24- and 26-homo-1,25-dihydroxyvitamin D3 analogs: potencies on in vitro bone resorption differ from those reported for cell differentiation.

It has been proposed that the stimulatory effects of 1,25-dihydroxyvitamin D on bone resorption may be mediated through actions on differentiation of marrow cells into monocytic osteoclast precursors. In human promyelocytic leukemia cells (HL-60), 24- and 26-homo-1,25-dihydroxyvitamin D3 and their delta 22 analogs and 24,24-dihomo-1,25-dihydroxyvitamin D3 are 10-fold more potent than 1,25-dihydroxyvitamin D3, and delta 22-24,24,24-trihomo-1,25-dihydroxyvitamin D3 is equipotent with 1,25-dihydroxyvitamin D3 in inducing differentiation into the monocytic phenotype. The effect of these 1,25-dihydroxyvitamin D3 analogous on resorption of fetal rat limb bones in vitro was determined in the present study. 1,25-Dihydroxyvitamin D3 was equipotent with 24-homo-1,25-dihydroxyvitamin D3, delta 22-24-homo-1,25-dihydroxyvitamin D3, 26-homo-1,25-dihydroxyvitamin D3, and delta 22-26-homo-1,25-dihydroxyvitamin D3 for in vitro bone resorption, whereas 24,24-dihomo-1,25-dihydroxyvitamin D3 and delta 22-24,24,24-trihomo-1,25-dihydroxyvitamin D3 were inactive. The failure of these analogs to show a higher bone-resorbing activity than 1,25-dihydroxyvitamin D3 were inactive. The failure of these analogs to show a higher bone-resorbing activity than 1,25-dihydroxyvitamin D3 provides evidence to suggest that the mechanism of 1,25-dihydroxyvitamin D3-induced bone resorption may not involve stimulation of monocytic cell differentiation.

Vitamin D metabolism during pregnancy.

Vitamin D metabolism is altered in the pregnant animal, presumably in response to fetal demands for calcium. Circulating levels of 1,25-dihydroxyvitamin D are elevated in the pregnant animal. The stimulus of this increase and the hydroxylase(s) (placental or renal) responsible are unknown. Maternal plasma 25-hydroxyvitamin D levels have been reported to be both unchanged and decreased during pregnancy but very much dependent upon exposure to ultraviolet light and vitamin D supplementation. The major vitamin D metabolites (25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D) circulate in fetal plasma but generally at lower concentrations than in the mother (exception is the sheep). All of these metabolites are able to cross the placenta. The fetal kidney and placenta both have 25-hydroxyvitamin D1 alpha- and 24-hydroxylase activity. However, the relative contribution of mother, fetus, and placenta to fetal vitamin D metabolism has yet to be fully determined.

A radioreceptor binding assay for measurement of platelet-activating factor synthesis by human neutrophils.

A new convenient method for the measurement of platelet-activating factor produced in vitro has been developed. This method involves incubation of neutrophils with stimuli, lipid extraction, purification of lipid extracts by normal phase high performance liquid chromatography (HPLC) and quantification of platelet-activating factor (PAF) by a competitive radioreceptor binding assay. The recovery of PAF from the extraction and purification procedures is 94.5 +/- 0.9%. The sensitivity of the assay is 10 pg/tube. Human neutrophils stimulated with 0, 0.25, 0.5, 1 and 2 microM A 23187 will produce ND, ND, 720, 840 and 900 pg of PAF, respectively (ND = not detectable). The values obtained for PAF produced by human neutrophils in the present assay are comparable to those obtained with the rabbit platelet aggregation assay.

Characterization of metabolites of Celecoxib in rabbits by liquid chromatography/tandem mass spectrometry.

