**2. Polymeric microparticles for tuberculosis treatment**

148 Polyurethane

form biologically acceptable degradation products; the rate of drug release from the carrier

The design of the PU controlled-release forms for therapeutic drug administration is the subject of intense interest. Such systems are being used for sustained and controlled delivery of various pharmaceutical agents such as prednisolon (Sharma et al., 1988), morphine, caffeine (Graham et al., 1988), prostaglandin (Embrey et al., 1986) and theophylline (Reddy et al., 2006). The PU carrier is utilized to deliver iodine-containing drugs (Touitou & Friedman, 1984). Urethane-based hydrogels were prepared based on the reaction of diisocyanates with amphilic ethylene oxide and triol crosslinker to deliver propranolol hydrochloride, an antihypertensive drug (Van Bos & Schacht, 1987). Drug delivery systems on a PU base with various antitumorous drugs, such as cyclophosphane, thiophosphamide and vincristine, have been prepared (Iskakov et al., 1998, 2000). An in vitro technique was used to determine the release characteristics of the drugs into model biological media. It was

PU-based drug delivery systems have considerable potential for treatment of tuberculosis. Tuberculosis is widely spread disease in most developing countries. The main method of tuberculosis treatment is chemotherapy. Although current chemotherapeutic agents for tuberculosis treatment are therapeutically effective and well tolerated, a number of problems remain. The chemotherapy is burden some, extends over long periods and requires continuous and repeated administration of large drug doses. Thus, traditional drug chemotherapy has serious limitations because of increasing microbial drug resistance and toxico-allergic side effects. One of the ultimate problems in effective treatment of tuberculosis is patient compliance. These problems of increasing drug resistance, toxicoallergic side effects, patient compliance can be approached by the use of long-acting polymeric drug delivery systems (Sosnik et al., 2010). The design of implantable systems containing the antituberculosis drugs in combination with biocompatible polymers would make possible to achieve the significant progress in treatment of this global debilitating

Biodegradable microsphere drug delivery systems have shown application for oral and parenteral administration. Administration of microparticles to the lungs (alveolar region) may provide the opportunity for the prolonged delivery active agent to tuberculosis infected macrophages. Microspheres can be produced to meet certain morphological requirements such as size, shape and porosity by varying the process parameters. However, the morphology of the lung is such that to achieve effective drug deposition it is necessary

The objective of the chapter is to develop an effective polymeric drug delivery systems based on PU for the treatment of tuberculosis. Polyurethane materials are investigated as carriers for sustained and controlled release of antituberculosis drugs. The synthesis and characterization of PU microcapsules are studied making use various molecular weight polyethylene glycol and tolylene-2,4-diisocyanate. Antituberculosis drug isoniazid (Is), rifampicin, ethionamide and florimicin were incorporated into the PU microcapsules and

must occur at an acceptable rate; the carrier must be able to be easily sterilized.

shown the drug release occurs in accordance with first-order kinetics.

disease *(*Shegokar et al., 2011).

to control the particle size of microparticles.

Recent trends in polymeric controlled drug delivery have seen microencapsulation of pharmaceutical substances in biodegradable polymers as an emerging technology. Extensive progressive efforts have been made to develop various polymeric drug delivery systems to either target the site of tuberculosis infection or reduce the dosing frequency (Toit et al., 2006). Carriers as microspheres have been developed for the sustained delivery of antituberculosis drugs and have demonstrated better chemotherapeutic efficacy when investigated in animal models. Antituberculosis drugs have been successfully entrapped in microparticles of natural and synthetic polymers such as alginate (ALG), ALG-chitosan, poly-lactide-co-glycolide and poly-butyl cyanoacrylate (Gelperina et al., 2005, Pandey & Khuller, 2006).

