James E. Evans

Norcocaine and N-Hydroxynorcocaine Formation in Human Liver Microsomes: Role of Cytochrome P-450 3A4

Cocaine was metabolized to norcocaine by microsomes prepared from lymphoblastoid cells expressing transfected human P-450 3A4. The specific activities of norcocaine formation by microsomes prepared from three human liver samples correlated with the amount of P-450 3A immunoreactive protein detected by immunoblot. Triacetyloleandomycin, a specific inhibitor of P-450 3A isoforms, inhibited formation of norcocaine from cocaine, but not formation of N-hydroxynorcocaine from norcocaine. The chemical identity of the norcocaine and N-hydroxynorcocaine produced by human liver microsomes was established by combination of gas chromatography and mass spectrometry. Thus, human P-450 3A4 is a cocaine demethylase, and P-450 isoforms of the 3A family are responsible for the majority of norcocaine production by human hepatic microsomes.

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain

High‐mobility‐group (HMG) proteins are a family of non‐histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP‐1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP‐1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin–sepharose and sulfatide‐octyl–sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP‐1. Characterization by high performance liquid chromatography–mass spectrometry (HPLC–MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG‐1 and HMG‐2. Isoelectric focusing, HPLC–MS and peptide sequencing data also suggested that HMG‐1 and SBP‐1 were identical. Similar to SBP‐1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC‐immuno‐overlay and solid‐phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti‐Leu7 hybridoma (HNK‐1) and anti‐SBP‐1 peptide antibodies, similar to SBP‐1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG‐1 and ‐2 proteins have similar biochemical and biological properties and HMG‐1 is most likely identical to SBP‐1.

Sulfoglucuronyl-Neolacto Series of Glycolipids in Peripheral Nerves Reacting with HNK-1 Antibody

Abstract: Novel sulfoglucuronyl‐neolacto series of glycolipids were detected in peripheral nerves of various species by TLC followed by immunostaining with HNK‐1 antibody. The major antigenic glycolipid, sulfoglucuronylneolactote‐traosylceramide, previously described in human nerves, was shown to be also present in the sciatic nerves of various species including rodents. A second slower migrating antigenic glycolipid present in the sciatic nerves of human and dog was isolated and purified. It was characterized by chemical and enzymatic degradation, sugar analysis after permethylation, and gas liquid chromatography‐mass spectrometry techniques as well as by fast atom bombardment‐mass spectrometry, as 3‐sulfogIucuronylneolactohexaosylceramide. During postnatal development of the rat sciatic and trigeminal nerves the concentration of these antigenic glycolipids increased with age.

Bioavailability of ACC-9653 (Phenytoin Prodrug)

Summary: The bioavailability of phenytoin from ACC‐9653 versus intravenously administered sodium phenytoin was determined using a crossover design for intravenous and intramuscular administration of ACC‐9653 to healthy volunteers. Absolute bioavailability of phenytoin derived from ACC‐9653 in each subject was calculated as the ratio of the phenytoin area under the plasma concentration time curve for time 0 to infinity [AUC(0‐inf)] after ACC‐9653 divided by the phenytoin AUC(O‐inf) after intravenous sodium phenytoin. The mean absolute bioavailability of ACC‐9653 was 0.992 after intravenous administration and 1.012 after intramuscular administration. These data establish that the bioavailability of ACC‐9653 is complete following intravenous or intramuscular administration in single‐dose volunteer studies. The absolute bioavailability of phenytoin derived from ACC‐9653 in subjects with therapeutic plasma phenytoin concentrations is being studied in patients given simultaneous infusions of stable isotope‐labeled tracer doses of ACC‐0653 and sodium phenytoin.

Carbamazepine increases phenytoin serum concentration and reduces phenytoin clearance.

Addition of carbamazepine to phenytoin monotherapy resulted in the following significant (p < 0.05) changes: (1) increased mean phenytoin serum concentration; (2) decreased phenytoin clearance, due to decreased production of phenytoin dihydrodiol and p-hydroxyphenyl-phenylhydantoin; (3) increased phenytoin elimination half-life; and (4) increased drug-related toxicity. Close monitoring is required after addition of carbamazepine to phenytoin.

