Spleen phenotype in autosomal dominant polycystic kidney disease.

AIM: To evaluate splenic phenotype in autosomal dominant polycystic kidney disease (ADPKD) including presence of cysts and splenomegaly to determine if these are ADPKD related or represent unrelated incidental findings. MATERIALS AND METHODS: The axial/coronal T2-weighted images of ADPKD patients (n=215) and age/gender-matched controls (n=215) were evaluated for the presence of T2-bright splenic lesions by three blinded observers. Spleen volume (SV) was evaluated in the context of clinical and imaging features as well as results of gene testing for PKD1 and PKD2 mutations. RESULTS: T2-bright splenic lesions were found in 16 of 215 (7%) ADPKD patients compared to 11 of 215 (5%) control patients (p=0.32) and their prevalence was similar in patients with either PKD1 or PKD2 mutations. Median SV was significantly higher in ADPKD patients than controls (236 [182; 313 ml] versus 176 [129; 264 ml], p<0.0001). In multivariable analysis, height-adjusted SV (htSV) was not associated with the presence of liver cysts, haemorrhagic cysts, or infections; however, htSV was directly associated with height-adjusted total kidney volume (htTKV), a biomarker for ADPKD disease severity. CONCLUSIONS: The prevalence of T2-bright splenic lesions is similar in ADPKD patients and non-ADPKD controls, suggesting no relation to the diagnosis of ADPKD; however, splenic enlargement in ADPKD compared to controls could not be explained by liver cystic involvement, by infection/inflammatory conditions, or by haemorrhagic renal cysts. This combined with direct correlation of htSV with htTKV, a biomarker of ADPKD severity, suggests splenomegaly may be related to the pathogenesis of ADPKD.

Seasonal and sex differences in cell proliferation, neurogenesis, and cell death within the dentate gyrus of adult wild-caught meadow voles.

Past research indicates that female meadow voles (Microtus pennsylvanicus) show decreased neurogenesis within the hippocampus during the breeding season relative to the non-breeding season, whereas male voles show no such seasonal changes. We expanded upon these results by quantifying a variety of endogenous cell proliferation and neurogenesis markers in wild-caught voles. Adult male and female voles were captured in the summer (breeding season) or fall (non-breeding season), and blood samples and brain tissue were collected. Four cellular markers (pHisH3, Ki67, DCX, and pyknosis) were labeled and then quantified using either fluorescent or light microscopy. The volume of the cell layers within the dentate gyrus (hilus and granule cell layer) was significantly larger in males than in females. In both sexes, all the cellular markers decreased significantly in the dentate gyrus during the breeding season relative to the non-breeding season, indicating decreased cell proliferation, neurogenesis, and pyknosis. Only the pHisH3 marker showed a sex difference, with females having a greater density of this cell proliferation marker than males. During the breeding season relative to the non-breeding season, males and females showed the predicted significant increases in testosterone and estradiol, respectively. Overall, these results suggest higher levels of neuronal turn-over during the non-breeding season relative to the breeding season, possibly due to seasonal changes in sex steroids.

Simultaneous assessment of the pharmacokinetics of a pleuromutilin, lefamulin, in plasma, soft tissues and pulmonary epithelial lining fluid.

BACKGROUND: Lefamulin is a pleuromutilin antibiotic under evaluation for the treatment of bacterial infections, including respiratory tract infections. Currently, there are no high-quality pharmacokinetic data on drug tissue concentrations of lefamulin available. METHODS: A single dose of intravenous lefamulin (150 mg) was given to 12 healthy men. The registered EudraCT number for this study was 2010-021938-54. Lefamulin concentrations were simultaneously measured in plasma, skeletal muscle tissue, subcutaneous adipose tissue and epithelial lining fluid (ELF) over 24 h, and corresponding pharmacokinetic parameters were calculated. Microdialysis was used to measure unbound lefamulin concentrations in skeletal muscle tissue and subcutaneous adipose tissue, which were similar to unbound lefamulin concentrations in plasma. Bronchoalveolar lavage was performed 1, 2, 4 and 8 h post-dose to determine lefamulin concentrations in ELF. RESULTS: Unbound lefamulin levels showed a 5.7-fold higher exposure in ELF compared with that in plasma, demonstrating good penetration to the target site. CONCLUSIONS: Lefamulin may be an addition to the therapeutic armamentarium for the treatment of infections. Simultaneous measurements of unbound drug concentration can guide target attainment for future therapeutic trials.

