ABSTRACT:Studies were designed to quantitatively assess the mRNA expression of 1) 10 cytochrome P450 (P450) enzymes in human cornea, iris-ciliary body (ICB), and retina/choroid relative to their levels in the liver, and of 2) 21 drug transporters in these tissues relative to their levels in human small intestine, liver, or kidney. Potential species differences in mRNA expression of PEPT1, PEPT2, and MDR1 were also assessed in these ocular tissues from rabbit, dog, monkey, and human. P450 expression was either absent or marginal in human cornea, ICB, and retina/choroid, suggesting a limited role for P450-mediated metabolism in ocular drug disposition. In contrast, among 21 key drug efflux and uptake transporters, many exhibited relative expression levels in ocular tissues comparable with those observed in small intestine, liver, or kidney. This robust ocular transporter presence strongly suggests a significant role that transporters may play in ocular barrier function and ocular pharmacokinetics. The highly expressed efflux transporter MRP1 and uptake transporters PEPT2, OCT1, OCTN1, and OCTN2 may be particularly important in absorption, distribution, and clearance of their drug substrates in the eye. Evidence of cross-species ocular transporter expression differences noted in these studies supports the conclusion that transporter expression variability, along with anatomic and physiological differences, should be taken into consideration to better understand animal ocular pharmacokinetic and pharmacodynamic data and the scalability to human for ocular drugs.

ABSTRACT:The recently introduced Clonetics human corneal epithelium (cHCE) cell line is considered a promising in vitro permeability model, replacing excised animal cornea to predict corneal permeability of topically administered compounds. The purpose of this study was to further characterize cHCE as a corneal permeability model from both drug metabolism and transport aspects. First, good correlation was found in the permeability values (P app ) obtained from cHCE and rabbit corneas for various ophthalmic drugs and permeability markers. Second, a previously established realtime quantitative polymerase chain reaction method was used to profile mRNA expression of drug-metabolizing enzymes (major cytochromes P450 and UDP glucuronosyltransferase 1A1) and transporters in cHCE in comparison with human cornea. Findings indicated that 1) the mRNA expression of most metabolizing enzymes tested was lower in cHCE than in excised human cornea, 2) the mRNA expression of efflux transporters [multidrug resistantassociated protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein], peptide transporters (PEPT1 and PEPT2), and organic cation transporters (OCTN1, OCTN2, OCT1, and OCT3) could be detected in cHCE as in human cornea. However, multidrug resistance (MDR) 1 and organic anion transporting polypeptide 2B1 was not detected in cHCE; 3) cHCE was demonstrated to possess both esterase and ketone reductase activities known to be present in human cornea; and 4) transport studies using probe substrates suggested that both active efflux and uptake transport may be limited in cHCE. As the first detailed report to delineate drug metabolism and transport characteristics of cHCE, this work shed light on the usefulness and potential limitations of cHCE in predicting the corneal permeability of ophthalmic drugs, including ester prodrugs, and transporter substrates.Topical instillation is the desired route of administration for ophthalmic drugs to treat diseases in the anterior segment of the eye including glaucoma, inflammations, infections, and dry eye. The primary pathway of drug permeation from the tear fluid to the anterior chamber of the eye is via the transcorneal route. The cornea has a multilayered structure constituted primarily of corneal epithelium, stroma, and endothelium. However, passage through the corneal epithelium is considered to be the rate-limiting step in the transcorneal penetration of most ophthalmic drugs (Maurice and Mishima, 1984).In the past decade, several in vitro corneal permeability models that can substitute for the isolated cornea to predict ocular absorption and facilitate ophthalmic drug discovery have been developed (Reichl and Becker, 2008). These models evolved from primary cultures using rabbit corneas and immortalized rabbit cell lines to the immortalized human corneal epithelial (HCE) cell lines, and even include complex systems such as human corneal constructs (summarized in Table 1). Given the pros and cons of each model, HCE cell lines seem to be most appealing owing to their human o...

Raloxifene (Evista) is a second generation selective estrogen receptor modulator used in the treatment of osteoporosis and for chemoprevention of breast cancer. It is bioactivated to reactive intermediates, which covalently bind to proteins and form GSH conjugates upon incubation with NADPH and GSH-supplemented human and rat liver microsomes. Despite these in vitro findings, no major raloxifene-related toxic events have been reported upon its oral administration to humans. This disconnect between safety of raloxifene and its in vitro bioactivation is attributed to its presystemic metabolism via glucuronidation. Current studies investigated the effect of hepatic and intestinal glucuronidation in modulating hepatic availability of raloxifene and its subsequent bioactivation, in vitro. The study design involved preincubation of raloxifene with intestinal microsomes followed by a sequential incubation with liver microsomes. The degree of bioactivation of raloxifene was assessed from the percentage of GSH conjugate formed in liver microsomal incubations or the amount of covalent binding of raloxifene-related material to liver microsomal proteins. The results indicated that human intestinal glucuronidation limited the hepatic exposure of raloxifene that underwent bioactivation in the liver. Similar experiments with rat microsomal preparations showed very little effect of intestinal glucuronidation. This effect of intestinal glucuronidation and the observed species difference were explained by comparing the efficiency (Cl(int)) of glucuronidation and oxidation in the two species. These findings suggested that even though the rate of bioactivation in the two species was similar, the Cl(int) of glucuronidation was 7.5-fold higher in the human intestine as compared to rats. These results support the hypothesis that intestinal glucuronidation modulates the amount of raloxifene undergoing bioactivation by liver and corroborate the importance of assessing other competitive metabolic pathways and species differences in metabolism prior to extrapolation of bioactivation results from rats to humans.

No abstract

ABSTRACT:Current studies explored the effect of structural changes on the aldehyde oxidase (AO)-mediated metabolism of zoniporide (1). Zoniporide analogs with modifications of the acylguanidine moiety, the cyclopropyl group on the pyrazole ring, and the quinoline ring were studied for their AO-catalyzed metabolism using the human S9 fraction. Analysis of the half-lives suggested that subtle changes in the structure of 1 influenced its metabolism and that the guanidine and the quinoline moieties were prerequisites for AO-catalyzed oxidation to 2-oxozoniporide (M1). In contrast, replacement of the cyclopropyl group with other alkyl groups was tolerated. The effect of structural variation on AO properties was rationalized by docking 1 and its analogs into the human AO homology model. These studies indicated the importance of electrostatic, -stacking and hydrophobic interactions of the three motifs with residues in the active site. Differences in substrate properties were also rationalized by comparing their half-lives with cLogD, electrophilicity parameters [electrostatic potential (ESP) charges and energy of lowest unoccupied molecular orbitals (E LUMO )], and the energies of formation of tetrahedral intermediates (J Med Chem 50:4642-4647, 2007). Whereas the success of energetics in predicting the AO substrate properties of analogs was 87%, the predictive ability of other descriptors was none (cLogD) to 60% (ESP charges and E LUMO ). Overall, the structuremetabolism relationship could be rationalized using a combination of both the energy calculations and docking studies. This combination method can be incorporated into a strategy for mitigating AO liabilities observed in the lead candidate or studying structuremetabolism relationships of other AO substrates.