N-acyloxymethyl carbamate linked prodrugs of pseudomycins are novel antifungal agents.

We describe herein the synthesis, bioconversion, antifungal activity, and preliminary toxicology evaluation of a series of N-acyloxymethyl carbamate linked triprodrugs of pseudomycins. The syntheses of these prodrugs (3-6) were achieved via simple N-acylation of PSB (1) or PSC' (2) with various prodrug linkers (7-9). As expected, upon incubation with mouse and/or human plasma, many of these prodrugs (3, 5, and 6) were converted to the parent compound within a few hours. Of particular significance, two pseudomycin triprodrugs (5 and 6) showed excellent in vivo efficacy against systemic Candidiasis without tail vein irritation being observed.

Prodrugs of 3-amido bearing pseudomycin analogues: novel antifungal agents.

With the aim of identifying safer pseudomycin derivatives, we synthesized and evaluated a number of N-acyloxymethyl carbamate linked prodrugs of 3-amido pseudomycin analogues. To our satisfaction, all of the prodrug-amide combinations prepared exhibited good in vivo efficacy against murine Candidiasis. When evaluated in a dose elevation study, all of the newly synthesized combinations (e.g., 4A, 6A, 8A, and 8B) demonstrated improved toxicity profiles in comparison to their corresponding 3-amides as well as the parent pseudomycin B.

Syntheses and antifungal activities of novel 3-amido bearing pseudomycin analogues.

As a result of our core SAR effort, we discovered a large number of 3-amido pseudomycin B (PSB) analogues (e.g., 4e LY448212 and 5b LY448731) that retain good in vitro and in vivo (IP) activities against Candida and Cryptococcus without inherent tail vein irritation. Several dimethylamino termini bearing 3-amides (e.g., 5b) also exhibited improved potency against Aspergillus in vitro. When evaluated in a two-week rat toxicology study, it was found that all animals receiving 4e (up to 75 mg/kg) were found to be normal. On the basis of these observations, we are convinced that it is possible to broaden the antifungal spectrum and improve the safety profile of pseudomycin analogues at the same time.

8-Amido-Bearing pseudomycin B (PSB) analogue: novel antifungal agents.

During the course of a structure-activity relationship (SAR) study on novel depsinonapeptide pseudomycin B, we synthesized a total of 12 8-amidopseudomycin analogues via standard two-step sequence from either ZPSB 2 or AllocPSB 3. A number of these amides exhibited good in vitro antifungal activities.

The synthesis of pseudomycin C' via a novel acid promoted side-chain deacylation of pseudomycin A.

The gamma hydroxyl present in the aliphatic side chain of the natural products pseudomycin A and C' provided a unique handle for the pH dependent side-chain deacylation. Low pH reaction conditions were used to cleave the side chain with minimal degradation of the peptide core. The pseudomycin nucleus intermediate obtained from the deacylation of pseudomycin A was pivotal in the synthesis of novel side-chain analogues. A practical synthesis of a minor fermentation factor pseudomycin C' and related analogues is reported.

Enhanced pneumocystis carinii activity of new primaquine analogues.

New analogues of the venerable antimalarial drug primaquine have been synthesized and bioassayed in vivo against Pneumocystis carinii, a life-threatening infection common among immunosuppressed patients. Two of these new compounds are significantly more active than primaquine itself, and provide new information for future drug design and development in this area.

LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy.

Semisynthetic echinocandins affect cell wall deposition of Pneumocystis carinii in vitro and in vivo.

Cyclic lipodepsipeptide compounds of the echinocandin class exhibit broad-spectrum antifungal activity and have been shown to be effective in the treatment of Pneumocystis carinii pneumonia in laboratory animal models. Previous studies have led investigators to propose that these compounds, active against fungal cell walls, are selectively active against the cyst forms of P. carinii. We demonstrate that a semisynthetic, water-soluble echinocandin analog, LY307853, is effective in reducing the number of all life cycle forms of P. carinii and is more effective in mice immunosuppressed with monoclonal antibody to L3T4+ cells than in mice immunosuppressed with dexamethasone. Treatment of P. carinii isolates with LY307853 in a short-term in vitro culture model resulted in cytoarchitectural alterations suggesting that this echinocandin may interfere with the export of surface glycoprotein and the formation of the tubular elements normally found on the surfaces of trophic forms. The cytoarchitectural changes in trophic forms treated in vitro with LY307853 were also observed in trophic forms in the lung tissue of rats treated with a closely related echinocandin analog, LY303366.

Comparison of corticosteroid- and L3T4+ antibody-immunosuppressed mouse models of Pneumocystis carinii pneumonia for evaluation of drugs and leukocytes.

