P.R.J. Gangadharam

Effect of polymer foam morphology and density on kinetics of in vitro controlled release of isoniazid from compressed foam matrices.

The purpose of this study was to compare the effect of polymer foam morphology and density prior to compaction on the kinetics of isoniazid (INH) release from the final high-density extruded matrices. The feasibility of preparing low density foams of several biopolymers, including poly(L-lactide) (PLLA), poly(glycolide) (PGA), poly(DL-lactide-co-glycolide) (PLGA), poly(gamma-benzyl-L-glutamate) (PBLG), and poly(propylene fumarate) (PPF), via a lyophilization technique was investigated. Low-density foams of PLGA, PBLG, and a mixture of PLGA and PPF were successfully fabricated by lyophilization of the frozen polymer solutions either in glacial acetic acid or in benzene. The morphology of these foams depends on the polymer as well as the solvent used in the fabrication process. Thus, PLGA produces a capillary structure when lyophilized from benzene solution and a leaflet structure from glacial acetic acid, but PBLG yields a leaflet structure from benzene. Matrices were prepared by impregnating these foams with aqueous solutions of INH, removing the water by a second lyophilization, and then compressing the low-density INH containing foams by compaction and high-pressure extrusion. The resulting nonporous matrices had densities of approximately 1.30 g/cm3. In vitro kinetics were in accord with the Roseman-Higuchi diffusion model and demonstrate that release rates depend on the initial foam density, while foam structure has little influence on the release kinetics.

Antituberculosis activity of clarithromycin.

Antituberculosis activity of clarithromycin (CLA), a macrolide antibiotic, was investigated in vitro, in macrophages, and in C57BL/6 mice, CLA showed high in vitro MICs (4 to > 16 micrograms/ml) for several strains of Mycobacterium tuberculosis and caused slight enhancement of activity of rifampin (RIF) against H37Rv but failed to increase the activity of either RIF or isoniazid (INH) against other strains. However, inside J774A.1 macrophages, CLA showed high activity and was synergistic with RIF against some strains of tubercle bacilli susceptible or resistant to INH and RIF. In the in vivo studies with a drug-susceptible strain (H37Rv), CLA protected mice from mortality due to tuberculosis for up to 8 weeks of observation. The CFU data for lungs and spleens revealed that the antituberculosis activity of CLA is inferior to those of INH and streptomycin. However, the activity of CLA when used alone or in combination was comparable to that of thiacetazone, indicating its potential usefulness as a secondary drug for the treatment of tuberculosis.

In vitro activity of the benzoxazinorifamycin KRM-1648 against drug-susceptible and multidrug-resistant tubercle bacilli.

We investigated the activity of benzoxazinorifamycin (KRM-1648) against several drug-susceptible and multidrug-resistant strains of tubercle bacilli. Since KRM-1648 is a rifamycin derivative, we included some strains of Mycobacterium tuberculosis resistant to rifampin (RIF) among the multidrug-resistant strains. For RIF-susceptible strains, the MIC of KRM-1648 was much lower than that of RIF (MICs of KRM-1648 and RIF at which 90% of strains are inhibited, < or = 0.015 and < or = 0.25 micrograms/ml, respectively). The MBC of KRM-1648 (range, 0.007 to 0.03 microgram/ml) was also much lower than that of RIF (range, 0.5 to 1.0 microgram/ml). Postantibiotic effect studies with KRM-1648 showed a rapid reduction in the CFU counts with an exposure of 24 h or more, and its sterilizing effect was maintained even up to 21 days thereafter. Parallel postantibiotic effect studies with RIF showed a less significant effect with a faster recovery of growth, and RIF failed to sterilize the organisms even after 72 h of exposure. KRM-1648 at 0.125 and 0.25 microgram/ml caused complete inhibition of intracellular growth of M. tuberculosis in J774 A.1 macrophages after 48 h of exposure. After a similar exposure time RIF at a concentration of 0.25 microgram/ml caused complete inhibition of growth, but a concentration of 0.125 microgram/ml caused only a 50% reduction in growth compared with that of controls at day 7. With 24 h of pulsed exposure of the intracellular organisms to 0.25 micrograms of the drugs per ml, KRM-1648 caused complete inhibition of intracellular growth, while RIF caused only moderate inhibition of intracellular growth. These findings suggest that KRM-1648 is a potentially useful drug for the treatment of tuberculosis.

