Physiological and subjective responses to controlled oral 3,4-methylenedioxymethamphetamine administration.

A randomized, within-subject, double-blind, inpatient study of the physiological and subjective effects of oral 3,4-methylenedioxymethamphetamine (MDMA) was conducted in human volunteers with previous MDMA experience. Placebo, low (1.0 mg/kg), and high (1.6 mg/kg) doses of oral MDMA were administered in a controlled inpatient setting at least 7 days apart to 6 African American (4 male, 2 female) and 2 white (both male) volunteers (mean [SE] age, 21.1 [0.8] years; weight, 77.2 [7.7] kg). 3,4-Methylenedioxymethamphetamine doses were 46 to 150 mg, in the range of typical recreational doses. Participants completed all sessions without clinically significant adverse events. 3,4-Methylenedioxymethamphetamine produced significant dose-dependent increases in heart rate (highest, 132 bpm), systolic (highest, 171 mm Hg) and diastolic (highest, 102 mm Hg) blood pressure, and subjective responses for energy level, closeness to others, mind racing, heightened senses, and high (evaluated by visual analog scales). Peak effects occurred 1 to 2 hours after dose, with no secondary peak. There were no significant effects on body temperature (measured at tympanic membrane), respiratory rate, or blood oxygen saturation (by pulse oximetry). Although most physiological and subjective parameters were significantly correlated with MDMA plasma concentrations, correlation coefficients were low and clinically insignificant, eliminating the ability to predict effects from single plasma concentrations. These findings suggest that oral MDMA in typical recreational doses produces short-term effects on cardiovascular function and subjective state but that temperature effects may result from interaction with environmental and subject factors.

Plasma pharmacokinetics of 3,4-methylenedioxymethamphetamine after controlled oral administration to young adults.

This study examines the plasma pharmacokinetics of 3,4-methylenedioxymethamphetamine (MDMA) and metabolites 4-hydroxy-3-methoxymethamphetamine (HMMA), 3,4-methylenedioxyamphetamine (MDA), and 4-hydroxy-3-methoxyamphetamine (HMA) in young adults for up to 143 hours after drug administration. Seventeen female and male participants (black, white, and Hispanic) received placebo, low (1.0 mg/kg), and high (1.6 mg/kg) oral MDMA doses (comparable to recreational doses) in a double-blind, randomized, balanced, within-subject design while residing on a closed research unit. Doses were separated by 1 week or more. A fully validated two-dimensional gas chromatography/mass spectrometry method simultaneously quantified MDMA, HMMA, MDA, and HMA. Calibration curves were MDA, 1 to 100 ng/mL; HMA, 2.5 to 100 ng/mL; and MDMA and HMMA, 2.5 to 400 ng/mL. Mean +/- standard deviation maximum plasma concentrations (C(max)) of 162.9 +/- 39.8 and 171.9 +/- 79.5 ng/mL were observed for MDMA and HMMA, respectively, after low-dose MDMA. After the high dose, mean MDMA Cmax significantly increased to 291.8 +/- 76.5 ng/mL, whereas mean HMMA C(max) was unchanged at 173.5 +/- 66.3 ng/mL. High intersubject variability in C(max) was observed. Mean MDA C(max) were 8.4 +/- 2.1 (low) and 13.8 +/- 3.8 (high) ng/mL. HMA Cmax were 3.5 +/- 0.4 and 3.9 +/- 0.9 ng/mL after the low and high doses, respectively. AUC infinity displayed similar trends to C(max), demonstrating nonlinear pharmacokinetics. Times of last plasma detection were generally HMA < MDA < MDMA < HMMA. Mean half-lives (t1/2) of MDMA, MDA, and HMMA were approximately 7 to 8 hours, 10.5 to 12.5 hours, and 11.5 to 13.5 hours, respectively. HMA t1/2 showed high variability. Mean MDMA volume of distribution was constant for low and high doses; clearance was significantly higher after the low dose. This study presents MDMA plasma pharmacokinetic data for the first time from blacks and females as well as measurement of HMMA and HMA concentrations after low and high MDMA doses and more frequent and extended plasma sampling than in prior studies.

