Karl Y Hostetler - Academia.edu (original) (raw)
Papers by Karl Y Hostetler
Antiviral Research, 1997
The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy... more The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy would be useful. We recently synthesized 1-O-octadecyl-sn-glycero-3-phosphonoformic acid (ODG-PFA) and noted a 93-fold increase in its anti-HCMV activity relative to PFA. In addition, the antiviral activity of ODG-PFA in herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1) infected cells was increased 40-fold relative to PFA (Hostetler et al., 1996. Antiviral Res. 31, 59). To evaluate structure-activity relationships further, we synthesized alkoxypropyl esters of foscarnet with varying alkyl chain lengths and degrees of saturation. These compounds were tested in vitro for activity and selectivity in comparison with PFA and ODG-PFA in cells infected with HCMV, HSV-1 or HIV-1. Antiviral activity was strongly dependent on chain length with alkyl ethers 14-18 carbon atoms long exhibiting the greatest antiviral activity against HCMV and HSV-1. In HIV-infected HT4-6C cells, optimal activity was observed at 18-22 carbon chain lengths. The antiviral activities of 1-octadecyloxypropane-3-PFA and 1-docosyloxypropane-3-PFA were 135- and 338-fold greater than that of PFA in HT4-6C cells infected with HIV-1. This also represents a 2.6-6-fold improvement in antiviral activity over ODG-PFA, the previously reported analog.
Virology, 1986
Influenza A virus entry into the host cell was studied by monitoring the fate of virion proteins ... more Influenza A virus entry into the host cell was studied by monitoring the fate of virion proteins in subcellular fractions of LLC-RMK2 cells under conditions of low multiplicity of infection in the absence of drugs. Adsorption but not entry could be demonstrated at 4 degrees. Restricted entry into a prelysosomal pool could be demonstrated at 20 degrees. At 37 degrees proteins entered the cell in the same relative distribution as present in virions arguing against the fusion hypothesis and for the endocytosis hypothesis. The hemagglutinin and matrix proteins were readily degraded intracellularly at 37 degrees, but the nucleoprotein was relatively resistant to degradation, also consistent with the latter hypothesis. Amantadine blocked neither entry into the host cell nor transfer between the prelysosomal and lysosomal compartments; however, it appeared to block exit from the prelysosomal and lysosomal pools. The addition of the drug reduced degradation of the matrix protein in these pools but not of the hemagglutinin, consistent with inhibition by amantadine of fusion of virion envelope and vesicle membrane.
The Journal of biological chemistry, Jan 5, 1992
Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthes... more Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthesized and found to have equal antiretroviral activity in HIV-infected HT4-6C cells. It was anticipated that the glycerol-1-P isomer would be less active because of slow metabolic conversion by cellular phospholipases A and C, but the antiretroviral results suggested that the human cell line (HT4-6C) may have phospholipases capable of hydrolyzing 2,3-dipalmitoyl-sn-glycerol-1-phospho-5'-azidothymidine (AZT). To evaluate this possibility, we purified lysosomal phospholipase A1, an enzyme known to play a major role in cellular phospholipid catabolism. This enzyme rapidly hydrolyzed both the sn-1 and sn-3 isomers of dipalmitoylphosphatidyl-AZT. We synthesized sn-2,3-dipalmitoyl-glycero-1-phosphocholine and found that it is also hydrolyzed readily by lysosomal phospholipase A1 although the Vmax, 59 mumol mg-1 h-1, is slightly lower than that of the sn-1,2-dipalmitoyl-glycero-3-phosphocholi...
Proceedings of the …, 1993
Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir {9-[(2-hydroxy... more Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir {9-[(2-hydroxyethoxy)methyl]guanine, Zovirax} is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovirresistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (ICso = 0.25 uM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 p.M). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3Hlacyclovir diphos
Antiviral Research, 2007
Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)pro... more Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide ® ) and its adenine counterpart (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and (S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the (S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.
