Trimetrexate for the Treatment of<i>Pneumocystis carinii</i>Pneumonia in Patients with the Acquired Immunodeficiency Syndrome

Allegra, Carmen J.; Chabner, Bruce A.; Tuazon, Carmelita U.; Ogata‐Arakaki, Debra; Baird, Barbara; Drake, James C.; Simmons, J T; Lack, Ernest E.; Shelhamer, James H.; Balis, Frank M.; Walker, Robert J.; Kovács, J.; Lane, H. Clifford; Masur, Henry

doi:10.1056/nejm198710153171602

Cited by 235 publications

(78 citation statements)

References 16 publications

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“…This is consistent with the fact that P. carini is a fungus. Very potent DHFR inhibitors such as trimetrexate and piritrexim are active as single agents against both murine and human PCP (1,23), but TMP alone appears to be ineffective in the treatment of rat PCP (16,26). While there is increasing evidence showing that the P. carinii dihydropteroate synthase is developing mutations that confer resistance to sulfa drugs (11,15,21; Mei et al, Letter, Lancet 351:1631, 1998), no mutations likely to confer drug resistance have been identified in the DHFR gene to date (21).…”

Section: Discussionmentioning

confidence: 99%

Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase

Kovacs

2000

Antimicrob Agents Chemother

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Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the ratderived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K m values for dihydrofolate and NADPH were 2.7 ؎ 0.3 and 14.0 ؎ 4.3 M, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K i values of 0.28 ؎ 0.08 and 0.065 ؎ 0.005 M, respectively, while trimetrexate and methotrexate were potent inhibitors, with K i values of 0.23 ؎ 0.03 and 0.016 ؎ 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for humanderived P. carinii DHFR.Pneumocystis carinii pneumonia (PCP) remains a leading cause of morbidity and mortality in AIDS. Currently, one of the most widely used agents for treatment and prophylaxis of this infection is the combination of trimethoprim (TMP) and sulfamethoxazole (SMX). TMP inhibits dihydrofolate reductase (DHFR) (EC 1.5.1.3), which catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate in the presence of NADPH and is essential for biosynthesis of thymidylate, purine nucleotides, and several amino acids. Despite its obvious efficacy, this combination is complicated by frequent toxic and allergic side effects (19); moreover, there are increasing concerns about whether TMP truly contributes to the activity of this combination against P. carinii. It has been shown in vitro that TMP is a poor inhibitor of rat-derived P. carinii DHFR (2,6,7,9,22,25) and that TMP alone is ineffective in the treatment of rat PCP (16,26). Recently, mutations in the P. carinii dihydropteroate synthase gene, the target of sulfamides, have been reported in the United States (15, 21; Q. Mei, S. Gurunathan, H. Masur, and J. A. Kovacs, Letter, Lancet 351:1631, 1998) and Europe (11) and have been associated with prophylaxis and/or treatment failures of TMP-SMX, suggesting that P. carinii is developing resistance to sulfa drugs. In contrast, the DHFR gene did not show any mutations suggestive of drug resistance (21). This may reflect an absence of drug pressure on DHFR and supports the concept that TMP contributes little to the efficacy of the TMP-S...

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Section: Discussionmentioning

confidence: 99%

Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase

Kovacs

2000

Antimicrob Agents Chemother

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“…Patients with P. carinii pneumonia are frequently treated with antifolates (inhibitors of tetrahydrofolate biosynthesis) such as bactrim (a combination of trimethoprim and sulfamethoxazole) (6,7), dapsone (6,13), and trimetrexate (1). The ability of P. carinii to synthesize tetrahydrofolate de novo has been reported recently (9).…”

