CPI-1189 attenuates effects of suspected neurotoxins associated with AIDS dementia: a possible role for ERK activation.

Individuals infected with the human immunodeficiency virus (HIV) often experience a dementia characterized by mental slowing and memory loss. Motor dysfunction may also accompany this condition. The pathogenesis of the dementia is not known, but microscopic examination of brain tissue from those afflicted shows evidence of chronic inflammation, reactive gliosis and cell death. Neurotoxic factors produced from activated macrophage or microglial cells such as tumor necrosis factor-alpha (TNFalpha), gp120 and quinolinic acid have been implicated as agents for the cell death which often appears to occur by an apoptotic mechanism. CPI-1189, a drug currently undergoing clinical evaluation as a treatment for the dementia associated with AIDS, is shown in this paper to mitigate apoptosis induced by TNFalpha, gp120, and necrosis induced by quinolinic acid. In addition, CPI-1189 mitigates the cell death produced by supernatants from cultured macrophages obtained from patients with AIDS dementia. The exact mechanism by which CPI-1189 prevents neurotoxicity is not known; however, protection from TNFalpha and supernatant-induced toxicity does not appear to involve NFkappaB translocation, and appears to be associated with an increase in activated ERK-MAP kinase. These findings may have implications for other neurological diseases where apoptotic cell death contributes to neurodegeneration.

CPI-1189 inhibits interleukin 1beta-induced p38-mitogen-activated protein kinase phosphorylation: an explanation for its neuroprotective properties?

The p38 mitogen-activated protein kinase (p38-MAPK) is a central enzyme in one of the major protein kinase cascades that regulate proapoptotic and proinflammatory signal transduction. p38-MAPK is activated by receptor/ligand recognition events or by exposure to extracellular stressors, including oxidative stress. Activation of p38-MAPK is affected by dual phosphorylation on a specific inhibitory domain. Dual phosphorylation causes a structural change in the p38-MAPK enzyme which allows binding of ATP and target substrate. Agents which block ATP docking to phosphoactivated p38-MAPK are being investigated for treatment of inflammatory diseases and neurodegenerative pathologies. An alternative strategy for p38-MAPK antagonism would be the inhibition of p38-MAPK phosphoactivation. We now report potent inhibition of p38-MAPK phosphorylation by a synthetic benzamide (CPI-1189) which displays protective action against tumor necrosis factor-alpha (TNFalpha)-induced neurodegeneration. In primary astrocytes treated with interleukin 1beta (IL1beta), CPI-1189 inhibits p38-MAPK phosphorylation at concentrations of 10 nM or less. While the precise molecular target of CPI-1189 remains unknown, these findings suggest a novel mechanism for the neuroprotective properties of the compound. These findings also indicate that antagonism of the p38-MAPK may be achieved through pharmacological inhibition of p38-MAPK phosphorylation, a strategy that is conceptually distinct from direct inhibition of ATP binding to the active enzyme.

CPI-1189 prevents apoptosis and reduces glial fibrillary acidic protein immunostaining in a TNF-alpha infusion model for AIDS dementia complex.

AIDS dementia complex (ADC) is characterized by increased apoptosis, gliosis, and oxidative stress in the CNS, as well as a compromised blood-brain barrier. TNF-alpha has been shown to be elevated in AIDS dementia complex brains and may contribute to AIDS dementia complex. To model elevated TNF-alpha in AIDS dementia complex, TNF-alpha was infused ICV bilaterally into rats for 3 days. TNF-alpha treatment increased apoptosis around the infusion site and selectively in the septum and corpus callosum. Co-administration of the synthetic antioxidant CPI-1189 prevented TNF-alpha induced apoptosis. Both TNF-alpha and CPI-1189 treatment suppressed glial fibrillary acidic protein (GFAP) staining at the infusion site. TNF-alpha did not significantly affect the integrity of the blood-brain barrier, but CPI-1189 treatment increased blood-brain barrier integrity at the infusion site. No effect of TNF-alpha or CPI-1189 treatment was found on measures of oxidative stress. These results support TNF-alpha as a key agent for increasing apoptosis in AIDS dementia complex. Additionally, CPI-1189 treatment may protect against TNF-alpha induced apoptosis and astrogliosis in AIDS dementia complex. Lastly, the toxic effect of TNF-alpha and the protective effect of CPI-1189 may not be mediated primarily through manipulation of classic reactive oxygen species.

Preventive actions of a synthetic antioxidant in a novel animal model of AIDS dementia.