Antiviral Chemistry and Chemotherapy, 2000
Biochimica et biophysica acta, Jan 23, 1972
1. 1. When added exogenously, both CDP- and dCDP-diglyceride supported diphosphatidylglycerol syn... more 1. 1. When added exogenously, both CDP- and dCDP-diglyceride supported diphosphatidylglycerol synthesis from phosphatidyl[1'-14C] glycerol in intact mitochondria. The maximum rate observed for CDP-diglyceride was about 2-fold greater than for dCDP-diglyceride. At optimal concentrations of CDP-diglyceride a 30-fold stimulation of diphosphatidylglycerol synthesis was observed. 2. 2. [14C] Glycerol was not formed in significant amounts during mitochondrial conversion of phosphatidyl[1'-14C] glycerol to diphosphatidylglycerol. 3. 3. In mitochondria, [2-3H]phosphatidyl[1'-14C] glycerol was converted in the presence of unlabeled CDP-diglyceride to a diphosphatidylglycerol having a nearly identical 3H/14C ratio. The above evidence confirms that diphosphatidylglycerol is formed from phosphatidylglycerol and CDP-diglyceride in mitochondria. 4. 4. In contrast, in preparations of membranes from Escherichia coli K12, the diphosphatidylglycerol formed at low concentrations of CDP-diglyceride had a 3H/14C ratio of 2.0 relative to its precursor [2-3H]phosphatidyl[1'-14C] glycerol. Furthermore, significant amounts of [14C] glycerol were isolated after incubation of the E. coli membranes with phosphatidyl[1'-14C]glycerol. These findings indicate that in E. coli diphosphatidylglycerol is formed from two molecules of phosphatidylglycerol. However, evidence is presented which suggests the operation of the CDP-diglyceride pathway in E. coli at higher concentrations of CDP-diglyceride.
Biochimica et biophysica acta, Jan 30, 1991
The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3&... more The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3'-deoxythymidine, 3'-deoxythymidine and 2',3'-dideoxycytidine from the corresponding nucleoside diphosphate diglycerides as a result of rat liver mitochondrial enzymatic activity is shown. The three analogs appeared to be about equally active as substrate for this pyrophosphatase activity which showed maximum conversion rates of 3-6 nmol min-1 mg protein-1 at substrate concentrations between 500 to 800 microM. These results may contribute to the biochemical explanation for the observed anti-HIV activity of this type of phospholipid conjugates in vitro.
Journal of lipid research, 1979
Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into pho... more Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidy...
Journal of lipid research, 1979
The subcellular distribution of neutral sphingomyelinase activity has been determined in rat live... more The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.
Molecular vision, 2011
To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) et... more To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) on ocular cell proliferation and collagen matrix contraction. For the antiproliferation studies, various ocular cell monolayers were exposed to HDP-PMEG, PMEG, 5-fluorouracil (5-FU), and daunorubicin (DNB). For the collagen contraction studies, retinal pigment epithelium (RPE) cells seeded onto type I collagen lattices were exposed for a single 5- or 50-min period to various concentrations of HDP-PMEG or 5-FU. For the cytotoxicity study, trypan blue exclusion tests were performed using a human Müller cell line. Cytotoxicity was determined up to 4 days after treatment. The proliferation of RPE cells, scleral fibroblasts, vessel endothelial cells, and ocular melanoma cells can all be significantly inhibited by HDP-PMEG. Its inhibitory effects on those cells were uniformly stronger than that of 5-FU. Contraction of the collagen matrix containing RPE cells was signifi...
Investigative ophthalmology & visual science, 1999
To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-s... more To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA), as a long-acting, nontoxic intravitreous injectable drug delivery system for cytomegalovirus (CMV) retinitis. ODG-PFA was synthesized by coupling the phosphonate residue of PFA to the 3 hydroxyl of 1-O-octadecyl-sn-glycerol and formulated as micelles and liposomes at concentrations so that, after injection into the rabbit vitreous, the resultant intravitreal concentrations were 0.2 mM, 0.63 mM, and 2 mM in micellar formulation and 0.02 mM, 0.063 mM, 0.2 mM, and 0.63 mM for liposomal formulation. The compounds were injected, and toxicology evaluations were performed. Intravitreal injections of micellar ODG-PFA resulted in aggregation of the material in vitreous and variable local retinal damage. Intravitreal injections of the liposomal ODG-PFA revealed even dispersion of the compounds and a clear vitreous, using final concentration in the vitreous ...
