Hemodynamic effects of potentially useful antineoplastic agents.

Adenosine (Ado) and Ado analogues produce multiple hemodynamic effects including coronary vasodilation, bradycardia, alterations in left ventricular contractility, and peripheral vasodilation or vasoconstriction depending on the vascular bed. The intact anesthetized Sprague-Dawley rat was examined in relation to electrocardiogram and blood pressure alterations induced by a series of potentially useful antineoplastic agents that are purine or pyrimidine analogues as part of a preclinical evaluation of these agents. The drugs tested were arabinosyladenine and its 5'-monophosphate derivative arabinosyladenine-5'-monophosphate (ara-AMP), the 2-fluoro derivative of ara-AMP, the pyrazolo[4,3-d]pyrimidines (formycin and formycin B), 8-azaadenosine, 6-methylmercaptopurine riboside, tricyclic nucleoside-5'-monophosphate, 5-fluorouracil, arabinosylcytosine, and 3-deazauridine. Those Ado analogues subject to deamination by adenosine deaminase (ADA) were also studied in the intact Sprague-Dawley rat after pretreatment with the ADA inhibitor 2'-deoxycoformycin. The results indicate that these agents have significant hemodynamic effects and should alert clinicians to potential adverse reactions when infusing these drugs.

Relative cytotoxicity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyladenine 5'-monophosphate.

The relative cytotoxicity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) were compared using wild-type and adenosine kinase (AK)-deficient baby hamster kidney somatic cell mutants. The cytotoxicity of ara-AMP to baby hamster kidney cells was dependent on the presence of AK activity since AK-deficient mutants were resistant to ara-AMP. On an equimolar basis, ara-AMP was consistently less cytotoxic than was 9-beta-D-arabinofuranosyladenine to wild-type and AK-deficient baby hamster kidney mutant cells. These findings are consistent with the common view that ara-AMP molecules do not enter mammalian cells as an intact nucleotide. Presumably, ara-AMP molecules were hydrolyzed by the nonspecific phosphatases and 5'-nucleotidase found in the serum or by the ecto-5'-nucleotidase on the outer surface of the membrane and only enter the mammalian cells as 9-beta-D-arabinofuranosyladenine.

Phase II trial of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate in non-Hodgkin's lymphoma: prospective comparison of response with deoxycytidine kinase activity.

A phase II study of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate was done in non-Hodgkin's lymphoma with a loading dose/continuous intravenous infusion schedule, consisting of a 20 mg/m2 loading dose followed by a continuous i.v. infusion of 30 mg/m2/24 h for 48 h. The loading dose was held constant while the continuous i.v. dose was escalated or decreased as appropriate for toxicity. Twenty-six patients were entered on the study; 25 are evaluable for response. The patients' median age was 61 years (range 25 to 73); their mean performance status was 1.1. They had received a mean of 2.6 prior chemotherapeutic regimens, and six also had prior radiation therapy. There was one complete response lasting 9+ months, and there were seven partial responses lasting 20, 13, 11, 11, 10, 5, and 2 months (response rate 32%). Toxicity was acceptable and consisted mainly of myelosuppression. 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate is dephosphorylated in vivo and then is thought to be activated intracellularly to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. The rate-limiting enzyme is deoxycytidine kinase. Deoxycytidine kinase activity was determined on pretreatment tumor samples for correlation with response. There was no difference between the values for responders and nonresponders. There was a trend for higher values in more malignant histological subtypes.

Phase I clinical investigation of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (NSC 312887), a new purine antimetabolite.

