantaneously,8 which were washed 23 times with distilled water before drying at room temperature for 12 h. These dried hydrogel microparticles were sized microscopically. The particle size of 3040 microparticles was measured with a micrometer and the mean particle size was determined as described by Takka & Acarturk.9 Alginate and water content associated with beads were assayed by measuring uronic acid production8 (degradation product of alginate) and the difference between the dry and wet weight of beads, respectively.9
Determination of drug content of hydrogel microparticles
The entrapment of drugs in alginate hydrogel microparticles was determined by lysing them in phosphate-buffered saline (pH 7.5). The drugs released in the supernatant were assayed by standard protocols. Isoniazid was estimated by the spectrofluorometric method of Scott & Wright10 with a sensitivity of 0.1 mg/L, rifampicin was measured spectrophotometrically as described by Deol et al.2 and pyrazinamide was estimated by a spectrophotometric method as described by Gurumurthy et al.11 with a sensitivity of 5 mg/L.
In vitro dissolution studies
In vitro release studies of drugs from the alginate microparticles were carried out at room temperature (2730°C) for up to 30 days by suspending 20 mg of drug-loaded alginate beads in simulated gastric (0.1 M HCl buffer, pH 1.2) and intestinal fluids (phosphate buffer, pH 7.4) prepared according to the US Pharmacopeia.9 Samples (5 mL) at appropriate intervals were withdrawn and an equal volume of dissolution medium was added to maintain constant volume. Drugs were assayed in the fluids by similar methods to those employed for determination of drug content in hydrogel microparticles.
In vivo drug disposition studies from alginate hydrogel microparticles
Guinea pigs were orally administered free drugs/drug-loaded alginate microparticles at doses of 10, 12 and 25 mg/kg body weight of isoniazid, rifampicin and pyrazinamide, respectively, through oral feeding cannula.
Guinea pigs were bled and killed at different time points post-administration of free drugs/drug-laden alginate hydrogel microparticles. Organ homogenates (20%) were prepared from lungs, liver, spleen and intestine in 0.9% NaCl. Isoniazid and pyrazinamide were estimated as reported earlier, whereas rifampicin concentration in plasma and tissue homogenates was determined by microbiological assay12 using Bacillus subtilis with a sensitivity of 0.01 mg/L. Results were expressed as concentration of drugs in mg/L obtained in plasma/tissue homogenates at various time intervals.
Pharmacokinetic analysis
Cmax (peak plasma concentration) and Tmax (time to reach peak concentration) were calculated from the actual timeconcentration curve of plasma. The elimination rate constant (kel) was calculated by least square regression analysis. Elimination half-life (t1/2e) was calculated using the formula 0.693/kel. The absorption rate constant (ka) was calculated by the residual method and absorption half-life (t1/2a) was determined using the formula 0.694/ka. The area under the concentrationtime curve (AUC)t was calculated by the trapezoidal rule and the AUC0
by dividing the last concentration of drug observed in plasma by the respective kel. The AUC0
is the sum of AUC0t and AUCt
. Relative bioavailability was calculated by dividing the AUC0
of alginate encapsulated drug by the AUC0
of free drug given by the same route.
Chemotherapeutic studies
Guinea pigs were infected with 1.0 mg of cells harvested from a 3 week culture of M. tuberculosis H37Rv in a volume of 0.1 mL of 0.9% sterile NaCl solution via the intramuscular route in the left thigh muscle.13 After 1520 days, infection was confirmed by Ziehl-Neelsen staining of tissue smears of spleen after the killing of two animals. The animals were then divided into three groups and each group had at least eight animals. Dosages of isoniazid, rifampicin and pyrazinamide used in this study were 10, 12 and 25 mg/kg body weight.
Group A. Served as controls (no therapy was given to them).
Group B. Orally administered drug-loaded alginate hydrogel microparticles (containing isoniazid, rifampicin and pyrazinamide) weekly for 8 weeks.
Group C. Orally administered free isoniazid, rifampicin and pyrazinamide in combination daily for 8 weeks.
Assessment of the number of viable bacilli
After 8 weeks of chemotherapy, guinea pigs were killed 6 days after the administration of the last therapeutic dose. One quarter of spleen tissue or caudal left lung lobe was homogenized in 5 mL of sterile normal saline and different dilutions were plated for cfu enumeration. One quarter of the liver was placed in 10% neutral buffered formalin for histopathological examination. Duplicate Lowenstein Jensen slopes were inoculated with 0.1 mL of diluted homogenates in normal saline, incubated at 37°C and after 4 weeks colonies were counted. Colony forming units counted were expressed as log10 cfu/mL.
