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NONLINEAR ORAL PHARMACOKINETICS OF THE -ANTAGONIST 4-AMINO-5-(4-FLUOROPHENYL)-6,7-DIMETHOXY-2-[4-(MORPHOLINOCARBONYL)-PERHYDRO-1,4-DIAZEPIN-1-YL]QUINOLINE IN HUMANS: USE OF PRECLINICAL DATA TO RATIONALIZE CLINICAL OBSERVATIONS

Departments of Pharmacokinetics, Dynamics and Metabolism (A.H., A.B., K.F., K.B., A.E., S.R.) and Clinical Sciences, Pfizer Global Research and Development, Sandwich, Kent, United Kingdom (J.D.); Department of Drug Safety Evaluation, Pfizer Global Research and Development, Amboise Laboratories, Amboise Cedex, France (P.C.); and Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Fresnes, France (P.M.)

(Received April 14, 2003; Accepted October 1, 2003)

	Abstract

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4-Amino-5-(4-fluorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline (UK-294,315) is an antagonist of the human ₁-adrenoceptor and exhibits nonlinear oral pharmacokinetics in humans. Superproportional increases in C_max occur (220-fold, over a 1- to 50-mg dose range), area under the curve increases linearly, but time to maximum concentration decreases with dose, suggesting variation in rate but not extent of absorption. Oral absorption in humans is extensive, with only 14% of an orally administered (20 mg) radiolabeled dose excreted unchanged in the feces. In rats and dogs, UK-294,315 is partially eliminated as unchanged drug in feces (29 and 14% of an intravenous dose, respectively). Oral bioavailability is low in rats (11%) and high in dogs (71%), in keeping with systemic clearance. Fecal elimination of unchanged drug was 60% after oral administration to rats, indicating incomplete absorption in this species, whereas absorption in dogs is complete. UK-294,315 is a P-glycoprotein (P-gp) substrate (K_m, 15 µM) exhibiting polarized flux in Caco-2 cell monolayers, saturable across a concentration range of 5 to 200 µM. Furthermore, the observations in vitro occurred at similar concentrations to those estimated in the gut lumen in clinical trials (dose range, 1-100 mg). It is considered that P-gp acts as a saturable absorption barrier to UK-294,315, slowing the rate of absorption at low doses, and is responsible for the observed nonlinearity in oral disposition in humans. Rat and dog pharmacokinetic studies offered limited insight into the process(es) driving nonlinear pharmacokinetics in humans. Our current understanding of the functional effects of P-gp in the human intestine, in combination with in vitro studies at clinically relevant concentrations, has helped rationalize the clinical data for UK-294,315.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Structure of UK-294,315 and UK-298,108 (internal standard). Asterisk denotes position of 14C in radiolabeled material.

Following progression to clinical studies, UK-294,315 exhibited nonlinear behavior in the rate, but not extent, of absorption over the dose range studied (1-100 mg). Nonlinear pharmacokinetics pose a challenge to the successful development of a drug candidate, especially when this behavior occurs across the therapeutic dose range. Such behavior complicates drug therapy due to extensive variability in exposure, which may lead to variable efficacy and narrow therapeutic window over potential adverse events. Indeed, nonlinear oral pharmacokinetics, within the commonly used dose range, of the recreational drug "ecstasy" are thought to result in cases of acute toxicity (De La Torre et al., 2000). A confident prediction of nonlinear oral pharmacokinetics before clinical studies would substantially reduce the time and research investment involved in getting a drug candidate from discovery through to the market and ultimately contribute to the development of safer drugs. Therefore, a mechanistic investigation of the nonlinear human pharmacokinetics of compounds such as UK-294,315 would provide valuable learning for the drug discovery and development process.

Nonlinearity in human oral pharmacokinetics may be a result of saturation of clearance processes [e.g., voriconazole (Roffey et al., 2003)] or effects on intestinal absorption. Saturation of intestinal transporter proteins (e.g., P-glycoprotein) has been implicated in the nonlinear oral absorption of several drugs, including talinolol, celiprolol, and UK-343,664 (Milne and Buckley, 1991; Wetterich et al., 1996; Abel et al., 2001). Preclinical studies demonstrated that UK-294,315 is a substrate for active transport proteins (e.g., P-glycoprotein), a property now recognized as potentially leading to nonlinear oral pharmacokinetics (Ayrton and Morgan, 2001). This paper aims to rationalize the nonlinear behavior observed for UK-294,315 in humans through a retrospective analysis using data derived from preclinical pharmacokinetic studies and in vitro P-glycoprotein (P-gp) affinity and cell transport studies.

	Materials and Methods

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Chemicals. 4-Amino-5-(4-fluorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline (UK-294,315) and internal standard 4-amino-5-(4-chlorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline (UK-298,108) (Fig. 1) were synthesized at Pfizer Global Research and Development (Sandwich, Kent, UK). [Quinoline-2-¹⁴C]UK-294,315 was synthesized by Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK) with a specific activity of 84 µCi/mg and radiochemical purity of >99%. Verapamil was obtained from Sigma Chemical (Poole, Dorset, UK). [4-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-6,7-dimethoxy-quinazolin-2-yl]-[2-(3,4-dimethoxy-phenyl)ethyl]-amine (CP-100,356) was obtained from Pfizer Global Research and Development (Groton Labs). Chemical purity of all reagents was >95%.

