Lack of a pharmacologic interaction between rifabutin and methadone in HIV-infected former injecting drug users.

Rifampin, an agent known to decrease the half-life of methadone, and rifabutin are two rifamycins that are structurally similar and share mechanisms of action. Hence the possibility of a drug-drug interaction between rifabutin and methadone was evaluated in 24 methadone-maintained, former injecting drug users infected with the human immunodeficiency virus. The study was an open-label, drug-drug interaction and safety trial in which patients were followed for 15 days. Each patient received rifabutin 300 mg as a single dose concomitantly with their individualized methadone dosage. No significant differences in methadone peak plasma concentration, time to peak plasma concentration, area under the plasma concentration-time curve, systemic clearance or renal clearance was observed in the presence of rifabutin. Seventy-five percent of the patients reported at least one symptom of narcotic withdrawal during the study, however, these symptoms were mild. A relationship between the development of narcotic withdrawal and methadone systemic exposure could not be established. Concurrent administration of rifabutin and methadone appeared to be safe in human immunodeficiency virus-infected injecting drug users maintained on stable doses of methadone and is not expected to produce any significant changes in the pharmacokinetics of methadone in these patients.

Drug abuse treatment programs as centers for HIV-related research and treatment.

The Observational Data Base (ODB) is a multicenter, longitudinal effort of the Community Programs for Clinical Research in AIDS (CPCRA) designed to collect HIV-related data on a large number of HIV-infected patients receiving primary care from community based physicians and health care groups. Over 400 patients from the Addiction Research and Treatment Corporation have enrolled in the ODB. Compared to the remainder of the CPCRA, ARTC ODB enrollees are more likely to be female, African American or Hispanic and to have injectable drug use and heterosexual contact with an injectable drug user as risk factors. The ARTC ODB patient profile closely resembles the fastest growing segments of the AIDS epidemic.

In vivo monitoring of changes in 5-fluorouracil metabolism induced by methotrexate measured by 19F NMR spectroscopy.

We have used in vivo 19F NMR spectroscopy to study the metabolism of 5-fluorouracil (FUra) in tumors with and without pretreatment with methotrexate (MTX). Using the CD8F1 murine mammary tumor as an in vivo model, we observed signals from FUra, alpha-fluoro-beta-alanine (F beta ALA), alpha-fluoro-beta-ureidopropionic acid (FUPA), and 5-fluorouracil-nucleotides (FUN) after intravenous or intraperitoneal injection of 150 mg/kg FUra. Formation of FUN was increased about 1.7-fold in CD8F1 tumor with methotrexate pretreatment as determined by acid extraction and HPLC analysis. A comparison of in vivo NMR spectra from FUra and sequential MTX + FUra-treated tumors showed a significantly higher ratio of the FUN signal to the initial total 19F signal in the MTX + FUra-treated tumors (p less than 0.001) for animals receiving FUra either intravenously or intraperitoneally. In addition, tumors treated with MTX + FUra had significantly longer time durations during which FUN was detected, independent of the mode of administration. These experiments indicate that in vivo 19F NMR spectroscopy can be used to noninvasively monitor alterations of 5-fluorouracil metabolism that occur with administration of modulating agents such as methotrexate. Further studies, in both murine tumor models and patients, are indicated to determine if these results can be correlated with tumor response.

Effect of uridine diphosphoglucose on levels of 5-phosphoribosyl pyrophosphate and uridine triphosphate in murine tissues.

The purpose of the present investigation was to determine whether a single bolus intravenous injection (2000 mg/kg) of uridine diphosphoglucose (UDPG) could affect levels of PRPP in a transplanted mammary adenocarcinoma and in liver of CD8FI mice. Six hours following a single intravenous injection of UDPG, 2000 mg/kg, tumor PRPP was lowered to 80 pmol/mg protein, a 53% decrease compared to saline control tumors. Liver was more sensitive than tumor to the 5-phosphoribosyl pyrophosphate (PRPP)-depleting effects of a single bolus intravenous injection of UDPG, since significantly lower levels of PRPP were found in liver, but not in tumor, at doses of 500-1000 mg/kg of UDPG. Maximal depression (30% of saline control) or PRPP occurred in liver 6 hr after intravenous UDPG at 1000-2000 mg/kg. Enhanced levels of UDPG in plasma (half-life less than 10 min) and tumor was detected at 30 min after intravenous UDPG at 2000 mg/kg. There was no detectable increase in endogenous levels of UDPG in liver at this time, probably as a result of rapid metabolism of UDPG by liver. At this same time, a twofold increase in uridine triphosphate (UTP) was measured in liver after intravenously administered UDPG. In contrast, the level of UTP was not increased significantly above control values in tumor. These data suggest the potential use of UDPG to elevate UTP pools in normal tissues in the delayed rescue of cancer chemotherapeutic drugs such as 5-fluorouracil which function as a uridine analogue in these tissues.

