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Treatment with fludarabine phosphate (9-β-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

Thiarabine is undergoing clinical trials. In support of that effort combination therapy of thiarabine plus six clinical anticancer agents was evaluated using various human tumor xenograft models. The antitumor activity of thiarabine in combination appeared to be greater than additive with irinotecan (DLD-1 colon), paclitaxel (PC-3 prostate), cisplatin (PC-3 prostate), or cyclophosphamide (RL lymphoma), additive with irinotecan (NCI-H460 NSCLC), cisplatin (NCI-H460 NSCLC) or methotrexate (CCRF-CEM leukemia), and less than additive with irinotecan (HT29 colon), paclitaxel (NCI-H460 NSCLC) or cisplatin (NCI-H23 NSCLC). Combining thiarabine with irinotecan, paclitaxel, cisplatin, or cyclophosphamide should receive consideration in the clinical treatment of cancer.

Many solid tumors and hematologic malignancies lack expression of the enzyme methylthioadenosine phosphorylase (MTAP), due either to deletion of the MTAP gene or to methylation of the MTAP promoter. In cells that have MTAP, its natural substrate, methylthioadenosine (MTA), generated during polyamine biosynthesis, is cleaved to adenine and 5-methylthioribose-1-phosphate. The latter compound is further metabolized to methionine. Adenine and methionine are further metabolized and hence salvaged. In MTAP-deficient cells, however, MTA is not cleaved and the salvage pathway for adenine and methionine is absent. As a result, MTAP-deficient cells are more sensitive than MTAP-positive cells to inhibitors of de novo purine synthesis and to methionine deprivation. The challenge has been to take advantage of MTAP deficiency, and the changes in metabolism that follow, to design a strategy for targeted treatment. In this review, the frequency of MTAP-deficiency is presented and past and recent strategies to target such deficient cells are discussed, including one in which MTA is administered, followed by very high doses of a toxic purine or pyrimidine analog. In normal host cells, adenine, generated from MTA, blocks conversion of the analog to its toxic nucleotide. In MTAP-deficient tumor cells, conversion proceeds and the tumor cells are selectively killed. Successful mouse studies using this novel strategy were recently reported.

The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2′-deoxyriboside (MeP-dR), 2-F-2′-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.

This study was designed to determine the in vitro and in vivo antitumor behaviour of three analogues of the natural product FR901464. All analogues demonstrated effective inhibition of cell proliferation. Minimal in vivo antitumor activity was observed, warranting further PK and antitumor efficacy studies.

4′-Thio-β-D-arabinofuranosylcytosine (4′-thio-ara-C), which has shown a broad spectrum of antitumor activity against human tumor systems in mice and is undergoing clinical trials, was evaluated for cross-resistance to seven clinical agents in order to identify potentially useful guides for patient selection for further clinical trials of 4′-thio-ara-C and possible noncross-resistant drug combinations with 4′-thio-ara-C. A drug resistance profile for 4′-thio-ara-C, which was administered intraperitoneally daily for nine consecutive days, was obtained using seven drug-resistant P388 and L1210 leukemias that were implanted intraperitoneally in mice. Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin, etoposide, or paclitaxel) exhibited no cross-resistance to 4′-thio-ara-C. Leukemias resistant to camptothecin, cisplatin, and 5-fluorouracil were also not cross-resistant to 4′-thio-ara-C. Only the leukemia resistant to 1-β-D-arabinofuranosylcytosine was cross-resistant to 4′-thio-ara-C. The data suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-β-D-arabinofuranosylcytosine and (2) the lack of cross-resistance seen with 4′-thio-ara-C may contribute to therapeutic synergism when 4′-thio-ara-C is combined with other agents.

As part of an ongoing program to develop novel antitumor agents over the years, we have synthesized and evaluated a number of 4'-C-substituted nucleosides. A few years ago, we reported the first synthesis of 4'-C-hydroxymethyl-2'-fluoro arabino nucleosides, which did not exhibit any cytotoxicity. In our exploration of related compounds, we synthesized and evaluated the 4'-C-methyl-2'-fluoro arabino nucleosides in both the purine and pyrimidine series. In the pyrimidine series, 1-(4-C-methyl-2-fluoro-beta-D-arabinofuranosyl) cytosine (13) was found to be highly cytotoxic and had significant antitumor activity in mice implanted with human tumor xenografts. The synthesis and anticancer activity of this series of nucleosides are reported.