9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-monophosphate (NSC 312887) is a new purine antimetabolite that has been evaluated in a Phase I clinical trial. The schedule of administration consisted of a single i.v. infusion over a period of 30 min once each day for 5 consecutive days, repeated at 4-week intervals. Thirteen patients received 30 courses of the drug in a dose range of 18 to 40 mg/sq m/day. Granulocytopenia and thrombocytopenia were dose limiting. Repeated courses produced similar degrees of granulocytopenia, but in 7 of 7 patients receiving 2 or more courses, the degree of thrombocytopenia was less severe during the first than during subsequent courses. Myelosuppression in humans was more severe than predicted from the mouse model. Lymphopenia was profound at all dose levels, but reversed within 3 weeks. Somnolence occurred during infusion in 8 of 13 patients, but quickly cleared after the infusion was completed. The infused drug was rapidly dephosphorylated in plasma and then cleared so there was no cumulation of drug in plasma when it was rapidly infused once each day in these doses. Phase II studies of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate are planned at a starting dose of 18 mg/ sq m/day for patients with prior chemotherapy or radiotherapy and 25 mg/sq m/day for those without prior therapy, as a single dose on each of 5 consecutive days repeated at 21- to 28-day intervals.

Chemotherapy of subcutaneous and intracranial human medulloblastoma xenografts in athymic nude mice.

The continuous human medulloblastoma cell line TE-671 was grown as s.c. and intracranial xenografts in athymic nude mice. Tumor-bearing animals were treated with chemotherapeutic agents at the 10% lethal dose; s.c. xenografts were sensitive to melphalan, 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea, and 5-azacytidine. No consistent response could be demonstrated to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate, and no response to methylglyoxal bis(guanyl hydrazone), N-trifluoroacetyl adriamycin-14-valerate, or to 1-beta-D-arabinofuranosylcytosine was observed. Melphalan produced a significant (P = less than or equal to 0.007) increase in the median survival of mice bearing intracranial xenografts, whereas no response was seen to 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea or 5-azacytidine. This model will allow analysis of the chemotherapeutic profile of human medulloblastoma, and provides a means to differentiate cellular sensitivity and resistance from drug access to the intracranial site.

Phase I and II study of fludarabine phosphate in leukemia: therapeutic efficacy with delayed central nervous system toxicity.

Fludarabine phosphate (9-beta-D-arabinofuranosyl-2-fluoroadenine), a novel purine nucleoside, has demonstrated excellent preclinical antitumor activity and little toxicity in phase I clinical trials. We evaluated the clinical use of fludarabine given as a continuous intravenous (IV) infusion for remission induction in patients with relapsed or refractory leukemia. Thirty infusions were administered to 25 patients. At doses less than or equal to 125 mg/m2/d for five days, only three of 17 patients cleared their bone marrow of leukemic cells, and none achieved complete remission (CR). Nine patients received doses of 150 mg/m2/d for five days or 125 mg/m2/d for seven days. Four of these patients achieved CR (three patients with acute nonlymphoblastic leukemia (ANLL), one patient with acute lymphoblastic leukemia (ALL]. However, severe CNS toxicity was encountered in five patients at the two highest dose levels. Initial symptoms of neurotoxicity were delayed from 21 to 43 days after starting treatment and consisted of optic neuritis, cortical blindness, altered mental status, and generalized seizure. Only one patient regained visual and neurologic function; four other patients experienced progressive neurologic deterioration and died. Clinicopathologic evaluation suggested widespread, severe demyelination as the etiology of these reactions. We conclude that fludarabine is an effective drug for remission induction in acute leukemia. However, doses required to achieve CR are associated with unacceptable CNS toxicity. In view of its potent antileukemic activity, further evaluation of fludarabine at lower doses (less than or equal to 75 mg/m2/d for five days) may be warranted in combination with other chemotherapeutic agents for the treatment of patients with acute leukemia.

In vivo gene therapy of cancer with E. coli purine nucleoside phosphorylase.

We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

Phase I clinical trials with fludarabine phosphate.

There have been six different phase I trials of Fludara I.V. (fludarabine phosphate) in patients with solid tumors and three different phase I trials of Fludara I.V. in patients with acute leukemia. In addition, one trial of the agent given intraperitoneally has also been published. In patients with solid tumors, the two most often used schedules are a daily bolus schedule of 18 to 25 mg/m2/d for 5 days repeated every 28 days, and a 20 mg/m2 loading dose followed by a 48-hour continuous infusion of the agent at a dose of 25 to 30 mg/m2/d. The dose-limiting toxicity in these studies has been myelosuppression. No dosage or schedule could be recommended for patients with acute leukemia because of the severe neurotoxicity (progressive dementia with blindness leading to coma) noted with doses greater than or equal to 96 mg/m2/d for 5 to 7 days. Other toxicities noted in phase I trials have included somnolence, mild to moderate nausea and vomiting, and a rare and reversible interstitial pneumonitis. Of greatest interest is that a profound lymphopenia has been noted as a side effect of Fludara I.V. That toxicity has proven to be of benefit for patients with chronic lymphocytic leukemia, low-grade lymphoma, and mycosis fungoides, all of which are responsive to Fludara I.V. therapy.

