Comprehensive in vitro analysis evaluating the variable drug-drug interaction risk of rifampicin compared to rifabutin.

Compared to rifampicin (600 mg/day), standard doses of rifabutin (300 mg/day) have a lower risk of drug-drug interactions due to induction of cytochrome P450 3A4 (CYP3A4) or P-glycoprotein (Pgp/ABCB1) mediated by the pregnane X receptor (PXR). However, clinical comparisons with equal rifamycin doses or in vitro experiments respecting actual intracellular concentrations are lacking. Thus, the genuine pharmacological differences and the potential molecular mechanisms of the discordant perpetrator effects are unknown. Consequently, the cellular uptake kinetics (mass spectrometry), PXR activation (luciferase reporter gene assays), and impact on CYP3A4 and Pgp/ABCB1 expression and activity (polymerase chain reaction, enzymatic assays, flow cytometry) were evaluated in LS180 cells after treatment with different rifampicin or rifabutin concentrations for variable exposure times and eventually normalized to actual intracellular concentrations. In addition, inhibitory effects on CYP3A4 and Pgp activities were investigated. While rifampicin is poorly taken up by LS180 cells, it strongly activates PXR and leads to enhanced expression and activity of CYP3A4 and Pgp. In contrast, rifabutin is a significantly less potent and less efficient PXR activator and gene inducer, despite sixfold to eightfold higher intracellular accumulation. Finally, rifabutin is a potent inhibitor of Pgp (IC50 = 0.3 µM) compared to rifampicin (IC50 = 12.9 µM). Together, rifampicin and rifabutin significantly differ by their effects on the regulation and function of CYP3A4 and Pgp, even when controlled for intracellular concentrations. Rifabutin's concurrent Pgp inhibitory action might partly compensate the inducing effects, explaining its weaker clinical perpetrator characteristics.

Activity of Rifabutin and Hemi-synthetic Derivatives Against Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus causes a wide range of clinical diseases that are difficult to treat. This microorganism is resistant not only to the classical antituberculosis agents but also to most of the antimicrobials that are currently available, resulting in limited therapeutic options and treatment failure. This scenario stresses the need to search for new drugs with activity against M. abscessus. OBJECTIVE: To evaluate in vitro the antimycobacterial activity and cytotoxicity of rifabutin (RFB 1) and ten derivatives (2-11) against M. abscessus ATCC 19977. METHOD: The minimum inhibitory concentration (MIC) of the molecules was determined by the microdilution broth method according to the guideline described in CLSI. The toxicity evaluation was carried in 96-well microplates, using the cell line J774A.1 (ATCC TIB-67). RESULT: From the eleven molecules tested, RFB 1 and RFB 4 were the compounds showing higher activities against M. abscessus, with MICs of 0.9 and 1.0 µM, respectively. The R1 and R2 moieties seem to have deciding influence over the final activity. Furthermore, N-oxide derivatives 9, 10, and 11 were also active against M. abscessus, with MICs of 7.2 µM, 1.8 µM and 3.8 µM, respectively. An explanatory hypothesis for the better activities of compounds RFB 1, RFB 4, RFB 10 and RFB 11 considers the likely hydrogen bonding between ligands and receptor, balancing the global flexibility and interaction energies. RFB 1 and its most effective derivatives were found to be not toxic. CONCLUSION: Besides RFB 1, its derivatives 4, 10 and 11 show potential for clinical development in the M. abscessus treatment.

Design of a nanostructured lipid carrier intended to improve the treatment of tuberculosis.

