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After traumatic brain injury (TBI), large amounts of red blood cells and hemolytic products are deposited intracranially creating debris in the cerebrospinal fluid (CSF). This debris, which includes heme and bilirubin, is cleared via the arachnoid granulations and lymphatic systems. However, the mechanisms by which erythrocytes and their breakdown products interfere with normal CSF dynamics remain poorly defined. The purpose of this study was to model in vitro how blood breakdown products affect arachnoid cells at the CSF-blood barrier, and the extent to which the resorption of CSF into the venous drainage system is mechanically impaired following TBI. Arachnoid cells were grown to confluency on permeable membranes. Rates of growth and apoptosis were measured in the presence of blood and lysed blood, changes in transepithelial electrical resistance (TEER) was measured in the presence of blood and hemoglobin, and small molecule permeability was determined in the presence of blood, lysed blood, bilirubin, and biliverdin. These results were directly compared with an established rat brain endothelial cell line (RBEC4) co-cultured with rat brain astrocytes. We found that arachnoid cells grown in the presence of whole or lysed erythrocytes had significantly slower growth rates than controls. Bilirubin and biliverdin, despite their low solubilities, altered the paracellular transport of arachnoid cells more than the acute blood breakdown components of whole and lysed blood. Mannitol permeability was up to four times higher in biliverdin treatments than controls, and arachnoid membranes demonstrated significantly decreased small molecule permeabilities in the presence of whole and lysed blood. We conclude that short-term (<24 h) arachnoid cell transport and long-term (>5 days) arachnoid cell viability are affected by blood and blood breakdown products, with important consequences for CSF flow and blood clearance after TBI.

Introduction: This study compares the performance of two neutral oral contrast agents in CT enterography (CTE). Mannitol 2.5%, an oral osmotic agent, is compared with psyllium fibre (Metamucil). Both these agents are commonly used, but to our knowledge, they have not been compared in CTE., Methods: CTE data were collected from 25 consecutive studies for both mannitol and psyllium fibre between 2011 and 2013. All images were reviewed by two radiologists and one registrar blinded to the oral contrast used. Each quadrant was assessed for maximum distension, proportion of bowel loops distended, presence of inhomogeneous content and bowel wall visibility. Overall subjective quality and whether the contrast agent reached the caecum were also assessed. Patients were invited to answer a questionnaire regarding tolerability of the preparations., Results: Wall visibility was rated good in 100% of the mannitol studies, compared with 71% of the psyllium fibre studies, in the right lower quadrant (P = 0.01). No statistically significant difference between groups was observed in either maximal distension or proportion of loops distended in any quadrant. Inhomogeneous material was observed in 12% of the mannitol cases and 86% of the psyllium fibre cases (P < 0.0001). In all mannitol cases, the contrast reached the caecum, compared with 50% of psyllium fibre cases (P < 0.0001), and 36% of the mannitol studies were considered excellent, compared with 20% of the psyllium fibre studies (P = 0.03)., Conclusion: Mannitol achieves studies of better quality and is now the preferred oral contrast for CTE studies at Auckland City Hospital., (© 2014 The Royal Australian and New Zealand College of Radiologists.)

Introduction: Brain oedema is a major complication after craniotomy. Hyperosmolar agents have been used as the medical treatment for this condition. Measurement and estimation of serum osmolality during hyperosmolar agent infusion is of clinical importance to evaluate clinical efficacy, adjust dosage and avoid side effects. However, several studies have shown that calculated serum osmolality may lead to a systematic bias compared with direct measurement. In the present study, mannitol or hypertonic saline (HS) will be used in patients after elective craniotomy. We aim to determine the accuracy of serum osmolality estimation during the application of hyperosmolar agent., Methods and Analysis: The study is a prospective, randomised, double-blinded, controlled, parallel-group design. Adult patients requiring the use of hyperosmolar agents for the prevention or treatment of postoperative brain oedema are enrolled and assigned randomly to one of the two treatment study groups, labelled as 'M group' and 'HS group'. Patients in the M and HS groups receive intravenous infusion of 125 mL of either 20% mannitol or 3.1% sodium chloride solution, respectively. Data will be collected immediately before the infusion of study agents, 15, 30, 60, 120, 240 and 360 min after the start of infusion of experimental agents, which includes serum osmolality, concentration of serum sodium, potassium, urea and glucose. Serum osmolality will be measured by means of freezing point depression. Estimated serum osmolality will also be calculated by using four formulas published previously. Osmole gap is calculated as the difference between the measured and the estimated values. The primary endpoint is the correlation of measured and estimated serum osmolality during hyperosmolar agent infusion., Ethics and Dissemination: The study was approved by the International Review Board (IRB) of Beijing Tiantan Hospital, Capital Medical University. Study findings will be disseminated through peer-reviewed publications and conference presentations., Trial Registration Number: ClinicalTrials.gov identifier: NCT02037815.