Population pharmacokinetic analyses for BC-3781 using phase 2 data from patients with acute bacterial skin and skin structure infections.

BC-3781, a pleuromutilin antimicrobial agent, is being developed for the treatment of patients with acute bacterial skin and skin structure infections (ABSSSI) and community-acquired bacterial pneumonia. Data from a phase 2 study of patients with ABSSSI were used to refine a previous population pharmacokinetic (PK) model and explore potential predictors of PK variability. The previously derived population PK model based on data from three phase 1 studies was applied to sparse sampling data from a phase 2 ABSSSI study and modified as necessary. Covariate analyses were conducted to identify descriptors (e.g., body size, renal function, age) associated with interindividual variability in PK. All population PK analyses were conducted by using Monte Carlo parametric expectation maximization implemented in S-ADAPT 1.5.6. The population PK data set contained 1,167 concentrations from 129 patients; 95% of the patients had 5 or more PK samples (median, 11). The previous population PK model (three-compartment model with first-order elimination and nonlinear protein binding) provided an acceptable and unbiased fit to the data from the 129 patients. Population PK parameters were estimated with acceptable precision; individual clearance values were particularly well estimated (median individual precision of 9.15%). Graphical covariate evaluations showed no relationships between PK and age or renal function but modest relationships between body size and clearance and volume of distribution, which were not statistically significant when included in the population PK model. This population PK model will be useful for subsequent PK-pharmacodynamic analyses and simulations conducted to support phase 3 dose selection. (This study has been registered at ClinicalTrials.gov under registration no. NCT01119105.).

Phase II clinical study of BC-3781, a pleuromutilin antibiotic, in treatment of patients with acute bacterial skin and skin structure infections.

This study investigated the potential of the novel systemic pleuromutilin antibiotic BC-3781 to treat patients with an acute bacterial skin and skin structure infection (ABSSSI) caused by a Gram-positive pathogen. Patients were randomized to intravenous BC-3781 100 mg, BC-3781 150 mg, or vancomycin 1 g every 12 h. Response to treatment was assessed daily and at test of cure (TOC). The primary endpoint was the clinical success rate at TOC in the modified intent-to-treat (MITT) and clinically evaluable (CE) analysis populations. Baseline characteristics, including the frequency of methicillin-resistant Staphylococcus aureus (MRSA), were comparable between the different treatment groups. Of 210 patients randomized, 186 (88.6%) patients completed the study. Clinical success at TOC in the CE population occurred in 54 (90.0%) patients in the BC-3781 100-mg group, 48 (88.9%) in the BC-3781 150-mg group, and 47 (92.2%) in the vancomycin group. At day 3, the clinical response rate was similar across the three treatment groups. Six patients discontinued study medication following an adverse event. The incidence rate for drug-related adverse events was lower for patients receiving BC-3781 (34.3% and 39.4% in the 100-mg and 150-mg groups, respectively) than those receiving vancomycin (53.0%). When BC-3781 was used to treat ABSSSIs caused by a Gram-positive pathogen, including MRSA, clinical success rates were comparable to those of the comparator, vancomycin. BC-3781 was generally well tolerated. These results provide the first proof of concept for the systemic use of a pleuromutilin antibiotic for the treatment of ABSSSIs.

Effects of testosterone on spatial learning and memory in adult male rats.

A male advantage over females for spatial tasks has been well documented in both humans and rodents, but it remains unclear how the activational effects of testosterone influence spatial ability in males. In a series of experiments, we tested how injections of testosterone influenced the spatial working and reference memory of castrated male rats. In the eight-arm radial maze, testosterone injections (0.500 mg/rat) reduced the number of working memory errors during the early blocks of testing but had no effect on the number of reference memory errors relative to the castrated control group. In a reference memory version of the Morris water maze, injections of a wide range of testosterone doses (0.0625-1.000 mg/rat) reduced path lengths to the hidden platform, indicative of improved spatial learning. This improved learning was independent of testosterone dose, with all treatment groups showing better performance than the castrated control males. Furthermore, this effect was only observed when rats were given testosterone injections starting 7 days prior to water maze testing and not when injections were given only on the testing days. We also observed that certain doses of testosterone (0.250 and 1.000 mg/rat) increased perseverative behavior in a reversal-learning task. Finally, testosterone did not have a clear effect on spatial working memory in the Morris water maze, although intermediate doses seemed to optimize performance. Overall, the results indicate that testosterone can have positive activational effects on spatial learning and memory, but the duration of testosterone replacement and the nature of the spatial task modify these effects.

