Development of a non-human primate model to support CNS translational research: Demonstration with D-amphetamine exposure and dopamine response.

BACKGROUND: Challenges specific to the discovery and development of candidate CNS drugs have led to implementation of various in silico, in vitro and in vivo approaches to improve the odds for commercialization of novel treatments. NEW METHOD: Advances in analytical methodology and microdialysis probe design have enabled development of a non-human primate model capable of measuring concentrations of drugs or endogenous chemicals in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF). Linking these to population modeling reduces animal numbers to support predictive translational sciences in primates. Application to measure D-amphetamine exposure and dopamine response in ECF and CSF demonstrate the approach. RESULTS: Following a 0.1 mg/kg intravenous bolus dose of D-amphetamine, a population approach was used to build a plasma compartmental-based and brain physiologic-based pharmacokinetic (PK) model linking drug concentrations in plasma to brain ECF and CSF concentrations. Dopamine was also measured in brain ECF. The PK model was used to simulate the relationship between D-amphetamine exposure and dopamine response in ECF over a wide dose range. COMPARISONS WITH EXISTING METHODS: Ability to co-sample and measure drug and endogenous substances in blood, brain ECF and/or CSF, coupled with population modeling, provides an in vivo approach to evaluate CNS drug penetration and effect in non-human primates. CONCLUSIONS: A method to measure drug and endogenous neurochemicals in non-human primate brain fluids is demonstrated. Its basis in non-human primates merits improved confidence regarding predictions of drug exposure and target engagement in human CNS.

Quantification of Methylphenidate, Dexamphetamine, and Atomoxetine in Human Serum and Oral Fluid by HPLC With Fluorescence Detection.

BACKGROUND: For psychostimulants, a marked individual variability in the dose-response relationship and large differences in plasma concentrations after similar doses are known. Therefore, optimizing the efficacy of these drugs is at present the most promising way to exploit their full pharmacological potential. Moreover, it seems important to examine oral fluid as less invasive biological matrix for its benefit in therapeutic drug monitoring for patients with hyperkinetic disorder. METHODS: A high-performance liquid chromatography method for quantification of methylphenidate (MPH), dexamphetamine (DXA), and atomoxetine in serum and oral fluid has been developed and validated. The analytical procedure involves liquid-liquid extraction, derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a label and chromatographic separation on a Phenomenex Gemini-NX C18 analytical column using gradient elution with water-acetonitrile. The derivatized analytes were detected at 330 nm (excitation wavelength) and 440 nm (emission wavelength). To examine the oral fluid/serum ratios, oral fluid samples were collected simultaneously to blood samples from patients with hyperkinetic disorder. RESULTS: The method allows quantification of all analytes in serum and oral fluid within 16 minutes under the same or similar conditions. Oral fluid/serum ratios for MPH and DXA were highly variable and showed an accumulation of these drugs in oral fluid. CONCLUSIONS: The developed method covers the determination of MPH, DXA, and atomoxetine concentrations in serum and oral fluid after the intake of therapeutic doses. Oral fluid samples are useful for the qualitative detection of MPH and DXA.

Impurity profiling of dexamphetamine sulfate by cyclodextrin-modified microemulsion electrokinetic chromatography.

A CD-modified microemulsion electrokinetic chromatography method has been developed and validated for dexamphetamine sulfate which allows the simultaneous determination of charged and uncharged impurities of the drug including the levorotary (R)-enantiomer. The optimized background electrolyte consisted of 1.5% w/w SDS, 0.5% w/w ethyl acetate, 3.5% w/w 1-butanol, 2.5% w/w 2-propanol and 92% w/w 50 mM sodium phosphate buffer, pH 3.0, containing 5.5% w/w sulfated ß-CD. Separations were performed in a 50.2/40 cm, 50 µm id fused silica capillary at a temperature of 20°C and an applied voltage of -14 kV. Carbamazepine was used as internal standard. The assay was validated in the range of 0.1-1.0% for the related substances and 0.1-5.0% for levoamphetamine based on a concentration of 3 mg/mL of dexamphetamine sulfate. The LOD of all analytes ranged between 0.05 and 0.2%. In commercial samples of dexamphetamine sulfate, levoamphetamine was found at concentrations between 3.2 and 3.8%, whereas none of the other impurities could be detected.