Engineered antibody Fc variants with enhanced effector function.

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

The basolateral complex of the amygdala mediates the modulation of intracranial self-stimulation threshold by drug-associated cues.

Learning and memory appear to be critical aspects of drug abuse; presumably playing an especially important role in craving and relapse. Thus, understanding the interaction of learning- and memory-related brain areas with the classical reward circuitry is of importance. Toward this goal, the effect of drug-associated contextual cues on intracranial self-stimulation (ICSS) behaviour was assessed in rats. We used a method that allows the establishment of baseline behaviour, the pairing of drug exposure with unique cues, and testing the effect of cue exposure within the same apparatus. ICSS thresholds were decreased by morphine (5 mg/kg, i.p.) or cocaine (10 mg/kg, i.p.) during five days of paired drug-cue training sessions. Subsequent presentation of the drug-associated cues decreased thresholds in the absence of drug. Cues associated with saline had no effect. These results suggest a Pavlovian conditioning phenomenon in which the functioning of brain reward circuitry is modulated by drug-associated cues. In a second experiment, we tested the hypothesis that the mechanism by which conditioning affects ICSS thresholds may include the basolateral complex of the amygdala (BLC) due to its known role in conditioning and anatomical linkage with classical reward circuitry. Lesions of the BLC abolished the ability of cocaine-associated cues to lower ICSS threshold. Lesions did not alter response capability or the unconditioned effect of cocaine. We conclude that the BLC is necessary for cues associated with previous drug exposure to modulate activity within or downstream from the classical reward circuitry of the medial forebrain bundle.

Electrical and chemical stimulation of the basolateral complex of the amygdala reinstates cocaine-seeking behavior in the rat.

RATIONALE: The basolateral complex of the amygdala (BLC) is part of a neural circuit that is activated in humans during cocaine craving elicited by exposure to drug-related environmental cues. In animals, the BLC is necessary for cocaine-seeking behavior elicited by cocaine-associated cues. It has not been determined whether BLC activation is sufficient to reinstate cocaine seeking. OBJECTIVES: To determine whether electrical or excitatory amino-acid stimulation of the BLC is sufficient to induce reinstatement of cocaine seeking in rats. METHODS: Rats were catheterized and trained to lever-press for intravenous cocaine infusions on a fixed ratio (FR)-1 schedule of reinforcement. Once baseline cocaine-taking criteria were met, lever-pressing behavior was extinguished by substitution of saline for cocaine. After meeting criteria for extinction, animals were subjected to brief electrical (20 Hz, 400 microA or 2 Hz, 400 microA; 200 pulses per stimulation) or N-methyl- d-aspartate (NMDA; 250 ng/0.5 microl) BLC stimulation and lever pressing behavior was monitored. RESULTS. Electrical BLC stimulation with 20-Hz produced reinstatement of lever pressing previously associated with cocaine self-administration, while 2-Hz stimulation did not. Electrical stimulation of cerebellar and medial forebrain bundle loci did not reinstate cocaine seeking. Hence, the reinstatement was frequency dependent and anatomically selective. NMDA microinjections into the BLC also reinstated cocaine-seeking behavior. CONCLUSIONS: BLC stimulation is sufficient to reinstate cocaine-seeking behavior in the rat. These results are congruent with the hypothesis that the basolateral complex of the amygdala is part of a neural system mediating drug-seeking behavior.

Cocaine treatment increases expression of a 40 kDa catecholamine-regulated protein in discrete brain regions.

