John J. McCormack

Amplification of antibody production by phosphorothioate oligodeoxynucleotides

A phosphorothioate oligodeoxynucleotide that is complementary (antisense) to the initiation region of the rev gene of HIV-1 causes hypergammaglobulinemia and splenomegaly in mice, and it induces B cell proliferation and differentiation in mouse spleen mononuclear cells (SMNCs) and human peripheral blood mononuclear cells in vitro. The current studies were performed to investigate the specificity of these immunomodulatory effects. Both the sense and antisense rev oligomers stimulated tritiated thymidine incorporation and secretion of immunoglobulin M (IgM) and immunoglobulin G (IgG) by mouse SMNCs in a concentration-dependent fashion, but the antisense oligomer produced greater immune effects. Studies comparing phosphorothioate oligomers (anti-rev, c-myc, and c-myb) either methylated or unmethylated at CpG dinucleotides showed that methylation effectively abrogated the proliferative effect and tended to reduce the immunoglobulin secretory activity, but the latter was not statistically significant except in the case of IgG in anti-rev oligomer-treated cultures. Mice were injected with the sense or antisense rev oligomers singly or in combination. The animals then were immunized with tetanus toxoid and received a booster 21 days later. Oligodeoxynucleotide-treated mice had significantly higher levels of IgM antibodies on days 28 and 35 and of IgG antibodies on days 14 and 35 as compared with mice that were immunized but received vehicle alone. There was no evidence for additive, synergistic, or antagonistic interactions of the sense and antisense rev oligomers. These results indicate that the unmethylated anti-rev oligomer is the most potent of the phosphorothioate oligomers tested at activating lymphocyte proliferation and differentiation and that a single intravenous injection of this oligodeoxynucleotide augments antibody production to a specific antigen as long as 35 days later.

Interaction of warfarin and nonsystemic gastrointestinal drugs

The absorption and pharmacologic action of warfarin may be influenced by such factors as gut motility, pH, solubility, and chemical binding. To investigate the possible interaction of warfarin with nonsystemic gastrointestinal drugs in clinical use, (1) cholestyramine, (2) an antacid mixture containing magnesium and aluminum hydroxide, and (3) psyllium hydrophyllic mucilloid were studied in 6 normal subjects. In a series of experiments average therapeutic doses of each drug were administered concurrently with a single oral dose of warfarin 40 mg. Cholestyramine was found to decrease significantly mean plasma warfarin levels and the hypoprothrombinemic effect of warfarin even after a 3 hour interval between ingestion of the two drugs. Neither the antacid nor psyllium colloid was found to alter plasma concentrations or pharmacologic action of warfarin. In vitro experiments demonstrated significant binding of warfarin to cholestyramine at a pH above its pKa of 5.5.

The synthesis and antitumor properties of a series of water soluble carboxylato-(1,2-diaminocyclohexane) platinum(II) complexes

Abstract Water soluble carboxylato-(1,2-diaminocyclohexane)- platinum(II) complexes have been synthesized and their mode of coordination characterized by elemental analysis and infrared spectra. Preliminary in vitro and in vivo screening tests for anti-tumor activity of these complexes against L1210 murine leukemia were performed. The results indicate that this class of complexes have good in vivo efficacy that can be greatly increased multiple drug administration.