Phase I trial of N-(phosphonacetyl)-L-aspartate, methotrexate, and 5-fluorouracil with leucovorin rescue in patients with advanced cancer.

Based on an animal model to improve the antitumor activity of 5-fluorouracil (FUra), a Phase I study of N-(phosphonacetyl)-L-aspartate, methotrexate, FUra, and leucovorin was conducted on 44 patients. Methotrexate was given in an intermediate dose (250 mg/m2) to overcome potential drug resistance, and N-(phosphonacetyl)-L-aspartate was given at a low dose (250 mg/m2) in order to allow escalation of FUra to toxicity. These two drugs were given 24 h before FUra to enhance maximal incorporation of FUra into RNA. Two schedules of administration were used; one every other week and one weekly for 2 weeks. The every other week schedule was well tolerated, with minimal gastrointestinal and hematological toxicity. However, the weekly for 2 weeks schedule was more toxic with increased mucositis, diarrhea requiring therapy, and decreased performance status of 20% in 4 of 6 patients. There were no responders in the every other week schedule. There was one partial response and three patients with stable disease in four evaluable patients on the weekly for 2 weeks schedule. At 24 h post-N-(phosphonacetyl)-L-aspartate-methotrexate treatment, PRPP levels were doubled in bone marrow biopsies, and increased 2.5- to 25-fold in tumor biopsies. We have currently added uridine rescue to this combination with the hope of further escalating the dose of FUra.

Inhibition by methotrexate of the stable incorporation of 5-fluorouracil into murine bone marrow DNA.

As a consequence of the inhibition of de novo purine synthesis by methotrexate (MTX) there is an increase in 5-phosphoribosyl-1-pyrophosphate (PRPP) concentration. In cells where 5-fluorouracil (FUra) is activated via orotate phosphoribosyltransferase (OPRtase), increased PRPP results in greater conversion of FUra to nucleotides. In the murine CD8F1 breast tumor system, MTX markedly enhances the antitumor activity of FUra, increasing both the activation of FUra to FUMP and the incorporation of FUTP into RNA. However, in contrast to reported tumor tissue culture studies, MTX pretreatment in vivo prevents the stable incorporation of FUra into CD8F1 bone marrow DNA. Pretreatment with MTX (300 mg/kg) 2.5 hr prior to [3H]FUra (100 mg/kg), with a 2-hr labeling, reduced the level of FUra in DNA from 921 pmol to 66 pmol/mg of DNA. Without MTX pretreatment, 59% of the total incorporation of FUra into nucleic acids was into DNA when FUra was administered. After MTX the percentage of incorporation into DNA was reduced to 9%. Additionally, the ratio of [3H]FUra to 32P in DNA when both were given simultaneously was reduced by greater than 90%, suggesting that MTX must be specifically blocking the incorporation of FUra rather than nonspecifically preventing its incorporation by inhibiting DNA synthesis. In contrast, MTX failed to reduce the formation of DNA containing fluorouracil residues from FdUrd. To test whether MTX prevents the initial incorporation of FUra into DNA, or acts to enhance removal by the DNA glycosylase repair system, DNA was prelabeled in vivo with [3H]FUra, and MTX or MTX plus dThd was then administered. The level of FUra in bone marrow DNA was not reduced by subsequent treatment with MTX, or MTX plus dThd, indicating that MTX does not enhance the removal of FUra from DNA. The level of total free fluorodeoxynucleotides formed from FUra was reduced by two-thirds following MTX pretreatment, suggesting that the action of MTX in preventing the stable incorporation of FUra into DNA was to reduce the availability of FdUTP.

Use of oral uridine as a substitute for parenteral uridine rescue of 5-fluorouracil therapy, with and without the uridine phosphorylase inhibitor 5-benzylacyclouridine.

