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Andrew L Finn1, WD Charlie Hill2, Ignacio Tagarro3, Larry N Gever41Product Development, BioDelivery Sciences International, Inc, Raleigh, NC, USA; 2Co-founding partner, InVisions Consultants, LLC, San Antonio, TX, USA; 3Marketing Centre CIP CNS, Meda Pharma S.A.U., Madrid, Spain; 4Medical Affairs, Meda Pharmaceuticals, Inc, Somerset, NJ, USAPurpose: Fentanyl buccal soluble film (FBSF) consists of a small, bilayered, water-soluble polymer film that adheres to the buccal mucosa and rapidly delivers fentanyl into the systemic circulation. The purpose of this study was to evaluate the absorption of fentanyl from FBSF in patients with cancer, with and without grade 1 oral mucositis, and to assess the tolerability of FBSF in this patient population.Patients and methods: In an open-label, single-dose study, two groups of opioid-naive patients (ie, not receiving opioids on a regular basis) with cancer received a 200 µg dose of FBSF. Patients in cohort I (n = 7) had grade 1 mucositis, and patients in cohort II (n = 7) were age- and gender-matched controls without mucositis. The FBSF dose was placed on the area of mucositis in cohort I and on a matching location in cohort II. Blood samples were collected up to 4 hours after administration, and safety assessments were made throughout the study.Results: Peak plasma concentration and area under the concentration–time curve from time 0 to 4 hours post-dose values of patients in the grade 1 mucositis cohort were lower than those observed in patients without mucositis. There was no application site irritation reported in any patient, regardless of mucositis status. Mild somnolence was reported by two patients with mucositis. There were no deaths or serious adverse events reported in this study.Conclusion: The results of this study indicate that application of FBSF to an area of grade 1 mucositis does not result in increased fentanyl exposure or irritation of the mucosa. The 200 µg dose of FBSF was well tolerated.Keywords: breakthrough cancer pain, clinical study, application site pain

A poem by Finn Ale Álvarez Contreras.

1 The plasma concentration of alpha 1-acid glycoprotein (AAG) was significantly greater in 27 epileptic subjects receiving anticonvulsants compared with 27 age- and sex-matched drug-free control subjects. 2 Increased AAG concentration was associated with enhanced lignocaine binding in the plasma of epileptics. 3 Increased AAG concentration was also associated with a redistribution of lignocaine out of red cells and into plasma thus lowering the blood to plasma concentration ratio. 4 Enhanced lignocaine binding in epileptics receiving anticonvulsant therapy may result in lower free (unbound) plasma concentrations of the drug compared to normal subjects with equivalent total plasma lignocaine concentrations. [ABSTRACT FROM AUTHOR]

Background and Objectives: Fentanyl buccal soluble film (FBSF) is a small, bilayered, water-soluble polymer film (BioErodible MucoAdhesive; BEMA™) that adheres to the buccal mucosa and rapidly delivers fentanyl into the systemic circulation. It is approved for the treatment of cancer breakthrough pain in adult opioid-tolerant patients. The objective of this study was to evaluate the dose proportionality of the pharmacokinetics of FBSF in healthy subjects across a range of doses., Methods: This was a phase I, open-label, single-dose, three-period, Latin-square crossover study in which 12 healthy subjects received single FBSF doses of 200, 600 and 1200 μg with 72 hours between doses. Oral naltrexone was administered to each subject prior to and after each study dose. Serial venous blood samples were collected for 48 hours after study drug administration. Adverse events were recorded throughout the study. Dose linearity was examined using a power model (P = a × Doseb), where P represents the dependent variable (maximum plasma drug concentration [C(max)], area under the plasma concentration-time curve [AUC] from time zero to time of the last measurable concentration [AUC(last)], or AUC from time zero to infinity [AUC(∞)]), and a and b are constants. A value of b≈1 indicated linearity., Results: Following administration of FBSF doses of 200-1200 μg, mean C(max) values increased in a linear manner with values ranging from 0.383 ng/mL to 2.19 ng/mL, respectively. Mean AUC(last) values increased from 3.001 ng·h/mL to 19.17 ng·h/mL and mean AUC(∞) increased in a linear manner from 3.456 ng·h/mL to 20.43 ng·h/mL. All reported adverse events were considered to be mild to moderate in severity., Conclusions: This study demonstrates that peak fentanyl plasma concentrations and overall exposure increase in a dose-proportional manner following administration of FBSF.

