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S F Queener

Publications and source records attributed to S F Queener.

At least 55 records · Page 3Linked to original sources
PubMed1996

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.

Anidulafungin↗
PubMed1995

Nonclassical 2,4-diamino-6-(aminomethyl)-5,6,7,8-tetrahydroquinazoline antifolates: synthesis and biological activities.

Twenty 6-substituted 2,4-diaminotetrahydroquinazolines were designed, synthesized, and biologically evaluated as novel nonclassical inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents. The 6-substituents included substituted anilinomethyls, with alkoxy (OCH3, and OCH2CH3) and halogen (Cl and Br) moieties on the phenyl ring; an indolinomethyl; and 1-naphthylaminomethyls. The compounds were synthesized from a protected key intermediate 2,4-bis(acetamido)-5,6,7, 8-tetrahydroquinazoline-6-carboxaldehyde (26) by reductive amination with the appropriate amine. Compound 26 was obtained via a Diels--Alder reaction of 2-(trimethylsiloxy) -1,3-butadiene with acrolein to afford cyclohexanone-4-carboxaldehyde dimethyl acetal (23) after deprotection of the silyloxy group and protection of the aldehyde in a single step. Cyclocondensation of 23 with dicyandiamide followed by protection of the 2,4-diamino groups and deprotection of the 6-acetal gave 26. The compounds were significantly potent ((7-330) x 10(-9) M) and selective against T. gondii (versus rat liver DHFR). The most selective analogue against T. gondii DHFR was 2,4-diamino-6-[[(2',5'-dimethoxyphenyl) methylamino]methyl]-5,6,7,8-tetrahydroquinazoline (5) which showed exceptionally high inhibitory activity against the growth of T. gondii cells in culture (IC50 = 5.4 x 10(-8) M). Selected analogues were evaluated as inhibitors of the growth of tumor cells in culture. The most active analogues inhibited the growth of tumor cells at GI50 = 10(-8) M.

Animals↗
PubMed1995

2,4-Diaminopyrido[3,2-d]pyrimidine inhibitors of dihydrofolate reductase from Pneumocystis carinii and Toxoplasma gondii.

Six previously unknown 2,4-diamino-6-(anilinomethyl)- and 2,4-diamino-6-[(N-methylanilino)-methyl]pyrido[3,2-d]pyrimidines (5-10) were synthesized from 2,4-diamino-6-(bromomethyl)-pyrido[3,2-d]pyrimidine hydrobromide (11.HBr) by treatment with the appropriate aniline or N-methylaniline in dimethylformamide at room temperature, with or without NaHCO3 present. Compounds 5-10 were tested as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, and rat liver as a part of a larger effort directed toward the discovery of lipophilic nonclassical antifolates combining high enzyme selectivity and high potency. Of the six analogues tested, the most potent and selective against T. gondii DHFR was 2,4-diamino-6-[(3',4',5'-trimethoxy-N-methylanilono)methyl]pyrido[ 3,2-d d pyrimidine (7), which had an IC50 of 0.0047 microM against this enzyme as compared with 0.026 microM against the rat liver enzyme. The potency of 7 against T. gondii DHFR was similar to that of trimetrexate (TMQ, 1) and piritrexim (PTX, 2) but was > 500-fold greater than that of trimethoprim (TMP, 3). However, while 7 was more selective than either TMQ (19x) or PTX (63x) against this enzyme, its selectivity in comparison with TMP was 8-fold lower. 2,4-Diamino-6-[3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyri midin e (6) was 17-fold less active than 7 and was also less selective. 2,4-Diamino-6-[(3',4'-dichloro-N-methylanilino)methyl]pyrido[3, 2-d]pyrimidine (10) had an IC50 of 0.022 microM against P. carinii DHFR and was comparable in potency to TMQ and PTX. The species selectivity of 10 for P. carinii versus rat liver DHFR was greater than that of either TMQ (21-fold) or PTX (31-fold). On the other hand, even though 10 was slightly more active than TMQ against the P. carinii enzyme, its selectivity was 7-fold lower than that of TMP. Thus, the goal of combining high enzyme binding activity, which is characteristic of the fused-ring compounds TMQ and PTX, with high selectivity for T. gondii and P. carinii DHFR versus rat liver DHFR, which is characteristic of the monocyclic compound TMP, remained unmet in this limited series.

