Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although the total concentration of cGMP in rod outer segments is thought to be substantially greater than the free concentration, no quantitatively relevant site for the bound cGMP has been described in mammalian photoreceptors. We have found that preparations of purified bovine rod photoreceptor cyclic nucleotide phosphodiesterase (PDE) contain 1.8 +/- 0.3 mol of tightly bound cGMP per mol of PDE. When subunits of the purified PDE were separated by reverse-phase HPLC in 0.1% trifluoroacetic acid and acetonitrile, a peak of material having spectral properties characteristic of a guanine ring was seen. This material was identified as cGMP by comigration with authentic cGMP on HPLC, conversion to 5-GMP by trypsin-activated rod PDE, and conversion to guanosine by a combination of trypsin-activated PDE and 5'-nucleotidase-containing snake venom. When incubated with 1 microM [3H]cGMP, only 0.1 mol of [3H]cGMP bound per mol of purified PDE, presumably because nearly all binding sites were occupied by tightly bound endogenous cGMP carried through the purification. Scatchard plots of [3H]cGMP binding have indicated that two classes of binding sites are present on the rod PDE. The off-rate of cGMP from the slowly dissociating site is extremely slow; it has a t1/2 of approximately 4 hr at 37 degrees C. At lower temperatures, very little cGMP dissociates; the amount of [3H]cGMP bound to rod PDE after 2 hr at 4 degrees C was essentially the same as at the beginning of the incubation. The observation that stoichiometric amounts of cGMP are tightly bound to PDE accounts for the inability to purify the bovine rod PDE on cGMP affinity columns or to demonstrate stoichiometric high-affinity binding sites with [3H]cGMP. More significantly, the tightly bound cGMP may resolve the apparent discrepancy between the free and total cGMP concentrations of photoreceptor outer segments.
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PMID:cGMP is tightly bound to bovine retinal rod phosphodiesterase. 254 68

A capillary zone electrophoretic (CZE) assay was developed for the determination of cis-5,6a,7,8,9,9a-hexahydro-2-[4-(trifluoromethyl)phenylmethyl]-5-methyl - cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one, SCH 51866 (I), a cyclic guanine monophosphate phosphodiesterase inhibitor, in rat serum using acetonitrile deproteination as a clean-up step before injection. The calibration curve was linear over a serum concentration range of 0.5 to 10 micrograms/ml serum with a correlation coefficient (r) > 0.99. The limit of quantitation (LOQ) was established at 0.5 micrograms/ml. Fifty microliters of serum were used for analysis, which allowed serial bleeding (8 samples) from a single rat to characterize the pharmacokinetic profile of I after either oral or intravenous drug administration. In traditional pharmacokinetic and toxicokinetic studies in rodents, one animal provides only one serum sample since 1 to 2 ml of sample volume is required for chromatographic analysis, resulting in the use of a large number of animals per study. This assay yields a significant reduction in the use of animals, hence providing a large reduction in resources and time in drug discovery and development.
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PMID:Determination of a cyclic guanine monophosphate phosphodiesterase inhibitor (SCH 51866) in rat serum using capillary zone electrophoresis. 758 78

A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.
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PMID:Determination of a new phosphodiesterase V inhibitor, DA-8159, in plasma and urine by high-performance liquid chromatography. 1236 77

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.
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PMID:Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine. 1452 22

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantitation of tadalafil (I) in human plasma, a new selective, reversible phosphodiesterase 5 inhibitor. The analyte and internal standard (sildenafil, II) were extracted by liquid-liquid extraction with diethyl ether/dichloromethane (70/30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on reverse phase Xterra MS C18 column with a mobile phase of 10mM ammonium formate/acetonitrile (10/90, v/v, pH adjusted to 3.0 with formic acid). The protonate of analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 390.4 --> 268.0 and m/z 475.5 --> 58.3 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 10-1000 ng/mL for tadalafil in human plasma. The lower limit of quantitation was 10 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. Run time of 1.2 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.
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PMID:Quantitation of tadalafil in human plasma by liquid chromatography-tandem mass spectrometry with electrospray ionization. 1531 72