This work aimed to design, develop, and characterize a lipid nanocarrier system for the selective delivery of rifabutin (RFB) to alveolar macrophages. Lipid nanoparticles, specifically nanostructured lipid carriers (NLC), were synthetized by the high-shear homogenization and ultrasonication techniques. These nanoparticles were designed to exhibit both passive and active targeting strategies to be efficiently internalized by the alveolar macrophages, traffic to the acidified phagosomes and phagolysosomes, and release bactericidal concentrations of the antituberculosis drug intracellularly. NLC that could entrap RFB were prepared, characterized, and further functionalized with mannose. Particles' diameter, zeta potential, morphology, drug% entrapping efficiency, and drug release kinetics were evaluated. The mannose coating process was confirmed by Fourier transform infrared. Further, the cytotoxicity of the formulations was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay in A549, Calu-3, and Raw 264.7 cells. The diameter of NLC formulations was found to be in the range of 175-213 nm, and drug entrapping efficiency was found to be above 80%. In addition, high storage stability for the formulations was expected since they maintained the initial characteristics for 6 months. Moreover, the drug release was pH-sensitive, with a faster drug release at acidic pH than at neutral pH. These results pose a strong argument that the developed nanocarrier can be explored as a promising carrier for safer and more efficient management of tuberculosis by exploiting the pulmonary route of administration.

Development and Evaluation of Chitosan Microparticles Based Dry Powder Inhalation Formulations of Rifampicin and Rifabutin.

BACKGROUND: The lung is the primary entry site and target for Mycobacterium tuberculosis; more than 80% of the cases reported worldwide are of pulmonary tuberculosis. Hence, direct delivery of anti-tubercular drugs to the lung would be beneficial in reducing both, the dose required, as well as the duration of therapy for pulmonary tuberculosis. In the present study, microsphere-based dry powder inhalation systems of the anti-tubercular drugs, rifampicin and rifabutin, were developed and evaluated, with a view to achieve localized and targeted delivery of these drugs to the lung. METHODS: The drug-loaded chitosan microparticles were prepared by an ionic gelation method, followed by spray-drying to obtain respirable particles. The microparticles were evaluated for particle size and drug release. The drug-loaded microparticles were then adsorbed onto an inhalable lactose carrier and characterized for in vitro lung deposition on an Andersen Cascade Impactor (ACI) followed by in vitro uptake study in U937 human macrophage cell lines. In vivo toxicity of the developed formulations was evaluated using Sprague Dawley rats. RESULTS: Both rifampicin and rifabutin-loaded microparticles had MMAD close to 5 µm and FPF values of 21.46% and 29.97%, respectively. In vitro release study in simulated lung fluid pH 7.4 showed sustained release for 12 hours for rifampicin microparticles and up to 96 hours for rifabutin microparticles, the release being dependent on both swelling of the polymer and solubility of the drugs in the dissolution medium. In vitro uptake studies in U937 human macrophage cell line suggested that microparticles were internalized within the macrophages. In vivo acute toxicity study of the microparticles in Sprague Dawley rats revealed no significant evidence for local adverse effects. CONCLUSION: Thus, spray-dried microparticles of the anti-tubercular drugs, rifampicin and rifabutin, could prove to be an improved, targeted, and efficient system for treatment of tuberculosis.

Human arylacetamide deacetylase is responsible for deacetylation of rifamycins: rifampicin, rifabutin, and rifapentine.

Rifamycins such as rifampicin, rifabutin, and rifapentine are used for the treatment of tuberculosis and induce various drug-metabolizing enzymes. Rifamycins have been reported to be mainly deacetylated by esterase(s) expressed in human liver microsomes (HLM) to 25-deacetylrifamycins, but the responsible enzyme remained to be determined. In this study, we found that recombinant human arylacetamide deacetylase (AADAC) could efficiently deacetylate rifamycins, whereas human carboxylesterases, which are enzymes responsible for the hydrolysis of many prodrugs, showed no activity. The involvement of AADAC in the deacetylation of rifamycins in HLM was verified by the similarities of the K(m) and K(i) values and the inhibitory characteristics between recombinant AADAC and HLM. Rifamycins exhibited potent cytotoxicity to HepG2 cells, but their 25-deacetylated metabolites did not. Luciferase assay using a reporter plasmid containing CYP3A4 direct repeat 3 and everted repeat 6 motifs revealed that 25-deacetylrifamycins have lesser potency to transactivate CYP3A4 compared with the parent drugs. Supporting these results, HepG2 cells infected with a recombinant adenovirus expressing human AADAC showed low cytotoxicity and induction potency of CYP3A4 by rifamycins. In addition, CYP3A4 induction in human hepatocytes by rifamycins was increased by transfecting siRNA for human AADAC. Thus, we found that human AADAC was the enzyme responsible for the deacetylation of rifamycins and would affect the induction rate of drug-metabolizing enzymes by rifamycins and their induced hepatotoxicity.