Fentanyl buccal soluble film (FBSF) is a rapidly absorbed transmucosal formulation of fentanyl for the management of breakthrough pain in opioid-tolerant patients with cancer. This open-label, 3-period, sequential dose study evaluated the dose-to-dose reproducibility of the pharmacokinetics of fentanyl following the administration of 600- or 1800-microg doses of FBSF in 12 naltrexone-blocked, healthy adult volunteers. Subjects received 3 study treatments: single doses of 600 microg of FBSF on day 1 and day 4 and three 600-microg doses administered at 1-hour intervals on day 7. Plasma fentanyl concentrations were measured over a 48-hour period after each single dose of FBSF and 72 hours after the 3-dose regimen. Peak plasma concentrations (mean C(max) = 1.08 and 1.01 ng/mL) and overall exposure (mean AUC(0-12) = 6.3 and 6.2 h.ng/mL; mean AUC(inf) = 9.14 and 9.60 h.ng/mL) were nearly identical after the 2 single doses (P >or= .1, all comparisons). C(max) and overall fentanyl exposure (AUC(inf)) increased approximately 3-fold with the 3-dose regimen compared with the single-dose periods. Fentanyl plasma concentrations following single doses of FBSF were reproducible, and 3 doses administered 1 hour apart produced a tripling in exposure and maximal concentration compared with a single dose.

Objective: The objectives of the study were to determine the absolute bioavailability of fentanyl from fentanyl buccal soluble film, estimate the percentage of a fentanyl dose absorbed through the buccal mucosa, and compare the bioavailability of equivalent doses administered either as single or multiple dose units., Design: Open-label, randomized, four-period, Latin-square crossover pharmacokinetic study., Setting: Inpatient phase 1 unit., Patients: Twelve healthy volunteers. Interventions. Injectable fentanyl citrate (200 microg) administered by intravenous infusion, injectable fentanyl citrate (800 microg/16 mL) administered orally, and fentanyl buccal soluble film (800 microg) administered as a single film and as four separate 200 microg films simultaneously., Outcome Measures: Plasma concentrations after fentanyl dosing; pharmacokinetic parameters., Results: The two buccal film treatments were bioequivalent and both had an absolute bioavailability of 71%. The percentage of an administered dose absorbed through the buccal mucosa was calculated to be 51%., Conclusions: Fentanyl buccal soluble film effectively delivers a high percentage of the administered fentanyl dose and nearly identical plasma profiles are obtained when equivalent doses are delivered by single or multiple dosage units.

Background: Fentanyl buccal soluble film (FBSF) has been developed as a treatment of breakthrough pain in opioid-tolerant patients with cancer. The objective of this study was to evaluate the efficacy of FBSF at doses of 200-1200 microg in the management of breakthrough pain in patients with cancer receiving ongoing opioid therapy., Patients and Methods: This was a multicenter, randomized, double-blind, placebo-controlled, multiple-crossover study that included opioid-tolerant adult patients with chronic cancer pain who experienced one to four daily episodes of breakthrough pain. The primary efficacy assessment was the sum of pain intensity differences at 30 min (SPID30) postdose., Results: The intent-to-treat population consisted of 80 patients with > or =1 post-baseline efficacy assessment. The least-squares mean (LSM +/- SEM) of the SPID30 was significantly greater for FBSF-treated episodes of breakthrough pain than for placebo-treated episodes (47.9 +/- 3.9 versus 38.1 +/- 4.3; P = 0.004). There was statistical separation from placebo starting at 15 min up through 60 min (last time point assessed). There were no unexpected adverse events (AEs) or clinically significant safety findings., Conclusions: FBSF is an effective option for control of breakthrough pain in patients receiving ongoing opioid therapy. In this study, FBSF was well tolerated in the oral cavity, with no reports of treatment-related oral AEs.

Background and Objectives: BioErodible MucoAdhesive (BEMA) is a new transmucosal drug delivery system designed to improve and ease the administration of drugs by this route. The first product that uses this novel delivery system contains fentanyl and is intended for the treatment of breakthrough pain in opioid-tolerant patients with cancer. The generic name is fentanyl buccal soluble film (FBSF). The objectives of this study were to compare the pharmacokinetic profile of FBSF formulations at three different pHs (pH 6, pH 7.25 and pH 8.5) and to understand the differences in the pharmacokinetics of fentanyl from FBSF compared with that of oral transmucosal fentanyl citrate (OTFC)., Methods: This was a randomized, open-label, single-dose, four-period, Latin-square crossover study consisting of a 9-day inpatient treatment period. The study was conducted at a phase 1 clinical research unit in Austin, TX, USA. Twelve healthy subjects were enrolled, nine males and three females, between the ages of 21 and 44 years. Each subject received four 800 microg doses of fentanyl: single doses of the three FBSF formulations (pH 6, pH 7.25 and pH 8.5) and OTFC, with concurrent naltrexone. Plasma fentanyl concentrations were measured over a 48-hour period after each study dose. Pharmacokinetic parameters were calculated and compared., Results: Peak plasma fentanyl concentrations (Cmax) and overall fentanyl systemic exposure (area under the plasma concentration-time curve from time zero extrapolated to infinity [AUCinfinity]) for each of the three FBSF formulations were greater than for OTFC. The pH 7.25 FBSF formulation provided the earliest time to reach Cmax (tmax), the highest Cmax value and the greatest AUC(infinity) value. Compared with OTFC, peak plasma fentanyl concentrations with pH 7.25 FBSF were significantly higher (mean Cmax 1.67 vs 1.03 ng/mL; p<0.05). Overall exposure was also greater with pH 7.25 FBSF than with OTFC (mean AUCinfinity 14.5 vs 10.3 ng . h/mL)., Conclusions: All three FBSF formulations produced greater peak plasma concentrations and overall exposure to fentanyl than OTFC. In particular, the pH 7.25 FBSF formulation showed the most favourable pharmacokinetic profile of the three FBSF formulations. In comparison with OTFC, the pH 7.25 FBSF formulation produced the fastest and most efficient fentanyl delivery and was selected for further clinical development.