Prosopis juliflora--a green solution to decontaminate heavy metal (Cu and Cd) contaminated soils.

Soil and plant samples (root and shoot) of Prosopis juliflora were collected in the vicinity of metal based foundry units in Coimbatore and assessed for their heavy metal content (Cu and Cd) to ascertain the use of P. juliflora as a green solution to decontaminate soils contaminated with Cu and Cd. The results showed that Cu and Cd content was much higher in plant components compared to their extractable level in the soil. Furthermore, there exist a strong correlation between the distance of the sources of industrial units and accumulation of heavy metals in plants. Accumulation of Cd in roots is comparatively higher than that of shoots. However, in case of Cu no such clear trend is seen. Considering the accumulation efficiency and tolerance of P. juliflora to Cd and Cu, this plant can be explored further for the decontamination of metal polluted soils. On the other hand, in view of heavy metal accumulate the practice of providing foliage and pods as fodder for live stock should be avoided.

Rational dose selection for a nonnucleoside reverse transcriptase inhibitor through use of population pharmacokinetic modeling and Monte Carlo simulation.

In order to choose a rational dose for GW 420867X, we first set a goal of therapy. We hypothesized that, for optimal antiretroviral activity, the trough free drug concentration should remain above the 90% effective concentration (EC90) of human immunodeficiency virus type 1. We performed population pharmacokinetic analysis on three different doses of GW 420867X (50, 100, and 200 mg). Monte Carlo simulation was performed, assuming a log-normal distribution for 1,000 simulated subjects for each dose, and was repeated three times. The trough concentrations were divided by 76 to account for protein binding and for the difference between EC50 and EC90. We then determined the fraction of the simulated population whose free drug trough concentrations would exceed an EC90 over a broad range of values. The target attainment for all three doses exceeded 95% out to a starting EC50 of 10 nM. For 16 viral isolates, the EC50 range encountered for GW 420867X did not exceed 8 nM, implying that the three doses could not be differentiated by effect in a clinical trial in naive patients. This prediction was shown to be correct in a randomized, double-blind trial with 1 week of monotherapy with GW 420867X.

GW420867X administered to HIV-1-infected patients alone and in combination with lamivudine and zidovudine.

PURPOSE: GW420867X is a nonnucleoside inhibitor of HIV-1 reverse transcriptase. The primary objective was to assess the safety of GW420867X in HIV-1-infected patients. The secondary objectives were to assess the effect of GW420867X on plasma HIV-1 RNA and viral genotype and phenotype and to examine the pharmacokinetics of GW420867X in HIV-1-infected patients. METHOD: HIV-1-infected patients were randomized to GW420867X 50 mg/day, 100 mg/day, or 200 mg/day from days 1-28 (n = 15 per group). Lamivudine (3TC) plus zidovudine (ZDV) was added from days 8-28. A control group (n = 15) received GW420867X, 3TC, and ZDV placebos. RESULTS: Plasma HIV-1 RNA and CD4+ counts improved in the GW420867X groups at days 8 and 28. No significant development of drug resistance was detected. Median observed peak GW420867X concentration (C(max)) generally occurred at 2 hours. The area under the curve over the dosing interval (AUCtau)on day 14 increased less than proportionally to dose, suggesting there was increased clearance and/or decreased absorption. Mean trough GW420867X concentrations were many fold above the in vitro IC(50) in the presence of human serum proteins. Seven of 15 patients on 50 mg GW420867X, 8/15 on 100 mg GW420867X, 12/15 on 200 mg GW420867X, and 8/15 on placebo reported drug-related adverse events. CONCLUSION: GW420867X was well tolerated and has potent antiretroviral activity alone and in combination with 3TC plus ZDV.