An immunologically immunosuppressed mouse model of Pneumocystis carinii pneumonia using antibody developed by Dialynas et al. (Immunol. Rev. 74:29-55, 1983) directed to L3T4+ T cells (referred to as L3T4+ antibody) was compared with a corticosteroid-immunosuppressed mouse model. Corticosteroid- or L3T4+ antibody-immunosuppressed BALB/c mice transtracheally inoculated with P. carinii developed severe infections within 5 weeks after inoculation and responded to treatments with an echinocandin B analog, LY302146, or trimethoprim plus sulfamethoxazole so that they had decreased numbers of P. carinii cysts and trophozoites. LY302146 appeared to be more effective in L3T4+ antibody-immunosuppressed mice than in dexamethasone-immunosuppressed mice. Leukocyte populations in lungs of both mouse models during development of infection and during treatment were compared by using immune cell-specific staining. Lungs of L3T4+ antibody-immunosuppressed mice had many more cells detected with pan-B antibody and pan-T antibody than dexamethasone-immunosuppressed mice and the lungs of successfully treated mice had about the same numbers of macrophages as those of nonimmunosuppressed uninfected mice. The immunologically immunosuppressed model will allow study of cytokines and other immune modulators alone and in combination with drugs.

An intestinal xenograft model for Cryptosporidium parvum infection.

Paired segments of near-term fetal rabbit small intestine were transplanted subcutaneously into athymic nude mice. At 5 weeks postsurgery, the xenografts were inoculated intraluminally with Cryptosporidium parvum sporozoites. Parasites rapidly and reliably infected the xenograft mucosal epithelium. Lesions typical of cryptosporidiosis were readily apparent by light microscopy and scanning and transmission electron microscopy. Xenografts are well suited to the study of the early events of C. parvum infection and are of potential value in the evaluation of anticryptosporidial chemotherapeutic agents.

Improved rat model of Pneumocystis carinii pneumonia: induced laboratory infections in Pneumocystis-free animals.

An immunosuppressed rat model of Pneumocystis carinii pneumonia is described that utilizes simple, noninvasive intratracheal (i.t.) inoculation of cryopreserved parasites and results in development of severe P. carinii pneumonia within 5 weeks. This is an improvement over the most commonly used models of P. carinii pneumonia that rely on immune suppression to activate latent P. carinii infections and that often require 8 to 12 weeks to produce heavy infections of P. carinii. It is also less labor intensive than more recent models requiring surgical instillation of parasites. Our report describes a series of preliminary studies to select an appropriate strain of rat; to determine suitable methods for inducing uniform immunosuppression, P. carinii inoculation, and laboratory maintenance of P. carinii; and to determine effective animal husbandry methods for maintaining animals free from serious secondary infections. Results of our more detailed studies demonstrate that animals receiving two or three i.t. inoculations of approximately 10(6) cryopreserved P. carinii organisms have a predictable course of disease progression which includes moderate P. carinii infections within 3 weeks, severe P. carinii pneumonia in 5 weeks, and a high percentage of mortality due to P. carinii pneumonia in 6 weeks. Parasites were distributed evenly between the right and left lungs, regardless of the number of P. carinii inoculations administered. Non-P. carinii-inoculated immunosuppressed control rats maintained in microisolator cages remained free of P. carinii, thus providing an important control that is missing from many P. carinii pneumonia models. Most non-P. carinii-inoculated control animals and P. carinii-inoculated rats treated with trimethoprim-sulfamethoxazole that were housed in open caging in the same room containing heavily infected animals had no detectable infections after 5 to 6 weeks of immunosuppression; however, some had a small number of P. carinii in their lungs. Because heavy, reproducible infections are achieved 5 weeks after i.t. inoculation, because few animals are lost to secondary infections, and because animals can be maintained as noninfected contemporaneous controls, this animal model is useful for the maintenance of P. carinii strains, for studies of the transmission and natural history of P. carinii, for the production of large numbers of organisms for laboratory studies, and for the evaluation of potential anti-P. carinii drugs.

Cryptosporidiosis.

Data suggest that C. parvum is now one of the three most commonly found enteropathogens causing diarrheal illness in humans worldwide. This article discusses the etiologic agents, epidemiology, clinical features, diagnosis, and treatment of cryptosporidiosis. To date, no effective therapy for cryptosporidiosis has been identified.

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.

An improved rat model of Pneumocystis carinii pneumonia: induced infections in Pneumocystis-free animals.

An immunosuppressed rat model of Pneumocystis carinii pneumonia (PCP) is described that results in a predictable course of disease development which includes moderate P. carinii (Pc) infections in 2 to 3 weeks, heavy infections in 4 to 5 wk, and a high percentage of mortality due to PCP in 6 wk. The model also provides uninfected, immunosuppressed contemporary controls, an experimental compartment that is needed to correctly interpret results obtained from many different studies. Non-invasive intratracheal inoculation of cryopreserved parasites into Pc- and virus-free rats immunosuppressed by weekly injections of methylprednisolone are key features of the model that result in the development of consistent heavy Pc infections and very few secondary infections by bacteria and fungi. This model is useful for (1) maintaining isolates or strains of Pc over time, (2) producing large numbers of parasites for laboratory studies, and (3) evaluating the anti-Pc activity of experimental compounds and approved drugs.