Background & Aims: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals., Methods: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody., Results: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody., Conclusions: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.

We have purified a protein from Necturus maculosus gallbladder cells that forms chloride channels in an artificial membrane. The same protein apparently can form channels that are highly selective for chloride but can have conductances varying from 9 to about 150 pS. The high-conductance channels are blocked by the monoclonal antibody used to purify the protein, but this antibody has no effect on the 9-pS channels. The observation that gating of the low- and high-conductance states is independent and that the antibody affects only the latter has implications regarding the control of chloride conductance in cell membranes and the different types of channels described in those cells.

We have used a monoclonal antibody (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder epithelial cells, to isolate a chloride channel protein by the use of an immunoaffinity column and FPLC. This protein (M(r) 219,000) has been reconstituted into a planar lipid bilayer, where it behaves as a chloride-selective channel (PCl/PNa = 20.2; PNa/PK = 1) whose unit conductance is 62.4 +/- 4.6 pS. Antibody added to the trans side (there is no effect from the cis side) causes channel open probability to drop to virtually zero, but has no effect on the conductance or the selectivity of single channels. To test the role of phosphorylation in the activity of the native channel, we studied the effects of the protein phosphatase inhibitor okadaic acid (OA) on intact gallbladders, and showed that channels opened by theophylline treatment and closed by antibody are reopened reversibly by OA (0.01-1.0 microM). Addition of the catalytic subunit of protein phosphatase 2A (PP-2A) to the cis side of a bilayer containing reconstituted chloride channels caused closure of the channels after a delay, and subsequent addition of ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA) caused immediate reopening. These data indicate that (a) this chloride channel protein inserts in a directed way into the bilayer such that the cis side is 'intracellular', (b) the purified channel protein is phosphorylated, and (c) gating from the cellular side is controlled by the direct phosphorylation and dephosphorylation of the channel protein.

The safety of ondansetron has been carefully evaluated through laboratory studies and clinical trials. Preclinical studies demonstrated that there is no end-organ toxicity in rats and dogs administered ondansetron doses 30 to 100 times those used in humans. At near-lethal doses of ondansetron, animals developed subdued activity, ataxia, and convulsions. Modest transient increases in serum transaminase values were observed. Concurrent administration of ondansetron with chemotherapy had no effect on tumor response in animals. The clinical safety of ondansetron has been evaluated in more than 2,500 cancer patients who received intravenous doses as large as 1.5 mg/kg. In adult patients receiving single-day chemotherapy, the incidence of adverse events was 36% with ondansetron (n = 647) and 50% with metoclopramide (n = 498). Diarrhea occurred in 9% of ondansetron patients and 19% of metoclopramide patients. Headache occurred in 14% of ondansetron patients and 8% of metoclopramide patients. Extra-pyramidal symptoms were reported in none of the ondansetron patients and 5% of the metoclopramide patients. The incidence of vascular occlusive events and seizure disorders was nearly identical with ondansetron and metoclopramide and similar to the cancer population in general. In a group of 209 pediatric patients receiving chemotherapy, the incidence of adverse events was 19% with ondansetron. Serum transaminase values increased significantly in 6% to 8% of ondansetron patients and 2% of metoclopramide patients. There was no apparent relationship between the cumulative dose of ondansetron administered and the incidence of increased transaminase values. However, there was an apparent relationship between the cumulative dose of cisplatin administered and the incidence of transaminase abnormalities. These data demonstrate that ondansetron is better tolerated than metoclopramide and is safe for intravenous administration to pediatric and adult patients receiving chemotherapy.