Antiviral chemistry & chemotherapy, 1998
In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93... more In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93 times more potent than free foscarnet (PFA) in human cytomegalovirus (HCMV)-, herpes simplex virus type 1 (HSV-1)- and human immunodeficiency virus type 1 (HIV-1)-infected cells. To evaluate the effect of substituting a 1-S-alkyl thioether for a 1-O-alkyl ether, we synthesized a series of PFA conjugates of 1-S-alkyl-sn-thioglycerols with varied 1-S-alkyl chain lengths. To establish structure-activity relationships we measured the in vitro antiviral activity of liposomal formulations of the drugs in cells infected with HCMV, HSV-1 or HIV-1. The optimum 1-S-alkyl chain length in the series was 16 to 18 carbon atoms. We compared the antiviral activity of 16- and 18-carbon alkyl thioglycerol versus alkylglycerol prodrugs and did not observe any significant differences in their antiviral activities. The series' most active member, 1-S-octadecyl-sn-glycero-3-foscarnet (ODSG-PFA) was 56-, ...
Biochimica et biophysica acta, Jan 7, 1993
Positional specificities in donor and acceptor phospholipids of the lysosomal phosphatidylcholine... more Positional specificities in donor and acceptor phospholipids of the lysosomal phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase have been determined. Comparison of the transfer of labelled fatty acid from sn-1 [14C]acyl and sn-2 [14C]acylphosphatidylcholines by extracts of rat liver lysosomes revealed that fatty acids in the sn-1 position were exclusively transferred. Degradation of the acylphosphatidylglycerol product by Rhizopus arrhizus lipase, highly specific for fatty acids esterified to sn-1 or sn-3 positions, indicated that sn-1 or sn-3 rather than sn-2 positions had been acylated. Assays of phospholipase A1, phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase, the conversion of lysophosphatidylglycerol to bis(monoacylglycero)phosphate and phospholipase A2 were performed at various steps in the purification of lysosomal phospholipase A1. After the penultimate step of chromatofocusing, there was a 1086-fold increase of phospholipase A1 specific ...
The Journal of biological chemistry, Jan 25, 1980
Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isola... more Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isolated from rat liver lysosomes. Lysosomal phospholipase C is active against all phospholipids tested, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine. It has an acid pH optimum, does not require divalent cations, and is not inhibited by EDTA. With [1-14C]dioleoylphosphatidylcholine as the substrate, 14C-labeled monoglyceride and diglyceride are the reaction products. Monoglyceride is formed rapidly from diglyceride by a lysosomal acid lipase, although some monoglyceride may be formed directly by phospholipase C hydrolysis of lysophosphatidylcholine. The other product, phosphocholine, has been identified by its behavior during Dowex 1-formate anion exchange chromatography. This appears to be the first demonstration in mammalian systems ofa phospholipase C which is active against all phosphoglycerides.
Journal of lipid research, 1980
Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethan... more Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethane (DH) to rats in oral doses of 100 mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver. To further characterize this drug-induced lipidosis, we have isolated and characterized the lipids of subcellular fractions from control rats and rats treated with chloroquine, DH, and Triton WR-1339. The phospholipid content of liver is increased 1.5-fold by chloroquine or DH treatment but is unaffected by Triton WR-1339. Acid phosphatase is increased by treatment with these three agents. Chloroquine and DH cause a shift of acid phosphatase from the light mitochondrial fraction (L) to the heavy mitochondrial fraction (M). Multilamellar bodies, an ultrastructural hallmark of chloroquine and DH-induced lipidosis, were isolated in a highly-purified form from the M fraction of chloroquine- or DH-treated rats. They are highly enriched in acid phosphatase indicating their lyso...