Animals↗
PubMed1995

Novel 2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines as classical and nonclassical antifolate inhibitors of dihydrofolate reductases.

Eight novel, nonclassical, antifolate 2,4-diamino-5-(anilinomethyl)pyrrolo[2,3-d]pyrimidines, 1-8, with 3',4',5'-trimethoxyphenyl, 3',4'-dimethoxyphenyl, 2',5'-dimethoxyphenyl, 4'-methoxyphenyl, 2',5'-diethoxyphenyl, 3',4'-dichlorophenyl, 1'naphthyl, and phenyl substituents were synthesized as potential inhibitors of dihydrofolate reductases (DHFRs). The classical analogue N-[4-[N-[(2,4-diaminopyrrolo[2,3-d]pyrimidin- 5-yl)methyl]amino]benzoyl]-L-glutamic acid (9) was also synthesized as an inhibitor of DHFR and an antitumor agent. The classical and nonclassical analogues were obtained via reductive condensations of the key intermediate 2,4-diamino-5-cyanopyrrolo[2,3-d]pyrimidine (12) with the appropriate substituted aniline or (p-aminobenzoyl)-L-glutamate followed by reduction of the intermediate Schiff bases with NaCNBH3. Compounds 1-9 were evaluated in vitro as inhibitors of rat liver (rl), Pneumocystis carinii (pc), and Toxoplasma gondii (tg) DHFRs. The nonclassical analogues were significantly selective against tgDHFR (vs rat liver DHFR), ranging from 7- to 92-fold. The inhibitory activity was lower in pcDHFR and rlDHFR (IC50s > 10(-5) M) than in tgDHFR (IC50s = 10(-6) M). The classical analogue had inhibitory activity similar to that of methotrexate (MTX) against the growth of human leukemia CCRF-CEM, A253, and FaDu squamous cell carcinoma (SCC) of the head and neck cell lines. Further evaluation of 9 against CCRF-CEM and its sublines having defined mechanisms of MTX resistance demonstrated that the analogue utilizes the reduced folate/MTX-transport system and primarily inhibits DHFR and poly-gamma-glutamylation plays a role in its mechanism of action. Compound 9 was found to be 3-fold more efficient than aminopterin as a substrate for human folylpolyglutamate synthetase.

Folic Acid Antagonists↗
PubMed1995

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents.

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.

Animals↗
PubMed1995

2,4-Diamino-5-substituted-quinazolines as inhibitors of a human dihydrofolate reductase with a site-directed mutation at position 22 and of the dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii.

2,4-Diaminoquinazoline antifolates with a lipophilic side chain at the 5-position, and in one case with a classical (p-aminobenzoyl)-L-glutamate side chain, were synthesized as potentially selective inhibitors of a site-directed mutant of human dihydrofolate reductase (DHFR) containing phenylalanine instead of leucine at position 22. This mutant enzyme is approximately 100-fold more resistant than native enzyme to the classical antifolate methotrexate (MTX), yet shows minimal cross resistance to the nonclassical antifolates piritrexim (PTX) and trimetrexate (TMQ). Although they were much less potent than trimetrexate and piritrexim, the lipophilic 5-substituted analogues were all found to bind approximately 10 times better to the mutant DHFR than to the wild-type enzyme. The potency of the analogue with a classical (p-aminobenzoyl)-L-glutamate side chain was similarly diminished in comparison with MTX, but the difference in its binding affinity to the two DHFR species was only 5-fold. Thus, by making subtle structural changes in the antifolate molecule, it may be possible to attack resistance due to mutational alterations in the active site of the target enzyme. Also, to test the hypothesis that DHFR from Pneumocystis carinii and Toxoplasma gondii may have a less sterically restrictive active site than the enzyme from mammalian cells, inhibition assays using several of the lipophilic analogues in the series were carried out against the P. carinii and T. gondii reductases in comparison with the enzyme from rat liver. In contrast to their preferential binding to mutant versus wild-type human DHFR, binding of these analogues to the P. carinii and T. gondii enzymes was weaker than binding to rat enzyme. It thus appears that, if the active site of the DHFR from these parasites is less sterically restrictive than the active site of the mammalian enzyme, this difference cannot be successfully exploited by moving the side chain from the 6-position to the 5-position.

Animals↗
PubMed1995

Diagnosis of Pneumocystis carinii pneumonia in AIDS patients by using polymerase chain reactions on serum specimens.