Administration of UK-294,315 to Animals. Single-Dose Studies. All animal work was carried out in compliance with UK or French law and was approved by a local ethical review process as appropriate. UK-294,315 was dissolved in 0.9% saline for intravenous administration and purified water for oral administration to rats and dogs. Four male Sprague-Dawley rats (250 g; Charles River, Margate, Kent, UK) were surgically prepared with an indwelling jugular vein cannula at least 2 days before administration of dose. Two rats received intravenous doses of UK-294,315 via the caudal vein (0.5 mg/kg, 1 ml/kg). Two rats received oral doses of UK-294,315 via gavage tube (4 mg/kg, 2 ml/kg). Blood samples (200 µl) were collected before dosing and at 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h postdose from the jugular vein cannula, which was flushed with heparinized saline (100 µl, 10 units/ml) after each sample. Samples were centrifuged (approximately 1500g, for 10 min), and the plasma was removed and stored at -20°C.

Four male beagle dogs (12-16 kg; Pfizer colony) were administered a single intravenous dose of UK-294,315 (0.5 mg/kg) by infusion (1 ml/min for 15 min) into the saphenous vein. The same animals received an oral solution dose of UK-294,315 (4 mg/kg, 1 ml/kg), administered by gavage tube, 1 week later. Blood samples (5 ml) were collected predose and at 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h postdose, from an indwelling saphenous vein cannula in the leg opposite that used for i.v. dose administration for the first 8 h, and by venipuncture of the cephalic vein for subsequent samples. Samples were centrifuged (approximately 1500g for 10 min), and the plasma was removed and stored at -20°C.

Administration of UK-294,315 to Animals. Multiple Dose Studies. UK-294,315 was administered to Sprague-Dawley rats (five male and five female per dose group as part of a toxicology study; mean body weight, 283 g and 204 g, respectively) as a suspension in 0.5% (w/v) aqueous methylcellulose (containing 0.1% w/v Tween 80), via oral gavage (5 ml/kg), at doses of 10, 25, and 75 mg/kg, daily for 159 days. On day 159 animals were anesthetized, and blood samples (1 ml) were collected from the orbital sinus at 1, 4, 7, and 24 h postdose. Samples were centrifuged (approximately 1500g for 10 min), and the plasma was removed and stored at -20°C.

UK-294,315 was administered to beagle dogs (four male and four female per dose group as part of a toxicology study; mean body weight, 9.3 and 7.6 kg, respectively) as a suspension in 0.5% w/v aqueous methylcellulose (containing 0.1% w/v Tween 80), via oral gavage (1 ml/kg), at doses of 2.5, 5, and 10 mg/kg, daily for 173 days. On day 173, blood samples (2.5 ml) were collected by puncture of the jugular vein at 1, 4, 7, and 24 h postdose. Samples were centrifuged (approximately 1500g for 10 min), and the plasma was removed and stored at -20°C.

Previous (14-day) toxicology studies in rat and dog have shown no changes in pharmacokinetics of UK-294,315 from single to multiple dosing (data not shown). Therefore, plasma samples were only taken at the end of the longer term toxicology studies described herein.

Dose Escalation Study in Humans. The human pharmacokinetic study was conducted according to the Association of the British Pharmaceutical Industry guidelines and to the revised Declaration of Helsinki. The clinical study protocol (single-blind, placebo-controlled, single escalating oral dose) was approved by a local ethics review committee, and the study was carried out at the Pfizer Clinical Research Unit at the Kent and Canterbury Hospital (Canterbury, UK). Written consent was obtained from 24 healthy male volunteers (aged 18 to 45 years, weighing 60-100 kg) who took part in the study. The study was divided into two cohorts of 12 subjects: cohort A, three-way crossover study investigating 1, 3, and 10 mg of UK-294,315, incorporating placebo substitution and cohort B, four-way crossover study investigating 30, 60, and 100 mg of UK-294,315 and placebo. The subjects were fasted for 12 h before dosing and for 4 h after dosing. UK-294,315 was administered in solution in water (250 ml). There was a minimum of 7 days between each dose. Blood samples (7 ml) were collected in heparinized tubes up to 24 to 48 h postdose. Samples were centrifuged (approximately 1500g at 4°C for 10 min) within 60 min of sample collection, and plasma was removed and stored in screw-capped polypropylene tubes at approximately -20°C.