Initial clinical trials have demonstrated that uridine (Urd) rescue given i.v. over at least 3 days can ameliorate 5-fluorouracil (FUra) toxicity; to avoid Urd-induced phlebitis in the peripheral veins of patients, a central vein is used. The latter necessity, along with the need for 3 days of i.v. administration, makes Urd rescue by parenteral means a cumbersome and complicated clinical procedure. It would appear preferable to use oral Urd; however, the oral Urd dose in the clinic is limited, as high doses cause diarrhea. Therefore, using a tumor-bearing murine model we investigated as to whether low doses of oral Urd coupled with a Urd phosphorylase inhibitor benzylacyclouridine (BAU), would effect safe rescue of FUra toxicity with preservation of antitumor activity. A high-dose FUra-containing drug combination that included parenteral Urd rescue was used as a control; other groups of tumor-bearing mice received the same drug combination, except that p.o. Urd was substituted for i.p. Urd. In the absence of BAU, p.o. Urd could effect rescue while maintaining an antitumor effect comparable to that obtained with i.p. Urd. When given concomitantly with BAU, a 50% reduction in the oral Urd dose (i.e., from 4,000 to 2,000 mg/kg) enabled the achievement of a comparable therapeutic index. Intraperitoneal Urd produces very high (6-8 mM) plasma and tissue Urd levels, which remain above 100 microM for at least 6 h. In contrast, neither oral Urd nor oral BAU alone raised plasma Urd concentrations above about 50 microM. However, the combination of oral Urd plus oral BAU gave a peak plasma Urd level of about 300 microM, and the level was maintained above 100 microM for 6 h. Following oral Urd administration, gut tissue levels of Urd were in the mM range and those of BAU were in the range of 10-20 micrograms/g tissue, a level sufficient to result in substantial inhibition of Urd phosphorylase. Oral Urd plus oral BAU appears to be a promising clinical alternative to parenteral administration of Urd for selective rescue of FUra toxicity.

Effect of L-histidinol on the metabolism of 5-fluorouracil in the BALB/c x DBA/8 F1 murine tumor system.

L-Histidinol, a structural analogue of histidine, which transiently inhibits proliferation, can protect cells from the toxic effects of proliferation-dependent chemotherapeutic agents such as 5-fluorouracil (FUra). In the BALB/c x DBA/8 F1 (hereafter called CD8F1) murine tumor system, L-histidinol protected mice from FUra-induced leukopenia, weight loss, and mortality; however, the therapeutic index was not improved since L-histidinol also protected the tumor against the toxic effects of FUra. In order to understand the mechanism of this protection, we examined the effects of L-histidinol on the metabolism of FUra. Results indicate that L-histidinol had no effect on the phosphoribosyl pyrophosphate levels in tumor, the activation of FUra to nucleotides or levels of free 5-fluorodeoxyuridine monophosphate in either tumor or bone marrow. L-Histidinol (7 mg/mouse, every 2 h for 5 doses) reduced RNA and DNA synthesis, as measured by 32P incorporation in vivo, by approximately one-half in tumor, and by 70% in bone marrow. This in turn resulted in reduced incorporation of FUra into RNA in both tumor and bone marrow. At 2 h, 4 h, and 24 h after FUra administration the level of FUra in RNA was 24-37% less in both tumor and bone marrow of mice that received L-histidinol with FUra. Using 32P as a monitor of overall RNA synthesis, the [3H]FUra/32P ratio remained unchanged, suggesting that the reduction of FUra incorporation into RNA was due to decreased RNA synthesis rather than a decrease in the number of FUra molecules per RNA chain. In contrast, L-histidinol had no effect on the in vivo inhibition of thymidylate synthetase by 5-fluorodeoxyuridine monophosphate as measured by the incorporation of [3H]-2'-deoxyuridine into DNA or on the percentages of thymidylate synthetase in the free versus 5-fluorodeoxyuridine monophosphate-bound state. We conclude that L-histidinol reduces FUra toxicity by reducing FUra incorporation into RNA. Since the major mechanism of action in the CD8F1 breast tumor system is the incorporation of FUra into RNA, the reduction in toxicity and antitumor activity observed when L-histidinol is combined with FUra is consistent with the observed reduction in tumor and bone marrow RNA containing incorporated FUra residues.

