Clinical and regulatory considerations in pharmacogenetic testing.

PURPOSE: Both regulatory science and clinical practice rely on best available scientific data to guide decision-making. However, changes in clinical practice may be driven by numerous other factors such as cost. In this review, we reexamine noteworthy examples where pharmacogenetic testing information was added to drug labeling to explore how the available evidence, potential public health impact, and predictive utility of each pharmacogenetic biomarker impacts clinical uptake. SUMMARY: Advances in the field of pharmacogenetics have led to new discoveries about the genetic basis for variability in drug response. The Food and Drug Administration recognizes the value of pharmacogenetic testing strategies and has been proactive about incorporating pharmacogenetic information into the labeling of both new drugs and drugs already on the market. Although some examples have readily translated to routine clinical practice, clinical uptake of genetic testing for many drugs has been limited. CONCLUSION: Both regulatory science and clinical practice rely on data-driven approaches to guide decision making; however, additional factors are also important in clinical practice that do not impact regulatory decision making, and these considerations may result in heterogeneity in clinical uptake of pharmacogenetic testing.

Prediction of Warfarin Dose in Pediatric Patients: An Evaluation of the Predictive Performance of Several Models.

OBJECTIVES: The objective of this study was to evaluate the performance of pediatric pharmacogenetic-based dose prediction models by using an independent cohort of pediatric patients from a multicenter trial. METHODS: Clinical and genetic data (CYP2C9 [cytochrome P450 2C9] and VKORC1 [vitamin K epoxide reductase]) were collected from pediatric patients aged 3 months to 17 years who were receiving warfarin as part of standard care at 3 separate clinical sites. The accuracy of 8 previously published pediatric pharmacogenetic-based dose models was evaluated in the validation cohort by comparing predicted maintenance doses to actual stable warfarin doses. The predictive ability was assessed by using the proportion of variance (R(2)), mean prediction error (MPE), and the percentage of predictions that fell within 20% of the actual maintenance dose. RESULTS: Thirty-two children reached a stable international normalized ratio and were included in the validation cohort. The pharmacogenetic-based warfarin dose models showed a proportion of variance ranging from 35% to 78% and an MPE ranging from -2.67 to 0.85 mg/day in the validation cohort. Overall, the model developed by Hamberg et al showed the best performance in the validation cohort (R(2) = 78%; MPE = 0.15 mg/day) with 38% of the predictions falling within 20% of observed doses. CONCLUSIONS: Pharmacogenetic-based algorithms provide better predictions than a fixed-dose approach, although an optimal dose algorithm has not yet been developed.

Discovery of non-oxime reactivators using an in silico pharmacophore model of reactivators for DFP-inhibited acetylcholinesterase.

Utilizing our previously reported in silico pharmacophore model for reactivation efficacy of oximes, we present here a discovery of twelve new non-oxime reactivators of diisopropylfluorophosphate (DFP)-inhibited acetylcholinesterase (AChE) obtained through virtual screening of an in-house compound database. Rate constant (kr) efficacy values of the non-oximes were found to be within ten-fold of pralidoxime (2-PAM) in an in vitro DFP inhibited eel AChE assay and one of them showed in vivo efficacy comparable to 2-PAM against brain symptoms for DFP induced neuropathology in guinea pigs. Short listing of the identified compounds were performed on the basis of in silico evaluations for favorable blood brain barrier penetrability, octanol-water partition (Clog P), toxicity (rat oral LD50) and binding affinity to the active site of the crystal structure of a OP- inhibited AChE.

Ethanol pharmacokinetics in neonates and infants.