Radiolabeled Studies in Animals. Formulations and dose administration for radiolabeled studies were as described above. [¹⁴C]UK-294,315 was administered to male (n = 2) and female (n = 2) Sprague-Dawley rats (250-300 g) intravenously (0.5 mg/kg, 10-15 µCi per rat), and to male (n = 2) and female (n = 2) rats orally (4 mg/kg, 10-15 µCi per rat). Beagle dogs (1 male and 1 female) also received single intravenous infusion and oral doses of [¹⁴C]UK-294,315 (0.5 and 4 mg/kg, respectively; approximately 60 µCi per dog) in a crossover study, with a period of 21 days between doses. Urine and feces were collected daily postdose for 4 days from rats and 7 days from dogs. Feces were homogenized with water (feces-water 1:1 w/v for rat and 4:1 w/v for dog). Fecal homogenates and urine were stored at -20°C before analysis. Radioactivity in urine (approximately 1 g) was measured by liquid scintillation counting of duplicate weighed samples in 4 ml of Starscint (Canberra Industries, Meriden, CT). Radioactivity in fecal homogenate (approximately 0.5 g) was measured by combustion of triplicate weighed samples in an Oximate sample oxidizer (Canberra Industries) and liquid scintillation counting of evolved [¹⁴C]O₂ in 10 ml of Permafluor E+ (Canberra Industries). Metabolite profiling of feces samples was carried out by pooling 0- to 48-h samples from rat and 0- to 168-h samples from dog. Rat samples were pooled to provide a single sample for male and female for each dose route. Radioactivity was separated from the fecal homogenate by sequential extractions with methanol (25 ml), methanol/0.1 M TRIS, pH 6 (24:1 v/v, 25 ml), and methanol/0.1 M TRIS, pH 9 (24:1 v/v, 25 ml). Between each extraction, the homogenate pellet was separated by centrifugation, and the supernatant was removed. Supernatants were then combined and evaporated to dryness under a stream of nitrogen at 37°C. Samples were resuspended in 2 ml of 0.1 M ammonium acetate, pH 6, for analysis by HPLC. Chromatography was carried out using reverse-phase HPLC with a HiRPB 25025 column (250 x 7.75 mm; Hichrom Ltd., Reading, UK) in conjunction with gradient elution using acetonitrile and 0.1 M ammonium acetate, pH 6 (0-60% acetonitrile over 10 min, 60-80% acetonitrile over 10 min, 80-100% acetonitrile over 5 min, 100% acetonitrile for 5 min), flow rate 1 ml/min. Detection was by radioflow detector (Ramona; LabLogic, Sheffield, UK) with data analysis using Laura software (LabLogic). Radiolabeled components were isolated from fecal extracts by collecting individual eluting peaks and evaporating the eluent to dryness under nitrogen at 37°C. Residue was then analyzed by mass spectrometry.

Radiolabeled Studies in Humans. Written consent and ethical approval for this study were obtained as detailed above, and the study was carried out at Pharma Bio Research Clinics (Assen, the Netherlands). Healthy male volunteers (n = 3, aged 45-65, 60-100 kg) were given UK-294,315 tablets, 20 mg, twice daily for 10 days. On day 6, the eleventh dose was administered as a solution of UK-294,315 containing approximately 60 µCi of [¹⁴C]UK-294,315. The next unlabeled dose was administered 12 h after the radiolabeled dose. Urine and feces were collected until samples were at background levels of radioactivity (144 and 216-336 h, respectively). Urine and feces radioactivity content was measured as described above. Radioactive material was extracted from fecal homogenates (approximately 10 g) and profiled by HPLC as described for rat and dog. The eluent gradient was 30% acetonitrile for 20 min, 35% acetonitrile for 10 min, then 100% acetonitrile for 5 min, and flow rate, 2 ml/min. Isolated radiolabeled components were analyzed by mass spectrometry.

Analysis of UK-294,315 in Animal and Human Plasma. The analysis of UK-294,315 in plasma from rats, dogs, and humans was carried out using mixed-phase extraction and a column-switching HPLC system with fluorescence detection. The analytical procedure was validated by the analysis of quality control samples. The method involved addition of internal standard (UK-298,108, 5 ng) to aliquots of plasma sample (0.1-1 ml), followed by addition of 3 ml of borate buffer (pH 10, Fischer Scientific, Ltd., Loughborough, UK). The sample mixture was added to a Chem Elut cartridge (3 ml; Varian Ltd., Walton-on-Thames, Surrey, UK) and allowed to soak for 3 min. Samples were eluted into polypropylene tapered tubes using 6 ml of ethyl acetate, and the eluent was evaporated to dryness under nitrogen at 37°C. Samples were reconstituted in 500 µl of 10% (v/v) acetonitrile in water (containing 0.1% trifluoroacetic acid). Three hundred microliters were injected onto the HPLC system. The HPLC consisted of a column-switching system comprising a strong cation ion exchange (SCX) cartridge and two reverse-phase base-deactivated columns. The sample was injected onto the SCX cartridge (S5SCX-10C5, 1 cm x 4.6 mm; HiChrom Ltd.) in a mobile phase of 10% (v/v) acetonitrile in water (containing 0.1% trifluoroacetic acid), at a flow rate of 1 ml/min. After 3 min, the SCX cartridge was backflushed onto a C₁₈ RPB cartridge (HiRPB-10C5, 1 cm x 4.6 mm, HiChrom Ltd.) using a mobile phase of 30% (v/v) acetonitrile, 23% (v/v) methanol in water (containing 0.1% trifluoroacetic acid), at a flow rate of 0.5 ml/min. The eluent from this cartridge was diverted to waste for 0.45 min and then switched onto the third column (C₁₈ RPB, 25 cm x 4.6 mm, HiRPB-250A; HiChrom Ltd.) for 1.5 min. UK-294,315 was eluted from the third column using a mobile phase of 30% (v/v) acetonitrile, 23% (v/v) methanol in water (containing 0.1% trifluoroacetic acid), at a flow rate of 0.5 ml/min. Detection was by fluorescence (_ex, 248 nm; _em, 388 nm). UK-294,315 had a retention time of approximately 7 min. The limit of detection was 0.1 ng/ml from 1 ml of plasma. Quality control samples were found to be within 20% of nominal concentration at the lower end of the calibration line (0.1 ng/ml) and within 15% at the middle and top (15 ng/ml), satisfying acceptance criteria for the assay. A modified method was used for analysis of toxicokinetic plasma samples; plasma (100-400 µl) was diluted with borate buffer pH 9 (1 ml) and then extracted with 4 ml of diethyl ether. The organic phase was removed and evaporated under nitrogen, and the residue was resuspended in 300 µl of the HPLC mobile phase detailed below (limit of detection was 5 ng/ml for dog and 10 ng/ml for rat toxicokinetic sample analysis). Samples were analyzed on an HPLC system comprising a HiRPB column (25 cm x 4.6 mm; HiChrom Ltd.), with a mobile phase of 45% acetonitrile/55% 0.01 M potassium dihydrogen orthophosphate containing 0.01 M octane sulfonic acid, pH 3, flow rate 1 ml/min, with UV detection (250 nM).

