QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.015933v1 35/10/1916    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qian, M. Articles by Christ, D. D. Search for Related Content PubMed PubMed Citation Articles by Qian, M. Articles by Christ, D. D.

Pharmacokinetics and Pharmacodynamics of DPC 333 ((2R)-2-((3R)-3-Amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)), a Potent and Selective Inhibitor of Tumor Necrosis Factor -Converting Enzyme in Rodents, Dogs, Chimpanzees, and Humans

Metabolism and Pharmacokinetics (M.Q., S.A.B., B.B., J.-T.W., M.J.F., C.E.G., Y.D., D.D.C.), Biology (R.-Q.L., M.B.C., K.V., R.C.N., J.T.), and Chemistry (T.M., J.J.-W.D., C.P.D.), Bristol-Myers Squibb Company, Princeton, New Jersey

(Received March 26, 2007; Accepted July 23, 2007)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) is a potent and selective inhibitor of tumor necrosis factor (TNF)--converting enzyme (TACE). It significantly inhibits lipopolysaccharide-induced soluble TNF- production in blood from rodents, chimpanzee, and human, with IC₅₀ values ranging from 17 to 100 nM. In rodent models of endotoxemia, DPC 333 inhibited the production of TNF- in a dose-dependent manner, with an oral ED₅₀ ranging from 1.1 to 6.1 mg/kg. Oral dosing of DPC 333 at 5.5 mg/kg daily for 2 weeks in a rat collagen antibody-induced arthritis model suppressed the maximal response by approximately 50%. DPC 333 was distributed widely to tissues including the synovium, the site of action for antiarthritic drugs. Pharmacokinetic and pharmacodynamic studies in chimpanzee revealed a systemic clearance of 0.4 l/h/kg, a V_ss of 0.6 l/kg, an oral bioavailability of 17%, and an ex vivo IC₅₀ for the suppression of TNF- production of 55 nM (n = 1). In a phase I clinical trial with male volunteers after single escalating doses of oral DPC 333, the terminal half-life was between 3 and 6 h and the ex vivo IC₅₀ for suppressing TNF- production was 113 nM. Measurement of the suppression of TNF- production ex vivo may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors. Overall, the pharmacological profiles of DPC 333 support the notion that suppression of TNF- with TACE inhibitors like DPC 333 may provide a novel approach in the treatment of various inflammatory diseases including rheumatoid arthritis, via control of excessive TNF- production.

View larger version (8K): [in this window] [in a new window]   FIG. 1. Chemical structure of DPC 333.

Human TNF- is synthesized as a biologically active 26-kDa membrane-bound precursor that is cleaved by a cell membrane-associated metalloprotease known as TNF--converting enzyme (TACE) to form a 17-kDa soluble TNF- (McGeehan et al., 1994; Black et al., 1997; Killar et al., 1999). Soluble TNF- exerts its biological effects by binding to cell surface receptors tumor necrosis factor receptors 1 and 2, and ultimately triggering a cascade of cellular activation producing inflammatory mediators like interleukin-6, matrix-degrading proteases, and active oxygen species that produce tissue damage (Feldmann et al., 1996; Kaufman and Choi, 1999). Inhibition of TACE and, therefore, the subsequent release of the soluble cytokine offer a potentially novel mechanism to modulate the activity of TNF-. Numerous studies have suggested that small-molecule TACE inhibitors under development may be as effective as other anti-TNF- therapies in the treatment of inflammatory diseases (Newton and Decicco, 1999; Conway et al., 2001; Newton et al., 2001; Beck et al., 2002; Zhang et al., 2004). DPC 333 (Fig. 1) ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) selectively inhibits TACE both in vitro and in vivo and is orally available (Duan et al., 2002). In the current article we report in detail the pharmacokinetics and pharmacodynamics of DPC 333 in mice, rats, dogs, and chimpanzees, as well as in a first-in-human clinical phase I trial in healthy volunteers.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. DPC 333 and the internal standards used in the analysis of DPC 333 were synthesized and characterized at Bristol-Myers Squibb (Princeton, NJ). Medical grade soluble TNF-R2:IgG fusion protein (etanercept) was obtained from Immunex Corporation (now Amgen, Thousand Oaks, CA). Antimurine TNF- antibody, formed in hamsters, and ELISA kits to measure TNF- in rodent plasma samples were obtained from Genzyme Corporation (Cambridge, MA). Bovine type II collagen, collagen monoclonal antibodies, and incomplete Freund's adjuvant were obtained from Chondrex LLC (Redmond, WA). Lipopolysaccharide (derived from Escherichia coli) and dinitrofluorobenzene were obtained from Sigma-Aldrich (St. Louis, MO). Alzet miniosmotic pumps were obtained from the Durect Corporation (Cupertino, CA). High-performance liquid chromatography-grade solvents used for liquid chromatography-mass spectrometry analysis and all other chemicals were obtained from commercial sources and were of analytical grade or higher.

Animals. BALB/c mice (18-20 g) and female CD rats (150-200 g) were obtained from Taconic Farms (Germantown, NY). Male and female Sprague-Dawley (SD), Dark Agouti (DA), and Lewis rats weighting approximately 225 to 350 g were obtained from Charles River Laboratories (Wilmington, MA). Male beagle dogs (11 ± 2 kg) were obtained from Marshall Farms (North Rose, NY). All animals were housed in temperature-control rooms with appropriate light/dark cycles and exercise regimens. The animals were fasted before dosing but were given access to water ad libitum. Food was provided to the rats and dogs 4 h after dosing. Some SD rats had jugular vein cannulas placed for sample collection. All animal studies were conducted according to protocols reviewed and approved by the Institutional Animal Care and Use Committee at the former DuPont Pharmaceutical Co. The in vivo chimpanzee (Pan troglodytes) studies were conducted according to protocols approved by the Animal Care and Use Committee at the New Iberia Research Center (New Iberia, LA). Four male adolescent chimpanzees, weighing between 59.0 and 78.6 kg, were used in these studies. A veterinarian observed each animal daily for signs of illness or distress during the study. Clinical and laboratory data were also monitored during the course of the study.