Therapy of Mycobacterium avium complex infections in beige mice with streptomycin encapsulated in sterically stabilized liposomes.

Mycobacterium avium complex (MAC) causes serious opportunistic infections in AIDS patients. Previous studies with MAC-infected beige mice have indicated that weekly administration of liposome-encapsulated streptomycin can reduce significantly the CFU in the liver and spleen. We examined whether streptomycin encapsulated in recently developed sterically stabilized liposomes with prolonged circulation times would have a therapeutic effect in this animal model. Two liposome types with prolonged circulation (polyethyleneglycol-distearoylphosphatidylethanolamine [PEG-DSPE]-distearoylphosphatidylcholine [DSPC]-cholesterol [chol] or phosphatidylinositol [PI]-DSPC-chol) and conventional liposomes (phosphatidylglycerol [PG]-phosphatidylcholine [PC]-chol) encapsulating streptomycin and administered twice weekly were bactericidal to MAC strain 101 in the spleen when the level of infection after treatment was compared with the level of infection before treatment. PI-DSPC-chol and PG-PC-chol liposomes encapsulating streptomycin were bactericidal in the liver. Although PG-PC-chol or PEG-DSPE-DSPE-chol liposomes encapsulating streptomycin were not bactericidal in the lungs, they reduced the level of MAC infection by more than 3 orders of magnitude compared with the level of MAC infection in untreated controls.

Antimycobacterial activity of a new rifamycin derivative, 3-(4-cinnamylpiperazinyl iminomethyl) rifamycin SV (T9).

The antimycobacterial activities of a new rifampin (RIF) derivative, 3-(4-cinnamylpiperazinyl iminomethyl) rifamycin SV (To), against 20 susceptible and multidrug-resistant strains of Mycobacterium tuberculosis and 20 Mycobacterium avium complex (MAC) strains were investigated. The radiometric MICs of T9 for M. tuberculosis were significantly lower than those of RIF. The MICs of T9 and RIF at which 90% of the RIF-susceptible strains were inhibited were < or = 0.25 and < or = 0.5 micrograms/ml, respectively. Interestingly, T9 had lower MICs against some RIF-resistant M. tuberculosis strains. T9 had better activity against MAC strains, and the MIC at which 90% of the MAC strains were inhibited was < or = 0.125 micrograms/ml, and that of RIF was < or = 2.0 micrograms/ml. T9 also showed high in vitro bactericidal and intracellular activities which were significantly superior to those of RIF against both M. tuberculosis, and MAC strains. More importantly, T9 showed excellent in vivo activity against M. tuberculosis H37Rv compared with that of RIF in both the lungs and spleens of C57BL/6 mice, indicating the potential therapeutic value of T9 in the treatment of mycobacterial infections.

In vitro controlled release of isoniazid from poly (lactide-co-glycolide) matrices

Abstract In vitro release kinetics of isoniazid (INH) from a biodegradable polymeric matrix has been investigated for systems prepared under varying fabrication conditions. Matrices were prepared either by evaporation of co-solutions followed by extrusion under controlled temperature and pressure, or by dry mixing INH with PLGA which had been preground and sieved to isolate specific particle size ranges prior to extrusion. The effect on release kinetics of loading, polymer particle size, extrusion pressure, and rod diameter was explored. Fractional release as a function of time is shown to fit the Roseman-Higuchi model in that plots of (1 − F )ln(1 − F ) + F are linear with time where F is the fraction of drug released at time t . The effect of varying fabrication parameter values on the slope (identified as the combined rate constant for drug release, K = 4 CD / Aa 0 2 ) is as follows: K increases with decreasing polymer particle size, lower extrusion pressure, lower loading. The effect of increasing the rod diameter results in a small increase in K . Other parameter values being equal, solvent cast systems released more rapidly than those prepared without the use of solvent.

