Cell therapy for full-thickness wounds: are fetal dermal cells a potential source?

The application of autologous dermal fibroblasts has been shown to improve burn wound healing. However, a major hurdle is the availability of sufficient healthy skin as a cell source. We investigated fetal dermal cells as an alternative source for cell-based therapy for skin regeneration. Human (hFF), porcine fetal (pFF) or autologous dermal fibroblasts (AF) were seeded in a collagen-elastin substitute (Novomaix, NVM), which was applied in combination with an autologous split thickness skin graft (STSG) to evaluate the effects of these cells on wound healing in a porcine excisional wound model. Transplantation of wounds with NVM+hFF showed an increased influx of inflammatory cells (e.g., neutrophils, macrophages, CD4(+) and CD8(+) lymphocytes) compared to STSG, acellular NVM (Acell-NVM) and NVM+AF at post-surgery days 7 and/or 14. Wounds treated with NVM+pFF presented only an increase in CD8(+) lymphocyte influx. Furthermore, reduced alpha-smooth muscle actin (αSMA) expression in wound areas and reduced contraction of the wounds was observed with NVM+AF compared to Acell-NVM. Xenogeneic transplantation of NVM+hFF increased αSMA expression in wounds compared to NVM+AF. An improved scar quality was observed for wounds treated with NVM+AF compared to Acell-NVM, NVM+hFF and NVM+pFF at day 56. In conclusion, application of autologous fibroblasts improved the overall outcome of wound healing in comparison to fetal dermal cells and Acell-NVM, whereas application of fetal dermal fibroblasts in NVM did not improve wound healing of full-thickness wounds in a porcine model. Although human fetal dermal cells demonstrated an increased immune response, this did not seem to affect scar quality.

Development of a population pharmacokinetic model to describe azithromycin whole-blood and plasma concentrations over time in healthy subjects.

Azithromycin (AZI), a broad-spectrum antibiotic, accumulates in polymorphonuclear cells and peripheral blood mononuclear cells. The distribution of AZI in proinflammatory cells may be important to the anti-inflammatory properties. Previous studies have described plasma AZI pharmacokinetics. The objective of this study was to describe the pharmacokinetics of AZI in whole blood (concentration in whole blood [Cb]) and plasma (concentration in plasma [Cp]) of healthy subjects. In this study, 12 subjects received AZI (500 mg once a day for 3 days). AZI Cb and Cp were quantified in serial samples collected up to 3 weeks after the last dose and analyzed using noncompartmental and compartmental methods. After the last dose, Cb was greater than Cp. Importantly, Cb, but not Cp, was quantifiable in all but one subject at 3 weeks. The blood area under the curve during a 24-h dosing interval (AUC24) was ∼2-fold greater than the plasma AUC24, but simulations suggested that Cb was not at steady state by day 3. Upon exploration of numerous models, an empirical 3-compartment model adequately described Cp and Cb, but Cp was somewhat underestimated. Intercompartmental clearance (CL; likely representing cells) was lower than apparent oral CL (18 versus 118 liters/h). Plasma, peripheral, and cell compartmental volumes were 439 liters, 2,980 liters, and 3,084 liters, respectively. Interindividual variability in CL was low (26.2%), while the volume of distribution variability was high (107%). This is the first report to describe AZI Cb in healthy subjects, the distribution parameters between Cp and Cb, and AZI retention in blood for up to 3 weeks following 3 daily doses. The model can be used to predict Cb from Cp for AZI under various dosing regimens. (This study has been registered at ClinicalTrials.gov under registration no. NCT01026064.).

Examining the associations between self-care practices and psychological distress among nursing students during the COVID-19 pandemic.