A polymerase chain reaction (PCR) that amplifies the internal transcribed spacers of rRNA genes of Pneumocystis carinii (Pc-ITS-PCR) was compared with dihydrofolate reductase (DHFR) gene PCR to determine its usefulness in the diagnosis of Pneumocystis carinii pneumonia. All specimens from non-P. carinii pneumonia AIDS patients were both Pc-ITS-PCR- and DHFR PCR-negative. Twenty-six (96%) of 27 bronchoalveolar lavage specimens from P. carinii pneumonia patients were Pc-ITS-PCR-positive, and all 27 of these patients had at least 1 serum sample positive by Pc-ITS-PCR. In contrast, DHFR PCR detected P. carinii in only 4 (17%) of 24 P. carinii-positive lavage specimens and 2 (10%) of the 20 sera from P. carinii pneumonia patients. These results suggest that serum Pc-ITS-PCR may be useful for the diagnosis of P. carinii pneumonia.

AIDS-Related Opportunistic Infections↗
PubMed1995

Structure-activity and structure-selectivity studies on diaminoquinazolines and other inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Twenty-eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, five 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4-diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14 and 29 microM, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than fivefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-fluoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fifty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed fivefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl) pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminop-teridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species.

Animals↗
PubMed1995

Evaluation of potent inhibitors of dihydrofolate reductase in a culture model for growth of Pneumocystis carinii.

Many antifolates are known to inhibit dihydrofolate reductase from murine Pneumocystis carinii, with 50% inhibitory concentrations (IC50s) ranging from 10(-4) to 10(-11) M. The relationship of the potency against isolated enzyme to the potency against intact murine P. carinii cells was explored with 17 compounds that had proven selectivity for or potency against P. carinii dihydrofolate reductase. Pyrimethamine and one analog were inhibitory to P. carinii in culture at concentrations two to seven times the IC50s for the enzyme, suggesting that the compounds may enter P. carinii cells in culture. Methotrexate was a potent inhibitor of P. carinii dihydrofolate reductase, but the concentrations effective in culture were more than 1,000-fold higher than IC50s for the enzyme, since P. carinii lacks an uptake system for methotrexate. Analogs of methotrexate in which chlorine, bromine, or iodine was added to the phenyl ring had improved potency against the isolated enzyme but were markedly less effective in culture; polyglutamation also lowered the activity in culture but improved activity against the enzyme. Substitution of a naphthyl group for the phenyl group of methotrexate produced a compound with improved activity against the enzyme (IC50, 0.00019 microM) and excellent activity in culture (IC50, 0.1 microM). One trimetrexate analog in which an aspartate or a chlorine replaced two of the methoxy groups of trimetrexate was much more potent and was much more selective toward P. carinii dihydrofolate reductase than trimetrexate; this analog was also as active as trimetrexate in culture. These studies suggest that modifications of antifolate structures can be made that facilitate activity against intact organisms while maintaining the high degrees of potency and the selectivities of the agents can be made.

Culture Media↗
PubMed1994

2,4-Diamino-5-chloroquinazoline analogues of trimetrexate and piritrexim: synthesis and antifolate activity.

Ten heretofore undescribed 2,4-diamino-5-chloroquinazoline analogues of trimetrexate (TMQ) and piritrexim (PTX) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) from rat liver, Pneumocystis carinii, and Toxoplasma gondii. The most active quinazolines against both the P. carinii and the T. gondii enzyme were those with an ArCH2-NH or ArNHCH2 side chain. Among ArNH(CH2)n compounds with n = 1-3 and either 2',5'-dimethoxyphenyl or 3',4',5'-trimethoxyphenyl as the Ar moiety, activity decreased in the order n = 1 > n = 2 > n = 3. The best inhibitor of P. carinii DHFR, 2,4-diamino-5-chloro-6-[(N-methyl-3',4',5'-trimethoxyanilino)methy l] quinazoline (10) had an IC50 of 0.012 microM and was slightly more potent than TMQ and PTX. Compound 10 was also the best inhibitor of T. gondii DHFR, with an IC50 of 0.0064 microM corresponding again to a minor increase in activity over TMQ and PTX. However, as with these standard agents, 10 showed no appreciable selectivity for either the P. carinii or T. gondii enzyme relative to the rat liver enzyme. The highest selectivity achieved in this limited series was with 2,4-diamino-5-chloro-6-[N-(3',4',5'-trimethoxybenzyl)-N-methylamino] quinazoline (17) against T. gondii DHFR. While 17 (IC50 = 0.016 microM) was somewhat less potent than 10, its selectivity, as defined by the ratio IC50(rat liver)/IC50(T. gondii) was ca. 30-fold higher than that of TMQ or PTX. Two compounds, 2,4-diamino-5-chloro-6-[(3',4',5'-trimethoxyanilino)methyl] quinazoline (9) and 2,4-diamino-5-chloro-6-[N-(3',4',5'-trimethoxybenzyl) amino]quinazoline (15), were also tested against human DHFR and were found to have an IC50/[E] of 0.5, indicating that their binding was near-stoichiometric.