Cisplatin may evoke both an acute emetic response during the first 24 hours following treatment and a less well-recognized syndrome of delayed emesis. While delayed emesis is usually less severe in terms of frequency of vomiting episodes, the problem continues to result in significant morbidity. In comparison with acute emesis, the exact pathogenesis of the delayed emesis syndrome remains unclear. Although a combination of oral metoclopramide and dexamethasone is effective in many patients in preventing delayed emesis, almost 50% continue to experience at least one emetic episode when treated with this regimen. A phase III multicenter study has evaluated oral ondansetron versus placebo in the prevention of the delayed-emesis syndrome in 50 patients during days 2 through 5 following high-dose cisplatin administration. Although the daily rates of complete emetic control, failure, and control of nausea favor ondansetron, this trial is statistically inconclusive in establishing efficacy of ondansetron as a single agent in the prevention of delayed emesis. Ondansetron was well tolerated in the dose and schedule used.

A native chloride channel in Necturus gallbladder epithelial cells is opened by a theophylline-induced rise in cellular cyclic AMP and is closed by removal of theophylline or by addition of specific antibody; however, it does not close if okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is added. The purified channel reconstituted into lipid bilayers closes upon the addition of protein phosphatase 2A and is reopened by the addition of Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. These results indicate that the channel protein is purified in a phosphorylated state and that its functional characteristics are at least partly controlled by direct phosphorylation and dephosphorylation.

The purpose of this study was to determine the effects of 150 mg ranitidine, 300 mg ranitidine or placebo, administered every 8 h, on gastro-oesophageal pH and heartburn parameters in reflux patients. Twelve symptomatic reflux patients received each of the three treatments in a randomized, double-blind, crossover fashion. Intragastric and oesophageal pH were monitored continuously for a 24 h period. Meals were standardized, consumed at set times and patients were allowed to recline and sleep from 23.00 hours until 06.00 hours only. The gastric record was analysed for the percentage of time that the pH was greater than or equal to 4. The oesophageal record was analysed for acid contact time (percentage time (%) pH less than or equal to 4.0) and reflux episode frequency. Finally, patients recorded each new episode of heartburn and graded daytime heartburn severity at the end of each hour. Ranitidine increased the median (%) time that the intragastric pH remained at or above 4, from 4.5 (placebo) to 33.9% (150 mg dose) and 33.3% (300 mg dose). Ranitidine dose-dependently reduced the median 24-hour oesophageal acid contact time from 13.3% (placebo) to 6.8% (150 mg dose) and 2.5% (300 mg dose). The 300 mg dose significantly reduced daytime heartburn episode frequency and severity while the 150 mg dose reduced heartburn severity only. We conclude that 150 and 300 mg doses of ranitidine administered every 8 h have major, sometimes dose-dependent effects on the objective parameters and symptoms of gastro-oesophageal reflux.

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.

In a multicenter trial, we evaluated the antiemetic efficacy of ondansetron, a selective serotonin type 3 (5-HT3) receptor antagonist, in 42 adult chemotherapy-naïve patients receiving a multiple-day cisplatin regimen (20-40 mg/m2 per day for 4-5 days). Thirty-one patients received 3 daily doses of ondansetron (0.15 mg/kg) given intravenously every 6 hours (first dose 30 minutes prior to cisplatin administration); 11 additional patients received an identical dosage and schedule except that a fourth daily dose was added 17.5 hours after cisplatin administration. No other antiemetics were administered. Forty patients were evaluable for efficacy response. Thirteen patients (33%) had no vomiting at any time during the 5-day study. When emetic episodes were evaluated on a daily basis, complete protection (zero emetic episodes) ranged from 50-75%, and major protection (less than or equal to 2 emetic episodes) ranged from 65-93%. The majority of therapy failures occurred on days 3 and 4. Side effects were minor and transient; no extrapyramidal side effects were observed. Ondansetron appears to be a safe and effective antiemetic when administered during a multiple-day cisplatin-containing chemotherapy regimen.

The control of nausea and emesis in cancer patients receiving chemotherapy poses a significant management problem. In this randomized, double-blind, placebo-controlled study, we evaluated the effect of serotonin S3 receptor blockade with ondansetron (GR 38032F) on the prevention of nausea and vomiting induced by cyclophosphamide-containing chemotherapy. Cyclophosphamide was given in doses of 500 to 600 mg/m2 and ondansetron as three intravenous (IV) doses of 0.15 mg/kg. Most patients had breast cancer. Cyclophosphamide was given in combination with doxorubicin (65% of patients) or with fluorouracil (85% of patients: 50% with Adriamycin [doxorubicin; Adria Laboratories, Columbus, OH] and 35% with methotrexate). All placebo-treated patients experienced vomiting, whereas 70% of patients treated with ondansetron did not vomit (P = .008). Median nausea scores were 8 mm on ondansetron and 65 mm on placebo (P less than .001). Seventy percent of patients treated with ondansetron retained their normal appetite, compared with 10% of placebo patients. Adverse events occurred in six placebo patients and one ondansetron patient. Diarrhea and headache were the most common events, both occurring more frequently in the placebo group. There were no extrapyramidal reactions, and the only significant biochemical change occurred in a placebo-treated patient. These results suggest that serotonin S3 receptor antagonists represent a novel, effective, and safe mode of therapy for nausea and emesis induced by cyclophosphamide-containing chemotherapies. In addition, our observations are compatible with the view that serotonin, acting on S3 receptors, mediates the nausea and emesis occurring after cyclophosphamide chemotherapy.