There is limited research regarding the impact of self-care practices on psychological distress, specifically on nursing students during a pandemic, such as COVID-19 (Corona Virus Disease- 2019). A 10-minute electronic survey was sent to nursing students at a large academic-medical center, and data from 285 student respondents were analyzed to assess psychological status, attitudes and behaviors in regards to the COVID-19 pandemic. Significant differences were found when comparing self-care practice scores by school grade for total scores (F = 4.48 [df = 4,250], p = .002), emotional subscale (F = 4.78 [df = 4,250], p = .001), and relationship subscale (F = 3.44 [df = 4,250], p = .009). While there were no significant differences in psychological distress by school grade, graduate students had the lowest self-care practice score compared to all the other grades. Finally, the subscale and total self-care practice scores were significantly and negatively associated with psychological distress. These findings suggest that utilization of self-care practices is associated with lower psychological distress, and should therefore be promoted among nursing student populations and integrated into curricula. Future studies should assess specific needs geared towards populations that may have poor self-care practices, such as graduate students, and understand ways to improve sleep quality to mitigate rates of psychological distress during a pandemic.

DHA and cholesterol containing diets influence Alzheimer-like pathology, cognition and cerebral vasculature in APPswe/PS1dE9 mice.

Cholesterol and docosahexenoic acid (DHA) may affect degenerative processes in Alzheimer's Disease (AD) by influencing Abeta metabolism indirectly via the vasculature. We investigated whether DHA-enriched diets or cholesterol-containing Typical Western Diets (TWD) alter behavior and cognition, cerebral hemodynamics (relative cerebral blood volume (rCBV)) and Abeta deposition in 8- and 15-month-old APP(swe)/PS1(dE9) mice. In addition we investigated whether changes in rCBV precede changes in Abeta deposition or vice versa. Mice were fed regular rodent chow, a TWD-, or a DHA-containing diet. Behavior, learning and memory were investigated, and rCBV was measured using contrast-enhanced MRI. The Abeta load was visualized immunohistochemically. We demonstrate that DHA altered rCBV in 8-month-old APP/PS1 and wild type mice[AU1]. In 15-month-old APP/PS1 mice DHA supplementation improved spatial memory, decreased Abeta deposition and slightly increased rCBV, indicating that a DHA-enriched diet can diminish AD-like pathology. In contrast, TWD diets decreased rCBV in 15-month-old mice. The present data indicate that long-term dietary interventions change AD-like pathology in APP/PS1 mice. Additionally, effects of the tested diets on vascular parameters were observed before effects on Abeta load were noted. These data underline the importance of vascular factors in the APP/PS1 mouse model of AD pathology.

Serum-free transient protein production system based on adenoviral vector and PER.C6 technology: high yield and preserved bioactivity.

Stable E1 transformed cells, like PER.C6, are able to grow at scale and to high cell densities. E1-deleted adenoviruses replicate to high titer in PER.C6 cells whereas subsequent deletion of E2A from the vector results in absence of replication in PER.C6 cells and drastically lowers the expression of adenovirus proteins in such cells. We therefore considered the use of an DeltaE1/DeltaE2 type 5 vector (Ad5) to deliver genes to PER.C6 cells growing in suspension with the aim to achieve high protein yield. To evaluate the utility of this system we constructed DeltaE1/DeltaE2 vector carrying different classes of protein, that is, the gene coding for spike protein derived from the Coronavirus causing severe acute respiratory syndrome (SARS-CoV), a gene coding for the SARS-CoV receptor or the genes coding for an antibody shown to bind and neutralize SARS-CoV (SARS-AB). The DeltaE1/DeltaE2A-vector backbones were rescued on a PER.C6 cell line engineered to constitutively over express the Ad5 E2A protein. Exposure of PER.C6 cells to low amounts (30 vp/cell) of DeltaE1/DeltaE2 vectors resulted in highly efficient (>80%) transduction of PER.C6 cells growing in suspension. The efficient cell transduction resulted in high protein yield (up to 60 picogram/cell/day) in a 4 day batch production protocol. FACS and ELISA assays demonstrated the biological activity of the transiently produced proteins. We therefore conclude that DeltaE1/DeltaE2 vectors in combination with the PER.C6 technology may provide a viable answer to the increasing demand for high quality, high yield recombinant protein.

