Identification and characterization of A-105972, an antineoplastic agent.

A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells. After screening more than 60,000 compounds, A-105972 was identified and selected for further testing. A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells. The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type. The potency of A-105972 is similar in cells expressing wild-type or mutant p53. A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle. Using [3H]A-105972, we found that A-105972 bound to purified tubulin. Unlabeled A-105972 competed with [3H]A-105972 binding with an IC50 value of 3.6 microL. Colchicine partially inhibited [3H]A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect. Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies. DNA ladder studies also indicated that A-105972 induced apoptosis. A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation. A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388. These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells. A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.

Synthesis and antimicrobial activity of 4H-4-oxoquinolizine derivatives: consequences of structural modification at the C-8 position.

The antibacterial 4H-4-oxoquinolizines were introduced recently to overcome bacterial resistance to fluoroquinolones. They exhibit potent antibacterial activity against Gram-positive, Gram-negative, and anaerobic organisms and are highly active against some quinolone-resistant bacteria including quinolone-resistant MRSA. Preliminary studies indicated that oxoquinolizines possess distinct activity and toxicity profiles as compared with their parent quinolones. In order to develop a potent antibacterial agent with the desired spectrum of activity, good tolerability, and balanced pharmacokinetic profile, we synthesized and evaluated a series of oxoquinolizines with various substituents at the C-8 position. Most compounds tested in this study demonstrated better activity against Gram-positive bacteria than ciprofloxacin and exhibited good susceptibility against ciprofloxacin- and methicillin-resistant S. aureus. While maintaining potent in vitro activity, several compounds showed improved in vivo efficacy over ABT-719 as indicated by the mouse protection test. As an example, the oral ED(50) values for the cis-3-amino-4-methylpiperidine analogue 3ss against S. aureus NCTC 10649M, S. pneumoniae ATCC 6303, and E. coli JUHL were 0. 8, 2.0, and 1.4 mg/kg, compared to 3.0, 10.0, and 8.3 mg/kg for ABT-719. The current study revealed that the steric and electronic environment, conformation, and absolute stereochemistry of the C-8 group are very important to the antibacterial profiles. Structural modifications of the C-8 group provide a useful means to improve the antibacterial activities, physicochemical properties, and pharmacokinetic profiles. Manipulation of the C-8 group also allows us to generate analogues with the desired spectrum of activity, such as analogues that are selective against respiratory pathogens.

New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Immune complexes in serum of rats during infection with Plasmodium berghei.

Large amounts of immune complexes were present in the serum of infected rats early in infection when parasitemias were low. As the infection progressed and parasitemia increased and then decreased, the amounts of immune complexes in the serum also fell. This result suggests that increased efficiency of complex clearance was an important factor in determining the levels of immune complexes in the serum. In high performance liquid chromatography (HPLC), the complexes in the serum migrated as a peak with material of 350 kDa and greater in mass. They sedimented in a sucrose gradient as a band with a sedimentation coefficient of 22 s, which was calculated to yield a mass of approximately 1100 kDa. Immunoelectrophoresis and radial immunodiffusion showed that IgG was the major immunoglobulin in the complexes. As the IgG content of the complexes increased, the levels of complexes in the serum generally decreased. HPLC analysis of precipitated complexes suggested that they contained loosely bound albumin. Serum proteins were affected by the infection. A depletion of free immunoglobulin was observed during the initial period of immune complex formation.

Effects of immune complexes on immunity to Plasmodium berghei infection.

The ELISA test titers and RIPA patterns of sera collected from vaccinated and non-vaccinated rats during P. berghei infection were similar. The sera collected just after clearance of parasitemia from the vaccinated rats, but not that from the non-vaccinated rats protected mice in passive protection tests. After precipitation to remove immune complexes, the sera from the non-vaccinated rats also protected mice. Administration of acute phase serum early in the course of infection aggravated parasitemia and delayed recovery from P. berghei infection in rats. Administration of hyperimmune serum early in the course of infection initially reduced parasitemia but then delayed recovery of rats from P. berghei infection. These results suggest that immune complexes may interfere with antibody mediated immunity to P. berghei and may also retard development of the induced immune response.