mentioning

confidence: 99%

Inhibition of Pneumocystis carinii dihydropteroate synthetase by sulfa drugs

Merali

Zhang

Sloan

et al. 1990

Antimicrob Agents Chemother

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A new reversed-phase high-pressure liquid chromatography assay procedure for dihydropteroate synthetase (DHPS) that involves the elution of the enzyme incubation solution with a series of three solvents of decreasing polarity (ammonium phosphate buffer, 10% methanol, and 50% methanol) was designed. By this procedure DHPS was detected in Escherichia coli and Pneumocystis carinii with specific activities of 450 and 14 U/mg, respectively. A comparison of the effects of five sulfa drugs on P. carinii DHPS activity revealed that dapsone is the most potent of these drugs.Pneumocystis carinii is the major life-threatening opportunistic agent in patients with acquired immunodeficiency syndrome (AIDS) and affects other immunocompromised patients. Approximately 60% of all patients with AIDS suffer from P. carinii pneumonia, and up to 40% of the episodes prove fatal (10).Patients with P. carinii pneumonia are frequently treated with antifolates (inhibitors of tetrahydrofolate biosynthesis) such as bactrim (a combination of trimethoprim and sulfamethoxazole) (6, 7), dapsone (6, 13), and trimetrexate (1). The ability of P. carinii to synthesize tetrahydrofolate de novo has been reported recently (9).Two enzymes involved in the biosynthesis of tetrahydrofolate that are important as targets of antifolates are dihydropteroate synthetase (DHPS) and dihydrofolate reductase. DHPS catalyzes the formation of dihydropteroate (DHP) from p-aminobenzoic acid (pAB) and 6-hydroxymethyldihydropterin pyrophosphate (MDHP-PP). Dihydrofolate reductase catalyzes the formation of tetrahydrofolate from dihydrofolate. Sulfones and sulfonamides act as competitive inhibitors of DHPS substrates, while drugs such as trimethoprim and pyrimethamine act as inhibitors of dihydrofolate reductase substrates. In this study we measured the inhibitory actions of several sulfonamides and sulfones on the parasite associated with DHPS using a new high-pressure liquid chromatography (HPLC) enzyme assay. MATERIALS AND METHODSGrowth and isolation of organisms. Male Sprague-Dawley rats (weight, 150 to 200 g) were fed ad libitum and administered tetracycline in their drinking water (1 g/liter). Subcutaneous injections of cortisone acetate (25 mg) were given twice a week. After 4 to 6 weeks of injections, those rats which showed signs of rapid breathing, blood in their noses, weight loss, and hair loss were used in the experiments.P. carinii was isolated by a modification of the method of Gradus and Ivey (4). Each rat was sacrificed by cervical dislocation. Their lungs were then quickly removed and kept at 4°C. Touch preparations were made from the lungs and stained with toluidine blue (3) bacteria were evident. Subsequent inoculation of the lung homogenate onto Luria broth agar plates confirmed the absence of contaminants. The lungs were homogenized by repeated passage through a 60-gauge wire screen. The resulting suspension was centrifuged at 2,000 x g for 10 min. The supernatant was discarded, and the pellet was suspended in phosphate-buffered saline. The suspensio...

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“…Since the adoption of pentamidine for prophylaxis, many reports of disseminated Pneumocystis infection in patients receiving aerosolized pentamidine prophylaxis suggest that it only suppresses development of pulmonary infection (142,151,170 (3), and the drugs were tested in animal models. Two potent dihydrofolate reductase inhibitors, trimetrexate and piritrexim, showed anti-Pneumocystis activity in animal models (100,144) and clinical trials (2,163). Trimetrexate was effective for therapy, but relapse after treatment was frequent.…”

Section: Treatmentmentioning

confidence: 99%

Pneumocystis carinii, an opportunist in immunocompromised patients

Bartlett

Smith

1991

Clin Microbiol Rev

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Pneumocystis carinii has been recognized as a cause of pneumonia in immunocompromised patients for over 40 years. Until the 1980s, Pneumocystis pneumonia (pneumocystosis) was most often seen in patients undergoing chemotherapy for malignancy or transplantation. Infection could be prevented by trimethoprim-sulfamethoxazole prophylaxis; thus, it was an uncommon clinical problem. With the onset of the AIDS epidemic, Pneumocystis pneumonia has become a major problem in the United States because it develops in approximately 80% of patients with AIDS and because almost two-thirds of patients have adverse reactions to anti-Pneumocystis drugs. Thus, physicians and laboratories in any community may be called upon to diagnose and provide care for patients with Pneumocystis pneumonia. The classification of the organism is currently controversial, but it is either a protozoan or a fungus. P. carinii appears to be acquired during childhood by inhalation and does not cause clinical disease in healthy persons but remains latent. If the person becomes immunosuppressed, the latent infection may become activated and lead to clinical disease. Damage of type I pneumocytes by Pneumocystis organisms leads to the foamy alveolar exudate which is characteristic of the disease. Diagnosis is established by morphologic demonstration of Pneumocystis organisms in material from the lungs. Current efforts to find better anti-Pneumocystis drugs should provide more effective therapy and prophylaxis.

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Trimetrexate for the Treatment ofPneumocystis cariniiPneumonia in Patients with the Acquired Immunodeficiency Syndrome

Cited by 235 publications

References 16 publications

Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase

Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase

Inhibition of Pneumocystis carinii dihydropteroate synthetase by sulfa drugs

Pneumocystis carinii, an opportunist in immunocompromised patients

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