Pharmacokinetic Analysis of Data. The terminal phase rate constant (k_el) was determined by linear regression of the log plasma concentration-time profile. The terminal elimination half-life (t_1/2) was calculated from 0.693/k_el. Area under the plasma concentration-time curve (AUC_0-T) was calculated using the linear trapezoidal rule and extrapolated to infinity (AUC_0-) using k_el. Systemic clearance was calculated from intravenous data using the relationship dose/AUC_0-. Volume of distribution was calculated using the relationship systemic clearance/k_el. Oral bioavailability was calculated from the ratio of AUC_0- values after oral and intravenous doses, after normalizing for dose.

Isolated Perfused Rat Liver Experiments. The disposition of UK-294,315 (1-mg dose) in the isolated perfused rat liver (male Sprague-Dawley rats, n = 2), was investigated according to previously described methodology (Gardner et al., 1995). Perfusate samples were collected at 0, 2, 5, 10, 25, 45, 60, 75, and 90 min postdose from the portal supply. Bile was collected predose and then continuously from 0 to 90 min. Perfusate (0.15 ml) and bile samples (0.01 ml) were analyzed as described above for rat plasma. Hepatic extraction was calculated from perfusate clearance divided by perfusate flow (15 ml/min).

Plasma Protein-Binding Determinations. Samples of rat, dog, and human plasma (1 ml, n = 5) containing [¹⁴C]UK-294,315 (200-500 ng/ml) were dialyzed (Spectrapor-1 dialysis membrane, 6000-8000 mol. wt. cut-off; Spectrum Medical Industries, Rancho Dominguez, CA) against isotonic Krebs-Ringer buffer (1 ml, pH 7.4) for 4 h at 37°C in a rotating dialyzer (DiaNorm, NBS Biologicals, Ltd., Huntingdon, UK). After dialysis, concentrations of drug in plasma (0.5 ml) and buffer (0.5 ml) were measured by liquid scintillation counting in Starscint (Canberra Industries). Plasma protein binding values were calculated using eq. 1:

(1)

Caco-2 Studies. Caco-2 cells (America Type Culture Collection, Manassas, VA) were seeded in 24-well Falcon Multiwell plates (polyethylene terephthalate membranes, pore size 1.0 µM) at 4.0 x 10⁴ cells/well. The cells were grown in culture medium consisting of minimum essential medium (21090-022; Invitrogen, Carlsbad, CA)-supplemented 20% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, and 2 mM sodium pyruvate. The culture medium was replaced three times every week, and the cells were maintained at 37°C, with 5% CO₂ and at 90% relative humidity. Permeability studies were conducted when the monolayers were between 15 and 18 days old. Cells were used between passages 23 and 40. UK-294,315 was prepared as 5, 10, 25, 50, 100, and 200 µM solutions in the transport buffer Hanks' balanced salt solution (HBSS) at pH 7.4. Before commencing the study, each monolayer was washed three times with HBSS. Control transport buffer was placed in each acceptor well, 250 µl on the apical side and 1 ml into the basolateral well. The study was commenced by adding UK-294,315 solution to each donor well, 250 µl to the apical wells, and 1 ml to the basolateral well. A Tecan Genesis robot (Tecan, Durham, NC) was used for all pipetting steps. After a 2-h incubation at 37°C, samples were removed from all wells and mixed with acetonitrile (final concentration 20% v/v) before analysis. The effects of the P-gp inhibitors verapamil and CP-100,356 (Wandel et al., 1999) on apical to basolateral (A-B) and basolateral to apical (B-A) flux of UK-294,315 (25 µM) were investigated by including the inhibitors in the apical and basolateral solutions at concentrations of 100 µM (verapamil) and 10 µM (CP-100,356). Membrane integrity was determined after the 2-h incubation with UK-294,315 using Lucifer yellow added to the apical wells at a concentration of 100 µM (Irvine et al., 1999). The concentration of UK-294,315 in each sample was analyzed by direct injection of HBSS onto a desalting column-switching chromatography system with a tandem mass spectrometry endpoint. The chromatography column used was Hypersil HS C18 (50 x 4.6 mm; Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA). The HBSS was directly injected onto the column, and the salts were then washed to waste with an aqueous mobile phase consisting of 90:10 water-methanol with added 0.027% formic acid and 2 mM ammonium acetate. The mobile phase was then switched to 90:10 methanol-water, again with added 0.027% formic acid and 2 mM ammonium acetate. Flow rate was constant at 2 ml/min. UK-294,315 was detected by an API 2000 mass spectrometer with an ionspray interface (PerkinElmerSciex Instruments, Boston, MA) using specific tandem mass spectrometry transitions. The amount measured in each sample was measured from a calibration line that was constructed from known standards. Apparent permeability (P_app; x 10^-6 cm/s) values were calculated using eq. 2. Q is the amount of compound in the acceptor well at the end of the experiment in nmol, t is the duration of the experiment in seconds, C_o is the initial concentration expressed in nmol/ml, and A is the surface area of each well in cm².