INTRODUCTION: Ethanol has been used for years in neonatal and infant liquid medications, yet the pharmacokinetics, pharmacodynamics, and safety of ethanol in this vulnerable population have not been well characterized. The purpose of this review is to raise awareness of ethanol use as an excipient in neonatal and infant medications and to provide insight, based on the available evidence, into clearance rates of ethanol in babies. We also discuss ethanol pharmacokinetics in adults, theoretical pharmacokinetic changes in neonates and infants as it may apply to ethanol disposition, and case reports involving ethanol exposure in neonates and infants. MATERIALS AND METHODS: This study was a narrative review in which relevant papers were selected using databases and scientific search engines such as PubMed with the key words ethanol, infant, and newborn infant. RESULTS: It remains unclear what ethanol exposure is safe for neonates and infants. The Food and Drug Administration and American Academy of Pediatrics have both taken action, by either setting limits of ethanol content in over-the-counter medications or by recommending restricted exposure to ethanol-containing pediatric formulations. CONCLUSIONS: Until the short- and long-term health effects of chronic ethanol administration can be further characterized, ethanol-containing medications should be used with caution.

Discovery of non-oxime reactivators using an in silico pharmacophore model of oxime reactivators of OP-inhibited acetylcholinesterase.

We earlier reported an in silico pharmacophore model for reactivation of oximes to tabun-inhibited AChE. Since DFP (diisopropylfluorophosphate) like tabun is a G-agent simulator, we utilized the model as a rational strategy to discover non-oxime reactivators of DFP-inhibited AChE in this study. The phramacophore was used for virtual screening of two commercial databases, Maybridge and ChemNavigator, to identify reactivators which lack the oxime functions. The procedure led us to identify several potent non-oxime compounds that reactivate DFP-inhibited AChE. These non-oxime reactivators contain a nucleophile group in lieu of the oxime moiety in the compound. Five of these novel non-oximes showed Kr values within ten-fold of 2-PAM in an in vitro assay. The pharmacophore model contained a hydrogen bond acceptor, a hydrogen bond donor, and an aromatic ring features distributed in a 3D space. Calculated stereoelectronic properties reported earlier with respect to the location of molecular orbitals and electrostatic potentials were consistent with the model and the newly identified compounds. Down selection of compounds after virtual screening was performed on the basis of fit score to the model, conformational energy, and in silico evaluations for favorable blood-brain barrier (BBB) penetrability, octanol-water partition (log P), and toxicity (rat oral LD(50)) assessments. In vitro reactivation efficacy of the compounds was evaluated in a DFP-inhibited eel acetylcholinesterase assay.

Pro-2-PAM therapy for central and peripheral cholinesterases.

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

3D-QSAR studies of 2,2-diphenylpropionates to aid discovery of novel potent muscarinic antagonists.

Muscarinic acetylcholine receptors (mAChRs) consisting of five known subtypes, are widely distributed in both central and peripheral nervous systems for regulation of a variety of critical functions. The present theoretical study describes correlations between experimental and calculated molecular properties of 15 alpha-substituted 2,2-diphenylpropionate antimuscarinics using quantum chemical and pharmacophore generation methods to characterize the drug mAChR properties and design new therapeutics. The calculated stereoelectronic properties, such as total energies, bond distances, valence angles, torsion angles, HOMO-LUMO energies, reactivity indices, vibrational frequencies of ether and carbonyl moieties, and nitrogen atom proton affinity were found to be well correlated when compared with experimentally determined inhibition constants from the literature using three muscarinic receptor assays: [(3)H]NMS receptor binding, alpha-amylase release from rat pancreas, and guinea pig ileum contraction. In silico predicted toxicity on rat oral LD(50) values correlated well with the [(3)H]NMS binding in N4TG1 cells and alpha-amylase release assays, but not the ileum contraction assay. Next, to explore the functional requirements for potent activity of the compounds, we developed a preliminary 3D pharmacophore model using the in silico techniques. The resulting model contained a hydrogen bond acceptor site on the carbonyl oxygen atom and a ring aromatic feature on one of the two aromatic rings in these compounds. This model was used as a template to search an in-house database for novel analogs. We found compounds equal in inhibition potency to atropine and, importantly, six not reported before as antimuscarinics. These results demonstrate that this QSAR approach not only provides a basis for understanding the molecular mechanism of action but a pharmacophore to aid in the discovery and design of novel potent muscarinic antagonists.