Dihydrofolate reductases within the genus Trypanosoma

Dihydrofolate reductase activity was detected in extracts of rat-adapted bloodstream forms of Trypanosoma (Trypanozoon) brucei, T. (T.)rhodesiense, T. (T.)equiperdum, T.(Duttonella)vivax, T.(Nannomonas) congolense (section Salivaria); T.(Herpetosoma)lewisi, and T.(Schizotrypanum)cruzi (section Stercoraria). This enzyme was also detected in extracts of culture forms of T.(T.)rhodesiense, T.(H.)lewisi, and T.(S.)cruzi. The trypanosomal dihydrofolate reductases shared the general properties of the analogous enzyme from diverse genera in their requirement for dihydrofolic acid as substrate and reduced nicotinamide adenine dinucleotide phosphate as cofactor. They also showed the characteristic extreme sensitivity to the inhibitory action of 4-amino analogs of folic acid such as methotrexate. On the other hand, the trypanosomal reductases exhibited a pattern of susceptibility to inhibition by certain 2,4-diaminopyrimidines and related heterocyclic compounds which was different from the patterns of susceptibility to these agents that have been described for reductases from bacterial and mammalian sources. Superimposed upon this distinctive pattern, moreover, was a further differential response, whereby the drug sensitivities of the reductases of salivarian trypanosomes were almost identical and, as a group, clearly distinguishable from those of the stercorarian species. Those properties of dihydrofolate reductases from culture and bloodstream forms of T.(T.)rhodesiense, T.(H.) lewisi, or T.(S.)cruzi that were compared were closely similar.

Xanthine oxidase-mediated oxidation of epinephrine

Abstract It has been reported [M. Oka, J. pharm. Soc. Japan 57, 566 (1961)] that epinephrine is capable of stimulating the oxidation of purines by xanthine oxidase (X.O.). The present investigation shows that such “stimulation” is, in fact, attributable to concomitant oxidation of epinephrine as well as of the purine substrate (hypoxanthine) by X.O. preparations.X.O. does not oxidize epinephrine under the conditions employed in the absence of an oxidizable substrate such as hypoxanthine. The oxidation of epinephrine, in the presence of X.O. and hypoxanthine is inhibited strongly by allopurinol [4-hydroxypyrazolo-(3,4-d)pyrimidine]. Spectroscopic and Chromatographic studies indicate that the product of epinephrine oxidation in the X.O. system is adrenochrome ( 2,3- dihydro -3- hydroxy -N- methylindole -5,6- quinone ). Oxidation of 2-amino-pteridine, 4-aminopteridine and 4-hydroxypteridine by X.O. also induced epinephrine oxidation. In the presence of 4-amino-6,7-dimethylpteridine and 2-amino-4-methyl-pteridine, which are not effective substrates for X.O., appreciable oxidation of epinephrine was not observed.

A phase I study of trimetrexate, an analog of methotrexate, administered monthly in the form of nine consecutive daily bolus injections

SummaryTrimetrexate glucuronate (TMTX) is a methotrexate (MTX) analog that is active against transport-deficient MTX-resistant tumor cells. We performed a phase I study of TMTX administered by daily bolus for 9 consecutive days since this schedule is one of the most active in experimental murine tumor models. The drug was administered in this fashion every 4 weeks for at least two cycles. Fifteen patients with refractory metastatic cancers were studied and all had received prior chemotherapy. The dose-limiting toxicity was a rapidly reversible thrombocytopenia first seen at a daily dose of 4.0 mg/m2 which occurred 7 days after the end of TMTX administration. There was great inter-and intrapatient variability in the platelet nadirs observed in the six patients treated at 4.0 mg/m2. One patient died of massive hemoptysis during a platelet nadir at that dose level. Granulocyte counts never dropped below 1500/mm3. Only one patient had significant non-hematological toxicity: a radiation recall skin toxicity along with a self-limited maculopapular rash. One patient with melanoma and lung metastases treated at 4.0 mg/m2 had a partial response. TMTX plasma levels were measured by HPLC every 3 days prior to daily dosing in patients receiving 4 mg/m2 to determine whether drug accumulation occurred during this prolonged administration schedule. Nadir drug levels varied from less than 0.02 to 0.35 μM and did not seem to increase during the 9-day schedule in individual patients. By comparison with other phase I trials, the hematologic toxicity of TMTX seems to be schedule-dependent, with less drug being tolerated and more severe thrombocytopenia observed with more protracted treatment protocols. A firm phase II starting dose for daily bolus x 9 schedules is difficult to recommend in view of the variable toxicity observed in the patients treated at 4.0 mg/m2 daily, who, in addition, had all been extensively pretreated. A reasonable starting dose might be 3.0 mg/m2 daily with built-in dosage increases or decreases.