The Journal of biological chemistry, Jan 10, 1980
Biochimica et biophysica acta, Jan 8, 1971
The Journal of biological chemistry, Jan 10, 1985
A method has been developed to measure the concentration of chloroquine in lysosomes isolated fro... more A method has been developed to measure the concentration of chloroquine in lysosomes isolated from the liver of rats. It employs 3H2O and [U-14C]sucrose to determine the intralysosomal water volume of purified lysosomes obtained by free flow electrophoresis. Twelve h after a single dose, the concentration of chloroquine in lysosomes was 6.3 mM and at 24 h it rose to 16.5 mM. With continued treatment, lysosomal chloroquine concentrations were 61 and 74 mM at 48 and 72 h. The lysosomal concentrations of chloroquine attained were sufficient to block intralysosomal phospholipase A1 activity. The lysosomal content of phospholipid rises 1.7-fold and 2.6-fold over that of control at 12 and 24 h, respectively. At 72 h, lysosomal phospholipid was 3.7-fold greater than that of control. Lysosomes show an increased negative surface charge with chloroquine administration which is due in part to an increased ratio of acidic to neutral phospholipids in the lysosomal membrane. The phosphatidylinosi...
Biochimica et biophysica acta, Jan 15, 1988
Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals ... more Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals and man. It has been proposed that this is due to its ability to inhibit lysosomal phospholipase A. To investigate this further, a crude lysosomal fraction from rat lung was prepared and phospholipase A was isolated and its positional specificity was determined. Analysis of the products formed after incubation with 2-[1-14C]oleoylphosphatidylcholine showed that only phospholipase A1 activity is present. This soluble preparation of lung lysosomal phospholipase A1 was used to study inhibition by amiodarone and desethylamiodarone, in vitro. Both were extremely potent inhibitors of the lung acid phospholipase A1. To evaluate the levels of amiodarone in lung lysosomes, rats were treated with the agent for 3 days and the combined mitochondrial/lysosomal fraction of lung tissue was prepared by differential centrifugation. This fraction had been shown previously to be highly enriched in amiodaro...
Antiviral Research, 1997
The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy... more The identification of more effective and less toxic foscarnet (PFA) analogs for antiviral therapy would be useful. We recently synthesized 1-O-octadecyl-sn-glycero-3-phosphonoformic acid (ODG-PFA) and noted a 93-fold increase in its anti-HCMV activity relative to PFA. In addition, the antiviral activity of ODG-PFA in herpes simplex virus type-1 (HSV-1) and human immunodeficiency virus type-1 (HIV-1) infected cells was increased 40-fold relative to PFA (Hostetler et al., 1996. Antiviral Res. 31, 59). To evaluate structure-activity relationships further, we synthesized alkoxypropyl esters of foscarnet with varying alkyl chain lengths and degrees of saturation. These compounds were tested in vitro for activity and selectivity in comparison with PFA and ODG-PFA in cells infected with HCMV, HSV-1 or HIV-1. Antiviral activity was strongly dependent on chain length with alkyl ethers 14-18 carbon atoms long exhibiting the greatest antiviral activity against HCMV and HSV-1. In HIV-infected HT4-6C cells, optimal activity was observed at 18-22 carbon chain lengths. The antiviral activities of 1-octadecyloxypropane-3-PFA and 1-docosyloxypropane-3-PFA were 135- and 338-fold greater than that of PFA in HT4-6C cells infected with HIV-1. This also represents a 2.6-6-fold improvement in antiviral activity over ODG-PFA, the previously reported analog.
Virology, 1986
Influenza A virus entry into the host cell was studied by monitoring the fate of virion proteins ... more Influenza A virus entry into the host cell was studied by monitoring the fate of virion proteins in subcellular fractions of LLC-RMK2 cells under conditions of low multiplicity of infection in the absence of drugs. Adsorption but not entry could be demonstrated at 4 degrees. Restricted entry into a prelysosomal pool could be demonstrated at 20 degrees. At 37 degrees proteins entered the cell in the same relative distribution as present in virions arguing against the fusion hypothesis and for the endocytosis hypothesis. The hemagglutinin and matrix proteins were readily degraded intracellularly at 37 degrees, but the nucleoprotein was relatively resistant to degradation, also consistent with the latter hypothesis. Amantadine blocked neither entry into the host cell nor transfer between the prelysosomal and lysosomal compartments; however, it appeared to block exit from the prelysosomal and lysosomal pools. The addition of the drug reduced degradation of the matrix protein in these pools but not of the hemagglutinin, consistent with inhibition by amantadine of fusion of virion envelope and vesicle membrane.