Accumulating evidence indicates that the mechanism for causing AIDS dementia complex (ADC) involves the release of damaging inflammatory-related agents by HIV-infected microglia in the brain resulting in CNS oxidative damage. One such agent, tumor necrosis factor alpha (TNF-alpha) is consistently elevated in the brains of ADC patients compared to non-demented HIV patients. To model this aspect of ADC in rats, chronic ventricular infusions of TNF-alpha were given and found to induce several aspects of ADC, including weight loss, learning/memory impairment, enlarged lateral ventricles, and increased apoptosis. Concurrent oral treatment with the antioxidant CPI-1189 prevented all of these TNF-alpha induced effects. The results support TNF-alpha as a key toxic agent in ADC and provide the first in vivo evidence that chronic treatment with a synthetic antioxidant may protect HIV-infected patients against ADC. Our findings may also have implications in other neurological diseases where brain TNF-alpha levels are elevated and inflammation/oxidative stress is suspected to be a contributing cause, such as Alzheimer's disease and Parkinson's disease.

A comparison of mass balance, pharmacokinetics and disposition of [14C(U)]- and [125I]recombinant human interleukin-2 in cynomolgus monkeys.

Recombinant human interleukin-2 (rHuIL-2) has been metabolically labeled with 14C amino acids in Escherichia coli and affinity purified on a rHuIL-2 receptor affinity column. The radiolabeled molecule had a specific radioactivity of 238 dpm/unit and the identical amino acid sequence and biological activity as unlabeled rHuIL-2. In this study, we used this labeled [14C(U)]rHuIL-2 and commercially available [125I]rHuIL-2 (identical in sequence to the [14C(U)]rHuIL-2) to compare the mass balance, pharmacokinetics, and disposition in cynomolgus monkeys. After a single intravenous bolus dose of 4 x 10(5) units/kg, serum samples were collected for 7 days and examined for biological activity, total radioactivity, and by molecular size exclusion chromatography. Urine and feces were analyzed for total radioactivity. When analyzed for biological activity, both [14C(U)]- and [125I]rHuIL-2 exhibited the following pharmacokinetic parameters: terminal elimination half-life of 1-2 hr, AUC0-infinity ranged from 2005 to 4659 units x hr/ml, clearance was 90-200 ml/hr/kg, and volume of distribution ranged from 103 to 163 ml/kg. Comparison of the pharmacokinetic profiles of the two radiolabels were very different from bioactivity, in that the elimination half-lives for radioactivity were approximately 8 days and 10 hr for [14C(U)]- and [125I]rHuIL-2, respectively. We conclude that the [14C(U)]rHuIL-2 was metabolized to constituent amino acids and recycled into newly synthesized proteins from our size exclusion chromatography studies.(ABSTRACT TRUNCATED AT 250 WORDS)

A 13-week toxicologic and pathologic evaluation of prolonged cytochromes P450 inhibition by 1-aminobenzotriazole in male rats.

The pharmacologic, toxicologic, and microscopic effects of 100 mg/kg/day of 1-Aminobenzotriazole (ABT), a suicide substrate inhibitor of cytochromes P450, were assessed in male Sprague-Dawley rats over a 13-week period. Hepatic cytochromes P450 levels and resorufin dealkylase activity were decreased to less than 30% of control values beginning at Day 2 and from Day 8 to Day 92. These decreases were not accompanied by overt clinical toxicity, e.g., changes in body weight, food consumption, or clinical appearance, during the study. Hemoglobin, hematocrit, and erythrocyte counts were slightly decreased at 8, 29, and 92 days and were accompanied by increased spleen weights and extramedullary hematopoiesis. Additionally, mean corpuscular hemoglobin concentration, mean corpuscular volume, red cell distribution width, and mean corpuscular hemoglobin were slightly increased at 92 days. Increases in liver weights at 8, 29, and 92 days were accompanied by centrilobular hypertrophy and intracytoplasmic vacuolization consistent with lipid accumulation. Thyroid stimulating hormone (TSH) was slightly elevated and triiodothyronine and thyroxine were slightly decreased at 29 days. TSH was also slightly elevated at 8 and 92 days, and thyroid gland weights were increased at 8, 29, and 92 days with microscopic evidence of hyperplasia and hypertrophy of thyroid gland follicular cells. Increased adrenal weights and hypertrophy of the zona fascicularis of the adrenal gland were observed at 8, 29, and 92 days. Kidney weights were also increased at these assessments. Changes in the thyroid gland, the thyroid hormone profile, and the liver may reflect increased synthesis of microsomal enzymes, an effect that is sometimes difficult to demonstrate directly with suicide substrate inhibitors of cytochromes P450. In general, the effects of daily ABT administration to male rats at a dose that significantly reduces oxidative metabolism over a 13-week period were considered to be well-tolerated under controlled laboratory conditions.

