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Bioavailability and food effect of naproxen after morning and evening administration of D-naproxen betainate sodium salt in steady state
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96 WELL PLATE ASSAY FOR METABOLISM BY HUMAN CYTOCHROME P450 EXPRESSING CELL LINES IN COMBINATION WITH ANALYSIS BY LC-MS/MS EM de Groene. FWP Schoutsen. RJ Vreeken. and RF Witkamu TN0 Phanna, P.O.Box 360.3700 AJ Zeist, The Netherlands
BRAIN PHARMACOKINETIC FOR FREE AND LIPOSOMEENCAPSULATED CATECHOLAMINES V. V. Yurasov, I. Zhigaltsev, V. Kuchcryanu, A. Kaplun, V. Shvetz. Institute of General Pathology and Pathophysiology. 125315; Baltiyskaya. 8, Moscow, Russia. Our previous data indicate that intraperitoneal (i.p.) administration of blood-brain-barrier (BBB)-impermeable dopamine in small unilamellar encapsulated egg CDA) phosphatydilcholynelcholesterol (ePC and Chol) liposomes (SUL) but not DA alone compensated disorders of motor activity and endogenous DA deficiency in Parkinsonian mice. So in order to verify if liposomes facilitate the penetration of DA through BBB we have examined brain pharmacokinetic of SUL-incapsulated - non metabolized by brain enzymes synthetic analogue of naturally occured DA _ 3,4-dihydroxybenzilamine (DHBA). Free DHBA (37.5 mgikg) or DHBA incapsulated in ePC/Chol (0.53 M10.024M) SUL (5.6 mglkg) were administered i.p. in a single injection. The brain pharmacokinetic parameters for SUL and free forms of DHBA were calculated after DHBA have been determined in the brain by using HPLC technique. Quantity of DHBA penetrate in striatum at I minute per I mg of tissue was equal 0.05 pmole for its SUL form and 0.005 - for its free form. The rates of DHBA penetration into the brain were 2.97x IO-7mlxmglxmip’ or S.94rl0~~~mlxmkm~~xmin~~for SUL form of DHBA and 2.88x10-9 mlxmg-lxmin-1 or 5.76~10-1~ mlxmkm%mi~’ for its free form. Number of SUL what contents penetrate in the brain at I min was 12 per I mkmz of endothelial surface or II80 per I endothelial cell. The results indicate that incorporation liposomal of catecholamines facilitates their penetration into the brain as much as IOO-fold. So the efficiency of SUL with DA in Parkinsonian mice (our previous findings) can be due to the ability of SUL relieve catecholamines’ penetration through BBB. (Work was ganted by the Russian Foundation for Basic Researches .
Combinatorial chemistry and high throughout screening can lead to the identification of relatively large numbers of compounds which might be potential new drugs. Knowledge of metabolism of these potential new drugs is essential in an early stage of drug discovery. We developed a 96 well plate assay in which metabolism by human cytochrome P450 expressing cell lines (CYPlAl, lA2,2ClO, 2D6,ZEl or 3A4) is directly assayed by LC-MS/MS, the sensitivity of this technique allows for this format. For example, the interaction of ibuprofen, tolbutamide and sulfaphenazole with diclofenac-hydroxylation by CYPZClO was studied. In this assay sulfaphenazole was the most potent inhibitor of diclofenac metabolism followed by tolbutamide and ibuprofen. This approach allows for the investigation metabolism of multiple compounds at once by individual P450 expressing cells or by combinations of these different cell lines and, thus, allows for an easy screening method for metabolic stability, drug-drug interactions as well as metabolite profiling of potential new drugs.
970449 I Il.)
370
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BIOAVAILABILITY AND FOOD EFFECT OF NAPROltgN AFTER MORNING AND EVENING ADMINISTRATION OF DNAPROXEN BETAINATE SODIUM SALT IN STEADY STATE
RAPID HIGH THROUGHPUT SCREENING OF DRUG COMPOUNDS WITH A J-DAY CACO-2 ASSAY SYSTEM. w., Becton Dickinson Labware, Bedford, MA 01730
A. Matzo”, N. CTpi Manti”, L. Dal Bo”, F. Verga”, F. Crivelli”, R. Aleotti Tettamanti ‘, S. Ismaili”, MR. Uhr”I-,C. Wool” R. Ce~tti*’ ” IPAS SA, Via Mastri, 6853 Ligornetto, Switzerkmd; ‘I Bracco S.p.A., Ethical Drugs, Medical Depamnent, Via Folli 50, Milan - Italy D-napmxen betainate sodium salt in 550 mg capsules (327 mg of napmxan as such) was administaed to 24 healthy volunteers (12 malts and 12 females) b.i.d. to steady state. Plasma concenUations of naproxm were measured by a well validated HPLC method with fluorimtaic
deteaion as a morning predose and in timed samples aft= the morning dose of day 7 ia fasting status, after the evening dose of day 7 after dinner and after the morning do% of day 8, half an hour after a high-fat content breakfast. From plasma concentrations target pharmacokinetic parameters were evaluated which were on average as follows: Gu tm9x Gl”l (my) (h) (mpn) 35.6 Morning fasting 1.3 75.0 3.2 36.6 Evening fee&i 34.0 61.8 6.3 Morning feeding
Au’+, (m&r .h)
PTF (%)
686.8 645.3 574.3
1.05 0.72 0.59
The steady state was reached early, namely after 2 days. The three situations tested did not ditTerfor the extent of absorption, whereas its rate was the fastest in fast@ conditions, the lowest with the evening dosing and interme&te afta the h&b-fat content break&t. The slow absorption rate of the evening dose would allow tberapeatieatly active levels early in the morning, when the arthritis pain is particularly tedious.