Plasmodium berghei malaria: effect of acute phase serum on immunity generated in rats by infection and by vaccination.

Acute phase serum (APS) given at the time of challenge with Plasmodium berghei inhibited the generation to immunity to the infecting plasmodia. Administered with a single dose of vaccine, it inhibited induction of immunity by the vaccine. Three weekly doses, the last given two weeks before infection, induced immunity. Administration of vaccine simultaneously with infection neither aggravated nor ameliorated the infection. These results indicate that the effect of administration of APS on immunity generated by immunization or infection is dose- and time-dependent. The depression of immunity induced by this serum is thus temporary, the host finally overcoming the depression and responding to the plasmodial antigen in the serum. The interaction of vaccine and infection observed indicates that the introduction of vaccine is not detrimental to the individual incubating infection; rather, the vaccine is rendered useless, the reducing the aggregate benefit of the immunization to the group.

Model of Streptococcus pneumoniae meningitis in adult rats.

The purpose of this study was to develop a model of bacterial meningitis in young adult rats for assessing the efficacy of antimicrobial agents. Sixty 200- to 300-g male Sprague Dawley CD rats were inoculated intracisternally with 5.78 log10 CFU of a clinical isolate of Streptococcus pneumoniae in 5% hog gastric mucin. Inoculated rats were assigned to six groups containing 10 animals each. Group-1 rats served as controls and did not receive antibiotics. Rats of groups 2 to 4 received (subcutaneously every 12 h) cefotaxime (25, 6.25, and 1.56 mg/kg of body weight respectively). Rats of groups 5 and 6 received ampicillin (50 and 12.5 mg/kg respectively) and gentamicin (2.0 and 0.5 mg/kg respectively). Five additional Sprague Dawley CD rats were inoculated with only gastric hog mucin and were assigned to group 7. At postinoculation day 4 all animals were euthanized. Cerebral spinal fluid was collected for culturing. Brains were harvested for histologic examination and culturing. Untreated, infected control (group-1) animals were culture-positive for S. pneumoniae in the brain and cerebral spinal fluid. Of the antibiotic regimens evaluated, only cefotaxime (25 mg/kg) eradicated bacteria from the cerebral spinal fluid and brain. Cefotaxime at 25 or 6.25 mg/kg significantly (P < or = 0.05) decreased the bacterial burden of S. pneumoniae, whereas cefotaxime at 1.56 mg/kg and ampicillin/gentamicin combinations did not. There was histopathological evidence of subacute meningitis in infected rats. No meningitis was observed in rats receiving 25 mg of cefotaxime/kg. This model demonstrates the ability to induce bacterial meningitis with S. pneumoniae in adult rats and the ability to clear infection in 90 to 100% of the animals by administration of cefotaxime at dosages of 6.25 and 25 mg/kg given subcutaneously every 12 h.

Relevance of the ferret model of Helicobacter-induced gastritis to evaluation of antibacterial therapies.

OBJECTIVES: The goals of the study were 1) to evaluate the efficacy of clinically relevant antibacterial therapies in the ferret model of Helicobacter-induced gastritis and 2) to compare these results to the efficacy achieved clinically in humans. METHODS: Ferrets were inoculated with H. mustelae, and gastritis was allowed to develop. The double therapy of clarithromycin and omeprazole and the triple therapies of clarithromycin or amoxicillin with metronidazole and omeprazole were administered. Efficacy was evaluated by Helicobacter burden cultured from biopsy samples and by histopathological evaluation of Helicobacter burden and gastric inflammation with pylorus and fundus samples obtained 4 wk after the end of antibacterial therapy. RESULTS: Clarithromycin-based double and triple therapies significantly reduced Helicobacter burden and decreased gastric inflammation. Clarithromycin-based double therapy was more effective than amoxicillin-based triple therapy. Reduction of the length of clarithromycin therapy from 14 to 7 days decreased efficacy. Antibacterial therapies in the ferret did not produce eradication rates comparable to clinical results, even though the serum concentrations of clarithromycin in ferret were in excess of concentrations used in humans. Relapse of Helicobacter infection after the end of therapy occurred in some cases. CONCLUSIONS: Although the ferret model of Helicobacter gastric infection underestimated the clinical efficacy of antibacterial treatments in humans, the model was valuable for comparing the relative efficacy of antibacterial therapies.