Cellular pharmacology of chloroquinoxaline sulfonamide and a related compound in murine B16 melanoma cells

Chloroquinoxaline sulfonamide (CQS), a chlorinated derivative of sulfaquinoxaline (SQ), inhibited proliferation of murine B16 melanoma cells, but only when relatively high drug concentrations (1 mM) were used. The inhibition of cell growth by CQS was at least partially reversible by incubation in drug-free medium. Incubation of melanoma cells with CQS was associated with an arrest of the cell cycle in G0/G1 as measured by flow cytometry. The drug slightly decreased uptake of radiolabeled deoxyuridine and thymidine after 24- and 48-hr incubation periods but increased nucleoside incorporation at 72 hr. No evidence of intercalation with DNA was found. Because SQ previously was reported to inhibit an aspect of folate metabolism, we investigated the possibility that CQS limits tumor cell growth by altering folate homeostasis. This appears unlikely, however, in view of the following observations: (1) the cytotoxic effects of CQS could not be reversed by folinic acid; (2) deoxyuridine suppression of thymidine incorporation was not affected by CQS treatment; (3) CQS did not inhibit dihydrofolate reductase from mammalian or bacterial sources; and (4) CQS toxicity in mice was not reduced by folinic acid. Experiments performed with analogues modified in the quinoxaline and para-amino phenyl functions indicated that tumor cell inhibition did not require preservation of the conventional sulfonamide structure.

Trypanocidal properties of 5′-O-sulfamoyladenosine, a close structural analog of nucleocidin☆

Abstract A rodent-adapted strain of Trypanosoma (Trypanozoon) rhodesiense , challenged in vivo by a series of purine and pyrimidine sulfamoyl nucleosides, was only sensitive to those containing the adenosine nucleus. The most active compound tested, 5′- O -sulfamoyladenosine, which differs structurally from the trypanocidal antibiotic nucleocidin only by the lack of a fluorine atom, was 100% curative with minimal toxicity to mice with early infections at a single orally administered dose of 2 mg/kg or at a dose of 0.1 mg/kg injected intraperitoneally daily for 3 consecutive days. The rate of cures fell if treatment was delayed for 24 hours or longer. 5′- O -Sulfamoyladenosine is less toxic to mice than nucleocidin, and its therapeutic index could be raised by manipulating the dose regimen. In vitro , 5′- O -sulfamoyladenosine inhibited by 50% the uptake of adenine-8- 14 C by bloodstream form T.(T.) rhodesiense at a concentration of 1 × 10 −8 M , and the incorporation of 14 C-phenylalanine into protein at 5 × 10 −8 M . In this concentration range, glucose utilization by the trypanosomes was inhibited only slightly, but progressively greater inhibition of glucose utilization was observed at higher concentrations.

A Rapid and Sensitive Method for Determination of Trimetrexate from Biological Fluids

Abstract Trimetrexate (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyllquinazoline), (Fig. 1), a potent inhibitor of dihydrofolate reductase (DHFR), has demonstrated antitumor activity against murine and human cell lines both in vitro and against a spectrum of murine tumors in vivo (1,Z).

Synthesis and antitumor activity of platinum complexes containing neutral and protonated amino-olefin ligands

Abstract A series of compounds containing N-substituted allyl amines bound to platinum are reported. Under acidic conditions, compounds of the general formula (protonated amino-olefin)PtCl 3 are formed. Infrared and 1 H NMR spectroscopy establish that these complexes contain a protonated nitrogen and that the olefin is coordinated to platinum. Complexes containing neutral allyl amines are apparently dimeric, with bridging ally amine. All of these complexes contain platinum-chloride bonds activated toward hydrolysis, and all were examined for potential antitumor activity. Most of the protonated ligand complexes exhibited significant cytotoxicity against both cis-platinum sensitive and resistant L1210 Leukemia in cell culture. Neutral ligand complexes were, in all cases, less cytotoxic. Preliminary studies in mice demonstrate only marginal activity against L1210 in vivo .