Peritoneal dialysis (PD) solutions dilate microvessels by undefined mechanisms. This vasodilation directly affects ultrafiltration and solute exchange during a PD dwell and is thought to account for the variable mass transfer area coefficient for small solutes during a glucose-based hypertonic dwell. We hypothesized that PD-mediated vasodilation occurs by endothelium-dependent mechanisms that involve endothelium energy-dependent K+ channels (K(ATP)), adenosine A1 receptor activation, and NO release. We used intravital videomicroscopy to study 3 levels of microvessels (A1 inflow arterioles about 100 microm diameter to pre-capillary A3 arterioles 10 - 15 microm diameter) in the terminal ileum of anesthetized rats under control conditions in vivo in a tissue bath. Ileum was bathed with hypertonic mannitol or 2.5% glucose-based PD solution (Delflex: Fresenius Medical Care North America, Waltham, MA, U.S.A.) with or without topical application of individual or combined specific inhibitors of the endothelium-dependent dilation pathways.: NO (L-NMMA), prostaglandin I2 (mefenamic acid), endothelium hyperpolarizing factor (glibenclamide), and adenosine A1 receptor antagonist (DPCPX). The mannitol and PD solutions induced rapid and sustained peritoneal vasodilation whose magnitude depended on microvascular level and osmotic solute. Combined inhibition of endothelium-dependent dilation pathways completely abolished the mannitol-induced hyperosmolality-mediated dilation at all microvascular levels, but selectively eliminated the PD solution-mediated A3 dilation. The K(ATP) and adenosine receptor antagonists, individually or combined, remarkably attenuated dilation in the smaller pre-capillary arterioles; NO inhibition, alone or combined with K(ATP) and adenosine receptor antagonists, eliminated the PD solution-induced dilation. The cyclooxygenase pathway is not involved in PD-induced dilation. Solutions for PD dilate the visceral peritoneal microvasculature by endothelium-dependent mechanisms, primarily the NO pathway. Adenosine receptor-activated NO release and K(ATP) channel-mediated endothelium hyperpolarization significantly contribute to vasodilation in the smaller peritoneal pre-capillary arterioles.

The blood-brain barrier (BBB) restricts the delivery of drugs into the brain. Different strategies have been developed to circumvent this obstacle. One such approach, the osmotic BBB disruption (BBBD), has been under pre-clinical study since the 70's. Typically, qualitative ex vivo assessment of the extent of BBBD has been performed using Evan's blue staining technique. In this study, we describe a simple quantitative technique based on albumin indirect immunohistochemistry to measure the extent of BBB breach. Thirty Fischer rats were assigned to one of 6 groups: a control group, and BBBD groups with escalation in IA mannitol infusion rate: 0.06, 0.08, 0.10, 0.12 and 0.15 cc/s. Fifteen minutes after the BBBD procedure, the animals were sacrificed, brain harvested and sections stained for albumin. Using an image analysis software, isolated albumin staining pixels were expressed as a fraction of the treated hemisphere. This ratio was used as a percentage value in the intensity of the BBB permeabilization. All sections studied harbored staining, averaging 0.37% for the controls (group 1), 5.69% for group 2 (0.06 cc/s), 10.44% for group 3 (0.08 cc/s), 6.99% for group 4 (0.1 cc/s), 18.50% for group 5 (0.12 cc/s) and reaching 61.70% for group 6 (0.15 cc/s). Important variations were observed between animals. A threshold effect was observed, and animals in group 6 presented a significant increase in BBB permeabilization compared to the other groups. We hereby detail a simple technique that can be applied to quantitatively measure the extent of the BBB breach notwithstanding the pathological process., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To evaluate intravenous mannitol during minimally invasive partial nephrectomy (PN) by comparing the renal function outcomes of the patients who received it versus those who did not., Methods: Of 285 consecutive elective minimally invasive PN cases from February 2005 to July 2010, 164 patients (58%) were treated with mannitol. We compared the renal function recovery using a multivariate generalized estimating equation linear model of estimated glomerular filtration rate (eGFR) controlling for nephrometry complexity, preoperative eGFR, American Society of Anesthesiologists score, ischemia time, estimated blood loss, age, and sex. Sensitivity analyses were performed to adjust for cold ischemia and individual surgeon differences corrected for year of surgery., Results: Of the 285 patients who underwent minimally invasive treatment, 164 received mannitol and 121 did not. Those who received mannitol had a better preoperative eGFR (median 72 vs 69 mL/min/m(2), P = .046), less complex nephrometry scores (P = 0.051), and were less likely to have an American Society of Anesthesiologists score of ≥ 3 (42% vs 54%, P = .005). Renal function recovery was similar in both groups (estimated effect of mannitol -0.7 mL/min/m(2), 95% confidence interval -3.6-2.2, P = .6). At no point in the postoperative period did mannitol make a significant difference in the eGFR according to the generalized estimating equation model after adjusting for multiple potential renal function confounders., Conclusion: Mannitol use did not influence renal function recovery within 6 months of minimally invasive PN as measured by the eGFR in our analysis. An appropriately designed prospective study of mannitol is being conducted to validate its use during PN., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency. All diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells. This movement of sodium into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na-K cotransporter on the basolateral side of the cell. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To better understand mannitol pharmacokinetics, the authors constructed and compared population models for high-versus low-dose bolus infusions in humans. Patients (aged 18-75, American Society of Anesthesiologists physical status 1-3) scheduled for elective craniotomy with an anticipated need for intraoperative mannitol were randomly assigned to receive either 0.5 (n = 10) or 1.0 (n = 12) g/kg of 20% mannitol over 15 minutes. Serial blood samples were collected at the predetermined intervals over 12 hours. Plasma mannitol concentrations were measured by gas chromatography and subjected to pharmacokinetic analysis; a 3-compartment model best described mannitol disposition characteristics. Weight and dose were the important covariates for rapid peripheral volume of distribution (V2) and central clearance (CL1), respectively. Estimated population means were 2.80, 8.86, and 12.0 L for central (V1), rapid (V2), and slow (V3) volumes of distribution, respectively. Clearances of the central compartments (CL1) were 0.07 versus 0.04 L/min in the high-versus low-dose group, respectively. Thus, mannitol kinetics can be considered as nonlinear. Clearances of the rapid peripheral (CL2) and slow peripheral compartments (CL3) were identical (2.07 and 0.16 L/min) in both. The current weight-based dosing guidelines yielded greater than expected plasma drug concentrations in obese patients.