Pharmacokinetics and tolerability of GW420867X, a nonnucleoside reverse transcriptase inhibitor, following single escalating doses in healthy male volunteers.

The aim of the current study was to characterize the pharmacokinetics of GW420867X, a new nonnucleoside reverse transcriptase inhibitor, using a single escalating dose protocol in healthy volunteers. Four dose levels were investigated in sequential order: 300, 600, 900, and 1200 mg, with a ratio of 4:1 subjects receiving active or placebo treatment, respectively. Following single-dose administration, GW420867X was readily absorbed with a median time to peak concentration of 3 to 5 hours. GW420867X plasma exposure (AUC) was dose proportional but variable within the 300 to 1200 mg dose range. Less than dose-proportional increases were observed for Cmax. The terminal elimination t(1/2) was 50 hours, which supports once-daily dosing in future studies. Plasma trough concentrations of GW420867X at 24 hours after dosing were many fold greater than the in vitro IC50 HIV-1(HXB2) in MT4 cells. GW420867X was generally well tolerated following single-dose administration up to 900 mg; increased central nervous system-related adverse events were observed at higher doses. GW420867X had a favorable pharmacokinetic and safety profile that would enable this drug to be explored in future clinical studies with HIV-1 infected patients at doses that would provide appropriate safety and efficacy.

The bioavailability of the novel nonnucleoside reverse transcriptase inhibitor GW420867X is unaffected by food in healthy male volunteers.

The effect of food on the bioavailability of GW420867X, a novel nonnucleoside reverse transcriptase inhibitor, was investigated in 15 young, healthy, male volunteers. A single oral dose of GW420867X 100 mg was administered in the fasted state, after a high-fat meal, and after a meal of normal fat composition. Tolerability and pharmacokinetic sampling were assessed at baseline and up to 600 hours. The median concentration-time plots for each treatment group were essentially superimposable. Neither the rate nor the extent of absorption of GW420867X was significantly affected by food. The median time to peak plasma concentration was 3 to 4 hours, irrespective of treatment. Pairwise comparisons using the fasted treatment as the comparator showed no impact of food on GW420867X pharmacokinetics. GW420867X was well tolerated. There were no serious or treatment-limiting adverse events; all episodes reported were rated as mild to moderate. The bioavailability of GW420867X was unaffected by food. GW420867X may be administered independently of food and fat intake.

Pharmacokinetics and safety of escalating single and repeat oral doses of GW420867X, a novel non-nucleoside reverse transcriptase inhibitor.

OBJECTIVE: To assess the pharmacokinetics, safety and tolerability of escalating oral doses of GW420867X, a non-nucleoside reverse transcriptase inhibitor, was investigated in healthy male volunteers in a randomized, double-blind placebo-controlled study. METHODS: Study subjects were divided into four groups of 12 subjects (10, 50, 100 and 200-mg dose groups) with eight subjects from each group receiving active treatment and the remaining four matched placebo. Subjects were initially administered a single dose of GW420867X or placebo, and following a 24- to 28-day washout period, re-exposed to the same treatment for 14 consecutive days. Safety measurements including clinical laboratory evaluations, ECG and vital signs were performed before, during and after dosing. RESULTS: Geometric mean GW420867X peak plasma concentrations (Cmax) following single oral doses of 10, 50, 100 and 200 mg were 160, 608, 1,000 and 1,662 ng/ml, respectively. Time to Cmax (tmax) increased from a median value of 1 h following the 10-mg dose, to 3 h after the 200-mg dose. Geometric mean plasma areas under the curves (AUC) were 4,325 (10 mg), 17,862 (50 mg), 35,295 (100 mg) and 62,338 ng/ml per hour (200 mg) and were proportionally less than the increase in the administered dose. Apparent terminal elimination half-life (t1/2) was approximately 50 h. Following repeat dosing, accumulation ratios based on plasma AUC were: 3.0+/-1.0 (10mg), 2.6+/-0.9 (50mg), 1.8+/-0.3 (100 mg) and 1.9+/-0.8 (200 mg) after 14 days of dosing compared to the corresponding single dose. In general, oral clearance (CL/F) was greater after 14 days and greater with higher doses except for the 10-mg dose group. Steady-state CL/F was 2.2, 3.4, 4.2, and 5.1 l/h for 10, 50, 100, and 200 mg, respectively. Steady-state was generally achieved within 7-10 days. Comparison of single and repeat dosing with GW420867X showed that Cmax increased by a factor of between 1.4 to 1.8, after 14 days of daily dosing to 288 (10 mg), 1,006 (50 mg), 1,401 (100 mg) and 2,613 (200 mg) ng/ml. These increases were proportionally less than the increase in the administered dose. GW420867X was well tolerated by subjects both after single and repeated dosing. Adverse effects reported by subjects on the active drug were similar to those receiving placebo. All episodes were rated as mild to moderate in severity and resolved spontaneously without further intervention. CONCLUSION: The pharmacokinetic findings of this study imply that systemic exposure to GW420867X decreases with increasing dose and displays time-variant pharmacokinetics, which suggests decreased absorption and/or increased clearance of GW420867X. The relatively long plasma half-life, of approximately 50 h, makes it suitable for once-daily dosing.

