Edward J. Kelly

Pharmacokinetics of Metformin during Pregnancy

Our objective was to evaluate the pharmacokinetics of metformin during pregnancy. Serial blood and urine samples were collected over one steady-state dosing interval in women treated with metformin during early to late pregnancy (n = 35) and postpartum (n = 16). Maternal and umbilical cord blood samples were obtained at delivery from 12 women. Metformin concentrations were also determined in breast milk samples obtained over one dosing interval in 6 women. Metformin renal clearance increased significantly in mid (723 ± 243 ml/min, P < 0.01) and late pregnancy (625 ± 130 ml/min, P < 0.01) compared with postpartum (477 ± 132 ml/min). These changes reflected significant increases in creatinine clearance (240 ± 70 ml/min, P < 0.01 and 207 ± 56 ml/min, P < 0.05 versus 165 ± 44 ml/min) and in metformin net secretion clearance (480 ± 190 ml/min, P < 0.01 and 419 ± 78 ml/min, P < 0.01 versus 313 ± 98 ml/min) in mid and late pregnancy versus postpartum, respectively. Metformin concentrations at the time of delivery in umbilical cord plasma ranged between nondetectable (<5 ng/ml) and 1263 ng/ml. The daily infant intake of metformin through breast milk was 0.13 to 0.28 mg, and the relative infant dose was <0.5% of the mother’s weight-adjusted dose. Our results indicate that metformin pharmacokinetics are affected by pregnancy-related changes in renal filtration and net tubular transport and can be roughly estimated by the use of creatinine clearance. At the time of delivery, the fetus is exposed to metformin concentrations from negligible to as high as maternal concentrations. In contrast, infant exposure to metformin through the breast milk is low.

Defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis.

Juvenile periodontitis (JP) is a disease characterized by severe gingival infections. PMN from some JP patients exhibit abnormal chemotactic responsiveness when challenged with the synthetic formyl peptide, FMLP. While investigating PMN function in JP, we found a patient in whom abnormal PMN chemotactic responses to FMLP were associated with a defective population of PMN formyl peptide receptor(s) (FPR). JP PMN failed to respond chemotactically when challenged with FMLP, but exhibited normal chemotactic responses upon exposure to purified human C5a. Furthermore, JP PMN were capable of degranulating and generating superoxide anion radicals as well as normal PMN upon exposure to FMLP. Binding studies demonstrated that JP PMN had a diminution in the number of high-affinity FPR. Studies in which FPR was radiolabeled by chemical cross-linking demonstrated that JP PMN FPR exhibited the same molecular weight and N-linked glycosylation as normal PMN FPR. JP PMN FPR, however, was more resistant to papain cleavage than normal PMN FPR. Autoradiograms obtained from 2D-PAGE of normal and JP PMN FPR demonstrated decreased amounts of FPR isoforms in JP PMN.

Identification of the C5a des Arg cochemotaxin. Homology with vitamin D-binding protein (group-specific component globulin).

The chemotactic activity of human C5a des Arg is enhanced significantly by an anionic polypeptide (cochemotaxin) in normal human serum and plasma. The cochemotaxin attaches to sialic acid residues within the oligosaccharide chain of native C5a des Arg to form a complex with potent chemotactic activity for human PMN. We investigated the nature of the cochemotaxin and found that vitamin D-binding protein is the putative cochemotaxin. Vitamin D-binding protein enhanced the chemotactic activity of native C5a des Arg, but had no effect on the chemotactic activity of either native C5a or FMLP. Sialic acid prevented both enhancement by vitamin D-binding protein of the chemotactic activity of native C5a des Arg and formation of C5a des Arg-vitamin D-binding protein complexes, detected by molecular sieve chromatography. Furthermore, vitamin D-binding protein and cochemotaxin exhibited identical molecular weights, isoelectric points, antigenic reactivity, and amino acid composition.