The Journal of biological chemistry, Jan 5, 1992
Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthes... more Glycerol-1-P and glycerol-3-P stereoisomers of dipalmitoylphosphatidylazidothymidine were synthesized and found to have equal antiretroviral activity in HIV-infected HT4-6C cells. It was anticipated that the glycerol-1-P isomer would be less active because of slow metabolic conversion by cellular phospholipases A and C, but the antiretroviral results suggested that the human cell line (HT4-6C) may have phospholipases capable of hydrolyzing 2,3-dipalmitoyl-sn-glycerol-1-phospho-5'-azidothymidine (AZT). To evaluate this possibility, we purified lysosomal phospholipase A1, an enzyme known to play a major role in cellular phospholipid catabolism. This enzyme rapidly hydrolyzed both the sn-1 and sn-3 isomers of dipalmitoylphosphatidyl-AZT. We synthesized sn-2,3-dipalmitoyl-glycero-1-phosphocholine and found that it is also hydrolyzed readily by lysosomal phospholipase A1 although the Vmax, 59 mumol mg-1 h-1, is slightly lower than that of the sn-1,2-dipalmitoyl-glycero-3-phosphocholi...
Proceedings of the …, 1993
Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir {9-[(2-hydroxy... more Infection with herpes simplex viruses (HSVs) resistant to treatment with acyclovir {9-[(2-hydroxyethoxy)methyl]guanine, Zovirax} is a growing clinical problem in patients with AIDS and other immunosuppressed states. Most virus isolates resistant to acyclovir are deficient or defective in virally coded thymidine kinase (TK), which converts acyclovir to acyclovir monophosphate in virus-infected cells. To restore acyclovir efficacy, we synthesized acyclovir diphosphate dimyristoylglycerol, an analog of a naturally occurring phospholipid, CDP-diacylglycerol. Its biological activity was tested in WI38 human lung fibroblasts infected with the acyclovirresistant DM21 strain of HSV, which is TK negative due to an 816-base-pair deletion in the TK coding region. Acyclovir diphosphate dimyristoylglycerol has substantial activity in DM21-infected cells (ICso = 0.25 uM), whereas acyclovir and acyclovir monophosphate were ineffective (IC50 > 100 p.M). Similar results were obtained in TK-altered and TK-deficient strains of HSV-1 and in acyclovir-resistant isolates of HSV-2 obtained from two AIDS patients. The phospholipid prodrug is active by means of TK-independent metabolic pathways that liberate acyclovir monophosphate inside the host cell. Acyclovir phosphates were 56 times greater in WI38 human lung fibroblasts incubated for 24 hr with [8-3Hlacyclovir diphos
Antiviral Research, 2007
Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)pro... more Acyclic nucleoside phosphonates (ANPs) and in particular (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC, cidofovir, CDV, Vistide ® ) and its adenine counterpart (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine [(S)-HPMPA] are highly active against orf virus infections. This parapoxvirus commonly causes infection in sheep, goats, but also humans. Alkoxyalkyl esters of CDV have an increased oral bioavailability and are more active against orthopoxviruses than the parent compounds. In the present study, the potency of several alkoxyalkyl esters of CDV, cyclic cidofovir (cCDV) and (S)-HPMPA was evaluated against different orf virus isolates in two cell types, human embryonic lung (HEL) fibroblast and primary lamb keratinocytes. Each prodrug was at least 10-fold more active than its parent compound in both cell types. Of all the compounds tested, the (S)-HPMPA alkoxyalkyl esters showed the highest activity and selectivity against orf virus. Our results support the development of alkoxyalkyl esters of ANPs as antivirals not only for the treatment of complicated human orf lesions, but also in the therapy and prophylaxis of contagious ecthyma in sheep and goats.
