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The effect of 3-deazauridine (DAUR) on the intracellular purine and pyrimidine nucleotide pools and on the metabolism of azacitidine (aza-CR) in L1210 cells, sensitive (L1210/0) and resistant (L1210/ara-C) to cytarabine (ara-C), was examined. The consequences of such a modulation were correlated with the therapeutic efficacy of this combination in mice bearing L1210 leukemia. In vitro and in vivo treatment of both L1210 sublines with DAUR produced a dose- and time-dependent reduction in the CTP and dCTP pools and an increase in the UTP pool. In addition to these changes in the pyrimidine nucleotide pools, DAUR produced a modest increase in the GTP pool and a marked expansion of the ATP pool in L1210/ara-C 12 hrs following in vivo drug treatment. These perturbations in nucleoside triphosphate pools were more pronounced in L1210/ara-C cells. Treatment of mice bearing L1210/ara-C with 100 mg/kg of DAUR reduced the CTP and dCTP pools in the leukemic cells by greater than 90% within 1-3 hrs after administration of the drug, with complete recovery of these pools occurring within 12 hrs. Fluctuation of the pyrimidine nucleoside pools after DAUR treatment was correlated with the subsequent increase in aza-CR metabolism and its incorporation into RNA and with the potentiation of the in vivo toxicity of aza-CR. In mice bearing L1210/0 or L1210/ara-C tumors, DAUR or aza-CR produced a less than or equal to 23% increase in life-span (ILS). Administration of aza-CR 3 hrs after DAUR, however, produced about an 80% ILS among mice bearing L1210/ara-C tumors, but no more than an approximately 20% ILS among mice bearing L1210/0 tumors. These data suggest that the therapeutic activity of the sequential combination of DAUR and aza-CR against mice bearing L1210/ara-C cannot be explained, per se, on the basis of the initial intracellular modulation of nucleotide pools, since DAUR affected these pools of the two tumors to approximately the same degree. What appears to be important, however, is that such a modulation by DAUR preferentially affected the metabolism of aza-CR in leukemic cells resistant to ara-C which are devoid of deoxycytidine kinase activity.

Thirteen experimental mouse neoplasms were tested by cytidine (CR)-deaminase and deoxycytidine (dCR)-kinase levels. Four neoplasms, Sarcoma T241, Adenocarcinoma E0771, Lewis lung carcinoma (LL), and Sarcoma 180 Japan (S180J), considered to have high deaminase and sufficient dCR-kinase activities, were tested in vivo for combination chemotherapy with cytosine arabinoside (ara-C) and the CR-deaminase inhibitor, tetrahydrouridine (THU). THU did not significantly improve the growth inhibition of ara-C in a wide range of combinations in T241, E0771, LL, and the solid form of S180J, but more than doubled the survival time of the S180J ascites-bearing animals. Toxicity in the form of weight loss and toxic deaths was observed in some but not all groups, especially at high dosages of ara-C and THU. Tissue distribution of [3H]-ara-C and [14C]-THU in T241-bearing mice revealed an accelerated clearance of ara-C-derived radioactivity under the influence of THU in the tumor and five host tissues, but not in the small intestines. With the exception of the small intestines, clearance of THU-derived radioactivity was faster in all tissues studied compared to the clearance of [3H]-ara-C-derived radioactivity. Intracellular CR-deaminase levels were inhibited significantly, ie, dose dependent, in tumor and host kidney after a single ip injection of THU to E0771--bearing mice. In the solid S180J, with or without simultaneous ip administration of THU, [3H]-ara-C was not converted to 5'-di- and tri-phosphates at all. In mice bearing the ascites form of S180J, [3H]-ara-C was extensively converted to ara-C 5'-di- and tri-phosphates. THU increased both overall ara-C-derived radioactivity and the relative amounts of ara-C 5'-di- and tri-phosphates.

