Development of an HPLC/UV assay for the evaluation of inhibitors of human recombinant monoacylglycerol lipase.

Monoacylglycerol lipase (MAGL) is a membrane-associated cytosolic serine hydrolase which catalyses the hydrolysis of the endocannabinoid 2-arachidonoylglycerol into arachidonic acid and glycerol. MAGL represents the link between the endocannabinoid and the eicosanoid system indeed its inhibition enhances endocannabinoid signalling and lowers eicosanoid production. Here we present a radioactive-free, sensitive and solid HPLC-UV based method to evaluate MAGL activity by using 4-nitrophenylacetate (4-NPA) as substrate. The enzymatic activity is measured by quantifying the 4-nitrophenol (PNP) (λ = 315 nm) formation on a C18 stationary phase. The method was validated by calculating IC50 values of the reference inhibitors JZL184, CAY10499 and JW642 and confirming the irreversible and non-competitive mechanism of inhibition for JZL184. Furthermore in order to resemble the catalytic conditions of MAGL at cell membrane level, the surfactant Triton X-100 was added, as a micelle forming agent and 4-nitrophenyldodecanoate (4-NPDo) was used as lipophilic substrate for MAGL. The data obtained confirmed that the HPLC method is an alternative, radioactive-free approach for the screening and characterization of new MAGL inhibitors. Finally this assay prevents, in an unequivocal manner, any interference related to the intrinsic absorbance of screened compounds or metabolites generated upon enzymatic cleavage which could seriously affect the assay readout.

Pharmacokinetics of intravenous and intramuscular parecoxib in healthy Beagles.

Parecoxib is an inactive pro-drug that is rapidly converted to valdecoxib, a selective cyclooxygenase (COX)-2 inhibitor registered for the management of post-operative pain in humans. Recent studies have suggested that parecoxib has excellent clinical efficacy and safety in veterinary species. The aim of the current study was to assess the pharmacokinetics of parecoxib and valdecoxib after intravenous (i.v.) and intramuscular (i.m.) administration. Seven healthy male Beagle dogs received 2.5 mg/kg parecoxib by either the i.v. or i.m. route in a cross-over design, with the alternative route of administration used 1 week later. The plasma concentrations of both analytes were detected according to a previously validated method using high performance liquid chromatography with fluorescence detection (HPLC-FL). No adverse effects were observed in any animal during the study. For both routes of administration, parecoxib was rapidly and almost completely converted to valdecoxib. The parecoxib/valdecoxib area under the curve (AUC) ratio for both routes of administration was 1.4. The half-life of valdecoxib was about 2 h, which was shorter than reported for humans, although the plasma concentrations following both routes of administration were likely to be effective for analgesia. The absolute bioavailability of parecoxib was 66%. The pharmacokinetic features of parecoxib make it suitable for treatment of acute pain in the canine species.

Simultaneous detection and quantification of parecoxib and valdecoxib in canine plasma by HPLC with spectrofluorimetric detection: development and validation of a new methodology.

Parecoxib is the injectable prodrug of valdecoxib, a cicloxygenase-2 selective drug, currently used in human medicine. Recent studies have suggested both its excellent clinical effectiveness and wide safety profile. The aim of the present study was to develop and validate a new high-performance liquid chromatography (HPLC) with spectrofluorimetric detection method to quantify parecoxib and valdecoxib in canine plasma. Several parameters both in the extraction and the detection method were evaluated. The applicability of the method was determined by administering parecoxib to one dog: the protocol provided the expected pharmacokinetic results. The final mobile phase was acetonitrile: AcONH(4) (10 mM; pH 5.0) 55:45, v/v, with a flow rate of 0.4 mL min(-1), and excitation and emission wavelengths of 265 and 375 nm, respectively. The analytical column was a reverse-phase C18 ODS2 3-µm particle size. Protein precipitation in acidic medium followed by two successive liquid-liquid steps was carried out. The best extraction solvent was cyclohexane:Et(2)O (3:2, v/v) that gave recoveries ranging from 81.1% to 89.1% and from 94.8% to 103.6% for parecoxib and valdecoxib, respectively. The limits of quantification were 25 and 10 ng mL(-1) for parecoxib and valdecoxib, respectively. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes, as confirmed by HPLC-mass spectrometry experiments. The other validation parameters were in agreement with the European Medicines Evaluation Agency and International Conference on Harmonisation guidelines. In conclusion, this method (extraction, separation and applied techniques) is simple and effective. This is the first time that use of a HPLC with spectrofluorimetric detection technique to simultaneously detect parecoxib and valdecoxib in plasma has been reported. This technique may have applications for pharmacokinetic studies.

Determination of tramadol and metabolites by HPLC-FL and HPLC-MS/MS in urine of dogs.

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC-MS/MS. A single oral dose of tramadol (4mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively. The present method allowed to obtain good selectivity, accuracy, precision and recoveries without the need of time consuming or expensive clean up steps. The short chromatographic time courses allowed this analysis to be proposed for routine purposes. The HPLC-MS/MS detected in the urine two metabolites (M6 and M7) considered negligible in humans. The low LOQ showed that the method could be useful for the determination of the illegal use of this drug in race-dogs' urine.