Expression and Characterization of CYP4V2 as a Fatty Acid ω-Hydroxylase

Biettis crystalline dystrophy is an ocular disease that is strongly associated with polymorphisms in the CYP4V2 gene. CYP4 enzymes are typically microsomal fatty acid ω-hydroxylases that function together with mitochondrial and peroxisomal β-oxidation enzymes to degrade cellular lipids. Indeed, ocular and peripheral cells cultured from patients with Biettis have been reported to exhibit abnormal lipid metabolism. However, CYP4V2 possesses low sequence homology to other members of the CYP4 family. Therefore, we cloned and expressed CYP4V2 and analyzed the functional characteristics of this new cytochrome P450 enzyme. We find that CYP4V2 is a selective ω-hydroxylase of saturated, medium-chain fatty acids with relatively high catalytic efficiency toward myristic acid. Moreover, N-hydroxy-N′-(4-n-butyl-2-methylphenyl formamidine) (HET0016) is a nanomolar inhibitor of the enzyme. Therefore, CYP4V2 exhibits catalytic functions typical of a human CYP4 enzyme, but with a distinctive chain-length selectivity coupled with high ω-hydroxylase specificity. Consequently, defective ω-oxidation of ocular fatty acids/lipids secondary to mutations in the CYP4V2 gene appears to be a plausible mechanism underlying Biettis crystalline dystrophy.

Expression of human metallothionein-III in transgenic mice

Transgenic mice that express human metallothionein-III (hMT-III) were generated. Human MT-III mRNA expression was prominent in brain, resulting in a 9-fold elevation of MT-III mRNA in cortex, a 3-5-fold elevation in hippocampus, thalamus, brainstem, and olfactory bulb, and a 1.4-fold elevation in cerebellum. Human MT-III protein was detected biochemically and accounted for a 3.4-fold increase in total brain MT. The concentration of zinc (but not copper) was elevated in those brain regions that expressed the most hMT-III mRNA. The histochemically reactive pool of zinc, as measured by Timms stain or TS-Q histofluorescence, was not appreciably altered. No changes in brain weight, morphology or histology have been noted; the mice breed normally and appear to have normal behavior.

Functional Coupling of Human Microphysiology Systems: Intestine, Liver, Kidney Proximal Tubule, Blood-Brain Barrier and Skeletal Muscle

Organ interactions resulting from drug, metabolite or xenobiotic transport between organs are key components of human metabolism that impact therapeutic action and toxic side effects. Preclinical animal testing often fails to predict adverse outcomes arising from sequential, multi-organ metabolism of drugs and xenobiotics. Human microphysiological systems (MPS) can model these interactions and are predicted to dramatically improve the efficiency of the drug development process. In this study, five human MPS models were evaluated for functional coupling, defined as the determination of organ interactions via an in vivo-like sequential, organ-to-organ transfer of media. MPS models representing the major absorption, metabolism and clearance organs (the jejunum, liver and kidney) were evaluated, along with skeletal muscle and neurovascular models. Three compounds were evaluated for organ-specific processing: terfenadine for pharmacokinetics (PK) and toxicity; trimethylamine (TMA) as a potentially toxic microbiome metabolite; and vitamin D3. We show that the organ-specific processing of these compounds was consistent with clinical data, and discovered that trimethylamine-N-oxide (TMAO) crosses the blood-brain barrier. These studies demonstrate the potential of human MPS for multi-organ toxicity and absorption, distribution, metabolism and excretion (ADME), provide guidance for physically coupling MPS, and offer an approach to coupling MPS with distinct media and perfusion requirements.

Cloning of a cDNA encoding a receptor related to the formyl peptide receptor of human neutrophils

We cloned a cDNA (RFP) encoding a receptor (RFP) related (70% overall nucleotide homology) to the formyl peptide receptor of human neutrophils (hFPR). RFP is a seven-transmembrane-domain receptor and its distribution is limited to myeloid cells. Domain sequence comparison with hFPR reveals highly conserved regions and provides clues to putative domains involved in ligand binding and receptor desensitization.

