The conjugation of xenobiotic carboxylic acids with b-Dglucuronic acid to yield 1-O-acyl-b-D-glucopyranuronates (1b-O-acyl glucuronides) is a major metabolic pathway for many compounds, including 2-arylpropionic acids (profens). The 1b-O-acyl glucuronides are labile and reactive in neutral and mildly alkaline aqueous solution, and undergo both hydrolysis and intramolecular acyl migration (Fig. 1). In acyl migration, the aglycone is transferred from the 1b position to the neighboring 2-hydroxyl group on the glucuronic acid moiety via an ortho-acid ester intermediate. 1,2) This process continues on to the OH-groups at C-3 and C-4, thereby forming several regioisomers. The glucuronide regioisomers readily ring-open and mutarotate to give a-and b-anomers. The acyl migrations are generally reversible, but acyl migration from C-2 to C-1 to reform the 1b-O-acyl glucuronide is not commonly observed. These acyl glucuronides are capable of reacting with protein nucleophiles to form covalent adducts.3-12) The protein adducts have been hypothesized as mediators of hypersensitive responses to carboxylate drugs. 1,13) Profens possess a chiral center and are marketed as racemates. Profens are metabolized predominantly to the diastereomeric acyl glucuronides with different physicochemical properties. Consequently, the diastereomeric acyl glucuronides might have distinct toxicological actions and be differentially eliminated. Although the stability of the diastereomeric acyl glucuronides has been investigated in several profens, 7,[14][15][16][17][18][19] little information is currently available on the factors that contribute to stereoselective differences in the degradation process.Enantiomeric 2-phenylpropionic acids (PAs) possess the fundamental structure of profens (Chart 1) and have been used as model substrates of profens to study the stereoselective metabolism and disposition of profens. PAs show similar pharmacokinetic properties to other profens, as observed in stereoselective isomerization and glucuronidation in rodents. The stereoselective acyl migration of diastereomeric 1b b-O-acyl glucuronides of (R)-and (S)-2-phenylpropionic acid [(R)-1PG and (S)-1PG, respectively] in phosphate buffer (pH 7.4) at 310 K was investigated using HPLC. The disappearance of (R)-1PG was faster than that of (S)-1PG according to pseudo first-order kinetics. A kinetic model describing the degradation reactions was constructed. The rate constant for acyl migration from the 1b b-O-isomer to the 2-O-acyl isomer (k 12 ) was about one order magnitude larger than that for hydrolysis from 1b b-O-acyl isomer to aglycone (k 10 ). The k 12 of (R)-1PG (0.377؎0.005 h ؊1 ) was about two times larger than that of (S)-1PG (0.184؎0.003 h ؊1 ). The results indicated that the stereoselectivity in the degradation of 1PG was apparently governed by the acyl migration from 1-isomer to 2-isomer. The kinetic parameters for acyl migration from 1-isomer to 2-isomer were estimated from temperature-dependent experiments using the transition state theory. The value o...

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Abstract. In the present study, changes in localization of nerve growth factor (NGF) and its receptors, trkA and p75 in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization of NGF, trkA and p75 in the ovary was also investigated. NGF and its receptors trkA and p75 were localized in oocytes, granulosa cells and theca cells of various stages of follicles throughout the estrous cycle. NGF and its two receptors were also present in numerous interstitial cells and luteal cells. The number of interstitial cells staining positively for NGF and its two receptors was greater in ovaries of day 1 (day 1=day of ovulation) than the other days during the estrous cycle. Treatment with the antiserum against luteinizing hormone releasing hormone (LHRH-AS) at 1100 h on day 4 completely blocked ovulation. There were few positive reactions for NGF and its two receptors in interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. The distinct widespread distribution of NGF and its two receptors in the ovary of golden hamsters suggest that NGF may be an important growth factor for regulation of ovarian function. Furthermore, the LH surge may be an important factor for inducing production of NGF and its two receptors in interstitial cells of the cyclic golden hamster. Key words: Estrous cycle, Golden hamster, Ovary, Nerve growth factor, TrkA, P75 (J. Reprod. Dev. 50: [605][606][607][608][609][610][611] 2004) he neurotrophins (NTs) are target-derived growth factor required for differentiation, survival and development of discrete neuronal populations in the central and peripheral nervous systems [1,2]. It is also believed that NTs not only have an effect on the nervous system, but also play an important role in regulating the development of non-neuronal cells, including cellular subsets of the immune, cardiovascular and endocrine systems [3][4][5]. The mammalian ovary is innervated by both sympathetic and sensory neurons of the peripheral nervous system [6]. It has been demonstrated that the survival and differentiation of the neurons i n n e r v a t i n g t h e o v a r y a r e s u p p o r t e d b y neurotrophic substances produced in the gland [7,8 ] . T o d a t e , b e s i d e s N G F , b r a i n -d e r i v e d