Disposition of [14C]acitretin in humans following oral administration.

The disposition of the antipsoriatic agent, acitretin, was investigated in six healthy human volunteers who each received a single, oral 50 mg dose of [14C]acitretin (50 microCi). plasma, urine, and feces were collected for 240 hr after administration. Mean values of 20.9 and 62.6% of the administered dose were recovered in the urine and feces, respectively. The terminal elimination half-life of total radioactivity from the plasma was approximately 120 hr. Extraction of pooled plasma samples followed by separation by HPLC and quantitation by liquid scintillation counting indicated that acitretin and its 13-cis-isomer, isoacitretin, were minor fractions of the total drug-related material in the plasma at most time points up to 72-hr postdose. The structures of acitretin, isoacitretin, and two other metabolites--(5E,7E)-8-(4-methoxy,2,3,6-trimethylphenyl)-2,6 -dimethyl-5,7- octadienoic acid (I) and (5E,7E)-8-(4-hydroxy-2,3,6-trimethylphenyl)-2,6-dimethyl-5,7 -octadienoic acid (II)--were confirmed by MS and cochromatography with authentic standards. I and II were major fractions of the drug-related material in the plasma at all time points. Other compounds, whose structures could not be confirmed, also account for a significant fraction of the circulating radioactivity.

Clinical pharmacology of oral all-trans retinoic acid in patients with acute promyelocytic leukemia.

All-trans retinoic acid (RA) induces leukemic cell differentiation and complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). However, remissions induced by all-trans RA tend to be brief, and relapses are associated with resistance to further treatment in vivo, although the leukemic cells appear to retain sensitivity to the cytodifferentiating effects of all-trans RA in vitro. The clinical pharmacology of all-trans RA was examined in 13 patients with APL. The drug was administered at a constant dose of 45 mg/m2/day, given as a single dose on the first day of therapy and in two divided doses thereafter. Plasma and urinary concentrations of the parent drug and metabolites were quantitated by reverse-phase high-performance liquid chromatography and, where required, by a combination of normal-phase liquid chromatography/negative chemical ionization mass spectrometry. In patients with APL, basal levels of endogenous retinol and natural retinoids were within the normal range. Peak plasma levels of all-trans RA (347 +/- 266 ng/ml, mean +/- SD) were reached 1-2 h after drug ingestion and decayed in a monoexponential fashion with a half-life of 0.8 +/- 0.1 h. The only drug metabolite detected in plasma or urine was 4-oxo-all-trans RA (present in urine as the glucuronide conjugate). This metabolite accounted for less than 10% of the circulating drug in plasma, and its cumulative urinary excretion accounted for less than 1% of the administered dose. The drug was not found in cerebrospinal fluid. Continued oral administration of all-trans RA was associated with a significant decrease in both the plasma peak levels and the area under the concentration-time curve (P = 0.01 and 0.004, respectively) when measured after 2-6 weeks of treatment. We previously reported that a decrease in plasma area under the concentration-time curve was highly correlated with clinical relapse. Observations in a subset of patients in this study suggested that, in fact, the major decrease occurred early, within the first 7 days of treatment. These changes were associated with a 10-fold increase in urinary excretion of 4-oxo-all-trans RA glucuronide, suggesting that the accelerated clearance from plasma was associated with increased drug catabolism. The rapid disappearance may explain early relapse from remissions induced by all-trans RA; clinical "resistance" to all-trans RA may either wholly or in part result from an inability to sustain effective plasma concentrations of all-trans RA during continuous treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

A computer program for the deconvolution of mass spectral peak abundance data from experiments using stable isotopes.

A computer program is described for deconvoluting the overlap which is often found in mass spectral peak abundance data from stable isotope experiments. Peak intensity data from calibration standards are corrected using parameters calculated from the analysis of separate external standard solutions of analytes and internal standard. If the calibration data are satisfactory, the same parameters and the slope and intercept values from the least squares analysis of the calibration data are used to correct and quantitate the mass spectral peak intensity data from the quality assurance and experimental samples. Reports and graphs appropriate to the process are produced. Applications are given for the analysis of plasma samples from stable isotope experiments with carprofen, cifenline, and midazolam.

Quantification of acitretin in human plasma by microbore liquid chromatography-negative chemical ionization mass spectrometry.