Chemotherapeutic evaluation using clinical criteria in spontaneous, autochthonous murine breast tumors.

Six drugs, three of which are considered to be active against human breast cancer [melphalan (PAM), cyclophosphamide (CTX), and 5-fluorouracil (FUra)] and three of which have failed to demonstrate activity against human breast cancer [N-phosphonacetyl-L-aspartate (PALA), cytarabine (ara-C), and 6-thioguanine (TG)], were tested at optimal weekly doses in (BALB/-cfC3H X DBA/8)F1 (CD8F1) mice bearing spontaneous, autochthonous breast tumors averaging 300 mg. When treatment was evaluated by laboratory criteria (i.e., tumor growth inhibition in comparison to vehicle-treated, size-matched controls), all six of the drugs tested were judged to be active. However, when the criteria for positive drug activity consisted of the attainment of tumor regressions of greater than or equal to 50% in greater than or equal to 20% of the treated individuals (i.e., analogous to clinical criteria), only the three drugs that are known to be active against human breast cancer (PAM, CTX, and FUra) were judged active against the spontaneous murine breast tumors. PALA, ara-C, and TG failed to demonstrate regressing activity against the spontaneous murine breast tumors. With a caveat concerning the limited spectrum of drugs evaluated in this study, it can be concluded that the CD8F1 breast tumor model demonstrated 100% correlation with human breast cancer in terms of both positive and negative drug sensitivity when the criteria for evaluation were parallel.

Failure of L-histidinol to improve the therapeutic efficiency of 5-fluorouracil against murine breast tumors.

It has been reported that L-histidinol, a structural analogue of the essential amino acid L-histidine, can transiently inhibit proliferative cycling in cells with normal phenotype while allowing continued cell cycle transit in tumor cells. Thus, in the presence of L-histidinol, the toxicity of a proliferation-dependent drug such as 5-fluorouracil (FUra) was found to be reduced in normal tissue cells of the DBA/2J mouse, but not in L1210 leukemia cells in the same mouse. Because of the potential clinical significance of this approach to reduce chemotherapy-associated host toxicity, we evaluated the L-histidinol-FUra combination in a nonleukemic, solid murine tumor model, the BALB/c X DBA/8 F1 (hereafter called CD8F1) breast tumor. The results of these studies indicate that the administration of L-histidinol can protect the CD8F1 mouse from FUra-associated leukopenia, body weight loss, and ultimately, from mortality. However, in contrast to results reported in the L1210 leukemic system, L-histidinol also reduced the cytotoxic activity of FUra against CD8F1 breast tumors. Therefore, although the dose of FUra that could be administered with safety was higher in mice receiving L-histidinol, the therapeutic results of the combination of FUra and L-histidinol were not superior to those obtained with FUra alone at a lower dose.

Decreased host toxicity in vivo during chronic treatment with 5-flourouracil.

Chronic weekly administration of FUra to CD8F1 female mice bearing spontaneous mammary tumors produced body weight loss during the first 2 weeks of treatment, which became less severe during subsequent weeks of therapy. To our knowledge, the development of such a decrease in FUra toxicity in vivo during chronic treatment with the drug has not been described previously, and a study of this phenomenon was therefore undertaken in tumor-free CD8F1 female mice. Weekly administration of FUra at 85 mg/kg resulted in toxicity expressed in body weight loss and in depressed peripheral WBC levels; however, the magnitude of these toxic effects decreased significantly by the 5th week of treatment. Pretreatment of normal mice with FUra for 7 weeks resulted in a dose-related shift in the LD50 of FUra administered as a subsequent challenge. Compared with an LD50 of 240 mg/kg for FUra in normal mice, the LD50 in mice pretreated with FUra at 50 or 85 mg/kg per week was found to be significantly elevated to 370 and 460 mg/kg, respectively. Pretreatment with FUra at 85 mg/kg for 7 weeks did not alter the activity of the enzymes responsible for the activation of FUra, namely uridine kinase or orotate phosphoribosyltransferase, in the intestinal epithelium or bone marrow, but it did decrease the 24-h urinary excretion of intact [3H]FUra by almost 40% (P less than 0.01). In addition, the FUra pretreatment schedule resulted in a 31% (P = 0.14) increase in the activity of dihydrouracil dehydrogenase in the liver. These results suggest that increased degradation of FUra can be induced by chronic treatment with the drug. Finally, knowledge of the development of increased drug catabolism was used to increase the therapeutic effectiveness of FUra by its incorporation into an increasing-dose regimen. Mice bearing 24-h transplants of the murine breast tumor were treated with a constant dose of FUra for 12 weeks or with a dose that was increased, after 7 weeks, to a dose normally causing a high degree of drug-related mortality. The group receiving the incremented FUra dose had a significantly slower tumor growth rate without an increase in drug-related toxicity. These results are discussed in light of their obvious clinical implications.

