Irving W. Wainer

NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

SummaryA new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.

Resolution of enantiomeric aromatic alcohols on a cellulose tribenzoate high-performance liquid chromatography chiral stationary phase: a proposed chiral recognition mechanism

Abstract The study investigates the chiral recognition mechanism for the stereochemical resolution of enantiomeric aromatic alcohols on a commercially available cellulose tribenzoate high-performance liquid chromatography chiral stationary phase. A series of chiral and achiral aromatic alcohols were chromatographed and their structural differences related to the chromatographic results. Series of primary and secondary alcohols were also used as mobile phase modifiers to investigate the effect of the steric structure of these modifiers on the capacity factor and stereoselectivity. The results of this investigation indicate that the chiral recognition mechanism for the enantiomeric aromatic alcohols studied involves: (1) the formation of diastereomeric solute-chiral stationary phase (CSP) complexes through a hydrogen bonding interaction between the solutes alcoholic hydrogen and an ester carbonyl on the CSP; (2) the stabilization of this complex through the insertion of the aromatic portion of the solute into a chiral cavity (or ravine) of the CSP; and (3) chiral discrimination between enantiomeric solutes due to difference in their steric fit in the chiral cavity.

Course of Improvement in Depressive Symptoms to a Single Intravenous Infusion of Ketamine vs Add-on Riluzole: Results from a 4-Week, Double-Blind, Placebo-Controlled Study

The N-methyl-D-aspartate antagonist ketamine has rapid antidepressant effects in patients with treatment-resistant major depression (TRD); these effects have been reported to last for 1 week in some patients. However, the extent and duration of this antidepressant effect over longer periods has not been well characterized under controlled conditions. Riluzole, a glutamatergic modulator with antidepressant and synaptic plasticity-enhancing effects, could conceivably be used to promote the antidepressant effects of ketamine. This study sought to determine the extent and time course of antidepressant improvement to a single-ketamine infusion over 4 weeks, comparing the addition of riluzole vs placebo after the infusion. Forty-two subjects (18–65) with TRD and a Montgomery–Asberg Depression Rating Scale (MADRS) score of ⩾22 received a single intravenous infusion of ketamine (0.5 mg/kg). Four to six hours post-infusion, subjects were randomized to double-blind treatment with either riluzole (100–200 mg/day; n=21) or placebo (n=21) for 4 weeks. Depressive symptoms were rated daily. A significant improvement (P<0.001) in MADRS scores from baseline was found. The effect size of improvement with ketamine was initially large and remained moderate throughout the 28-day trial. Overall, 27% of ketamine responders had not relapsed by 4 weeks following a single ketamine infusion. The average time to relapse was 13.2 days (SE=2.2). However, the difference between the riluzole and placebo treatment groups was not significant, suggesting that the combination of riluzole with ketamine treatment did not significantly alter the course of antidepressant response to ketamine alone.

Characterization of the protein binding of chiral drugs by high-performance affinity chromatography interactions of R- and S-ibuprofen with human serum albumin

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3 x 10(5) M-1 at pH 6.9 and 25 degrees C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1 x 10(5) M-1 and 1.2 x 10(5) M-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. The chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.

Intensive Therapy With Growth Factor Support for Patients With Ewing Tumor Metastatic at Diagnosis: Pediatric Oncology Group/Children's Cancer Group Phase II Study 9457—A Report From the Children's Oncology Group