CE assay for simultaneous determination of charged and neutral impurities in dexamphetamine sulfate using a dual CD system.

A CE assay for the simultaneous determination of charged and uncharged potential impurities (1S,2S-(+)-norpseudoephedrine, 1R,2S-(-)-norephedrine, phenylacetone and phenylacetone oxime) of dexamphetamine sulfate including the stereoisomer levoamphetamine was developed and validated. The optimized background electrolyte consisted of a 50 mM sodium phosphate buffer, pH 3.0, containing 80 mg/mL sulfobutylether-beta-CD and 25 mg/mL sulfated beta-CD. Separations were performed in 40.2/35 cm, 50 mum id fused-silica capillaries at a temperature of 20 degrees C and an applied voltage of -10 kV. 1R,2S-(-)-ephedrine was used as internal standard. The assay was validated in the range of 0.05-1.0% for the related substances and in the range of 0.05-5.0% for levoamphetamine. The LOD was 0.01-0.02% depending on the analyte. The assay also allowed the separation of the E,Z-stereoisomers of phenylacetone oxime. The effect of the degree of substitution of sulfobutylether-beta-CD was investigated. In commercial samples of dexamphetamine sulfate between 3.2 and 3.7% of levoamphetamine were found. Furthermore, phenylacetone and phenylacetone oxime could be observed at the LOD, indicating the synthetic origin of the investigated samples.

Profiling of levoamphetamine and related substances in dexamphetamine sulfate by capillary electrophoresis.

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity of dexamphetamine as well as the analysis of 1R,2S-(-)-norephedrine and 1S,2S-(+)-norpseudoephedrine as potential impurities has been developed and validated. Heptakis-(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin was chosen as chiral selector upon a screening of neutral and charged cyclodextrin derivatives. Separation of the analytes was achieved in a fused-silica capillary at 20 degrees C using an applied voltage of 25 kV. The optimized background electrolyte consisted of a 0.1 M sodium phosphate buffer, pH 2.5, containing 10 mg/ml of the cyclodextrin. The assay was linear in the range of 0.06-5.0% of the impurities based on a concentration of 2.0 mg/ml dexamphetamine sulfate in the sample solution. Analysis of commercial dexamphetamine sulfate samples revealed the presence of 3-4% of levoamphetamine while norephedrine or norpseudoephedrine could not be detected, indicating that the compound was prepared by fractionated crystallization of racemic amphetamine. Comparison with polarimetric measurements indicated that dexamphetamine with an enantiomeric excess as low as 80% still passes the pharmacopeial test of specific rotation while an amount of 0.06% of levoamphetamine can be detected by capillary electrophoresis.

A convenient derivatization method for gas chromatography/mass spectrometric determination of phenmetrazine in urine using 2,2,2-trichloroethyl chloroformate.

