Novel erythromycin derivatives with aryl groups tethered to the C-6 position are potent protein synthesis inhibitors and active against multidrug-resistant respiratory pathogens.

A novel series of erythromycin derivatives has been discovered with potent activity against key respiratory pathogens, including those resistant to erythromycin. These compounds are characterized by having an aryl group tethered to the C-6 position of the erythronolide skeleton. Extensive structural modification of the C-6 moiety led to the discovery of several promising compounds with potent activity against both mef- and erm-mediated resistant Streptoccoccus pneumoniae. Preliminary mechanistic studies indicated that the new macrolides are potent protein synthesis inhibitors, which interact with methylated ribosomes isolated from resistant organisms. In experimental animal models, these compounds exhibited excellent in vivo efficacy and balanced pharmacokinetic profiles.

Efficacies of ABT-773, a new ketolide, against experimental bacterial infections.

ABT-773 is a novel ketolide effective against antibacterial-resistant respiratory tract pathogens. The pharmacokinetic profile of ABT-773 was studied in rats and consisted of a mean peak concentration in plasma of 1.07 microg/ml and an area under the concentration-time curve (AUC) of 12.03 microg. h/ml when the compound was delivered at a dose of 25 mg/kg of body weight. It concentrated in rat lung tissue, with a lung tissue-to-plasma ratio of 29 based on the AUC. In acute systemic infections in mice, ABT-773 showed efficacy against macrolide-susceptible strains of Staphylococcus aureus, Streptococcus pneumoniae, S. pyogenes, and Listeria monocytogenes. Additionally, ABT-773 improved the survival of mice infected with resistant S. pneumoniae containing either the ermB gene, the mefE gene, or altered penicillin binding protein genes. In a rat lung model of infection, ABT-773 demonstrated 50% effective doses lower than those of comparator macrolides when evaluated against the following strains of S. pneumoniae: a macrolide-lincosamide-streptogramin B-susceptible strain, an ermB strain, and an mefE strain. ABT-773 was also effective against Haemophilus influenzae lung infections in rats. Thus, ABT-773 may prove to be a useful new antibacterial agent for the treatment of respiratory tract infections.

Total synthesis and antifungal evaluation of cyclic aminohexapeptides.

The need for new therapies to treat systemic fungal infections continues to rise. Naturally occurring hexapeptide echinocandin B (1) has shown potent antifungal activity via its inhibition of the synthesis of beta-1,3 glucan, a key fungal cell wall component. Although this series of agents has been limited thus far based on their physicochemical characteristics, we have found that the synthesis of analogues bearing an aminoproline residue in the 'northwest' position imparts greatly improved water solubility (> 5 mg/mL). The synthesis and structure-activity relationships (SAR) based on whole cell and upon in vivo activity of the series of compounds are reported.

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.

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.

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.

Synthesis and structure-activity relationships of 2-pyridones: a novel series of potent DNA gyrase inhibitors as antibacterial agents.

Two novel series of 2-pyridones were synthesized by transposition of the nitrogen of 4-quinolones to the bridgehead position. This subtle interchange of the nitrogen atom with a carbon atom yielded two novel heterocyclic nuclei, pyrido[1,2-alpha]pyrimidine and quinolizine, which had not previously been evaluated as antibacterial agents and were found to be potent inhibitors of DNA gyrase. Quinolizines with a methyl group at the 9-position such as (S)-45a (ABT-719) demonstrate exceptional broad spectrum antibacterial activity. Most notably, they are active against resistant bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of enterococci, and ciprofloxacin-resistant organisms. In addition, 2-pyridones also possess favorable physiochemical and pharmacokinetic properties. These 2-pyridones were synthesized from the commercially available starting materials by 10-17 linear transformations. The structure of an adduct yielded by this sequence, (S)-45a (ABT-719), was determined by X-ray crystallographic analysis.

Efficacies of ABT-719 and related 2-pyridones, members of a new class of antibacterial agents, against experimental bacterial infections.

The 2-pyridones are a new class of broad-spectrum orally bioavailable antibacterial agents. These compounds are potent bacterial DNA gyrase inhibitors which differ from fluoroquinolones by placement of the nitrogen atom in the ring juncture. ABT-719 is an S isomer and a representative 2-pyridone. ABT-719 administered orally or subcutaneously was 4- to 10-fold more effective than ciprofloxacin against Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes infections in normal mice. ABT-719 was equivalent in efficacy to ciprofloxacin for treatment of gram-negative bacterial infections caused by Pseudomonas aeruginosa or Escherichia coli. The racemate and R forms of ABT-719 produced similar results against gram-positive and gram-negative bacterial infections. The 50% effective doses of ABT-719 were at least threefold lower than those of ciprofloxacin for therapy of intracellular infections caused by Salmonella typhimurium or Listeria monocytogenes. In immunosuppressed mice, ABT-719 was more effective than ciprofloxacin against quinolone-sensitive S. aureus, Enterococcus faecalis, and Enterococcus faecium. The pharmacokinetic properties of ABT-719 were consistent with its relative efficacy. The 2-pyridones are potent, orally available antibacterial agents with efficacy against gram-positive and gram-negative bacterial infections in mice.

