-
Annals of Oncology : Official Journal... Jan 1992To increase the complete remission (CR) rate achieved with two cycles of cisplatin, continuous infusion fluorouracil (5-FU) and oral leucovorin (PFL) we added two... (Clinical Trial)
Clinical Trial
To increase the complete remission (CR) rate achieved with two cycles of cisplatin, continuous infusion fluorouracil (5-FU) and oral leucovorin (PFL) we added two antifolate drugs, methotrexate (MTX) and the lipophilic piritrexim (PTX), to the combination (PFL-MP). Twenty-eight patients with previously untreated Stage IV squamous cell carcinoma of the head and neck received 2 cycles of cisplatin 100 mg/m2 on day 1 followed by a 5-day continuous infusion of 5-FU at 800 mg/m2/day and 100 mg of leucovorin administered orally every 4 hours. MTX was administered at 40 mg/m2 IV on day 15 and PTX at 75 mg orally twice daily on days 22 to 26, with cycle 2 starting on day 36. After 2 of the first 5 patients had tumor progression between days 15 and 35, the regimen was intensified to MTX 50 mg/m2, PTX 100 mg twice daily and a cycle duration of 28 days. Local therapy consisted of surgery and/or radiotherapy with concomitant 5-FU and hydroxyurea (FHX) administered every other week. Eleven patients (39%, 95% confidence intervals 21-57%) had a CR, 9 (32%) had a PR, and four patients had no response. Four patients were unevaluable for response to PFL-MP. Patients with poor performance status or N3 disease were less likely to achieve a CR. Mucositis following PFL was the dose-limiting toxicity. Local therapy included surgery in 15 patients and FHX chemoradiotherapy in 19 patients. The administration of FHX in this setting proved feasible and the regimen was given near the intended dose intensity in the majority of patients.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cisplatin; Combined Modality Therapy; Drug Administration Schedule; Drug Evaluation; Female; Fluorouracil; Head and Neck Neoplasms; Humans; Leucovorin; Male; Methotrexate; Middle Aged; Pyrimidines
PubMed: 1606074
DOI: 10.1093/oxfordjournals.annonc.a058079 -
The Journal of Protozoology 1991A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii...
A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.
Topics: 4-Aminobenzoic Acid; Animals; Drug Evaluation, Preclinical; Folic Acid; Male; Pneumocystis; Radiometry; Rats; Rats, Inbred Strains; Tritium
PubMed: 1818144
DOI: No ID Found -
The disposition and metabolism of [14C]piritrexim in dogs after intravenous and oral administration.Drug Metabolism and Disposition: the... 1991The disposition of [14C]piritrexim ([14C]PTX) in male dogs after iv and po doses of 1.8 mg/kg was examined. After either route of administration, greater than 90% of the...
The disposition of [14C]piritrexim ([14C]PTX) in male dogs after iv and po doses of 1.8 mg/kg was examined. After either route of administration, greater than 90% of the dose was recovered in the exreta within 72 hr; approximately 20% was recovered in urine and 70% in feces. [14C]PTX was extensively metabolized by dogs; unchanged drug accounted for less than 15% of the dose in the excreta. The O-demethylated metabolites, 2'- and 5'-demethyl PTX, the glucuronide conjugate of 2'-demethyl PTX, and the sulfate conjugate of 5'-demethyl PTX were the major metabolites. Unchanged drug accounted for a large proportion of the drug-related radiocarbon in plasma. The average plasma half-life of PTX after iv administration was 2.6 +/- 0.3 hr, and the average total body clearance was 0.33 +/- 0.13 liter/hr/kg. After po administration, peak plasma concentrations of 0.9 +/- 0.3 micrograms/ml occurred about 1.1 hr after the dose; the absolute oral bioavailability of PTX was 0.63 +/- 0.14. Because the O-demethyl metabolites were active dihydrofolate reductase inhibitors, 2'- and 5'-demethyl PTX were synthesized, and the pharmacokinetics and bioavailability of these compounds in dogs after iv and po administration (5 mg/kg) were examined. The plasma concentration-time data for both compounds after iv doses were described by a two-compartment model, with t1/2 beta = 1.3 and 0.8 hr for the 2'- and 5'- demethyl compounds, respectively. Neither compound showed significant advantages over PTX in terms of pharmacokinetics or bioavailability.
Topics: Administration, Oral; Animals; Antineoplastic Agents; Biological Availability; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dogs; Feces; Injections, Intravenous; Male; Pyridines; Pyrimidines; Tetrahydrofolate Dehydrogenase
PubMed: 1687022
DOI: No ID Found -
Protein Expression and Purification 1991Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity...
