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P P Saunders

Publications and source records attributed to P P Saunders.

At least 19 recordsLinked to original sources
PubMed1996

Purification and properties of a human nicotinamide ribonucleoside kinase.

Nicotinamide ribonucleoside kinase (NRK) phosphorylates at least two nucleoside analogs of potential clinical interest, tiazofurin and 3-deazaguanosine. In this study NRK has been purified to near homogeneity from human placenta. The purification procedure consists of several chromatographic steps including salt precipitation, DE-52 chromatography, sucrose density gradient fractionation, hydroxylapatite chromatography, and anion exchange FPLC. The final enzyme preparation is homogeneous as judged by a single silver-stainable band on both nondenaturing and denaturing polyacrylamide gels. The molecular weight of the enzyme, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75 HR 10/30, is approximately 29 and 32 kDa, respectively. The isoelectric pH for NRK is 5.6. The reaction requires ATP. The pH optimum is in the region 6.5-9.0. NRK in the purified preparations, with added bovine serum albumin, was stable for days at 4 degrees C and for months at -70 degrees C. The enzyme is very unstable at low protein concentration. NRK phosphorylated several substrates including nicotinamide ribonucleoside, guanosine, tiazofurin, and 3-deazaguanosine with apparent Km values of 9.6, 115, 90, and 16.5 microM, respectively.

Chromatography, DEAE-Cellulose↗
PubMed1996

Metabolism and action of benzamide riboside in Chinese hamster ovary cells.

Benzamide riboside (3-(1-deoxy-beta-D-ribofuranosyl)benzamide, BR) a new analog of nicotinamide riboside, is toxic to Chinese hamster ovary cells and inhibits guanine nucleotide synthesis in a manner comparable to that of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). Adenosine kinase deficient cells demonstrate slight resistance but retain the ability to form the NAD analog, benzamide adenine dinucleotide (BAD). HPLC analysis of BAD containing cells is described. A BR resistant cell line was isolated that demonstrates cross-resistance to both tiazofurin and 6-aminonicotinamide, suggesting a common metabolic step; enzymatic analysis indicates reduced levels of NAD pyrophosphorylase in these cells. BR toxicity was only partially reversed or prevented by the presence of guanosine, suggesting either that BR inhibits guanine salvage to some extent or, more probably, that BR can, at high concentration, inhibit cell growth by another mechanism in addition to inhibition of guanine nucleotide synthesis. Cells incubated with BR for several hours retain the ability to salvage exogenously provided guanosine. The demonstration that BAD can be phosphorylated by NAD kinase, presumably to form BADP, suggests that this metabolite may be formed in cells and may have inhibitory activity at high concentrations of BR.

Animals↗
PubMed1994

Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2'-deoxy-5-fluorouridine 5'-monophosphate (FdUMP): a strategy to introduce nucleotides into cells.

The bis[(pivaloyloxy)methyl] [PIV2] derivative of 2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP) was synthesized as a potential membrane-permeable prodrug of FdUMP. The compound was designed to enter cells by passive diffusion and to revert to FdUMP after removal of the PIV groups by hydrolytic enzymes. The most convenient preparation of PIV2FdUMP was by condensation of 2'-deoxy-5-fluorouridine (FUdR) with PIV2 phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV2FdUMP was stable in the pH range 1.0-4.0 (t1/2 > 100 h). It was also fairly stable at pH 7.4 (t1/2 = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t1/2 < 2 min). In the presence of hog liver carboxylate esterases, PIV2FdUMP was converted quantitatively to the mono-[(pivaloyloxy)methyl] [PIV1] analogue PIV1FdUMP. After a 24 h incubation, only trace amounts of FdUMP (1-3%) were observed, indicating that PIV1FdUMP is a poor substrate for carboxylate esterases. In mouse plasma, PIV2FdUMP was rapidly metabolized, first to PIV1FdUMP and then to FdUMP. With continued incubation, FUdR was formed, presumably due to further catabolism of FdUMP by plasma phosphatases or 5'-nucleotidases. Since PIV1FdUMP is a poor substrate for carboxylate esterase, the cleavage of the second PIV group is most likely mediated by plasma phosphodiesterases. The rate of degradation of PIV2FdUMP in the presence of acid and alkaline phosphatase, 5'-nucleotidase, or spleen phosphodiesterase was the same as that in buffer controls, indicating that the compound is not a substrate for these nucleotide catabolizing enzymes. The concentration of PIV2FdUMP and its 3'-O-acetyl ester (PIV2 3'-O-Ac-FdUMP) required to inhibit the growth of Chinese hamster ovary (CHO) cells in vitro to less than 50 cells per colony was 5 x 10(-6) M, the same as that required for 5-fluorouracil (FU). Both nucleotide prodrugs showed the same growth-inhibitory potency against a mutant CHO cell line that was 20-fold resistant to FU (CHO/FU). Administered intraperitoneally at optimal dosage for 5 consecutive days, PIV2FdUMP and PIV2 3'-O-Ac-FdUMP were as effective as FU at prolonging the life spans of mice bearing intraperitoneally implanted P388 leukemia. Both prodrugs retained full therapeutic activity against a P388 subline resistant to FU. Collectively, these data indicate that PIV2FdUMP and PIV2 3'-O-Ac-FdUMP are effective membrane-permeable prodrugs of FdUMP.