Purpose The purpose of this investigation was to synthesize a series of carbonate and carbamate derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma non-toxic metabolite of penclomedine (PEN) seen in patients. DM-PEN has been observed to be an active antitumor agent in mouse human xenograft tumor models and non-neurotoxic in a rat model, however, activity in intracranially implanted human glioma xenograft models have not been reported. The major goal was to identify derivatives that are active in brain tumors. Methods Derivatives were prepared from DM-PEN and evaluated in vivo against human U251 glioblastoma, D54 glioblastoma and MX-1 breast tumor xenografts and mammary tumor 16/C that were implanted in the mammary fat pad or intracranially (IC). Results Carbonate and carbamate derivatives were found to be superior to DM-PEN against IC growing human glioblastoma xenografts. Conclusion The activity of the carbonates and carbamates against human tumor xenografts in vivo suggests consideration of these two series of derivatives of DM-PEN for clinical development.

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-alpha-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-alpha-L-talofuranosyl]-2-F-adenine, and 9-[alpha-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.

Pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136 (NSC 694501) selectively cross-links guanine residues located on opposite strands of DNA, and exhibits potent in vitro cytotoxicity. In addition, SJG-136 is highly active in vivo in hollow fiber assays. In the current investigation, SJG-136 was evaluated for in vivo efficacy in 10 tumor models selected on the basis of sensitivity of cells grown in the hollow fiber and in vitro time course assays: LOX IMVI and UACC-62 (melanomas); OVCAR-3 and OVCAR-5 (ovarian carcinomas); MDA-MB-435 (breast carcinoma); SF-295 and C-6 (gliomas); LS-174T (colon carcinoma); HL-60 TB (promyelocytic leukemia); and NCI-H522 (lung carcinoma). SJG-136 was active against small (150 mg) and large (250–400 mg) xenografts with tumor mass reductions in all 10 models. In addition, significant growth delays occurred in nine models, cell kill in six models ranged between 1.9 and 7.2 logs, and there were 1 to 4/6 tumor-free responses in six models. SJG-136 is active following i.v. bolus injections, as well as by 5-day continuous infusions. Of all of the schedules tested, bolus administrations for 5 consecutive days (qd×5) conferred the greatest efficacy. SJG-136 is active over a wide dosage range in athymic mouse xenografts: on a qd×5 schedule, the maximum-tolerated dose was ∼120 μg/kg/dose (total dose: 0.6 mg/kg = 1.8 mg/m2) and the minimum effective dose in the most sensitive model (SF-295) was ∼16 μg/kg/dose (total dose: 0.08 mg/kg = 0.24 mg/m2). Results of this study extend the initial in vivo observations reported in the reference above and confirm the importance of expediting more detailed preclinical evaluations on this novel agent in support of phase I clinical trials in the United Kingdom and the United States, which are planned to commence shortly.

Purpose: The purpose of this investigation was to compare the antitumor activities of a series of acyl derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) observed to be an active antitumor agent in vivo and non-neurotoxic in a rat model with that of DM-PEN. Methods: Acyl derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted subcutaneously (s.c.) or intracerebrally (i.c.). Several derivatives were also evaluated against other human tumor xenografts and murine P388 leukemia cell lines. Results: Several of the acyl derivatives were found to be superior to DM-PEN against MX-1, human ZR-75-1 breast tumor, human U251 CNS tumor and the P388 leukemia parent cell line and lines resistant to cyclophosphamide and carmustine. 4-Demethyl-4-methoxyacetylpenclomedine showed inferior activity to current clinical brain tumor drugs against a glioma cell line, superior activity to temozolomide and procarbazine against the derived mismatch repair-deficient cell line, and superior activity to cyclophosphamide and carmustine but inferior activity to temozolomide against two ependymoma cell lines, all of which were implanted s.c. Conclusion: Proposed mechanisms of activation and action of DM-PEN and the acyl derivatives support the potential clinical superiority of the acyl derivatives.