Studies with stable isotopes II: Phenobarbital pharmacokinetics during monotherapy.

Six healthy adults receiving no other medications were given tracer doses of 90 mg of stable isotope‐labeled phenobarbital (PB) intravenously before, and four weeks after, and 12 weeks after beginning therapy. Serum samples were collected for 96 hours after each injection, and the concentration of stable isotope‐labeled PB in each sample was determined by gas chromatographic mass spectrometry. The volume of distribution, elimination half‐life, and total clearance of PB did not differ significantly on any of the three occasions measured. Phenobarbital clearance did not correlate significantly with total PB serum concentration. Clearances determined from single‐dose studies before beginning PB therapy accurately predicted steady‐state PB serum concentrations. Therefore, it is not necessary to adjust PB dosage for time‐dependent or dose‐dependent changes in clearance during monotherapy. In addition, clearance or serum concentration determined at one dosing rate directly predicts serum concentration at another dosing rate.

High pressure liquid chromatography of neutral glycosphingolipids.

Abstract A system for the separation of neutral glycosphingolipids by high pressure liquid chromatography of their benzoylated derivatives has been devised. Serum mono-, di-, tri-and tetraglycosylceramides are completely resolved within 25 min. A full scale recorder response can be obtained with approximately 1.0 nmole of glycolipid.

Studies With Stable Isotopes I: Changes in Phenytoin Pharmacokinetics and Biotransformation During Monotherapy

Six patients were given tracer doses of 13C15N2‐phenytoin (PHT) before and four and 12 weeks after beginning monotherapy. The following significant (P < .05) changes occurred during monotherapy: (1) Apparent (from tracer doses) PHT total clearance by linear method decreased; (2) apparent PHT elimination half‐life increased; (3) apparent mean PHT serum concentration per unit dose increased; (4) apparent rate of excretion of p‐hydroxyphenyl‐phenylhydantoin (p‐HPPH) decreased; (5) apparent rate of excretion of PHT dihydrodiol increased; and (6) apparent PHT total clearance and elimination half‐life and apparent p‐HPPH rate of excretion were dose dependent. Phenytoin apparent pharmacokinetic and biotransformation values undergo a typical series of changes after beginning monotherapy at typical dosing rates, because PHTs dose‐dependent pharmacokinetics result in differing apparent vaiues as the serum concentration rises to steady state. Stable iostope methods are particularly suitable for investigating such phenomena.

Long-term statin therapy and CSF cholesterol levels: implications for Alzheimer's disease.

Background/Aims: It is not yet established whether statins (lipophilic or hydrophilic) reduce the risk of Alzheimer’s disease and, if so, by differentially modifying brain lipid levels. Our aim was to assess changes in brain cholesterol metabolism as reflected in the cerebrospinal fluid (CSF) before and after treatment with either atorvastatin or simvastatin. Methods: We carried out a longitudinal analysis of CSF cholesterol, lathosterol and 24(S)-hydroxycholesterol before and after treatment with maximum doses of statins in 10 asymptomatic subjects, 8 of whom were heterozygous for apolipoprotein E ε4, and in 6 presymptomatic PS1 subjects. Results: Statins initially reduced CSF lathosterol cholesterol and 24(S)-hydroxycholesterol in both PS1 and non-PS1 subjects reaching a nadir at 6–7 months, followed by a return to baseline at 15 months with an overshoot at 2 years, tending to return to baseline thereafter. Conclusions: Possible long-term protective effects of statins are not likely largely related to the temporally-dependent biphasic effects of statin therapy upon the magnitude and direction of changes in CSF lipid levels and their subsequent return to baseline levels.

Phenobarbital does not alter phenytoin steady‐state serum concentration or pharmacokinetics

Phenytoin pharmacokinetics and biotransformation were studied with stable isotope tracer techniques in six patients before and after addition of phenobarbital. No significant (p < 0.05) changes in phenytoin serum concentration, clearance, elimination half-life, volume of distribution, or clearance via production of p-hydroxyphenyl-phenylhydantoin or phenytoin dihydrodiol occurred after addition of phenobarbital. Thus, frequent phenytoin serum concentration determinations or a change in phenytoin dosing rate are probably not necessary after adding phenobarbital.