Cam-2 cells are being increasingly used as an in vitro model system for screening drug candidatea for their intwtinal absorption potential. The traditional method of culturing Caeo-2 cells to obtain a differentiated phenotype rquires up to 3 we&s culture and involves many labor intensive steps. Reocntly, the BIOCOAP Caco-2 Assay System (which providtJ monolayer of Caco-2 cells ready for compound permeability screening in 3 days) has baea reported to be a more productive and ef&ient system for generating compound permeability data’.‘.‘. To hntha characterize the BIOCOAT systemsadcompare its performance to the 21-day system, Caco-2 cell differentiation wa teated with both methods. Caco-2 cells cultured in either the 3&y BIOCOAT or 21.day cultura system wue te&d for barrier function, brash border enzyme activity, transporter fimetion, and morphological characteristics indicative of cell d&ret&ion Our results indicate that Caco-2 cells cultured in the BIOCOAT 3day system possesses many of the.cell diiereatiation features found in the traditional 2lday Caco-2 system. Many ofthe biological and morphological markers indicatk of Caco-2 cell differentiation compare favorably between the 3-day BIOCOAT and 21-day Cm-2 culture systems. Therefore, the BIOCOAT Caco-2systemprovidesa moreconvenient andproductivealtanativeto the2 I day Caco-2system.By developing and chamcterizing a more efficient system for Gee-2 cultme, we believe that the posaibilitiea for the use of Caeo-2 cells as an in vitro model for intestinal compound permeability ate greatly enhanced. 1. Chong, S., ef.aL, PharmRes. (1997),Vol. 14, pp 11351837. 2. Asa, D. and LaRocca, P. Poster presentation at 4s’International Confetemx on Drug Absorption, Edinburgh, (1997) 3. Woods, W. and Aaa, D. Poster presentation at AAPS Annual Meeting, Boston, (1997)
369
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96 WELL PLATE ASSAY FOR METABOLISM BY HUMAN CYTOCHROME P450 EXPRESSING CELL LINES IN COMBINATION WITH ANALYSIS BY LC-MS/MS EM de Groene. FWP Schoutsen. RJ Vreeken. and RF Witkamu TN0 Phanna, P.O.Box 360.3700 AJ Zeist, The Netherlands
BRAIN PHARMACOKINETIC FOR FREE AND LIPOSOMEENCAPSULATED CATECHOLAMINES V. V. Yurasov, I. Zhigaltsev, V. Kuchcryanu, A. Kaplun, V. Shvetz. Institute of General Pathology and Pathophysiology. 125315; Baltiyskaya. 8, Moscow, Russia. Our previous data indicate that intraperitoneal (i.p.) administration of blood-brain-barrier (BBB)-impermeable dopamine in small unilamellar encapsulated egg CDA) phosphatydilcholynelcholesterol (ePC and Chol) liposomes (SUL) but not DA alone compensated disorders of motor activity and endogenous DA deficiency in Parkinsonian mice. So in order to verify if liposomes facilitate the penetration of DA through BBB we have examined brain pharmacokinetic of SUL-incapsulated - non metabolized by brain enzymes synthetic analogue of naturally occured DA _ 3,4-dihydroxybenzilamine (DHBA). Free DHBA (37.5 mgikg) or DHBA incapsulated in ePC/Chol (0.53 M10.024M) SUL (5.6 mglkg) were administered i.p. in a single injection. The brain pharmacokinetic parameters for SUL and free forms of DHBA were calculated after DHBA have been determined in the brain by using HPLC technique. Quantity of DHBA penetrate in striatum at I minute per I mg of tissue was equal 0.05 pmole for its SUL form and 0.005 - for its free form. The rates of DHBA penetration into the brain were 2.97x IO-7mlxmglxmip’ or S.94rl0~~~mlxmkm~~xmin~~for SUL form of DHBA and 2.88x10-9 mlxmg-lxmin-1 or 5.76~10-1~ mlxmkm%mi~’ for its free form. Number of SUL what contents penetrate in the brain at I min was 12 per I mkmz of endothelial surface or II80 per I endothelial cell. The results indicate that incorporation liposomal of catecholamines facilitates their penetration into the brain as much as IOO-fold. So the efficiency of SUL with DA in Parkinsonian mice (our previous findings) can be due to the ability of SUL relieve catecholamines’ penetration through BBB. (Work was ganted by the Russian Foundation for Basic Researches .
