Human pharmacokinetic prediction of UDP-glucuronosyltransferase substrates with an animal scale-up approach.

The aim of the current study was to evaluate the accuracy of allometric scaling methods for drugs metabolized by UDP-glucuronosyltransferases (UGTs), such as ketoprofen, imipramine, lorazepam, levofloxacin, zidovudine, diclofenac, furosemide, raloxifene, gemfibrozil, mycophenolic acid, indomethacin, and telmisartan. Human plasma clearance (CL) predictions were conducted from preclinical in vivo data by using multiple-species allometry with the rule of exponents and single-species allometric scaling (SSS) of mice, rats, monkeys, or dogs. Distribution volume at a steady state (V(ss)) was predicted by multiple-species allometry or SSS of V(ss). Oral plasma clearance (CL(po)) was calculated under the assumption that F(a) × F(g) was equivalent across species. Each of the results was compared with the observed parameter calculated from the clinical data after intravenous or oral administration. Multiple-species allometry and SSS of mice, rats, and dogs resulted in a similar accuracy of CL and CL(po) predictions. Monkeys tended to provide the most accurate predictions of human CL and CL(po). The ability to predict the half-life, which was determined from CL and V(ss) predictions, was more accurate in SSS of rats and monkeys. The in vivo fraction metabolized by glucuronidation (f(m,UGT)) in bile duct-cannulated monkeys was relatively similar to that of humans compared with other animal species, which likely contributed to the highest accuracy of SSS prediction of monkeys. On the basis of the current results, monkeys would be more reliable than other animal species in predicting human pharmacokinetics and f(m,UGT) for drugs metabolized by UGTs.

Investigation of the intestinal permeability and first-pass metabolism of drugs in cynomolgus monkeys using single-pass intestinal perfusion.

To clarify the causes of low oral bioavailability (BA) of drugs in cynomolgus monkeys, the experimental method to evaluate the drug permeability and the metabolism in the intestine of cynomolgus monkeys was established. An in situ intestinal perfusion method was performed with blood sampling from both portal and peripheral veins to calculate the intestinal permeability and the metabolism of drugs simultaneously. In all experiments, antipyrine was co-perfused with test drugs as a non-metabolized reference to calculate the individual portal vein blood flow. The effective permeability coefficient (P(eff)) of acetaminophen and piroxicam were high, and the fraction of dose absorbed from the gastrointestinal tract (Fa) thought to be 1. The intestinal availability (Fg) of acetaminophen and piroxicam were calculated to be 0.39 and 1.09, respectively. The Fa*Fg values of these drugs calculated from the perfusion study almost coincided with those obtained from the in vivo PK analysis in the previous report. In addition, the Fg values of verapamil and midazolam were calculated as 0.16 and 0.26, respectively, suggesting these drugs were metabolized extensively in the intestine after oral administration to cynomolgus monkey. Furthermore, the Fg values of these drugs were increased to 0.8-0.85 in the presence of 1-aminobenzotriazole, a typical cytochrome P450 (CYP) inhibitor. In conclusion, it was clarified that acetaminophen, verapamil and midazolam were metabolized extensively in the intestine of cynomolgus monkeys. This intestinal perfusion method is considered to be useful to identify the factors of species difference in the oral absorption of drugs.

Depletion of substance P, a mechanism for inhibition of mouse scratching behavior by tacrolimus.