Previous reports from our laboratory have described brain-specific catecholamine-regulated proteins, which bind dopamine and related catecholamines. Evidence from the molecular cloning of a 40 kDa catecholamine-regulated protein (CRP40) revealed that CRP40 is dopamine-inducible and has properties similar to those of the 70 kDa heat shock protein (HSP70) family. The present study investigates the effects of acute and chronic cocaine treatment on CRP40 expression in the striatum, nucleus accumbens, prefrontal cortex, and medulla. Acute treatment with cocaine increased CRP40 expression in the nucleus accumbens and striatum, whereas chronic treatment with cocaine increased CRP40 expression in the nucleus accumbens only. Neither of these treatments affected CRP40 levels in the prefrontal cortex or medulla. In addition, pretreatment with the spin-trapping agent alpha-phenyl-tert-butylnitrone did not attenuate cocaine-induced expression of CRP40, suggesting that the observed increases in CRP40 levels were not caused by free radicals. On the other hand, pretreatment with anisomycin, a protein synthesis inhibitor, blocked the cocaine-induced expression of CRP40. Thus, protein synthesis may be involved in the observed CRP40 level increases. Furthermore, neither acute nor chronic cocaine treatment affected levels of inducible or constitutively expressed HSP70, which indicates a specificity of cocaine's effects on CRP40. Since cocaine has been shown to increase extracellular dopamine levels, these findings suggest that increased expression of CRP40 is associated with high extracellular levels of dopamine (or its metabolites). Elevated levels of CRP40 could play a protective role for dopamine neurons in response to increased oxidative stress that has been shown to be induced by cocaine and that can lead to apoptosis and neurodegeneration.

Combining computational and experimental screening for rapid optimization of protein properties.

We present a combined computational and experimental method for the rapid optimization of proteins. Using beta-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space. By eliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of approximately equal 200,000 mutants. These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime. In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance. To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM beta-lactamases, the most prevalent type of Gram-negative beta-lactamases. This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.

Computational stabilization of human growth hormone.

Recombinant human growth hormone (hGH) is used worldwide for the treatment of pediatric hypopituitary dwarfism and in children suffering from low levels of hGH. It has limited stability in solution, and because of poor oral absorption, is administered by injection, typically several times a week. Development has therefore focused on more stable or sustained-release formulations and alternatives to injectable delivery that would increase bioavailability and make it easier for patients to use. We redesigned hGH computationally to improve its thermostability. A more stable variant of hGH could have improved pharmacokinetics or enhanced shelf-life, or be more amenable to use in alternate delivery systems and formulations. The computational design was performed using a previously developed combinatorial optimization algorithm based on the dead-end elimination theorem. The algorithm uses an empirical free energy function for scoring designed sequences. This function was augmented with a term that accounts for the loss of backbone and side-chain conformational entropy. The weighting factors for this term, the electrostatic interaction term, and the polar hydrogen burial term were optimized by minimizing the number of mutations designed by the algorithm relative to wild-type. Forty-five residues in the core of the protein were selected for optimization with the modified potential function. The proteins designed using the developed scoring function contained six to 10 mutations, showed enhancement in the melting temperature of up to 16 degrees C, and were biologically active in cell proliferation studies. These results show the utility of our free energy function in automated protein design.

Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening.

Granulocyte-colony stimulating factor (G-CSF) is used worldwide to prevent neutropenia caused by high-dose chemotherapy. It has limited stability, strict formulation and storage requirements, and because of poor oral absorption must be administered by injection (typically daily). Thus, there is significant interest in developing analogs with improved pharmacological properties. We used our ultrahigh throughput computational screening method to improve the physicochemical characteristics of G-CSF. Improving these properties can make a molecule more robust, enhance its shelf life, or make it more amenable to alternate delivery systems and formulations. It can also affect clinically important features such as pharmacokinetics. Residues in the buried core were selected for optimization to minimize changes to the surface, thereby maintaining the active site and limiting the designed protein's potential for antigenicity. Using a structure that was homology modeled from bovine G-CSF, core designs of 25-34 residues were completed, corresponding to 10(21)-10(28) sequences screened. The optimal sequence from each design was selected for biophysical characterization and experimental testing; each had 10-14 mutations. The designed proteins showed enhanced thermal stabilities of up to 13 degrees C, displayed five-to 10-fold improvements in shelf life, and were biologically active in cell proliferation assays and in a neutropenic mouse model. Pharmacokinetic studies in monkeys showed that subcutaneous injection of the designed analogs results in greater systemic exposure, probably attributable to improved absorption from the subcutaneous compartment. These results show that our computational method can be used to develop improved pharmaceuticals and illustrate its utility as a powerful protein design tool.