Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.     

Preliminary animal pharmacokinetics of the parenteral antifungal agent MK-0991 (L-743,872)

MK-0991 (L-743,872) is a potent antifungal agent featuring long half-life pharmacokinetics. The pharmacokinetics of MK-0991 administered intravenously to mice, rats, rhesus monkeys, and chimpanzees is presented. Unique to MK-0991 is its consistent cross-species performance. The range of values for the pharmacokinetic parameters were as follows: clearance, 0.26 to 0.51 ml/min/kg; half-life, 5.2 to 7.6 h; and distributive volume, 0.11 to 0.27 liters/kg. The level of protein binding of MK-0991 was determined to be 96% in mouse and human serum. The compound exhibited high affinities for human serum albumin and at least two lipid components. The rationale for the selection of MK-0991 as a drug development candidate was based on its two- to threefold superior pharmacokinetic performance in chimpanzees over the performance of an otherwise equivalent analog, L-733,560. Once-daily dosing for MK-0991 is indicated by a graphical comparison of levels in the circulations of chimpanzees and mice. In a study of the pharmacokinetics of MK-0991 in mouse tissue, the organs were assayed following intraperitoneal administration. The area under the concentration-versus-time curves (AUC) segregated the tissues into three exposure categories relative to plasma. The tissues with greater exposure than that for plasma were liver (16 times), kidney (3 times), and large intestine (2 times). The exposure for small intestine, lung, and spleen were equivalent to that for plasma. Organs with lower levels of exposure were the heart (0.3 times that for plasma), thigh (0.2 times), and brain (0.06 times). Kinetically, drug was cleared more slowly from all tissues than from plasma, indicating that terminal-phase equilibrium had not been achieved by 24 h. Thus, some measure of accumulation is predicted for all tissues. Single daily doses of MK-0991 should provide adequate systemic levels of fungicidal activity as a result of its long half-life pharmacokinetics, wide distribution, and slowly accumulating concentrations in tissue.     

Mu opioid agonists potentiate amphetamine- and cocaine-induced rotational behavior in the rat

Opioids modulate brain dopaminergic function in various experimental paradigms. This study used the rotational model of behavior in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway to investigate this interaction. Doses of two presynaptically acting dopaminergic drugs, amphetamine and cocaine, were coadministered with several doses of the mu opioid agonist, morphine. Morphine, at 3.0 mg/kg, potentiated rotational behavior induced by each dose of the stimulants. To determine the receptor specificity of the actions of morphine, the mu opioid agonists buprenorphine, fentanyl, levorphanol, meperidine, and methadone, and dextrorphan, the non-opioid isomer of levorphanol, were administered alone and with 1.0 mg/kg amphetamine. Each of these drugs, as well as morphine, produced circling behavior on its own. All of the mu opioid agonists and dextrorphan increased amphetamine-induced turning; the coadministration of dextrorphan, levorphanol, meperidine, methadone and morphine with amphetamine produced turning greater than predicted by simple additivity. To determine whether an opioid receptor was involved in these interactions, the opioid antagonist, naltrexone, was administered before the amphetamine/mu opioid receptor agonist combination. Naltrexone blocked the potentiating effects of morphine, but not those of the other drugs. Moreover, naltrexone alone dose-dependently increased amphetamine-induced rotational behavior. These studies show that some mu opioid receptor agonists can potentiate stimulant-induced rotational behavior and that blockade of opioid receptors can also produce a potentiation. The role of mu opioid receptors in these effects remains unclear.     