Pharmacokinetics of tramadol and metabolites after injective administrations in dogs.

The aim of this study was to determine the pharmacokinetics of tramadol and its main metabolites after i.v. and i.m. injections. The pharmacokinetic cross-over study was carried out on 6 healthy male beagle dogs. Tramadol was administered by intravenous (i.v.) and intramuscular (i.m.) injection at 4 mg/kg. Tramadol and its main metabolites O-desmethyl-tramadol (M1), N-,N-didesmethyl-tramadol (M2) and N-,O-didesmethyl-tramadol (M5) concentrations were measured in plasma samples by a HPLC coupled with fluorimetric detection; pharmacokinetic evaluations were carried out with a compartmental and non-compartmental model for tramadol and its metabolites, respectively. The bioavailability of the drug, ranging between 84-102% (mean 92%), was within the generally accepted values for a positive bioequivalence decision of (80-125%). After the i.m. injection the mean plasma drug concentration peak was reached after a T(max) of 0.34 h with a C(max) of 2.52 microg/mL. No therapeutic relevant differences were observed between i.m. and i.v. administration. The minimal effective plasma concentration was reached after a few minutes and maintained for about 6-7 h in both administrations. M1 plasma concentration was low and the amounts of the other metabolites produced were analogous in both routes of administration. In conclusion, tramadol was rapidly and almost completely absorbed after i.m. administration and its systemic availability was equivalent to the i.v. injection. The different onset time and duration of action observed were very small and probably therapeutically irrelevant. The i.m. injection is a useful alternative to i.v. injection in the dog.

Pharmacokinetics of tramadol and its major metabolites following rectal and intravenous administration in dogs.

AIM: To compare the rectal and I/V administration of tramadol in dogs, to assess both its pharmacokinetic properties and absolute bioavailability. METHODS: After rectal administration via suppositories and I/V injection of tramadol (4 mg/kg), the concentration of tramadol and its main metabolites, O-desmethyl-tramadol (M1), N-desmethyl-tramadol (M2) and N,O-didesmethyl-tramadol (M5), were determined in plasma, using high-performance liquid chromatography (HPLC). A balanced cross-over study was used, involving six male Beagle dogs. RESULTS: Plasma concentrations after rectal and I/V administration were fitted on the basis of mono- and bi-compartmental models, respectively. Following rectal administration tramadol was detected from 5 minutes up to 10 hours, in lesser amounts than M5 and M2, while M1 was detected in negligible amounts. Following I/V administration tramadol was detected up to 10 hours, M2 and M5 were detected at similar concentrations, and M1 was present at low concentrations. The area under the curve (AUC) of the three metabolites did not differ significantly after either route of administration of tramadol. The absolute bioavailability of tramadol via rectal administration was 10 (SD 4)%. CONCLUSIONS: After rectal administration of tramadol suppositories, absorption of the active ingredient was rapid, but its metabolism quickly transformed the parent drug to high levels of M2 and M5. CLINICAL RELEVANCE: In the dog, rectal pharmaceutical formulation of tramadol would have a different pharmacokinetic behaviour than in humans.

Pharmacokinetic and urine profile of tramadol and its major metabolites following oral immediate release capsules administration in dogs.

The aim of the present paper was to test the oral administration of oral immediate release capsules of tramadol in dogs, to asses both its pharmacokinetic properties and its urine profile. After capsules administration of tramadol (4 mg/kg), involving eight male Beagle dogs, the concentration of tramadol and its main metabolites, M1, M2 and M5, were determined in plasma and urine using an HPLC method. The plasma concentrations of tramadol and metabolites were fitted on the basis of mono- and non-compartmental models, respectively. Tramadol was detected in plasma from 5 min up to 10 h in lesser amounts than M5 and M2, detected at similar concentrations, while M1 was detected in negligible amounts. In the urine, M5 and M1 showed the highest and smallest amount, respectively; M1 and M5 resulted widely conjugate with glucuronic acid. In conclusion, after oral administration of tramadol immediate release capsules, the absorption of the active ingredient was rapid, but its rapid metabolism quickly transformed the parental drug to high levels of M5 and M2, showing an extensive elimination via the kidney. Hence, in the dog, the oral immediate release pharmaceutical formulation of tramadol would have different pharmacokinetic behaviour than in humans.

Synthesis and antiplatelet activity of some 2,7-di(N-cycloamino)-3-phenyl-1,8-naphthyridine derivatives.

Several 2,7-di(N-cycloamino)-3-phenyl-1,8-naphthyridine derivatives were synthesized and tested for their ability to inhibit human platelet aggregation in vitro induced by arachidonic acid, collagen and ADP. Only five compounds showed any appreciable activity, and the results of all the active derivatives were similar to those of papaverine in the test with arachidonic acid and collagen. Moreover, the most active compounds were investigated in the test with ADP and again showed a significant activity. The tested compounds that possessed the best activity were also shown to increase the c-AMP level significantly without involving the adenylyl cyclase system.