Because of the excellent in vivo activity of 4'-thio-beta-D-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of beta-D-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5'-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5'-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5'-monophosphate of araC was a better substrate for deoxycytidine 5'-monophosphate deaminase than was the 5'-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

Four 5-substituted (chloro, fluoro, bromo, methyl) 1-(4-thio-beta-D-arabinofuranosyl)cytosines and their alpha anomers were synthesized by a facile route in high yields. All of these nucleosides were evaluated for cytotoxicity against a panel of human tumor cell lines in vitro. Only 5-fluoro-1-(4-thio-beta-D-arabinofuranosyl)cytosine was found to be highly cytotoxic in all the cell lines and was further evaluated in vivo.

Cl-F-ara-A, an analog of fludarabine, was evaluated against a spectrum of tumor systems in culture and in mice. The compound exhibited significant cytotoxicity against a variety of human tumor cell lines. More importantly, the compound showed selectivity in vivo, with excellent activity being demonstrated against human colon and renal tumors. Human nonsmall cell lung and prostate tumors were also sensitive in vivo to the compound, albeit at a reduced level.

4'-thio-beta-D-arabinofuranosylcytosine was synthesized by a facile route in high yields. It was evaluated for antitumor activity against a panel of human tumors, both in vitro and in vivo.

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.

Thiarabine was evaluated for antitumor activity in seven human leukemia, lymphoma, and myeloma xenograft models to explore the activity in hematological malignancies. Thiarabine was active against all of the human leukemia and lymphoma lines tested, being curative against HL-60 leukemia and AS283 lymphoma and effecting tumor regressions in CCRF-CEM, MOLT-4, and K-562 leukemia and RL lymphoma models, but did not exhibit any appreciable activity against RPMI-8226 myeloma. For the leukemia/lymphoma models, thiarabine was more efficacious than ara-C/palmO-ara-C (four models), clofarabine (three models), fludarabine monophosphate (five models), cladribine (four models), and gemcitabine (six models). Thiarabine warrants future clinical trials with leukemias/lymphomas.

Pd(IV)-Mediated cross-coupling reactions of ribofuranosylpurine (I) with organozinc halides (II) and subsequent deprotection afford the C-6 substituted purine ribosides (III) in satisfactory to high yields.

A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.

A series of C-6 alkyl, cycloalkyl, and aryl-9-(β- d -ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph3P)4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β- d -ribofuranosyl)-purine (6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β- d -ribofuranosyl)purine derivatives 7–11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β- d -ribofuranosyl)purine derivatives 12–16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β- d -arabinofuranosyl)purine (18) was prepared and evaluated.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC50 values in the range of 1−10 μg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4,6-tetra-O-acetyl-1-α,β-d-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

Four 5-substituted (chloro, fluoro, bromo, methyl) 1-(4-thio-P-Darabinofuranosy1) cytosines and their a anomers were synthesized by a facile route in high yields. All of these nucleosides were evaluated for cytotoxicity against a panel of human tumor cell lines in vitro. Only 5-fluoro-1 -(4-thio-β-D-arabinofuranosyl)cytosine was found to be highly cytotoxic in all the cell lines and was further evaluated in vivo.

As part of an ongoing program to develop novel antitumor agents over the years, we have synthesized and evaluated a number of 4'-C-substituted nucleosides. A few years ago, we reported the first synthesis of 4'-C-hydroxymethyl-2'-fluoro arabino nucleosides, which did not exhibit any cytotoxicity. In our exploration of related compounds, we synthesized and evaluated the 4'-C-methyl-2'-fluoro arabino nucleosides in both the purine and pyrimidine series. In the pyrimidine series, 1-(4-C-methyl-2-fluoro-beta-D-arabinofuranosyl) cytosine (13) was found to be highly cytotoxic and had significant antitumor activity in mice implanted with human tumor xenografts. The synthesis and anticancer activity of this series of nucleosides are reported.