Efficacy and side effects of praziquantel treatment against Schistosoma haematobium infection among primary school children in Zimbabwe.

We examined the efficacy of praziquantel against Schistosoma haematobium among primary school children during a school-based deworming programme in the Burma Valley commercial farming area and the Nyamaropa rural areas in Zimbabwe, where the disease is highly endemic. Among 767 individuals infected with S. haematobium, 675 (88.0%) received treatment. Two single oral doses of 40mg/kg praziquantel were given 6 weeks apart. Of the 675 participants, heavy infection intensity was more common in males than females (chi(2)=6.61, P=0.010). Six weeks later, 624 participants (92.4%) were successfully followed up. The overall cure rate was 88.5% and the egg reduction rate was 98.2%. The highest cure rate was among those individuals with light infection. Seventy-two individuals remained infected at 6 weeks post treatment, among which 3 and 69 individuals had heavy and light infection, respectively. Forty-six of these children resolved following a second round of treatment at 6 weeks follow-up. Of the remaining children successfully followed-up, 22 resolved after a third round of treatment 6 months later. A wide range of observed mild and transient side effects were not associated with egg intensity. The parasitological cure rate was not associated with gender or age. Our study demonstrates that praziquantel is efficacious against S. haematobium in Zimbabwe, although low levels of persistent infection warrant further investigation.

In vitro-in vivo correlation of hepatobiliary drug clearance in humans.

The biliary clearance (Cl(biliary)) of three compounds was estimated using sandwich-cultured human hepatocytes (SCHH) and compared with Cl(biliary) values measured in vivo. Tc-99m sestamibi (MIBI) Cl(biliary) was determined in seven healthy volunteers using an oroenteric catheter to aspirate duodenal secretions, and gamma scintigraphy to determine gallbladder contraction; this technique was used previously to determine Tc-99m mebrofenin (MEB) and piperacillin (PIP) in vivo Cl(biliary). In vitro Cl(biliary) of MEB, MIBI, and PIP was quantified in SCHH as the ratio of mass excreted into bile canaliculi and area under the blood concentration-time curve (AUC) in medium. MIBI Cl(biliary) in vivo was 5.5+/-1.2 mL/min/kg (mean+/-SD). The rank order of Cl(biliary) predicted from SCHH corresponded well with the in vivo Cl(biliary) values in mL/min/kg for MEB (7.44 vs 16.1), MIBI (1.20 vs 5.51), and PIP (0.028 vs 0.032). In conclusion, the methods developed allowed for reproducible quantification of Cl(biliary) of drugs in healthy humans and prediction of Cl(biliary) from in vitro data.

First molecular surveillance report of HIV type 1 in injecting drug users and female sex workers along the U.S.-Mexico border.

HIV prevalence is increasing among high-risk populations in the Mexican-U.S. border cities of Tijuana and Ciudad Juarez. In 2005, the molecular epidemiology of HIV-1 was studied among injecting drug users (IDU) and female sex workers (FSW) in these cities, which are corridors for over two-thirds of the migrant flow between Mexico and the United States. Eleven samples (eight IDU and three FSW) were successfully amplified, sequenced, and analyzed. The results revealed that all 11 samples were subtype B. There was no phylogenetic clustering or separation of the strains between IDU and FSW or between Tijuana and Ciudad Juarez nor was the Mexican strain phylogenetically distinct from other subtype B strains. Two of three drug naive FSWs had low-level HIV-1 resistance mutations. This community-based study demonstrated that HIV-positive IDUs and FSWs in Ciudad Juarez and Tijuana were predominantly infected with subtype B. Further prevalence studies on HIV-1 resistance mutations among FSWs in these border cities are warranted.

Pitfalls in determining knee alignment: a radiographic cadaver study.