Animals↗
PubMed1992

Determination of nicotinamide-adenine dinucleotide and thiazole-4-carboxamide-adenine dinucleotide in human leukocytes by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for cellular nicotinamide-adenine dinucleotide and thiazole-4-carboxamide-adenine dinucleotide is presented that is appropriate for analysis of these dinucleotides in extracts of Ficoll-purified human leukemic cells. The separation, which is effected by reversed-phase chromatography, is highly reproducible and the limit of quantitation is as low as 10-15 pmol. The stability of these compounds in neutralized perchloric acid extracts is addressed and the applicability of the procedure to clinical specimens is demonstrated.

Adenine Nucleotides↗
PubMed1992

Enhanced killing of Acanthamoeba cysts in vitro using dimethylsulfoxide.

PURPOSE: Acanthamoeba keratitis is difficult to treat and requires prolonged therapy despite the well-documented in vitro effectiveness of a variety of drugs. The authors propose that this may be due to the cysts formed by the organism in response to hostile conditions. Consequently, the study concentrates on increasing penetration of drugs effective against the parasite into the cysts using dimethylsulfoxide (DMSO). METHODS: The organism is forced to encyst in vitro on solid media by nutrient deprivation. In the first set of experiments, serial dilutions of a standard treatment regimen are applied to the organisms, and these treated cysts are then subcultured onto nutrient-rich material and observed for growth. The experiments are then repeated with DMSO added to the serially diluted standards. In a second set of experiments, the effects of retreatment on a larger concentration of organisms is examined. RESULTS: When applied to a cyst-only population of Acanthamoeba, none of three standard drugs, propamidine isethionate 0.1%, neomycin 1%, or miconazole 1%, was cysticidal. When combined with DMSO 30%, propamidine isethionate was clearly cysticidal even in low dilution. This was confirmed by the retreatment experiments using a larger, standardized cyst population. CONCLUSION: The authors propose that DMSO is acting as a "carrier" for the propamidine isethionate and increases its penetration into the normally drug-resistant cyst form of the organism. Because DMSO has been used topically in the past and shown to be quite safe, this may be a viable new therapy for this difficult condition.

Acanthamoeba↗
PubMed1992

Bilateral malignant glaucoma.

Two cases of bilateral malignant glaucoma are presented. In one case the condition developed sequentially in the two eyes; pars plana vitrectomy was eventually needed in the operated eye, whereas the condition responded to medical treatment in the fellow eye. In the second case the two eyes were involved simultaneously nearly 1 year after surgery, and the glaucoma responded to medical treatment.

Female↗
PubMed1991

Metabolism and action of purine nucleoside analogs.

Recent investigations have identified many new purine nucleoside analogs that act as antimetabolites. This article focuses on the metabolism and mechanisms of action of tiazofurin, 3-deazaguanosine, neplanocin A, arabinosyladenine in combination with inhibitors of adenosine deaminase, arabinosyl-2-fluoroadenine, and 2-chloro-2'-deoxyadenosine, drugs that are either currently being evaluated in clinical trials or are close to that stage. The diverse metabolic requirements for activation, unique mechanisms of action, and differential biological activities of these compounds are characterized and evaluated for prospective therapeutic application.

2-Chloroadenosine↗
PubMed1990

Tiazofurin is phosphorylated by three enzymes from Chinese hamster ovary cells.