ALG, a natural polymer, has attracted researchers owing to its ease of availability, compatibility with hydrophobic as well as hydrophilic molecules, biodegradability under physiological conditions, lack of toxicity and the ability to confer sustained release potential. The ability of ALG to co-encapsulate multiple antitubercular drugs and offer a controlled release profile is likely to have a major impact in enhancing patient compliance for better management of tuberculosis (Ahmad & Khuller, 2008).

Spherical microspheres able to prolong the release of Is were produced by a modified emulsification method, using sodium ALG as the hydrophilic carrier (Rastogi et al., 2007). The particles were heterogeneous with the maximum particles of an average size of 3.719 μm. Results indicated that the mean particle size of the microspheres increased with an increase in the concentration of polymer and the cross-linker as well as the cross-linking time. The entrapment efficiency was found to be in the range of 40-91%. Concentration of the cross-linker up to 7.5% caused increase in the entrapment efficiency and the extent of drug release. Optimized Is-ALG microspheres were found to possess good bioadhesion. The bioadhesive property of the particles resulted in prolonged retention in the small intestine. Microspheres could be observed in the intestinal lumen at 4h and were detectable in the intestine 24h post-oral administration. Increased drug loading (91%) was observed for the optimized formulation suggesting the efficiency of the method. Nearly 26% of Is was released in simulated gastric fluid pH 1.2 in 6h and 71.25% in simulated intestinal fluid pH 7.4 in 30h.

ALG microparticles were developed as oral sustained delivery carriers for antituberculosis drugs in order to improve patient compliance (Qurrat-ul-Ain et al., 2003). Pharmacokinetics and therapeutic effects of ALG microparticle encapsulated Is, rifampicin and pyrazinamide were examined in guinea pigs. ALG microparticles containing antituberculosis drugs were evaluated for in vitro and in vivo release profiles. These microparticles exhibited sustained

release of Is, rifampicin and pyrazinamide for 3-5 days in plasma and up to 9 days in organs. Peak plasma concentration, elimination half-life and infinity of ALG drugs were significantly higher than those of free drugs. The encapsulation of drug in ALG microparticles resulted in up to a nine-fold increase in relative bioavailability compared with free drugs. Chemotherapeutic efficacy of ALG drug microspheres against experimental tuberculosis not detectable at 1:100 and 1:1000 dilutions of spleen and lung homogenates. Histopathological studies further substantiated these observations, thus suggesting that application of ALG-encapsulated drugs could be useful in the effective treatment of tuberculosis.

Polyurethane as Carriers of Antituberculosis Drugs 151

pigs, administration of a therapeutic dose of microspheres spaced 10 days apart produced a

Polylactide-co-glycolide (PLG) polymers are biodegradable and biocompatible, they have been the most commonly used as carriers for microparticle formulations. Monodispersed poly-lactic-co-glycolic acid (PLGA) microspheres containing rifampicin have been prepared by solvent evaporation method (Makino et al., 2004, Yoshida et al., 2006). The microspheres were spherical and their average diameter was about 2 μm. The loading efficiency of rifampicin was dependent on the molecular weight of PLGA. The higher loading efficiency was obtained by the usage of PLGA with the lower Mw, which may be caused by the interaction of the amino groups of rifampicin with the terminal carboxyl groups of PLGA. PLGA with the monomer compositions of 50/50 and 75/25, of lactic acid/glycolic acid, were used in this study. From rifampicin-loaded PLGA microspheres formulated using PLGA with the Mw of 20,000, rifampicin was released with almost constant rate for 20 days after the lag phase was observed for the initial 7 days at pH 7.4. On the other hand, from rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 5000 or 10,000, almost 90% of rifampicin-loaded in the microspheres was released in the initial 10 days. Highly effective delivery of rifampicin to alveolar macrophages was observed by the usage of rifampicin-loaded PLGA microspheres. Almost 19 times higher concentration of rifampicin was found to be incorporated in alveolar macrophages when rifampicin-loaded PLGA microspheres were added to the cell culture medium than when

