Phase I dose-escalating trial of Escherichia coli purine nucleoside phosphorylase and fludarabine gene therapy for advanced solid tumors.

BACKGROUND: The use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation. PATIENTS AND METHODS: A first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m(2)) and escalating virus in the fourth (10(11)-10(12) viral particles, VP). RESULTS: All 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy. CONCLUSIONS: This first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted. CLINICALTRIALSGOV IDENTIFIER: NCT01310179.

Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

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-ß-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.

Synthesis and biological activity of 2-fluoro adenine and 6-methyl purine nucleoside analogs as prodrugs for suicide gene therapy of cancer.

A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5-position have been synthesised These compounds bear a 5'-heterocycle such as triazole or a imidazole with a two carbon chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl purine nucleosides, their corresponding alpha-linker nucleosides with L-xylose and L-lyxose were also synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP.

Gene therapy of cancer: activation of nucleoside prodrugs with E. coli purine nucleoside phosphorylase.

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.

Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells.

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

Polyamine pools in HIV-infected cells.

The polyamines putrescine, spermine, and spermidine, present in all living cells, have been implicated in the replication of some herpesviruses and retroviruses, and elevated levels of these polyamines have been found in the lymphocytes of patients infected with HIV-1. We have examined the effect of HIV-1 infection on polyamine pools in cell culture. HIV-1 did not significantly affect the polyamine pools in CEM cells. Consistent with this observation, inhibitors of the two key enzymes of this pathway, ornithine decarboxylase and S-adenosylmethionine decarboxylase, did not prevent viral-induced cytopathic effects (CPE) in this cell line. Our results indicate that inhibitors of this pathway will not be therapeutically useful in the treatment of AIDS.

Cell to cell contact is not required for bystander cell killing by Escherichia coli purine nucleoside phosphorylase.

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

Toxicity of cordycepin in combination with the adenosine deaminase inhibitor 2'-deoxycoformycin in beagle dogs.

For 3 consecutive days, the nucleoside cordycepin (3'-deoxyadenosine) was administered as 1-hr iv infusions (0, 1, 4, 8, 10, or 20 mg/kg/day) to dogs. These doses were given 1 hr after a bolus iv injection (0.25 mg/kg/day) of 2'-deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase. The hypothesis was that dCF would affect the toxicity of cordycepin. Plasma adenosine deaminase activity was strongly inhibited during the dose period and for 5 days following the final dose of dCF. Dogs given cordycepin alone showed no drug-related toxicities. In dogs given only dCF, drug-related toxicity to lymphoid tissue (lymphopenia and thymus lymphoid depletion), thrombocytopenia, and decreases in food consumption were observed. Cordycepin in combination with dCF produced symptoms associated with severe gastrointestinal toxicity (decreased body weights, emesis, diarrhea, decreased food consumption, and necrosis of the gastrointestinal tract) and bone marrow toxicity (lymphopenia, thrombocytopenia, and depletion of hematopoietic cells). The gastrointestinal tract and bone marrow were sites associated with dose-limiting toxicities. In surviving dogs, most of the effects were reversible by Day 30. The maximum tolerated dose of cordycepin administered in combination with dCF was 8 mg/kg/day (160 mg/m2/day) given daily for 3 days.

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.

Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines.

Inefficiency of gene delivery, together with inadequate bystander killing, represent two major hurdles in the development of a toxin-mediated gene therapy for human malignancy. The product of the Escherischia coli DeoD gene (purine nucleoside phosphorylase, PNP) differs from the mammalian enzyme in its substrate specificity and is capable of catalyzing the conversion of several non-toxic deoxyadenosine analogs to highly toxic adenine analogs. We have found that expression of E. coli PNP in < 1% of a human colonic carcinoma cell line leads to the death of virtually all bystander cells after treatment with 6-methyl-purine-2'-deoxyribonucleoside, a deoxyadenosine analog that is a substrate for E. coli PNP but not human PNP. Minimal toxicity was observed in non-transfected or E. coli LacZ transfected cells that were treated with this compound. These results establish a rational approach to achieve significant bystander killing, even after gene transfer to only a small fraction of tumor cells.