Phenmetrazine is a central nervous system stimulant currently used as an anorectic agent. The drug is abused and is reported to cause death from overdose. We describe a new derivatization method for phenmetrazine using 2,2,2-trichloroethyl chloroformate. Quantitation of urinary phenmetrazine can be easily achieved by using N-propylamphetamine as an internal standard. The phenmetrazine 2,2,2-trichloroethyl carbamate showed a molecular ion isotope cluster at m/z 351, 353, 355, and 357 (isotope effect of three chlorine atoms in the derivatized molecule) and other peaks at m/z 247, 245, 204, 114, and 70 in the electron ionization mass spectrometry, thus aiding in unambiguous identification. The underivatized phenmetrazine showed a relatively weaker molecular ion at m/z 177 and a base peak at m/z 71. The N-propylamphetamine 2,2,2-trichloroethyl carbamate (internal standard) showed a very weak molecular ion at m/z 351 and a base peak at m/z 260. Another strong characteristic peak at m/z 91 was also observed. The retention time of derivatized phenmetrazine (9.5 min) was substantially longer than the retention time of the underivatized molecule (2.5 min). Moreover, underivatized phenmetrazine showed poor peak shape (substantial tailing) while derivatized phenmetrazine had excellent chromatographic property. The within-run and between-run precisions of the assay were 1.9% and 3.2% at a urinary phenmetrazine concentration of 20 micrograms/mL. The assay was linear for urinary phenmetrazine concentration of 1 microgram/mL to 100 micrograms/mL with a detection limit of 0.5 microgram/mL.

Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid.

An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method.

Evaluation of the Abuscreen ONLINE assay for amphetamines on the Hitachi 737: comparison with EMIT and GC/MS methods.

The performance of the ONLINE Assay for Amphetamines on the Hitachi 737 was compared to the Syva Emit d.a.u. Assay and GC/MS. Randomly screened (n = 2964) patient urine samples were assayed using ONLINE and Emit d.a.u. assays concurrently, using d-amphetamine, 1000 ng/mL and d-methamphetamine, 1000 ng/mL as the screening cutoff for ONLINE and Emit d.a.u. assays, respectively. All presumptive positives were confirmed by GC/MS. The specificity was 99% (2937/2964) for ONLINE and 97% (2873/2964) for Emit. Agreement with GC/MS was 80% (110/137) for ONLINE and 55% (110/201) for Emit.

Aromatic ring oxidation of N-n-butylamphetamine is enhanced in the rat by prior treatment with quinidine.

Prior administration of quinidine is known to reduce aromatic oxidation of amphetamine and its analog methoxyphenamine by inhibiting the cytochrome P450IID6 results in isozyme. In contrast, it is now shown that prior administration of quinidine results in a significant increase in the aromatic oxidation of N-n-butylamphetamine in rat, suggesting that the P450IID6 isozyme is not involved in this metabolic reaction.

Liquid chromatographic chiral stationary phases in pharmaceutical analysis: determination of trace amounts of the (-)-enantiomer in (+)-amphetamine.

A rapid and accurate method was developed for the determination of the enantiomeric composition of amphetamine preparations. Amide derivatives of the amphetamine enantiomers are first formed by using achiral 2-naphthoyl chloride. The resulting enantiomeric amides are then chromatographed on a commercially available chiral stationary phase. The capacity factors (k') of (-)- and (+)-2-naphthoylamphetamine are 20 and 22, respectively, and the separation factor (alpha) for the two enantiomers is 1.08. The method allows detection of as little as 0.5% of the (-)-enantiomer in (+)-amphetamine and is applicable to both bulk drug and single-tablet analyses.

Application of chiral lanthanide nuclear magnetic resonance shift reagents to pharmaceutical analysis. II. Determination of dextro- and levoamphetamine mixtures.

Optically pure d- and l-amphetamine sulfate, as well as various mixtures of the 2 enantiomers, were analyzed using a europium chiral nuclear magnetic resonance shift reagent. The enantiomeric shift difference (delta delta delta) exhibited by the doublet associated with the alpha-methyl protons was large enough to differentiate between the levo- and dextro-isomers. The alpha-methyl protons were decoupled and the enantiomeric composition was determined by using the peak heights of the resulting singlets. As little as 5% of the levo-isomer in the presence of the dextro-isomer can be determined using this method. The method is applicable to the analysis of bulk drug and pharmaceutical preparations.

Quantitative determination of enantiomeric compounds. I--Simultaneous measurement of the optical isomers of amphetamine in human plasma and saliva using chemical ionization mass spectrometry.