Controlled release rifampicin-loaded microspheres were evaluated in nonhuman primates (Quenelle et al., 2004). These microsheres were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Animals received either 2.0 g of a large formulation (10–150 mcm, 23 wt% rifampicin) injected subcutaneously at Day 0 (118–139 mg rifampin/kg), 4.0 g of a small formulation (1–10 mcm, 5.8 wt% rifampicin) administered intravenously in 2.0 g doses on Day 0 and 7 (62.7–72.5 mg rifampicin/kg), or a combination of small and large microspheres (169–210 mg rifampin/kg). Extended rifampicin release was observed up to 48 days. Average rifampicin concentrations remaining in the liver, lung, and

PLG nanoparticles encapsulating streptomycin were prepared by the multiple emulsion technique and administered orally to mice for biodistribution and chemotherapeutic studies (Pandey & Khuller, 2007). The mean particle size was 153.12 nm with 32.12±4.08% drug encapsulation and 14.28±2.83% drug loading. Streptomycin levels were maintained for 4 days in the plasma and for 7 days in the organs following a single oral administration of PLG nanoparticles. There was a 21-fold increase in the relative bioavailability of PLGencapsulated streptomycin compared with intramuscular free drug. In *Mycobacterium tuberculosis (M.tuberculosis)* H37Rv infected mice, eight doses of the oral streptomycin formulation administered weekly were comparable to 24 intramuscular injections of free

spleen at 30 days were 14.03, 4.09, and 1.98 μg/g tissue, respectively.

clearance of bacilli equivalent to conventional treatment for 6 weeks.

rifampicin solution was added.

streptomycin.

Pharmacokinetics and tissue distribution of free and ALG-encapsulated antituberculosis drugs were evaluated in mice at different doses (Ahmad et al., 2006). ALG nanoparticles encapsulating Is, rifampicin, pyrazinamide and ethambutol were prepared by controlled cation-induced gelification of ALG. The formulation was orally administered to mice at two dose levels. A comparison was made in mice receiving free drugs at equivalent doses. The relative bioavailabilities of all drugs encapsulated in ALG nanoparticles were significantly higher compared with free drugs. Drug levels were maintained at or above the minimum inhibitory concentration until 15 days in organs after administration of encapsulated drugs, whilst free drugs stayed at or above 1 day only irrespective of dose. The levels of drugs in various organs remained above the minimum inhibitory concentration at both doses for equal periods, demonstrating their equiefficiency.

Chemotherapeutic potential of ALG nanoparticle-encapsulated econazole and antituberculosis drugs were studied against murine tuberculosis (Ahmad et al., 2007). Econazole (free or encapsulated) could replace rifampicin and Is during chemotherapy. Eight doses of ALG nanoparticle-encapsulated econazole or 112 doses of free econazole reduced bacterial burden by more than 90% in the lungs and spleen of mice infected with Mycobacterium tuberculosis. ALG nanoparticles reduced the dosing frequency of azoles and antitubercular drugs by 15-fold.

Is was encapsulated into microspheres of ALG-chitosan by means of a complex coacervation method in an emulsion system (Lucinda-Silva & Evangelista, 2003). The particles were prepared in three steps: preparation of a emulsion phase and adsorption of the drug. The results showed that microspheres of ALG-chitosan obtained were of spherical shape. The emulsion used for microparticle formation allows the preparation of particles with a narrow size distribution. The adsorption observed is probably of chemical nature, i.e. there is an ionic interaction between the drug and the surface of the particles.

ALG-chitosan microspheres encapsulating rifampicin, Is and pyrazinamide, were formulated (Pandey & Khuller, 2004). A therapeutic dose and a half-therapeutic dose of the microsphere-encapsulated were orally administered to guinea pigs for pharmacokinetic and chemotherapeutic evaluations. The drug encapsulation efficiency ranged from 65% to 85% with a loading of 220-280 mg of drug per gram microspheres. Administration of a single oral dose of the microspheres to guinea pigs resulted in sustained drug levels in the plasma for 7 days and in the organs for 9 days. In Mycobacterium tuberculosis H37Rv-infected guinea pigs, administration of a therapeutic dose of microspheres spaced 10 days apart produced a clearance of bacilli equivalent to conventional treatment for 6 weeks.