Inhibition of the deamination of 14C-cytosine arabinoside by two lots of tetrahydrouridine was studied in monkey serum. The average inhibition of deaminase activity was 78% for tetrahydrouridine lot AJ39 (1.0 muM) when the concentration of cytosine arabinoside ranged from 44.2 to 170.7 muM; under the same conditions tetrahydrouridine lot AJ22 inhibited deamination by an average of 68%. Apparent Ki values were 0.26 muM for AJ39 and 0.43 muM for AJ22. The assay may be used to check the relative biologic activity of various lots of tetrahydrouridine.

A new technique using immunofluorescence and autoradiography is described, in which the DNA of cells in S phase are labeled with two different probes. This method makes it possible to study the relationship between DNA synthesis and the uptake and/or incorporation of chemotherapeutic agents into normal or neoplastic cells. An example is provided in which the incorporation of 3H-cytarabine into DNA is demonstrated to occur only in cells which were synthesizing DNA during exposure to 3H-cytarabine. Other radioactively labeled probes can be used as well.

Two potent new ribonucleotide reductase inhibitors, 3,4,5-trihydroxybenzohydroxamic acid (VF 122) and 3,4-dihydroxybenzohydroxamic acid (VF 147), were investigated for their ability to modulate the cellular pharmacology of cytarabine (ara-C) in HL-60 cells. VF 122 and VF 147 increased the total cellular uptake of ara-C by a mean (+/- SE) of 8% +/- 3% and 29% +/- 3%, respectively, when measured 2 hours after the start of exposure to 0.1 microM ara-C. This effect was evident after only 10 minutes of exposure to the riboNucleotide reductase inhibitor and did not vary significantly over the concentration range of 10-100 microM for either agent. VF 122 enhanced the incorporation of ara-CTP into DNA by 3.6-fold; VF 147 produced a 5.6-fold increase. In comparison, the maximum enhancement achievable with hydroxyurea was 2.1-fold, and with thymidine was 1.8-fold. These results provide a biochemical rationale for further investigation of these agents in combination with ara-C.

We studied the effect of deoxyguanosine (dGuo) on cellular cytarabine (ara-C) nucleotide accumulation of the human leukemia cell line K562 and of bone marrow blast cells derived from patients with acute nonlymphocytic leukemia. Exposure of cells in culture to dGuo increased ara-C nucleotide accumulation measured in cell lysate, with an average increase of 386% (range, 242%-537%) of control in the presence of 500 microM dGuo. Maximal elevation occurred after 8 hours of exposure and remained constant through 48 hours. dGuo also enhanced deoxycytidine nucleotide accumulation, but dGuo enhancement favored accumulation of ara-C nucleotides over dCyd nucleotides. In cell cycle kinetic studies using flow cytometry, dGuo slowed accumulation of cells with apparent S-phase DNA content in a concentration-dependent fashion. However, neither the rate nor the magnitude of this effect correlated with the increase in ara-C nucleotide accumulation. Since the increase in ara-C nucleotide accumulation caused by dGuo could be prevented by 5 micrograms/ml of cycloheximide, this process appears to require new protein synthesis. Although these data suggest that the elevation of ara-C nucleotide accumulation caused by dGuo may represent induction of enzyme synthesis, other possibilities are discussed. Exposure of bone marrow blast cells obtained from patients with acute nonlymphocytic leukemia to dGuo for 16 hours in liquid culture also increased ara-C nucleotide accumulation. In six of seven studies, exposure to dGuo in concentrations from 50 to 500 microM increased ara-C nucleotide accumulation from 160% to 3400%. These data suggest that dGuo may alter ara-C metabolism in a clinically useful fashion.

[14C]-tetrahydrouridine (THU), a strong inhibitor of cytidine (CR) deaminase, was, after iv administration, rapidly and quantitatively cleared from the blood with a plasma half-life of about 1 hour. The main pathway of excretion was through the kidneys: most of a dose of 50 mg/kg was excreted within 12 hours and excretion was essentially complete within 48 hours. Oral administration of the same dose revealed absorption of about 10% from the gastrointestinal tract. THU at 10, 25, and 50 mg/kg given 15 minutes before [3H]-cytosine arabinoside (ara-C) at a dose of 0.003 mg/kg produced about a two fold increase in ara-C blood levels at all times measured from 5 minutes to 4 hours, with only slight increases in the half-life of ara-C. A dose-related effect of THU upon the deamination of ara-C was obvious only during the time from 15 minutes to 1 hour after the injection of 3H-ara-C. The inhibitory effect of THU upon CR deaminase was also reflected in a considerably increased ratio of ara-C/uracil arabinoside in the urine.