Efficacy of ABT-719, a 2-pyridone antimicrobial, against enterococci, Escherichia coli, and Pseudomonas aeruginosa in experimental murine pyelonephritis.

ABT-719 is a 2-pyridone antimicrobial which inhibits DNA gyrase activity. It has considerable subcutaneous (sc) and oral efficacy in the treatment of experimental pyelonephritis induced in carrageenan-treated mice by clinical isolates of Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Pseudomonas aeruginosa. Therapeutic ED50s, defined here as producing a 2 log10 reduction in kidney bacterial burden, provide a reliable end point for comparison of drug efficacy in this experimental infection. Therapeutic ED50s for ABT-719 against these infections were equal to or up to ten-fold lower than those for ciprofloxacin, used as a reference because of similarity in mode of action. Against E. faecalis, the therapeutic ED50s for ABT-719 were 4.5-13.6 mg/kg.day for sc administration and 6.8-8.9 mg/kg.day for oral administration. ABT-719 was more potent than ciprofloxacin and vancomycin against the E. faecalis strains, which showed ciprofloxacin and vancomycin resistance covering a range of MICs. Against E. faecium, the therapeutic ED50s for ABT-719 were 8.8 mg/kg.day (sc) and 9.4 mg/kg.day (oral). Against an isolate of E. faecium showing ciprofloxacin and vancomycin resistance the ED50 for ABT-719 to achieve a 1 log10 reduction in kidney bacterial burden was 17.9 mg/kg.day by sc administration. While ABT-719 had lower efficacy against this isolate than against others, ciprofloxacin and vancomycin failed to show efficacy. Against E. coli, the therapeutic ED50 for ABT-719 was 1.1 mg/kg.day (oral), and against P. aeruginosa, this value was 2.7 mg/kg.day (oral) with values against both of these pathogens similar to those for ciprofloxacin. ABT-719, which represents the new 2-pyridone compound class, has promise for the treatment of urinary tract infections, as suggested by the significant efficacy seen against experimental pyelonephritis caused by E. coli, P. aeruginosa and susceptible and resistant enterococci.

Immune T cells sorted by flow cytometry confer protection against infection with Treponema pallidum subsp. pertenue in hamsters.

The role of cell-mediated immunity against infection with Treponema pallidum subsp. pertenue in humans or experimental animals is unclear. Hamsters injected subcutaneously in the hind paws with 4 x 10(6) unfractionated lymph node cells or enriched lymph node T cells (immunoglobulin negative, Ia negative) from T. pallidum subsp. pertenue-immune hamsters were resistant to challenge with T. pallidum subsp. pertenue. The popliteal lymph nodes of hamsters that received immune cells weighed less and had significantly fewer treponemes than did lymph nodes from hamsters infused with cells from nonimmune donors. Furthermore, recipients of immune T cells failed to develop antitreponemal antibodies 21 days after challenge. Enriched T cells were obtained by flow cytometric separation by using monoclonal anti-Ia antibody 14-4-4s, which identified hamster B cells. Flow cytometric analysis by two-color immunofluorescent staining with anti-hamster-immunoglobulin and monoclonal anti-Ia antibody 14-4-4s confirmed that monoclonal anti-Ia antibody 14-4-4s recognized B cells. In addition, lymph node cells obtained after treatment with anti-Ia monoclonal antibody 14-4-4s and complement were 97% T cells, as determined by monoclonal antibody 20, a hamster T-cell marker. These results demonstrated that highly enriched T cells (immunoglobulin negative, Ia negative) from T. pallidum subsp. pertenue-immune hamsters conferred partial protection on hamsters against infection with T. pallidum subsp. pertenue.

Phagocytosis of opsonized Treponema pallidum subsp. pallidum proceeds slowly.

