Preclinical Drug Metabolism and Pharmacokinetic Evaluation of GW844520, A Novel Anti-Malarial Mitochondrial Electron Transport Inhibitor HONG XIANG,1 JEANELLE MCSURDY-FREED,1 GANESH S. MOORTHY,1 ERIN HUGGER,1 RAMESH BAMBAL,1 CHAO HAN,1 SANTIAGO FERRER,2 DOMINGO GARGALLO,2 CHARLES B. DAVIS1 1
Drug Metabolism and Pharmacokinetics, Microbial, Musculoskeletal and Proliferative Diseases, Center of Excellence for Drug Discovery, GlaxoSmithKline R&D, 1250 South Collegeville Rd, Collegeville, PA 19426 2
Center for Diseases of the Developing World, GlaxoSmithKline R&D, 28760 Tres Cantos, Madrid, Spain
Received 8 November 2005; revised 2 May 2006; accepted 3 May 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20681
ABSTRACT: GW844520 is a potent and selective inhibitor of the cytochrome bc1 complex of mitochondrial electron transport in P. falciparum, the parasite primarily responsible for the mortality associated with malaria worldwide. GW844520 is fully active against the parasite including resistance isolates, showing no cross resistance with agents in use. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-malarial. GW844520 had low blood clearance of about 0.5–4% of hepatic blood flow and a steady-state volume of distribution of 2–4 times total body water in mouse, rat, dog, and monkey. Oral bioavailability was high (51–100%). Consistent with the in vivo data, GW844520 had low intrinsic clearance in liver microsomes and hepatocytes of animal and human origin, high passive cellular permeability and was not a P-glycoprotein substrate. GW844520 did not associate appreciably with blood cells but was highly bound to plasma proteins (>99%) in all species. GW844520 was a substrate and inhibitor of human CYP2D6 but not of CYP1A2, 2C9, 2C19, and 3A4. This conjunctive analysis supports continued evaluation of this compound in definitive pre-IND studies and exemplifies our strategy supporting the discovery of novel agents to treat diseases of the developing world. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:2657–2672, 2006
Keywords: pharmacokinetics; clearance; metabolism; Cytochrome P450; protein binding; bioavailability; permeability; antimalarial; 4(1H)-pyridone; blood to plasma partitioning
INTRODUCTION
GW844520 was awarded ‘‘Project of the Year’’ 2003 by Medicines for Malaria Venture Abbreviations: CL, blood clearance; Vss, volume of distribution at steady-state; T1/2, apparent terminal half-life; Cmax, maximum observed blood concentration; Tmax, time to observed Cmax; AUC0–1, total area under the blood concentration-time curve from time zero to infinity; MDR, multidrug resistance. Correspondence to: Hong Xiang (Telephone: þ610-917-5198; Fax: þ610-917-7005; E-mail:
[email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 2657–2672 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association
Malaria is endemic to the poorest countries in the world, causing 300–500 million clinical cases and more than 1 million deaths directly each year. More than 90% of the cases and the deaths occur in Sub-Saharan Africa and almost all the deaths are in children under the age of 5 years. Plasmodium falciparum is the species of malaria parasite that predominates in Africa and is the cause of most of the mortality from malaria worldwide. Effective treatment options are often restricted due to drug resistance, particularly for
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covery of novel agents to treat diseases of the developing world.
MATERIALS AND METHODS Chemicals Figure 1. Structure of GW844520.