Treatment of experimental Toxoplasma gondii infection by clarithromycin-based combination therapy with minocycline or pyrimethamine.

The efficacy of clarithromycin combined with either pyrimethamine or minocycline for treatment of experimental Toxoplasma gondii infection was investigated. Mice were infected intraperitoneally with 2 x 10(3) to 2 x 10(4) T. gondii strain RH or TS4 tachyzoites. Mortality was recorded for 35 days postinfection. Latency was evaluated by inoculation of brain homogenates from surviving mice into naive untreated mice. The combination of clarithromycin and pyrimethamine therapy caused a significantly greater reduction in mortality than did either drug alone. Similar synergy was observed between clarithromycin and minocycline. A 100% cure rate of active and latent infection was achieved in mice treated with the clarithromycin based combinations. Clarithromycin in combination with either pyrimethamine or minocycline produced efficacy comparable to combined therapy of pyrimethamine with sulfamethoxazole. The in vitro potency of clarithromycin, pyrimethamine, or minocycline against T. gondii on a mouse macrophage monolayer was not predictive of the in vivo efficacy in mice. Clarithromycin combined with minocycline or pyrimethamine could allow greater flexability for treatment of patients predisposed to the toxicity associated with standard pyrimethamine-sulfonamide or pyrimethamine-nonsulfonamide therapy. This therapy could be especially useful since clarithromycin-based therapy provides safe and effective treatment against Mycobacterium avium complex infections associated with AIDS patients.

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.

An immunosuppressed rat model of respiratory cryptosporidiosis.

A rat model is described in which animals develop respiratory cryptosporidiosis, a disease which is well documented in immunocompromised patients, especially those with AIDS. Our present lack of knowledge of the pathophysiology and immunology of Cryptosporidium parvum respiratory infections warrants the development of a laboratory animal model. Lewis rats immunosuppressed by subcutaneous injection of methylprednisolone acetate and inoculated intratracheally with 10(6) C. parvum oocysts developed a reproducible infection consisting of all known developmental stages in the epithelium lining airways from the trachea to the terminal bronchioles. Developmental stages were morphologically indistinguishable from those seen in gut epithelium. Infections were apparent at 4 days post-inoculation, and at 10-14 days post-inoculation, rats exhibited respiratory distress and severe weight loss and had enlarged, elastic lungs. Increased mucus production and exfoliative necrosis of the epithelium resulted in accumulation of large amounts of mucocellular exudate throughout the airways and patchy alveolitis involving alveoli emerging from respiratory bronchioles.

Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies.

Microspheres composed of biocompatible, biodegradable poly(DL-lactide-co-glycolide) (DL-PLG) and staphylococcal enterotoxin B (SEB) toxoid were evaluated as a vaccine delivery system when subcutaneously injected into mice. As measured by circulating immunoglobulin G (IgG) antitoxin titers, the delivery of SEB toxoid via DL-PLG microspheres, 1 to 10 microns in diameter, induced an immune response which was approximately 500 times that seen with nonencapsulated toxoid. The kinetics, magnitude, and duration of the antitoxin response induced with microencapsulated toxoid were similar to those obtained when an equal toxoid dose was administered as an emulsion with complete Freund adjuvant. However, the microspheres did not induce the inflammation and granulomata formation seen with complete Freund adjuvant. The adjuvant activity of the microspheres was not dependent on the superantigenicity of SEB toxin and was equally effective at potentiating circulating IgG antitrinitrophenyl levels in response to microencapsulated trinitrophenyl-keyhole limpet hemocyanin. Empty DL-PLG microspheres were not mitogenic, and SEB toxoid injected as a mixture with empty DL-PLG microspheres was no more effective as an immunogen than toxoid alone. Antigen-containing microspheres 1 to 10 microns in diameter exhibited stronger adjuvant activity than those greater than 10 microns, which correlated with the delivery of the 1- to 10-microns, but not the greater than 10-microns, microspheres into the draining lymph nodes within macrophages. The antibody response induced through immunization with microencapsulated SEB toxoid was protective against the weight loss and splenic V beta 8+ T-cell expansion induced by intravenous toxin administration. These results show that DL-PLG microsphere vaccine delivery systems, which are composed of pharmaceutically acceptable components, possess a strong adjuvant activity for their encapsulated antigens.

Biodegradable microspheres as a vaccine delivery system.