A highly sensitive liquid chromatographic-mass spectrometric procedure has been developed to quantitate plasma concentrations of acitretin, a dermatologic agent used to treat severe psoriasis. The assay utilizes the combination of normal-phase microbore high-performance liquid chromatography, negative chemical ionization mass spectrometry, selective ion monitoring and stable isotope dilution. The method has been used to measure acitretin and its metabolite, 13-cis-acitretin, over a range of 1-20 ng/ml in human plasma. The inter-assay precision was 5.3% for acitretin and 3.9% for 13-cis-acitretin, while the intra-assay precisions for acitretin and 13-cis-acitretin were 10.8 and 12.7%, respectively. Reproducibility of the assay for acitretin and 13-cis-acitretin, which was determined by the relative standard deviation of multiple analyses of the same quality assurance sample, was 5.9 and 8.1%, respectively.

Quantification of recombinant interleukin-2 in human serum by a specific immunobioassay.

A sensitive and specific assay for recombinant interleukin-2 (rIL-2) in human serum is described. The assay is based on a sequential sandwich immunobioassay that uses a microtiter plate coated with anti-rIL-2 monoclonal antibody (specific for recombinant human IL-2) to capture rIL-2 from serum, and an IL-2 dependent T-cell line that proliferates in a dose-dependent fashion. The lower limit of quantitation of the assay is 2 units/mL (1 unit = approximately 50 pg) using 0.1 mL of serum and the calibration curves ranged from 2 to 50 units/mL. Data are reported on the sensitivity, precision, reproducibility, and specificity of the assay; the stability of rIL-2 in serum; and the recovery of rIL-2 from serum. We also report on the use of the procedure to assay clinical samples from patients with AIDS undergoing treatment with rIL-2.

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b.

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

Quantification of dideoxycytidine in human plasma by gas chromatography/mass spectrometry.

A gas chromatographic/mass spectrometric procedure has been developed for the quantification in plasma of dideoxycytidine (DDC), a candidate anti-AIDS drug. The assay uses an extraction with ethyl acetate containing 10% methanol followed by three derivatization steps: (i) reaction with t-butyl dimethyl chlorosilane to silylate the 5'-hydroxyl group; (ii) pentafluorobenzoylation of the amino group with pentafluorobenzoyl chloride; (iii) methylation of the ring nitrogen adjacent to the amino group with diazomethane. The resulting derivative is quantified using stable isotope dilution, selective ion monitoring and methane negative chemical ionization mass spectrometry. Plasma concentrations of DDC were measured over a range of 2-200 ng ml-1 using 1 ml plasma for extraction.

Quantitation of the enantiomers of rimantadine in human plasma and urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric procedure has been developed for the quantitation in plasma and urine of the enantiomers of rimantadine, an antiviral drug effective against type A influenza. The assay utilizes derivatization with an optically active reagent, selective ion monitoring, methane negative-ion chemical ionization (NICI) mass spectrometry and stable isotope dilution. The method has been used to measure concentrations of each rimantadine enantiomer over a range of 2.5-250 and 12.5-1250 ng/ml in the plasma and urine, respectively, of four male volunteers administered rimantadine. In plasma and urine, no differences were observed in the disposition of the unconjugated enantiomers. In urine, one enantiomer, but not both, was released following enzymatic hydrolysis.

Quantitative selected ion monitoring processing system: software and hardware for the automated collection and analysis of selected ion monitoring data acquired for use in pharmacokinetic studies.

The Quantitative Selected Ion Monitoring Processing System (QSIMPS) is a collection of software and hardware which was designed with the capacity to analyze 30,600 samples per year in support of pharmacokinetic studies. On a per sample basis, QSIMPS was designed to inject a sample into the GC, control the GC divert valve, collect selected ion monitoring data, identify the peaks for the drug and one metabolite and each compound's reference standard, fit the peaks to a relevant chromatographic model, calculate chromatographic features of merit, calculate the peak heights and ratio of the drug and its reference standard and the metabolite and its reference standard, and, using calibration data, convert the ratio to an amount of drug. On a per tray (batch) basis, QSIMPS was designed to fit all the peak height ratios from the calibration standards to either a linear equation, or a generalized nonlinear isotope dilution equation, report a statistical analysis of the fit, and, using aliquot factors, convert the measured amount of drug into concentrations. On a per project basis, QSIMPS prints reports summarizing statistical data on the calibration standards and the quality assurance samples, and prints reports presenting the concentration data as a function of, for examples, subject, drug treatment, time postdose, etc., along with other ancillary data such as subject sex, weight, species, etc. In addition, QSIMPS can fit the concentration data to a number of common pharmacokinetic model-derived equations, and report the resulting pharmacokinetic parameters along with a statistical comparison of the parameters.