A sensitive and specific method for the quantitative determination of the optical isomers of amphetamine from human plasma and saliva is described. Amphetamine was extracted from basified plasma or saliva with hexane and then derivatized by reaction with N-pentafluorobenzoyl-S-(-)-prolyl-1-imidazolide. The reaction of the amphetamine enantiomers with this chiral reagent yields diasteriomers which are easily resolved by gas liquid chromatography. The resolved diasteriomers upon elution from the gas chromatograph were measured quantitatively by chemical ionization mass spectrometry. Plasma and saliva levels achieved following the oral administration of 10 mg of dl-, d- and l-amphetamine in man are reported.

Punch sampling technique for quantitative identification of tritiated D-amphetamine.

A punch sampling, tissue solubilization, and scintillation counting technique has been devised for the quantitative identification of tritiated d-amphetamine in monkey brain tissue. The method is superior to autoradiography in identifying this water soluble radiochemical, and is easier and more anatomically precise than radiochemical assay of brain homogenates. This rapid and simple technique allows one to survey the whole brain by sampling unit volumes of individual structures verified by light microscopy. The procedure consists of six steps which may be spaced as the laboratory schedule permits: obtaining tissue blocks, preparation for sectioning, punch sampling and sectioning, preparing samples for scintillation counting, histological verification of sample sites, and analysis of scintillation count data. The technique has been successfully used to demonstrate a gradient of radioactivity following experimental intraventricular injections of 3H amphetamine.

Radioimmunoassay of amphetamines in rat parotid saliva.

Amphetamines, a commonly abused class of drugs, have been detected in various biological specimens, in particular, urine and blood. However, little information is available concerning the detection of these drugs in saliva. This investigation, utilizing the rat salivary secretions, has been attempted to establish the ability of amphetamines to be secreted in saliva and to determine the feasibility of using radioimmunoassay (RIA) for drug detection in saliva. The results of this investigation showed that (1) d-amphetamine and methamphetamine decreased salivary flow, (2) after d-amphetamine RIA tests were demonstrated in both saliva and plasma for a period of fifty minutes, and (3) positive RIA reactions were obtained by the following metamphetamine metabolites: amphetamine, 4-hydroxynorephedrine and 4-hydroxyamphetamine. Methamphetamine and 4-hydroxy-N-methylamphetamine were found to be non-reactive in the radioimmunoassay procedure. The results indicate that saliva could be radioimmunoassayed for the detection of amphetamine or amphetamine derivatives after the administration of either d-amphetamine and methamphetamine.

Single-tablet enantiomeric purity assay of amphetamine by rotation enhancement.

Enhancement of the optical rotation of dextroamphetamine by production of an optically active chromophore through reaction with 1-fluoro-2,4-dinitrobenzene to give alpha-methPYL-N-(2,4-dinitrophenyl)-beta-phenylethylamine is reported. This reaction forms the basis of an assay for both the content and optical purity of dextroamphetamine sulfate at single-dose levels (5 mg). Results of assays of standard solutions and of commercial tablets demonstrate the suitability of the method for the determination of enantiomeric purity and amphetamine content.

Elevation of amphetamine levels in rat brain after administration of the choline acetyltransferase inhibitor 4-(1-Naphthylvinyl) pyridine (NVP).

Following amphetamine administration both NVP and SKF-525A cause elevations in whole brain levels of amphetamine compared with saline treated controls. NVP was found to be about 3 times more active than SKF-525A; significant effects were obtained after doses of 0.625mg/kg and above. NVP also inhibited brain choline acetyltransferase in vitro (I50 of 4.0 x 10-5 M). Following the administration of NVP (20-200 MG/KG, I.P), there was a dose-dependent inhibition of this enzyme in brain. These results suggest that potentiation of the behavioral effects of amphetamine by NVP, which occur after doses of 5 mg/kg of NVP, are probably related to its inhibitory effects on amphetamine metabolism.