Pneumocystis carinii dihydrofolate reductase (DHFR) expressed in Escherichia coli was purified to homogeneity in a single step using methotrexate-Sepharose affinity chromatography. The purified enzyme migrated as a single 24-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the first 26 amino acids from the N-terminus of the purified enzyme was in accord with that predicted from the DNA sequence. The enzyme showed a broad pH optimum with maximum activity over the pH range 6 to 7. The enzyme was activated by salts, with maximal twofold activation at 50 to 150 mM KCl and 50 to 200 mM NaCl. Urea at 2.5 M also increased the enzyme activity twofold. Kinetic analysis of the purified enzyme revealed that the Km values for dihydrofolate and NADPH were 1.8 and 1.4 microM, respectively, and that the kcat was 70 s-1. Inhibition studies verified that trimethoprim and pyrimethamine were poor inhibitors of P. carinii DHFR and showed little selectivity over the human DHFR. Trimetrexate and piritrexim were much more potent inhibitors of the P. carinii enzyme, but these inhibitors are also potent inhibitors of human DHFR.
Topics: Cloning, Molecular; Escherichia coli; Gene Expression; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Pneumocystis; Recombinant Proteins; Tetrahydrofolate Dehydrogenase
PubMed: 1821803
DOI: 10.1016/1046-5928(91)90088-z -
Antimicrobial Agents and Chemotherapy Oct 1991Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA)....
Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.
Topics: 4-Aminobenzoic Acid; Anti-Infective Agents; Chromatography, High Pressure Liquid; Microbial Sensitivity Tests; Pneumocystis; Temperature
PubMed: 1759815
DOI: 10.1128/AAC.35.10.1965 -
Cancer Research Jun 1991Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which...
Trimetrexate, a lipid-soluble analogue of methotrexate, appears to enter mammalian cells by passive diffusion, thus circumventing the methotrexate transport system which is frequently a subject for alterations leading to methotrexate resistance. Using a single-step selection protocol with trimetrexate, we have isolated 45 clonal variants and found the majority of them to be selectively resistant to lipophilic antifolates while retaining their sensitivity to methotrexate and drugs involved in multidrug resistance. The majority of spontaneously induced trimetrexate-resistant clones showed a change in neither the mRNA levels of dihydrofolate reductase (24 of 30) and P-glycoprotein (26 of 30) nor their gene copy numbers, whereas a small fraction of clones (4 of 30) showed multidrug resistance gene amplification and P-glycoprotein mRNA overexpression. gamma-Irradiation prior to selection markedly enhanced the frequency of trimetrexate resistance (100-fold after 1000 rads). None of the gamma-ray-induced trimetrexate-resistant clones (0 of 15) had evidence of dihydrofolate reductase and multidrug resistance gene amplification and/or overexpression. Flow cytometry data on trimetrexate-resistant clones showed no defect in the transport of trimetrexate. Verapamil, a modulator of the multidrug resistance phenotype, had no cytotoxic effect on parental and trimetrexate-resistant clones. However, when present with trimetrexate, verapamil (0.3-0.6 microM) reversed the lipophilic antifolate-resistant phenotype in clones that had invariant levels of P-glycoprotein and dihydrofolate reductase. This selective resistance to lipid-soluble antifolates was initially unstable but became stable after continued drug-selective growth. Two-dimensional gel electrophoresis showed some differences in protein(s) that may potentially be associated with this phenotype of selective resistance to lipophilic antifolates. We conclude that a gamma-radiation-enhanceable, verapamil-reversible, stable phenotype of selective resistance to lipid-soluble antifolates frequently emerges which requires neither the amplification nor the overexpression of dihydrofolate reductase or multidrug resistance genes.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Binding, Competitive; Cell Line; Cricetinae; Drug Resistance; Folic Acid Antagonists; Gene Amplification; Membrane Glycoproteins; Methotrexate; Phenotype; Pyrimidines; Quinazolines; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Trimetrexate; Verapamil
PubMed: 1674447
DOI: No ID Found -
Cancer May 1991Thirty patients with recurrent and/or metastatic head and neck cancer were treated with sequentially administered methotrexate (MTX) and piritrexim (PTX). The treatment... (Clinical Trial)
Clinical Trial
Thirty patients with recurrent and/or metastatic head and neck cancer were treated with sequentially administered methotrexate (MTX) and piritrexim (PTX). The treatment schedule consisted of intravenous (IV) MTX (50 mg/m2) administered on day 1 and oral PTX (75 mg/m2) administered twice daily on days 8 to 12. Courses were repeated every 21 days with dose escalation in subsequent courses aimed at achieving Grade 1 toxicity. Two patients were not evaluable for response, 5 (17%; 95% confidence interval, 4% to 30%) had a partial response (PR), 10 had stable disease, and 13 had progressive disease. All five responses were seen in patients with regional lymph nodes as measurable disease. The median time to progression for all patients was 1.4 months, and the median survival was 6.7 months. Generally, this regimen was well tolerated with only mild toxicity seen during cycle 1 in the majority of patients. Dose escalation in subsequent cycles was possible in a high percentage of patients. Although the overall response rate and survival figures in this Phase II trial were disappointing, the doses and schedule used in this trial may have been suboptimal as reflected by the low incidence of moderate to severe toxicity. Additional evaluation of this combination of drugs in a more aggressive schedule may be warranted.
Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Blood Cell Count; Drug Administration Schedule; Drug Evaluation; Female; Head and Neck Neoplasms; Humans; Lymphatic Metastasis; Male; Methotrexate; Middle Aged; Neoplasm Recurrence, Local; Pyrimidines
PubMed: 2013031
DOI: 10.1002/1097-0142(19910501)67:9<2253::aid-cncr2820670907>3.0.co;2-y -
Biochemistry International May 1991Polyglutamated dihydrofolate, accumulated as a result of potent inhibition of dihydrofolate reductase (DHFR), has been postulated to directly inhibit the purine pathway...
Polyglutamated dihydrofolate, accumulated as a result of potent inhibition of dihydrofolate reductase (DHFR), has been postulated to directly inhibit the purine pathway at 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (reaction 9) in leukemia cells exposed to methotrexate (MTX). We have observed that 25 microM MTX or piritrexim, a "non-classical" antifolate, induce several-fold accumulations of AICAR and N-succino-AICAR to a combined cellular concentration of 89 microM in mouse L1210 leukemia cells after 2 h. By contrast, complete inhibition of reaction 4 by 25 microM azaserine results in accumulation of N-formyl-glycinamide ribotide (FGAR) polyphosphates to a combined cellular concentration of greater than 10 mM. MTX prevented azaserine-induced accumulation of FGAR polyphosphates. Hence, these antifolates induce primary inhibition of the de novo purine pathway at, or prior to, glycinamide ribotide transformylase (reaction 3).
Topics: Acyltransferases; Animals; Azaserine; Folic Acid Antagonists; Hydroxymethyl and Formyl Transferases; Leukemia L1210; Methotrexate; Phosphoribosylaminoimidazolecarboxamide Formyltransferase; Purine Nucleotides; Purines; Pyrimidines; Tumor Cells, Cultured
PubMed: 1768258
DOI: No ID Found -
The disposition and metabolism of [14C]piritrexim in rats after intravenous and oral administration.Drug Metabolism and Disposition: the... 1991The disposition of [14C]piritrexim in male rats after iv (5 and 10 mg/kg) and po (5, 10, and 20 mg/kg) doses was studied. After an iv dose of 10 mg/kg, rats excreted an...
The disposition of [14C]piritrexim in male rats after iv (5 and 10 mg/kg) and po (5, 10, and 20 mg/kg) doses was studied. After an iv dose of 10 mg/kg, rats excreted an average of 57% of the dose in feces and 32% in urine; after a po dose of 10 mg/kg, 84% of the dose was excreted in feces and 9% in urine. After iv doses, the elimination of unchanged drug from plasma was first order, with a t1/2 of 0.6 hr; at any time point, unchanged drug accounted for less than 50% of the total radiocarbon in the plasma. Oral bioavailability of unchanged drug was less than 5%. O-Demethylation and subsequent conjugation were the main pathways of metabolism; the demethyl metabolites of piritrexim were potent inhibitors of dihydrofolate reductase and were cytotoxic to cells in culture. Concentrations of radiocarbon were highest in liver 24 hr after an iv dose, but less than 1% of the radiocarbon was unchanged drug. Concentrations of radiocarbon in liver after po doses were approximately 40% of those attained after equivalent iv doses.
Topics: Administration, Oral; Animals; Biological Availability; Biotransformation; Cell Survival; Folic Acid Antagonists; Injections, Intravenous; Iodine Radioisotopes; Male; Pyrimidines; Rats; Tetrahydrofolate Dehydrogenase; Tissue Distribution
PubMed: 1680625
DOI: No ID Found -
Archives of Dermatology Apr 1991Methotrexate is an effective and convenient treatment for severe psoriasis whose use is limited by the development of hepatic fibrosis and cirrhosis in a small number of... (Clinical Trial)
Clinical Trial
Methotrexate is an effective and convenient treatment for severe psoriasis whose use is limited by the development of hepatic fibrosis and cirrhosis in a small number of patients. The mechanism of hepatotoxicity is unknown, but it is believed to be the result of intracellular polyglutamation and prolonged retention of methotrexate within the cell. Piritrexim isethionate is a lipid-soluble antifolate that has a mechanism of action similar to that of methotrexate. Since it is not polyglutamated, piritrexim could be effective in the treatment of psoriasis without the associated long-term hepatotoxicity. A 12-week phase I/II clinical trial of severe chronic plaque psoriasis assessed the safety and efficacy of oral piritrexim therapy. Based on experience gained from oncologic trials, each patient received a twice-daily dosage for 5 consecutive days every 2 weeks. Dosages ranged from 25 to 100 mg twice a day. Improvement in both lesion scores and percentage of body involvement was significant at a dose of 50 mg or more twice daily. Fifteen of 19 patients who completed 12 weeks of therapy demonstrated greater than 50% improvement in lesion scores. Improvement was limited by recrudescence of lesions over the 9-day rest period. Adverse experiences were minimal and dose related. Piritrexim is efficacious in the treatment of psoriasis.
Topics: Adult; Aged; Drug Administration Schedule; Female; Folic Acid Antagonists; Humans; Male; Middle Aged; Psoriasis; Pyrimidines
PubMed: 2006875
DOI: No ID Found