PURPOSE Prognosis is poor for Ewing sarcoma patients with metastasis at diagnosis. We intensified a five-drug therapy (ifosfamide, etoposide alternated with vincristine, doxorubicin, and cyclophosphamide) using filgrastim but not stem-cell support. We studied topotecan alone and combined with cyclophosphamide in therapeutic windows before the five-drug therapy. A randomly assigned proportion of patients received amifostine as a cytoprotective agent. PATIENTS AND METHODS Eligible patients were < or = 30 years old and had histologically proven Ewing sarcoma or primitive neuroectodermal tumor (PNET) and metastasis at diagnosis. Chemotherapeutic cycles began every 21 days, after recovery from toxicities. RESULTS One hundred ten of the 117 patients enrolled were eligible. Thirty-six patients received initial topotecan. Three had partial responses (PRs), and 17 had progressive disease (PD). Thirty-seven patients were administered topotecan and cyclophosphamide; 21 of these patients achieved PR, and one patient had PD. In a randomly assigned group of 69 patients, amifostine did not provide myeloprotection, which was measured by absolute neutrophil count, platelet count, or cycle intervals. The best responses to the overall therapy included 45 complete responses, 41 PRs, stable disease in 14 patients, and PD in five patients. For all patients, the 2-year event-free survival (EFS) rate was 24% (+/- 4%), and the overall survival rate was 46% (+/- 5%). For the 39 patients with isolated pulmonary metastases, the 2-year EFS rate was 31% (+/- 7%) compared with 20% (+/- 5%) for patients with more widespread disease. CONCLUSION Topotecan had limited activity in patients with Ewing sarcoma or PNET metastatic at diagnosis. The topotecan-cyclophosphamide combination was active. Amifostine was not myeloprotective. Overall results showed no improvement compared with previous studies.

Investigation of the enantioselective separations of α-alkylarylcarboxylic acids on an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase using quantitative structure-enantioselective retention relationships. Identification of a conformationally driven chiral recognition mechanism

Abstract A series of 28 chiral α-alkyl arylcarboxylic acids were chromatographed on an amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase (AD-CSP). The retention data were correlated to a series of molecular descriptors to produce quantitative structure-enantioselective retention relationships (QSERR) incorporating the hydrogen bonding ability and aromaticity of the solutes. The QSERR equations were used to guide molecular modelling experiments designed to investigate the chiral recognition mechanism responsible for the observed enantioselective separations. The results of the study indicate that unlike the standard “three-point interaction” model of chiral recognition, enantioselectivity was due to a “conformationally driven” chiral recognition process.

Stereochemical resolution of enantiomeric 2-aryl propionic acid non-steroidal anti-inflammatory drugs on a human serum albumin based high-performance liquid chromatographic chiral stationary phase

SummaryThe native enantioselectivity in binding of human serum albumin (HSA) towards 2-aryl propionic acid non-steroidal anti-inflammatory drugs (2-APA-NSAIDs, the “profens”) was found to be preserved when the protein was immobilized within a commercially available diol high-performance liquid chromatographic column. High capacity factors were obtained, reflecting the previously observed extensive binding of the 2-APA-NSAIDs to free HSA. The capacity factors were modified by the addition of octanoic acid to the mobile phase. Chiral resolution of the enantiomers of all nine 2-APA-NSAIDs studied was achieved. Preliminary studies show that in addition to being a useful chiral analytical tool for this therapeutically important series of compounds, the HSA chiral stationary phase may provide useful information on the affinity and binding mechanism of small molecules to HSA.

Dextromethorphan as an in vivo probe for the simultaneous determination of CYP2D6 and CYP3A activity

Dextromethorphan (DM) is O-demethylated into dextrorphan (DEX) in humans by the cytochrome P450 designated as CYP2D6 and N-demethylated into 3-methoxymorphinan (3MM) via CYP3As. Clinically, DM has been successfully used as an index of CYP2D6 and this paper describes analytical and clinical data that will help evaluate the use of DM hydrobromide as a probe of CYP3A activity. DM and its three demethylated metabolites were measured in a 4-h spot urine sample using a HPLC method employing solid-phase extraction (C(18)), analysis on a phenyl column [mobile phase, methanol-acetonitrile-phosphate buffer (10 mM, pH 3.5, 20:25:55, v/v)] and fluorescence detection (excitation at lambda=228 nm, no emission cut-off filter). The urinary molar ratio DM-DEX was used to assess CYP2D6 activity while DM-3MM was used for CYP3As. The DM-3MM ratios were sensitive to the co-administration of selective CYP3A inhibitors grapefruit juice and erythromycin. In addition, in healthy volunteers and cancer patients, the N-demethylation of DM correlated with the CYP3A-mediated metabolism of verapamil and tamoxifen. DM appears to be a promising way to simultaneously phenotype patients for CYP2D6 and CYP3As.