Mannitol is widely considered the hyperosmolar therapy of choice in routine neurosurgical practice for the reduction of intracranial pressure (ICP). The authors present a unique case of a patient with a large meningioma treated with mannitol, in which mannitol accumulation within the tumor and its surrounding parenchyma was shown using in vivo magnetic resonance spectroscopy (MRS). This rare appearance of mannitol on MRS was characterized by a wide-based peak at 3.8 ppm, which remained detectable several hours after the last dose. These findings provide the first in vivo evidence in support of the prevailing theory that mannitol leakage into the peritumoral edematous region may contribute to rebound increases in ICP and suggest that this phenomenon has the potential to occur in extraaxial tumors. Judicious use of mannitol in the setting of elevated ICP due to tumor may be indicated to avoid potentially deleterious side effects caused by its accumulation.

An understanding of the cellular physiology of cardiac myocytes (MCs) and non-myocytes (NMCs) may help to explain the mechanisms underlying cardiac hypertrophy. Despite numerous studies using MC/NMC co-culture systems, it is difficult to precisely evaluate the influence of each cell type because of the inherent cellular heterogeneity of such a system. Here we developed a co-culture system using Wistar rat neonatal MCs and NMCs isolated by discontinuous Percoll gradient and adhesion separation methods and cultured on either side of insert well membranes. Co-culture of MCs and NMCs resulted in significant increases in [3H]-leucine incorporation by MCs, in the amount of protein synthesized by MCs, and in the secretion of natriuretic peptides, while the addition of MCs to NMC cultures significantly reduced [3H]-thymidine incorporation by NMCs. Interestingly, the percentage of the brain natriuretic peptide (BNP) component of total natriuretic peptide secreted (atrial natriuretic peptide+BNP) increased as the number of NMCs placed in the MC/NMC co-culture system increased. However, MCs did not affect production of angiotensin II (Ang II) by NMCs or secretion of endothelin-1 and transforming growth factor-beta1 into the MC/NMC co-culture system. This finding was supported by the anti-hypertrophic and anti-fibrotic actions of RNH6270, an active form of olmesartan, on MCs in the MC/NMC co-culture system and on NMCs that may synthesize Ang II in the heart. The present data indicate that cardiac fibrosis may not only facilitate MC hypertrophy (possibly through the local angiotensin system) but may also change particular pathophysiological properties of MCs, such as the secretory pattern of natriuretic peptides.