The metabolism of the anti-inflammatory drug Celecoxib in rabbits was characterized using liquid chromatography (LC)/tandem mass spectrometry (MS/MS) with precursor ion and constant neutral loss scans followed by product ion scans. After separation by on-line liquid chromatography, the crude urine samples and plasma and fecal extracts were analyzed with turbo-ionspray ionization in negative ion mode using a precursor ion scan of m/z 69 (CF(3)) and a neutral loss scan of 176 (dehydroglucuronic acid). The subsequent product ion scans of the [M - H] ions of these metabolites yielded the identification of three phase I and four phase II metabolites. The phase I metabolites had hydroxylations at the methyl group or on the phenyl ring of Celecoxib, and the subsequent oxidation product of the hydroxymethyl metabolite formed the carboxylic acid metabolite. The phase II metabolites included four positional isomers of acyl glucuronide conjugates of the carboxylic acid metabolite. These positional isomers were caused by the alkaline pH of the rabbit urine and were not found in rabbit plasma. The chemical structures of the metabolites were characterized by interpretation of their product ion spectra and comparison of their LC retention times and the product ion spectra with those of the authentic synthesized standards.

Dermal inflammation in primates, mice, and guinea pigs: attenuation by second-generation leukotriene B4 receptor antagonist, SC-53228.

Granulocyte infiltration is a prominent feature of human psoriasis. Psoriatic lesional skin contains abnormally high amounts of immunoreactive leukotriene B4 (LTB4), a potent granulocyte chemotaxin in vivo and in vitro. SC-53228 [(+)-(S)-7-(3-}2-(cyclopropylmethyl)-3-methoxy-4- [(methylamino)carbonyl]phenoxy}propoxy}-3,4-dihydro-8-propyl-2H-1- benzopyran-2-propanoic acid], a second-generation LTB4 receptor antagonist, was tested topically and orally in phorbol ester-induced dermal inflammation in three species. Skin inflammation was induced by topical application of phorbol-12-myristate-13-acetate-(PMA/TPA) and assessed by ear thickness, levels of the neutrophil marker enzyme myeloperoxidase (MPO) and histological examination. In mice, SC-53228 inhibited inflammation with a topical ED50 value of 200 +/- 18 micrograms. When applied to guinea pigs, SC-53228 (100 micrograms) inhibited the MPO increase by 86%, while 1000 micrograms abrogated inflammation in rhesus macaques with no plasma accumulation of the drug. A 1% gel formulation was also efficacious in guinea pig PMA-induced epidermal inflammation. Furthermore, single oral dose administration to mice was efficacious (ED50 < 2.5 mg/kg) as was multidose administration to rhesus macaques. PMA-induced skin inflammation possesses some of the attributes of human psoriasis and an agent such as SC-53228 may have utility in the medical management of this condition.

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist, SC-53228: impact upon leukotriene B4- and 12(R)-HETE-mediated events.

Leukotriene B4 (LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig. LTB4 and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihy dro-8-propyl-2H - 1-benzopyran-2-carboxylic acid), a first-generation LTB4 receptor antagonist, inhibited the chemotactic actions of LTB4 when given orally with an ED50 value of 1.7 mg/kg. The second-generation LTB4 receptor antagonist, SC-53228 [(+)-(S)-7-(3-(2-(cyclopropylmethyl)-3-methoxy-4- [(methylamino)carbonyl]phenoxy)propoxy)-3,4-dihydro-8-propyl-2H-1- benzopyran-2-propanoic acid], inhibited LTB4-induced chemotaxis when given intragastrically with an ED50 value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED50 value of 5.8 mg/kg. When dosed orally over a range of 0.03-100 mg/kg, SC-53228 gave Cmax plasma concentrations of 0.015-41.1 micrograms/ml. SC-53228 inhibited LTB4-primed membrane depolarization of human neutrophils with an IC50 value of 34 nM. As a potent LTB4 receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB4 and/or 12(R)-HETE are implicated as inflammatory mediators.

Survey of vitamin D metabolite levels during growth and development in Japanese quail.

Plasma calcium and vitamin D metabolite levels were monitored during growth and development in male and female Japanese quail. In male Japanese quail, plasma calcium levels were constant (range 9.0 to 10.9 mg/dl); the 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels remained undetectable in all age groups. Plasma calcium levels in the females rose at 6 weeks of age and remained elevated. The female 1,25-(OH)2D levels increased from undetectable levels at 3 to 5 weeks to a peak of 297 pg/ml at 8 weeks and remained elevated at 12 weeks of age in ovulating birds. Twelve-week old female without an egg in their oviduct had plasma 1,25-(OH)2D levels similar to males and immature females. No marked changed occurred in plasma 25-(OH)D levels due to sex or age. Plasma 24, 25-(OH)2D levels stayed relatively constant at about 2 ng/ml for both male and females at all ages studied.