Drug targeting in antiviral chemotherapy. A chemically stable conjugate of 9-beta-D-arabinofuranosyl-adenine 5'-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells.

With the aim of improving the chemotherapeutic index of 9-beta-D-arabinofuranosyl-adenine 5' monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes. We used a L-SA-ara-AMP conjugate which, in contrast to those previously employed, has the advantage of remaining soluble after lyophilization. We found in mice that: (I) this new conjugate was quite stable in the bloodstream where only a small part of ara-AMP was released; (II) after administration of the conjugate labelled in the drug moiety both acid insoluble and soluble radioactivities were several times higher in liver than in other organs; (III) in mice with Ectromelia virus hepatitis, the conjugate inhibited virus DNA synthesis in liver without affecting cellular DNA synthesis in intestine and bone marrow; (IV) the conjugate did not display any recognizable sign of acute toxicity even at doses several fold higher than those pharmacologically active; and (V) when prepared with homologous albumin it was not immunogenic.

The bioavailability of oral fludarabine phosphate is unaffected by food.

INTRODUCTION: A prospective, open and randomized, two-way crossover study was conducted to evaluate the pharmacokinetics and bioavailability of oral fludarabine phosphate when taken on a full versus an empty stomach. The effectiveness of therapy was also assessed after two cycles of treatment, four weeks apart MATERIALS AND METHODS: Patients with chronic lymphocytic leukemia or low-grade non-Hodgkin's lymphoma were randomly assigned to two groups, both of which received two cycles of treatment with 90 mg of oral fludarabine phosphate administered when either fed or fasted. Patients in Group 1 (n = 8) received oral treatment on a full stomach for the first cycle then on a fasted stomach for the second, while those in Group 2 (n = 10) received their treatment in the reverse sequence. Oral fludarabine phosphate was administered on the first day of the two study cycles and intravenous fludarabine phosphate was administered on days 3-6. RESULTS AND CONCLUSION: Of 22 patients recruited, 18 (CLL n = 10; NHL n = 8) were eligible for efficacy and safety evaluation, and 16 for bioavailability and pharmacokinetic analyses. The response to oral 2-F-ara-AMP was rapid: by two treatment cycles, 12 out of 18 patients (66.7%) had achieved partial response. Of the six patients who did not respond, five patients (27.7%) had stable disease. There was no notable difference in the rate of response between patients with B-CLL and lg-NHL. There was a marginal increase in total systemic availability of fludarabine phosphate when administered orally on a fed stomach (2-F-ara-A AUC((0-24 h)) = 3.28 +/- 1.48 microg.h/ml) compared to a fasted stomach (2-F-ara-A AUC((0-24 h)) = 3.05 +/- 1.56 microg.h/ml). Time to peak plasma concentration was slightly extended by the presence of food (2.2 +/- 1.0 versus 1.3 +/- 0.74 h) but the terminal half-life was unaffected. The minor differences in the pharmacokinetics of oral fludarabine phosphate when taken after food were not statistically significantly different and seem unlikely to be clinically relevant. The efficacy and safety data closely paralleled previous experience with the intravenous formulation.

Phase I clinical trial of fludarabine phosphate (F-ara-AMP).