Sc administration of cytarabine entrapped in multivesicular liposomes to BDF1 mice resulted in slow first-order release from skin, with a half-life of 4 days. The half-life was 10 minutes for unencapsulated free drug. A single-dose sc treatment of BDF1 mice, inoculated iv with 10(5) L1210 leukemia cells 24 hours previously, resulted in long-term survivors over a wide dose range. This study demonstrates that encapsulation of cytarabine in multivesicular liposomes results in slow release from skin and that a single sc dose is often curative for mice with systemic leukemia.

The effect of dipyridamole, an inhibitor of membrane nucleoside transport, on the uptake and toxicity of cytarabine was examined in normal and malignant tissues. Preliminary pharmacokinetic data were obtained in mice and humans to determine appropriate dipyridamole dosage ranges for in vitro testing. At concentrations achievable in man, dipyridamole produced 75% and 94% reductions in cytarabine uptake in freshly harvested normal mouse and human bone marrow cells, respectively. Under the same conditions, greater than 90% reductions in cytarabine uptake were also seen in both L1210 murine leukemia and HL-60 human leukemia cells. In addition, treatment with dipyridamole also reduced the growth inhibitory effects of cytarabine on HL-60 cells in culture and protected mice from toxic doses of this antimetabolite. These results demonstrate the ability of dipyridamole to modulate the activity of cytarabine in both murine and human cells.

The nobel metal "cage" complex, rhodium(II) acetate, was found to inhibit the deamination of the fraudulent nucleoside, arabinosylcytosine. Potent inhibition of a purified cytidine deaminase from mouse kidney was observed when either cytidine or arabinosylcytosine was used as substrate.

2,2'-Anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (anhydro-ara-FC) was compared with cytosine arabinoside (ara-C) in the treatment of ic implanted murine Glioma 261. Both drugs given in ip doses of 500 mg/kg immediately inhibited the uptake and incorporation of tritiated thymidine into the DNA of brain tumor, small intestine, and spleen. Inhibition of DNA synthesis in the tumor recovered within 12 hours of anhydro-ara-FC administration, yet it remained depressed greater than 50% of control 12 hours after ara-C administration. Inhibition in the small intestine recovered within 24 hours of drug administration with either agent while inhibition in the spleen remained depressed greater than 24 hours. Anhydro-ara-FC administered ip in single doses less than or equal to 1500 mg/kg or in multiple doses less than or equal to 200 mg/kg three times a week for ten doses failed to prolong the survival of tumor-bearing mice, and minimal increased survival followed drug administration of 200 mg/kg every 24 hours for five doses. In contrast, ara-C in doses of 50, 100, or 200 mg/kg three times weekly for ten doses significantly increased the survival of tumor-bearing animals between 17% and 36%.

For a full exploitation of the 1000-fold ip pharmacokinetic advantage obtained when cytarabine is administered ip, the drug exposure must be prolonged because the drug is a cell cycle phase-specific agent. Multivesicular liposomes made from nontoxic components were studied as biodegradable, slow-release microcapsules for intracavitary therapy. No attempt was made to increase efficacy by fusing liposomal membrane with that of tumor cells or by selective targeting of liposomes to cancer cells other than simple administration into a cavity. The presence of HCl with cytarabine at the time of encapsulation was found to decrease the leakage rate in human plasma dramatically. After ip administration of encapsulated cytarabine to mice, the total intracavitary drug concentration initially increased tenfold in 15 hours, then fell to the original concentration at 87 hours. The total amount of the drug in the peritoneal cavity decreased with a 21-hour half-life. All mice treated 24 hours after ip inoculation with 1 X 10(6) L1210 cells were cured when 32 mg/kg or 16 mg/kg of cytarabine in liposomes was given ip every 4 days for a total of three doses. Multivesicular liposomes do prolong intracavitary exposure to cytarabine and are efficacious in this model system.