Synthesis and evaluation of antihypertensive activity of 1,8-naphthyridine derivatives. Part X.

A series of 4-(N-methylencycloalkylamino)-1,8-naphthyridine derivatives variously substituted in positions 2 and 7 were synthesized and pharmacologically investigated for possible antihypertensive activity. These compounds were tested to determine a possible vasodilator mechanism of action. Compounds 22, 23, 27-29, 47 and 48 showed satisfactory levels of potency (pIC(50)>5), which in one case (compound 23) reached a really interesting value (pIC(50) 6.92). Furthermore, for some selected compounds (19, 22, 23, 26, 28, 29, 47), the vasorelaxing activity was also evaluated in the presence of the guanylate cyclase blocker ODQ or of the adenylate cyclase blocker SQ 22536, and some of these can be considered as possible guanylate-cyclase inhibitors. Finally, compounds 19, 22 and 23 were also tested in the presence of the ATP-sensitive potassium channel blocker glybenclamide and seem to possess activating properties on these potassium channels.

Synthesis and beta-blocking activity of (R,S)-(E)-oximeethers of 2, 3-dihydro-1,8-naphthyridine and 2,3-dihydrothiopyrano[2, 3-b]pyridine:potential antihypertensive agents - part IX.

The synthesis of oximeethers of 2,3-dihydro-1,8-naphthyridine and 2, 3-dihydrothiopyrano[2,3-b]pyridine is described. These compounds exhibit a selective beta-blocking activity, with a selectivity towards beta(2)-receptors. Groups in the N(1) position giving rise to a considerable steric hindrance led to a higher beta(2)-blocking selectivity, whereas groups creating a moderate hindrance caused a weak but significant decrease in beta(2)-antagonist potency. Substitution of the N(1)-R group with a sulfur atom led to compounds possessing beta(1)-, beta(2)- and beta(3)-blocking properties. Compounds 9c(1) and 10a(1) showed a beta(3)-antagonist activity slightly lower than that of propranolol.

A novel class of highly potent and selective A1 adenosine antagonists: structure-affinity profile of a series of 1,8-naphthyridine derivatives.

A series of 1,8-naphthyridine derivatives (12-36), bearing a phenyl group in position 2 and various substituents in positions 4 and 7, were synthesized in an attempt to obtain potent, selective antagonists for the A1 adenosine receptor subtype. The compounds were tested to evaluate their affinity for A1 compared with A2A and A3 adenosine receptor subtypes. In binding studies in bovine brain cortical membranes, most of the compounds showed an affinity for A1 receptors in the low nanomolar range and two in the subnanomolar range with an interesting degree of A1 versus A2A and A3 selectivity. Comparison of the 4-substituted derivatives indicated that 4-OH substitution, with a 4-quinoid structure, causes an increase in the A1 and A2A affinity and generally also in A1 selectivity. The kind of substitution in position 7 can greatly modulate the affinity: the most interesting substituents in this position seemed to be electron-withdrawing groups; in particular the 7-chloronaphthyridine 25d showed a remarkable selectivity (A2A/A1 ratio of 670, A3/A1 ratio of 14,000) associated with a higher A1 affinity (Ki = 0.15 nM). NMR studies on these compounds 12-36 indicated that the 4-OH-substituted ones prefer the tautomer in which the oxygen in position 4 is in the quinoid form and the nitrogen in position 1 is protonated. Theoretical calculations are in agreement with the NMR data.

Synthesis and antiplatelet activity of some 3-phenyl-1,8-naphthyridine derivatives.

A series of 2-cycloalkylamino-3-phenyl-1,8-naphthyridine derivatives, variously substituted in the 6- and 7-positions were synthesized and tested for their ability to inhibit human platelet aggregation in vitro induced by arachidonate, collagen and ADP. Compounds 5a,b, 7a,b, 8a and 10c,d showed a remarkable activity similar to that of indomethacin in the test with arachidonate and collagen. In the test with ADP only compound 8a showed a significant activity. The presence of a morpholinyl or piperidinyl group in position 2 and of a chloro or methoxy group in position 7 of the 1,8-naphthyridine nucleus seem to favour a higher activity. However on the basis of the pharmacological results, no structure-activity relationship can be deduced. Compounds 5b and 7b, which possess the best activity in the arachidonate test, were also shown to increase the c-AMP level significantly, without involving the adenylyl cyclase system.

Synthesis and evaluation of antimycobacterial activity of 4-phenyl-1,8-naphthyridine derivatives.

Some 4-phenyl-1,8-naphthyridine derivatives with a piperazino group in the 2- and/or 7-position have been synthesized and evaluated for their tuberculostatic activity. The compounds 1, 6, 10, 17b,c and 19i showed a marked activity against Mycobacterium tuberculosis H37Rv. For this series of compounds, submitted to biological screening, no structure-activity relationship can be deduced.