150 Polyurethane

tuberculosis.

equal periods, demonstrating their equiefficiency.

and antitubercular drugs by 15-fold.

release of Is, rifampicin and pyrazinamide for 3-5 days in plasma and up to 9 days in organs. Peak plasma concentration, elimination half-life and infinity of ALG drugs were significantly higher than those of free drugs. The encapsulation of drug in ALG microparticles resulted in up to a nine-fold increase in relative bioavailability compared with free drugs. Chemotherapeutic efficacy of ALG drug microspheres against experimental tuberculosis not detectable at 1:100 and 1:1000 dilutions of spleen and lung homogenates. Histopathological studies further substantiated these observations, thus suggesting that application of ALG-encapsulated drugs could be useful in the effective treatment of

Pharmacokinetics and tissue distribution of free and ALG-encapsulated antituberculosis drugs were evaluated in mice at different doses (Ahmad et al., 2006). ALG nanoparticles encapsulating Is, rifampicin, pyrazinamide and ethambutol were prepared by controlled cation-induced gelification of ALG. The formulation was orally administered to mice at two dose levels. A comparison was made in mice receiving free drugs at equivalent doses. The relative bioavailabilities of all drugs encapsulated in ALG nanoparticles were significantly higher compared with free drugs. Drug levels were maintained at or above the minimum inhibitory concentration until 15 days in organs after administration of encapsulated drugs, whilst free drugs stayed at or above 1 day only irrespective of dose. The levels of drugs in various organs remained above the minimum inhibitory concentration at both doses for

Chemotherapeutic potential of ALG nanoparticle-encapsulated econazole and antituberculosis drugs were studied against murine tuberculosis (Ahmad et al., 2007). Econazole (free or encapsulated) could replace rifampicin and Is during chemotherapy. Eight doses of ALG nanoparticle-encapsulated econazole or 112 doses of free econazole reduced bacterial burden by more than 90% in the lungs and spleen of mice infected with Mycobacterium tuberculosis. ALG nanoparticles reduced the dosing frequency of azoles

Is was encapsulated into microspheres of ALG-chitosan by means of a complex coacervation method in an emulsion system (Lucinda-Silva & Evangelista, 2003). The particles were prepared in three steps: preparation of a emulsion phase and adsorption of the drug. The results showed that microspheres of ALG-chitosan obtained were of spherical shape. The emulsion used for microparticle formation allows the preparation of particles with a narrow size distribution. The adsorption observed is probably of chemical nature, i.e. there is an

ALG-chitosan microspheres encapsulating rifampicin, Is and pyrazinamide, were formulated (Pandey & Khuller, 2004). A therapeutic dose and a half-therapeutic dose of the microsphere-encapsulated were orally administered to guinea pigs for pharmacokinetic and chemotherapeutic evaluations. The drug encapsulation efficiency ranged from 65% to 85% with a loading of 220-280 mg of drug per gram microspheres. Administration of a single oral dose of the microspheres to guinea pigs resulted in sustained drug levels in the plasma for 7 days and in the organs for 9 days. In Mycobacterium tuberculosis H37Rv-infected guinea

ionic interaction between the drug and the surface of the particles.