CYP4V2 in Bietti's Crystalline Dystrophy: Ocular Localization, Metabolism of ω-3-Polyunsaturated Fatty Acids, and Functional Deficit of the p.H331P Variant

Biettis crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

Interindividual Differences in Response to Chemoprotection Against Aflatoxin-Induced Hepatocarcinogenesis: Implications for Human Biotransformation Enzyme Polymorphisms

It is now evident that most, if not all, of the remarkable species differences in susceptibility to AFB hepatocarcinogenesis is due in large part, if not exclusively, to differences in biotransformation. Certainly the relative rate of oxidative formation of the proximate carcinogen, AFB-8,9-exo-epoxide, is an important determinant of species and interindividual differences in susceptibility to AFB. However, mice produce relatively large amounts of exo-AFBO, yet are highly resistant to AFB-hepatocarcinogenesis because they express a particular form of GST with remarkably high catalytic activity toward the exo-epoxide of AFB. Rats, which are highly susceptible to AFB hepatocarcinogenesis,can be made resistant through dietary induction of an orthologous form of GST that is normally expressed in only very small amounts. Based on these findings in laboratory animal models, there is great interest in identifying chemicals and/or specific dietary constituents that could offer protection against AFB-hepatocarcinogenesis to humans. Current experimental strategies have focused on the antiparasitic drug, oltipraz, which induces protection in rats and has also shown some promise in humans. The mechanism of protection in rats appears to be via induction of an alpha class GST with high catalytic activity toward AFBO (rGSTA5-5). vet human alpha class GST proteins that are constitutively expressed in the liver (hGSTA1 and hGSTA2) have little, if any activity toward AFBO. Rather, it appears that mu class GSTs may be responsible for the very low, but potentially significant, detoxification activity toward AFBO. Oltipraz and certain dietary constituents may induce mu class GSTs in human liver, and this could afford some protection against the genotoxic effects of AFBO. However, it also appears that oltipraz, and perhaps certain dietary constituents, act as competitive inhibitors of human CYP1A2. As CYP1A2 appears to mediate most of the activation of AFB to exo-AFBO in human liver at low dietary concentrations of AFB encountered in the human diet, much of the putative protective effects of oltipraz could be mediated via inhibition of CYP1A2 rather than induction of GSTs. There is now evidence that human microsomal epoxide hydrolase (mEH) could play a role in protecting human DNA from the genotoxic effects of AFB, although the importance of this detoxification pathway, relative to mu class GSTs, remains to be elucidated. Oltipraz is an effective inducer of mEH in rats (Lamb Franklin, 2000), and thus induction of this pathway in humans could also potentially contribute to the protective effects of this drug toward AFB genotoxicity. Because the dihydrodiol of AFB may contribute indirectly to the carcinogenic effects of AFB via protein adduction and subsequent hepatotoxicity, the recently characterized human aflatoxin aldehyde reductase (AFAR) may also offer some protection against AFB-induced carcinogenicity in humans. Current and future dietary and/or chemointervention strategies aimed at reducing the carcinogenic effects of AFB in humans should consider all of the possible mechanistic approaches for modifying AFB-induced genotoxicity.

Pharmacogenetics of Intravenous and Oral Busulfan in Hematopoietic Cell Transplant Recipients

Kinetics‐based dose targeting is often conducted in hematopoietic cell transplant (HCT) patients conditioned with intravenous (IV) or oral busulfan to lower rates of rejection, nonrelapse mortality, and relapse. Using the candidate gene approach, the authors evaluated whether busulfan clearance was associated with polymorphisms in the genes regulating the predominant metabolizing enzymes involved in busulfan conjugation, specifically glutathione S‐transferase (GST) isoenzymes A1 (GSTA1) and M1 (GSTM1). Busulfan clearance was estimated after the morning dose on days 1, 2, and 3; each patients average clearance was used for analyses. The average (± standard deviation) busulfan clearance was 3.2 ± 0.56 mL/min/kg in the separate population of 95 patients who received oral busulfan and 103 ± 24 ml/min/m2 in the 57 patients who received IV busulfan. Oral busulfan clearance was associated with GSTA1 (P = .008) but not GSTM1 (P = .57) genotypes. However, among the GSTA1 haplotypes (ie, *A*A, *A*B, *B*B), there was significant overlap in the observed oral busulfan clearance and similar rates of achieving the target busulfan exposure. Clearance of IV busulfan was not associated with GSTA1 (P = .21) or GSTM1 (P = .99). These data suggest that personalizing either IV or oral busulfan dosing cannot be simplified on the basis of GSTA1 or GSTM1 genotype.