Animals↗
PubMed1994

Simultaneous determination of primaquine and carboxyprimaquine in plasma using high-performance liquid chromatography with electrochemical detection.

A selective and sensitive high-performance liquid chromatographic method with electrochemical detection is described for the simultaneous quantitation of primaquine and carboxyprimaquine, its primary metabolite, in plasma. After addition of internal standard, plasma was deproteinized by addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/ml with quantitation limits of 5 and 20 ng/ml, respectively. None of 47 tested antimicrobial agents interfered. In contrast to previously reported methods, the assay sensitivity and specificity are sufficient to permit quantitation of primaquine in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and Pneumocystis carinii pneumonia.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Classical and nonclassical furo[2,3-d]pyrimidines as novel antifolates: synthesis and biological activities.

Classical antifolate analogues containing a novel furo[2,3-d]pyrimidine ring system which include N-[4-[N-[(2,4-diaminofuro[2,3-d]pyrimidin-5- yl)methyl]amino]benzoyl]-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Four nonclassical antifolates, 2,4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, and 2,5-dimethoxy substituents, respectively, in the phenyl ring, were also synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms responsible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements of the key intermediate 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino-6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T. gondii, and Lactobacillus casei DHFRs. The classical analogues showed moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) with the N-CH3 analogue 2 about twice as potent as 1. The nonclassical analogues were inactive with IC50S > 3 x 10(-5) M. The classical analogues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferase, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical analogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were marginally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanisms of methotrexate (MTX) resistance, demonstrated that the analogues utilize the reduced folate/MTX-transport system and primarily inhibit DHFR and that poly-gamma-glutamylation was crucial to their mechanism of action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin or the combination of thymidine plus hypoxanthine. Furthermore, for compounds 1 and 2, in contrast to MTX, the FaDu cells were better protected by thymidine alone than hypoxanthine alone, suggesting a predominantly antithymidylate effect.

AIDS-Related Opportunistic Infections↗
PubMed1994

Albendazole inhibits Pneumocystis carinii proliferation in inoculated immunosuppressed mice.

Albendazole, a benzimidazole derivative widely used for treating helminth infections, was successfully used to treat and prevent development of Pneumocystis carinii pneumonia in transtracheally inoculated immunosuppressed mice. For treatment, 3 weeks postinoculation, albendazole at 300 and 600 mg/kg of body weight per day was administered in food for 3 weeks. For prophylaxis, albendazole was begun on the same day as inoculation at 300 mg/kg/day for 7 days, and then the dose was reduced to 150 mg/kg/day for 35 additional days. With these regimens, albendazole was effective both for treatment and prophylaxis. Both dexamethasone-immunosuppressed and L3T4+ monoclonal antibody-immunosuppressed mouse models were used, and albendazole inhibited P. carinii infection in both.

Albendazole↗
PubMed1994

Pneumocystis carinii is resistant to imidazole antifungal agents.

Because Pneumocystis carinii is closely related to fungi, drugs useful for treating mycoses have been considered for use in the treatment of P. carinii pneumonia. Six antifungal imidazole drugs were tested for their activities against P. carinii in a short-term culture screen and in animals. None of the imidazoles tested was effective in inoculated infected rats, and only miconazole showed slight effects in culture at the high concentration of 10 micrograms/ml. Analysis of cell membranes from culture-grown P. carinii showed that ergosterol, the target for this class of antifungal agents, was absent, so that the lack of effect of these agents is rational.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

Typing of Pneumocystis carinii strains that infect humans based on nucleotide sequence variations of internal transcribed spacers of rRNA genes.