Internal acyl migration reactions of drug 1-O-acyl-beta-D-glucopyranuronates (1beta-acyl glucuronides) are of interest because of their possible role in covalent binding to proteins and consequent adverse effects. The reactivity of the synthetic probenecid 1beta-acyl glucuronide (PRG), the principal metabolite of probenecid (PR) in humans, has been investigated in terms of acyl migration, hydrolysis, and covalent binding to proteins in phosphate buffer (pH 7.4) and human plasma at 37 degrees C. PRG primarily degraded by acyl migration according to apparent first-order kinetics and the 2-, 3-, and 4-acyl isomers sequentially appeared as both alpha- and beta-anomeric forms. In addition, small amounts of PRG and extremely labile 1alpha-acyl isomer existed in the equilibrated mixture favoring the 2alpha/beta-acyl isomer, that provided significant information regarding the mechanism of acyl migration. All of the positional isomers and anomers were characterized using preparative HPLC and NMR spectroscopy. Acyl migration was observed to predominate over hydrolysis in both media although the extent of hydrolysis in plasma was larger than that in the buffer. The overall degradation half-lives (h) in the buffer and plasma were 0.27 +/- 0.003 and 0.17 +/- 0.007, respectively. The covalent binding rapidly proceeded mainly via the Schiff's base mechanism and reached a plateau after 2 h of incubation. The maximal binding was 146 +/- 4.8 pmol/mg of protein, and ca. 10% of the initial concentration of PRG. These results indicated that PRG is most labile and susceptible to acyl migration of all the drug acyl glucuronides reported to date in the physiological conditions, and highly reactive to plasma proteins, that could provide a possible explanation for the immunologically based adverse effects of PR.

High resolution 19F NMR spectroscopy has been used to investigate the kinetics of internal acyl migration and hydrolysis of the synthetic beta -1-O-acyl-D-glucopyranuronates of 2-, 3-, and 4-(trifluoromethyl) benzoic acids (TFMBAs) in phosphate buffer solutions at 30 degrees C as models of drug ester glucuronides. Apparent first-order degradation of the 1-O-acyl glucuronide and the sequential appearance of 2-, 3-, and 4-O-acyl isomers as both alpha- and beta-anomeric forms were observed for each TFMBA isomer. The overall degradation rate constants of the 2-, 3-, and 4-TFMBA 1-O-acyl isomers were 0.065 h-1, 0.25 h-1, and 0.52 h-1. In order to probe the reasons for these differences in reactivity, theoretical structural and electronic parameters for the beta-anomers of the 1-O-acyl glucuronides, their beta-2-O-acyl isomers, and both structures of the postulated ortho-acid ester intermediate were computed using semiempirical molecular orbital (AM1 and PM3) methods. The distinction between the slowly reacting 2-TFMBA glucuronide and the much faster reacting 3- and 4-TFMBA glucuronides could be observed by calculation of the relative bond order of the C-O bonds in the ortho-acid ester intermediates. The slow internal acyl migration rate of the 2-TFMBA isomer was also partly attributed to the high degree of steric hindrance of the trifluoromethyl group obstructing attack by the glucuronic acid 2-hydroxy group on the carbonyl carbon to form the ortho-acid ester intermediate. Some calculated molecular orbital properties, namely, dipole moment, energy of the lowest unoccupied molecular orbital (LUMO), LUMO density, and nucleophilic frontier density on the carbonyl carbon, were also shown to be related to the measured half-lives. This work gives insight into the molecular physicochemical properties that influence the acyl migration kinetics of simple model drug glucuronides and is of potential importance in understanding more complex drug glucuronide acyl migration reactions of toxicological interest.