Full-length anteroposterior (AP) radiographs provide the basis for evaluating leg alignment. A cadaver study was performed to determine the effect of knee flexion and hip rotation on projected angles on full-length AP radiographs. The outcomes were verified mathematically. The results of the cadaver study were similar to the mathematical results. Flexion of the knee without rotation of the lower extremity has little effect on angles as projected on full-length AP radiographs. Rotation of the lower extremity without flexion of the knee also has little effect. Simultaneous flexion of the knee and rotation of the leg, however, cause large changes in projected angles. Full-length radiographs can be taken without fluoroscopic control as long as the knee can be fully extended. In flexion contracture, a full-length AP radiograph taken under lateral fluoroscopic control is necessary to obtain accurate determination of the mechanical axis.

Focally positive margins in breast conserving surgery: Predictors, residual disease, and local recurrence.

BACKGROUND: Re-excision after breast conserving surgery (BCS) for invasive breast cancer (IBC) can be omitted for focally positive margins in the Netherlands, but this guideline is not routinely followed. Focally positive and extensively positive margins have rarely been studied separately and compared to negative margins regarding clinicopathological predictors, residual disease incidence, and local recurrence. METHODS: All females with BCS for Tis-T3, without neo-adjuvant chemotherapy between 2005 and 2014 at one university hospital were included. Clinicopathological and follow-up information was collected from electronic patient records. Index tumor samples from all patients with re-excision were reviewed by one pathologist. Margins were classified as negative (≥2 mm width), close (<2 mm width), focally positive (≤4 mm length of tumor touching inked margin), or extensively positive (>4 mm length). RESULTS: From 499 patients included, 212 (43%) had negative, 161 (32%) had close, 59 (12%) had focally positive, and 67 (13%) had extensively positive margins. Increasingly involved margins were associated with lobular type, tumor size, and adjacent DCIS in IBC patients and lesion size in purely DCIS patients. In IBC patients, 17%, 49%, and 77% had re-excision after close, focally positive, and extensively positive margins and residual disease incidence was 55%, 50%, and 70% respectively. In purely DCIS patients, 26 (65%), 13 (87%), and 16 (94%) had re-excision after close, focally positive, and extensively positive margins and residual disease incidence was 39%, 46%, and 90% respectively. CONCLUSION: Incidence of residual disease after focally positive margins was not different from close margins, but was significantly higher after extensively positive margins. We recommend quantifying extent of margin involvement in all pathology reports.

Assessing blood flow, microvasculature, erythema and redness in hypertrophic scars: A cross sectional study showing different features that require precise definitions.

BACKGROUND: In hypertrophic scar assessment, laser Doppler imaging (LDI), colorimetry and subjective assessment (POSAS) can be used to evaluate blood flow, erythema and redness, respectively. In addition, the microvasculature (i.e. presence of microvessels) can be determined by immunohistochemistry. These measurement techniques are frequently used in clinical practice and/or in research to evaluate treatment response and monitor scar development. However, until now it has not been tested to what extent the outcomes of these techniques are associated, whilst the outcome terms are frequently used interchangeably or replaced by the umbrella term 'vascularization'. This is confusing, as every technique seems to measure a specific feature. Therefore, we evaluated the correlations of the four measurement techniques. METHODS: We included 32 consecutive patients, aged ≥18 years, who underwent elective resection of a hypertrophic scar. Pre-operatively, we performed LDI (measuring blood flow), colorimetry (measuring erythema) and the POSAS (subjective redness) within the predefined scar area of interest (∼1.5cm). Subsequently, the scar was excised and the area of interest was sent for immunohistochemistry, to determine the presence of microvessels. RESULTS: Only a statistically significant correlation was found between erythema values (colorimetry) and subjective redness assessment (POSAS) (r=0.403, p=0.030). We found no correlations between the outcomes of LDI, immunohistochemistry and colorimetry. CONCLUSIONS: Blood flow, the presence of microvessels and erythema appear to be different hypertrophic scar features because they show an absence of correlation. Therefore, in the field of scar assessment, these outcome terms cannot be used interchangeably. In addition, we conclude that the term 'vascularization' does not seem appropriate to serve as an umbrella term. The use of precise definitions in research as well as in clinical practice is recommended.