Rapid assessment of mycobacterial growth inside macrophages and mice, using the radiometric (BACTEC) method

SETTING Conventional methods of determining the growth and drug susceptibility of mycobacteria either in macrophages or in vivo are time consuming and laborious. OBJECTIVE We sought to develop a simple and rapid method for assessing growth and susceptibility of mycobacteria in macrophages and in vivo. DESIGN The radiometric (BACTEC) method was compared with the conventional method for rapid assessment of growth and susceptibility of Mycobacterium avium complex (MAC) to clofazimine (CFM) and its two analogues (B4100 and B4101) in macrophages and in beige mice. RESULTS AND CONCLUSIONS A linear relationship was observed between growth index (GI) readings by the BACTEC method and the colony forming unit (CFU) counts obtained by conventional plating on 7H11 agar in vitro of lysates from infected macrophages and of homogenates from the infected organs. There was also a good agreement between the two methods with respect to susceptibility of MAC to CFM and its analogues both in macrophages and in spleen. The BACTEC method, in addition to being quick and simple, was found to be more consistent. Our studies thus demonstrate that the radiometric method has potential application in rapid screening of drugs for antimycobacterial activity in macrophages and in animals besides its established value in in vitro screening.

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

AbstractWe review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macr...

In vitro and in vivo synergistic effect of isoniazid with streptomycin and clofazimine against Mycobacterium avium complex (MAC).

SETTING Isoniazid (INH), the powerful antituberculosis drug, has also been used in regimens for treating disease caused by Mycobacterium avium complex (MAC), an important opportunistic pathogen encountered in AIDS patients. Its use for treatment of MAC disease has also been endorsed by the American Thoracic Society. However some controversy has emerged recently in medical literature, discounting its role and even implying that its use is contraindicated in chemotherapy of MAC disease. OBJECTIVE In view of the controversy, we investigated its in vitro and in vivo activity in combination with streptomycin (SM) and clofazimine (CFM) against MAC. DESIGN In the in vitro studies, the minimal inhibitory concentrations (MIC) of individual drugs or combinations of INH and SM as well as INH and CFM were determined in a checker-board type study by both conventional and radiometric (BACTEC) methods. In vivo studies assessed the efficacy of chemotherapy with INH alone or in combination with either SM or CFM against MAC infection in beige mice. RESULTS AND CONCLUSIONS While MICs of INH and SM were 12.5 micrograms/ml and 6.25 micrograms/ml respectively, complete inhibition of growth was seen at 1.56 micrograms/ml with the combination of both drugs. The synergistic effect was observed both in conventional and BACTEC methods. In vivo studies demonstrated elevated activity when INH was given along with SM or CFM. Based on these observations we stress that isoniazid has still a place in chemotherapy of MAC disease, at least until other potent drugs are discovered.

Original articleMaintenance of therapeutically active levels of isoniazid for prolonged periods in rabbits after a single implant of biodegradable polymer

SETTING Poor patient compliance is the serious limiting factor in the chemotherapy of tuberculosis. OBJECTIVE To solve this problem we have been developing depot-drug delivery of antimycobacterial drugs. Earlier studies with mice using isoniazid in polylactic-co-glycolic acid (PLGA) co-polymer have shown that a single implant of the polymer could ensure sustained levels of free isoniazid for up to 8 weeks. Similar studies were not undertaken in rabbits. DESIGN The biodegradable PLGA polymer rods containing isoniazid were implanted on the back of the rabbits under anaesthesia in an isoniazid dose of 90 mg/kg. Concentrations of isoniazid and acetylisoniazid in serum and urine were determined by the high-performance liquid chromatography (HPLC) method at close intervals up to 96 h to study the burst-size, and later at weekly intervals up to 9 weeks to study the sustained levels. RESULTS AND CONCLUSIONS There was no abnormal release of isoniazid in the earlier periods. Concentrations of isoniazid > or = 0.2 microgram/ml were found both in serum and urine up to 63 days after implant. Urine specimen obtained at 6 weeks after giving the implant inhibited the growth of Mycobacterium tuberculosis in vitro as measured by the radiometric (Bactec) method. These findings in non-rodent animal species confirm the usefulness of the depot-drug delivery method of drug administration and warrant hopes for the successful treatment of tuberculosis avoiding the problem of non-compliance.