Two areas of investigation are discussed: (a) the identification of critical biochemical parameters that may lead to prediction of response of tumor cells to antimetabolites, such as 1-beta-D-arabinofuranosyl-cytosine (ara-C) and 5-FU, and (b) the potential use of normal purine and pyrimidine metabolites in the selective and specific modulation of critical parameters related to ara-C and 5-FU activity. The results presented indicate a strong correlation between ara-CTP formation and retention in leukemic cells and response of animals with leukemias or patients with acute myelocytic leukemia (AML) treated with ara-C or a protocol containing ara-C. Furthermore, thymidine was found to be an effective modulator of the intracellular pools of dCTP and ara-CTP in rats, bone marrow, small intestine, and colon tumor cells. The mechanism of this effect is unclear, but initial evidence indicates that specific scheduling of the sequential administration of the metabolite and the antimetabolites may be important in determining increased selectivity of antitumor effects. In vivo studies with fluorinated pyrimidines have focused on quantitating 5-fluorodeoxyuridine monophosphate (FdUMP) pools and their retention, and on the incorporation of fluorouridine (FUR) into RNA of sensitive and resistant L1210 cells. The results suggest that the retention of intracellular FdUMP pools above a threshold may be a more critical determinant in selectivity than the initial FdUMP levels achieved in target cells. Thymidine has been demonstrated to alter the pharmacokinetic parameters of 5-FU in man and in mice, but a selective modification of the antitumor activity of 5-FU has not yet been unequivocally demonstrated.

Most studies using high-dose cytarabine (ara-c) for the therapy of acute leukemia have employed intermittent short infusions. In this study, we have evaluated the pharmacology and toxicity of high-dose ara-c by 72-hour continuous infusion. Plasma ara-c concentrations varied from 3.6 microM at the starting dose of 4 g/m2/72 hours to 22.6 microM at 18 g. Plasma clearance appeared to decrease progressively at doses greater than 10 g, suggesting that the route of elimination was saturable. CSF ara-c concentrations ranged from 1.2 microM at 4 g to 4.1 microM at 18 g; the ratio of CSF to plasma ara-c decreased progressively from 0.33 at 4 g to 0.18 at 18 g. The toxic effects were significant and included myelosuppression, nausea, and vomiting in all patients. No single dose-limiting toxicity was identified. Further dose escalation was precluded by combined organ system effects, which included hepatic, pulmonary, renal, and gastrointestinal toxic effects. Attempts to incorporate a 72-hour infusion of ara-c into a combination chemotherapy regimen should proceed cautiously with a starting dose of 6 g/m2/72 hours.

To investigate the possibility that a significant kinetic basis exists for the improved clinical response often obtained using drug combinations, Chinese hamster ovary cells were exposed to cytarabine and daunorubicin in combination. Cell survival was determined by a colony-counting assay; kinetic perturbations were monitored by flow cytometry. No major synergistic effects of the conditions, each drug appeared to provide some protection against the lethal effects of the other. By pairing a moderately toxic concentration of one drug with a nontoxic concentration of the other, we observed sequence-dependent differences in survival. These differences could not be attributed simple to alteration of the cell cycle phase positions by the nontoxic drug, combined with phase-specific lethality of the toxic drug. It is likely, however, that daunorubicin provided partial protection from cytarabine-related cytotoxicity through its effect on the DNA synthesis rate.