In Vitro Studies. Whole blood TNF assay. Blood was drawn from mice, rats, dogs, chimpanzees, and human volunteers into heparinized tubes (BD Biosciences, Franklin Lakes, NJ), and a 225-µl aliquot of blood was pipetted directly into Bio-Rad (Hercules, CA) polypropylene tubes. Test compounds, including DPC 333, were added at varying concentrations and preincubated with the blood cells for 10 min. LPS (Calbiochem, La Jolla, CA) was added to achieve a final concentration of 100 ng/ml and the blood was incubated for 5 h at 37°C. A 750-µl aliquot of AIM-V serum-free medium (Invitrogen, Carlsbad, CA) was added, and the cell pellets were centrifuged at 1500 rpm for 15 min. A 500-µl aliquot of supernatant was collected and assayed for TNF- using a standard sandwich ELISA. Blood collected from animals dosed with DPC 333 was also used to determine the amount of TNF- generated after LPS stimulation. The degree of inhibition after addition or administration of DPC 333 was calculated in relation to the pretreatment values.

Serum protein binding. The in vitro protein binding of DPC 333 in mouse, rat, dog, chimpanzee, and human serum, as well as with purified human serum albumin and 1-acid glycoprotein was determined by ultrafiltration using Centrifree micropartition devices with a molecular mass cutoff of >30,000 Da (Millipore, Billerica, MA). DPC 333 was added to serum and purified protein samples in triplicate to yield final concentrations of 1 µM. Human serum albumin and 1-acid glycoprotein solutions were prepared in deionized water at physiological concentrations of 40 mg/ml and 0.5 mg/ml, respectively. After incubation at 37°C for 1 h, the devices were centrifuged at 1700g for 10 min at 37°C. Aliquots of the serum ultrafiltrate were extracted on a C₈ solid phase extraction column and analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The unbound fraction (f_u) was calculated as the concentration of DPC 333 in the serum or purified protein ultrafiltrate divided by the nominal concentration in the serum or purified protein reservoir. Values are reported as means ± S.D. for three determinations.

In Vivo Studies. Dose administration and sample collection. All doses were administered as solutions in either isotonic saline (i.v., i.p., and oral) or 1% Tween 80 in 0.5% methylcellulose (oral). Blood samples were collected in EDTA Vacutainers and the plasma was obtained by centrifugation. Urine samples, when collected, were obtained via a collection pan placed under the cage that drained into a vessel maintained at 4°C by wet ice. All biological samples were frozen immediately after collection at -20°C until they were analyzed. Specifics of each study are detailed below.

Single dose pharmacokinetic studies in mice and rats. DPC 333 was administered to BALB/c mice, or SD or DA rats intravenously, orally, or intraperitoneally at doses ranging from 3.4 mg/kg to 68 mg/kg. Blood samples (0.5 ml) were collected from mice in a composite study design (three mice pooled per time point) and from individual rats (n = 3 for each dose route) at predose and at specific times up to 24 h postdose.

Synovial pharmacokinetic study in rats. The pharmacokinetic profile of DPC 333 in synovial fluid was determined using female Lewis rats (200 g, n = 18), a strain used as the rodent model for collagen-induced arthritis. The rats were given a single oral dose of 51 mg/kg DPC 333 in 1% Tween 80 and 0.5% methylcellulose. Three animals were sacrificed at each time point: predose, and 1, 2, 4, 8, and 24 h postdose. Blood samples (0.5 ml) were collected by cardiac puncture. Synovial fluid samples were collected under surgical microscopy according to the following procedure. The patellar ligament of the stifle knee joint was cut and the synovial cavity was incised and opened. Forty microliters of normal saline containing a structurally similar internal standard, TA034 (0.5 µM), was injected into the joint space of the knee, and then aspirated back to get approximate 40 µl of synovial fluid sample after rinsing the space carefully and thoroughly. The collection process was completed within 5 min to minimize diffusion and drug loss. The use of the internal standard allowed a more precise estimate of the actual volume of synovial fluid collected, which was used for calculating the final concentrations of DPC 333 in each sample. Plasma concentrations of DPC 333 were determined as described previously.

Mouse and rat models of endotoxemia. Male BALB/c mice (18-20 g) or female CD rats were randomly assigned to groups of 5 to 10 animals per group. All groups except the negative control group received E. coli-derived LPS i.p. (10 µg/mouse or 50 µg/rat). For the dose-response studies, vehicle (saline) or different doses of DPC 333, ranging from 0.2 mg/kg to 68 mg/kg, were administered p.o. or i.p. along with LPS. The mice or rats were sacrificed 1 h after dosing, and blood was obtained by cardiac puncture and placed in EDTA for the determination of plasma TNF- concentrations. Results were expressed as mean TNF- concentration for all the animals in the group or as percentage inhibition compared with the vehicle-treated group.

Collagen-induced arthritis rat model. Animals were treated as described by Holmdahl et al. (1989). In brief, DA rats were immunized by two intradermal injections at the base of the tail on day 0 and again on day 7 with bovine type II collagen emulsified in incomplete Freund's adjuvant. Five days later, all rats were given a single i.p. injection of LPS (50 µg/rat). Signs of arthritis such as erythema and swelling of the hind paws are typically observed between 5 and 10 days after the LPS injection. Rats that developed the disease (5-7/group) were randomly assigned to four treatment groups: vehicle control, DPC 333 treatment twice daily with oral doses of 5.5 or 10.5 mg/kg, and treatment with etanercept at 2.5 mg/rat twice a week by intraperitoneal administration. Paw swelling and the clinical signs were monitored three times a week and clinical scores were assigned based on a scale of 0 to 3, with 3 being most severe. Results were expressed as total arthritic score per rat from all rats in the group. To determine the percentage suppression of disease, the areas under the curve (AUCs) of the clinical score from the different treatment groups were compared. The AUCs of the clinical score were calculated as described previously (Bendele et al., 2000). To determine a DPC 333 concentration-effect relationship of DPC 333 in the rat CIA model, blood was collected from a cannulated jugular vein and at different time points after the final dose and analyzed for DPC 333.

Single dose pharmacokinetics in dogs. DPC 333 HCl salt was administered to male beagles (n = 3 for each dose route) as single intravenous and oral doses of 5 mg/kg. Blood samples (2 ml) were collected by jugular venipuncture into EDTA containing tubes at predose, 2, 5, 10, 15, and 30 min, and 1, 1.5, 2, 4, 6, 8, 10, 12, and 16 h postdose.