F-Ara-AMP (fludarabine phosphate) is an adenosine analogue that is resistant to deamination; it is a more potent cytotoxic compound than ara-A in experimental tumor systems. F-Ara-AMP was given by continuous IV infusion over 5 days once every 4 weeks to 27 evaluable adult patients with advanced cancer. The median Karnofsky performance status was 70% (range 50%-90%), and the median age was 58 years (range 41-74). In addition to adequate blood counts, a creatinine clearance of at least 60 ml/min was required. The initial dose level was 35 mg/m2/day. Dose-limiting myelosuppression was seen in the first patient. Subsequent patients were treated at lower doses. Myelosuppression was the only major toxicity. Leukopenia was generally more prominent than thrombocytopenia, but 2 patients experienced prolonged thrombocytopenia which prevented further therapy. Nausea was minimal, and neither renal nor neurologic toxicity was encountered. In patients with good renal function a dose of 25 mg/m2/day can be safely administered. However, because of apparent cumulative myelosuppressive effects a lower dose is more appropriate for patients who have had extensive prior chemotherapy or radiotherapy.

Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine. Synthesis of the carrier and pharmacological properties of the conjugate.

BACKGROUND/AIMS: The hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine (L-Poly(Lys)) must have a high solubility in order to be injected in a small volume compatible with the intramuscular route. In this paper the molecular weights of Poly(Lys) which allowed the synthesis of conjugates with the properties of high solubility and limited loss by the kidney were determined and a procedure for obtaining Poly(Lys) preparations with the required range of polymerization has been described. METHODS: Conjugates were prepared using Poly(Lys) of different molecular weights obtained by the procedure described here or purchased from a commercial source. Their solubility and renal loss in mice was determined. RESULTS: Poly(Lys) with molecular weights ranging from 45,000 and 65,000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuck hepatitis virus-infected woodchucks for 37 days at a daily dose of 5.8 mg/kg exerted a strong antiviral activity. These preparations were devoid of acute toxicity in rat and caused no toxic effects when injected intramuscularly daily for 28 days at a dose ten times higher than that active in woodchucks. CONCLUSIONS: The results support the possibility of a clinical use of L-Poly(Lys) to obtain liver targeting of adenine arabinoside monophosphate for the treatment of chronic hepatitis B virus infection.

Phase II trial of fludarabine phosphate (F-Ara-AMP) in patients with advanced head and neck cancer.

Thirteen patients with advanced head and neck cancer were entered into a phase II study of fludarabine phosphate. Fludarabine phosphate was given by continuous infusion for 5 days, at a starting dose of 20 mg/m2 per day for patients previously treated with one regimen and 25 mg/m2 per day for previously untreated patients; therapy was repeated every 3-4 weeks. Of the 13 patients, 3 had undergone one prior regimen and 10 patients were previously untreated by chemotherapy. No responses were observed. Myelosuppression was the most common toxicity observed. Four patients developed mild nausea, vomiting and seven developed bleeding stomatitis that resolved in one week. In addition, four patients developed headaches which resolved spontaneously. No renal, hepatic, or neurotoxicity was observed. Our study demonstrates that in previously treated and untreated patients, fludarabine phosphate given on this schedule has little activity in patients with advanced head and neck cancer.

Fludarabine phosphate for the treatment of low grade lymphoid malignancy.

Thirty-four patients with previously treated, advanced, low grade NHL were treated with Fludarabine, a deamination-resistant analogue of adenosine arabinoside, at a dose of 25 mg m-2 intravenously, daily for 5 days (median number of cycles = 3, range 1-10). Complete remission (CR) was achieved in six and partial remission (PR) in a further seven. Overall, responses were seen in 11/23 patients (48%) with follicular lymphoma and in 2/11 (18%) with low grade, diffuse NHL. Fifteen patients with previously treated CLL and one patient with prolymphocytic leukaemia (PLL) were also treated as above (median no. of cycles = 3, range 1-6). A partial response was seen in three of the 11 evaluable patients with CLL and CR was achieved in the patient with PLL. There were four deaths due to infection and 19 further episodes requiring admission to hospital. No other significant toxicity was reported in a total of 164 cycles of Fludarabine. This agent is active in advanced low grade lymphoid malignancy. Further studies are required to assess its role in newly diagnosed patients.

Ineffectiveness of adenine arabinoside and adenine arabinoside 5'-monophosphate in simian varicella infection.