As an in vitro model for the chemotherapy of acute myeloid leukemia with cytarabine (ARA-C), the cytotoxicity of this drug was investigated using a human promyelocytic cell line (HL-60) and different drug schedules. The continuous exposure to ARA-C was shown to be more cytotoxic than the intermittent exposures at identical concentrations or under conditions where the concentrations multiplied by time of exposure were the same. In a comparison of exposures, as the intervals between drug exposures were reduced, the cytotoxicity of ARA-C increased. Flow microfluorometric analysis of DNA content showed that a 3-hour exposure to ARA-C every 6 hours produced a greater accumulation of cells in S phase than the same exposure repeated every 12 hours. After a 3-hour exposure to 20 micrograms/ml of ARA-C, the cells recovered 38% and 85% of their capacity to synthesize DNA within 4 and 8 hours, respectively. These findings can be explained in part by the short half-life of the intracellular nucleotide pool of ARA-CTP in these cells (about 60 minutes). These data indicate that when there is a prolongation of the interval between exposures to ARA-C, a greater fraction of cells recover from the inhibitory effects of this drug and escape its cytotoxic action. These observations may be important with respect to the design of more optimal schedules of high-dose ARA-C therapy for the treatment of patients with acute leukemia.

In conventional clinical use, cytosine arabinoside (ara-C) is rapidly deaminated by pyrimidine nucleoside deaminase to the nontoxic compound uracil arabinoside. Tetrahydrouridine (THU) effectively inhibits this enzymatic degradation but is by itself nontoxic. This study demonstrates that concomitant administration of THU markedly increases the myelosuppressive potency of ara-C. When 25 or 50 mg/kg of THU iv and 0.1--0.2 mg/kg of ara-C iv are given daily x 5 days, they produce moderate-to-severe leukopenia and mild-to-moderate thrombocytopenia. A dose of 25 mg/kg of THU with 0.1 mg/kg of ara-C iv daily x 5 days appears appropriate for phase II studies; it produces myelosuppression equivalent to that produced by 3 mg/kg/day x 5 days of ara-C alone. No toxicity occurred with this combination that would not have been expected from ara-C given alone in an equitoxic dose. Although THU and ara-C produced a reduction in peripheral blood and bone marrow blast cells in eight of nine patients with acute leukemia, bone marrow remission did not occur in any of these heavily pretreated patients.

An integrated mathematic computer-based model of the pharmacokinetics, intracellular enzyme kinetics, and cell kinetics of the treatment of L1210 leukemia by cytosine arabinoside (ara-C) is described. The compartment model of Bischoff and Dedrick is extended to the intracellular level by inclusion of equations describing the phosphorylation, dephosphorylation, and deamination of ara-C with enzymatic feedback control. The activities of kinase, deaminase, and phosphatase are explicitly included in the models and are estimated from relevant data. Cell proliferation is described by a continuous-flow mathematic model in which cellular maturation and cell-to-cell variability in maturation rates are key variables. Cell proliferation is related to intracellular biochemistry through mathematic expressions which relate cell lethality and progression delay to the time course of intracellular ara-CTP. In vitro and in vivo experiments performed in a number of laboratories are compared by simulation. The most sensitive parameters in dose-response and cell-survival simulations are deoxycytidine kinase activity, ara-CTP half-life, renal clearance of ara-C, and cell-kinetic parameters for proliferation and cell killing. Progression delay is vital to the realistic simulation of divided-dose schedules. By comparative simulation we have identified areas of uncertainty which can be classified by a few additional measurements. The applications of simulations combining pharmacokinetic, biochemical, and cell-kinetic data in vitro and in vivo are discussed, exploring consistency among different measurements, and relating experimental protocols to clinical treatment.

High-dose cytarabine (HDARA-C) at doses ranging from 1000 to 3000 mg/m2 administered as 30-min iv infusions was used in 12 patients with acute leukemia. HDARA-C toxicity was marked by nausea, vomiting, and somnolence; fever occurred in one patient. Myelosuppression was brief and reversible; the wbc count nadir occurred between Days 10 and 15 after treatment. In this study of a limited number of patients, no reliable conclusions could be drawn about antileukemic activity. However, (a) HDARA-C appeared to be a well-tolerated regimen in acute myeloblastic leukemia in complete remission; (b) a clear improvement was obtained in a patient with central nervous system leukemia; and (c) a sharp but transient decrease in peripheral blast cell counts was seen in two patients with acute myeloblastic leukemia. Cytarabine distribution was bi- or tri-compartmental; plasma final half-life was greater than 4 hrs in six patients. Pharmacokinetic parameters were not correlated with serum deoxycytidine deaminase activity. HDARA-C crosses the blood-brain barrier and may be useful in the prophylaxis against and treatment of central nervous system leukemia.