(2)

P-gp Binding Affinity. Affinity of UK-294,315 for human P-glycoprotein (P-gp) was determined using a previously described method of measuring phosphate release from P-gp membranes (Abel et al., 2001). Apparent kinetic parameters K_m and V_max were determined using Grafit 3.0 and the Michaelis-Menten equation (eq. 3; where, and V_max are the observed and maximal rates of inorganic phosphate release, respectively, K_m is the Michaelis constant equal to the concentration at half the maximal rate of inorganic phosphate release, and [S] is the concentration of UK-294,315).

(3)

	Results

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Plasma Protein Binding. UK-294,315 was highly bound to plasma proteins in dog and human, with values of 95 and 93%, respectively. Binding was lower in rat plasma at 83%.

Caco-2 and P-gp Affinity Studies. Caco-2 flux data are presented in Fig. 2. A-B P_app values increased with increasing UK-294,315 concentrations, from 6.7 x 10^-6 cm/s at 5 µM to 15.7 x 10^-6 cm/s at 200 µM. B-A flux was not markedly affected by increasing substrate concentration. Despite an efflux ratio (B-A/A-B) of 4 at low drug concentrations (5 µM)², A-B and B-A fluxes were similar at high drug concentrations (100 and 200 µM) (Fig. 2). Efflux ratio at 25 µM was reduced from 2.4 to 1.1 and 1.3, respectively, by the P-gp inhibitors CP-100,356 and verapamil, although only the CP-100,356 result showed statistical significance (p < 0.05). A corresponding increase in A-B P_app values was observed, from 10 x 10^-6 cm/s (control) to 15 x 10^-6 cm/s (CP-100,356) and 13 x 10^-6 cm/s (verapamil). The K_m for UK-294,315 in the P-gp binding affinity assay was 15 µM (n = 3).

View larger version (19K): [in this window] [in a new window]   FIG. 2. Caco-2 monolayer flux of UK-294,315 in A-B and B-A directions over a concentration range of 5 to 200 µM (n = 3).

Pharmacokinetics. Single-dose plasma pharmacokinetic data for rat and dog are presented in Table 1. Based on the physicochemistry of UK-294,315, blood-plasma partitioning was assumed to be 1:1. After intravenous administration, UK-294,315 exhibited high clearance of 91 ml/min/kg in rat and moderate clearance of 15 ml/min/kg in dog. Volume of distribution was moderately large, with values of 5.6 liters/kg in rat and 4.8 liters/kg in dog. The lower clearance in dog resulted in a longer half-life in this species (6.3 h) than in rat (1.0 h). Oral bioavailability in rat was 11%. This value is in keeping with the high systemic clearance in this species (approximately 90% liver blood flow), resulting in extensive first-pass extraction. Oral bioavailability in dogs was high at 71%, which is also in keeping with the moderate systemic clearance observed in this species (approximately 30% liver blood flow), resulting in a lower first-pass effect than observed in the rat.

In toxicology studies there were no sex-dependent pharmacokinetics, and therefore male and female data were combined. In the rat, mean C_max values were 116 (± 47), 322 (± 152), and 1411 (± 659) ng/ml for the 10, 25, and 75 mg/kg/day dose groups, respectively. Corresponding AUC_0-T values were 1031 (± 316), 3106 (± 989), and 21,779 (± 11,173) ng · h/ml, respectively. Thus, exposure in the rat increased in a dose-proportional manner from 10 to 25 mg/kg/day but increased superproportionally from 25 to 75 mg/kg/day. In the dog, mean C_max and AUC_0-T values were 710 ± 137 ng/ml and 3548 ± 759 ng · h/ml, 1743 ± 1036 ng/ml and 8335 ± 4223 ng · h/ml, and 3930 ± 2192 ng/ml and 14,487 ± 8017 ng · h/ml for the 2.5, 5, and 10 mg/kg/day dose groups, respectively. Exposure in the dog increased in a dose-proportional manner across the dose range studied.

In humans, across an oral dose range of 1 to 100 mg, UK-294,315 exhibited a mean elimination half-life of 11.6 ± 1.4 h (Table 2), with approximately 80% of total AUC_0-T obtained within 18 h. Although AUC_0-T increased proportionally with dose from 1 to 100 mg, peak plasma concentration (C_max) increased superproportionally up to a dose of 50 mg, with a 220-fold increase in C_max for a 50-fold increase in dose. Between 50 and 100 mg, C_max increased in a dose-proportional manner (Fig. 3). The time to reach C_max (T_max) was variable, decreasing from 3.8 h at 1 mg to a plateau of 0.6 ± 0.2 h for all doses above and including 30 mg (Fig. 4).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Mean plasma exposure after administration of single oral solution doses of UK-294,315 to healthy volunteers; AUC0-T versus dose (a) and Cmax versus dose (b). Dotted line indicates the regression line, based on 1- to 10-mg doses.