Effect of multidrug resistance modulators on the hepatobiliary disposition of doxorubicin in the isolated perfused rat liver.

P-Glycoprotein (P-gp)-mediated multidrug resistance (MDR) in cancer cells may be modulated by competitive inhibitors of P-gp. In the liver, P-gp is localized on the canalicular membrane of hepatocytes. Quinidine and GF120918 inhibit the transport of P-gp substrates, including doxorubicin. Competitive inhibition of P-gp transport may alter biliary excretion of substrates. This study was designed to examine the effects of MDR modulators on the hepatobiliary disposition of doxorubicin and to elucidate the site(s) of drug-modulator interaction using pharmacokinetic modeling techniques. Livers from male Sprague Dawley rats were isolated and perfused for 2 h at 37 degrees C with recirculating male rat blood. MDR modulator (16.8-480 microg of GF120918 or 0.3-3.0 mg of quinidine) or vehicle (buffer or DMSO, respectively) was administered as a bolus to the perfusate reservoir 5 min prior to the addition of doxorubicin (464 microg). Perfusate and bile were collected during the perfusion, the liver was homogenized after the perfusion, and samples were analyzed by high-pressure liquid chromatography for doxorubicin and the major metabolite doxorubicinol. In the presence of GF120918, the biliary excretion of doxorubicin and doxorubicinol was decreased significantly without alterations in doxorubicin perfusate concentrations or doxorubicin and doxorubicinol liver concentrations. In the presence of quinidine, the biliary excretion of doxorubicin was reduced significantly; however, doxorubicinol recovery in bile was not altered. The perfusate and liver concentrations of doxorubicin were not altered by quinidine; doxorubicinol liver concentrations were increased. A series of pharmacokinetic models were evaluated incorporating perfusate, liver, and bile compartments to describe the disposition of doxorubicin and doxorubicinol in the isolated perfused rat liver. The model that best described these data, based on goodness-of-fit criteria, included first-order rate constants for all disposition processes. On the basis of this model, the rate-limiting process for doxorubicin and doxorubicinol elimination was biliary excretion. In the presence of GF120918, rate constants associated with doxorubicin and doxorubicinol canalicular egress were decreased, and other doxorubicinol disposition pathways were increased slightly. Quinidine was associated with a decrease in doxorubicin canalicular egress, doxorubicinol formation, and other doxorubicinol pathways. Pharmacokinetic modeling of the data supported the hypothesis that decreased biliary excretion of doxorubicin in the isolated perfused rat liver, as determined by mass-balance analysis, was due to interactions at the canalicular membrane. The present study further supports the utility of pharmacokinetic modeling in identifying sites of drug interactions within the hepatobiliary system. This approach may be particularly useful in predicting the effects of perturbations in hepatic translocation processes on the hepatobiliary disposition of drugs and derived metabolites.

Increased oral bioavailability of paclitaxel by GF120918 in mice through selective modulation of P-glycoprotein.