P. falciparum. There is an immediate need to develop novel anti-malarial agents that provide rapid and effective treatment.1 GW844520 (Fig. 1), a non-chiral 4(1H)-pyridone derivative, is a potent, selective inhibitor of Plasmodium mitochondrial function blocking the electron transport chain machinery. The site of action of this class of compounds is cytochrome b, a critical element of the respiratory complex III or bc1 complex. This complex is the target of atovaquone which has been in clinical use for many years.2 The IC50 of GW844520 against cytochrome bc1 complex of P. falciparum was 2 nM, 10-fold higher than that for atovaquone (data on file). However, GW844520 is fully active against isolates carrying resistance determinants to marketed agents (IC50 of GW844520 against various resistant strains of P. falciparum was 2.5– 7.6 nM, data on file) and shows no cross-resistance with agents in current use, including atovaquone. Lack of synergy with proguanil further suggests a novel mechanism of action for GW844520. GW844520 is effective in murine models of P. falciparum and P. yoelli infection following oral administration (data on file). In preparation for preclinical safety testing and clinical evaluation, we have investigated the drug metabolism and pharmacokinetic properties of this novel anti-malarial. The in vivo pharmacokinetics were evaluated following single intravenous and oral administration to the mouse, rat, dog, and monkey. Protein binding and blood-cell association were investigated in the plasma or whole blood from preclinical species and human, in vitro. The routes and rates of metabolism of GW844520 were studied in animal and human liver microsomes and hepatocytes. Concentration- and timedependent human cytochrome P450 inhibition, permeability and efflux transport were investigated in vitro. The results of these comprehensive studies are herein presented. A conjunctive analysis supports continued evaluation of this compound in definitive pre-IND studies and exemplifies our strategy for supporting the dis-
GW844520 was obtained from the Center for Diseases of the Developing World, GlaxoSmithKline (Tres Cantos, Spain). Male CD1 mouse, male Sprague–Dawley rat, male beagle dog, male cynomolgus monkey and mixed gender human liver microsomes were obtained from XenoTech LLC, Kansas City, KS. Microsomes were stored at approximately 808C, then thawed in a 378C water bath for 5 min just prior to use. Hepatocytes from male CD1 mouse, male Sprague– Dawley rat, male Beagle dog, male cynomolgus monkey, and human were from Cedra (Austin, TX). b-nicotinamide adenine dinucleotide phosphate (NADP), uridine 50 -diphosphoglucuronic acid (UDPGA), D-glucose-6-phosphate and glucose6-phosphate dehydrogenase were from Sigma (St Louis, MO). Basic William’s medium E and Hank’s Balanced Salt Solution (HBSS) were from JRH Biosciences (Lenexa, KS). Microsomes prepared from human lymphoblast cells expressing individual recombinant human CYP450 isozymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were obtained from BD Gentest (Woburn, MA) and stored approximately 808C. MDR1MDCKII cells were obtained through a material transfer agreement as described previously.3 Twelve-multiwell Transwell1 systems (catalog No. 3401; 0.4 mm pore size, 1.134 cm2 surface area) were obtained from Corning Inc. (Corning, NY). GF120918 and amprenavir were prepared at GSK. Dulbecco’s Modified Eagle Medium (DMEM) with glutamine, 10% fetal bovine serum, 50 U/mL penicillin and 50 mg/mL streptomycin was from Invitrogen (Grand Island, NY). Lucifer yellow was from Sigma. Cavitron (hydroxypropyl b-cyclodextrin) was purchased from Cerestar USA, Inc. (Hammond, IN). All other chemicals and reagents were of standard laboratory reagent grade or better. Animal Husbandry and Handling Male Sprague–Dawley rats were obtained from Charles River Laboratories (Kingston, NY) and housed individually in polycarbonate cages in unidirectional air flow rooms (25 28C, relative humidity 50 10%, 12-h light/dark cycle).