Effect of dietary mineral and vitamin D content and parathyroidectomy on the plasma disappearance rate of 1,25-dihydroxyvitamin D3 in rats.

The plasma disappearance rate of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was determined in rats after a single intravenous injection of the tritiated hormone. Tritiated 1,25-(OH)2D3 (120 Ci mmol-1) was administered to rats at a dose of 400 000 dpm kg-1 body weight and the animals were bled between 0 and 8 hours. The dose was estimated to produce negligible perturbations in endogenous plasma levels of 1,25-(OH)2D3. The plasma disappearance of 3H-1,25-(OH)2D3 occurred in two phases. The second phase plasma half-life of 1,25-(OH)2D3 in immature 25- to 35-day-old animals (4.7 hours) was significantly shorter than the second phase plasma half-life of maturing 49- to 65-day-old animals (8.0 hours). Phosphorus deprivation for 12 days significantly prolonged the second phase plasma half-life of 1,25-(OH)2D3 from a control value of 4.9 hours to 10.4 hours. Parathyroidectomy, regardless of the plasma calcium concentrations, shortened the second phase plasma half-life of 1,25-(OH)2D3 from control values of 9.1 hours to 5.0 hours. Calcium deprivation for 7 days did not alter the second phase plasma half-life of 1,25-(OH)2D3. Vitamin D deprivation for 5 weeks increased the second phase plasma half-life from 11.0 to 19.9 hours but the difference was not significant.

Subcellular location and properties of rat renal 25-hydroxyvitamin D3-1 alpha-hydroxylase.

The subcellular location and some properties of the rat kidney 25-hydroxyvitamin D3-1 alpha-hydroxylase are described. Enzyme activity can be measured as previously discussed (Tanaka, Y., and DeLuca, H.F. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 196-199) using saturating substrate (25-hydroxyvitamin D3) concentrations. The reaction is linear with time for up to 30 min at a substrate concentration of 80 microM and 9-11 mg/ml mitochondrial protein. The enzyme, located in the mitochondria, requires molecular oxygen and a source of NADPH. Succinate supplies NADPH for 1 alpha-hydroxylation through reversal of electron transport and transhydrogenation as shown by inhibition with antimycin A and dinitrophenol. Malate supplies NADPH for the reaction via the mitochondrial malic enzyme or malate dehydrogenase and transhydrogenase as indicated by the lack of inhibition by antimycin A but inhibition with dinitrophenol. Metyrapone and carbon monoxide both inhibit 1 alpha-hydroxylation indicating the involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, has no effect on 1 alpha-hydroxylation.

Plasma vitamin D metabolite levels in pregnant and nonpregnant ewes.

1. Maternal calcium homeostasis during pregnancy is strained due to fetal mineral requirements for bone formation. 2. In most species, the mother adjusts to the mineral requirements of the fetus with alterations in her metabolism of vitamin D that include a decrease in plasma 25-(OH)D levels and an increase in circulating levels of the hormone, 1,25-(OH)2D. 3. Plasma 25-(OH)D and 1,25-(OH)2D levels in adult male, adult female and pregnant sheep were measured by specific radioreceptor binding assays. 4. Pregnancy did not alter circulating levels of 25-(OH)D or 1,25-(OH)2D in the sheep. 5. The pregnant ewe differs from all species studied to date in that maternal plasma 1,25-(OH)2D levels do not rise as a result of pregnancy.

Pregnancy does not alter the metabolic clearance of 1,25-dihydroxyvitamin D in rats.