GR 38032F (ondansetron) is a selective serotonin subtype-3 receptor antagonist with reported antiemetic efficacy in patients receiving cisplatin. This study evaluated the safety and efficacy of ondansetron in three consecutive nonrandomized groups of patients who were receiving a 4- or 5-day regimen of cisplatin (20 to 40 mg/m2/d) combination chemotherapy. Thirty-six patients were enrolled. Thirty-five patients were assessable for efficacy. All patients received three daily intravenous doses of 0.15 mg/kg of ondansetron. Twenty-four patients had received no prior chemotherapy. Twelve of these received ondansetron every 2 hours and 12 received ondansetron every 6 hours. Twelve additional patients who had received at least one prior course of chemotherapy were administered ondansetron every 6 hours. All patients were monitored for emetic episodes (vomiting or retching), adverse events, and laboratory safety parameters. Ten patients (29%) had no vomiting or retching throughout the entire study period and 18 patients (51%) experienced two or fewer emetic episodes during the entire study period. The greatest antiemetic efficacy was on day 1 when 27 patients (77%) had no emesis. The chemotherapy-naive patients responded better than the nonnaive patients on all study days. Reported adverse events were minor, with the most common possibly drug-related event being headache (14% of patients). No extrapyramidal symptoms were observed. Transient increases in total SGOT, and SGPT were observed in some patients.

We compared the efficacy and safety of ondansetron (GR 38032F), a selective antagonist of serotonin S3 receptors, with that of placebo in controlling the nausea and vomiting induced by cisplatin treatment in 28 patients with cancer. The patients received either three intravenous doses of ondansetron (0.15 mg per kilogram of body weight) or normal saline (placebo) at four-hour intervals, beginning 30 minutes before the administration of cisplatin. Nausea and vomiting were markedly diminished in the group given ondansetron. The median time to the first episode of emesis was 2.8 hours in the placebo group and 11.6 hours in the ondansetron group (P less than 0.001); the median number of episodes in 24 hours was 5.5 in the placebo group and 1.5 in the ondansetron group (P less than 0.001); the mean (+/- SEM) number of regurgitations or dry heaves per episode was 3.2 +/- 0.5 in the placebo group and 1.17 +/- 0.1 in the ondansetron group (P less than 0.001). None of the 14 patients given ondansetron, but 12 of 14 given placebo, required treatment with antiemetic-rescue agents for the control of nausea and vomiting. There were no adverse effects attributable to ondansetron. The urinary excretion of 5-hydroxyindoleacetic acid, the main metabolite of serotonin, was increased in all patients two to six hours after they received cisplatin chemotherapy, and the increases paralleled the episodes of emesis. We conclude that ondansetron is an effective and safe agent for controlling the nausea and vomiting induced by cisplatin treatment. We suggest that cisplatin treatment increases the release of serotonin from enterochromaffin cells, and that ondansetron acts by blocking S3 receptors for serotonin.

Necturus gallbladder epithelium transports sodium and chloride by a process that first involves the cellular entry of each ion across the apical membrane in an electrically silent process. In this paper we present results from cell volume and fluid flux measurements in the presence of different inhibitors and at normal and reduced sodium concentrations, which bear on the process by which ionic entry is effected. We find that reduction of mucosal sodium to a concentration of 10 mM has no effect on either cell volume or on the rate of transepithelial fluid transport, whereas the complete removal of sodium causes a significant decrease in cell volume in addition to its known inhibitory effect on fluid transport. Amiloride had no effect on cell volume at normal sodium concentrations but markedly reduced it when the sodium concentration was reduced to 10 mM. Amiloride, bumetanide, and dipyridamole markedly and reversibly inhibited fluid transport. Finally, the addition of ouabain to the serosal medium induced cell swelling, which was prevented by the removal of potassium from the mucosal medium. These results indicate that the process of sodium entry at the apical membrane is complicated and likely includes both cotransport (NaCl or Na-K-2Cl) and parallel exchange (Na-H and Cl-HCO3) transport mechanisms, and that the proportion of NaCl transported by the different mechanisms varies with the conditions.