Mannosylated nanoparticulate carriers of rifabutin for alveolar targeting.

The objective of the present study was to evaluate the prospective of engineered nanoparticles for selective delivery of an antituberculosis drug, rifabutin, to alveolar tissues. Drug-loaded solid lipid nanoparticles (SLNs) were synthesized and efficiently mannosylated. The formation of uncoated and coated SLNs was characterized by FTIR spectroscopy and SEM studies. A variety of physicochemical parameters such as drug loading, particle size, polydispersity index, zeta potential, and in vitro drug release were determined. The toxicity and targeting potential of the prepared formulation were assessed with alveolar macrophage uptake, hematological studies, and in vivo studies of uncoated and coated SLNs. Ex vivo cellular uptake studies of SLNs formulations in alveolar macrophages depicted almost six times enhanced uptake due to mannose coating. The hematological studies proved mannose-conjugated system to be less immunogenic and suitable for sustained delivery as evaluated against uncoated formulation. Further, the serum level and organ distribution studies demonstrated efficiency of the system for prolonged circulation and spatial delivery of rifabutin to alveolar tissues. Finally, it was concluded that mannose-conjugated SLNs can be exploited for effective and targeted delivery of rifabutin compared to its uncoated formulation and ultimately increasing the therapeutic margin of safety while reducing the side effects.

Rifabutin accumulates in the lens and reduces retinal function in the rabbit eye.

PURPOSE: To study the toxicology of rifabutin in the rabbit eye with emphasis on retinal function and histopathology. METHODS: Seven rabbits received a daily dose of rifabutin during 15 months. Six rabbits receiving only the vehicle were used as controls. Repeated standardized full-field electroretinograms (ERG) were assessed. After discontinuing treatment, the rabbits were killed and the cornea, the lens, and the sectioned retina was studied. Immunhistochemistry directed against vimentin, glial fibrillaryacidic protein (GFAP), protein kinase C (PKC), and peanut agglutinin (PNA) was performed. RESULTS: Rifabutin was detected in serum of the treated rabbits. During treatment, the full-field ERG demonstrated significantly reduced b-wave amplitudes in the total rod-cone response as well as in the isolated rod and cone response compared with the recordings before treatment. The control rabbits did not demonstrate a reduction of the ERG amplitudes. The treated rabbits developed a discoloration of the lens, not seen in the control group. No retinal pathology was demonstrated using immunohistochemical methods. CONCLUSION: Rifabutin causes a discoloration of the lens and reduces both rod and cone function in rabbits, but does not alter retinal morphology. Previous reports on ocular side effects caused by rifabutin are supported by the results of this study.

Enzymatic reduction and glutathione conjugation of benzoquinone ansamycin heat shock protein 90 inhibitors: relevance for toxicity and mechanism of action.

