npg Acta Pharmacologica Sinica (2012) 33: 1372–1378 © 2012 CPS and SIMM All rights reserved 1671-4083/12 $32.00 www.nature.com/aps Original Article Pharmacokinetic-pharmacodynamic modeling of diclofenac in normal and Freund’s complete adjuvant-induced arthritic rats Jing ZHANG, Pei LI, Hai-fang GUO, Li LIU, Xiao-dong LIU* Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Aim: To characterize pharmacokinetic-pharmacodynamic modeling of diclofenac in Freund’s complete adjuvant (FCA)-induced arthritic rats using prostaglandin E2 (PGE2) as a biomarker. Methods: The pharmacokinetics of diclofenac was investigated using 20-day-old arthritic rats. PGE2 level in the rats was measured using an enzyme immunoassay. A pharmacokinetic-pharmacodynamic (PK-PD) model was developed to illustrate the relationship between the plasma concentration of diclofenac and the inhibition of PGE2 production. The inhibition of diclofenac on lipopolysaccharide (LPS)-induced PGE2 production in blood cells was investigated in vitro. Results: Similar pharmacokinetic behavior of diclofenac was found both in normal and FCA-induced arthritic rats. Diclofenac significantly decreased the plasma levels of PGE2 in both normal and arthritic rats. The inhibitory effect on PGE2 levels in the plasma was in proportion to the plasma concentration of diclofenac. No delay in the onset of inhibition was observed, suggesting that the effect compartment was located in the central compartment. An inhibitory effect sigmoid Imax model was selected to characterize the relationship between the plasma concentration of diclofenac and the inhibition of PGE2 production in vivo. The Imax model was also used to illustrate the inhibition of diclofenac on LPS-induced PGE2 production in blood cells in vitro. Conclusion: Arthritis induced by FCA does not alter the pharmacokinetic behaviors of diclofenac in rats, but the pharmacodynamics of diclofenac is slightly affected. A PK-PD model characterizing an inhibitory effect sigmoid Imax can be used to fit the relationship between the plasma PGE2 and diclofenac levels in both normal rats and FCA-induced arthritic rats. Keywords: pharmacokinetic-pharmacodynamic modeling; diclofenac; prostaglandin E2 (PGE2); Freund’s complete adjuvant (FCA)-induced arthritis; lipopolysaccharide; blood cells Acta Pharmacologica Sinica (2012) 33: 1372–1378; doi: 10.1038/aps.2012.67; published online 30 Jul 2012 Introduction illustrate the relationship between the plasma concentration- Diclofenac sodium is a non-steroidal anti-inflammatory drug time profiles of NSAIDs and the pharmacological activity of (NSAID) that is widely prescribed and used for relieving the these drugs using two types of traditional pharmacological pain and edema associated with inflammatory conditions, such endpoints: (i) those exploring the inflammatory response as osteoarthritis and rheumatoid arthritis[1–3]. It is commonly and having a mechanistic interest, such as central and local known that diclofenac acts by potent cyclo-oxygenase (COX) hyperthermia (body and skin temperature), hyperalgesia inhibition, which decreases the formation of proinflammatory (pain score) and edema (paw volume) and (ii) the hybrid mediators, such as prostaglandins (PGs)[4]. The prediction of endpoints that have direct clinical relevance and reflect both safety and long-term efficacy has become the main challenge in the pain and functional impairments[2, 6–11]. However, these the evaluation of NSAIDs for the treatment of pain in chronic studies have disregarded the direct relationship between the inflammatory conditions. Pharmacokinetic-pharmacodynamic drug concentration and the pharmacological response to COX (PK-PD) modeling represents a powerful tool to quantitatively inhibition. In addition, the lack of direct correlation between describe the pharmacokinetic, pharmacodynamic and system- the plasma drug concentrations and the analgesic or adverse related processes[5]. Several attempts have been made to effects in chronic inflammatory conditions has made it difficult to predict the appropriate dosing regimen for the treatment of [12] * To whom correspondence should be addressed. chronic inflammatory pain . E-mail [email protected] Prostaglandin E2 (PGE2 ) is associated with acute and chronic Received 2012-03-13 Accepted 2012-05-09 inflammatory pain, and NSAIDs exert analgesic effects via www.chinaphar.com npg Zhang J et al 1373 the inhibition of COX-2 activity. As a biomarker of COX the ankle. Scores were given for the left hindpaw and both activity, it is well known that PGE2 can reflect a downstream forepaws for each rat, yielding a maximum possible score of process on the causal pathway between target occupancy and 12 on d 20 after treatment with adjuvant. The total score was analgesic response. This indicates that PGE2 can be used as a defined as the secondary inflammation index (arthritic index). specific biomarker to explain and understand the variability Body weight and food intake were monitored every