Increased circulating levels of 1,25-dihydroxyvitamin D [1,25(OH)2D] during pregnancy could be due to an increase in production or decrease in the metabolic clearance rate of 1,25(OH)2D. To answer this question an isotope dilution method was used to determine the clearance rate of 1,25(OH)2D in pregnant and aged-matched nonpregnant female rats. A bolus of 0.146 muCi 1,25(OH)2[3H]D3 was given to 60 pregnant and 60 aged-matched nonpregnant rats and the disappearance of the isotope was followed in these animals over the next 48 h. In 12 pregnant rats vs. 14 nonpregnant controls not injected with tracer, plasma calcium (9.6 +/- 0.41 vs. 10.7 +/- 0.17 mg/ml) and 25(OH)D (17.1 +/- 1.15 vs. 25.4 +/- 1.58 ng/ml) levels were significantly lower (P less than 0.01 and P less than 0.001), whereas plasma 1,25(OH)2D levels (110 +/- 16.1 pg/ml vs. 77 +/- 6.0 pg/ml) were significantly higher (P less than 0.05). Clearance rates of 1,25(OH)2D of 25.8 +/- 1.31 microliters/min in pregnant rats and 20.2 20.2 +/- 1.38 microliters/min in nonpregnant aged-matched rats were not significantly different. Similarly, the apparent volume of distribution of 1,25(OH)2D in the pregnant rats (15 +/- 1.0 ml) was not significantly different from that in the nonpregnant control animals (18 +/- 2.1 ml). Production rates of.1,25(OH)2D were elevated in the pregnant rats (2.83 pg/min) compared with the nonpregnant controls (1.55 pg/min). In conclusion, the elevated maternal plasma 1,25(OH)2D level during pregnancy is a result of increased production and is not due to a decreased clearance.

Assay and properties of rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase.

An in vitro assay has been developed for the rat yolk sac 25-hydroxyvitamin D3 1 alpha-hydroxylase (1 alpha-hydroxylase). The subcellular location and some properties of the enzyme are described. 1,25-Dihydroxyvitamin D3 produced from incubations of yolk sac homogenates was extracted, purified by Sephadex LH-20 chromatography and straight- and reverse-phase high-performance liquid chromatography (HPLC), and measured by a competitive binding assay using chick intestinal receptor. The reaction is linear with time for up to 45 min at a substrate concentration of 80 microM and 4-6 mg/mL microsomal protein. The enzyme, located in the microsomes, requires molecular oxygen and NADPH. Metyrapone (1 X 10(-3) M) was found to inhibit 1-hydroxylation, but a 90% carbon monoxide-10% oxygen atmosphere did not, leaving open the question of involvement of cytochrome P-450. Diphenyl-p-phenylenediamine, a lipid peroxidase inhibitor, inhibited 1 alpha-hydroxylation.

Plasma levels of vitamin D metabolites in fetal and pregnant ewes.

The plasma concentrations of calcium; inorganic phosphorus; 25-hydroxyvitamin D; 24,25-dihydroxyvitamin D; and 1,25-dihydroxyvitamin D were determined in sheep maternal and fetal arterial circulations. In addition, plasma concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were determined simultaneously across the uterine and umbilical circulations. Fetal arterial levels of calcium (r = 0.560); inorganic phosphorus (r = -0.095); and 1,25-dihydroxyvitamin D (r = 0.040) were significantly higher than and did not correlate with maternal arterial levels. Maternal levels of 25-hydroxyvitamin D were significantly higher than and correlated (r = 0.693) with fetal 25-hydroxyvitamin D levels. No significant difference existed between maternal and fetal arterial levels of 24,25-dihydroxyvitamin D. No significant difference was detected in the concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D across the uterine or umbilical circulations.

Do you know that one of your employees is looking for a new employer?

Turnover of employees is a natural event. It is sometimes a relief when a particular employee leaves the office. On the other hand, the cost of losing any employee can be high because of the costs related to lost productivity, training, and the time required for recruiting a replacement. Because of these cost elements, it makes sense to try to delay an employee's departure. By "buying time," the manager can minimize the time required to recruit a replacement and possibly even redistribute the workload among current employees before the job-hunting employee leaves the organization. This approach allows the manager to control the situation. Sometimes it is better to let the employee leave immediately upon submitting a resignation if this will avoid disruption in work flow or the "chemistry" among the rest of the employees. Some employees become negative in their behavior patterns once they decide to leave the organization. The knowledge that an employee is looking for a new position is vitally important information to a manager. The clues provided by the job-hunting employee can go a long way toward maintaining the stability of the work force and the effectiveness of the manager in achieving the goals of the organization.