Brain and CSF entry of the novel non-nucleoside reverse transcriptase inhibitor, GW420867X.

(S)-2-ethyl-7-fluoro-3-oxo-3, 4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) inhibits HIV-1 reverse transcriptase and could be used for the treatment of HIV infection. This study quantified the movement of [14C]GW420867X into the CNS by means of a guinea-pig brain perfusion technique. Results indicated that [14C]GW420867X can enter the brain (Kin: 38.4+/-7.7 microl min(-1) g(-1)) and cerebrospinal fluid (CSF; Kin: 1.2+/-0.1 microl min(-1) g(-1)). Self-inhibition studies also suggested the presence of a saturable transport system for [14C]GW420867X at the blood-brain barrier (BBB). Thus [14C]GW420867X can enter the brain via the BBB and, compared with the blood-CSF barrier, this route is the predominant pathway for the brain entry of this drug. This would suggest that GW420867X is a promising drug for the treatment of HIV infection within the brain.

Use of drug effect interaction modeling with Monte Carlo simulation to examine the impact of dosing interval on the projected antiviral activity of the combination of abacavir and amprenavir.

The delineation of optimal regimens for combinations of agents is a difficult problem, in part because, to address it, one needs to (i) have effect relationships between the pathogen in question and the drugs in the combination, (ii) have knowledge of how the drugs interact (synergy, antagonism, and additivity), and (iii) address the issue of true between-patient variability in pharmacokinetics for the drugs in the population. We have developed an approach which employs a fully parametric assessment of drug interaction using the equation of W. R. Greco, G. Bravo, and J. C. Parsons (Pharmacol. Rev. 47:331-385, 1995) to generate an estimate of effects for the two drugs and have linked this approach to a population simulator, using Monte Carlo methods, which produce concentration-time profiles for the drugs in combination. This software automatically integrates the effect over a steady-state dosing interval and produces an estimate of the mean effect over a steady-state interval for each simulated subject. In this way, doses and schedules can be easily evaluated. This software allows for a rational choice of dose and schedule for evaluation in clinical trials. We evaluated different schedules of administration for the combination of the nucleoside analogue abacavir plus the human immunodeficiency virus type 1 protease inhibitor amprenavir. Amprenavir was simulated as either 800 mg every 8 h (q8h) or 1,200 mg q12h, each along with 300 mg q12h of abacavir. Both regimens produced excellent effects over the simulated population of 500 subjects, with average percentages of maximal effect (as determined from the in vitro assays) of 90.9%+/- 11.4% and 80.9%+/-18.6%, respectively. This difference is statistically significant (P<<0.001). In addition, 68.8 and 46.0% of the population had an average percentage of maximal effect which was greater than or equal to 90% for the two regimens. We can conclude that the combination of abacavir plus amprenavir is a potent combination when it is given on either schedule. However, the more fractionated schedule for the protease inhibitor produced significantly better effects in combination. Clinicians need to explicitly balance the improvement in antiviral effect seen with the more fractionated regimen against the loss of compliance attendant to the use of such a regimen. This approach may be helpful in the preclinical evaluation of multidrug anti-infective regimens.

Abacavir: absolute bioavailability, bioequivalence of three oral formulations, and effect of food.