Itching is the most important problem in atopic dermatitis and tacrolimus has been suggested to attenuate the itching by topical application. However, the anti-itch mechanism of tacrolimus has not been well elucidated. In the present study, an allergic dermatitis accompanied by frequent scratching behaviors was induced by repeated paintings with 2,4-dinitrofluorobenzene (DNFB) acetone solution onto the mouse ear and the effects of tacrolimus and dexamethasone on the dermatitis and associated scratching behavior were comparatively examined. Repeated DNFB paintings caused a typical dermatitis accompanied by elevated serum immunoglobulin E (IgE) and frequent scratching behaviors. Both tacrolimus and dexamethasone given topically for 10 days before the final challenge significantly inhibited the ear swelling and reduced the expression of interferon-gamma mRNA. Dexamethasone inhibited the accumulation of eosinophils completely, although tacrolimus did not. Both drugs did not affect the elevation of serum IgE levels. Tacrolimus significantly inhibited the scratching behavior, whereas dexamethasone failed to affect it. Repeated DNFB challenge depleted substance P in the dermis. Treatment with tacrolimus before the final challenge completely inhibited the recovery of substance P content, whereas dexamethasone facilitated the recovery. DNFB-induced ear swelling and scratching behavior were significantly inhibited by FK888, a tachykinin NK(1) receptor antagonist. Therefore, substance P seems to participate in the induction of ear swelling and scratching behavior upon final challenge with DNFB, and depletion of substance P by tacrolimus in the dermis contributes to its inhibition of ear swelling and scratching behavior at least in part.

The species differences of intestinal drug absorption and first-pass metabolism between cynomolgus monkeys and humans.

In order to elucidate the causes of the species differences in the oral bioavailability (BA) between cynomolgus monkeys and humans, the contributions of first-pass metabolism and intestinal absorption were investigated. Typical substrates of cytochrome P450 enzymes, UDP-glucuronosyltransferase enzymes and efflux transporters were selected, and the BA, the hepatic availability (Fh) and the fraction dose absorbed from gastro-intestinal tract (Fa*Fg) were calculated from pharmacokinetic analysis after oral and intravenous administration in cynomolgus monkeys. In addition, in vitro metabolism was investigated using liver and intestinal microsomes to evaluate the relationship between in vivo and in vitro results. The BA of cynomolgus monkeys was low compared with that in humans with most of the drugs tested, and not only Fh but also Fa*Fg contributed significantly to the low BA in cynomolgus monkeys. When Fh was evaluated in in vitro experiments, it correlated well with the in vivo Fh. However, although the metabolic activities of CYP3A4 substrates were high in cynomolgus monkey intestinal microsomes, those of the other substrates were low or not detected. These findings suggested that the species differences and low BA in cynomolgus monkeys could be mostly attributed not only to hepatic first-pass metabolism but also to the intestinal absorption process.

Characterization of gastrointestinal drug absorption in cynomolgus monkeys.

Possible factors of species differences in gastrointestinal drug absorption between cynomolgus monkeys and humans were examined using several commercial drugs. Oral bioavailability (BA) of acetaminophen, furosemide, and propranolol in cynomolgus monkeys was significantly lower than that in humans. From the pharmacokinetic analysis, these drugs were found to show the low fraction absorbed into portal vein (FaFg), suggesting that the low BA in cynomolgus monkeys was attributed mainly to the gastrointestinal absorption processes. The gastric emptying rate (GER) calculated from plasma concentration profiles after oral administration of acetaminophen in cynomolgus monkeys was similar in humans. The gastrointestinal transit time (GITT) in cynomolgus monkeys was only slightly shorter than that in humans. On the other hand, it was demonstrated that the apparent intestinal permeability (Papp) of five drugs to cynomolgus monkey intestine was lower than that to rat intestine; especially propranolol and furosemide showed the remarkably low Papp. The expression levels of mRNAs of efflux transporters analyzed by real-time RT-PCR indicated that mRNA expression levels of MDR1, MRP2, and BCRP in monkey intestine were significantly higher than those in human intestine. This result suggested that low oral absorption of furosemide in cynomolgus monkeys was attributed to the high activities of efflux transporters in its intestinal membrane. Results of in vivo PK analysis clearly showed that FaFg values of propranolol and acetaminophen in cynomolgus monkeys were markedly lower than those in humans. Since propranolol and acetaminophen were the drug with high membrane permeability, it was considered that the high first-pass metabolism in the enterocytes was a main factor of their low FaFg in cynomolgus monkeys. In conclusion, it was demonstrated that the high activities of efflux transporters and/or metabolizing enzymes in the intestinal membrane are possible factors to cause poor oral absorption of drugs in cynomolgus monkeys.