The growth inhibitory activity of tiazofurin toward adenosine kinase deficient Chinese hamster ovary (CHO) cells was partially reversed by the presence of nicotinamide riboside. Similarly, the formation of tiazofurin 5'-monophosphate and the active metabolite, tiazofurin 5'-adenine dinucleotide could be partially inhibited by 100 microM nicotinamide riboside in CHO cells and substantially inhibited (80-90%) in adenosine kinase deficient cells. Tiazofurin phosphorylating activity from CHO cell extracts was resolved into two peaks by DEAE-cellulose chromatography. The first peak of activity was identified as adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). The second peak of activity correlated with a previously described 3-deazaguanosine phosphorylating activity that was identified as a nicotinamide ribonucleoside kinase. Contaminating purine nucleoside phosphorylase was removed by sedimentation through a sucrose density gradient which also resolved the tiazofurin phosphorylating activity into two peaks, one requiring just ATP and the other requiring both ATP and IMP. Of the substrates tested with the lower density peak, nicotinamide riboside was most efficient and was the only natural substance that competed well with tiazofurin for phosphorylation, substantiating its suggested identity as a nicotinamide ribonucleoside kinase. The apparent Km value for nicotinamide riboside (2 microM) was significantly less than that for tiazofurin (13.6 microM). ATP was the best phosphate donor; CTP and UTP were utilized less efficiently and IMP did not support the reaction. The best substrate for the higher density peak of tiazofurin phosphorylation was inosine and both ATP and IMP were required for the reaction, suggesting its identity as a 5'-nucleotidase. In summary, it appears that adenosine kinase, nicotinamide ribonucleoside kinase, and 5'-nucleotidase may all contribute to the phosphorylation of tiazofurin in CHO cells.

Adenosine Kinase↗
PubMed1990

Nonpigmented congenital iris stromal cyst.

A nonpigmented iris stromal cyst was observed in the right eye of a 6-week-old girl. There was no past history of trauma or use of topical miotics, and the remainder of the ocular examination was normal. The pathophysiology and management of this rare developmental anomaly are controversial.

Cysts↗
PubMed1989

Phosphorylation of 3-deazaguanosine by nicotinamide riboside kinase in Chinese hamster ovary cells.

The growth inhibitory activity of 3-deazaguanosine toward a mutant line (TGR-3) of Chinese hamster ovary cells deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was substantially reversed by the simultaneous addition of nicotinamide riboside. The activities of most other ribonucleoside analogues tested were unaffected. The formation of cellular 3-deazaGMP and 3-deazaGTP from the ribonucleoside analogue, as measured by high-pressure liquid chromatography, was inhibited by the presence of nicotinamide riboside. The inhibition was dependent on concentration of 3-deazaguanosine and could also be demonstrated by following the metabolism of 3-deazaguanosine, labeled with 14C in the ribose moiety, to [14C]3-deazaGTP. In the presence of 100 microM nicotinamide riboside formation of the labeled triphosphate derivative of 3-deazaguanosine was undetectable. A 3-deazaguanosine phosphorylating activity was separated from other cellular kinases by DEAE-cellulose chromatography. Contaminating purine nucleoside phosphorylase (EC 2.4.2.1) was subsequently removed by sucrose density gradient centrifugation. The resulting enzyme preparation demonstrated the greatest activities with nicotinamide riboside and 3-deazaguanosine and, in addition, could also phosphorylate tiazofurin and guanosine to lesser, but significant, degrees. These and other observations suggest that 3-deazaguanosine, and perhaps other agents such as tiazofurin, may, at least in part, be phosphorylated by a nicotinamide ribonucleoside kinase in these cells. If so, it is possible that the activity of this agent in other types of cells in vivo could be dependent upon the presence of this enzyme and that it could be influenced by cellular concentrations of the natural pyridine nucleoside.

Animals↗
PubMed1987

Use of tiazofurin to enhance the metabolism and cytotoxic activities of analogues of guanine, guanosine, and deoxyguanosine.

An effective modulator of cellular guanine nucleotide pools, 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) was tested for its ability to affect utilization of certain guanine, guanosine, and deoxyguanosine analogues by Chinese hamster ovary cells and hypoxanthine guanine phosphoribosyltransferase (HGPRTase)-deficient variants. The nucleoside analogues investigated were chosen for their potential to be metabolized to the nucleotide level by pathways other than those requiring the action of HGPRTase. Exposure of tiazofurin-treated (500 microM for 3 h) cells to 3-deazaguanosine (200 microM for 3 h) resulted in enhanced 3-deazaGTP formation and an increase (5-10-fold) in the ratio 3-deazaGTP/GTP. Tiazofurin treatment also stimulated [3H]deoxyguanosine utilization (8-fold) by HGPRTase-deficient cells, and accordingly, greatly increased the cytotoxicity of 2'-deoxy-3-deazaguanosine and arabinosylguanine. This study emphasizes the potential usefulness of tiazofurin in sequential combination with appropriate analogues of guanosine and deoxyguanosine in a clinical setting and as a tool in studying the metabolism of these agents.

Animals↗
PubMed1986

3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.

Alkaline Phosphatase↗
PubMed1986

Mechanisms of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (Dacarbazine) cytotoxicity toward Chinese hamster ovary cells in vitro are dictated by incubation conditions.

