No abstract

EM-652 (SCH 57068) is a new orally active antiestrogen that demonstrates pure antagonistic effects in the mammary gland and endometrium. In vivo studies have shown that EM-652 is primarily glucuronidated at the 7-hydroxy position in rats and that the metabolite is present in the plasma of female monkeys and human subjects after EM-800 (SCH 57050) or EM-652.HCl oral administration. Using hepatic microsomes from rat, monkey, and human, the formation of two EM-652 monoglucuronides at positions 4' and 7 was demonstrated by a liquid chromatographic tandem mass spectrometric method. Although no difference in EM-652 conjugation was observed between male and female monkey livers, an interindividual variation of hepatic EM-652 glucuronidation was shown with female human donors. Using microsome preparations from human embryonic kidney 293 cells stably expressing each of the 12 human and 11 monkey UGT enzymes cloned to date, the two EM-652-monoglucuronides were detected after incubation with microsomes containing human UGT1A1, UGT1A3, UGT1A8, UGT1A9, and monkey monUGT1A01, monUGT1A03, and monUGT1A09. Despite human UGT1A1 and monkey monUGT1A09 favored formation of EM-652-7-glucuronide, other active UGT1A enzymes formed both 4'- and 7-glucuronide derivatives in equal amounts. Kinetic analysis of EM-652 glucuronidation by these enzymes showed Michaelis constant (K(m)) values between 36 and 302 microM for EM-652-4'-glucuronide and 19 and 233 microM for EM-652-7-glucuronide. The present results demonstrate the importance of UGT1A isoforms, mainly UGT1A1, for EM-652 metabolism in humans.

TH9507, an analogue of human growth hormone-releasing factor (hGRF(1-44)NH 2 ) minimally modified by addition of a trans -3-hexenoyl moiety to Tyr 1 of the amino acid sequence, was found to be resistant to dipeptidyl aminopeptidase-IV deactivation. Compared to natural hGRF(1-44)NH 2 , the modification slowed the in vitro degradation of the peptide in rat, dog and human plasma and prolonged the in vivo plasma elimination kinetics of immunoreactive TH9507. Plasma growth hormone and insulin-like growth factor-1 (IGF-1) markedly increased in pigs, rats and dogs after daily repeat intravenous or subcutaneous injections of TH9507 at doses up to 600 µ g /kg. Subchronic toxicity studies in rats and dogs with TH9507 treatment for up to 4 months showed a significant, but not dose-related, increase in body weight gain associated with increased biomarker response. Although TH9507 was well tolerated by both rats and dogs, a more pronounced anabolic effect and more evident (reversible) adverse effects (liver and kidney findings, anaemia, clinical chemistry changes, organ weight effects) were observed in dogs after repeat daily subcutaneous injections, which were attributed to prolonged exposure to supraphysiological levels of growth hormone and/or IGF-1. In both rats and dogs, toxicokinetic evaluations indicated that exposure to immunoreactive TH9507 was dose related after both routes of administration. The apparent elimination t 1/2 in dogs ranged from 21 to 45 min. In conclusion, TH9507 is a modified hGRF peptide having enhanced potency and duration of action. The adverse treatment-related effects in dogs appear to be associated with sustained exposure to supraphysiological levels of growth hormone and IGF-1 induced by prolonged TH9507 treatment.

The primary structure of metallothionein (MT) and MT-like proteins (MLP) contains a high proportion of cysteine residues which are capable of complexing a variety of metals. Due to the unique metal-binding properties of MT and MLP, metal saturation assays have been used as an indirect method of quantifying the concentration of these proteins in various samples. In adapting Cd-saturation methodologies of MT from mammalian sources for MLP from freshwater mussels, the redox state of the MLP cysteine residues was indirectly monitored by adding Cd in the presence, or absence, of a reducing agent followed by high-performance liquid chromatography (HPLC)-microatomization (MA)-atomic absorption spectroscopy (AAS) and/or HPLC-inductively-coupled plasma-mass spectrometry (ICP-MS). The presence of 2-mercaptoethanol(2-MCE) in freshwater mussel extract results in a higher concentration of Cd present in the MLP fraction, indicating that these proteins may be partially oxidized in vivo and/or during isolation under anoxic conditions. Standard preparations of rabbit MT obtained by induced synthesis due to controlled exposure to Cd are fully reduced and saturated with metal (Cd and Zn) and, therefore, did not respond similarly to the reduction treatment used in these studies. Thus, as conventional metal saturation assays exclude a reduction step, they may be unsuitable for the quantitative analysis of MLP from molluscan tissues without further modification.

Abstract-Zebra mussels (Dreissena polymorpha) are freshwater mollusks that have recently infested the Great Lakes ecosystem. Possessing a large capacity for filtration, these mussel populations act as bioconcentrators for contaminants, such as heavy metals, found in the Great Lakes ecosystem. Metallothionein is a low-molecular-weight, heavy metal-binding protein found in most living organisms. Characterization and partial purification of metallothionein-like Cd-binding proteins from zebra mussels were performed. Zebra mussels were exposed to 500 g/L Cd for 14 d. During the exposure period, two mussels were removed on alternate days for analysis of Cd-binding proteins. Gel-filtration high-performance liquid chromatography-microatomization-atomic absorption spectrophotometry results showed a single Cd-binding molecular weight protein fraction after 2 d of Cd exposure. After 10 d of Cd exposure, however, mussels exhibited an additional, higher molecular weight, Cd-binding protein fraction. The lower molecular weight metallothionein-like Cd-binding protein was further isolated and purified by acetone fractionation, Sephadex G75, and diethylaminoethyl anion-exchange chromatography. The quantities of Zn, Cu, and Cd in the anion-exchange metallothionein-like protein isoforms were determined by inductively coupled plasma-mass spectrometry. The ability to bioconcentrate heavy metals in a metallothionein-like form coupled with their large population in the Great Lakes make zebra mussels suitable for use in a freshwater biomonitoring program for aquatic metal contamination.