Combinatorial chemistry and high throughout screening can lead to the identification of relatively large numbers of compounds which might be potential new drugs. Knowledge of metabolism of these potential new drugs is essential in an early stage of drug discovery. We developed a 96 well plate assay in which metabolism by human cytochrome P450 expressing cell lines (CYPlAl, lA2,2ClO, 2D6,ZEl or 3A4) is directly assayed by LC-MS/MS, the sensitivity of this technique allows for this format. For example, the interaction of ibuprofen, tolbutamide and sulfaphenazole with diclofenac-hydroxylation by CYPZClO was studied. In this assay sulfaphenazole was the most potent inhibitor of diclofenac metabolism followed by tolbutamide and ibuprofen. This approach allows for the investigation metabolism of multiple compounds at once by individual P450 expressing cells or by combinations of these different cell lines and, thus, allows for an easy screening method for metabolic stability, drug-drug interactions as well as metabolite profiling of potential new drugs.
970449 I Il.)
370
372
BIOAVAILABILITY AND FOOD EFFECT OF NAPROltgN AFTER MORNING AND EVENING ADMINISTRATION OF DNAPROXEN BETAINATE SODIUM SALT IN STEADY STATE
RAPID HIGH THROUGHPUT SCREENING OF DRUG COMPOUNDS WITH A J-DAY CACO-2 ASSAY SYSTEM. w., Becton Dickinson Labware, Bedford, MA 01730
A. Matzo”, N. CTpi Manti”, L. Dal Bo”, F. Verga”, F. Crivelli”, R. Aleotti Tettamanti ‘, S. Ismaili”, MR. Uhr”I-,C. Wool” R. Ce~tti*’ ” IPAS SA, Via Mastri, 6853 Ligornetto, Switzerkmd; ‘I Bracco S.p.A., Ethical Drugs, Medical Depamnent, Via Folli 50, Milan - Italy D-napmxen betainate sodium salt in 550 mg capsules (327 mg of napmxan as such) was administaed to 24 healthy volunteers (12 malts and 12 females) b.i.d. to steady state. Plasma concenUations of naproxm were measured by a well validated HPLC method with fluorimtaic
deteaion as a morning predose and in timed samples aft= the morning dose of day 7 ia fasting status, after the evening dose of day 7 after dinner and after the morning do% of day 8, half an hour after a high-fat content breakfast. From plasma concentrations target pharmacokinetic parameters were evaluated which were on average as follows: Gu tm9x Gl”l (my) (h) (mpn) 35.6 Morning fasting 1.3 75.0 3.2 36.6 Evening fee&i 34.0 61.8 6.3 Morning feeding
Au’+, (m&r .h)
PTF (%)
686.8 645.3 574.3
1.05 0.72 0.59
The steady state was reached early, namely after 2 days. The three situations tested did not ditTerfor the extent of absorption, whereas its rate was the fastest in fast@ conditions, the lowest with the evening dosing and interme&te afta the h&b-fat content break&t. The slow absorption rate of the evening dose would allow tberapeatieatly active levels early in the morning, when the arthritis pain is particularly tedious.
Cam-2 cells are being increasingly used as an in vitro model system for screening drug candidatea for their intwtinal absorption potential. The traditional method of culturing Caeo-2 cells to obtain a differentiated phenotype rquires up to 3 we&s culture and involves many labor intensive steps. Reocntly, the BIOCOAP Caco-2 Assay System (which providtJ monolayer of Caco-2 cells ready for compound permeability screening in 3 days) has baea reported to be a more productive and ef&ient system for generating compound permeability data’.‘.‘. To hntha characterize the BIOCOAT systemsadcompare its performance to the 21-day system, Caco-2 cell differentiation wa teated with both methods. Caco-2 cells cultured in either the 3&y BIOCOAT or 21.day cultura system wue te&d for barrier function, brash border enzyme activity, transporter fimetion, and morphological characteristics indicative of cell d&ret&ion Our results indicate that Caco-2 cells cultured in the BIOCOAT 3day system possesses many of the.cell diiereatiation features found in the traditional 2lday Caco-2 system. Many ofthe biological and morphological markers indicatk of Caco-2 cell differentiation compare favorably between the 3-day BIOCOAT and 21-day Cm-2 culture systems. Therefore, the BIOCOAT Caco-2systemprovidesa moreconvenient andproductivealtanativeto the2 I day Caco-2system.By developing and chamcterizing a more efficient system for Gee-2 cultme, we believe that the posaibilitiea for the use of Caeo-2 cells as an in vitro model for intestinal compound permeability ate greatly enhanced. 1. Chong, S., ef.aL, PharmRes. (1997),Vol. 14, pp 11351837. 2. Asa, D. and LaRocca, P. Poster presentation at 4s’International Confetemx on Drug Absorption, Edinburgh, (1997) 3. Woods, W. and Aaa, D. Poster presentation at AAPS Annual Meeting, Boston, (1997)