Small portions of the 18S and the 26S rRNA genes, the entire 5.8S rRNA gene, and internal transcribed spacers ITS1 and ITS2 (located between the 18S and 5.8S rRNA genes and between the 5.8S and 26S rRNA genes, respectively) of Pneumocystis carinii that infect humans were cloned and sequenced. The nucleotide sequences of the 18S, 5.8S, and 26S rRNA genes determined in the study were approximately 90% homologous to those of P. carinii that infect rats, while the sequences of ITS1 and ITS2 of P. carinii from the two different hosts were only 60% homologous. The 18S, 5.8S, and 26S rRNA gene sequences of P. carinii from 15 patient specimens were determined and were found to be identical to each other, whereas the ITS sequences were found to be variable. With the observed sequence variation, it was possible to classify the ITS1 sequences into two types and the ITS2 sequences into three types. P. carinii strains that had the same type of ITS1 sequence could have a different type of ITS2 sequence. On the basis of the sequence types of the two ITS regions, P. carinii from the 15 patients were classified into four groups. P. carinii from three patient specimens were found to contain two different ITS sequence patterns. More surprisingly, one additional specimen was found to have one ITS sequence typical of P. carinii isolates that infect humans and another typical of P. carinii isolates that infect rats. The studies indicate that it is possible to type P. carinii strains on the basis from one patient, suggesting that coinfection with more than one strain of P. carinii may occur in the same patient.

Animals↗
PubMed1993

Synthesis of 5-methyl-5-deaza nonclassical antifolates as inhibitors of dihydrofolate reductases and as potential antipneumocystis, antitoxoplasma, and antitumor agents.

A series of 2,4-diamino-5-methyl-6-(anilinomethyl)pyrido[2,3-d]pyrimidines 4-9 were synthesized as 5-deaza nonclassical antifolates containing trimethoxy, dichloro-, or trichlorophenyl substitutions and a N-H, N-CH3, or N-CHO at the 10-position. The compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii), rat liver (RL), and Lactobacillus casei (L. casei); as inhibitors of T. gondii and P. carinii cell growth in culture; and as antitumor agents. The compounds were prepared by modifications of procedures for classical 5-deaza folates. 2,4-Diamino-5-methyl-6-[(3',4',5'-trimethoxy-N- methylanilino)methyl]pyrido[2,3-d]pyrimidine (5a) exhibited high potency as well as selectivity (compared to RL DHFR) for P. carinii and T. gondii DHFR. Compound 5a is one of the most potent and selective nonclassical folate inhibitors of T. gondii DHFR known. The N-10 formyl analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]pyrido-[2,3-d]pyrimidine (6a) had decreased potency, but it maintained high selectivity for T. gondii DHFR. The corresponding chloro-substituted analogues maintained potency or had decreased potency; N-10 substitution did not increase potency or selectivity to the extent observed in the 3',4',5'-trimethoxy series. Partial reduction of the B ring to afford the dihydro analogue 2,4-diamino-5-methyl-6-[(N-formyl-3',4',5'-trimethoxyanilino) methyl]-5,8-dihydropyrido[2,3-d]pyrimidine (7), its 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine analogue 8, and 2,4-diamino-5-methyl-6-[(3',4',5'-trimethoxyanilino)methyl]-5,6,7, 8- tetrahydropyrido[2,3-d]pyrimidine (9) resulted in a significant decrease in potency. In T. gondii cell culture inhibitory studies, 2,4-diamino-5-methyl-6-[(3',4',5'- trimethoxyanilino)methyl]pyrido[2,3-d]pyrimidine (4a), 5a, and 6a were less potent compared to their DHFR inhibitory potencies. Against P. carinii cells in culture, 4a and 5a at 10 micrograms/mL were as effective as the clinically used combination of trimethoprim/sulfamethoxazole (50/250 micrograms/mL). With the exception of the B ring reduced analogues 7-9, all of the compounds were significantly cytotoxic to leukemia CCRF-CEM cells in culture. The chloro-substituted analogues, in general, were more potent against a variety of other tumor cells in culture than the trimethoxy analogues. These results were corroborated by the preclinical tumor screening program at the National Cancer Institute where the most potent compound 2,4-diamino-5-methyl-6-[(3',4'-dichloroanilino)methyl]pyrido[2,3- d]pyrimidine (4b) was found to inhibit the growth of 26 tumor cell lines at an IG50 < 1.00 x 10(-8) M.

Animals↗