Glass microelectrodes were used to measure membrane potentials and the ratio of apical to basolateral membrane resistances before and after the passage of current from the potential-recording microelectrode to ground, in toad urinary bladder epithelium, in order to iontophorese cations into the cell. After application of the current, there was a transient change in the tip potential of the microelectrode. This artifact was measured with the microelectrode in the mucosal medium and was subtracted from the potential recorded in the cell. The serosal medium was bathed by Ringer's solution containing 51.5 mM K+ to minimize any current-induced increase of K+ in the unstirred layer. Under those conditions, both Na+ and K+ iontophoresis caused a significant hyperpolarization of basolateral membrane potential (Vcs) and a significant increase in the ratio of apical to basolateral membrane resistances (Ra/Rb). When bladders were exposed to amiloride in the mucosal solution, Na+ iontophoresis caused the basolateral membrane to hyperpolarize, but no significant changes were observed in Ra/Rb. When Na+ was injected in the presence of serosal ouabain, Vcs depolarized and Ra/Rb increased. K+ iontophoresis caused the basolateral membrane potential to hyperpolarize in the presence of ouabain but Ra/Rb did not change significantly. These results indicate that the Na+ pump in toad bladder is rheogenic, that apical Na+ conductance is sensitive to the cell levels of Na+ and K+ and that the basolateral membrane is K+ permeable.

The electrical properties of the transepithelial pathways of Necturaus gallbladder were studied with intracellular microelectrode techniques under control conditions and after exposure to hyperosmotic mucosal bathing media (addition of sucrose or other solutes). Doubling mucosal osmolality produces a large increase in the resistance of the intercellular shunt pathway (from 420 +/- 50 to 700 +/- 70 omega - cm2, P is less than 0.001) and a significant decrease in the resistance of the apical membrane of the cells (from 3,510 +/- 420 to 1,540 +/- 380 omega p cm2, P is less than 0.001). The basolateral membrane resistance remains unchanged. Both the apical and basolateral membranes depolarize (from 59.5 +/- 1.8 to 36.6 +/- 2.8 mV, P less than 0.0001, and from 61.8 +/- 1.7 to 32.2 +/- 3.0 mV,P is less than 0.001, respectively). The transepithelial diffusion potential resulting from NaCl concentration gradients due to the osmotic water flow does not explain the cell potential changes. Mucosal solution ion substitution experiments demonstrate increases of gk, gcl, and gna (ca. 2.5-fold, 4-fold, and 7-fold, respectively). Therefore, cell depolarization appears to be produced mainly by a reduction of apical membrane K permselectivity. The increase in the resistance of the shunt is attributable to reduction of the width of the lateral intercellular spaces, because of the osmotic serosa-to-mucosa water flow.

Phenytoin is a highly effective anticonvulsant medication that is considered to be the treatment of choice for generalized major motor and focal epileptic seizures. An understanding of the pharmacokinetic properties of phenytoin greatly facilitates the management of seizure patients. The judicious use of serum-monitoring techniques coupled with careful clinical evaluations enable one to obtain the optimal anticonvulsant effect, freedom from dose-related toxicity, and the avoidance of unnecessary polypharmacy.

In Necturus gallbladder epithelium, elevation of mucosal K+ to 95 mM in the presence of 10 mM Na+ resulted in cell swelling at a rate of 3.2% original volume per minute, followed by volume-regulatory shrinking. When Na+ was completely removed from or when amiloride (10(-4) M) was added to the mucosal medium, K+-induced cell swelling was abolished. In the presence of 10 mM Na+, 1 mM Ba2+ abolished and substitution of mucosal Cl- by NO-3 had no effect on K+-induced swelling. Thus solute entry following elevation of mucosal K+ is effected by separate K+ and Cl- pathways. Furthermore, substitution of 95 mM K+ for Na+ in the mucosal bathing medium leads to the development of a Cl- conductance in the basolateral membrane as long as some Na+ remains in the medium. However, cell swelling induced by mucosal dilution does not lead to the appearance of a Cl- conductance. Thus the activation of this conductance requires both swelling and membrane depolarization. These results show that 1) high mucosal K+ leads to cell swelling due to the entry of Cl- along with K+ and the Cl- can enter across either membrane, 2) the Cl- pathways require the presence of mucosal Na+, and 3) cell volume regulation is activated by an increase in volume per se, i.e., a hyposmotic exposure is not required for volume regulation to occur.