STUDY OBJECTIVES: Study A: to determine the absolute bioavailability of a single 300-mg abacavir hemisulfate tablet. Study B: to determine the bioequivalence of two oral abacavir formulations (300-mg hemisulfate tablet, 100-mg succinate caplet), the effect of food on the bioavailability of the 300-mg hemisulfate tablet, and the bioavailability of the hemisulfate tablet relative to the hemisulfate solution. DESIGN: Phase I, randomized, open-label, balanced two- (study A) and three- or four-period (study B), crossover studies. SETTING: Two clinical research centers. SUBJECTS: Six men infected with the human immunodeficiency virus (HIV), aged 27-39 years (study A), and 18 HIV-infected men and women, aged 21-50 years (study B). INTERVENTIONS: In study A, all subjects received a single, oral 300-mg tablet of abacavir hemisulfate or a single, intravenous infusion of abacavir hemisulfate 150 mg over 60 minutes. In study B, all subjects received each of three single-dose treatments: three 100-mg abacavir succinate caplets in a fasted state, one 300-mg abacavir hemisulfate tablet in a fasted state, and one 300-mg abacavir hemisulfate tablet with a high-fat breakfast. Twelve subjects in study B also received a fourth treatment of abacavir hemisulfate 300 mg as an oral solution in a fasted state. Plasma samples collected for 24 hours (study A) or 12 hours (study B), and urine samples collected for 12 hours (study A) were analyzed by validated high-performance liquid chromatographic methods. MEASUREMENTS AND MAIN RESULTS: Abacavir pharmacokinetic parameters were calculated using standard, noncompartmental methods. In study A, the geometric least square (GLS) mean absolute bioavailability of oral abacavir was 83% (range 65-107%). In study B, the hemisulfate tablet was bioequivalent to the succinate caplet, but its time to maximum concentration (Tmax) occurred 30 minutes earlier. Administration of the abacavir hemisulfate tablet with food had no effect on area under the curve from time zero to infinity (AUC0-infinity), decreased maximum concentration (Cmax) by 26%, and delayed Tmax by 38 minutes. The relative bioavailability (GLS mean AUC0-infinity ratio) of the 300-mg abacavir hemisulfate tablet to solution was 101%, Cmax was 11% lower, and Tmax was unchanged. The most common drug-related adverse events associated with abacavir were nausea, vomiting, abdominal pain, and headache, all of which were mild. CONCLUSION: Based on our results, abacavir is safe and well tolerated and can be administered with or without meals.

Evidence of anaphylaxy after alteplase infusion.

BACKGROUND AND PURPOSE: Although alteplase, a recombinant tissue plasminogen activator (tPA), is structurally identical to endogenous tPA and therefore should not induce allergy, single cases of acute hypersensitivity reactions have been reported. Until now, specific antibodies against alteplase were not detected in blood samples obtained in these patients. CASE DESCRIPTION: We report an anaphylactic reaction in a 70-year-old white female who was treated with intravenous alteplase for thrombolysis of acute ischemic stroke 160 minutes after onset of a right-sided hemiparesis. Thirty minutes after infusion of alteplase had been started, the patient suffered acute severe sinus tachycardia and hypotension, followed by cyanosis and loss of consciousness. The alteplase infusion was stopped, and following antiallergic therapy, tachycardia and hypotension resolved within 1 hour. The hemiparesis remained unaltered, but additional harm resulting from the hemodynamic complication was not observed. Serum samples analyzed with a radioimmunoprecipitation assay were negative for total antibodies to alteplase, but in a subsequent ELISA, both samples were positive for IgE antibodies to alteplase. CONCLUSIONS: The detection of specific IgE antibodies reactive with alteplase in this patient could provide the first evidence of an anaphylactic-type reaction to alteplase in man. Because previous exposure to alteplase can be excluded, the results suggest that this patient had preexisting antibodies that were cross-reactive with one or more epitopes of alteplase and therefore precipitated the anaphylactic-type reaction.

Safety, pharmacokinetics, and antiretroviral activity of the potent, specific human immunodeficiency virus protease inhibitor nelfinavir: results of a phase I/II trial and extended follow-up in patients infected with human immunodeficiency virus.