Histopathology
Formalin fixed tissues embedded in paraffin and sectioned at 5 µm were mounted on glass slides and stained with haematoxylineosin and acid fast stains.
Toxicity studies
The activities of alkaline phosphatase and alanine transaminase were determined in plasma samples of guinea pigs (on the seventh day post-administration of last therapeutic dose) using Boehringer-Mannheim kits, and results were expressed as U/L.
Statistical analysis
The data were analysed by Students unpaired t-test. Free drugs and alginate drug treatment groups were compared with untreated groups.
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Results |
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The alginate microspheres were almost spherical particles. The mean particle size of all formulations was between 90 and 100 µm. The entrapment of isoniazid, rifampicin and pyrazinamide was found to be 2535%, 4070% and 3343%, respectively (the percentages given are of the initial drug concentrations); 70% water content and 10.5% uronic acid content (degradation product of alginate) were found to be associated with 100 mg of drug-loaded alginate microparticles.
In vitro release profiles
Release of isoniazid, rifampicin and pyrazinamide from drug-loaded alginate microparticles was studied in simulated intestinal fluid (SIF), pH 7.4 and simulated gastric medium (SGM), pH 1.2. All the drugs showed sustained release for 25 days in SIF, whereas limited drug release was observed in SGM. Approximately 2736% of the drugs were released in gastric fluid over a period of 25 days, whereas 8590% of the drugs were released in simulated intestinal fluid over a period of 20 days. This is because at acidic pH, alginate microparticles shrink due to tightening of gel meshwork, whereas at alkaline pH, alginate erodes and releases the contents in a sustained manner.14,15
In vivo drug disposition studies from alginate hydrogel microparticles
In vivo drug disposition studies were carried out in guinea pigs by oral administration of a single dose of alginate microparticles containing individual or a combination of drugs. Subsequently, the plasma and organ levels of isoniazid, rifampicin and pyrazinamide were monitored at different time intervals. Figure 1(a) depicts the plasma concentration of free isoniazid and alginate entrapped isoniazid at different time intervals. Both the formulations exhibited comparable peak concentration (Cmax), but the time to reach peak level was 6 h for free isoniazid whereas isoniazid in alginate peaked later. However, entrapped isoniazid was detected up to 96 h whereas free isoniazid disappeared from plasma after 20 h.
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Although free pyrazinamide levels were higher at 2, 4, 6 and 8 h, it could only be detected up to 48 h. Entrapped pyrazinamide had a Tmax of later than 6 h, had significantly higher drug levels at 12, 24 and 48 h, and remained in circulation for 108 h (Figure 1c). Thus, it is clear that all three drugs when administered orally in alginate hydrogel particles exhibited a sustained release pattern of drugs in plasma for up to 35 days. Similar observations were made when alginate-encapsulated isoniazid, rifampicin and pyrazinamide were administered in combination.
When the tissue disposition of drugs was examined, sustained release of isoniazid, rifampicin and pyrazinamide was detected in all the organs up to 9 days (Figure 2). The concentrations of isoniazid, rifampicin and pyrazinamide obtained at all the time points were found to be higher than the MICs of respective drugs. When the same dose of free drugs was administered to guinea pigs, isoniazid, rifampicin and pyrazinamide levels were seen in only liver and spleen until 48 h.
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Therapeutic efficacy
The chemotherapeutic efficacy of free/alginate-encapsulated isoniazid (10 mg/kg body weight), rifampicin (12 mg/kg body weight) and pyrazinamide (25 mg/kg body weight) in the lungs and spleens of guinea pigs infected with M. tuberculosis was evaluated (data not shown). Free drugs were administered daily for 8 weeks, whereas alginate-encapsulated drugs were given weekly for the same time period. Treatment in all the groups resulted in no detectable bacilli in spleens and lungs at 1:100 and 1:1000 dilutions of infected tissue homogenates. At the same dilutions, controls exhibited 5.1 log units of M. tuberculosis in lungs and 5.0 log units in spleens. Daily therapy with isoniazid, rifampicin and pyrazinamide exhibited the same results as that of 8 weeks of alginate-encapsulated drugs administered weekly.