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Extruded diet #5L35 was available ad libitum. Male CD-1 mice were obtained from Charles River Laboratory (Raleigh, NC) and housed as were the rats. Certified rodent chow No. 5001 was available ad libitum. Male Beagle dogs were obtained from Marshall Farms (North Rose, NY) or Covance Research Products (Cumberland, VA), grouphoused until placed on study, then individually housed in stainless-steel cages in an environmentally controlled room (18–298C; relative humidity 30–70%, 12-h light/dark cycle). Dogs received 2.5–3 cups certified canine diet No. 5007 once daily. Male cynomolgus monkeys were obtained from Primate Products Incorporated (Miami, FL) or Covance Research Products Inc. (Alice, TX) and individually housed in stainless-steel cages in a controlled environment (18–298C, relative humidity 30–70%, 12-h light/dark cycle). Each monkey received 6–8 biscuits certified primate chow No. 5048 from Purina Mills twice daily plus two pieces of produce. All animal food was purchased from Purina Mills (St. Louis, MO). In all cases, filtered tap water was available ad libitum. In all studies, and for each study leg where appropriate, animals were fasted overnight (12–14 h) and food was provided 4 h post-dose. Animals were appropriately acclimated to handling procedures and restraint devices prior to study. Pharmacokinetics in the Mouse Thirty-six mice (20–30 g) received GW844520 (0.25 mg/kg, 5 mL/kg) by bolus injection in the tail vein. Thirty animals (fasted) received GW844520 (0.5 mg/kg, 10 mL/kg) by oral gavage. The formulation for iv and oral administration was 1% (v/v) DMSO, 20% (v/v) Cavitron, 7.5% (v/v) PEG 400 in isotonic saline. Blood samples (0.4 mL) were collected terminally by cardiac puncture following anesthetization in a CO2 chamber at 5, 10, 30, 60 min, 2, 4, 8, 24, 36, 48, 72, and 96 h following iv administration or at 30, 60 min, 2, 4, 8, 24, 36, 48, 72, and 96 h following oral administration. Blood samples were collected into tubes containing heparin and placed on crushed ice promptly after collection. An aliquot of each blood sample was diluted with an equal volume of de-ionized water, snap frozen and stored at about 308C prior to analysis.
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anesthetized using ketamine (87 mg/kg im; Ketaset, Aveco Co., Fort Dodge, IA)/xylazine (13 mg/kg im; Rompun, Haver, Shawnee, KS) or isoflurane (3% in oxygen; IsoFlo1, Abbott Laboratories, North Chicago, IL). General anesthesia was maintained with the same agents. The rats were allowed to recover from surgery for at least 48 h. The formulation was the same as that described for the mouse. Five animals received GW844520 (0.25 mg/kg, 5 mL/kg) by iv bolus injection and five animals (fasted) received GW844520 (0.5 mg/kg; 10 mL/kg) by oral gavage. Blood samples (0.15 mL) were collected from the femoral arterial catheter of each rat prior to dosing and at 5, 10, 30, 60 min, 2, 4, 8, 24, 36, 48, 72, 96, and 120 h following iv administration. Following oral administration, blood samples were collected from the arterial catheter of each rat prior to dosing and 10, 30, 60 min, 2, 4, 8, 24, 48, 72, 96, and 120 h following administration. Samples were processed and stored as described above for the mouse studies. Pharmacokinetics in the Dog The study was conducted using a crossover design (n ¼ 3, 9–14 kg) with a 6-day washout period between dose sessions. Dogs were restrained in slings for dosing and sample collection only. On study Day 1, each animal received GW844520 (0.25 mg/kg, 5 mL/kg) as a 60-min infusion into a cephalic vein. On study Day 2, each animal received GW844520 (0.4 mg/kg, 8 mL/kg) by oral gavage. The formulation was the same as that described for the mouse studies (iv dose solutions were sterile-filtered). Blood samples were collected by venipuncture of either the contralateral cephalic or jugular vein (0.4 mL) prior to dosing and at 20, 40, 60, 70, 90 min, 2, 4, 8, 24, 36, 48, 72, 96, and 120 h following iv administration. Urine samples were collected prior to dosing and at 0– 12, 12–24, 24–48, 48–72, 72–96, 96–120 h following iv administration. For the oral leg, blood was collected prior to dosing (144 h after the initial iv dose) and at 10, 30, 60 min, 2, 4, 8, 24, 36, 48, 72, 96, 120, 509, and 768 h following iv administration. Blood samples were processed and stored as described above for the mouse studies. Urine samples were snap frozen and stored at 308C prior to analysis.