Polylactide-co-glycolide (PLG) polymers are biodegradable and biocompatible, they have been the most commonly used as carriers for microparticle formulations. Monodispersed poly-lactic-co-glycolic acid (PLGA) microspheres containing rifampicin have been prepared by solvent evaporation method (Makino et al., 2004, Yoshida et al., 2006). The microspheres were spherical and their average diameter was about 2 μm. The loading efficiency of rifampicin was dependent on the molecular weight of PLGA. The higher loading efficiency was obtained by the usage of PLGA with the lower Mw, which may be caused by the interaction of the amino groups of rifampicin with the terminal carboxyl groups of PLGA. PLGA with the monomer compositions of 50/50 and 75/25, of lactic acid/glycolic acid, were used in this study. From rifampicin-loaded PLGA microspheres formulated using PLGA with the Mw of 20,000, rifampicin was released with almost constant rate for 20 days after the lag phase was observed for the initial 7 days at pH 7.4. On the other hand, from rifampicin-loaded PLGA microspheres formulated using PLGA with the molecular weight of 5000 or 10,000, almost 90% of rifampicin-loaded in the microspheres was released in the initial 10 days. Highly effective delivery of rifampicin to alveolar macrophages was observed by the usage of rifampicin-loaded PLGA microspheres. Almost 19 times higher concentration of rifampicin was found to be incorporated in alveolar macrophages when rifampicin-loaded PLGA microspheres were added to the cell culture medium than when rifampicin solution was added.

Controlled release rifampicin-loaded microspheres were evaluated in nonhuman primates (Quenelle et al., 2004). These microsheres were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Animals received either 2.0 g of a large formulation (10–150 mcm, 23 wt% rifampicin) injected subcutaneously at Day 0 (118–139 mg rifampin/kg), 4.0 g of a small formulation (1–10 mcm, 5.8 wt% rifampicin) administered intravenously in 2.0 g doses on Day 0 and 7 (62.7–72.5 mg rifampicin/kg), or a combination of small and large microspheres (169–210 mg rifampin/kg). Extended rifampicin release was observed up to 48 days. Average rifampicin concentrations remaining in the liver, lung, and spleen at 30 days were 14.03, 4.09, and 1.98 μg/g tissue, respectively.

PLG nanoparticles encapsulating streptomycin were prepared by the multiple emulsion technique and administered orally to mice for biodistribution and chemotherapeutic studies (Pandey & Khuller, 2007). The mean particle size was 153.12 nm with 32.12±4.08% drug encapsulation and 14.28±2.83% drug loading. Streptomycin levels were maintained for 4 days in the plasma and for 7 days in the organs following a single oral administration of PLG nanoparticles. There was a 21-fold increase in the relative bioavailability of PLGencapsulated streptomycin compared with intramuscular free drug. In *Mycobacterium tuberculosis (M.tuberculosis)* H37Rv infected mice, eight doses of the oral streptomycin formulation administered weekly were comparable to 24 intramuscular injections of free streptomycin.

PLG nanoparticle-encapsulated econazole and moxifloxacin have been evaluated against murine tuberculosis (drug susceptible) in order to develop a more potent regimen for tuberculosis (Ahmad et al., 2008). PLG nanoparticles were prepared by the multiple emulsion and solvent evaporation technique and were administered orally to mice. A single oral dose of PLG nanoparticles resulted in therapeutic drug concentrations in plasma for up to 5 days (econazole) or 4 days (moxifloxacin), whilst in the organs (lungs, liver and spleen) it was up to 6 days. In comparison, free drugs were cleared from the same organs within 12- 24h. In *M. tuberculosis*-infected mice, eight oral doses of the formulation administered weekly were found to be equipotent to 56 doses (moxifloxacin administered daily) or 112 doses (econazole administered twice daily) of free drugs. Furthermore, the combination of moxifloxacin+econazole proved to be significantly efficacious compared with individual drugs. Addition of rifampicin to this combination resulted in total bacterial clearance from the organs of mice in 8 weeks. PLG nanoparticles appear to have the potential for intermittent therapy of tuberculosis, and combination of moxifloxacin, econazole and rifampicin is the most potent.