Single dose pharmacokinetics and pharmacodynamics of DPC 333 in chimpanzees. The single oral dose pharmacokinetics and pharmacodynamics, which was assessed as the percentage inhibition from predose levels of ex vivo LPS-stimulated TNF- production of DPC 333, were determined in three male chimpanzees. The chimpanzees were given DPC 333 as a single dose of 2.7 mg/kg (as free base) formulated in aqueous Tang orange drink. Blood samples were collected predose and at 1, 2, 3, 6, 9, 12, 24, and 36 h after DPC 333 administration. Blood samples were stimulated with LPS at 37°C for 5 h to produce TNF-, which was measured by ELISA as described previously, and the percentage inhibition from the predose sample was calculated. Plasma concentrations of DPC 333 were determined by LC/MS/MS. Plasma concentration-effect data were only available for one of the chimpanzees because ketamine was used to sedate the other two animals to facilitate the collection of blood samples. Ketamine has been shown to potentially inhibit the production of TNF- (Takenaka et al., 1994). In a separate cassette dose PK study using one male chimpanzee, the intravenous pharmacokinetics of DPC 333 was determined along with five other structurally similar compounds after a 30-min infusion of 0.5 mg/kg each. Blood samples were obtained predose and at 0.08, 0.25, 0.5, 1, 2, 3, 4, 6, 10, 12, 18, 24, 36, and 48 h after starting the infusion. Urine samples were also collected from 0 to 6, 6 to 12, 12 to 24, and 24 to 48 h postdose. All plasma and urine samples were kept frozen at -20°C until analysis.

Single dose pharmacokinetic and pharmacodynamic study in humans. The safety, tolerability, pharmacokinetics and ex vivo TNF- concentrations after single oral doses of DPC 333 (15, 25, 40, 80, 120, 225, 345, and 530 mg) were determined in a double-blinded, randomized, placebo-controlled (6 active/2 placebo) phase I study in healthy subjects. The study was reviewed and approved by an authorized Institutional Review Board, and all subjects provided informed consent before participation. Among the subjects evaluated, 90.0% (72 of 80) of subjects were male and 10.0% (8 of 80) were female; the majority of subjects were Caucasian (96.3%, 77 of 80). The mean age, height, weight, and body-mass index were 32.0 years, 175.7 cm, 77.5 kg, and 25.0 kg/m², respectively. Safety assessments include adverse experiences, clinical laboratory tests (including hematology, serum chemistry, and urinalysis), vital sign measurements, electrocardiograms, physical examinations, and tolerability. DPC 333 was administered after an overnight fast as either an oral solution (dosages of 15 and 25 mg) or as a suspension (dosages 40 mg). Blood samples for the determination of plasma DPC 333 concentrations were drawn from indwelling catheters or by direct venipuncture into Vacutainer tubes (BD Biosciences) containing EDTA before dosing and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 18, 24, 36, and 48 h after DPC 333 administration. Blood samples for the determination of DPC 333 concentrations were centrifuged for 10 min at 1500g at approximately 4°C and plasma was stored at -20°C or below until analysis. Whole blood samples (5 ml) were collected into heparinized Vacutainer tubes at predose, and at 2, 4, 6, and 8 h after dose administration for the determination of TNF- concentrations in plasma using the ELISA Quantikine TNF- kit (R&D Systems, Minneapolis, MN). The validated assay range using 200 µl of cell-free supernatant was 15.6 to 1000 pg/ml. Interassay precision and accuracy ranged from 1.6 to 8.3% and 93.4 to 106.9%, respectively.

Analytical Methods. Analysis of DPC 333 by LC/MS/MS. DPC 333 concentrations in all biological samples were determined by a LC/MS/MS-based method. Briefly, an aliquot of plasma or serum (0.2-0.5 ml) was mixed with a structural analog internal standard (TA034) or a deuterium-labeled isotope of DPC 333, TA769-d₅, and loaded onto preconditioned Isolute C₈ cartridges. The cartridges were washed with two 1-ml aliquots of water, dried, and then eluted with 1 ml of 2% ammonium hydroxide in methanol. The eluent was evaporated to dryness under nitrogen at 40°C and the residue was reconstituted with 0.10 to 0.15 ml of acetonitrile/aqueous formic acid (0.1%) (5:95 v/v). Urine samples were diluted with acetonitrile/aqueous formic acid (0.1%) (5:95 v/v) containing the internal standard and analyzed directly. The chromatographic separation of DPC 333 and the internal standard TA034 was accomplished on a reversed-phase column (Metachem AQ C₁₈ column, 2.1 x 50 mm, 5 µm; Varian, Inc., Palo Alto, CA), using a gradient elution at a flow rate of 0.3 ml/min. The gradient solvents used were 0.1% formic acid in water and 0.1% formic acid in acetonitrile for mobile phases A and B, respectively. The initial solvent composition was 7% solvent B, which was increased to 60% in 3.5 min and maintained at 60% for 1 min. The content of solvent B was then decreased to 7% in 0.5 min and maintained at 7% for 3 min. The total run time was 8 min. DPC 333 was detected using a Micromass Quattro (Waters, Milford, MA) LC/MS/MS apparatus with positive electrospray interface in the multiple reaction monitoring mode. The parent/daughter ion transitions monitored were m/z 477157 for DPC 333 and 466276 for TA034. The linear range for the standard curves in plasma was 1 to 1000 nM, and the lower limit of quantification was 1 nM. A similar validated LC/MS/MS method was used to determine DPC 333 concentrations in human plasma. The validated assay concentration range for DPC 333 using 200 µl of plasma was 1.05 to 2098 nM. Interassay precision and accuracy ranged from 4.56 to 5.88% and 0.86 to 8.53%, respectively.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Plasma and synovial concentration-time profiles of DPC 333 after a single oral dose of 60 mg/kg to Lewis rats.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Suppression of TNF- production in mouse and rat models of endotoxemia by DPC 333. Dose-dependent suppression of TNF- production in the mouse (A) and rat (B) endotoxemia model after p.o. (dotted line) and i.p. (solid line) administration of DPC 333.

A preliminary pharmacokinetic/pharmacodynamic (PK/PD) model was also developed for the clinical phase I data using the observed concentrations of TNF- and DPC 333. The actual TNF- concentrations were used in this model (rather than percentage inhibition from baseline) to avoid potential transformation errors. The model was then used to generate percentage inhibition curves after the fitting procedure. The pharmacodynamic model used in this analysis was:

(1)

where TNF_t is the concentration of TNF- at time t, TNF₀ is the baseline (predose) TNF- concentration, C(t) is the plasma concentration of DPC 333 at time t,IC₅₀ is the concentration of DPC 333 that causes a 50% inhibition of TNF-, and is a shape factor that describes the slope of the concentration-response curve.