Macrophages were found to phagocytize Treponema pallidum subsp. pallidum attached to polycarbonate filters. This environment simulated the in vivo interaction of surface-adherent treponemes with macrophages. The phagocytosis of T. pallidum subsp. pallidum was found to proceed slowly. Heat-killed T. pallidum subsp. pallidum were susceptible to opsonization with 2% immune serum, whereas live treponemes were resistant to this concentration of antibody. High concentrations of immune serum were found to increase phagocytosis of the spirochetes. Live T. pallidum subsp. pallidum had bound limited quantities of immunoglobulin G in vivo, and only opsonization with 20% immune serum resulted in a detectable increase in surface-bound immunoglobulin in vitro. Kinetic studies suggested a steady rate of phagocytosis that is considerably slower than with other bacteria. Scanning electron microscopy studies of the phagocytizing macrophages showed that the treponemes were detached from the membrane filters and scooped onto the ruffled portion of the macrophage surface. This lengthy physical process, along with the lack of a dramatic increase in ingestion after opsonization, may account for the slow rate of phagocytosis.

Borreliacidal activity of sera from hamsters infected with the Lyme disease spirochete.

An in vitro borreliacidal assay that accurately reflects the levels of protective antibody determined by passive transfer of immunity studies was developed. Borreliacidal antibody in sera obtained from normal hamsters infected with Borrelia burgdorferi was readily detected. When immune serum containing complement was incubated with B. burgdorferi organisms, spirochetes were killed within 2 h. Treating immune serum with anti-hamster immunoglobulin G abrogated the borreliacidal activity. Killing of B. burgdorferi in serum was detected 1 week after infection; it peaked at week 3 and gradually declined. Relatively high levels of borreliacidal antibody were found, especially in week 3 immune serum, which could be diluted 1,280-fold. The decrease in borreliacidal antibody after infection may account for occurrences of reinfection and the remitting course of Lyme disease.

Induction of interleukin-1 release by high- and low-passage isolates of Borrelia burgdorferi.

Low-passage isolates of Borrelia burgdorferi induced arthritis when injected into the hind paws of irradiated hamsters, while high-passage isolates did not. To examine a possible mechanism for induction of arthritis, peritoneal exudate cells were coincubated with high- and low-passage isolates of B. burgdorferi, and the resultant conditioned medium was assayed for interleukin-1 (IL-1) activity. Comparable amounts of IL-1 activity were detected in culture supernatants generated by high- and low-passage spirochetes and were dependent on the number of spirochetes added. Live B. burgdorferi stimulated greater release of IL-1 activity than did heat-killed organisms. No evidence of release of IL-1 due to shedding of soluble components from spirochetes was obtained. A recombinant human IL-1 receptor antagonist blocked the proliferative activity of conditioned medium in a murine thymocyte assay for IL-1 activity. The greater ability of low-passage spirochetes to survive in vivo may be more important than the ability to induce IL-1 production in the pathogenesis of Lyme arthritis.

Role of L3T4+ and 38+ T-cell subsets in resistance against infection with Treponema pallidum subsp. pertenue in hamsters.

The protective immunity conferred by T-cell subsets against infection with Treponema pallidum subsp. pertenue was studied. We demonstrated that hamster T cells can be separated into two subsets by monoclonal antibody (MAb) GK 1.5 (anti-L3T4) and MAb 38. Eighty-five percent of hamster thymocytes were L3T4+ and 87% were 38+ cells; 84% were dual positive for MAbs anti-L3T4 and 38. In the peripheral lymph nodes, however, the L3T4+ and 38+ T cells were mutually exclusive according to two-color immunofluorescence analysis. The two T-cell subsets were found to be functionally distinct according to their secretion of interleukin 2 (IL-2) when stimulated with concanavalin A. The L3T4+ cells secreted IL-2 and had characteristics of T helper cells, while the 38+ cells did not secrete IL-2 and appeared to be T cytotoxic-suppressor cells. Transfer of 4 x 10(6) helper or cytotoxic-suppressor T lymphocytes from T. pallidum subsp. pertenue-immune hamsters protected irradiated naive hamsters against challenge with this subspecies. IL-2 production could still be detected in the irradiated recipients 12 days after irradiation of naive recipients, although at a low level. This suggests that the remaining lymph node cells could support the survival and expansion of the infused cytotoxic-suppressor T cells. No accumulation of macrophages was observed in regional lymph nodes of immune T-cell recipients within 10 days of infection. Instead, there was an influx of polymorphonuclear neutrophils in all animals injected with T. pallidum subsp. pertenue. This report demonstrates that hamster T cells can be separated into two phenotypically and functionally distinct subsets and that both T-cell subsets confer protection against challenge with T. pallidum subsp. pertenue.