Evidence from the peritoneal dialysis literature suggests that the peritoneal permeability of a number of hydrophilic anticancer drugs may be considerably less than plasma clearance. Pharmacokinetic calculations indicate that such drugs administered ip in large volumes are expected to maintain a significantly greater concentration in the peritoneal space than in the plasma. This concentration difference offers a potentially exploitable biochemical advantage in the treatment of patients with presumed microscopic residual ovarian cancer confined to the peritoneal cavity.

A computer-based model of the pharmacokinetics of cytosine arabinoside (ara-C) and its active metabolite, ara-CTP, has been developed. This model, which is an intracellular extension of the Bischoff-Dedrick model of multi-organ pharmcokinetics gives predictions which are in agreement with the recent measurements of Chou et al on tissue concentrations of ara-CTP and Borsa et al on blood levels of ara-C. It is shown that the ara-CTP halving time in tissue is much greater than the ara-C halving time in blood because of low tissue levels of phosphatase. For a single dose at the LD10 level in mice, significant splenic DNA inhibition is calculated to occur for 26 hours, while the ara-C levels are negligible in 6 hours. The calculated duration of cytostatic effect at lower dosages (2.5 mg/kg) is 10 or 12 hours, while ara-C blood levels are negligible within 3 hours. Implications for cell kinetics and scheduling studies are also briefly described.

The distribution of cyclocytidine and cytosine arabinoside has been studied in normal BDF mice and in mice bearing 6-day solid L1210 lymphocytic leukemia by whole-body radioautography, bioassay, and radiochemical techniques. Radioactivity was widely distributed throughout the tissues between 15 minutes and 12 hours after a single intravenous dose of either cyclocytidine-2-14C or cytosine arabinoside-2-14C. Whole-body radioautograms demonstrated that for most tissues, cytosine arabinoside-derived 14C was uniformly excreted by 48 hours; cyclocytidine-derived 14C, however, was localized in certain tissues as early as 15 minutes after drug administration and was retained in these sites for 48 hours. Depot loci of 14C included salivary and adrenal glands, fat, cardiac muscle, gastrointestinal tract, and L1210 tumor. The distribution and persistence of cyclocytidine-derived radioactivity is consistent with other reports of toxicity induced by the drug in these tissues. Radiochromatography and bioassay data from BDF mice dosed intraperitoneally with cyclocytidine demonstrated that 65%-95% of the 14C-radioactivity in a number of tissues was the parent compound itself. Thus, cyclocytidine contributed in large measur to the generation of the radioautograms. This study demonstrates that the retention of cyclocytidine in body tissues may serve to effect the sustained release of the deaminase-resistant chemotherapeutic drug from these depot sites and thus prolong cytotoxic levels of drug in tumor tissue.

Enzymatic and clinical pharmacologic studies indicate that 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (AAFC) is relatively resistant to enzymatic deamination and is not phosphorylated but slowly releases 1-beta-D-arabinofuranosyl-5-fluorocytosine. The latter is deaminated rapidly to 1-beta-D-arabinofuranosyl-5-fluorouracil. After iv injection of labeled cytosine arabinoside (Ara-C), 2,2'-anhydro-1-beta-D-arabinofuranosylcytosine, and AAFC into patients, radioactivity appears in substantial amounts and persists for a prolonged time in the saliva. AAFC slowly penetrates into the cerebrospinal fluid, reaches significant levels, and is retained there for a long time. Ara-C, due to rapid deamination in plasma, does not provide sustained levels of Ara-C in the cerebrospinal fluid. It is likely that this difference between AAFC and Ara-C is due to reduced polarity of the former compound.