Clofarabine increases the activation of 1-beta-D-arabinofuranosyl cytosine (araC) in tumor cells, and combination of these two drugs has been shown to result in good clinical activity against various hematologic malignancies. 1-beta-D-[4-thio-arabinofuranosyl] cytosine (T-araC) is a new cytosine analog that has exhibited excellent activity against a broad spectrum of human solid tumors and leukemia/lymphoma xenografts in mice and is currently being evaluated in patients as a new drug for the treatment of cancer. Since T-araC has a vastly superior preclinical efficacy profile in comparison to araC, we have initiated studies to determine the potential value of clofarabine/T-araC combination therapy.In vitro studies have been conducted to determine the effect of clofarabine on the metabolism of T-araC, and in vivo studies have been conducted to determine the effect of the clofarabine/T-araC combination on five human tumor xenografts in mice.Initial studies with various tumor cells in culture indicated that a 2-h incubation with clofarabine enhanced the metabolism of T-araC 24 h after its removal by threefold in three tumor cell types (HCT-116 colon, K562 leukemia, and RL lymphoma) and by 1.5-fold in two other tumor cell types (MDA-MB-435 breast (melanoma), and HL-60 leukemia). Pretreatment with clofarabine resulted in a slight decrease in metabolism of T-araC in RPMI-8226 myeloma cells (65% of control) and inhibited metabolism of T-araC in CCRF-CEM leukemia cells by 90%. In vivo combination studies were conducted with various human tumor xenografts to determine whether or not the modulations observed in vitro were reflective of the in vivo situation. Clofarabine and T-araC were administered on alternate days for five treatments each (q2dx5) with the administration of T-araC 24 h after each clofarabine treatment. Combination treatment of HCT-116, K562, HL-60, or RL tumors with clofarabine and T-araC resulted in dramatically superior anti-tumor activity than treatment with either agent alone, whereas this combination resulted in antagonism in CCRF-CEM tumors. The in vivo antitumor activity of clofarabine plus T-araC against HCT-116 tumors was much better than the activity seen with clofarabine plus araC.These studies provide a rationale for clinical trials using this combination in the treatment of acute leukemias as well as solid tumors and suggest that this combination would exhibit greater antitumor activity than that of clofarabine plus araC.

In silico chemical library screening (virtual screening) was used to identify a novel lead compound capable of inhibiting S-adenosylmethionine decarboxylase (AdoMetDC). AdoMetDC is intimately involved in the biosynthesis of polyamines, which are essential for tumor progression and are elevated in numerous types of tumors. Therefore, inhibition of this enzyme provides an attractive target for the discovery of novel anticancer drugs. We performed virtual screening using a computer model derived from the X-ray crystal structure of human AdoMetDC and the National Cancer Institute's Diversity Set (1990 compounds). Our docking study suggested several compounds that could serve as drug candidates since their docking modes and scores revealed potential inhibitory activity toward AdoMetDC. Experimental testing of the top-scoring compounds indicated that one of these compounds (NSC 354961) possesses an IC50 in the low micromolar range. A search of the entire NCI compound collection for compounds similar to NSC 354961 yielded two additional compounds that exhibited activity in the experimental assay but with significantly diminished potency relative to NSC 354961. In this report, we disclose the activity of NSC 354961 against AdoMetDC and its probable binding mode based on computational modeling. We also discuss the importance of virtual screening in the context of enzymes that are not readily amenable to high-throughput assays, thereby demonstrating the efficacy of virtual screening, combined with selective experimental testing, in identifying new potential drug candidates.

To develop a rapidly dissolving porous particle formulation of paclitaxel without Cremophor EL that is appropriate for quick intravenous administration.A rapidly dissolving porous particle formulation of paclitaxel (AI-850) was created using spray drying. AI-850 was compared to Taxol following intravenous administration in a rat pharmacokinetic study, a rat tissue distribution study, and a human xenograft mammary tumor (MDA-MB-435) model in nude mice.The volume of distribution and clearance for paclitaxel following intravenous bolus administration of AI-850 were 7-fold and 4-fold greater, respectively, than following intravenous bolus administration of Taxol. There were no significant differences between AI-850 and Taxol in tissue concentrations and tissue area under the curve (AUC) for the tissues examined. Nude mice implanted with mammary tumors showed improved tolerance of AI-850, enabling higher administrable does of paclitaxel, which resulted in improved efficacy as compared to Taxol administered at its maximum tolerated dose (MTD).The pharmacokinetic data indicate that paclitaxel in AI-850 has more rapid partitioning from the bloodstream into the tissue compartments than paclitaxel in Taxol. AI-850, administered as an intravenous injection, has been shown to have improved tolerance in rats and mice and improved efficacy in a tumor model in mice when compared to Taxol.