Antiviral Chemistry and Chemotherapy, 2000
Biochimica et biophysica acta, Jan 23, 1972
1. 1. When added exogenously, both CDP- and dCDP-diglyceride supported diphosphatidylglycerol syn... more 1. 1. When added exogenously, both CDP- and dCDP-diglyceride supported diphosphatidylglycerol synthesis from phosphatidyl[1'-14C] glycerol in intact mitochondria. The maximum rate observed for CDP-diglyceride was about 2-fold greater than for dCDP-diglyceride. At optimal concentrations of CDP-diglyceride a 30-fold stimulation of diphosphatidylglycerol synthesis was observed. 2. 2. [14C] Glycerol was not formed in significant amounts during mitochondrial conversion of phosphatidyl[1'-14C] glycerol to diphosphatidylglycerol. 3. 3. In mitochondria, [2-3H]phosphatidyl[1'-14C] glycerol was converted in the presence of unlabeled CDP-diglyceride to a diphosphatidylglycerol having a nearly identical 3H/14C ratio. The above evidence confirms that diphosphatidylglycerol is formed from phosphatidylglycerol and CDP-diglyceride in mitochondria. 4. 4. In contrast, in preparations of membranes from Escherichia coli K12, the diphosphatidylglycerol formed at low concentrations of CDP-diglyceride had a 3H/14C ratio of 2.0 relative to its precursor [2-3H]phosphatidyl[1'-14C] glycerol. Furthermore, significant amounts of [14C] glycerol were isolated after incubation of the E. coli membranes with phosphatidyl[1'-14C]glycerol. These findings indicate that in E. coli diphosphatidylglycerol is formed from two molecules of phosphatidylglycerol. However, evidence is presented which suggests the operation of the CDP-diglyceride pathway in E. coli at higher concentrations of CDP-diglyceride.
Biochimica et biophysica acta, Jan 30, 1991
The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3&... more The release of the 5'-monophosphates of the antiretroviral nucleoside analogs 3'-azido-3'-deoxythymidine, 3'-deoxythymidine and 2',3'-dideoxycytidine from the corresponding nucleoside diphosphate diglycerides as a result of rat liver mitochondrial enzymatic activity is shown. The three analogs appeared to be about equally active as substrate for this pyrophosphatase activity which showed maximum conversion rates of 3-6 nmol min-1 mg protein-1 at substrate concentrations between 500 to 800 microM. These results may contribute to the biochemical explanation for the observed anti-HIV activity of this type of phospholipid conjugates in vitro.
Journal of lipid research, 1979
Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into pho... more Mitochondria from the 7777 hepatoma incorporate substantial amounts of l-[U-(14)C]serine into phospholipid by a Ca(2+)-dependent base-exchange reaction. This reaction is virtually absent in normal liver mitochondria. The finding cannot be attributed to microsomal contamination of the sucrose gradient-purified 7777 hepatoma mitochondria. The reaction is also absent in the rapid-growth controls, fetal rat liver and regenerating rat liver. [(14)C]Serine incorporation into 7777 hepatoma mitochondrial phospholipid by base-exchange requires Ca(2+) and is inhibited by EDTA. Ca(2+) cannot be replaced by Mg(2+), Mn(2+), or Co(2+). The reaction is inhibited by a sulfhydryl reagent and by detergents and is abolished by heating to 70 degrees C for 10 min. Product analysis indicates that phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, are formed by 7777 hepatoma mitochondria, while phosphatidylserine is the sole product with microsomes. The conversion of phosphatidy...
Journal of lipid research, 1979
The subcellular distribution of neutral sphingomyelinase activity has been determined in rat live... more The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.