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor.

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.

Metabolism and excretion of [(14)C]celecoxib in healthy male volunteers.

We determined the disposition of a single 300-mg dose of [(14)C]celecoxib in eight healthy male subjects. The [(14)C]celecoxib was administered as a fine suspension reconstituted in 80 ml of an apple juice/Tween 80/ethanol mixture. Blood and saliva samples were collected at selected time intervals after dosing. All urine and feces were collected on the 10 consecutive days after dose administration. Radioactivity in each sample was determined by liquid scintillation counting or complete oxidation and liquid scintillation counting. Metabolic profiles in plasma, urine, and feces were obtained by HPLC, and metabolites were identified by mass spectrometry and NMR. [(14)C]Celecoxib was well absorbed, reaching peak plasma concentrations within 2 h of dosing. [(14)C]Celecoxib was extensively metabolized, with only 2.56% of the radioactive dose excreted as celecoxib in either urine or feces. The total percentage of administered radioactive dose recovered was 84.8 +/- 4.9%, with 27.1 +/- 2.2% in the urine and 57.6 +/- 7.3% in the feces. The oxidative metabolism of celecoxib involved hydroxylation of celecoxib at the methyl moiety followed by further oxidation of the hydroxyl group to form a carboxylic acid metabolite. The carboxylic acid metabolite of celecoxib was conjugated with glucuronide to form the 1-O-glucuronide. The percentages of the dose excreted in the feces as celecoxib and the carboxylic acid metabolite were 2.56 +/- 1.09 and 54.4 +/- 6.8%, respectively. The majority of the dose excreted in the urine was the carboxylic acid metabolite (18.8 +/- 2.1%); only a small amount was excreted as the acyl glucuronide (1.48 +/- 0.15%).

Pharmacokinetics, metabolism and excretion of an inhibitor of inducible nitric oxide synthase, L-NIL-TA, in dog.

1. The pharmacokinetics, metabolism and excretion of L-NIL-TA, an inducible nitric oxide synthase inhibitor, were investigated in dog. 2. The dose of [14C]L-NIL-TA was rapidly absorbed and distributed after oral and intravenous administration (5 mg kg-1), with Cmax of radioactivity of 6.45-7.07 microg equivalents g-1 occurring at 0.33-0.39-h after dosing. After oral and intravenous administration, radioactivity levels in plasma then declined with a half-life of 63.1 and 80.6-h, respectively. 3. Seven days after oral and intravenous administrations, 46.4 and 51.5% of the radioactive dose were recovered in urine, 4.59 and 2.75% were recovered in faeces, and 22.4 and 22.4% were recovered in expired air, respectively. The large percentages of radioactive dose recovered in urine and expired air indicate that [14C]L-NIL-TA was well absorbed in dogs and the radioactive dose was cleared mainly through renal elimination. The mean total recovery of radioactivity over 7 days was approximately 80%. 4. Biotransformation of L-NIL-TA occurred primarily by hydrolysis of the 5-aminotetrazole group to form the active drug L-N6-(1-iminoethyl)lysine (NIL or M3), which was further oxidized to the 2-keto acid (M5), the 2-hydroxyl acid (M1), an unidentified metabolite (M2) and carbon dioxide. The major excreted products in urine were M1 and M2, representing 22.2 and 21.2% of the dose, respectively.

Pharmacokinetics of celecoxib after oral administration in dogs and humans: effect of food and site of absorption.