Cryptosporidiosis.

Before 1982, only eight case reports of human cryptosporidiosis and fewer than 30 papers on Cryptosporidium spp. appeared in the biomedical literature. At that time, cryptosporidiosis was thought to be an infrequent infection in animals and rarely an opportunistic infection in humans. The concept of Cryptosporidium spp. as pathogens has changed dramatically within the past 8 years because of improved diagnostic techniques, increased awareness within the biomedical community, and the development of basic research programs in numerous laboratories. Presently, greater than 1,000 publications including over 400 case reports in the biomedical literature address Cryptosporidium spp. and cryptosporidiosis. Cryptosporidium parvum is now thought to be one of the three most common enteropathogens causing diarrheal illness in humans worldwide, especially in developing countries. It is likely that cryptosporidiosis was previously included in the 25 to 35% of diarrheal illness with unknown etiology. Because of the severity and length of diarrheal illness and because no effective therapy has been identified, cryptosporidiosis is one of the most ominous infections associated with AIDS. The role of C. parvum as an enteropathogen is well established; documentation of its role as a cause of hepatobiliary and respiratory diseases is now appearing in the literature. Our present understanding of the natural history, epidemiology, biology, and immunology of Cryptosporidium spp. as well as the clinical features, pathogenicity, and treatment of cryptosporidiosis are reviewed here.

Use of mouse macrophage cell lines for in vitro propagation of Toxoplasma gondii RH tachyzoites.

In most laboratories, Toxoplasma gondii is maintained in mice and is studied in vitro using nonlymphoid cell lines or primary mouse macrophages. In this study, three rapidly dividing mouse macrophage cell lines (J774 A.1, P388D1, RAW264.7) were evaluated for their suitability for studying the RH strain of T. gondii. For comparison, tachyzoites were also grown in two slowly dividing epithelial cell types: a rat lung cell line (L2) and a bovine turbinate cell line (BT). Various inocula of T. gondii were added to the above cells and tachyzoites were harvested from the culture supernatants after 2-8 days of infection. The mouse macrophage cell lines supported rapid growth of T. gondii RH allowing up to a 300-fold increase of the inoculum in 2-4 days. L2 and BT supported slower growth of T. gondii (10- to 90-fold increase of inoculum in 5 to 8 days) and, thus, may be more suitable for assessment of host cell-parasite interactions and drug activity. Toxoplasma gondii RH isolated from each of the cell cultures described were able to multiply in all cell types used. Protein profiles of whole tachyzoite isolated from mice or cell cultures and protein profiles of the corresponding soluble and membrane fractions of the intraphagosomal membrane network were similar as seen after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In mice, intraperitoneal injection of 10(6), 10(5), and 10(3) tachyzoites isolated from the cell cultures or from infected mice caused death after 4, 5, and 8 days, respectively, indicating that parasites grown in vitro retained virulence.

Comparative activity of macrolides against Toxoplasma gondii demonstrating utility of an in vitro microassay.

The utility of spiramycin for preventing transplacental transmission of toxoplasmosis and the efficacy of conventional macrolides against Toxoplasma gondii are subjects of active debate. An in vitro microassay was developed to determine the relative inhibitory activity against T. gondii of 24 conventional macrolides derived from erythromycin and tylosin (14- and 16-membered macrolides, respectively). Macrolides and T. gondii RH tachyzoites were added to monolayers of BT cells grown in 96-well plates. Plates were incubated for 20 h at 37 degrees C, and the growth of T. gondii was then measured by the selective incorporation of [3H]uracil in trichloroacetic acid-precipitable material during an additional incubation of 20 h. Dose-response curves and 50 and 90% inhibitory concentrations (IC50 and IC90, respectively) were determined for each drug. Microscopic examination was performed on stained replicates of the infected monolayers, and the relative toxicities of the drugs for host cells were determined. Spiramycin and tylosin showed only limited activity against T. gondii (IC50 of 20.16 and 20.00 micrograms/ml, respectively). Erythromycin and azithromycin had a better anti-Toxoplasma activity with IC50 of 14.38 and 8.61 micrograms/ml, respectively, whereas drugs like desmycosin, dirithromycin, and roxithromycin had no detectable activity. Although many macrolides inhibited intracellular proliferation of T. gondii, azithromycin was the only macrolide demonstrating prolonged inhibitory activity on the replication of intracellular tachyzoites. We conclude that conventional 14- and 16-membered macrolides often interfere with the growth of, but may not kill, T. gondii RH tachyzoites in vitro.