Two-electron reduction of benzoquinone ansamycin (BA) heat shock protein (Hsp) 90 inhibitors by NAD(P)H:quinone oxidoreductase 1 (NQO1) to hydroquinone ansamycins (BAH2s) leads to greater Hsp90 inhibitory activity. BAs can also be metabolized by one-electron reductases and can interact with glutathione, reactions that have been associated with toxicity. Using a series of BAs, we investigated the stability of the BAH2s generated by NQO1, the ability of BAs to be metabolized by one-electron reductases, and their conjugation with glutathione. The BAs used were geldanamycin (GM), 17-(allylamino)-17-demethoxygeldanamycin (17AAG), 17-demethoxy-17-[[2-(dimethyl amino)ethyl]amino]-geldanamycin (17DMAG), 17-(amino)-17-demethoxygeldanamycin (17AG), and 17-demethoxy-17-[[2-(pyrrolidin-1-yl)ethyl]amino]-geldanamycin (17AEP-GA). The relative stabilities of BAH2s at pH 7.4 were GM hydroquinone>17AAG hydroquinone>17DMAG hydroquinone>17AG hydroquinone and 17AEP-GA hydroquinone. Using human and mouse liver microsomes and either NADPH or NADH as cofactors, 17AAG had the lowest rate of one-electron reduction, whereas GM had the highest rate. 17DMAG demonstrated the greatest rate of redox cycling catalyzed by purified human cytochrome P450 reductase, whereas 17AAG again had the slowest rate. GM formed a glutathione adduct most readily followed by 17DMAG. The formation of glutathione adducts of 17AAG and 17AG were relatively slow in comparison. These data demonstrate that GM, the most hepatotoxic BAs in the series had a greater propensity to undergo redox cycling reactions catalyzed by hepatic one-electron reductases and markedly greater reactivity with thiols when compared with the least hepatotoxic analog 17AAG. Minimizing the propensity of BA derivatives to undergo one-electron reduction and glutathione conjugation while maximizing their two-electron reduction to stable Hsp90 inhibitory hydroquinones may be a useful strategy for optimizing the therapeutic index of BAs.

Evidence for the mode of action of the highly cytotoxic Streptomyces polyketide kendomycin.

The macrocyclic polyketide kendomycin exhibits antiosteoporotic and antibacterial activity, as well as strong cytotoxicity against multiple human tumor cell lines. Despite the promise of this compound in several therapeutic areas, the cellular target(s) of kendomycin have not been identified to date. We have used a number of approaches, including microscopy, proteomics, and bioinformatics, to investigate the mode of action of kendomycin in mammalian cell cultures. In response to kendomycin treatment, human U-937 tumor cells exhibit depolarization of the mitochondrial membrane, caspase 3 activation, and DNA laddering, consistent with induction of the intrinsic apoptotic pathway. To elucidate possible apoptotic triggers, DIGE and MALDI-TOF were used to identify proteins that are differently regulated in U-937 cells relative to controls. Statistical analysis of the proteomics data by the new web-based application GeneTrail highlighted several significant changes in protein expression, most notably among proteasomal regulatory subunits. Overall, the profile of altered expression closely matches that observed with other tumor cell lines in response to proteasome inhibition. Direct assay in vitro further shows that kendomycin inhibits the chymotrypsin-like activity of the rabbit reticulocyte proteasome, with comparable efficacy to the established inhibitor MG-132. We have also demonstrated that ubiquitinylated proteins accumulate in kendomycin-treated U-937 cells, while vacuolization of the endoplasmic reticulum and mitochondrial swelling are induced in a second cell line derived from kangaroo rat epithelial (PtK(2)) cells, phenotypes classically associated with inhibition of the proteasome. This study therefore provides evidence that kendomycin mediates its cytotoxic effects, at least in part, through proteasome inhibition.

In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin in rats.

The differential toxicity of two anticancer agents is described using the in vitro rat liver slice culture model. Liver slices from F-344 rats were cultured for 5 days in Waymouth's-based medium with exposure to a range of geldanamycin (GEL) or 17-allylaminogeldanamycin (17-AAG) concentrations. GEL induced concentration-dependent reduction of alkaline phosphatase and of gamma-glutamyl transferase levels, which are indicators of biliary epithelial cell(s) (BEC) viability, and exhibited hepatocellular toxicity at higher concentrations. Histologically, BEC cell injury was evident at the lowest GEL concentration (0.1 microM) and progressed to overt bile duct necrosis at 5 microM, a level at which hepatocellular damage was also more prominent. Slices exposed to the same concentrations were more sensitive to toxic effects of GEL than of 17-AAG. 17-AAG at the lowest concentration had more slice biomarker retention than GEL, and histological analysis revealed minimal toxic effect on BEC. With increasing concentration, BEC were progressively lost, and BEC proliferation was completely inhibited at 5 microM 17-AAG. Hepatocellular injury was evident only at high dose exposures. This is believed to be the first use of an in vitro liver tissue model to accurately predict the differential and concentration-dependent toxicities of these compounds.