Effect of 9-benzyl-9-deazaguanine, a potent inhibitor of purine nucleoside phosphorylase, on the cytotoxicity and metabolism of 6-thio-2'-deoxyguanosine.

6-Thio-2'-deoxyguanosine (T-dGuo) has been reported to be both phosphorylated by deoxycytidine kinase and converted to 6-thioguanine by purine nucleoside phosphorylase (PNP). Combination of T-dGuo with an inhibitor of PNP would be expected to generate the 5'-triphosphate of T-dGuo and limit or prevent the formation of 6-thioguanosine triphosphate. Because the incorporation of 6-thioguanine into DNA is believed to be primarily responsible for the antitumor activity of the thiopurines, this treatment might result in enhanced activity against certain tumors, particularly those of T-cell origin. We have evaluated the metabolic basis of this strategy by examining the effects of 9-benzyl-9-deazaguanine (BDG), a potent inhibitor of PNP, on the metabolism of T-dGuo in CEM cells. The concentration of T-dGuo required to inhibit cell growth by 50% was approximately 50-fold greater in the presence of 8.0 microM BDG than in its absence. As expected, the addition of BDG to cells treated with T-dGuo prevented the metabolism of T-dGuo to 6-thio-guanine-containing ribo-nucleotides, but, unexpectedly, no 6-thio-2'-deoxyguanosine 5'-triphosphate was detected. In cells treated with T-dGuo plus BDG, the major phosphorylated metabolite was T-dGMP. These results indicated that even in the absence of PNP activity, T-dGuo cannot be phosphorylated directly to 6-thio-2'-deoxyguanosine 5'-triphosphate due to the inability of guanylate kinase to utilize T-dGMP as a substrate.

Purine nucleoside phosphorylase inhibitors: biochemical and pharmacological studies with 9-benzyl-9-deazaguanine and related compounds.

Certain derivatives of 9-deazaguanine that contain arylmethyl, heteroarylmethyl or cycloalkylmethyl groups at the 9-position are potent inhibitors of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). To determine whether these agents can produce metabolically significant inhibition of PNP in cells and in animals, the authors performed pharmacological studies with a representative member of the series, 9-benzyl-9-deazaguanine (BzDAG). BzDAG was a potent inhibitor of PNP from calf spleen (Ki = 12 nM). It was also an effective inhibitor of PNP in cells and in animals as shown by the findings that it 1) inhibited the conversion of inosine to nucleotides in L1210 cells in culture at concentrations that had little effect on the utilization of hypoxanthine; 2) potentiated the toxicity of deoxyguanosine to CCRF-CEM cells in culture; 3) increased the pools of deoxy GTP in CCRF-CEM, Molt-3 and Molt-4 cells that had been treated with deoxyguanosine; 4) prevented the toxicity of 6-thioguanosine to HEp-2 cells in culture; 5) increased the plasma levels of endogenous inosine in rats; and 6) increased the plasma levels of 2',3'-dideoxyinosine in rats that had received BzDAG and dideoxyinosine in combination. Pharmacokinetic analysis of BzDAG in the rat showed it to be 48% orally bioavailable (at a dose of 5 mg/kg). About 95% of BzDAG was protein bound. After i.v. administration of BzDAG (5 mg/kg), more than 50% of the erythrocyte PNP was inhibited for 40 min. These results indicate that the 9-substituted-9-deazaguanines are potent orally active PNP inhibitors and are therefore of potential clinical interest as immunosuppressive and anti-inflammatory agents.

Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds. Role of adenylate deaminase.

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis and biochemical properties of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine.

The synthesis and characterization of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine (3a) are presented. This compound is a substrate for adenosine deaminase and adenosine kinase. In L1210 cells 3a is converted to 8-aminoinosine monophosphate (4b), apparently by the action of AMP deaminase on the monophosphate of 3a, as well as to the triphosphate derivative of 3a. Pentostatin was used to inhibit adenosine deaminase, and coformycin was used to inhibit AMP deaminase in experiments designed to delineate the metabolic fate of 3a. Pentostatin was without influence on the cytotoxicity of 3a, but coformycin potentiated the cytotoxicity. The potentiation was associated with an increased cellular concentration of phosphates of 3a and a decreased concentration of 4b.