Previous studies in mice with disrupted mdr1a P-glycoprotein genes have shown that the oral bioavailability of paclitaxel is very low because of the presence of this drug-transporting protein in the intestinal wall. Additional studies with cyclosporin A have shown that this P-glycoprotein-inhibiting agent is able to increase the bioavailability of paclitaxel in mouse models and in patients. However, the potential immune-suppressive side effects of cyclosporin A renders this compound less suitable for chronic use in cancer patients. In this paper we present the results obtained with GF120918, an experimental P-glycoprotein inhibitor, on the oral bioavailability of paclitaxel in both wild-type and mdrlab knockout mice. GF120918 (25 mg/kg) was administered p.o. by gavage 15 min or 2 h before oral or i.v. dosing of paclitaxel, respectively. Paclitaxel plasma levels were quantified by high-performance liquid chromatography. GF120918 increased the plasma values for areas under the concentration-time curve of oral paclitaxel in wild-type mice by 6.6-fold from 408 to 2701 ng x ml(-1) h. Calculated relative to their respective values for area under the concentration-time curve after i.v. administration, GF120918 increased the oral bioavailability of paclitaxel in wild-type mice from 8.5 to 40.2%. The plasma pharmacokinetics of paclitaxel in mdr1ab knockout mice was not altered by GF120918, whereas the pharmacokinetics of paclitaxel in wild-type mice receiving GF120918 became comparable with mdr1ab knockout mice. This result indicates that GF120918 at this dose-level selectively and completely blocks P-glycoprotein in the intestines and does not notably interfere in the elimination of paclitaxel by metabolism or other transporters. On the basis of this result, GF120918 has been selected for additional study in humans.

Altered morphine glucuronide and bile acid disposition in patients with nonalcoholic steatohepatitis.

The functional impact of altered drug transport protein expression on the systemic pharmacokinetics of morphine, hepatically derived morphine glucuronide (morphine-3- and morphine-6-glucuronide), and fasting bile acids was evaluated in patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH) compared to healthy subjects. The maximum concentration (Cmax ) and area under the concentration-time curve (AUC0-last ) of morphine glucuronide in serum were increased in NASH patients (343 vs. 225 nM and 58.8 vs. 37.2 µM*min, respectively; P ≤ 0.005); morphine pharmacokinetics did not differ between groups. Linear regression analyses detected an association of NASH severity with increased morphine glucuronide Cmax and AUC0-last (P < 0.001). Fasting serum glycocholate, taurocholate, and total bile acid concentrations were associated with NASH severity (P < 0.006). Increased hepatic basolateral efflux of morphine glucuronide and bile acids is consistent with altered hepatic transport protein expression in patients with NASH and may partially explain differences in efficacy and/or toxicity of some highly transported anionic drugs/metabolites in this patient population.

Human Ontogeny of Drug Transporters: Review and Recommendations of the Pediatric Transporter Working Group.

The critical importance of membrane-bound transporters in pharmacotherapy is widely recognized, but little is known about drug transporter activity in children. In this white paper, the Pediatric Transporter Working Group presents a systematic review of the ontogeny of clinically relevant membrane transporters (e.g., SLC, ABC superfamilies) in intestine, liver, and kidney. Different developmental patterns for individual transporters emerge, but much remains unknown. Recommendations to increase our understanding of membrane transporters in pediatric pharmacotherapy are presented.

Frequency analysis of multidrug resistance-1 gene transfer into human primitive hematopoietic progenitor cells using the cobblestone area-forming cell assay and detection of vector-mediated P-glycoprotein expression by rhodamine-123.