Purpose: The purpose of this investigation was to synthesize a series of thiolo-, thiono- and dithiocarbonate and thiocarbamate derivatives of 4-demethylpenclomedine (DM-PEN), the major plasma metabolite of penclomedine (PEN) in patients observed subsequently to be an active antitumor agent and non-neurotoxic in a rat model, in order to compare their antitumor activity with that of DM-PEN. Methods: Derivatives were prepared from DM-PEN and evaluated in vivo against human MX-1 breast tumor xenografts implanted in the mammary fat pad, several of which were also evaluated against human brain tumor xenografts. Results: Thiolocarbonate and thiocarbamate derivatives were found to be superior to DM-PEN against MX-1 tumor and modestly active against glioblastoma. Conclusion: The activity of the thiolocarbonates and thiocarbamates against human tumor xenografts in vivo suggests consideration of these two series of derivatives of DM-PEN for clinical development.

Isophosphoramide mustard (IPM) is the cytotoxic alkylating metabolite of Ifosfamide (IFOS). IPM is being readied for a phase I clinical trial. In the present preclinical study, IPM was evaluated for usage in multidose intravenous (IV) infusion protocols.Mice and dogs received IV IPM daily for 3 days. Single-day dosing-oral and IV-to mice, rats, and monkeys is also reviewed for comparison. Complete toxicology studies were completed in the mice and dogs. For mice, dogs and monkeys, IV pharmacokinetic studies were conducted and compared.For mice, the LD(10) for the 3-day IV schedule for IPM was calculated to be 119 mg/kg (with 95% confidence limits of 87-134 mg/kg) (combined sexes), and for adult male dogs the maximum tolerated dose (MTD) was 5 mg/kg. Pharmacokinetic studies in mice, dogs and monkeys were compared and projected to human dosing. For dogs that received 10 mg/kg of IPM, T(1/2beta) was 0.99 h, and clearance was constant (1.01 l/h/kg). IPM was detected from 0 h to 1.5 h after the 5 mg/kg dose and from 0 h to 2 h after the 10 mg/kg dose; none was detected after 2 h. The IV MTD in dogs was 5 mg/kg per day for 3 days. Renal tubular necrosis and bone marrow failure were the causes of death. Transient liver, renal and bone marrow toxicity and gastrointestinal dysfunction were seen at low doses (5 mg/kg) in dogs. In mice (receiving 100 mg/kg IV) plasma concentrations disappeared in less than 1 h (T(1/2alpha) 2 min), with a clearance of 8.44 l/h/kg. For monkeys, the mean T(1/2) was 4.2 h. Median clearance was 1.65 l/h/kg and no IPM was detected 4 h after dosing. No potential IPM metabolites could be detected in any of the studies. In vitro, plasma protein bound 90% of IPM within 5 min of incubation.Predictions for human pharmacokinetic parameters and dosing are made from allometric analysis using the above three species. Data predicted an acceptable starting dose of 30 mg/m(2) with a clearance of 39.5 l/h, and a T(1/2) of 1 h 45 min for a 70-kg patient.

The main objective of this investigation was to identify the reactive pharmacophore in penclomedine (PEN, 3,5-dichloro-4,6-dimethoxy-2-(trichloromethyl) pyridine) for in vivo antitumor activity and also to discover related ring structures and sulfur analogues that might exhibit superior antitumor activity in vivo. Several new analogues of PEN and related structural variants have been synthesized and evaluated in vivo against MX-1 human breast tumor xenograft implanted subcutaneously (sc), although none of them demonstrated significant activity.

The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine (MeP-dR) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2'-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5'-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC(50) values in the range of 1-10 microg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4, 6-tetra-O-acetyl-1-alpha,beta-D-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