Adenine arabinoside and adenine arabinoside 5'-monophosphate (ara-AMP) have been evaluated for antiviral activity against simian varicella virus infection in monkeys. In a preliminary study for toxicity, intramuscular injection of ara-AMP at 15 mg/kg per day as a single injection for 5 days to two normal patas monkeys caused no detectable local reaction, no weight loss or changes in serum transaminase levels, and no hematological abnormalities. When this dose was given in the treatment of four simian varicella virus-infected patas monkeys, no effect was observed on the clinical course of infection, as compared with four infected monkeys which received phosphate-buffered saline. Treatment was begun 43 h after virus inoculation and was continued for 16 days. Toxicity of intravenously administered ara-AMP at 100 to 50 mg/kg per day for 5 days to pairs of uninfected patas monkeys was evident by hematological and hepatic histological alterations, as well as the death of one monkey in each pair. No gross evidence of toxicity occurred in two monkeys which received 20 mg of ara-AMP per kg per day. The antiviral efficacy of intravenous treatment was studied in groups of four African green monkeys which received adenine arabinoside at 15 mg/kg per day or ara-AMP at 18.4 mg/kg per day. Drug administration began 48 h after inoculation with simian varicella virus and continued for 10 days. The monkeys that received treatment did not respond to infection differently from four infected control monkeys similarly treated with phosphate-buffered saline.

Fludarabine therapy in macroglobulinemic lymphoma.

Fludarabine, a fluorinated analogue of adenine, was given to 11 patients with macroglobulinemic lymphoma, all but one having failed prior standard chemotherapy. Five patients (45%) responded with more than a 50% reduction of immunoglobulin M (IgM) tumor mass for a projected median duration of longer than 1 year. The onset of remission was usually slow, with a median tumor halving time of 5.2 months in responding patients, emphasizing the importance of repeated courses of treatment. Fludarabine is an important new agent effective against macroglobulinemic lymphoma, and should be evaluated further in combination with other active modalities.

Pharmacodynamics and proposed mechanism of therapeutic action and host toxicity of 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP) in P388 murine leukemia-bearing mice.

The cellular metabolism of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'monophosphate (F-araAMP), a soluble nucleoside analog with proven antileukemic activity in animal and human tumors, has been studied in mice bearing P388 leukemia. Earlier studies showed markedly less in vivo accumulation of F-ara ATP the principal active metabolite, in gastrointestinal mucosa (GI) and bone marrow (BM) compared with P388 after F-araA or F-araAMP administration. To elucidate the mechanism of toxicity this work has examined the pharmacodynamics of F-araAMP anabolites, F-araATP and F-ATP, in P388 cells, BM and GI mucosa tissues after nontoxic (LD1) and toxic (LD50) doses of F-araAMP. F-araATP was the major triphosphate metabolite in acid-soluble extracts from P388 cells, BM, and GI mucosa tissues. F-araATP accumulated to approximately 1 mM in P388 cells after either LD1 or LD50 treatment of F-araAMP and was eliminated with a t1/2,el of approximately 5 h. The ratio of the area under the concentration-time curve (AUC 0----infinity) of F-araATP was 1.01 after the LD50 over the LD1 doses of F-araAMP. BM and GI mucosa tissues accumulated 40-fold less F-araATP than the concentration in P388 cells. 2-Fluoro-ATP, a second toxic anabolite, accumulated in P388 cells to 156 +/- 39 microM and 447 +/- 79 microM after the two doses of the drug, respectively. The ratio of area under the curve (AUC) of F-ATP in P388 cells after the two doses of F-araAMP was 38.77, which approaches the ratio of % lethality (LD50/LD1 = 50). F-ATP was also quantitated in BM and GI mucosa reaching one-fifth to one-half the concentration of F-araATP after the LD50 dose of F-araAMP. The AUC values of F-ATP (0----24 h) were 9.5- to 12.5-fold higher after the LD50 than after the LD1 dose of F-araAMP. These results suggest that there is a selective therapeutic action of F-araAMP against P388 and that the increased cellular concentration of F-ATP in both the tumor cells and the host tissues (BM and GI mucosa) could explain the mode of toxicity of F-araAMP.