Incorporation of 5-fluorouracil into murine bone marrow DNA in vivo.

Although 5-fluorouracil (FUra) is readily incorporated into RNA, the possibility of its being incorporated into DNA in substantial amounts has only recently been recognized. Examination of nucleic acids prepared from tumor-bearing BALB/c X DBA/8 F1 mice labeled with [3H]FUra in vivo revealed very little alkali-stable, acid-precipitable radioactivity in tumor and only small amounts in intestine. However, substantial amounts were detected in bone marrow. Pretreatment of mice with low-dose thymidine (500 mg/kg) increased the incorporation of FUra into RNA but did not change the amount incorporated into alkali-stable material. The net result was a reduction in the fraction of the total in an alkali-stable form. Formation of DNA containing FUra residues is substantially reduced if the mice receive very high doses of thymidine along with the labeled FUra, presumably through competition from an expanded deoxythymidine triphosphate pool. Bone marrow nucleic acids labeled with 32P and [3H]FUra were analyzed by cesium sulfate gradients. Two distinct peaks of tritium radioactivity were observed that band with 32P radioactivity at the densities of RNA and DNA. Pretreatment with alkali destroyed the (32P/3H)RNA peak, but not the DNA peak. Cesium sulfate-purified DNA containing FUra residues was digested with pancreatic DNase and venom phosphodiesterase. The resulting nucleotides were analyzed by high-pressure liquid chromatography. The majority of the radioactivity cochromatographed with 5-fluorodeoxyuridine monophosphate marker. No radioactivity was detected in the regions corresponding to fluorouridine monophosphate or deoxyuridine monophosphate, although radioactivity was detected cochromatographing with deoxythymidine monophosphate. After digestion with alkaline phosphatase, the majority of the radioactivity cochromatographed with fluorodeoxyuridine (and some thymidine). These results confirm previous observations of FUra incorporation into DNA of tissue culture cells.

Effect of Uridine on the Metabolism of 5-Fluorouracil in the CD8F 1 Murine Mammary Carcinoma System.

The effect of uridine on the incorporation of 5-fluorouracil into RNA and the inhibition of DNA synthesis by the FdUMP block of thymidylate synthetase was studied in the CD8F1 murine mammary carcinoma system. The administration of exogenous uridine resulted in about a one third reduction of 5-fluorouracil in RNA of tumor and normal tissues. However, unlike thymidine, uridine was unable to reverse the early, partial inhibition of DNA synthesis. The amount of fluorouridine nucleotides and (5-fluorouracil)RNA formed in various tissues correlates with the level of orotate phosphoribosyl transferase activity suggesting that the major pathway for activation of 5-fluorouracil to nucleotide form in these tissues is via phosphoribosyl transferase. Enzyme preparations from three different murine tumors convert about 15 times as much 5-fluorouracil to FUMP as they do uracil to UMP. In contrast, the ratio of FUMP to UMP formed in enzyme preparations from gut and bone marrow is lower, 2-6 fold. However, in none of these tissues was the in vitro conversion of 5-fluorouracil to FUMP or incorporation into RNA substantially inhibited by uracil. Examination of tumor, gut and bone marrow uridine nucleotide pools showed that the thymidine-uridine-5-fluorouracil schedule does increase uridine nucleotide pools. Thus, the reduction in 5-fluorouracil in RNA is probably not due to inhibition of the conversion of 5-fluorouracil to FUMP by uracil (derived from phosphorylase cleavage of uridine) but, rather, is probably due to the elevated levels of UTP. We conclude that the protection from 5-fluorouracil toxicity afforded by the addition of uridine is due to the reduction in 5-fluorouracil in RNA rather than by reversal of the FdUMP block on thymidylate synthetase.