The safety, antiretroviral activity, and pharmacokinetic profile of nelfinavir, a potent and specific inhibitor of human immunodeficiency virus (HIV) protease, were assessed in a small open-label phase I/II dose-ranging study in protease inhibitor-naive HIV-positive men. A total of 22 patients with baseline plasma HIV RNA > or = 20,000 copies/mL and CD4+ counts between 200 and 500 cells/mm3 were enrolled in the study. Of the 22 patients, 20 were evaluated for activity; 10 patients assigned to 771 mg/day base equivalent (300 mg three times daily) and 10 patients assigned to 1,026 mg/day base equivalent (600 mg twice daily) given monotherapy. A capsule formulation of nelfinavir was used. The initial study period was 28 days; patients showing a virologic response of 1 log10 reduction were eligible for enrollment in an extension phase and addition of nucleoside analogues. A maximally tolerated dose of nelfinavir was not established. A dose-response relationship was observed for four (40%) patients in the 771-mg group and six (60%) patients in the 1,026-mg group experiencing a reduction from baseline in plasma HIV RNA of at lest 1 log during the 28-day study. Of these patients, five sustained the reduction in plasma HIV RNA beyond day 28 (2 patients receiving 771 mg/day and 3 patients receiving 1,026 mg/day). Median increases from baseline in CD4+ counts at day 28 were 216 cell/mm3 and 86 cell/mm3 in the 771-mg and 1,026-mg groups, respectively. After oral administration, median nelfinavir plasma concentrations on day 28 reached a maximum at 1 hour (2,966 ng/mL) in the 771-mg group and at 3 hours (3,157 ng/mL) in the 1,026-mg group. Data for 22 patients were included in the safety analysis; 12 patients (55%) reported at least one grade 2 or worse (moderate, severe, or very severe) adverse event. The most common grade 2 or worse adverse event was diarrhea, reported by two patients (20%) receiving 771 mg/day and seven patients (70%) receiving 1,026 mg/day; followed by nausea, flatulence, asthenia, and headache (each reported in 1 patient [10%] in the 771-mg group) and dizziness (reported in 1 patient [10%] receiving 1,026 mg/day). In the small subgroup (n = 6) who continued taking nelfinavir for longer periods (between 8 and 15 months), virologic responses were sustained in the majority of patients with good tolerability. Nelfinavir is an active HIV-protease inhibitor with favorable pharmacokinetics, good tolerability, and sustained antiviral effects. Results of this early phase I/II dose-ranging study provided data for the safety and antiretroviral activity of nelfinavir and led to the selection of higher doses for phase II/III trials to further optimize virologic and immunologic responses.

Pharmacodynamics of recombinant human DNase I in serum.

Recombinant human deoxyribonuclease I (rhDNase) may be an effective therapeutic for the treatment of systemic lupus erythematosus (SLE). The pharmacodynamics of rhDNase in serum was investigated using two activity assays: one based on hydrolysis of a radiolabelled phage DNA and the other based on hydrolysis of human chromatin. The concentration of endogenous immunoreactive DNase in sera from 16 normal subjects was 3.2 +/- 1.4 ng/ml (mean +/- s.d.); however, low levels or no nuclease activity were detected in the same sera, suggesting the presence of DNase inhibitors. We assessed the ability of rhDNase to degrade DNA in undiluted serum, since the observed inhibition of endogenous DNase was reversed upon dilution. Addition of rhDNase to undiluted serum at a concentration of 50-100 ng/ml was necessary for degradation of radiolabelled phage DNA. The activity of rhDNase added to serum from normal subjects and SLE patients was similar. rhDNase degraded human chromatin and chromatin/anti-DNA immune complexes in serum with similar potency (EC50 approximately 100-200 ng/ml). A 500-fold variation in the chromatin/anti-DNA stoichiometry did not significantly affect the digestion of these immune complexes by rhDNase in buffer. These results indicate that a minimum rhDNase concentration of 50-100 ng/ml in serum was required to achieve detectable catalytic activity and that the presence of antibodies to DNA did not inhibit the degradation of DNA/anti-DNA immune complexes.

Improved potency of hyperactive and actin-resistant human DNase I variants for treatment of cystic fibrosis and systemic lupus erythematosus.

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a approximately 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was approximately 100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of approximately 400-fold over wild type for the +2 variant E13R/N74K. The +3 enzymes have approximately 4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the +6 enzyme has approximately 30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.