In vitro detection of differential and cell-specific hepatobiliary toxicity induced by geldanamycin and 17-allylaminogeldanamycin using dog liver slices.

Experiments on rat liver slices demonstrated the differential hepatobiliary toxic potency of two anticancer agents, geldanamycin (GEL) and 17-allylaminogeldanamycin (17-AAG), over a 5-day period. This report describes the pattern of toxicity of these agents in dog liver tissue, using the in vitro liver slice culture model. Liver slices (200 microm thick) from male beagle dogs were cultured for 5 days in chemically defined culture medium containing a range of GEL and 17-AAG concentrations (0.1-5 microM). Tissues were evaluated using a panel of clinically relevant biomarkers and histological endpoints. GEL-induced reduction of alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) slice levels, indicators of biliary epithelial cell (BEC) viability, was supported by histological findings showing an increasing loss of BEC as higher concentrations were applied. At the highest concentrations studied, GEL caused both hepatocellular necrosis and BEC loss. Biomarker pattern results in the medium concurred with those from slice biochemistry measurements and histology. 17-AAG, a less potent compound in vivo, elicited more biomarker retention at higher concentrations than did GEL. Histological analysis revealed higher BEC viability and significant retention of BEC proliferation as compared with GEL. However, at the highest concentration, the toxic insult caused a marked decrease in BEC viability and proliferation. Comparison of responses with both compounds indicated that slices exposed to the same concentrations were more sensitive to GEL than to 17-AAG. Dog liver slices can thus be used to evaluate species-, compound-, and concentration-dependent differences in toxicity.

17-Allylamino-17-demethoxygeldanamycin activity against thyroid cancer cell lines correlates with heat shock protein 90 levels.

Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes growth factor receptors and signaling molecules. Disruption of this action inhibits the MAPK and phosphatidylinositol-3 kinase cascades and can induce cancer cell death. The goal of this study was to determine whether thyroid cancer cells are sensitive to the cytotoxic effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor in clinical trials, and to determine predictors of this response. Papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid cancers were incubated with 17-AAG in vitro. Surprisingly, the ARO cells were most sensitive to the cytotoxic effects of this agent. Conversely, all cell lines displayed similar responses to specific blockers of phosphatidylinositol-3 kinase and MAPK kinase (LY294002 and U0126, respectively). Western blot demonstrated that the NPA cells that were most resistant to 17AAG-induced cytotoxicity had the lowest levels of Hsp90 and were the only cells with persistent levels of Akt protein. Interestingly, even the WRO and ARO cell lines that were sensitive to 17-AAG-induced cell death did not undergo apoptosis. These data suggest that sensitivity of thyroid cancer cells to 17-AAG-induced cytotoxicity relates to Hsp90 levels rather than histological subtype and that thyroid cancer cells have a reduced apoptotic response to 17-AAG.

Plasma pharmacokinetics and tissue distribution of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) in CD2F1 mice1.