View larger version (9K): [in this window] [in a new window]   FIG. 4. Mean Tmax versus dose after administration of single oral solution doses of UK-294,315 to healthy volunteers. Error bars indicate standard deviation of the mean.

Radiolabeled Studies with [¹⁴C]UK-294,315 in Animals. After administration of [¹⁴C]UK-294,315 to rats, excretion balance and metabolite profiling data were both quantitatively and qualitatively similar between the sexes, and therefore, data for the four animals were combined. Recovery of radioactivity was complete after intravenous and oral administration, with the majority of the dose being excreted in the feces (Table 3). Renal elimination of radioactivity was low (<6% of dose). The majority of radioactivity was recovered within 24 h. Of the radioactivity excreted in the feces, 29.0 ± 2.9% of dose was characterized by mass spectrometry as unchanged UK-294,315 after intravenous administration, indicating that a substantial proportion of systemic clearance is mediated via secretion of unchanged drug into the gut. The remainder of the dose in feces was present as a mixture of oxidative metabolites. After oral administration (4 mg/kg), a higher proportion of the dose was excreted in feces as unchanged UK-294,315 (60.1 ± 21.0% of dose) (Table 4). Assuming that approximately 29% of whatever is absorbed will be excreted unchanged in the feces (based on intravenous data), the amount of unchanged drug in the feces after oral administration suggests that approximately 56% of the administered dose was absorbed in the rat (i.e., 60% unchanged drug in the feces can be accounted for by 44% not being absorbed, in combination with [56% absorbed x 0.29 =] 16% via absorption and then biliary and/or gut wall secretion into the feces). These data indicate that oral absorption of UK-294,315 in the rat is incomplete at a dose of 4 mg/kg.

In the dog, recovery of radioactivity was complete after intravenous or oral administration of [¹⁴C]UK-294,315, and the majority of radioactivity was recovered within 48 h. Elimination was mainly fecal after intravenous and oral administration (Table 3). Renal elimination accounted for <9% of the dose. Radiometric HPLC confirmed that UK-294,315 was excreted unchanged in the feces to a similar extent after intravenous and oral administration (13.7%, range 10.9-16.5; and 15.0%, range 13.6-16.3, respectively) (Table 4), indicating complete absorption in the dog. The identity of UK-294,315 was confirmed by mass spectrometry.

Isolated Perfused Rat Liver Experiments. Clearance of UK-294,315 in the isolated perfused rat liver preparations was 7.4 to 8.4 ml/min. Hepatic extraction was 49 to 56%, which is in keeping with the high in vivo clearance in rats (91 ml/min/kg). Bile flow was 1 ml/h in both preparations, which is consistent with physiological parameters in vivo (Davies and Morris, 1993). Elimination of unchanged drug in the bile was low (<5% of dose).

Radiolabeled Studies with [¹⁴C]UK-294,315 in Humans. After oral administration of 20 mg of [¹⁴C]UK-294,315 to healthy volunteers, recovery of radioactivity was complete (101.8 ± 5.9% of dose), the majority of which was excreted within 96 h postdose. As observed in rat and dog, elimination was mainly fecal (97.5 ± 5.9% of dose) with <5% of dose eliminated in the urine. Radioactivity in feces comprised several components, including unchanged UK-294,315 which accounted for 14% of the dose.

	Discussion

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UK-294,315 is rapidly cleared in the rat, resulting in a short half-life (1 h), despite a relatively large volume of distribution. Absorption of UK-294,315 in the rat is incomplete (approximately 56%). In this species, approximately 60% of the dose was found as unchanged drug in the feces after an oral dose as a result of secreted drug and dose not absorbed. It is likely that a significant proportion of this is due to intestinal secretion, since 29% of the dose appears unchanged in feces after an intravenous dose, and biliary secretion in an isolated perfused rat liver preparation was low. This intestinal secretory process may act as an active barrier to absorption across the gut wall. In keeping with this hypothesis, and observations in the rat for the intestinally secreted compound paclitaxel (Kimura et al., 2002), absorption of UK-294,315 in the rat was slow (T_max, 4 h). It is unlikely that solubility limits the absorption of UK-294,315 in the rat, given that aqueous solubility in water is 0.74 mg/ml, increasing to >40 mg/ml at pH 5. Indeed, oral exposure in toxicology studies increased proportionally up to 25 mg/kg. On increasing the dose from 25 to 75 mg/kg, a superproportional increase in both C_max and AUC_0-T was observed, suggesting saturation of a first-pass process. This is probably an effect on hepatic metabolism, due to very high oral doses used in this study (50-fold greater than the highest dose studied in humans), but may also reflect saturation of an absorption barrier as discussed above.