For interspecies scaling of the pharmacokinetics, the mean values of CL_s and V_ss obtained from mouse, rat, dog, and chimpanzee, as well as from cynomolgus monkey (unpublished data), were plotted against mean body weight (kg) on a log-log scale. The following allometric equations were used for the analysis:

(2)

(3)

where a and b are the allometric coefficients, BW the body weight, and x and y the allometric exponents for CL_s and V_ss, respectively. The allometric equations obtained were used to estimate human pharmacokinetic parameters CL_s and V_ss. A 70-kg body and an oral bioavailability of 17% (derived from the chimpanzee study) were assumed in the human study. Human plasma AUC was estimated using the following equation:

(4)

	Results

Top Abstract Materials and Methods Results Discussion References

 
Anti-TNF- Activity of DPC 333 in Blood. The in vitro IC₅₀ values for the inhibition of TNF- production were 39, 30, 17, and 100 nM using blood from mice, Lewis rats, SD rats, and humans, respectively; and complete inhibition of TNF- production was observed at 190, 300, 134, and 1860 nM in these respective species.

Serum Protein Binding of DPC 333. Protein binding results of DPC 333 are summarized in Table 1. The binding of DPC 333 to purified human albumin and 1-acid glycoprotein, the two major drug-binding proteins in blood, was less extensive than binding in serum, 18.6 and 18.1%, respectively, at 1 µM.

Pharmacokinetics of DPC 333 in Mice and Rats. After an i.v. bolus dose in mice and rats, DPC 333 plasma concentrations declined rapidly, with an average terminal half-life of approximately 1 h (Table 2). The mean systemic plasma clearance was 6.2 and 2.7 l/h/kg and the mean V_ss was 1.5 and 0.6 l/kg in mice and rats, respectively. After oral or intraperitoneal dosing, DPC 333 was rapidly absorbed, with maximal plasma concentrations (C_max) observed within 30 min postdose. The apparent oral bioavailability was 11% and 17% in mice and SD rats, respectively; however, the dose studied in the mouse was high, 68 mg/kg. The bioavailability after intraperitoneal dosing was high in the two species, 98% and 78%, respectively.

Synovial Pharmacokinetics of DPC 333 after a Single Oral Dose to Rats. The plasma and synovial fluid concentration-time profiles of DPC 333 after a single oral dose of 60 mg/kg to Lewis rats are shown in Fig. 2. Mean peak plasma and synovial concentrations of 13.489 and 2.970 µM, respectively, were observed 1 h after oral dosing. Concentrations in plasma and synovium declined in parallel to 0.394 and 0.267 µM 4 h postdose and were still measurable at 24 h. The estimated half-life of DPC 333 was approximately 4 h. DPC 333 exposure, measured as the AUC, in the synovial fluid was 26.4% of the plasma exposure.

Suppression of Plasma TNF- Levels by DPC 333 in Rodent Models of Endotoxemia. The effect of increasing oral and i.p. doses of DPC 333 on the TNF- response after LPS challenge in a mouse model of endotoxemia is shown in Fig. 3A. The suppression of TNF- production was dose-dependent, with a calculated ED₅₀ of 6.1 and 1.9 mg/kg after oral and i.p. administration, respectively. Similar experiments using rats revealed an ED₅₀ of 1.1 mg/kg after i.p. administration (Fig. 3B).

Efficacy and PK-PD Relationship of DPC 333 in the Rat Model of Collagen-Induced Arthritis. The effect of DPC 333 on the average clinical score in the rat collagen-induced arthritis model is shown in Fig. 4. At doses of 5.5 mg/kg and 10.5 mg/kg given twice daily, DPC 333 markedly decreased the severity of the arthritis, with a mean reduction of the clinical score of 56% and 65%, respectively (Table 3). The magnitude of this reduction was indistinguishable from that produced by etanercept, the positive control.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Efficacy of DPC 333 and etanercept in a rat model of collagen-induced arthritis. Results are expressed as group mean (±S.D.) of clinical score/rat.

To determine the PK-PD relationship of DPC 333 in this rat model of arthritis, plasma concentration-time data on day 14 of the experiment were obtained after the 5.5- or 10-mg/kg doses of DPC 333. As shown in Table 3, TNF- production was reduced by 56% relative to control animals when the plasma AUC was 358 nM · h and the C_max was 444 nM. Suppression of the clinical score by the 10-mg/kg dose was similar, 65%.

Pharmacokinetics of DPC 333 in Dogs. Pharmacokinetic parameters of DPC 333 in dogs are summarized in Table 2. Systemic plasma clearance was high, 1.8 l/h/kg, a value close to hepatic blood flow in this species. The volume of distribution at steady state was 2.4 l/kg, approximately 4 times the total body water in the dog (0.6 l/kg), suggesting extensive tissue distribution. The terminal half-life of DPC 333 was 3.9 h. After oral dosing, DPC 333 was rapidly absorbed in dog, with mean maximal plasma concentration of 740 nM occurring approximately 45 min postdose. The mean oral bioavailability of a 5-mg/kg dose of DPC 333 was 15%.

Pharmacokinetics and Pharmacodynamics of DPC 333 in Chimpanzees. Plasma concentrations of DPC 333 declined polyexponentially after termination of an i.v. infusion of 0.5 mg/kg. The calculated systemic plasma clearance was 0.4 l/h/kg, and the volume of distribution at steady state and terminal half-life were 0.6 l/kg and 1.8 h, respectively (Table 2). The fraction of DPC 333 excreted in urine unchanged was 1.6% of total dose, with most appearing within the first 24 h after dose. The small percentage of dose recovered in the urine suggests that the renal clearance plays a minor role in the elimination of DPC 333 in the chimpanzee. The apparent oral bioavailability was low, averaging 15% from three chimpanzees. The mean peak plasma concentration (C_max) was 589 nM and occurred approximately 2 h postdose.