The utility of biodegradable and biocompatible microspheres as a vaccine delivery system for the induction of systemic and disseminated mucosal antibody responses was investigated. Intraperitoneal (ip) injection into mice of 1-10 microns microspheres, constructed of the copolymer poly(DL-lactide-coglycolide) (DL-PLG) which contained approximately 1% by weight a formalinized toxoid vaccine of staphylococcal enterotoxin B (SEB), dramatically potentiated the circulating IgG anti-toxin antibody response as compared to the free toxoid. The initiation of vaccine release was delayed in larger microspheres, and a mixture of 1-10 and 20-50 microns microspheres stimulated both a primary and an anamnestic secondary anti-toxin response following a single injection. However, neither free nor microencapsulated SEB toxoid induced a detectable mucosal IgA anti-toxin response following systemic injection. In contrast, three peroral immunizations with toxoid-microspheres stimulated circulating IgM, IgG and IgA anti-toxin antibodies and a concurrent mucosal IgA response in saliva, gut washings and lung washings. Systemic immunization with microencapsulated toxoid primed for the induction of disseminated mucosal IgA responses by subsequent oral or intratracheal (it) boosting in microspheres, while soluble toxoid was ineffective at boosting. These results indicate that biodegradable and biocompatible microspheres represent an adjuvant system with potentially widespread application in the induction of both circulating and mucosal immunity.

Vaccine-containing biodegradable microspheres specifically enter the gut-associated lymphoid tissue following oral administration and induce a disseminated mucosal immune response.

Biodegradable and biocompatible microspheres have been investigated for their usefulness as a vaccine delivery system for both parenteral and enteral immunization. Microspheres composed of poly(DL-lactide-co-glycolide) which contained a toxoid vaccine of Staphylococcal enterotoxin B were found to strongly potentiate the circulating anti-toxin antibody response following intraperitoneal injection. Following oral administration, microspheres less than 10 microns in diameter were specifically taken up into the Peyer's patches of the gut-associated lymphoid tissue, where those greater than or equal to 5 microns remained fixed for an extended period. Microspheres less than 5 microns were disseminated within macrophages to the mesenteric lymph nodes, blood circulation and spleen. Oral immunization with enterotoxoid-containing microspheres induced circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin response in saliva, gut wash fluids and bronchial-alveolar wash (BAW) fluids. In contrast, soluble enterotoxoid was completely ineffective as an oral immunogen.

Biodegradable microspheres: vaccine delivery system for oral immunization.

The potential of biocompatible and biodegradable microspheres as a controlled release oral vaccine delivery system has been examined. Orally-administered 1-10 micron microspheres composed of poly (DL-lactide-co-glycolide) were specifically taken up into the Peyer's patch lymphoid tissue of the gut, where those greater than or equal to 5 micron remained for up to 35 days. Microspheres less than 5 micron disseminated within macrophages to the mesenteric lymph nodes and spleen. In contrast to soluble staphylococcal enterotoxin B toxoid, oral immunization with enterotoxoid in microspheres induced circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin response in saliva and gut fluid.

Simultaneous expression of multiple immune complex receptors on murine thymocytes and spleen cells.

Mixed rosette studies were performed to evaluate the coexpression of IgG Fc. IgM Fc, and complement receptors (C3R) by thymocytes obtained from mice 7 days after cortisone injection and by spleen cells. Indicator cells coated with IgM, IgG, or C3 independently were mixed and could be distinguished by morphology or by a fluorescein label. In double-marker studies, 36% of spleen cells formed rosettes with IgG- and/or IgM-sensitized red blood cells. Among this population there was a 24% overlap of cells binding IgM and IgG complexes simultaneously. Of the spleen cells, 78% bound IgM- and/or C3-sensitized cells. Of the spleen cells forming rosettes with IgM and C3 indicator cells, 15% coexpressed these receptors. With IgG and C3 indicator cells, 58% of spleen cells bound to one or both kinds of complexes with an 18% overlap. Of cortisone-resistant thymocytes, 14% formed rosettes with IgM- and/or IgG-sensitized red blood cells; within this population there was an overlap of 21%. With IgM- or C3-sensitized cells, 19% of cortisone-resistant thymocytes bound to one or both, among which there was a coexpression of 21%. With IgG- or C3-sensitized cells, there was a 14% overlap of rosette-forming cells binding both. In triple-marker studies 79% of spleen cells formed rosettes with C3-, IgG-, and/or IgM-sensitized indicator cells, out of which 11% coexpressed IgM and IgG FcR, 20% coexpressed IgG and C3R, and 10% coexpressed IgM FcR and C3R. Of rosette-forming cells, 13% coexpressed all three receptors. With cortisone-resistant thymocytes, 19% bound one or more kinds of immune complexes. Among these, 9% coexpressed IgG FcR and C3R, 14% coexpressed IgM FcR and C3R, and 14% bound IgG and IgM complexes. We could not detect the simultaneous expression of all three receptors on cortisone-resistant thymocytes. Using Isopaque-Ficoll fractionation of cells binding C3-sensitized cells, cortisone-resistant thymocytes were enriched and depleted of C3-receptor-bearing cells and their Lyt phenotypes were determined by immunofluorescence microscopy. The C3-receptor-enriched population contained 56% C3R+ cells which were 79% Lyt-1 positive and 100% Lyt-2 positive. The C3R-depleted population contained 1.3% C3R+ cells with 10% Lyt-1 positive and 22% Lyt-2 positive among the total. Surface phenotypic expression of normal and cortisone-resistant thymocytes was also evaluated by direct and indirect fluorescence by fluorescence-activated cell sorter (FACS).(ABSTRACT TRUNCATED AT 400 WORDS)