In the dog, UK-294,315 exhibited a similar volume of distribution as observed in the rat. This is indicative of good tissue penetration, which is in keeping with the moderately lipophilic and basic nature of the compound (log of octanol/water partition coefficient at pH 7.4, 1.8; pK_a, 8.5). However, in marked contrast to the rat, rapid and complete oral absorption is indicated in the dog. This observation is based upon short T_max (0.75 h) and comparison of oral bioavailability (71%) with systemic clearance (approximately 30% liver blood flow) and low levels of unchanged UK-294,315 present in feces after oral administration (15% dose). Indeed, the extent of fecal elimination of unchanged UK-294,315 in dogs was similar after intravenous and oral administration, suggesting that drug clearance, rather than incomplete absorption, is the source of parent drug in feces after oral dosing. In addition to complete and rapid oral absorption, exposure in dogs (C_max and AUC_0-T) increases proportionally with dose from 2.5 to 10 mg/kg, demonstrating linear oral pharmacokinetics in this species. However, absorption of drugs in dogs is often high, due to large aqueous pore size and frequency of pores in the intestinal epithelium (He et al., 1998), allowing for increased paracellular absorption compared with other species (e.g., rat and human). Therefore, transcellular barriers (e.g., poor membrane permeability, intestinal secretion processes) that may limit oral absorption in rat or human are unlikely to be limiting in the dog due to an alternative facile route across the gut wall in this species. This is likely to be the underlying factor responsible for the dog's propensity for markedly overpredicting human absorption in some cases (Chiou et al., 2000).

UK-294,315 appears to be extensively absorbed in humans, with only a small proportion of [¹⁴C]UK-294,315 excreted unchanged in feces after oral administration, accounting for approximately 14% of the dose. Assuming no metabolism by gut microflora, the extent of absorption of UK-294,315 in humans is therefore estimated to be at least 86%. However, a nonproportional relationship between dose and oral absorption parameters was observed in clinical studies. In humans, the shape of the pharmacokinetic profile changed dramatically from a flat, prolonged profile at lower doses to a sharp peak with rapid decline in concentrations at higher doses (Fig. 5). A decrease in T_max from 3.8 to 0.5 h was observed with increasing dose (1-100 mg), which is indicative of slower absorption at lower doses (Fig. 4), in contrast to the good intrinsic membrane permeability of the compound. At doses 30 mg in humans, T_max of approximately 0.5 h was consistently observed, suggesting saturation of the limiting process had occurred. The decrease in T_max was accompanied by a superproportional increase in C_max as dose increased from 1 to 50 mg. However, increases in C_max became dose-proportional from 50 to 100 mg (Fig. 3), in line with changes in T_max, again suggesting saturation of a process that was limiting absorption rate at lower doses. Regardless of the nonlinearity observed in C_max and T_max, systemic exposure (AUC_0-T) was dose-proportional across the whole dose range (Fig. 3). Therefore, saturation of first-pass metabolism is unlikely to contribute significantly to the nonlinearity observed in the oral disposition of UK-294,315, where rate but not extent of absorption is affected at the lower end of the dose range.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Mean pharmacokinetic profiles after oral administration of UK-294,315 in solution to man at doses of 3 mg () and 100 mg (). Profiles are normalized to a dose of 100 mg.

The nonlinear behavior of UK-294,315 in humans was reflected in vitro in Caco-2 cells, where flux in the A-B direction was found to be concentration-dependent, increasing from 6.7 x 10^-6 cm/s at 5 µMto 15.7 x 10^-6 cm/s at 200 µM. Correspondingly, the efflux ratio (B-A/A-B) significantly decreased from 4-fold at 5 µM to near unity at 100 and 200 µM. The higher efflux ratio at low drug concentrations, with saturation as concentrations increase, is indicative of active transport-mediated efflux. UK-294,315 is a substrate for the transport protein P-gp (K_m, 15 µM), and it is likely that this is involved in transporting the drug out of the cells. This is supported by saturation of efflux occurring at concentrations spanning the P-gp K_m. Furthermore, studies in Caco-2 monolayers with the P-gp inhibitors verapamil and CP-100,356 (Kajiji et al., 1994; Pauli-Magnus et al., 2000) resulted in a reduction of efflux ratio to near unity. Since UK-294,315 is a P-gp substrate and this property is considered responsible for nonlinearity in Caco-2 experiments, it is likely that this is also the case in humans. P-gp is expressed on the apical membrane of intestinal epithelial cells and can act as an efflux pump, intercepting the drug molecule on its passage through the cell membrane and returning it to the gut lumen (Hunter and Hirst, 1997).

Nonlinear behavior in humans is observed at doses below approximately 30 mg, corresponding to estimated gut concentrations of 10 to 235 µM.³ Interestingly, this overlaps with the concentration range studied in Caco-2 experiments (5-200 µM) (Fig. 6), linking the effects seen in clinical trials to saturation of efflux in Caco-2 cells, which in turn is related to concentrations exceeding the K_m for UK-294,315 versus P-gp. These data suggest that P-gp delays absorption of UK-294,315 by recycling the compound between the gut lumen and gut epithelium. These data also suggest that UK-294,315 does not undergo major gut wall metabolism since systemic exposure (AUC_0-T) remains linear over the entire dose range, despite variability in absorption rate. Saturation of P-gp by a substrate for cytochrome P450-mediated gut wall metabolism can lead to nonlinear increases in the extent of absorption (e.g., UK-343,664; Abel et al., 2001). -Adrenergic receptor antagonists as a class are generally administered using a gastrointestinal, modified release formulation to improve the safety and/or pharmacokinetic profile of the drugs (e.g., doxazosin, prazosin, alfuzosin) (Elliott et al., 1988; Cases, 2000; Van Kerrebroeck et al., 2002). The fundamental principle of modified release is to delay the absorption rate of a drug, and therefore, such formulations would attenuate nonlinearity in absorption rate, allowing development of compounds such as UK-294,315.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Comparison of UK-294,315 Caco-2 efflux ratio/apical chamber concentration relationship (), with estimated gut lumen concentrations after single oral solution doses of UK-294,315 to humans (gray bars).