Figure 5 shows the relationship between plasma concentrations of DPC 333 and the percentage inhibition of ex vivo LPS-stimulated TNF- production in whole blood from the one chimpanzee dosed orally who was not sedated with ketamine. The inhibition of TNF- production was dependent on the plasma concentrations of DPC 333 in this conscious chimpanzee. The inhibition of TNF- production by DPC 333 was maximal from 1 to 3 h, when total plasma concentrations were greater than 336 nM, and was sustained over approximately 6 to 7 h. Negligible inhibition was observed 9 h postdose when the plasma concentrations were at or below 3 nM. The whole blood assay IC₅₀ of 55.1 nM was estimated and maximal suppression was seen at concentrations greater than 92.5 nM using a sigmoidal E_max model with a sigmoidicity factor of 3.54. In the other two chimpanzees sedated with ketamine for blood collection, no clear relationship between DPC 333 concentrations and percentage TNF- suppression was observed. This is consistent with the potent suppressive effect of ketamine on LPS-induced TNF- production in vitro and in vivo (Takenaka et al., 1994). Thus, the two animals were excluded from the PD analysis.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Time course of DPC 333 plasma concentrations () and the effect on ex vivo inhibition of TNF- by DPC 333 () after a single oral administration of 2.7 mg/kg to an alert chimpanzee.

Single Oral Dose Pharmacokinetics and Pharmacodynamics of DPC 333 in Humans. Overall, DPC 333 was generally safe and well tolerated. A total of 32 (48.5%) subjects who received DPC 333 in the study had an adverse experience compared with 8 (40.0%) placebo-treated subjects. The most commonly observed adverse experience was taste disturbance (28.3%, 17 of 60 DPC 333 subjects versus 20.0%, 4 of 20 placebo). For all laboratory parameters, vital sign measurements, and electrocardiogram parameters, no dose- or gender-related mean trends were apparent.

The mean plasma concentration-time profiles from single escalating oral doses of DPC 333 in healthy male volunteers are shown in Fig. 6, while a summary of the pharmacokinetics results is shown in Table 4. DPC 333 was rapidly absorbed after oral dosing, with T_max values ranging from 0.25 to 1 h postdose. The mean half-life for each dose cohort ranged between 2 and 6 h. The C_max and AUC values increased in a dose-proportional manner from 15 to 225 mg; however, doses larger than 225 mg produced greater-than-proportional increases in both C_max and AUC.

View larger version (23K): [in this window] [in a new window]   FIG. 6. Plasma concentrations-time profiles of DPC 333 after a single oral dose to human volunteers in the phase I clinical trial.

The compiled pharmacodynamic data from all healthy male subjects given DPC 333 is illustrated in Fig. 7. The degree of inhibition for the LPS-stimulated TNF- release closely followed DPC 333 plasma concentrations. Total inhibition of TNF- release was seen 2 h after the 225-mg dose. Near-maximal inhibition of TNF- release was achieved over the entire 8-h sampling period at the 345-mg dose. Pharmacodynamic fitting of these data using a sigmoid E_max model (Fig. 7) resulted in a calculated IC₅₀ value (total drug concentration) of 113 nM (95% CI of 95-131 nM), a value indistinguishable from that obtained from in vitro studies conducted with human whole blood (100 nM).

View larger version (30K): [in this window] [in a new window]   FIG. 7. Ex vivo percentage inhibition of TNF- production as a function of DPC 333 plasma concentrations after a single oral dose to human volunteers in the phase I clinical trial.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Interspecies allometric scaling for systemic clearance (CLs) and volume of distribution at steady state (Vss).The regression line was determined by least-squares analysis of the five-animal data, excluding the projected human data.

View larger version (22K): [in this window] [in a new window]   FIG. 9. Comparison of predicted and actual systemic exposure to DPC 333 in various human doses.

	Discussion

Top Abstract Materials and Methods Results Discussion References

The plasma clearance of DPC 333 in rodents and in dogs is high, approximately the hepatic blood flow in these species, and is consistent with the low oral bioavailability (<20%) seen in these species. The clearance of DPC 333 in the chimpanzee, however, is approximately one-third the apparent hepatic blood flow in humans. It is reasonable to make this comparison between chimpanzee and human because of the similarities in body size and allometric relationships between size and organ perfusion. However, these differences in clearances between the chimpanzee and the other animal species did not translate into a greater oral bioavailability for DPC 333. The oral bioavailability of DPC 333, given as either a solution or as a suspension, in all of the preclinical species tested ranged from 11 to 17%. It is unknown whether the lower than expected oral bioavailability in the chimpanzee is due to gastrointestinal tract metabolism, pharmaceutical issues with the formulation that was used, or the effects of the anesthesia used to collect blood samples from the chimpanzees. DPC 333 is rapidly absorbed in all species after oral dosing in solution, with T_max values of less than 2 h and, in some cases, less than 30 min.

DPC 333 is moderately bound to serum proteins in all species tested, although there are notable species variations in the binding. The free fraction is 2 to 3 times greater in rodents than in dog, chimpanzee, and human. The reason for this difference is not clear. The unbound fraction was the same using purified human albumin and 1-acid glycoprotein, approximately 18 to 19%, and was greater than the free fraction determined with human serum, 7%. These data suggest that other proteins in serum, perhaps a globulin fraction, also bind DPC 333. Because 1-acid glycoprotein levels can increase in rheumatoid arthritis (Belpaire and Bogaert, 1990), the significance of DPC 333 bound to 1-acid glycoprotein should be determined in sera from these patients.

Pharmacokinetic data for the chimpanzee was used to predict the oral human exposure in the single ascending-dose "first-in-human" trial with DPC 333, although there are some notable limitations of the chimpanzee data, such as the low number of animals used and the small dose range studied. Wong et al. (2004) and Riska et al. (1999) have shown that the pharmacokinetics of a wide range of xenobiotics in chimpanzees closely mimics those seen in humans. Although the mechanisms responsible for systemic clearance of DPC 333 are unknown, the excellent correlation of the projected CL_s and Vd_ss from the allometry suggests that the biochemical differences in chimpanzee cytochrome P450 isozymes (Wong et al., 2004) may not be important and that the chimpanzee seems to be an appropriate model. Furthermore, our human exposure estimate using a simple weight-derived allometric scaling also supports the use of the chimpanzee as a suitable model for the human pharmacokinetics of DPC 333 (Figs. 8 and 9).