Celecoxib pharmacokinetics was evaluated after single and multiple oral dosing; after dosing in a solution and as a solid; with and without food; and after administration into different sites of the GI tract using dog. After oral dosing in a solution, celecoxib was rapidly absorbed and reached maximum concentrations by 1 h; absorption was delayed another 1 to 2 h when administered as a solid. The absolute bioavailability of celecoxib was higher when given as a solution (64--88%) compared with capsule (22--40%). The absorption of celecoxib given in a capsule was delayed by food, although systemic exposure increased by 3- to 5-fold. The systemic availability of celecoxib given intragastrically in solution was similar to that obtained following direct instillation into the duodenum, jejunum, or colon through a chronic intestinal access port. Collectively, these data suggest that celecoxib is a highly permeable drug that can be absorbed throughout the GI tract and that dissolution may be a rate-limiting factor for absorption from solid dosage forms. Unlike dogs, celecoxib given to humans with a high fat meal exhibits only a slight increase in AUC(0--infinity) (11%) that is not clinically significant with regard to safety or efficacy. In humans, a lower dose and a longer GI residence time may promote the opportunity for absorption of a poorly soluble drug such as celecoxib that can be absorbed throughout the GI tract. This would minimize the effect of food on absorption; as such, patients with arthritis can be given celecoxib with or without food.

Evidence for polymorphism in the canine metabolism of the cyclooxygenase 2 inhibitor, celecoxib.

The pharmacokinetics of celecoxib, a cyclooxygenase-2 inhibitor, was characterized in beagle dogs. Celecoxib is extensively metabolized by dogs to a hydroxymethyl metabolite with subsequent oxidization to the carboxylic acid analog. There are at least two populations of dogs, distinguished by their capacity to eliminate celecoxib from plasma at either a fast or a slow rate after i.v. administration. Within a population of 242 animals, 45.0% were of the EM phenotype, 53.5% were of the PM phenotype, and 1.65% could not be adequately characterized. The mean (+/-S.D.) plasma elimination half-life and clearance of celecoxib were 1.72 +/- 0.79 h and 18.2 +/- 6.4 ml/min/kg for EM dogs and 5.18 +/- 1.29 h and 7.15 +/- 1.41 ml/min/kg for PM dogs. Hepatic microsomes from EM dogs metabolized celecoxib at a higher rate than microsomes from PM dogs. The cDNA for canine cytochrome P-450 (CYP) enzymes, CYP2B11, CYP2C21, CYP2D15, and CYP3A12 were cloned and expressed in sf 9 insect cells. Three new variants of CYP2D15 as well as a novel variant of CYP3A12 were identified. Canine rCYP2D15 and its variants, but not CYP2B11, CYP2C21, and CYP3A12, readily metabolized celecoxib. Quinidine (a specific CYP2D inhibitor) prevented celecoxib metabolism in dog hepatic microsomes, providing evidence of a predominant role for the CYP2D subfamily in canine celecoxib metabolism. However, the lack of a correlation between celecoxib and bufuralol metabolism in hepatic EM or PM microsomes indicates that other CYP subfamilies besides CYP2D may contribute to the polymorphism in canine celecoxib metabolism.

Plasma protein binding of celecoxib in mice, rat, rabbit, dog and human.

The plasma protein binding of celecoxib was determined for animals and humans using in vitro and ex vivo methods. Eight, healthy, human volunteers (three male, five female, 20-39 years) received celecoxib (600 mg) BID for 7 days, blood samples were collected and concentrations of bound and unbound celecoxib determined. The fraction of bound drug in the volunteers was constant (97.4 +/- 0.1%) at total celecoxib plasma concentrations ranging from 0.01 to 4.02 microg/mL. The ex vivo plasma protein binding of celecoxib in the animals was concentration-independent up to approximately 12, 8 and 10 microg/mL for mouse, rat and dog, respectively. The plasma protein binding of celecoxib after a single oral dose of 10 and 300 mg/kg to mice was 98.3 +/- 0.2%, of 1 and 400 mg/kg to rats was 98.3 +/- 0.2% and of 1 and 100 mg/kg to dogs was 98.5 +/- 0.1%. The percent binding of celecoxib to plasma proteins in vitro was slightly lower than those values determined ex vivo. The in vitro binding of celecoxib to plasma protein was constant over the concentrations of 0.1-10 microg/mL for all species, except rat.