( S )-10-(2,6-Dimethyl-4-pyridinyl)-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7 H -pyrido[1,2,3 de ][1,4] benzothiazine-6-carboxylic acid (WIN 58161) is an enantiomerically pure quinolone with outstanding bacterial topoisomerase II (DNA gyrase, EC 5.99.1.3) inhibitory and antibacterial activity. Unlike most quinolones, WIN 58161 also exhibits significant inhibitory activity against mammalian topoisomerase II (EC 5.99.1.3). DNA gyrase and topoisomerase II inhibitory activities are enantioselective. Consequently, WIN 58161 and its enantiomer (WIN 58161-2) provide useful tools to probe the contribution of topoisomerase II inhibition to the mechanism of cytotoxicity of quinolones and the potential utility of quinolone-topoisomerase II inhibitors as antitumor agents. WIN 58161 inhibited both highly purified Escherichia coli DNA gyrase and HeLa cell topoisomerase II by the promotion of enzyme-DNA covalent complexes. WIN 58161 did not bind stably to DNA via intercalation and did not enhance the formation of topoisomerase I (EC 5.99.1.2)-DNA covalent complexes. At drug concentrations that are cytotoxic to P388 murine leukemia cells, WIN 58161 promoted intracellular DNA single-strand breaks (SSBs) that exhibited the hallmarks of being mediated by topoisomerase. DNA fragments were complexed with protein, and SSBs were readily resealed at 37 ° following drug removal. WIN 58161-2 was neither cytotoxic nor did it promote intracellular SSBs in P388. These observations suggest that the mechanism of cytotoxicity of WIN 58161 is predominantly, if not exclusively, a result of topoisomerase II inhibition. When studied in tumor-bearing mice, WIN 58161 exhibited a significant antitumor effect against each of five tumors tested, whereas neither toxicity nor antitumor activity was observed with WIN 58161-2. We conclude from these studies that WIN 58161 represents the prototype of a novel chemical class of topoisomerase II inhibitor with potential clinical utility in treating cancer.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

A series of halogen analogs of phosphoramide mustard, isophosphoramide mustard, and triphosphoramide mustard, the cytotoxic metabolites of the antitumor drugs cyclophosphamide, ifosfamide, and trofosfamide, respectively, was evaluated in vitro against human tumor cell lines and in vivo against experimental tumors to investigate the effect of replacement of chlorine with bromine or fluorine on the antitumor activity of the parent phosphoramide mustards. In the experimental tumors L1210 leukemia, B16 melanoma, mammary adenocarcinoma 16/C, and ovarian sarcoma M5076, the antitumor activity of the analogs was observed to be generally comparable with that of the parent mustards when chlorine was replaced by bromine but uniformly lower when chlorine was replaced by fluorine. Furthermore, the monobromo analog of isophosphoramide mustard displayed equal or somewhat greater activity in comparison with cyclophosphamide when evaluated against subcutaneously implanted L1210 leukemia with intraperitoneal drug treatment and against mammary adenocarcinoma 16/C.

HOOI is a hydroperoxy-derivative of ifosfamide (IFOS) and a pro-drug of isophosphoramide mustard (IPM) [the active metabolite of IFOS] – a bi-functional alkylator that cross-links with G/C DNA base sequences resulting in irreparable inter-strand DNA cross-linking and cell death. HOOI has been an unstable laboratory curiosity for years; however, HOOI.L-lysine is a stable salt complex that has allowed the development of HOOI for clinical trials. HOOI spontaneously releases acrolein and chloroacetaldehyde in situ in cancer cells not extracellular in the general circulation (as does IFOS) and has not been associated with cystitis, renal tubular necrosis and/or CNS toxicity. HOOI has been screened in 20+ human xenograft tumor and murine tumor models and has significantly improved %ILS in intracranially implanted human xenografts – MX-1, U251; ZR-75-1 and in P399/CPA leukemia vs. IFOS and IPM. The U251 data (%ILS – 84+) is impressive considering – BCNU produced a 72% ILS. The ZR-75-1 breast cancer had a %ILS – 83. Bone marrow failure was the DLT in mice – LD10 100 mg/kg (for M/F). Dogs (M/F) were treated with 10, 15, 20 and 30 mg/kg. For both sexes, the LD10 was calculated to be 17.2, while the LD50 was calculated to be 17.3 mg/kg. PK values in dogs revealed the following profile for groups dosed with 30 mg/kg: AUC0- t = 1.53 (mg h/L), T1/2α = 0.93 (h), T1/2β = 6.1 (h) & CL = 19.5 (L/ h) [a two compartment model]. The AUC was linear for the 10 and 30 mg/kg doses. HOOI did not generate any detectable plasma chloroacetaldehyde vs. IFOS (2.12 µg/mL from 400 mg/kg) and 25% of the acrolein generated by the MTD of CPA & IFOS. No convulsions, neuropathies or renal dysfunction were observed in either specie. Unlike IFOS/IPM, HOOI is lipophilic, activated intracellular (with < extracellular acrolein and no chloroacetaldehyde released); no IFOS/IPM-associated CNS or GU toxicity was noted. HOOI may increase the safety and efficacy margins of this class of alkylators in advanced CPA- and IFOS-resistant cancers and broaden the target-range (CNS-gliomas). The L-lysine salt stability supports developing HOOI for clinical trials. Supported by grant R44 CA094566 from the NCI/SBIR program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2011-3222