There is close agreement between the predicated and actual systemic exposure after oral doses studied in the phase I clinical trial, especially at the lower doses. This agreement validates the rationale and importance of using allometric scaling or the chimpanzee model to estimate starting clinical dose of DPC 333, one of the most daunting challenges during the translational phase from preclinical to the first-in-human trial. At higher doses, the predicted exposure underestimated the actual human data, as seen in Fig. 9. This finding is most likely due to the fact that the predicted data assume linear kinetics in humans, which may not be the case, since the AUC and C_max values increase modestly in a greater than dose-proportional manner, approximately 2-fold. This implies that with an increase in dose in human volunteers, either the clearance of DPC 333 fell or the absorption of the compound increased. Any change in clearance is difficult to detect without i.v. doses for humans. That the data available are limited does not rule out the possibility that the actual clearance in humans may be greater than, and hence oral bioavailability more susceptible to, saturation of presystemic extraction. Another possible reason for the greater than dose-proportional increase in AUC with oral doses may be, in part, saturation of efflux pump/transporter throughout the intestinal tract at the higher oral doses. Recently, Luo et al. (2007) have reported that DPC 333 may be a substrate for P-glycoprotein and that glucuronide conjugates of DPC 333 may inhibit the Mrp-2-mediated transport of methotrexate.

The rodent models of inflammation represent simple yet versatile systems to assess the in vivo activity of TNF- inhibitors (Williams et al., 1992; Michie, 1998). After the injection of mice or rats with bacterial endotoxin, systemic TNF- levels are routinely elevated 100-fold over pretreatment levels within 1 to 2 h and remain elevated above baseline values for an additional 4 h (Bemelmans et al., 1993). Similar concentration-time profiles for TNF- levels have been shown in humans after a LPS challenge (van der Poll et al., 1997; Dekkers et al., 1999). After single escalating p.o. and i.p. doses of DPC 333 to LPS-treated mice, the ED₅₀ values of DPC 333 to lower TNF- level are 6.1 and 1.9 mg/kg, respectively. These results demonstrate that DPC 333 can effectively reduce induced TNF- levels in vivo. The 3-fold difference between the ED₅₀ values derived from p.o. and i.p. routes of administration in mice is most likely due to differences in the systemic availability between the two routes of administration. The apparent bioavailability of DPC 333 was between 11 and 17% after oral administration, whereas after i.p. doses, it ranged from 78 to 98%. Bioavailability from the mouse experiments must be interpreted cautiously, however, since the oral dose was high (68 mg/kg) and the linearity of the pharmacokinetics is unknown.

The anti-inflammatory potential of DPC 333 was also assessed in the collagen-induced arthritis (CIA) rat. This animal model is well characterized and has a number of pathological similarities to human rheumatoid arthritis (Anthony and Haqqi, 1999). Like human rheumatoid arthritis, synovitis and erosions of cartilage and bone are hallmarks of the CIA rat model. In patients with RA, Deleuran et al. (1992) found that up-regulated TNF- and TNF- receptor (TNF--R) expression are present in the synovium and particularly in the cartilage-pannus junction. The target site of antiarthritic drugs, such as DPC 333, is mainly in the joint space. DPC 333 appears to distribute well into the synovial space in the rat since the exposure in the synovial fluid is approximately 26% of that in plasma after oral administration. More importantly, DPC 333 treatment of CIA rats dramatically reduces the clinical signs of arthritis to an extent similar to that seen in the rats treated with the positive control etanercept (Fig. 4; Table 3).

Despite a clear response to the LPS challenge that produced large amounts of TNF- from animal species and a highly consistent TNF- inhibition profile by DPC 333 in our study, using the rat CIA model (Table 3), there seems to be lack of direct correlation between DPC 333 doses (or plasma concentrations) and clinical scores (percentage of suppression of disease). The reasons for the lack of a dose-response relationship remain unclear but may be in part because of the fact that the lower dose and resulting plasma concentrations had already produced the maximal effect (E_max), i.e., 50 to 65% decrease from control. In a number of clinical trials, a similar no-response rate (2050%) was found with anti-TNF therapeutics (Olsen and Stein, 2004; Feldmann and Steinman, 2005). The lack of response to anti-TNF- treatment has been associated with genetic variation in the production and effector pathways of its targets (Ulfgren et al., 2000; de Vries and Tak, 2005). Because of the complexity of the down-stream cytokine cascade and signal transduction triggered by elevated TNF-, it is reasonable to use the rat CIA model only to demonstrate the proof of concept in efficacy, rather than to predict appropriate dose regimens, of TNF inhibitors for treatment of human rheumatic arthritis.

The ability of DPC 333 to inhibit ex vivo LPS-stimulated TNF- production/release from whole blood was assessed as a biomarker for anti TNF- activity. A preliminary experiment using one unanesthetized chimpanzee demonstrated that the pharmacodynamic response (percentage inhibition of TNF-) could be fitted to an E_max pharmacodynamic model, with an apparent IC₅₀ of 55 nM. Similarly, the apparent IC₅₀ in humans is 113 nM, calculated using this pharmacodynamic model to fit the more extensive ex vivo human data. This value is almost identical to the in vitro IC₅₀ value obtained with human blood (100 nM). The similarity between the in vitro and ex vivo human plasma IC₅₀ suggests that contributions from any potential active metabolites to inhibit TNF- production or release are negligible. Consistently, in preliminary in vitro studies on the biotransformation of DPC 333, no metabolites retaining significant pharmacological activity have been identified (data not presented). A close examination of the in vivo pharmacodynamic relationship in humans between inhibition of soluble TNF- in vivo and plasma concentrations of DPC 333 reveals that the peak concentrations (C_max) achieved in most subjects at the lowest dose (15 mg) are generally greater than the IC₅₀ values measured in vitro for soluble TNF- release. Concentrations approaching or exceeding the IC₅₀ for inhibition of TNF- production (113 nM) are easily reached and last approximate 4 h postdose, after single doses of 25 to 40 mg. The results show a dose-dependent maximum inhibition of TNF- release with greater than 90% inhibition at the 225-mg dose, along with a C_max of 3139 nM and an AUC of 3000 nM · h. These values are quite similar to those observed in the LPS mouse infusion study. Measurement of TNF- level using the whole blood assay as described in these studies may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors.

Because TNF- is a key cytokine that acts by inducing several other proinflammatory cytokines, chemokines, adhesion molecules, and matrix-degrading enzymes (Feldmann et al., 1996), diminishing TNF- level for a short duration with DPC 333 may be sufficient to provide long-lasting anti-inflammatory effects. Alternatively, with a relatively short half-life (<6 h) compared with the anti-TNF- biologics (>4 days to 2 weeks), DPC 333 may offer the ability to more precisely control regulation of TNF- levels, as well as the duration of TNF- blockade, allowing for timely discontinuation in the event of intercurrent illness. This could minimize the risk of infection and other adverse events seen with some of the anti-TNF- biologics with long half-lives (Wallis et al., 1998; Feldmann and Steinman, 2005).