day. A in the therapeutic of these drugs[12]. Recently, efforts have sensitized animal was considered to have arthritis when at been made to establish the relationship between biomarkers, least one non-injected paw was inflamed, and these rats were pain measurement and safety[13–16]. However, information on then used for the following experiments. the integrated pharmacokinetic-pharmacodynamic profiles of these drugs under normal and chronic inflammatory condi- In vivo experiments tions is still limited. On d 20, after FCA injection, the FCA-induced arthritic rats The aim of this study was to characterize the PK-PD pro- and age-matched normal rats were fasted overnight and given files of diclofenac in normal and Freund’s complete adjuvant a dose of diclofenac sodium intravenously (10 mg/kg) in the (FCA)-induced arthritic rats using PGE2 as a biomarker. The tail vein. Blood samples (approximately 200 µL) were col- inhibitory effect of diclofenac on PGE2 release induced by lected under light ether anesthesia via the oculi chorioideae lipopolysaccharide (LPS) was also measured in vitro in the vein before dosing, as well as at 5, 15, 30, 60, 120, 180, 240, and blood cells of normal and FCA-induced arthritic rats. 360 min after dosing. The blood sample taken prior to dosing was used for measuring the basal levels of PGE2. The plasma Materials and methods samples were obtained by centrifugation at 5000 rounds per Chemicals minute for 10 min and stored at -80 °C for assaying diclofenac Diclofenac sodium and Bacillus Calmette Guerin (BCG) were and PGE2 levels. purchased from the National Institute for the Control of Phar- maceutical and Biological Products (Beijing, China), pento- In vitro experiments in whole blood barbital and lipopolysaccharide (LPS) were purchased from For the in vitro experiments, the blood samples of FCA-induced Sigma Chemical Co (St Louis, MO, USA), and the ELISA kits arthritic rats and normal rats were collected under anesthesia for PGE2 were from Cayman Chemical Co (Ann Arbor, MI, via the abdominal aorta with intraperitoneal administration USA). All other reagents were of analytical grade and were of pentobarbital (60 mg/kg). The PGE2 released by LPS in commercially available. the blood samples was documented according to a previously described method[20]. A 200-µL blood sample was added to a Animals tube containing different levels of diclofenac sodium, heparin Male Sprague Dawley rats (110–120 g) were purchased from (0.3%) and aspirin (10 µg/mL). After adding LPS (final levels B&K Universal Group Ltd (Shanghai, China). The rats were 10 µg/mL), the blood samples were incubated for 24 h at 37 °C maintained in air-conditioned animal quarters at a tempera- in a gently stirring water bath. The plasma was separated by ture of 22±2 °C with a relative humidity of 50%±10% and a centrifugation at 5000 rounds per minute for 10 min and was 12-h light/dark cycle. The rats received a standard diet (labo- stored at -80 °C for assessing PGE2 levels. ratory rodent chow; Nanjing, China) and water ad libitum. The studies were approved by the Animal Ethics Committee of HPLC analysis of diclofenac China Pharmaceutical University. The concentration of diclofenac in the plasma was analyzed by a validated HPLC procedure using ultraviolet (UV) detec- Induction of Freund’s complete adjuvant (FCA)-induced arthritic tion that has been previously described[21]. Briefly, plasma rats samples were spiked with 10 µL of internal standard (50 FCA-induced arthritic rats were developed according to a µg/mL naproxen in methanol). Then, 200 µL acetonitrile was previously described method[17]. Rats were acclimated for 1 added, and the samples were vortex-mixed. After centrifuga- week before the experiments and randomly divided into two tion at 15 000 rounds per minute for 10 min, the organic layer groups. Freund’s complete adjuvant (FCA) was prepared by was transferred to a clean tube and evaporated to dryness grinding 60 mg of heat-killed BCG in a mortar and adding under a stream of nitrogen gas in a water bath at 45 °C. The a mixture of liquid paraffin and lanolin (2:1, v/v) so that the residue was reconstituted in 100 µL of mobile phase and cen- final concentration of BCG was 10 mg/mL. Rats received a trifuged (1000 rounds per minute, 10 min). Next, 20 µL of the single intradermal injection of 0.1 mL of FCA in the right hind- supernatant was injected into an HPLC system equipped with paw by inserting a 25-gauge, 0.5-inch needle between the sec- an LC-10AD pump, a CTO-10ASvp column oven, and a SPD- ond and third digits into the dorsum of the hind paw on d 0[18]. 10A UV absorbance detector (Shimadzu, Kyoto, Japan) set to For clinical evaluation of FCA-induced arthritis, the polyar- a wavelength at 276 nm. Chromatography was performed on [19] thritis severity was graded on a scale of 0–4 : 0, no swelling; a Diamonsil C18 5-µm column (150 mm×4.6 mm, Dikma, Tech- 1, isolated phalanx joint involvement; 2, involvement of the nologies, Beijing, China).