Differences in the metabolism and metabolic effects of the carbocyclic adenosine analogs, neplanocin A and aristeromycin.

Neplanocin A and aristeromycin are carbocyclic adenosine analogs that differ only in that neplanocin A contains a double bond in the carbocyclic ring, whereas this ring in aristeromycin is saturated. We have compared the metabolism and some of the metabolic effects of neplanocin A and synthetic (+/-)-aristeromycin (C-Ado) in murine leukemia L1210 cells in culture. C-Ado, as shown earlier, was not only converted to its own phosphates but also was metabolized to phosphates of carbocyclic guanosine. Both rapidly proliferating and slowly proliferating or resting cells phosphorylated C-Ado, but C-Ado was not converted to phosphates of carbocyclic guanosine in detectable amounts in cells whose growth had reached a plateau. When the metabolism of neplanocin and C-Ado was examined in the same experiment, both analogs were converted to the triphosphate analogs of ATP; no conversion of neplanocin A to the corresponding carbocyclic analogs of guanine nucleotides was detected, whereas C-Ado was converted to the carbocyclic analog of GTP in amounts that approximated the GTP pool. This difference in metabolism was associated with a marked difference in effects of the two analogs on the utilization of hypoxanthine and guanine which was inhibited by C-Ado but not by neplanocin. The failure of neplanocin A to be converted to analogs of guanine nucleotides apparently is the result of poor capacity of its monophosphate to serve as a substrate for AMP deaminase; the Vmax for deamination of neplanocin-5'-monophosphate by this enzyme was only 5% of that for C-Ado monophosphate. In contrast, neplanocin A was a better substrate than C-Ado for adenosine deaminase.

Inhibition of utilization of hypoxanthine and guanine in cells treated with the carbocyclic analog of adenosine. Phosphates of carbocyclic nucleoside analogs as inhibitors of hypoxanthine (guanine) phosphoribosyltransferase.

In cell cultures treated with the carbocyclic analog of adenosine (C-Ado, (+/-)-aristeromycin), the utilization of hypoxanthine and guanine has been observed to be blocked. In an attempt to define the mechanism of this inhibition, we have reexamined the metabolism of C-Ado and its effects on the metabolism of guanine and hypoxanthine. In cultures of L1210 cells, C-Ado at a concentration of 25 microM inhibited the utilization of hypoxanthine and guanine for nucleotide synthesis by more than 90% but produced little or no inhibition of the utilization of these bases in cultures of L1210/MeMPR cells which lack adenosine kinase and cannot phosphorylate C-Ado. In cultures of mammalian cells (L1210, HEp-2, and colon-26 cells), C-Ado was converted to the triphosphate (as previously observed) and also to the triphosphate of the carbocyclic analog of guanosine. The presence of coformycin in the medium at a concentration sufficient to inhibit AMP deaminase almost completely prevented the formation of carbocyclic GTP; thus, the deamination of C-Ado monophosphate is essential for the formation of phosphates of carbocyclic guanosine. Since hypoxanthine (guanine) phosphoribosyltransferase is known to be subject to end product inhibition, it was considered likely that phosphates of carbocyclic guanosine or carbocyclic inosine, present in C-Ado-treated cells, were responsible for inhibition of utilization of hypoxanthine and guanine. The 5'-phosphates of the carbocyclic analogs of inosine and guanosine were synthesized and found to be effective inhibitors of the phosphoribosyltransferase. Carbocyclic GMP was a better inhibitor than carbocyclic IMP and was also superior to GMP and IMP; the concentration of C-GMP that produced a 50% inhibition of GMP formation was approximately 1 microM. It is probable that the presence of phosphates of carbocyclic guanosine accounts for the inhibition of utilization of hypoxanthine and guanine in C-Ado-treated cells.