In summary, DPC 333 is a potent inhibitor of TACE and shows a dose (concentration)-dependent reduction of LPC-induced TNF- in blood in vitro, in vivo, and ex vivo from mouse, rat, chimpanzee, and human. Pharmacokinetic and pharmacodynamic characteristics of DPC 333 support the concept that TACE inhibitors may provide a novel approach in the treatment of various diseases, including RA, for which a precise control of excessive soluble TNF- production is deemed desirable.

	Acknowledgments

 
We acknowledge the contributions of the former members of the Drug Metabolism and Pharmacokinetics Department and TACE program of DuPont Pharmaceuticals Company who played a role in generating the data in this article. We are grateful to Hang Zeng, Bing Lu, Foster Brown, Bob Collins, Tracy Taylor, and Sherrill Nurnberg for excellent technical assistance in some of these studies. The support provided by Drs. T. J. Rowell and J. Moran and the staff of the New Iberia Research Center was critical to the completion of chimpanzee studies.

	Footnotes

 
¹ Current affiliations: ¹ Genentech, Inc., South San Francisco, California; ² Endo Pharmaceuticals, Chadds Ford, Pennsylvania; ³ Adolor Corporation, Exton, Pennsylvania; ⁴ Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts; ⁵ Incyte Corporation, Newark, Delaware; ⁶ GlaxoSmithKline, King of Prussia, Pennsylvania; ⁷ AstraZeneca Plc, Waltham, Massachusetts; ⁸ SNC Partners LLC, Newark, Delaware.

doi:10.1124/dmd.107.015933.

ABBREVIATIONS: TNF-, tumor necrosis factor-; AUC, area under the plasma concentration-time curve; CIA, collagen antibody-induced arthritis; CL_s, systemic clearance; C_max, maximum observed concentration achieved after oral dosing; DA, Dark Agouti; DPC 333, (2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide); ELISA, enzyme-linked immunosorbent assay; F, extravascular or oral bioavailability; IC₅₀, concentration producing 50% of maximal inhibition; LC/MS/MS, liquid chromatography-tandem mass spectrometry; LPS, lipopolysaccharide; PK, pharmacokinetics; PD, pharmacodynamics; RA, rheumatoid arthritis; SD, Sprague-Dawley; TACE, TNF--converting enzyme; T_max, time to reach maximum observed concentration; t, terminal half-life; V_ss, steady-state volume of distribution.

Address correspondence to: Dr. Mingxin Qian, Pharmacokinetics, Pharmacodynamics and Bioanalytical Sciences, Genentech Inc., One DNA Way, South San Francisco, CA 94080. E-mail: mqian{at}gene.com

	References

Top Abstract Materials and Methods Results Discussion References

 


Alamanos Y and Drosos AA (2005) Epidemiology of adult rheumatoid arthritis. Autoimmune Rev 4: 130-136.[CrossRef]

Anthony DD and Haqqi TM (1999) Collagen-induced arthritis in mice: an animal model to study the pathogenesis of rheumatoid arthritis. Clin Exp Rheumatol 17(2): 240-244.[Medline]

Beck G, Bottomley G, Bradshaw D, Brewster M, Broadhurst M, Devos R, Hill C, Johnson W, Kim H, Kirtland S, et al. (2002) (E)-2(R)-[1(S)-(Hydroxycarbamoyl)-4-phenyl-3-butenyl]-2'-isobutyl-2'-(methanesulfonyl)-4-methylvalerohydrazide (Ro 32-7315), a selective and orally active inhibitor of tumor necrosis factor-alpha convertase. J Pharmacol Exp Ther 302: 390-396.[Abstract/Free Full Text]

Belpaire FM and Bogaert MG (1990) Binding of diltiazem to albumin, alpha 1-acid glycoprotein and to serum in man. J Clin Pharmacol 30: 311-317.[Abstract]

Bemelmans MH, Gouma DJ, and Buurman WA (1993) Influence of nephrectomy on tumor necrosis factor clearance in a murine model. J Immunol 150: 2007-2017.[Abstract]

Bendele AM, Chlipala ES, Scherrer J, Frazier J, Sennello G, Rich WJ, and Edwards CK 3rd (2000) Combination benefit of treatment with the cytokine inhibitors interleukin-1 receptor antagonist and PEGylated soluble tumor necrosis factor receptor type I in animal models of rheumatoid arthritis. Arthritis Rheum 43: 2648-2659.[CrossRef][Medline]

Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, et al. (1997) A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385: 729-733.[CrossRef][Medline]

Conway JG, Andrews RC, Beaudet B, Bickett DM, Boncek V, Brodie TA, Clark RL, Crumrine RC, Leenitzer MA, McDougald DL, et al. (2001) Inhibition of tumor necrosis factor-a production and arthritis in the rat by GW3333, a dual inhibitor of TNF--converting enzyme and matrix metalloproteinases. J Pharmacol Exp Ther 298: 900-908.[Abstract/Free Full Text]

Dekkers PEP, Lauw FN, ten Hove T, te Velde AA, Lumley P, Becherer D, van Deventer SJH, and van der Poll T (1999) The effect of a metalloproteinase inhibitor (GI5402) on tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha receptors during human endotoxemia. Blood 94: 2252-2258.[Abstract/Free Full Text]

Deleuran BW, Chu CQ, Field M, Brennan FM, Mitchell T, Feldmann M, and Maini RN (1992) Localization of tumor necrosis factor receptors in the synovial tissue and cartilage pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor alpha. Arthritis Rheum 135: 1170-1178.

de Vries N and Tak PP (2005) Editorial. The response to anti-TNF-alpha treatment: gene regulation at the bedside. Rheumatology (Oxf) 44: 705-707.[CrossRef]

Duan JJ-W, Chen L, Lu L, Wassermen Z, Maduskuie T, Liu R-Q, Covington MB, Vaddi K, Qian M, Voss M, et al. (2002) Discovery of selective and orally bioavailable TACE inhibitors. Abstracts of Papers, 224th National Meeting of the American Chemical Society, Boston, MA, Aug 18-22, 2002. American Chemical Society, Washington DC.