Antitumor activity in mice was observed for the oxime of the previously reported ethyl [6-amino-4-[(1-methyl-2-phenyl-2-oxoethyl)amino]-5-nitropyridin -2-yl] carbamate (8) and several related compounds. These compounds are precursors of the active ethyl pyrido[3,4-b]pyrazin-7-ylcarbamates (e.g., 4), which are potent antimitotic agents. In the 5-nitropyridine series overall biological activity was reduced by replacement of the oxime moiety with a keto or alcohol group and by replacement of the 1-methyl group of the side chain with hydrogen. Reduction of the nitro group of the 5-nitropyridines containing an alcohol in the side chain to the corresponding 5-aminopyridines increased biological activity. Preliminary studies showed that the 5-nitropyridine oximes were considerably less potent than the pyridopyrazines as antimitotic agents and that the former are apparently not converted to the latter in vivo. The inhibition of the incorporation of pyrimidine nucleosides into DNA and RNA was identified as another possible mode of action of the 5-nitropyridine oximes.

Since 1978, over 50 clinically useful antitumor drugs or new candidate antitumor agents have been evaluated in vivo against cisplatin-resistant P388 leukemia (P388/DDPt) in our laboratories. Analysis of this data base has yielded insights into the cross-resistance, collateral sensitivity, and mechanisms of resistance of P388/DDPt. P388/DDPt was cross-resistant or marginally cross-resistant to eight agents [carmethizole.HCl, rhizoxin, dibromodulcitol, spirohydantoin mustard, hepsulfam, arabinosyl-5-azacytosine (ara-AC), tiazofurin, and deoxyspergualin]. Of these eight agents, the latter six have entered various phases of clinical trials. For these trials, it may be important to exclude or to monitor with extra care patients who have previously been treated with cisplatin. P388/DDPt was collaterally sensitive to six agents [fludarabine phosphate (2-F-ara-AMP), amsacrine (AMSA), mitoxantrone, etoposide (VP-16), batracylin, and flavone acetic acid] and, possibly, to two others (merbarone and echinomycin). These observations of collateral sensitivity suggest that a combination of cisplatin plus any one of these drugs might exhibit therapeutic synergism. Therapeutic synergism has been observed in animal models for combinations of cisplatin plus VP-16, AMSA, or mitoxantrone. The observation of collateral sensitivity for P388/DDPt to four agents (AMSA, mitoxantrone, merbarone, and VP-16) that have been reported to interact with DNA topoisomerase II suggests the possible involvement of the latter in cisplatin resistance. Both the increased sensitivity of P388/DDPt to these agents and a portion of its resistance to cisplatin could be the result of an increase in DNA topoisomerase II activity.

Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increased CAR RNA levels and was associated with a 5-10 fold increase in adenovirus transgene expression following adenovirus infection of FK228-treated cells. Since treatment of cultured cells with 1 ng/ml FK228 caused increased acetylated histone H3, the mechanism for the FK228 effect in vitro may be through protein acetylation. This potential mechanism was also suggested because treatment with the histone deacetylase inhibitors sodium butyrate or trichostatin A also caused increased levels of CAR. Increases in CAR levels, transgene expression, and histone H3 acetylation were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment in vivo was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI human melanoma xenografts. Treatment efficacy was determined by analyzing CAR RNA levels by semi- quantitative RT-PCR analysis or by analyzing CAR protein by western blot analysis. A maximum human CAR increase of 13.6- fold (|[plusmn]|4.3) compared to untreated xenografts was observed 24 h following i.v. injection of mice with 3.6 mg/kg FK228. By comparison, murine CAR RNA levels in livers, kidneys and lungs from the same animals remained unchanged. Xenograft protein from mice treated with 3.6 mg/kg FK228 and analyzed at 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR levels. Based on these results xenograft-bearing mice treated with 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h later. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228-treated mice compared to untreated controls. In order to determine the mechanism for this effect, the levels of histone H3 acetylation in treated compared to control xenografts were analyzed. Western blot analysis of xenografts from mice showed that 6 h following treatment with 3.6 mg/kg FK228, there was an average 28.2-fold (|[plusmn]|22.9) increase in histone H3 acetylation that remained at 20.4-fold (|[plusmn]|12.6) 24 h following treatment. These studies suggest that the mechanism by which FK228 increases the efficiency of adenovirus gene therapy in vivo is through protein acetylation.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD+-dependent dehydrogenase (type D) and as the O2-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD+ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The Km for xanthine is not significantly different for the two forms.