Pressure support ventilation in adult respiratory distress syndrome: short-term effects of a servocontrolled mode

PURPOSE: To assess the short-term effects of pressure support ventilation in adult respiratory distress syndrome (ARDS), we studied 17 patients with moderate to severe ARDS using mandatory rate ventilation (MRV), a servocontrolled mode of PSV having respiratory rate as the targeted parameter. MATERIALS AND METHODS: Based on the duration of ARDS, the patients were divided into two groups: Group 1, early ARDS (duration up to 1 week), 10 patients; Group 2, intermediate ARDS (duration between 1 and 2 weeks). The patients were initially ventilated with assisted mechanical ventilation then with MRV, and finally with controlled mechanical ventilation. After a 20-minute period allowed for stabilization in each mode, ventilatory variables, gas exchange, hemodynamics, and patient's inspiratory effort were evaluated. RESULTS: During MRV blood gases, airway pressures and hemodynamic variables remained within acceptable limits in all patients. Compared with assisted mechanical ventilation, during MRV, patients of group 1 decreased their VT and V (from 0.64 +/- 0.04 to 0.42 +/- 0.03 L/sec) and increased their TI/TT (from 0.39 +/- 0.03 to 0.52 +/- 0.03). f did not change. PAO2 - PaO2 and QS/QT decreased (from 306 +/- 16 to 269 +/- 15 mm Hg, and from 20.2 +/- 1.4 to 17.5 +/- 1.1, respectively), while PaCO2 increased (from 44 +/- 3 to 50 +/- 3 mm Hg). On the contrary, patients of group 2 increased their VT (from 0.69 +/- 0.02 to 0.92 +/- 0.09 L), decreased their f (from 22.3 +/- 0.5 to 19.3 +/- 0.3 b/min), although they did not change their V and TI/TT. PAO2 - PaO2 and QS/QT remained stable. PaCO2 diminished (from 39 +/- 3 to 34 +/- 3 mm Hg). Pressure support level was higher in group 2 than in group 1 (29.4 +/- 3.0 v 19.8 +/- 2.9 cm H2O). CONCLUSIONS: We conclude that (1) PSV delivered by MRV may adequately ventilate patients with moderate to severe ARDS, preserving gas exchange and hemodynamics, at least for the short period tested; (2) early and intermediate ARDS respond in a different manner to MRV in terms of breathing pattern, gas exchange, and level of pressure assistance; and (3) patients with early ARDS are those who have an improvement in intrapulmonary oxygenation probably due, at least in part, to alveolar recruitment augmented by active diaphragmatic contraction.     

Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.     

There are no racial, age, sex, or weight differences in the effect of salt on blood pressure in salt-sensitive hypertensive patients

STUDY DESIGN: A bench-top trauma sled was used to apply four intensities of whiplash trauma to human cadaveric cervical spine specimens and to measure resulting intervertebral rotations using high-speed cinematography. OBJECTIVES: To determine the cervical spine levels most prone to injury from whiplash trauma and to hypothesize a mechanism for such injury. SUMMARY OF BACKGROUND DATA: Whiplash injuries traditionally have been ascribed to hyperextension of the head, but other mechanisms such as hypertranslation also have been suggested. METHODS: Six occiput to T1 (or C7) fresh cadaveric human spines were studied. Physiologic flexion and extension motions were recorded with an Optotrak motion analysis system by loading up to 1.0 Nm. Specimens then were secured in a trauma sled, and a surrogate head was attached. Flags fixed to the head and individual vertebrae were monitored with high-speed cinematography (500 frames/sec). Data were collected for 12 traumas in four classes defined by the maximum sled acceleration. The trauma classes were 2.5 g, 4.5 g, 6.5 g, and 8.5 g. Significance was defined at P < 0.01. RESULTS: In the whiplash traumas, the peak intervertebral rotations of C6-C7 and C7-T1 significantly exceeded the maximum physiologic extension for all trauma classes studied. The maximum extension of these lower levels occurred significantly before full neck extension. In fact, the upper cervical levels were consistently in flexion at the time of maximum lower level extension. CONCLUSIONS: In whiplash, the neck forms an S-shaped curvature, with lower level hyperextension and upper level flexion. This was identified as the injury stage for the lower cervical levels. A subsequent C-shaped curvature with extension of the entire cervical spine produced less lower level extension.