PURPOSE: 17-(Allylamino)-17-demethoxygeldanamycin (17AAG) is a benzoquinone ansamycin compound agent that has entered clinical trials. Studies were performed in mice to: (1) define the plasma pharmacokinetics, tissue distribution, and urinary excretion of 17AAG after i.v. delivery; (2) to define the bioavailability of 17AAG after i.p. and oral delivery; and (3) to characterize the concentrations of 17AAG metabolites in plasma and tissue. MATERIALS AND METHODS: All studies were performed in female CD2F1 mice. Preliminary toxicity studies used 17AAG i.v. bolus doses of 20, 40 and 60 mg/kg. Pharmacokinetic studies used i.v. 17AAG doses of 60, 40, and 26.67 mg/kg and i.p. and oral doses of 40 mg/kg. The plasma concentration versus time data were analyzed by compartmental and noncompartmental methods. The concentrations of 17AAG were also determined in brain, heart, lung, liver, kidney, spleen, skeletal muscle, and fat. Urinary drug excretion was calculated until 24 h after treatment. RESULTS: A 60 mg/kg dose of 17AAG, in its initial, microdispersed formulation, caused no changes in appearance, appetite, waste elimination, or survival of treated animals as compared to vehicle-treated controls. Bolus i.v. delivery of 60 mg/kg microdispersed 17AAG produced "peak" plasma 17AAG concentrations between 5.8 and 19.3 micrograms/ml in mice killed 5 min after injection. Sequential reduction of the 17AAG dose to 40 and 26.67 mg/kg resulted in "peak" plasma 17AAG concentrations between 8.9 and 19.0 micrograms/ml, and 4.8 and 6.1 micrograms/ml, respectively. Noncompartmental analysis of the plasma 17AAG concentration versus time data showed an increase in AUC from 402 to 625 and 1738 micrograms/ml.min when the 17AAG dose increased from 26.67 to 40 and 60 mg/kg, respectively. Across the range of doses studied, 17AAG total body clearance varied from 34 to 66 ml/min per kg. Compartmental modeling of the plasma 17AAG concentration versus time data showed that the data were fitted best by a two-compartment, open, linear model. In each study, substantial concentrations of a material, subsequently identified as 17-(amino)-17-demethoxygeldanamycin (17AG), were measured in plasma. A subsequent, lyophilized formulation of 17AAG proved excessively toxic when delivered i.v. at 60 mg/kg. A repeat i.v. study using a 40 mg/kg dose of this new formulation produced peak plasma 17AAG concentrations of 20.2-38.4 micrograms/ml, and a 17AAG AUC of 912 micrograms/ml.min, which was approximately 50% greater than the AUC produced by a 40 mg/kg dose of microdispersed 17AAG. The bioavailabilities of 17AAG after i.p. and oral delivery were 99% and 24%, respectively. Minimal amounts of 17AAG and 17AG were detected in the urine. After i.v. bolus delivery to mice, 17AAG distributed rapidly to all tissues, except the brain. Substantial concentrations of 17AG were measured in each tissue. CONCLUSIONS: 17AAG has excellent bioavailability when given i.p. but only modest bioavailability when given orally and is metabolized to 17AG and other metabolites when given i.v., i.p., or orally. 17AAG is widely distributed to tissues. These pharmacokinetic data generated have proven relevant to the design of recently initiated clinical trials of 17AAG and could be useful in their interpretation.

Inducing properties of rifampicin and rifabutin for selected enzyme activities of the cytochrome P-450 and UDP-glucuronosyltransferase superfamilies in female rat liver.

Important species differences have been reported concerning the induction properties of rifampicin towards enzymes of the P-450 superfamily. Mice, rabbits and humans are far more responsive than rats and guinea pigs. In the present study a strong induction of cytochrome P-450 3A-dependent enzyme activities was observed in female rat liver microsomes after high dose treatment (> or = 250 mg/kg/day for 9 days) with rifampicin, resulting in an up to 30-fold enhanced hydroxylation rate of testosterone in the 2 beta-, 6 beta- and 15 beta-position in vitro. Other cytochrome P-450 isozyme-selective reactions were not, or only marginally, affected. A steep increase in cytochrome P-450 3A activity on a moderate elevation of the dose administered, together with the previously observed lack of efficient induction with doses below 200 mg/kg/day demonstrated that there is a threshold in enzyme induction by rifampicin. For rifabutin such a threshold was not apparent. Induction by rifabutin showed an isoenzyme-selectivity profile similar to that produced by rifampicin, but the maximally achievable induction of cytochrome P-450 3A by rifabutin was about two-fold lower compared with rifampicin. Rifampicin and rifabutin enhanced the glucuronidation of 1-naphthol, 4-hydroxybiphenyl and beta-estradiol by a factor of two to three. The potential implications of the enzyme induction by rifampicin derivatives in terms of possible drug-drug interactions are discussed.

Overview of toxicological data on rifabutin.