It is possible that the variable absorption rate of UK-294,315 in humans is a consequence of transporter proteins other than P-gp. Indeed, a range of transporters has been identified which are expressed in both Caco-2 cells and are abundant in human jejunal tissue (e.g., BCRP and MRP2) (Taipalensuu et al., 2001), although regional variation in expression and function of these proteins in the human intestine is unknown.

Once in solution, the absorption of a drug candidate is dependent on a number of factors. These can combine to produce various undesirable absorption characteristics; e.g., UK-224,671, where a combination of P-gp affinity (resulting in high efflux from Caco-2 cells) and low intrinsic membrane permeability is a major factor driving low absorption (<10%) of the drug in humans (Beaumont et al., 2000a,b), and UK-343,664 where saturation of P-gp leads to nonlinearity in extent of absorption due to effects on gut wall metabolism of this compound. These examples, including UK-294,315, serve to demonstrate that the actual absorption profile depends on intrinsic membrane permeability, transporter affinity, and cytochrome P450 affinity.

In conclusion, these studies demonstrate that P-gp (and/or other transport proteins) is likely to be responsible for the nonlinearity in rate of absorption of UK-294,315 but does not act to limit the extent of absorption in humans due to the favorable physicochemical properties of the drug and possible regional variation in functional activity of this transporter protein through the gut. Our current understanding of efflux due to P-gp in the intestine, from regional intestinal strips and cell lines, helps to rationalize the clinical data for UK-294,315. Preclinical pharmacokinetic studies in dogs (and, to a lesser extent, rats) provided little insight prospectively or retrospectively in predicting or understanding the nonlinear oral disposition of UK-294,315 in human. UK-294,315 exhibits linear oral pharmacokinetics in dogs (over the dose range 2.5-10 mg/kg), albeit at higher doses than studied in humans. Furthermore, the reported overprediction of human oral absorption from dog data (Chiou et al., 2000) reduces confidence in using this species for evaluating absorption potential. Although nonlinear oral pharmacokinetics were observed in rat studies, it is unclear whether this is a result of saturation of first-pass metabolism or saturation of the absorption-limiting process in this species. Additionally, doses at which this occurred in the rat were >50-fold higher than the top dose studied in humans, again questioning the relevance of the observation in this species to the human situation. For future drug candidates, it is unlikely that the preclinical studies carried out with UK-294,315 in rat and dog could contribute further to predicting nonlinear oral absorption without increasing the usage of animals to allow wider dose ranges (allowing lower doses comparable with those studied in humans) and characterization of animal transporter proteins to assess relevance to humans. Although the animal studies were not predictive of absorption in humans, they were pivotal in identifying the potential for UK-294,315 to have a suitable duration of action (based on plasma half-life). Indeed, the half-life in dog was indicative of that observed in humans (6.1 and 12 h, respectively). Pharmacokinetic estimates for humans (i.e., half-life) based primarily on dog pharmacokinetic studies played a key role in the decision to develop UK-294,315.

The studies that we have undertaken suggest that the use of in vitro data alone, incorporating physicochemical measurements, P-gp affinity, and Caco-2 flux experiments across a clinically relevant concentration range, can highlight potential absorption problems (in particular, nonlinear pharmacokinetics) in humans with compounds before progression to clinical trials. This type of analysis can therefore provide a powerful tool for the drug discovery/development process in selecting drug candidates with suitable absorption properties.

	Acknowledgments

 
We gratefully acknowledge many Pfizer colleagues for their scientific and practical input to this paper. In particular, we thank Joanne Bennett for initial Caco-2 observations, Sarah Kempshall and Dawn Halton for Caco-2 and P-gp affinity data, Mark Savage and Pat Wright for metabolite profiling and mass spectrometry analysis, Jenny Gedge for radiolabeled study data, Tim Letby for generating preclinical pharmacokinetic data, Fidelma Atkinson for isolated perfused rat liver data, and Don Walker for scientific discussion and guidance in preparation of the manuscript.

	Footnotes

 
¹ Abbreviations used are: UK-294,315, 4-amino-5-(4-fluorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline; P-gp, P-glycoprotein; UK-298,108, 4-amino-5-(4-chlorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline; CP-100,356, [4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-6,7-dimethoxy-quinazolin-2-yl]-[2-(3,4-dimethoxy-phenyl)-ethyl]-amine; HPLC, high-performance liquid chromatography; k_el, terminal elimination rate constant; AUC_0-T, area under the plasma concentration-time curve; AUC_0-, area under the plasma concentration-time curve, extrapolated to infinity; HBSS, Hanks' balanced salt solution; A-B, apical to basolateral; B-A, basolateral to apical;P_app, apparent permeability;T_max, time to maximum concentration.

² In this laboratory, the P-gp substrate talinolol exhibits an A-B flux of <1 and B-A flux of 13 x 10^-6 cm/s across Caco-2 cells at a substrate concentration of 25 µM.

³ Estimated from dose dissolved in a combination of dose solution volume (250 ml) and upper gastrointestinal tract fluid volume (120-350 ml; taken from Dressman et al., 1998). Maximum concentration is calculated from the dose solution alone.

Address correspondence to: Dr. Anthony Harrison, Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK. E-mail: anthony_harrison{at}sandwich.pfizer.com

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