Molecular vision, 2011
To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) et... more To investigate the safety and inhibitory effects of hexadecyloxypropyl 9-[2-(phosphonomethoxy) ethyl] guanine (HDP-PMEG) on ocular cell proliferation and collagen matrix contraction. For the antiproliferation studies, various ocular cell monolayers were exposed to HDP-PMEG, PMEG, 5-fluorouracil (5-FU), and daunorubicin (DNB). For the collagen contraction studies, retinal pigment epithelium (RPE) cells seeded onto type I collagen lattices were exposed for a single 5- or 50-min period to various concentrations of HDP-PMEG or 5-FU. For the cytotoxicity study, trypan blue exclusion tests were performed using a human Müller cell line. Cytotoxicity was determined up to 4 days after treatment. The proliferation of RPE cells, scleral fibroblasts, vessel endothelial cells, and ocular melanoma cells can all be significantly inhibited by HDP-PMEG. Its inhibitory effects on those cells were uniformly stronger than that of 5-FU. Contraction of the collagen matrix containing RPE cells was signifi...
Investigative ophthalmology & visual science, 1999
To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-s... more To determine intraocular toxicity and efficacy of the lipid prodrug of foscarnet, 1-O-octadecyl-sn-glycerol-3-phosphonoformate (ODG-PFA), as a long-acting, nontoxic intravitreous injectable drug delivery system for cytomegalovirus (CMV) retinitis. ODG-PFA was synthesized by coupling the phosphonate residue of PFA to the 3 hydroxyl of 1-O-octadecyl-sn-glycerol and formulated as micelles and liposomes at concentrations so that, after injection into the rabbit vitreous, the resultant intravitreal concentrations were 0.2 mM, 0.63 mM, and 2 mM in micellar formulation and 0.02 mM, 0.063 mM, 0.2 mM, and 0.63 mM for liposomal formulation. The compounds were injected, and toxicology evaluations were performed. Intravitreal injections of micellar ODG-PFA resulted in aggregation of the material in vitreous and variable local retinal damage. Intravitreal injections of the liposomal ODG-PFA revealed even dispersion of the compounds and a clear vitreous, using final concentration in the vitreous ...
Antiviral chemistry & chemotherapy, 1998
In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93... more In a previous study, we reported that 1-O-octadecyl-sn-glycero-3-foscarnet (ODG-PFA) was 40 to 93 times more potent than free foscarnet (PFA) in human cytomegalovirus (HCMV)-, herpes simplex virus type 1 (HSV-1)- and human immunodeficiency virus type 1 (HIV-1)-infected cells. To evaluate the effect of substituting a 1-S-alkyl thioether for a 1-O-alkyl ether, we synthesized a series of PFA conjugates of 1-S-alkyl-sn-thioglycerols with varied 1-S-alkyl chain lengths. To establish structure-activity relationships we measured the in vitro antiviral activity of liposomal formulations of the drugs in cells infected with HCMV, HSV-1 or HIV-1. The optimum 1-S-alkyl chain length in the series was 16 to 18 carbon atoms. We compared the antiviral activity of 16- and 18-carbon alkyl thioglycerol versus alkylglycerol prodrugs and did not observe any significant differences in their antiviral activities. The series' most active member, 1-S-octadecyl-sn-glycero-3-foscarnet (ODSG-PFA) was 56-, ...
Biochimica et biophysica acta, Jan 7, 1993
Positional specificities in donor and acceptor phospholipids of the lysosomal phosphatidylcholine... more Positional specificities in donor and acceptor phospholipids of the lysosomal phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase have been determined. Comparison of the transfer of labelled fatty acid from sn-1 [14C]acyl and sn-2 [14C]acylphosphatidylcholines by extracts of rat liver lysosomes revealed that fatty acids in the sn-1 position were exclusively transferred. Degradation of the acylphosphatidylglycerol product by Rhizopus arrhizus lipase, highly specific for fatty acids esterified to sn-1 or sn-3 positions, indicated that sn-1 or sn-3 rather than sn-2 positions had been acylated. Assays of phospholipase A1, phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase, the conversion of lysophosphatidylglycerol to bis(monoacylglycero)phosphate and phospholipase A2 were performed at various steps in the purification of lysosomal phospholipase A1. After the penultimate step of chromatofocusing, there was a 1086-fold increase of phospholipase A1 specific ...