Transfer of the multidrug resistance-1 (MDR1) gene into hematopoietic progenitor cells may reduce myelotoxicity of MDR1-related cytotoxic agents and therefore allow dose intensification. Mobilized peripheral blood progenitor cells (PBPC) can be obtained in ample quantity and are a suitable target cell population. CD34-selected PBPC samples (n = 6) were transduced with cell-free supernatant (SNT) of a cell line producing recombinant retrovirus containing the human MDR1 gene. Limiting-dilution long-term cultures were employed that allow continuous monitoring of stroma-adherent cobblestone areas (CA) and comparison of their frequency in a 5-log range over time. MDR1 provirus integration in CA-containing wells followed single-hit kinetics. According to Poisson statistics, proviral DNA was contained in 22% of unselected cobblestone area-forming cells (CAFC) at week 6, which represent primitive hematopoietic precursors. In comparison, 1.0 +/- 0.44% (mean +/- SEM) of week-6 CAFC were expressing P-glycoprotein at sufficient levels to convey vincristine resistance, suggesting low expression of the retroviral vector or splicing of the vector-drived mRNA in hematopoietic progenitor cells. Next we analyzed lineage-committed progenitors. The proviral DNA was detectable in 20-66% of colony-forming units granulocyte-macrophage (CFU-GM) while corresponding percentages (25-52%) of CD34+ PBPC were in the S/G2M phase of the cell cycle at the end of the transduction period. The proportion of vincristine-resistant CFU-GM was similar to the CAFC data and no significant differences were found between various MDR1-SNT transduction schedules whereas MDR1 co-cultivation, which served as a positive control, yielded significantly higher proportions of resistant colonies (5.3 +/- 1.4%, IL-3, 96 hr, p < or = 0.05). Assessment of rhodamine-123 (Rh-123) efflux in the myelo-monocytic progeny of MDR1-transduced cells mirrored the colony assay results in the SNT and co-cultivation groups. Less culture effort was required in the Rh-123 assay and functional characterization of the transferred P-glycoprotein was possible using cyclosporin A. Further development toward an effective MDR1 gene therapy should be facilitated by the CAFC assay, which allows estimation of the retroviral gene transfer frequency into primitive hematopoietic cells, and by the Rh-123 assay, which permits tractable side-by-side assessments of numerous MDR1 transduction protocols or different MDR1-SNT lots.

Effects of probenecid on the pharmacokinetics and pharmacodynamics of adinazolam in humans.

The effects of probenecid (2 gm) on the pharmacokinetics, pharmacodynamics, and uricosuric effects of adinazolam and N-desmethyladinazolam were assessed after single dose administration of adinazolam mesylate sustained-release tablets (60 mg) in a randomized, four-way crossover, double-blind study involving 16 healthy male volunteers. Probenecid decreased adinazolam oral clearance, renal N-desmethyladinazolam clearance, and the amount of N-desmethyladinazolam excreted in the urine. Probenecid increased the N-desmethyladinazolam/adinazolam AUC ratio, adinazolam maximum concentration (Cmax), N-desmethyladinazolam Cmax, and N-desmethyladinazolam time to reach Cmax. Uric acid renal clearance was increased significantly by adinazolam or probenecid administration compared with placebo; however, coadministration of adinazolam plus probenecid had no additive effect on uric acid clearance. Psychomotor performance was decreased in the adinazolam plus probenecid treatment compared with the adinazolam treatment. Probenecid potentiated the psychomotor effects of adinazolam after coadministration of the compounds, predominantly because of alterations in N-desmethyladinazolam pharmacokinetics. Therefore the adinazolam dose may need to be reduced when coadministered with probenecid.

Hepatic disposition of acetaminophen and metabolites. Pharmacokinetic modeling, protein binding and subcellular distribution.

Successful pharmacokinetic modeling often requires the ability of a simple model to describe a complex series of physiological processes. However, a simple model may be inappropriate. Physiologically-relevant modeling may offer a more appropriate description, but requires further support from in vitro/in vivo data. A well-stirred hepatic model with linear processes was proposed to describe in vivo disposition of acetaminophen and metabolites after a 100 mg/kg bolus of acetaminophen to vehicle- or phenobarbital-pretreated, renal-ligated rats. Model simulations underpredicted acetaminophen glucuronide (AG) concentrations at early time points in serum, and were inconsistent with AG biliary excretion-rate profiles. Intracellular binding of AG by ligandin was hypothesized, and a cytosolic compartment with reversible binding was incorporated into the model. In this second model, only AG bound in the cytosolic compartment was available for excretion into bile. Model 2 better described the AG biliary excretion rate-time profiles based on calculated Akaike's information criterion values. However, no apparent change was observed in the underprediction of AG serum concentrations. Parameter estimates derived from the two models also were different. The rate constants regulating AG formation and sinusoidal egress were increased significantly after phenobarbital pretreatment according to model 1, while the AG biliary excretion rate constant was decreased significantly. Parameter estimates based on model 2 suggested that phenobarbital pretreatment impaired the cytosolic binding of AG but increased significantly the AG biliary excretion rate constant. The physiologic relevance of model 2 was not supported by a subsequent investigation of the protein binding and subcellular distribution of acetaminophen and metabolites. Acetaminophen, AG and acetaminophen sulfate (AS) were not bound extensively in hepatic cytosol (mean +/- SD unbound fractions were 0.90 +/- 0.08, 0.97 +/- 0.08, and 0.88 +/- 0.06, respectively). Phenobarbital pretreatment did not alter significantly the unbound fractions of acetaminophen, AG or AS in hepatic cytosol. Acetaminophen was distributed to a greater extent in lysosomes than in the nuclear, mitochondrial, microsomal and cytosolic fractions. Distribution of AS predominated in cytosolic and lysosomal fractions. AG was detected only in cytosol. Phenobarbital pretreatment decreased the content of acetaminophen, AG and AS in all hepatic fractions. This study demonstrates the utility of pharmacokinetic modeling in exploring mechanistic hypotheses. However, these results underscore the importance of obtaining pivotal data from in vitro/in vivo studies to validate hypothesized mechanisms.