Intragastric pH-metry was utilized to assess the effect of the time of meal ingestion and ranitidine administration on 24-h intragastric acidity. Twelve volunteers with a documented history of duodenal ulcer were studied in a four-way crossover design. Subjects randomly received ranitidine at 18.00 and 22.00 hours, with and without food. Serial blood samples were collected and analysed for ranitidine by high pressure liquid chromatography. Over the interval of 18.00-0.700 hours, the mean hydrogen-ion activity was significantly lower with the 18.00 hour dose than with the 22.00 hour dose (P less than or equal to 0.05). There were no differences between the four treatments in median pH or mean hydrogen-ion activity over the 23-h study interval. There were no differences between treatments in peak ranitidine concentrations, time to peak concentration, area under the serum-concentration time curve or elimination half-life.

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.

In toad and frog urinary bladder, electrophysiological data suggest that inhibition of transepithelial sodium transport by mucosal amiloride results in a decrease in basolateral membrane conductance. These results were confirmed by showing that amiloride addition caused a decrease in basolateral membrane potassium permeability.

Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

The electrical resistances of the transcellular and paracellular pathways across the toad urinary bladder epithelium (a typical "tight" sodium-transporting epithelium) were determined by two independent sets of electrophysiological measurements: (a) the measurement of the total transepithelial resistance, the ratio of resistance of the apical to the basal cell membrane, and cable analysis of the voltage spread into the epithelium; (b) the measurement of the total transepithelial resistance and the ratio of resistances of both cell membranes before and after replacing all mucosal sodium with potassium (thus, increasing selectively the resistance of the apical membrane). The results obtained with both methods indicate the presence of a finite transepithelial shunt pathway, whose resistance is about 1.8 times the resistance of the transcellular pathway. Appropriate calculations show that the resistance of the shunt pathway is almost exclusively determined by the zonula occludens section of the limiting junctions. The mean resistance of the apical cell membrane is 1.7 times that of the basal cell membrane. The use of nonconducting materials on the mucosal side allowed us to demonstrate that apparently all epithelial cells are electrically coupled, with a mean space constant of 460 microm, and a voltage spread consistent with a thin sheet model.

Conventional microelectrode and tracer flux techniques were used to study the effects of reduction in serosal chloride concentration ([Cl]s) on the electrical properties of toad urinary bladder epithelium. Reduction in [Cl]s resulted in a transient change in transepithelial potential (Vms) (and of apical and basolateral membrane potentials) that was inversely dependent on the base-line values of those potentials. In all cases, however, there was a decrease in transepithelial resistance (Rt) that was explained by an increase in the sodium conductance of the apical membrane. In tissues in which the transepithelial potential increased, there was a rise in the active mucosal-to-serosal sodium flux. The increase in conductance was directly related to the increase in short-circuit current. The changes in Vms and Rt brought about by reduction in [Cl]s were prevented by agents known to modify sodium transport, including low mucosal sodium concentration, addition of amiloride or amphotericin B to the mucosal solution, or of ouabain to the serosal solution. The results are best explained by a primary effect of chloride reduction on sodium extrusion across the basolateral membrane, with a secondary increase in apical sodium conductance. In addition, the data provide new evidence for the existence of a basolateral chloride conductance pathway.

Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that permitted rapid changes in the ion composition of the serosal solution. The transepithelial electrical properties exhibited a marked seasonal variation that could be attributed to variations in the conductance of the shunt pathway, apical membrane selectivity, and basolateral Na+ transport. In contrast, the passive electrical properties of the basolateral membrane remained constant throughout the year. The apparent transference numbers (Ti) of the basolateral membrane for K+ and Cl- were determined from the effect on the basolateral membrane equivalent electromotive force of a sudden increase in the serosal K+ concentration from 2.5 to 50 mM/liter or a decrease in the Cl- concentration from 101 to 10 mM/liter. TK and TCl were 0.71 +/- 0.05 and 0.04 +/- 0.01, respectively. The basolateral K+ conductance could be blocked by Ba2+ (0.5 mM), Cs+ (10 mM), or Rb+ (10 mM), but was unaffected by 3,4-diaminopyridine (100 microM), decamethonium (100 microM), or tetraethylammonium (10 mM). We conclude that a highly selective K+ conductance dominates the electrical properties of the basolateral membrane and that this conductance is different from those found in nerve and muscle membranes.

A model for improving physician prescribing that utilizes computerized feedback was studied in a family medicine residency practice. Resident and faculty physicians were stratified by level of experience and randomized into two groups. For 9 months the experimental group received monthly printouts identifying drugs they had prescribed by brand name with estimates of cost savings that might have been realized by prescribing generic drugs. The control group received no feedback. Prescription monitoring of both groups continued for 12 months after all feedback had ceased. Median weighted rates of generic prescribing for the experimental physicians were 14% for the baseline, 67% for the feedback, and 54% for the follow-up periods. Rates for the control physicians for the three periods were 32%, 37% and 31%, respectively. The increase in generic prescribing by physicians in the experimental group was significantly greater than for control physicians (P = 0.01). The feedback model improved rates of generic prescribing but should be evaluated for broader areas of physician prescribing.

