Barbara A. Evans

Uric acid is a mediator of the Plasmodium falciparum-induced inflammatory response.

Background Malaria triggers a high inflammatory response in the host that mediates most of the associated pathologies and contributes to death. The identification of pro-inflammatory molecules derived from Plasmodium is essential to understand the mechanisms of pathogenesis and to develop targeted interventions. Uric acid derived from hypoxanthine accumulated in infected erythrocytes has been recently proposed as a mediator of inflammation in rodent malaria. Methods and Findings We found that human erythrocytes infected with Plasmodium falciparum gradually accumulate hypoxanthine in their late stages of development. To analyze the role of hypoxanthine-derived uric acid induced by P. falciparum on the inflammatory cytokine response from human blood mononuclear cells, cultures were treated with allopurinol, to inhibit uric acid formation from hypoxanthine, or with uricase, to degrade uric acid. Both treatments significantly reduce the secretion of TNF, IL-6, IL-1β and IL-10 from human cells. Conclusions and Significance Uric acid is a major contributor of the inflammatory response triggered by P. falciparum in human peripheral blood mononuclear cells. Since the inflammatory reaction induced by P. falciparum is considered a major cause of malaria pathogenesis, identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.

Bioavailability of ACC-9653 (Phenytoin Prodrug)

Summary: The bioavailability of phenytoin from ACC‐9653 versus intravenously administered sodium phenytoin was determined using a crossover design for intravenous and intramuscular administration of ACC‐9653 to healthy volunteers. Absolute bioavailability of phenytoin derived from ACC‐9653 in each subject was calculated as the ratio of the phenytoin area under the plasma concentration time curve for time 0 to infinity [AUC(0‐inf)] after ACC‐9653 divided by the phenytoin AUC(O‐inf) after intravenous sodium phenytoin. The mean absolute bioavailability of ACC‐9653 was 0.992 after intravenous administration and 1.012 after intramuscular administration. These data establish that the bioavailability of ACC‐9653 is complete following intravenous or intramuscular administration in single‐dose volunteer studies. The absolute bioavailability of phenytoin derived from ACC‐9653 in subjects with therapeutic plasma phenytoin concentrations is being studied in patients given simultaneous infusions of stable isotope‐labeled tracer doses of ACC‐0653 and sodium phenytoin.

Carbamazepine increases phenytoin serum concentration and reduces phenytoin clearance.

Addition of carbamazepine to phenytoin monotherapy resulted in the following significant (p < 0.05) changes: (1) increased mean phenytoin serum concentration; (2) decreased phenytoin clearance, due to decreased production of phenytoin dihydrodiol and p-hydroxyphenyl-phenylhydantoin; (3) increased phenytoin elimination half-life; and (4) increased drug-related toxicity. Close monitoring is required after addition of carbamazepine to phenytoin.

Increased activity of indoleamine 2,3-dioxygenase in serum from acutely infected dengue patients linked to gamma interferon antiviral function

The depletion of l-tryptophan (L-Trp) has been associated with the inhibition of growth of micro-organisms and also has profound effects on T cell proliferation and immune tolerance. The enzyme indoleamine 2,3-dioxygenase (IDO) catalyses the rate-limiting step in the catabolic pathway of L-Trp. Gene expression analysis has shown upregulation of genes involved in L-Trp catabolism in in vitro models of dengue virus (DENV) infection. To understand the role of IDO during DENV infection, we measured IDO activity in sera from control and DENV-infected patients. We found increased IDO activity, lower levels of L-Trp and higher levels of l-kynurenine in sera from DENV-infected patients during the febrile days of the disease compared with patients with other febrile illnesses and healthy donors. Furthermore, we confirmed upregulation of IDO mRNA expression in response to DENV infection in vitro, using a dendritic cell (DC) model of DENV infection. We found that the antiviral effect of gamma interferon (IFN-gamma) in DENV-infected DCs in vitro was partially dependent on IDO activity. Our results demonstrate that IDO plays an important role in the antiviral effect of IFN-gamma against DENV infection in vitro and suggest that it has a role in the immune response to DENV infections in vivo.

Studies with stable isotopes II: Phenobarbital pharmacokinetics during monotherapy.

Six healthy adults receiving no other medications were given tracer doses of 90 mg of stable isotope‐labeled phenobarbital (PB) intravenously before, and four weeks after, and 12 weeks after beginning therapy. Serum samples were collected for 96 hours after each injection, and the concentration of stable isotope‐labeled PB in each sample was determined by gas chromatographic mass spectrometry. The volume of distribution, elimination half‐life, and total clearance of PB did not differ significantly on any of the three occasions measured. Phenobarbital clearance did not correlate significantly with total PB serum concentration. Clearances determined from single‐dose studies before beginning PB therapy accurately predicted steady‐state PB serum concentrations. Therefore, it is not necessary to adjust PB dosage for time‐dependent or dose‐dependent changes in clearance during monotherapy. In addition, clearance or serum concentration determined at one dosing rate directly predicts serum concentration at another dosing rate.