Pharmacokinetics in the Rat Ten rats (300–360 g) had cannulae surgically implanted in the femoral vein and artery aseptically. On the day of surgery, the rats were DOI 10.1002/jps
Pharmacokinetics in the Monkey The study was conducted using a crossover design (n ¼ 3, 2.5–5 kg) with a 2-week washout period
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between dose sessions. On study Day 1, each animal received GW844520 (0.25 mg/kg, 5 mL/kg) as a 60-min infusion into a cephalic vein. On study Day 2, each animal received GW844520 (0.4 mg/kg, 8 mL/kg) by oral gavage. The formulation was the same as that described for the mouse studies (iv dose solutions were sterilefiltered). Blood samples (0.1 mL) were collected by a venous catheter and vascular access port prior to dosing and at 20, 40, 60, 70, 90 min, 2, 4, 8, 24, 36, 48, 72, 96, 120, and 240 h following iv administration. Urine samples were collected prior to dosing and at 0–12, 12–24, 24–48, 48–72, 72– 96, 96–120 h following iv administration. For the oral leg, blood was collected prior to dosing and at 10, 30, 60 min, 2, 4, 8, 24, 36, 48, 72, 96, 120, 192, 264, 360, and 432 h following administration. Blood samples were processed and stored as described above for the mouse studies. Urine samples were snap frozen and stored at 308C prior to analysis. Pharmacokinetic Analysis GW844520 blood concentration-time data were analyzed by non-compartmental methods using the computer program WinNonlin Professional (version 3.2). Area under the blood concentrationtime curve was calculated using the linear trapezoidal rule for each incremental trapezoid up to Cmax, and the log trapezoidal rule for each trapezoid thereafter. The terminal elimination rate constant (lz) was derived from the log-linear disposition phase of the concentration-time curve using least-squares regression analysis with visual inspection of the data to determine the appropriate number of terminal data points for regression analysis. AUC0–1 was estimated as the sum of AUC0–t and Ct/lz, where Ct was the predicted concentration from the log-linear regression analysis at the last measurable time point. The elimination half-life (T1/2) was calculated as ln 2/lz. Following iv administration, the mean residence time (MRT), total body clearance (CL), and the volume of distribution at steady-state (Vss) were also estimated. CL was calculated by dividing the dose by AUC0–1. MRT was estimated as ([AUMC0–1/AUC0–1]—infusion time/2). Vss was calculated using the following equation: Vss ¼ CL MRT. Oral bioavailability was estimated using the ratio of dose-normalized AUC0–1 values following oral and iv administration. Due to the long half-life of GW844520 in the dog and monkey, there was drug in the pre-dose samples in the oral
dose sessions of the crossover. Oral AUC was corrected for carryover from the iv administration as appropriate. In the dog study, the terminal elimination phase following iv administration was not completely characterized with 144 h blood sampling. Therefore, the half-life from the oral data was used to estimate pharmacokinetic parameters from the iv data. Specifically, the half-life from oral data for each individual animal was used to calculate AUC that was carried over into the second dosing session (AUClast-1 ¼Clast0.693/T1/2, where Clast ¼ last measurable concentration). The total AUC following the single iv dose was estimated from AUClast and the carry-over AUC (AUClast–1). The rat and mouse PK studies were conducted with a non-crossover design. The oral bioavailability was calculated using mean iv and oral data therefore the true variability of the oral bioavailability could not be estimated. In addition, the mouse PK study used composite sampling (n ¼ 3 per time point). Data from three animals per time point were averaged for the pharmacokinetic analysis and thus variability of PK parameters could not be estimated. Renal clearance in the dog and monkey was calculated using the cumulative amount of unchanged drug in the urine (0–120 h) divided by AUC0–120 h. The percentage of the dose excreted was estimated using the cumulative amount of unchanged drug in the urine divided by the administered dose. In order to predict the clearance in humans, three allometric scaling methods were used.4 The first method employed the following