Microelectrode techniques were employed to study the ionic permeability of the apical cell membrane of Necturus gallbladder epithelium. Results obtained from continuous records in single cells, and from several cellular impalements shortly after a change in solution, were similar and indicate that both the apical membrane equivalent electromotive force (Va) and electrical resistance (Ra) strongly depend on external [K]. Cl substitutions produced smaller effects, while the effects of Na substitutions with N-methyl-D-glucamine on both Va and Ra were minimal. These results indicate that the permeability sequence of the apical membrane is PKgreater thanPClgreater than PNa. From the calculated absolute value of PNa it is possible to estimate the diffusional Na flux from the mucosal solution into the cells (from the cell potential and an assumed intracellular Na concentration). The calculated flux is roughly three orders of magnitude smaller than the measured net transepithelial flux in this tissue and in gallbladders of other species. Thus, only a minimal portion of Na entry can be attributed to independent diffusion. From estimations of the electrochemical potential gradient across the apical membrane, Cl transport at that site must be active. At the serosal cell membrane, Na transport takes place against both chemical and electrical potentials, while a significant portion of the Cl flux can be passive, if this membrane has a significant Cl conductance. The changes in shunt electromotive force and in transepithelial potential after mucosal substitutions were very similar, indicating that transepithelial bi-ionic potentials yield appropriate results on the properties of shunt pathway.

We have studied the problem of cell volume homeostasis in toad and frog urinary bladder by using electrophysiological measurements and an optical measure of cell volume. After osmotically induced swelling, urinary bladder cells spontaneously regulate their volume through a net loss of potassium, chloride, and water. During inhibition of sodium transport by amiloride the cells swell to the same extent as controls, but the volume-regulatory process is blocked. Electrophysiological results under isosmotic conditions indicate that basolateral membrane resistance increases simultaneously with the amiloride-induced rise in apical membrane resistance during transport inhibition. These independent observations indicate that inhibition of apical membrane sodium entry results in a secondary decrease in basolateral membrane potassium permeability. When cells are exposed to calcium-free, hyposmotic Ringer's solution, cell volume regulation is blocked; subsequent addition of the calcium ionophore A23187 is ineffective in restoring the regulatory process. The ionophore does induce volume regulation, however, in amiloride-inhibited, osmotically swollen cells in the presence of external calcium. Calcium thus seems to control basolateral membrane potassium permeability and may be the intracellular mediator of apical and basolateral membrane interactions.

Microelectrode techniques were employed to study the mechanisms of the transepithelial voltage transients (deltaVms) observed during transmural current clamps in the isolated Necturus gallbladder. The results indicate that: a) part of deltaVms is due to a transepithelial resistance change (deltaRt), and part to a tissue emf change. b) deltaRt is entirely caused by changes of the resistance of the paracellular pathway. At all current densities employed, the measured changes are probably due to changes in both fluid conductivity and width of the lateral intercellular spaces. At high currents, in addition to the effects on the lateral spaces, the resistance of other elements of the pathway (probably the limiting junction) drops, regardless of the direction of the current. c) The magnitude and polarity of the deltaRt-independent transepithelial and cell membrane potential transients indicate that the largest emf change takes place at the basolateral membrane (deltaEb), with smaller changes at the luminal membrane (deltaEa) and the paracellular (shunt) pathway (deltaEs). It is shown that two-thirds of the transient are caused by deltaEs, and one-third by delta(Eb--Ea). deltaEs can be explained by a diffusion potential generated by a current-dependent NaCl concentration gradient across the tissue. deltaEa and deltaEb are caused by [K] changes, mainly at the unstirred layer in contact with the basolateral membrane.

1. The potential profile and the cellular and paracellular transepithelial resistances of the toad urinary bladder were measured, by means of micro-electrode techniques, as functions of the osmolality of the serosal solution. 2. Reductions in serosal osmolality (that increase the rate of active sodium transport) produced proportional decreases in the electrical resistances of the apical and basal-lateral cell membranes, while the changes in resistance of the paracellular pathway were more complex. The apical membrane potential increased. 3. Increases in serosal osmolality (that decrease sodium transport) produced increases in the electrical resistances of both cell membranes, and moderate reduction in the paracellular resistance. The polarity of the apical membrane potential reversed. 4. These results indicate that reductions in serosal solution osmolality stimulate sodium transport by increasing both the sodium permeability of the luminal cell membrane (thus increasing sodium entry), and the electromotive force generated at the serosal border of the cell, thus enhancing the rate of sodium pumping. Conversely, increases in osmolality reduced sodium transport by reducing both the sodium permeability of the luminal membrane and the serosal membrane electromotive force.