Polyurethane as Carriers of Antituberculosis Drugs 153

PU microparticles could be interesting matrices for controlled drug delivery. Aliphatic PU Tecoflex was evaluated as microsphere matrix for the controlled release of theophylline (Subhaga et al., 1995). PU microspheres were prepared using solvent evaporation technique from a dichloromethane solution of the polymer containing the drug. A dilute solution of poly(vinyl alcohol) served as the dispersion medium. Microspheres of good spherical geometry having theophylline content of 35% could be prepared by the technique. The release of the drug from the microspheres was examined in simulated gastric and intestinal fluids. While a large burst effect was observed in gastric fluid, in the intestinal fluid a close

Microencapsulation of theophylline in PU was developed with 4, 4'-methylenediphenylisocyanate, castor oil and ethylene diamine as chain extender (Rafienia et al., 2006). PU microspheres were prepared in two steps pre-polymer preparation and microspheres formation. Particle size investigation with optical microscopy revealed size distribution of 27–128 μm. Controlled release experiment of theophylline was performed in phosphate buffered saline at pH 7.4 with UV-spectrometer at 274 nm. Drug release profiles showed

The effect of chain-extending agent on the porosity and release behavior of biologically active agent diazinon from PU microspheres were studied (Jabbari & Khakpour, 2000). Microsphere was prepared using a two-step suspension polycondensation method with methylene diphenyl diisocyanate, polyethylene glycol 400 and 1,4-butanediol as the chainextending agent. Chain-extending agent was used to increase the ratio of hard to soft segments of the PU network, and its effect on microsphere morphology was studied with SEM. According to the results, porosity was significantly affected by the amount of chainextending agent. The pore size decreased as the concentration of chain-extending agent increased from zero to 50 mole%. With further increase of chain-extending agent to 60 and 67%, PU chains became stiffer and formation of pores was inhibited. Therefore, pore morphology was significantly affected by variations in the amount of chain-extending agent. The release behavior of microspheres was investigated with diazinon as the active agent. After an initial burst, corresponding to 3% of the incorporated amount of active agent, the

PU polymers and poly(ether urethane) copolymers were chosen as drug carriers for alphatocopherol (Bouchemal et al., 2004). This active ingredient is widely used as a strong antioxidant in many medical and cosmetic applications, but is rapidly degraded, because of its light, heat and oxygen sensitivity. PU and poly(ether urethane)-based nanocapsules were synthesized by interfacial reaction between two monomers. Interfacial polycondensation combined with spontaneous emulsification is a new technique for nanoparticles formation. Nanocapsules were characterized by studying particle size (150-500 nm), pH, yield of encapsulation and morphologies. Polyurethanes were obtained from the condensation of isophorone diisocyanate and 1,2-ethanediol, 1,4-butanediol , 1,6-hexanediol. Poly(ether urethane) copolymers were obtained by replacing diols by polyethylene glycol oligomers Mw 200, 300, 400 and 600. Mw of di- and polyols have a considerable influence on

initial release of 2–40% and further release for more than 10 days.

to zero-order release was seen.

release rate was zero order.

Antituberculosis drugs Is, rifampicin, streptomycin and moxifloxacin have been encapsulated in poly(butyl cyanoacrylate) nanoparticles (Anisimova et al., 2000, Kisich et al, 2007). Incorporation of drugs in polymeric nanoparticles not only increased the intracellular accumulation of these drugs in the cultivated human blood monocytes but also produced enhanced antimicrobial activity of these agents against intracellular *M. tuberculosis* compared with their activity in extracellular fluid. Encapsulated moxifloxacin accumulated in macrophages approximately three-fold times more efficiently than the free drug, and was detected in the cells for at least six times longer than free moxifloxacin at the same extracellular concentration.

This brief review suggested that micro- and nanoparticles based delivery systems have a considerable potential for treatment of tuberculosis. Their major advantages, such as improvement of drug bioavailability and reduction of the dosing frequency, may create a sound basis for better management of the disease, making directly observed treatment more practical and affordable.