Preclinical in vivo efficacy of two 9-dihydrotaxane analogues against human and murine tumours.

Two 9-dihydrotaxane analogues were synthesised and tested for in vitro potency and in vivo efficacy against murine and human tumour xenografts in mice. The in vitro potency of 9-dihydrotaxol (9-DH-t) and 10-deacetyl-9-dihydrotaxol (10-DeAc-9-DH-t) was generally less than that of paclitaxel against human and murine tumour cells. However, both analogues were at least 20-fold more soluble than paclitaxel in water. The analogues yielded cure rates > or = 60% against human MX-1 solid tumour xenografts in mice, compared with a cure rate of 10% for mice treated with paclitaxel. Both of the analogues were more effective than paclitaxel for treatment of murine M109 solid tumour in mice. 10-DeAc-9-DH-t was as effective as paclitaxel against murine B16 ascites tumour, while 9-DH-t was less effective. Both 10-DeAc-9-DH-t and 9-DH-t were demonstrably less toxic than paclitaxel. At equal dosages 9-DH-t produced serum concentrations greater than paclitaxel, while 10-DeAc-9-DH-t yielded serum concentrations less than paclitaxel. However, the decrease in toxicity of 9-DH-t and 10-DeAc-9-DH-t allowed a 4-fold increase in daily dosage. These two 9-dihydrotaxane analogues yielded favourable preclinical data and demonstrated good potential for further development.

Phagocytosis of Treponema pallidum pertenue by hamster macrophages on membrane filters.

The role of the macrophage in destruction of virulent treponemes is disputed. A major obstacle has been the inability to demonstrate quantitative phagocytosis of treponemes by macrophages. Treponema pallidum pertenue was attached to polycarbonate filters for assessment of treponemal phagocytosis by macrophages. The disappearance of treponemes due to phagocytosis was measured by enumeration with immunofluorescence. Resident and lipopolysaccharide-activated macrophages were found to phagocytize treponemes equally well. The phagocytosis of killed treponemes by macrophages was enhanced by opsonization with immune serum. Macrophages successfully phagocytized Staphylococcus aureus organisms when they were incubated on filters under identical conditions. Treatment of macrophages with cytochalasin B, a known inhibitor of phagocytosis, prevented the disappearance of treponemes and phagocytosis of S. aureus. In addition, fluorescent treponemal debris was observed only inside macrophages cultured with treponemes. These results demonstrate that macrophages can phagocytize pathogenic treponemes on polycarbonate filters.

Enteral formula composition does not affect response to lethal infectious challenge in mice.

The effects of enteral formulations on the response of mice to infectious challenge with Listeria monocytogenes, influenza A or Candida albicans were studied to test the efficacy of specialized ingredients. CF-1 outbred female mice (12-15 g) were fed nonpurified diet (Purina No. 5002) or commercially available liquid formulas: Osmolite HN, Perative or Impact. There were no differences between the groups fed the liquid formulas with regards to mean survival time or percentage of survivors in any of these models of infection. Examination of spleens from the groups challenged with L. monocytogenes, lungs from mice infected with Influenza A and kidneys from the groups challenged with C. albicans revealed no differences in cure rate of survivors. Pre-feeding periods of up to 8 d before infection produced similar results for mice fed enteral formulations compared to nonpurified diet. Contrary to previous reports, the use of Impact did not improve resistance to disease in mice challenged with lethal doses of L. monocytogenes, as compared with mice fed Osmolite HN. Additionally, mice fed Impact, Perative, or nonpurified diet responded similarly to challenge with L. monocytogenes, C. albicans or influenza A. The results indicate that these acute lethal animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas.