Inhibition of scratching behavior associated with allergic dermatitis in mice by tacrolimus, but not by dexamethasone.

Itching is the most important problem in many allergic and inflammatory skin diseases especially in atopic dermatitis. However, animal models for allergic dermatitis useful for the study of itching have rarely been established. We established a mouse allergic dermatitis model involving frequent scratching behavior by repeated painting with 2,4-dinitrofluorobenzene (DNFB) acetone solution onto the mouse skin, and comparatively examined the effects of tacrolimus and dexamethasone on the dermatitis and associated scratching behavior. Repeated DNFB painting caused typical dermatitis accompanied by elevated serum immunoglobulin E (IgE) and frequent scratching behavior. An apparent thickening of the epidermis and dermis, and the significant accumulation of inflammatory cells were observed. Increased interferon (IFN)-gamma mRNA expression and the induction of interleukin (IL)-4 and IL-5 mRNA expression were also observed in the skin lesion. The scratching behavior was inhibited by dibucaine and naloxone. Although tacrolimus reduced the increased expression of IFN-gamma and IL-4 mRNA, dexamethasone potently depressed that of IFN-gamma, IL-4 and IL-5 mRNA. Dexamethasone inhibited the accumulation of lymphocytes and eosinophils, although tacrolimus did not. Both drugs failed to inhibit the elevation of serum IgE levels. Tacrolimus significantly inhibited the scratching behavior that was associated with the inhibition of nerve fiber extension into the epidermis, whereas dexamethasone failed to have any effect. The mouse dermatitis model seems to be beneficial for the study of itching associated with allergic dermatitis, such as atopic dermatitis, and tacrolimus seems to exhibit an anti-itch effect through the inhibition of nerve fiber extension at least in part.

Involvement of unique mechanisms in the induction of scratching behavior in BALB/c mice by compound 48/80.

Compound 48/80 induced scratching behavior in BALB/c mice, and the role of mast cell mediators in this behavior was examined. Mouse scratching behavior was detected and evaluated using a new apparatus, MicroAct. Compound 48/80 increased the incidence of scratching behavior and scratching time in a dose-dependent manner, accompanied by a potent activation of mast cells and a potent increase in vascular permeability. Dibucaine and mu-opioid receptor antagonists inhibited the scratching behavior. Although histamine H(1) receptor antagonists potently inhibited the vascular permeability increase, they did not affect the scratching behavior. Methysergide inhibited the scratching behavior slightly without affecting the vascular permeability increase, whereas cyproheptadine inhibited both. A cyclooxygenase inhibitor, a 5-lipoxygenase-activating protein inhibitor and a PAF receptor antagonist did not affect the scratching behavior. High doses of serotonin induced scratching behavior less frequently than did compound 48/80. Furthermore, mast cell-deficient WBB6F1-W/W(v) mice exhibited frequent scratching behavior after injection of compound 48/80. These results clearly indicate that compound 48/80 can induce scratching behavior in mice independent of mast cell mediators.

Highly water-soluble matrix metalloproteinases inhibitors and their effects in a rat adjuvant-induced arthritis model.

A new series of succinate-based dual inhibitors against matrix metalloproteinases (MMPs) and tumor necrosis factor alpha converting enzyme (TACE) possessing highly-water solubility was designed, synthesized, and evaluated for enzyme inhibition. Incorporating of acidic or basic functional groups at the P(2)' position afforded sufficient water solubility without significant loss of inhibitory potencies. Compound 18e, which had a guanidino group at the P(2)' position as the basic functional group, exhibited broad inhibition against target enzymes for a relatively long period in rat plasma (beta t(1/2); 2.0h) after sc administration when compared with compounds possessing acidic functional groups (18a and 18b). Consequently, the representative compound 18e together with compound 18b, Marimastat and Trocade were evaluated in the rat adjuvant-induced arthritis model, a model of chronic cartilage destruction. It is concluded that the newly synthesized highly water-soluble compound 18e showed significant activity in suppressing hindpaw swelling and the bone destruction with a minimal administration period (days 3-7).