equation: CL ¼ a(W)b, where W is the body weight, a and b are the coefficient and exponent factor of the allometric equation, respectively. The second method involved a correction of the clearance values for maximum life-span potential5 (MLP) using the following equations: CL MLP ¼ a(W)b and MLP (years) ¼ 185.4 (BW)0.636 W0.225, where BW ¼ brain weight. The third method employed brain weight to adjust clearance values6: BW CL ¼ a(W)b. Values for human body weight and brain weight were taken from the literature.13 Actual animal body weights were used throughout. The allometric equations were transformed to log scale and linear regression analysis was performed on the log-transformed data (clearance or modified clearance vs. body weight) to predict human clearance. The allometric exponent b is the slope of the linear regression analysis. The significance of the slope from zero was assessed
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using Splus version 6.2 (Insightful Inc., Seattle, WA). A p-value less than 0.05 was considered statistically significant for the allometric relationship and for two-tailed t-test used in the comparison of half-lives in rat and monkey. Blood Partitioning The in vitro blood partitioning of GW844520 was determined in fresh blood from rat, mouse, dog, monkey, and human (single donor) at target concentrations ranging from 0.25 to 5 mg/mL (0.5% (v/v) DMSO final). Spiked blood samples were mixed gently then incubated at 378C for 30 min. Aliquots of blood were taken and mixed with an equal volume of water and the remaining blood was centrifuged to collect plasma. Blood and plasma samples were stored frozen at approximately 308C prior to analysis. Protein Binding The in vitro plasma protein binding of GW844520 was investigated by equilibrium dialysis (3 cells/ species) in fresh rat, mouse, dog, monkey, and human plasma at 0.5 and 5 mg/mL (0.5% (v/v) DMSO final). Spiked plasma samples were mixed gently and triplicate aliquots were collected to verify initial concentrations. Following assembly of dialysis cells (Spectra/Por 4 dialysis membrane discs, molecular weight cut off limit of 12,000– 14,000 Da), spiked plasma was placed in the donor compartment of the cell and phosphate-buffered saline, pH 7.4, in the receiver compartment. Cells were incubated in a water bath at 378C and mixed continuously for 6 h. Triplicate aliquots (volume determined gravimetrically) from donor and receiver compartments were snap frozen and stored at 308C prior to analysis. Percent binding was estimated using standard equations which accounted for volume changes due to Donnan effects.7 In order to study the potential impact of high protein binding on drug efficacy across species, rodent and human protein binding in diluted plasmas (20% v/v) was also investigated. Intrinsic Clearance in Liver Microsomes and Hepatocytes Microsomal intrinsic clearance was measured as described by Clarke and Jeffrey.8 The compound (0.5 mM) was incubated with 0.5 mg/mL microsomal protein, 0.34 mg/mL NADP, 1.56 mg/mL DOI 10.1002/jps
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glucose-6-phosphate and 1.2 units/mL glucose-6phosphate dehydrogenase, 2.6 mg/mL UDPGA, 0.5% (v/v) methanol in 50 mM potassium phosphate buffer, pH 7.4, at 378C. Fifty microliters aliquots of the incubation mixture were withdrawn at various time-points over 30 min and added to 100 mL stop solution (80:20:1 (v/v/v) acetonitrile:ethanol:acetic acid) containing internal standard. Samples were snap frozen and stored at 808C until analyzed by LC/MS/MS. Prior to analysis, samples were thawed at room temperature, vortexed, then centrifuged, and the supernatant taken for analysis. No cofactor controls were also included to assess non-P450 dependent clearance. Clearance was estimated for ethoxycoumarin and testosterone in parallel to assure integrity of the microsomal preparations and acceptable inter-assay variability. pNitrophenol was used as a positive control for glucuronidation. For hepatocyte intrinsic clearance, incubations were performed with 0.5 mM GW844520 in a 0.2 million cell/mL suspension in William’s Medium E, pH 7.4. Culture plates were placed into a 378C, 5% (v/v) CO2 incubator on a shaker at 40 rpm. At various time-points up to 240 min, 50 mL aliquots were removed and added to 100 