Synergistic effect of macrophage activation and immune serum, especially IgG2, on resistance to infection with Treponema pallidum ssp. endemicum in hamsters.

Experimental studies have indicated that macrophages are involved in the pathogenesis of syphilis. Whether macrophages alone or with immune serum are ultimately responsible for killing of treponemes is disputed. We have demonstrated that BCG-vaccinated hamsters administered normal serum contained fewer treponemes in the inguinal and popliteal lymph nodes than did the nonvaccinated controls. When BCG-vaccinated hamsters were injected with syphilitic immune serum and challenged with Treponema pallidum ssp. endemicum, treponemicidal activity was enhanced. Treponemicidal activity was also detected in BCG-vaccinated hamsters challenged with treponemes treated in vitro with immune serum and its immunoglobulin fractions, especially IgG2. The immune IgG2 fraction had more treponemicidal activity than did the immune IgG1 fraction and the unfractionated immune serum. Our observations indicate an important synergistic role for macrophages and immune serum, especially IgG2, for elimination of T. pallidum ssp. endemicum from the host.

In vivo efficacy of ABT-255 against drug-sensitive and -resistant Mycobacterium tuberculosis strains.

Current therapy for pulmonary tuberculosis involves 6 months of treatment with isoniazid, pyrazinamide, rifampin, and ethambutol or streptomycin for reliable treatment efficacy. The long treatment period increases the probability of noncompliance, leading to the generation of multidrug-resistant isolates of Mycobacterium tuberculosis. A treatment option that significantly shortened the course of therapy, or a new class of antibacterial effective against drug-resistant M. tuberculosis would be of value. ABT-255 is a novel 2-pyridone antibacterial agent which demonstrates in vitro potency and in vivo efficacy against drug-susceptible and drug-resistant M. tuberculosis strains. By the Alamar blue reduction technique, the MIC of ABT-255 against susceptible strains of M. tuberculosis ranged from 0.016 to 0.031 microg/ml. The MIC of ABT-255 against rifampin- or ethambutol-resistant M. tuberculosis isolates was 0.031 microg/ml. In a murine model of pulmonary tuberculosis, 4 weeks of oral ABT-255 therapy produced a 2- to 5-log10 reduction in viable drug-susceptible M. tuberculosis counts from lung tissue. Against drug-resistant strains of M. tuberculosis, ABT-255 produced a 2- to 3-log10 reduction in viable bacterial counts from lung tissue. ABT-255 is a promising new antibacterial agent with activity against M. tuberculosis.

Dynamics of clarithromycin and azithromycin efficacies against experimental Haemophilus influenzae pulmonary infection.

The dynamics of clarithromycin and azithromycin efficacy against pulmonary Haemophilus influenzae infection in rats were evaluated. Efficacy was measured by reduction in pulmonary H. influenzae burden on days 3 and 7 postinoculation. Clarithromycin therapy was effective on day 3 or 7 of therapy, while azithromycin was effective on day 7 but not on day 3 of therapy. Both macrolides produced marked efficacy against all six strains of H. influenzae tested, including four strains for which MICs were above the susceptible breakpoint (8 microgram/ml) concentration of clarithromycin. The two macrolides demonstrated markedly different pharmacokinetic characteristics, with clarithromycin present in both blood and tissue, while azithromycin was concentrated primarily in tissue. During pulmonary infection in rats, H. influenzae was found in both intracellular locations and an extracellular location in the lung. Blood concentrations of clarithromycin and azithromycin approximated human pharmacokinetics, and the blood concentrations for either macrolide rarely exceeded MICs for H. influenzae. At dosages producing blood concentrations similar to values achieved clinically, clarithromycin produced efficacy on day 3 of therapy, while both clarithromycin and azithromycin were equally effective on day 7. The different dynamics of clarithromycin and azithromycin suggest that length of therapy should be considered as a key parameter in evaluations of drug efficacy.