A new purification procedure for bovine milk xanthine oxidase is reported. The enzyme so obtained is of the highest purity and shows little evidence of degradation. The enzyme displays a single protein band on either polyacrylamide gels or on sodium dodecyl sulfate-urea polyacrylamide gels. Sedimentation equilibrium studies indicate a native molecular weight of 303,000 and a subunit molecular weight of approximately 150,000. The latter value is in good agreement with the minimum molecular weight of 157,000 calculated from dry weight determination and flavin analysis. In contrast, purification of xanthine oxidase from pancreatin-treated cream yields a protein which displays two subunits corresponding to molecular weights of 92,000 and 39,000 as determined by dodecyl sulfate-urea polyacrylamide gel electrophoresis. Pancreatinized enzyme has a greater mobility than unproteolyzed enzyme on polyacrylamide gels. Exposure of milk xanthine oxidase to pancreatin before isolation or after purification yields the same result.

Rat liver xanthine dehydrogenase, type D, has been isolated directly from crude extracts as an antibody complex and its properties compared with those of two oxidase forms of the enzyme, heat-treated type O and trypsin-treated type O, also isolated as antibody complexes. The type D antibody complex displays electron acceptor specificities and electron paramagnetic resonance properties characteristic of an NAD+-dependent dehydrogenase whereas the trypsin-treated type O complex behaves as an O2-utilizing oxidase. The heat-treated type O complex displays intermediate behavior. After electrophoresis in dodecyl sulfate-urea-acrylamide gels, type D and heated type O enzymes show single polypeptide bands, each of approximately 150,000 molecular weight. The trypsinized type O also shows one major band but with an approximate molecular weight of 130,000. Purified type D enzyme, when proteolytically treated, is converted to an oxidase with increased mobility on polyacrylamide gels. The 150,000 molecular weight subunit is cleaved into smaller subunits during proteolysis. Treatment with 5,5′-dithiobis-(2-nitrobenzoic acid) converts the type D enzyme, whether isolated as the purified enzyme or as the immune precipitate, to type O enzyme in a time-dependent manner. Titration of type D and the two type O antibody complexes with 5,5′-dithiobis-(2-nitrobenzoic acid) reveals that type D and heated type O each has approximately 28 reactive sulfhydryls, whereas the trypsinized type O has only 8 such groups. Many of the free sulfhydryls are vicinal and form disulfide bonds during the conversion to an oxidase by this reagent. Unproteolyzed preparations of type O rat liver enzyme and milk xanthine oxidase are converted to type D enzymes by treatment with dithiothreitol. The converted enzymes display electron acceptor specificities and epr properties characteristic of an NAD+-dependent dehydrogenase molecule.

Combined deficiency of sulfite oxidase and xanthine oxidase as a result of defective synthesis of molybdenum-cofactor. A girl is presented with congenital abnormalities including asymmetry of the skull and slight mediofacial dysplasia. The eye lenses were dislocated. Severe neurological abnormalities and deep mental retardation were observed. On routine screening a low serum urate (0.01-0.07 mmol/1) was observed. Chromatography of urinary purines revealed xanthinuria. In addition the sulfite test in the urine was repeatedly positive. Sulfite oxidase deficiency was suggested by high excretions of S-sulfocysteine, taurine and thiosulfate and low levels of urinary sulfate. Deficiencies of both xanthine oxidase and sulfite oxidase were demonstrated in a liver biopsy specimen. Because sulfite oxidase and xanthine oxidase are known to require an activated form of molybdenum (Mo-cofactor), investigations of Mo metabolism were carried out. A complete absence of hepatic Mo-cofactor activity was demonstrated and analysis by atomic absorption revealed a severe depletion of hepatic Mo as well. A near normal amount of inactive xanthine oxidase protein was present, but several immunological techniques failed to detect any sulfite oxidase apo-enzyme. It is suggested that a defective synthesis of Mo-cofactor causes the biochemical abnormalities. Treatment with oral supplements of molybdenum did not result in biochemical or clinical improvement.