Decomposition of the antitumor agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC, Dacarbazine) produces several potentially toxic compounds, the concentration of which depend on incubation parameters such as pH, temperature and illumination. The action of DTIC on chinese hamster ovary (CHO) cell clone formation in the dark (7-8-day incubation) reflects the slow formation of 2-azahypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8)-deficient cells are resistant to DTIC under these conditions, reflecting their inability to utilize 2-azahypoxanthine. The toxicity of DTIC in conventional survival experiments (1-2-h exposure to drug) is dependent upon illumination and is highly influenced by the pH of the medium. Toxicity of DTIC in these experiments appears to reflect rapid accumulation of the immediate photodecomposition product of the drug, 4-diazoimidazole-5-carboxamide (DZC), since HGPRT-deficient cells are not resistant to DTIC under these conditions. The biologically initiated pathway of DTIC action (enzymatic hydroxylation) has little, if any, role in the action of this agent toward cultured CHO cells.

Animals↗
PubMed1985

Metabolism and action of neplanocin A in Chinese hamster ovary cells.

Neplanocin A is a naturally occurring carbocyclic analog of adenosine which contains a cyclopentene moiety in place of ribose and has demonstrated antitumor and antimicrobial activity. This compound was highly toxic to Chinese hamster ovary (CHO) cells; the approximate minimum inhibitory concentration of neplanocin A for inhibition of clone formation was 0.1 microM. The toxicity of the agent was greatly reduced by prior treatment with adenosine deaminase. [3H]Uridine incorporation into perchloric acid insoluble material in growing cells was inhibited by neplanocin A more dramatically than that of [3H]thymidine or [3H]leucine. Treatment with the drug resulted in a marked depression of ATP pool levels. High pressure liquid chromatographic analysis of cellular nucleotide pools from cells treated with neplanocin A revealed the formation of an apparent drug metabolite (NpcTP) that eluted in the triphosphate region of the chromatographic profile. Treatment of NpcTP with alkaline phosphatase produced a nucleoside with properties similar to neplanocin A. An adenosine-kinase-deficient cell line formed little, if any, NpcTP but demonstrated only slight resistance to the agent. These observations suggest that neplanocin A was efficiently metabolized to the triphosphate level but that this metabolite was responsible for only a fraction of the observed toxicity.

Adenosine↗
PubMed1984

Synthesis and biological evaluation of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine).

The synthesis of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine, 2) has been accomplished from 3-(ethoxycarbonyl)pyrrole-2-acetonitrile. In contrast to 3-deazaguanine, compound 2 did not show any antitumor, antiviral, or antibacterial properties. Furthermore, it was not a substrate for hypoxanthine-guanine phosphoribosyltransferase or purine nucleoside phosphorylase.

Animals↗
PubMed1983

Nucleoside kinase activities of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells and appropriate drug-resistant mutants derived from them have been analyzed for nucleoside kinase activities relevant to the phosphorylation of adenosine, deoxyadenosine, deoxyguanosine and deoxycytidine and for resistance to a variety of nucleoside analogs. Fractionation of extracts by DEAE-cellulose chromatography revealed three major peaks of activity. Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20), the first to elute from the column is responsible for the majority of the deoxyadenosine phosphorylation in cell extracts and, according to resistance data, appears to phosphorylate most adenosine analogs tested, including 9-beta-D-arabinosyladenine (ara-A). A deoxyguanosine kinase, the second enzyme to elute from the column, was responsible for the majority of deoxyguanosine and deoxyinosine phosphorylation in cell extracts. The function of this enzyme in cell metabolism is unclear. 2-Chlorodeoxyadenosine, on the other hand, appeared from resistance data to be phosphorylated, at least in part, by deoxycytidine kinase (ATP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74), which in cell extracts could also phosphorylate deoxyguanosine and deoxyadenosine, though much less efficiently than deoxycytidine.

Animals↗
PubMed1983

Action of 2-beta-d-Ribofuranosylthiazole-4-carboxamide (tiazofurin) in Chinese hamster ovary and variant cell lines.

The metabolism and inhibitory activity of 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin, RTC, NSC 286193) was studied in Chinese hamster ovary (CHO) cells and a series of mutants derived from them. Isotope incorporation experiments indicate dramatic inhibition of DNA and RNA synthesis by tiazofurin but little or no effect on protein synthesis. In CHO cells, tiazofurin is more toxic than ribavirin and also demonstrates a stronger inhibition of guanine nucleotide synthesis. Tiazofurin is phosphorylated by crude extracts of CHO cells. Variant cell lines lacking adenosine kinase (EC 2.7.1.20) or deoxycytidine kinase (EC 2.7.1.74) show no resistance to tiazofurin, suggesting that these enzymes are not responsible for the phosphorylation of tiazofurin. The inhibitory activity of this agent can be reversed by exogenously supplied guanine, suggesting that the inhibition is due, at least in part, to guanylate deficiency.

Animals↗