The Journal of biological chemistry, Jan 25, 1980
Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isola... more Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isolated from rat liver lysosomes. Lysosomal phospholipase C is active against all phospholipids tested, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine. It has an acid pH optimum, does not require divalent cations, and is not inhibited by EDTA. With [1-14C]dioleoylphosphatidylcholine as the substrate, 14C-labeled monoglyceride and diglyceride are the reaction products. Monoglyceride is formed rapidly from diglyceride by a lysosomal acid lipase, although some monoglyceride may be formed directly by phospholipase C hydrolysis of lysophosphatidylcholine. The other product, phosphocholine, has been identified by its behavior during Dowex 1-formate anion exchange chromatography. This appears to be the first demonstration in mammalian systems ofa phospholipase C which is active against all phosphoglycerides.
Journal of lipid research, 1980
Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethan... more Administration of chloroquine or 4,4'-bis(diethylaminoethoxy)alpha, beta-diethyldiphenylethane (DH) to rats in oral doses of 100 mg/kg for 7 days causes phospholipid and cholesteryl ester accumulation in liver. To further characterize this drug-induced lipidosis, we have isolated and characterized the lipids of subcellular fractions from control rats and rats treated with chloroquine, DH, and Triton WR-1339. The phospholipid content of liver is increased 1.5-fold by chloroquine or DH treatment but is unaffected by Triton WR-1339. Acid phosphatase is increased by treatment with these three agents. Chloroquine and DH cause a shift of acid phosphatase from the light mitochondrial fraction (L) to the heavy mitochondrial fraction (M). Multilamellar bodies, an ultrastructural hallmark of chloroquine and DH-induced lipidosis, were isolated in a highly-purified form from the M fraction of chloroquine- or DH-treated rats. They are highly enriched in acid phosphatase indicating their lyso...
The Journal of biological chemistry, Jan 10, 1980
Biochimica et biophysica acta, Jan 8, 1971
The Journal of biological chemistry, Jan 10, 1985
A method has been developed to measure the concentration of chloroquine in lysosomes isolated fro... more A method has been developed to measure the concentration of chloroquine in lysosomes isolated from the liver of rats. It employs 3H2O and [U-14C]sucrose to determine the intralysosomal water volume of purified lysosomes obtained by free flow electrophoresis. Twelve h after a single dose, the concentration of chloroquine in lysosomes was 6.3 mM and at 24 h it rose to 16.5 mM. With continued treatment, lysosomal chloroquine concentrations were 61 and 74 mM at 48 and 72 h. The lysosomal concentrations of chloroquine attained were sufficient to block intralysosomal phospholipase A1 activity. The lysosomal content of phospholipid rises 1.7-fold and 2.6-fold over that of control at 12 and 24 h, respectively. At 72 h, lysosomal phospholipid was 3.7-fold greater than that of control. Lysosomes show an increased negative surface charge with chloroquine administration which is due in part to an increased ratio of acidic to neutral phospholipids in the lysosomal membrane. The phosphatidylinosi...
Biochimica et biophysica acta, Jan 15, 1988
Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals ... more Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals and man. It has been proposed that this is due to its ability to inhibit lysosomal phospholipase A. To investigate this further, a crude lysosomal fraction from rat lung was prepared and phospholipase A was isolated and its positional specificity was determined. Analysis of the products formed after incubation with 2-[1-14C]oleoylphosphatidylcholine showed that only phospholipase A1 activity is present. This soluble preparation of lung lysosomal phospholipase A1 was used to study inhibition by amiodarone and desethylamiodarone, in vitro. Both were extremely potent inhibitors of the lung acid phospholipase A1. To evaluate the levels of amiodarone in lung lysosomes, rats were treated with the agent for 3 days and the combined mitochondrial/lysosomal fraction of lung tissue was prepared by differential centrifugation. This fraction had been shown previously to be highly enriched in amiodaro...