We developed and validated a reversed-phase high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of YM-64227 [4-cyclohexyl-1-ethyl-7-methylpyrido(2,3-d)pyrimidin-2-(1H)-one], a novel and selective phosphodiesterase type 4 inhibitor, and its fi ve hydroxylated metabolites in dog plasma. The plasma samples were extracted with tert-butyl methyl ether under alkali conditions. The analytes were well separated on a phenyl ethyl column (5 microm, 250 x 4.6 mm i.d.), opreating at 40 degrees C and using an acetonitrile-acetic acid gradient at a fl ow rate of 1.0 mL/min. The fluorescence signal was monitored at an excitation and emission wavelength of 330 and 400 nm, respectively. No interfering peak was observed at the retention time of YM-64227, its metabolites or the internal standard. The validated quantitation range of the method was 0.4-200 ng/mL for all analytes using 0.5 mL of the plasma sample. The recovery of analytes in the extraction process was more than 65.5%. The intra- and inter-assay precision was less than 5.1 and 12.6%, respectively, and the intra- and inter-assay accuracy ranged from -8.1 to 11.8% and -8.0 to 9.9%, respectively. Using this assay, the plasma concentration of YM-64227 and metabolites can be determined after the oral administration of YM-64227 to beagle dogs.
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PMID:Simultaneous determination of YM-64227, a phosphodiesterase type 4 inhibitor, and its five metabolites in dog plasma by high-performance liquid chromatography with fluorescence detection. 1538

A liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) method was developed to screen for the presence of synthetic phosphodiesterase type 5 (PDE-5) inhibitors including sildenafil, tadalafil and vardenafil. The method was applied to the analysis of dietary supplements and bulk herbal materials. Bulk powders or composites of tablets, capsules or liquids were prepared and an extraction of PDE-5 inhibitors was performed using a mixture of acetonitrile and water with sonication. Identification of sildenafil, vardenafil or tadalafil was accomplished using a single quadrupole mass spectrometer coupled to a liquid chromatograph with an electrospray interface. Positive ion detection in the full scan mode was used while in-source collision induced dissociation (CID) provided several structurally significant fragment ions to aid in the mass spectral identification. Approximately half of the 40 botanical products analyzed were found to contain undeclared synthetic PDE-5 inhibitors. For products found to contain one of these three compounds by LC-MS, HPLC with UV detection was used for quantitation.
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PMID:Analysis of undeclared synthetic phosphodiesterase-5 inhibitors in dietary supplements and herbal matrices by LC-ESI-MS and LC-UV. 1552 26

Tadalafil is a potent reversible phosphodiesterase-5 inhibitor used for the treatment of erectile dysfunction. This study describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the determination of tadalafil in 50 microl of rat plasma. Tadalafil and the internal standard lamotrigine were extracted with 0.5 ml of tert-butyl methyl ether, after the samples alkalinized with 20 microl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a C18 column with the mobile phase of acetonitrile-water containing 20 mM phosphate buffer (pH 7) (35/65, v/v), at a flow rate of 1 ml/min. The eluant was detected at 290 nm. The retention time was about 4.5 min for lamotrigine and 15 min for tadalafil. No endogenous substances were found to interfere. Calibration curves were linear from 10 to 2000 ng/ml. The recovery of tadalafil from plasma was greater than 77%. The limit of quantitation was 10 ng/ml. The intra- and inter-day imprecision (expressed as coefficient of variation, C.V.) did not exceed 10.7%, and the accuracy was within 5.9% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring tadalafil concentration.
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PMID:Determination of tadalafil in small volumes of plasma by high-performance liquid chromatography with UV detection. 1599 35

MSP001 is a newly synthesized piperidine analog of the lead antihypertensive compound MS23 that dually targets cAMP-specific type 4 phosphodiesterase and L-type calcium channels. We validated an analytical protocol for MSP001 in rat plasma using high performance liquid chromatographic method. A C18 column and a phosphate/acetonitrile buffer were used to perform the chromatographic separation. UV detection was carried out at 307 nm, a wavelength at which an absorption peak was detected for this group of compounds. The calibration curve for MSP001 was linear in the range from 25 to 10,000 ng/ml. The limit of quantification (LOQ) was 25 ng/ml. The results demonstrate that this method has high linearity (R=0.99995), compound specificity, and acceptable precision/accuracy. The protocol is suitable for in vivo pharmacokinetic studies on the compound.
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PMID:High performance liquid chromatographic analysis of MS23 piperidine analog MSP001 in rat plasma. 1681 62

SK3530.2HCl, (2-(5-(4-(2-hydroxyethyl)piperazin-1-ylsulfonyl)-2-n-propoxyphenyl)-5-ethyl-7-n-propyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one dihydrochloride), is a novel a new phosphodiesterase type V (PDE V) inhibiting agents. The pharmaceutical development of SK3530 necessitated the availability of an assay for the quantification and purity determination of SK3530 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase high performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with a CapcellPack MG (4.6 mm x 150 mm, 5 microm) column with ammonium acetate buffer (pH 4.0, 20 mM)-acetonitrile (60:40, v/v) as the isocratic mobile phase and UV detection at 250 nm. The method has been shown good chromatographic separation for SK3530 and the other related substances. The method was found to be linear 200-300 microg/ml, precise and accurate. Stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indication capacity. Moreover, the use of LC-MS and on-line diode array detection enabled us to propose structures for degradation products.
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PMID:Validation of a HPLC method for the quantification and purity determination of SK3530 in drug substance and tablet. 1713 66


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