mL stop solution as described for the microsomal incubations. No cell and no drug control incubations were performed as was a positive control for activity and inter-assay variability (ethoxycoumarin). Samples were snap frozen and stored at 808C until analyzed by LC/MS/ MS. Concentrations of ethoxycoumarin were determined by HPLC (Agilent 1100, Agilent, Piscataway, NJ). The intrinsic clearance was calculated based on the method published by Obach et al.,9 using the first-order elimination rate constant for disappearance of parent compound. This was calculated from the slope of the log-transformed concentration-time curve using Grafit Version 4.0.13 (Erithacus, Middlesex, UK). Intrinsic clearance was estimated using the actual volume of the incubation and assuming 52.5 mg microsomal protein/g liver8 or 1.2 108 cells/g liver for hepatocytes (2.4 108 for dog hepatocytes); clearances were expressed in units of mL/min/g liver. The clearance was predicted based on the assumption that the drug concentration (0.5 mM) was most likely well below the Km.10 The lower limit of quantification was 0.5 mL/min/g liver and this corresponded to 5:1. The lower limit of quantification was typically 5 ng/mL and the assays were typically linear over a 100- to a 1000-fold drug concentration range depending on the matrix and the expected concentration range of the analyte. For intrinsic clearance and permeability studies, the ratio of analyte to internal standard peak area was used to estimate relative drug concentration. For biotransformation studies, the analytical HPLC system consisted of Agilent 1100 series solvent delivery system, degasser, diode array detector (Agilent), and a CTC Analytics HTS DOI 10.1002/jps
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PAL autosampler (Leap Technologies). Chromatography was performed on a 2 mm 150 mm, 3 mm, Aqua C18 column (Phenomenex, Torrance, CA) with a mobile phase containing a mixture of water with 0.1% (v/v) formic acid (solvent A) and acetonitrile (solvent B). The initial mobile phase composition was 95:5 (v/v) solvent A: solvent B (first 5 min) then the gradient progressed linearly to 100% solvent B in 15 min. The mobile phase composition was returned to the starting solvent mixture in 0.1 min and the system equilibrated for approximately 10 min between runs. A flow rate of 0.2 mL/min was employed. LC/MS/MS was conducted with a Finnigan LCQ Deca XP (Thermo Finnigan, San Jose, CA) equipped with an electrospray ion source. The effluent from the HPLC column was introduced into a diode array detector followed by the mass spectrometer atmospheric ionization source. The diode array detector response was recorded in real time by the mass spectrometer data system, which provided simultaneous detection of absorbance and MS data. The electrospray interface was operated at 5,500 V, and the mass spectrometer was operated in the positive ion mode. Data were processed with a VX1120 Gateway computer operating Xcalibur 1.2 (Thermo Finnigan). Qualitative assessment of the extent of metabolism was based on the peak intensity relative to parent.
RESULTS Pharmacokinetics Following iv administration of GW844520 to the mouse, mean blood concentrations declined in a mono-phasic fashion with a half-life of 25 h (Fig. 2, Tab. 1). Blood clearance was 0.74 mL/min/kg, which is 1% of liver blood flow in this species.13 Volume of distribution at steady-state in the mouse was 1.6 L/kg, 2-times total body water in the mouse, suggesting there was substantial penetration of GW844520 into tissues. The apparent oral bioavailability of a solution formulation in the mouse was high (72%, Tab. 2). Following oral administration, Cmax was attained at 4 h; thereafter, blood concentrations declined mono-exponentially with a half-life indistinguishable from that observed after iv administration (24 vs. 25 h, Tabs. 1 and 2, respectively). In the mouse, there was limited association of drug with blood cells as blood to plasma concentration ratios
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Figure 2. Mean (SD) blood concentration versus time profiles after iv (*) or oral (*) administration of 0.25 mg/kg GW844520 to male CD-1 mice (composite sampling, n ¼ 3 per time-point). Data from the oral leg (0.5 mg/kg) were dose-normalized for comparison with the iv data.