Erzurum SC (2006) Inhibition of tumor necrosis factor alpha for refractory asthma. N Engl J Med 354: 754-758.[Free Full Text]

Feldmann M, Brennan FM, and Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14: 397-440.[CrossRef][Medline]

Feldmann M and Steinman L (2005) Design of effective immunotherapy for human autoimmunity. Nature 435: 612-619.[CrossRef][Medline]

Finckh A, Simard JF, Gabay C, and Guerne PA (2006) SCQM physicians. Evidence for differential acquired drug resistance to anti-tumour necrosis factor agents in rheumatoid arthritis. Ann Rheum Dis 65: 746-752.[Abstract/Free Full Text]

Gibaldi M and Perrier D (1982) Pharmacokinetics, pp 409-416. Marcel Dekker, New York.

Holmdahl R, Andersson ME, Goldschmidt TJ, Jansson L, Karlsson M, Malmstrom V, and Mo J (1989) Collagen induced arthritis as an experimental model for rheumatoid arthritis. Immunogenetics, pathogenesis and autoimmunity. Acta Pathol Microbiol Immunol Scand 97: 575-584.

Kaufman DR and Choi Y (1999) Signaling by tumor necrosis factor receptors: pathways, paradigms and targets for modulation. Int Rev Immunol 18: 405-427.[Medline]

Killar L, White J, Black R, and Peschon J (1999) Adamalysins. A family of metzincins including TNF-alpha converting enzyme (TACE). Ann N Y Acad Sci 878: 442-452.[CrossRef][Medline]

McGeehan GM, Becherer JD, Bast RC, Boyer CM, Champion B, Connolly KM, Conway JG, Furdon P, Karp S, Kidao S, et al. (1994) Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor. Nature 370: 558561.

Luo G, Garner CE, Xiong H, Hu H, Richards LE, Brouwer KL, Duan J, Decicco CP, Maduskie T, Shen H, et al. (2007) Effect of DPC 333 ([(2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide], a human tumor necrosis factor -convertase enzyme inhibitor, on the disposition of methotrexate: a transporter-based drug-drug interaction case study. Drug Metab Dispos 35: 835-840.[Abstract/Free Full Text]

Michie HR (1998) The value of animal models in the development of new drugs for the treatment of the sepsis syndrome. J Antimicrob Chemother 41 (Suppl A): 47-49.[Abstract/Free Full Text]

Newton RC and Decicco CP (1999) Therapeutic potential and strategies for inhibiting tumor necrosis factor. J Med Chem 42: 2295-2314.[CrossRef][Medline]

Newton RC, Solomon KA, Covington MB, Decicco CP, Haley PJ, Friedman SM, and Vaddi K (2001) Biology of TACE inhibition. Ann Rheum Dis 60: 25-32.

Olsen NJ and Stein CM (2004) Drug therapy: new drugs for rheumatoid arthritis. N Engl J Med 350: 2167-2179.[Free Full Text]

Riska PS, Joseph DP, Dinallo RM, Davidson WC, Keirns JJ, and Hattox SE (1999) Biotransformation of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, in mice, rats, rabbits, dogs, monkeys, and chimpanzees. Drug Metab Dispos 27: 1434-1447.[Abstract/Free Full Text]

Takenaka I, Ogata M, Koga K, Matsumoto T, and Shigematsu A (1994) Ketamine suppresses endotoxin-induced tumor necrosis factor alpha production in mice. Anesthesiology 80: 402-408.[Medline]

Ulfgren AK, Grondal L, Lindblad S, Khademi M, Johnell O, Klareskog L, and Andersson U (2000) Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment. Ann Rheum Dis 59: 439-447.[Abstract/Free Full Text]

van der Poll T, Coyle SM, Levi M, Jansen PM, Dentener M, Barbosa K, Buurman WA, Hack CE, ten Cate JW, Agosti JM, et al. (1997) Effect of a recombinant dimeric tumor necrosis factor receptor on inflammatory responses to intravenous endotoxin in normal humans. Blood 89: 3727-3734.[Abstract/Free Full Text]

Wallis WJ, Furst DE, Strand V, and Keystone E (1998) Biologic agents and immunotherapy in rheumatoid arthritis. Progress and perspective. Rheum Dis Clin North Am 24: 537-565.[CrossRef][Medline]

Williams RO, Feldmann M, and Maini RN (1992) Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc Natl Acad Sci U S A 89: 9784-9788.[Abstract/Free Full Text]

Wong H, Grossman SJ, Bai SA, Diamond S, Wright MR, Grace JE Jr, Qian M, He K, Yeleswaram K, and Christ DD (2004) The chimpanzee (Pan troglodytes) as a pharmacokinetic model for selection of drug candidates: model characterization and application. Drug Metab Dispos 32: 1359-1369.[Abstract/Free Full Text]

Zhang Y, Xu J, Levin J, Hegen M, Li G, Robertshaw H, Brennan F, Cummons T, Clarke D, Vansell N, et al. (2004) Identification and characterization of 4-[[4-(2-butynyloxy)phenyl]sulfonyl]-N-hydroxy-2,2-dimethyl-(3S) thiomorpholinecarboxamide (TMI-1), a novel dual tumor necrosis factor-alpha-converting enzyme/matrix metalloprotease inhibitor for the treatment of rheumatoid arthritis. J Pharmacol Exp Ther 309: 348-355.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. P. Jia, D. C. Look, P. Tan, L. Shi, M. Hickey, L. Gakhar, M. C. Chappell, C. Wohlford-Lenane, and P. B. McCray Jr Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia Am J Physiol Lung Cell Mol Physiol, July 1, 2009; 297(1): L84 - L96. [Abstract] [Full Text] [PDF]

				X. Deng, J. Lu, L. D. Lehman-McKeeman, E. Malle, D. L. Crandall, P. E. Ganey, and R. A. Roth p38 Mitogen-Activated Protein Kinase-Dependent Tumor Necrosis Factor-{alpha}-Converting Enzyme Is Important for Liver Injury in Hepatotoxic Interaction between Lipopolysaccharide and Ranitidine J. Pharmacol. Exp. Ther., July 1, 2008; 326(1): 144 - 152. [Abstract] [Full Text] [PDF]

				C. E. Garner, E. Solon, C.-M. Lai, J. Lin, G. Luo, K. Jones, J. Duan, C. P. Decicco, T. Maduskuie, S. E. Mercer, et al. Role of P-Glycoprotein and the Intestine in the Excretion of DPC 333 [(2R)-2-{(3R)-3-Amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] in Rodents Drug Metab. Dispos., June 1, 2008; 36(6): 1102 - 1110. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.107.015933v1 35/10/1916    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qian, M. Articles by Christ, D. D. Search for Related Content PubMed PubMed Citation Articles by Qian, M. Articles by Christ, D. D.