Autacoid binding to serum proteins. Interaction of platelet activating factor (PAF) with human serum alpha-1-acid glycoprotein (AAG).

Platelet activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent bioactive phospholipid released from platelets, neutrophils, basophils and macrophages that has been proposed as a mediator of anaphylaxis, acute lung injury and other disorders. Specific factors which stabilize PAF and/or regulate PAF activity in body fluids are largely unknown. As part of a general autacoid-serum protein binding screen, the platelet activating factor-alpha-1-acid glycoprotein (PAF-AAG) interaction was indirectly characterized by examining the ability of PAF to displace propranolol from AAG. Both PAF and its deacetylated metabolite (lyso-PAF), at 20 microM, doubled the fraction unbound of propranolol (0.4 microM) from purified human AAG (20 microM). None of the other autacoids that were studied (epinephrine, serotonin, spermidine, putrescine, leu-enkephalin or phenethylamine) exhibited any propranolol displacement activity. Scatchard analysis indicated that PAF competitively displaced propranolol from AAG, causing the apparent affinity constant for propranolol-AAG to decrease from 1.8 X 10(5) M to 6.9 X 10(4) M. PAF behaved qualitatively like chlorpromazine (a documented inhibitor of propranolol binding to AAG), but PAF was less effective at displacing propranolol. The apparent binding to AAG may help stabilize and transport extracellular PAF. Furthermore, the interaction of PAF and AAG suggests that serum AAG, which fluctuates in a number of diseases, may function to regulate PAF activity during acute and chronic disease states.

Protein binding and hepatobiliary distribution of valproic acid and valproate glucuronide in rats.

The protein binding and hepatobiliary distribution of valproic acid (VPA) and its glucuronide conjugate (V-G) were examined in rats with a combination of in vitro and ex vivo protocols. VPA was moderately bound to proteins in both serum and hepatic cytosol, and the degree of binding was lower ex vivo than in vitro. V-G, which was more highly bound than VPA ex vivo in serum, may have displaced the parent drug from its binding sites when VPA was administered in vivo. Examination of ex vivo hepatic subcellular distribution revealed that VPA localization tended to be high in cytosol and low in the microsomal fraction; V-G appeared to be distributed evenly throughout the cell although V-G concentrations within the liver were very low. The steady-state elimination rate of VPA did not increase proportionately with increasing steady-state concentrations of unbound VPA in serum, consistent with saturable systemic elimination of the drug. In contrast, steady-state VPA elimination was related linearly to unbound cytosolic VPA concentrations. Moreover, a nonlinear relationship between the unbound concentrations of VPA in hepatic cytosol and serum was observed, consistent with saturable distribution of the unbound drug between the two compartments in vivo. These observations suggest that the nonlinear elimination of VPA in rats may be due to concentration-dependent penetration of the drug into the liver as opposed to saturable biotransformation.