Studies With Stable Isotopes I: Changes in Phenytoin Pharmacokinetics and Biotransformation During Monotherapy

Six patients were given tracer doses of 13C15N2‐phenytoin (PHT) before and four and 12 weeks after beginning monotherapy. The following significant (P < .05) changes occurred during monotherapy: (1) Apparent (from tracer doses) PHT total clearance by linear method decreased; (2) apparent PHT elimination half‐life increased; (3) apparent mean PHT serum concentration per unit dose increased; (4) apparent rate of excretion of p‐hydroxyphenyl‐phenylhydantoin (p‐HPPH) decreased; (5) apparent rate of excretion of PHT dihydrodiol increased; and (6) apparent PHT total clearance and elimination half‐life and apparent p‐HPPH rate of excretion were dose dependent. Phenytoin apparent pharmacokinetic and biotransformation values undergo a typical series of changes after beginning monotherapy at typical dosing rates, because PHTs dose‐dependent pharmacokinetics result in differing apparent vaiues as the serum concentration rises to steady state. Stable iostope methods are particularly suitable for investigating such phenomena.

Long-term statin therapy and CSF cholesterol levels: implications for Alzheimer's disease.

Background/Aims: It is not yet established whether statins (lipophilic or hydrophilic) reduce the risk of Alzheimer’s disease and, if so, by differentially modifying brain lipid levels. Our aim was to assess changes in brain cholesterol metabolism as reflected in the cerebrospinal fluid (CSF) before and after treatment with either atorvastatin or simvastatin. Methods: We carried out a longitudinal analysis of CSF cholesterol, lathosterol and 24(S)-hydroxycholesterol before and after treatment with maximum doses of statins in 10 asymptomatic subjects, 8 of whom were heterozygous for apolipoprotein E ε4, and in 6 presymptomatic PS1 subjects. Results: Statins initially reduced CSF lathosterol cholesterol and 24(S)-hydroxycholesterol in both PS1 and non-PS1 subjects reaching a nadir at 6–7 months, followed by a return to baseline at 15 months with an overshoot at 2 years, tending to return to baseline thereafter. Conclusions: Possible long-term protective effects of statins are not likely largely related to the temporally-dependent biphasic effects of statin therapy upon the magnitude and direction of changes in CSF lipid levels and their subsequent return to baseline levels.

Phenobarbital does not alter phenytoin steady‐state serum concentration or pharmacokinetics

Phenytoin pharmacokinetics and biotransformation were studied with stable isotope tracer techniques in six patients before and after addition of phenobarbital. No significant (p < 0.05) changes in phenytoin serum concentration, clearance, elimination half-life, volume of distribution, or clearance via production of p-hydroxyphenyl-phenylhydantoin or phenytoin dihydrodiol occurred after addition of phenobarbital. Thus, frequent phenytoin serum concentration determinations or a change in phenytoin dosing rate are probably not necessary after adding phenobarbital.

Variable Absorption of Carbidopa Affects Both Peripheral and Central Levodopa Metabolism

Carbidopa (CD), a competitive inhibitor of aromatic l‐amino acid decarboxylase that does not cross the blood‐brain barrier, is routinely administered with levodopa (LD) to patients with Parkinson disease (PD) to reduce the peripheral decarboxylation of LD to dopamine. Using a stable isotope‐labeled form of LD, the authors examined in 9 PD patients the effects of variable CD absorption on peripheral and central LD metabolism. Subjects were administered orally 50 mg of CD followed in 1 hour by a slow bolus intravenous infusion of 150 mg stable isotope‐labeled LD (ring 1′,2′,3′,4′,5′,6′‐13C). Eight patients underwent a lumbar puncture 6 hours following the infusion. Blood and cerebrospinal fluid (CSF) samples were analyzed for labeled and unlabeled metabolites using a combination of high‐performance liquid chromatography and mass spectrometry. When patients were divided into “slow” and “rapid” CD absorption groups, significantly greater peripheral LD decarboxylation (as measured by area under the curve [AUC]‐labeled serum HVA) was noted in the poor absorbers (p = 0.05, Mann‐Whitney U test). Elimination half‐lives for serum LD did not differ between groups, suggesting a further capacity for decarboxylation inhibition in the “rapid” absorbers. A significant correlation between AUC serum CD and percent‐labeled HVA in CSF was found for all patients (R = 0.786, p = 0.02). “Rapid” as compared to “slow” CD absorbers had significantly more percent‐labeled CSF HVA (60 vs. 49, p = 0.02, Mann‐Whitney U test), indicating greater central‐labeled DA production in the better CD absorbers. The data suggest that peripheral aromatic l‐amino acid decarboxylase activity is not saturated at CD doses used in current practice. The authors believe that future studies to better examine a dose dependence of CD on peripheral LD decarboxylation and LD brain uptake are warranted.

Studies With Stable Isotopes III: Pharmacokinetics of Tracer Doses of Drug

Stable isotope labeled tracer doses of phenytoin (PHT) and phenobarbital (PB) were given intravenously before and four and 12 weeks after beginning monotherapy in two groups of six patients. Phenytoin demonstrated nonlinear pharmacokinetics, while PB demonstrated linear pharmacokinetics. Each of the 36 sets of tracer dose serum concentration versus time data points appeared linear during the elimination phase on semilog plots, and each demonstrated a high degree of linearity using semilog regression analysis (r2 = .977‐.999, P < .001, for PHT; r2 = .791‐.996, P < .005, for PB). We conclude tracer doses administered at steady‐state serum concentration will exhibit linear serum concentration versus time relationships on semilog plots regardless of whether the steady‐state serum concentration is in the linear or the nonlinear portion of a drugs dose versus steady‐state serum concentration relationship. The mechanism and implications of this conclusion are discussed.