Evaluation of the in vivo toxicity of the drugs
In vivo hepatotoxicity induced (in terms of alkaline phosphatase and alanine transaminase) by the antitubercular drugs given in free/alginate-entrapped form was evaluated on day 7 after the last therapeutic dose. Free drugs were capable of inducing toxicity in infected guinea pigs as reflected by the increased activity of alanine aminotransaminase (152.0 ± 39.5 U/L), whereas encapsulated drugs did not show apparent change in activity of this enzyme (46.5 ± 5.7 U/L) compared with normal animals (45.2 ± 1.2 U/L). Alkaline phosphatase levels were almost normal in untreated (58.7 ± 5.6 U/L), free drug (71.6 ± 6.4 U/L) and encapsulated drug treated groups (61.5 ± 15.3 U/L).
Histopathological studies
Histopathology of liver in the control group showed multiple granulomas in portal tract, Langhans type of giant cell infiltration and microscopic foci of necrosis. The free drug treatment group revealed no granulomas in liver, very little inflammation in portal tract and no infiltration in portal veins but fibrosis was observed in the tissues. Fatty steatosis was also seen in the portal veins. The alginate-encapsulated drug treatment group had the same histopathological findings as that of the free drug treated group but calcification was observed in portal tract and liver tissue. Furthermore, rod-shaped acid fast bacilli were seen in liver tissue in controls, whereas no bacilli were seen in liver of treated groups.
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Discussion |
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Alginate microparticles have been examined for in vitro studies of proteins and oral delivery of drugs.6 Insulin has been entrapped to 70% and delivered effectively to mice to control hypoglycaemia. In our study, we observed 2570% entrapment efficiencies of antitubercular drugs in alginate microspheres of size 90100 µm, whereas with PLG microspheres, the maximum antitubercular drug loading was from 8% to 18% as reported earlier,3 thus indicating that alginate hydrogel microparticles have better drug loading capacity than PLG polymers. Furthermore, alginate microspheres containing antitubercular drugs exhibited sustained release in simulated intestinal fluid and limited release in the gastric medium, thereby surviving the harsh gastric environment and providing an excellent system for delivering drugs to the intestine where they are absorbed in the usual manner.15
When drug-loaded alginate microparticles/free drugs were administered orally to guinea pigs, higher Cmax, Tmax, delayed elimination rate and higher bioavailability were observed compared with free drugs. Previously, these drugs in PLG microspheres exhibited five-fold increased relative bioavailability compared with free drugs;16 however, with alginate microspheres the relative bioavailability observed was increased nine-fold. Also, alginate has been found to have bio-adhesive properties that help in delaying the intestinal transit time of the encapsulated compound.5 Therefore, it is suggested that alginate microspheres probably adhere to the intestine mucosa for a prolonged period where they release drug in a sustained manner before being eroded off. In this study, AUC0 was increased significantly for alginate-encapsulated isoniazid; however, the gain in the AUC with rifampicin was small (45125 µg·h/mL). In contrast, substantially higher AUC for rifampicin (367386 µg·h/mL) have been reported in man following the same dose per kg;17 this difference may reflect differences in absorption between species.
The chemotherapeutic efficacy of alginate (encapsulated isoniazid, rifampicin and pyrazinamide) microparticles showed no apparent bacilli in lungs and spleens of guinea pigs after 8 weeks of weekly administration. Comparable clearance of bacilli was observed in the organs of guinea pigs treated with alginate-entrapped/free drugs; however, free drugs were administered daily for 8 weeks and alginate-entrapped drugs were given weekly for the same time. In our earlier reports, the chemotherapeutic potential of liposomal drugs2 and PLG-encapsulated antitubercular drugs18 has been evaluated in mice where the bacterial load in organs was reduced only by 12 log units, whereas in the present study we were able to achieve an apparent sterilization in lungs and spleens of guinea pigs with 8 weeks of therapy with alginate-entrapped drugs administered weekly. Entrapped drug groups showed normal liver morphology, whereas untreated animals exhibited characteristic histopathological changes in the liver tissue. Few reports of complete clearance of bacilli from organs of mice after treatment with isoniazid, rifabutin and pyrazinamide are available, which is probably due to pyrazinamide, a potent sterilizing agent.19,20 Our results clearly demonstrate that 57 doses of free drugs can be replaced with eight doses of alginate-entrapped drugs to achieve almost complete clearance of bacilli within 8 weeks in guinea pigs. Earlier reports2123 indicated that isoniazid in slow release preparations when administered at higher doses gave adverse side effects. In our study we did not observe toxicity with alginate-encapsulated isoniazid when administered in combination with rifampicin and pyrazinamide at therapeutic doses; however, free drugs at the same dose indicated biochemical toxicity in guinea pigs.
In brief, alginate-entrapped antitubercular drugs can be delivered orally weekly to improve antimycobacterial therapy, thus resulting in improved compliance by the patients.
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Acknowledgements |
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Footnotes |
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References |
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