(B/P) were estimated to be 0.5–0.6 from in vitro incubations (Tab. 3). In the rat, following iv administration, blood concentrations declined biphasically, with
an initial half-life of 8 h (Fig. 3, Tab. 1). After 30 h, however, concentrations appeared to decline much more slowly, hovering near the limit of detection of the assay for up to 120 h (data on file). This secondary phase was not well-described in the current study, however, given that blood concentrations had declined about 50-fold during the initial 30 h, it is unlikely that this secondary phase contributed substantially to the total exposure in the rat. Mean blood clearance in the rat was 2.3 mL/min/kg (4% of liver blood flow in the rat) and the mean volume of distribution at steadystate was 2.0 L/kg (3-times total body water). As in the mouse, there was no significant association of GW844520 with blood cells, in vitro (B/P 0.5– 0.6, Tab. 3). The absorption in the rat was complete (oral bioavailability of 118% in a non-crossover study design). Cmax was attained 2 h after administration; thereafter, blood concentrations declined with a half-life similar to that observed after iv administration (mean values of 7.9 vs. 7.1 h, p > 0.1, Tabs. 1 and 2, respectively, Fig. 3). Following iv administration of GW844520 to the dog, blood concentrations declined very slowly over the initial 144 h of blood sampling decreasing 0.1, Tabs. 1 and 2, respectively). In the monkey, as in other preclinical species as well as human, there was limited association of drug with blood cells in vitro (B/P 0.6–0.7 compared to 0.5–0.6 in other species, Tab. 3).
Table 3. In Vitro Cell Partitioning of GW844520 in Animal and Human Blood*
Species Mouse Rat Dog Monkey Human
Concentration (mg/mL)
Blood to plasma ratio
0.25 2.5 0.25 2.5 0.25 2.5 0.25 2.5 0.5 5.0
0.56 0.01 0.48 0.02 0.50 0.02 0.56 0.03 0.52 0.01 0.52 0.01 0.69 0.06 0.62 0.01 0.54 0.06 0.60 0.03
*Data are expressed as the mean and standard deviation. DOI 10.1002/jps
Figure 3. Mean (SD) blood concentration versus time profiles after iv (*) or oral (*) administration of 0.25 mg/kg GW844520 to male Sprague–Dawley rats (n ¼ 5/group). Data from the oral leg (0.5 mg/kg) were dose-normalized for comparison with the iv data. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 12, DECEMBER 2006
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Figure 4. Mean (SD) blood concentration versus time profiles after iv (*) and oral (*) administration of 0.25 mg/kg GW844520 to male beagle dogs (n ¼ 3, crossover study design). Data from the oral leg (0.4 mg/kg) were dose-normalized for comparison with the iv data.
Figure 5. Mean (SD) blood concentration versus time profiles after iv (*) and oral (*) administration of 0.25 mg/kg GW844520 to male cynomolgus monkeys (n ¼ 3, crossover study design). Data from the oral leg (0.4 mg/kg) were dose-normalized for comparison with the iv data.
Three different methods were used for allometric scaling of the pharmacokinetics to human. The results are summarized in Table 4. The various approaches applied (see below) predict a low human in vivo blood clearance ranging from 0.074 to 0.14 mL/min/kg depending on the method employed (p < 0.05 for methods 2 and 3). The pvalue for the significance of the slope for these methods is listed in Table 4. Using a similar approach (method 1), the Vss in human was estimated to be 3 L/kg (significance of the slope from zero, p < 0.001).
exceeded 99.8% at a drug concentration of 0.5 mg/mL (99.8 97.5 0.2 99.7 0.1 >99.8 98.3 0.1 99.8 0.1 99.6 0.1 99.4 0.1 >99.8 99.6 0.1 >99.8 97.8 0.3 99.9 0.1
Mouse
Rat
Dog Monkey Human
*Data are expressed as the mean and standard deviation where appropriate. Protein binding was performed in undiluted plasma and for selected studies following a fivefold dilution in phosphate-buffered saline as described in the text. DOI 10.1002/jps
parent in vitro. Nonetheless, based on the in vitro human protein binding data, an intrinsic clearance value of 0.5 mL/min/g liver (the limit of quantification) and standard extrapolation techniques (well-stirred model14), hepatic clearance in humans was predicted to be