Improved therapeutic index with sequential N-phosphonacetyl-L-aspartate plus high-dose methotrexate plus high-dose 5-fluorouracil and appropriate rescue.

Although clinical trials of high-dose methotrexate (MTX) sequenced before 5-fluorouracil (FUra) with leucovorin (LV) rescue apparently have resulted in increased numbers of tumor responses, this increased antitumor activity often has been accompanied with toxicity. The present report describes an attempt to improve therapeutic results with this drug combination by appropriate metabolic modulation in the preclinical BALB/c X DBA/8 F1 murine breast tumor model. A LV rescue schedule consisting of 300 mg/kg administered at 4.5 and 19.5 hr after high-dose MTX (300 mg/kg/week for 3 weeks) prevented MTX toxicity. When FUra was administered 2.5 hr after MTX (with LV rescue), the dose of FUra had to be decreased, and we could not obtain convincing evidence for a differential cytotoxic effect on tumor versus normal host tissue. However, when a delayed uridine rescue schedule was added to protect the host from the toxic activity of FUra, the FUra dose could be increased even in the presence of high-dose MTX, and the therapeutic result was enhanced significantly without an increase in host toxicity. Finally, it was possible to add N-phosphonacetyl-L-aspartate to this drug combination (in the appropriate sequence: N-phosphonacetyl-L-aspartate before high-dose MTX-before high-dose FUra, followed by double rescue with LV and uridine) without producing increased toxicity to yield a significant increase in partial tumor regression rate. The biochemical rationale for the selection and sequence of administration of these agents is discussed.

Therapeutic utility of utilizing low doses of N-(phosphonacetyl)-L-aspartic acid in combination with 5-fluorouracil: a murine study with clinical relevance.

Doses of N-(phosphonacetyl)-L-aspartic acid (PALA) lower than those required for therapeutic activity against the spontaneous murine breast tumor (BALB/c X DBA/8 F1) were found to produce significant depression in uridine triphosphate pools in the tumor. Using such low, nontherapeutic, but biochemically active doses of PALA in combination with 5-fluorouracil (FUra(, it was possible to maintain the dose of FUra at its full maximum tolerated single agent dose. In comparison with the maximum tolerated dose of FUra alone, the combination of FUra plus low-dose PALA produced a significant increase in the level of tumor FUra-containing RNA, only a slight increase in intestinal FUra-containing RNA, and no increase in bone marrow FUra-containing RNA. These biochemical results correlated with the therapeutic findings of significantly increased antitumor activity without an increase in host toxicity. A review of currently reported PALA plus FUra clinical protocols reveals that the experimental parameters which have produced successful therapeutic results in the laboratory (i.e., a low-PALA:high-FUra dosage ratio) have not yet been translated into clinical trial. Final judgment on the clinical efficacy of PALA:FUra combinations must await the results of proposed low-PALA:high-FUra clinical trials.

Modulation of 5-fluorouracil-induced toxicity in mice with interferon or with the interferon inducer, polyinosinic-polycytidylic acid.

Partially purified preparations of mouse interferon, administered during the 2-day period following the administration of a toxic dose of 5-fluorouracil (FUra), yielded significant protection from mortality in BALB/c X DBA/2 F1 mice. Protection against FUra-induced toxicity was also observed when the interferon inducer polyinosinic-polycytidylic acid (poly I X poly C) was administered with FUra. The temporal relationship between the administration of poly I X poly C and FUra was found to be a critical determinant of the intensity of toxic manifestations. In relation to FUra alone, poly I X poly C could enhance (when administered 48 hr before FUra), diminish (when administered together with FUra), or not affect (when administered 48 hr after FUra) the degree of resultant toxicity. Cytofluorometric analysis of the DNA content of bone marrow cells indicated a transient period (about 42 hr) of inhibition of cell cycling following the administration of poly I X poly C, followed by reentry into cycle (between 42 and 66 hr) and a return to normal cycle phase distribution by 90 hr. This disturbance of the kinetic pattern of cell cycling in bone marrow would explain the administration time-dependent variability of the effect of poly I X poly C on FUra toxicity, since FUra is known to be a cell cycle-specific cytotoxic drug. Potential practical application of this observation to the clinical use of FUra in cancer therapy is discussed.