Rifabutin is a wide spectrum antibiotic particularly active on atypical and rifampicin-resistant mycobacteria. Rifabutin is more potent than rifampicin on Mycobacterium tuberculosis in vitro. Its mode of action is characterized by a high intracellular penetration in treated individuals. Clinical trials have proven the therapeutic value of rifabutin especially in AIDS patients with concomitant MAC. The preclinical safety evaluation of this compound included single and repeated dose toxicity studies of up to one year in rodents and non-rodents, reproduction and carcinogenicity studies and mutagenicity tests. During toxicological studies the most significant finding after repeated administration of rifabutin was the presence of multinucleated hepatocytes (MNH) in rats. This is a species specific finding which did not affect the life span of the hepatocytes. As shown in carcinogenicity studies, there was no tendency to further proliferative changes. Another specific histological feature among the species studied was the presence of a lipofuscin-like brown pigment, which was seen in many organs. This is a common finding with amphipilic compounds, such as rifabutin, which bind lipids and proteins, forming membrane-bound complexes. Even in carcinogenicity studies this change did not constitute a stimulus to cell proliferation and did not cause any secondary changes. In rodents, there was a mild hemolytic anemia at doses higher than 10 mg/kg/day. At doses ranging from 160-200 mg/kg/day rifabutin inhibited the functions of the male gonads in rats. This effect was reflected in a reduction of implantations observed in the fertility studies. Doses of 40 mg/kg/day did not induce any embryotoxic effects or changes in reproductive performance.(ABSTRACT TRUNCATED AT 250 WORDS)

Multinucleated hepatocytes induced by rifabutin in rats.

Rifabutin is an antibiotic of the rifamycin class, which is particularly active against mycobacteria, including those that occur in AIDS patients. Because clinical use will include long-term therapy, an extensive battery of long-term toxicity studies was carried out by the oral route, including carcinogenicity studies. An interesting feature was the occurrence of multinucleated hepatocytes (MNHs) in the rat. In some instances, as many as 25 nuclei occurred in a single cell. Light microscopy revealed a large hepatocyte with normal eosinophilic staining. The multiple nuclei stained like those present in the surrounding normal cells. Electron microscopy showed no abnormalities of the nuclei and no cell membranes within the cytoplasm. The customary organelles were present. MNHs were dose- and sex-related, starting from 10 mg/kg/day and being more evident in males. They began to appear after 5 wk of treatment and persisted over long periods of recovery (12 mo), without showing any tendency for cell proliferation. The life-span of MNHs was similar to that of normal hepatocytes. MNHs were present in the carcinogenicity study, but there was no increase in liver tumors. MNHs did not occur in mice or monkeys treated with rifabutin, nor did they occur in response to treatment with rifampin. The effect appears to be specific to the rat.

Myelotoxicity of rifabutin and 3'-azido-3'-deoxythymidine, alone and in combination, to human hematopoietic progenitor cells in vitro.

Mycobacterial infection is a common complication of acquired immunodeficiency syndrome (AIDS) frequently requiring antimycobacterial medication. It was of interest to determine if one such agent, rifabutin, could be tolerated by AIDS patients in conjunction with 3'-azido-3'-deoxythymidine (AZT) therapy. We evaluated the in vitro myelotoxic effects of rifabutin on human hematopoietic progenitor cells, alone and in combination with AZT (rifabutin: AZT, 1:10 ratio) over a range of concentrations in a microcapillary assay. Both rifabutin and AZT at 5 microM were moderately toxic to hematopoietic progenitors, inhibiting colony formation by 57-65% and 59-63%, respectively. The combination of rifabutin (5 microM) and AZT (50 microM) inhibited colony formation by 59-73%. Granulocyte-macrophage progenitors were less sensitive to this combination than erythroid progenitors. The combination of ribabutin and AZT did not exceed the in vitro myelotoxicity to human progenitors of AZT alone. These results suggest that rifabutin may be tolerated in AIDS patients, with no anticipated increase in myelotoxicity when given with AZT.