The osmodiuretic mannitol can be potentially misused in sports, owing to its urine diluting effect and the possibility to decrease bodyweight. To reveal a doping offence, resulting urinary mannitol concentrations after a prohibited intravenous application and a permitted oral intake have to be differentiated. Therefore, a reliable gas chromatography-mass spectrometry (GC-MS) method was established based on peracetyl derivatives of the analytes. All possible hexitols (allitol, galactitol, iditol, altritols, sorbitol and mannitol) that can occur in human urine were separated and identified on a phenyl-methylpolysiloxane column (HP-5MS) within 10.75 min, and the method demonstrated its capability for quantification purposes. The lower limit of detection and lower limit of quantification were estimated at 0.9 microg mL(-1) and 2.4 microg mL(-1), respectively, and the assay was validated for mannitol and sorbitol regarding the parameters specificity, linearity, intra- (<10%) and inter-day precision (<15%) and accuracy (92-102%). To investigate urinary mannitol concentrations after oral intake the method was applied to an excretion study, providing a mean urinary excretion of mannitol of 19.5%. Comparison of theoretically expected urinary levels after a common therapeutic dose of mannitol and preliminary results on physiological urinary mannitol levels were promising, regarding a threshold level for mannitol that can be utilised for doping control purposes.

Aim: To determine if small bowel involvement at diagnosis could predict early relapse in children with ulcerative colitis., Methods: Children with newly diagnosed ulcerative colitis were evaluated prospectively at three time points: within 1 month, 6 months and 1 year after diagnosis. Clinical activity indices were used to measure disease activity. Laboratory studies were performed at each visit and/or at the time of relapse. At diagnosis, all patients underwent colonoscopy and a cellobiose/mannitol small intestinal permeability study. Some children were further investigated with an upper gastrointestinal endoscopy., Results: Thirty-three patients completed the 1-year study. Overall, nine patients (27.3%) relapsed within 6 months of diagnosis, one patient (3%) within 1 year, whereas 23 patients (69.7%) did not relapse. The mean clinical activity indices, laboratory parameters, extent of colonic involvement, upper and lower gastrointestinal histological features were not predictive of early relapse. Results of the cellobiose/mannitol small intestinal permeability study were significantly higher in children who relapsed within 6 months compared with children who did not relapse (P < 0.013). The cellobiose/mannitol small intestinal permeability study was abnormal in 77.8% of early relapsers compared with only 8.3% of non-relapsers., Conclusion: Abnormal small intestinal permeability in children with ulcerative colitis could predict a more relapsing disease.

Intestinal glucose and amino acid transport is stimulated by the serum- and glucocorticoid-inducible kinase isoforms SGK1, SGK2, and SGK3 and protein kinase B which are, in turn, stimulated following activation of the phosphoinositol-3 kinase (PI3 kinase). The present study has been performed to explore whether pharmacological inhibition of the PI3 kinase affects electrogenic jejunal transport of glucose and amino acids. In Ussing chamber experiments, glucose (20 mM), phenylalanine (20 mM), glutamine (20 mM), cysteine (20 mM), and proline (20 mM) generated lumen negative currents (I (glc), I (phe), I (gln), I (cys), and I (pro)), respectively, which gradually declined following application of the PI3 kinase inhibitor Wortmannin (1 muM). Within 40 min, Wortmannin treatment significantly decreased I (glc) by 39 +/- 10% (n = 5), I (phe) by 70 +/- 7% (n = 4), I (gln) by 69 +/- 8% (n = 4), I (cys) by 67 +/- 8% (n = 6), and I (prol) by 79 +/- 12% (n = 3). A similar decline of I (glc) was observed following application of the PI3 kinase inhibitor LY294002 (50 microM). Exposure to the inhibitors did not significantly alter transepithelial potential difference and resistance in the absence of substrates for electrogenic transport. The observations suggest that the electrogenic transport of glucose and several amino acids requires the continued activity of PI3 kinase.

To address the hypothesis that functional changes in tissue transport can be related to structural alterations, we combined mathematical modeling with in vivo experimentation. The model concept includes interstitial diffusion and removal by a distributed microvasculature. Transport of solute and water across the peritoneum is measured via a plastic chamber affixed to the abdominal wall of anesthetized Sprague-Dawley rats. Solutions containing [(14)C]mannitol, with or without vasoactive compounds [control (C; n = 10), C + nitroprusside (NP; n = 10), C + norepinephrine (NE; n = 10)], were infused into the chamber, and the volume and tracer concentrations were determined over 60 min to calculate the mass transfer coefficient (MTC) and the water flux. At 60 min, FITC-dextran (500 kDa) was given to mark the perfused vasculature. After euthanasia, the tissue under the chamber was frozen, dried, sliced with a cryomicrotome, and examined with fluorescent microscopy and quantitative autoradiography. The microvessel density (x10(3)/cm(2): NE, 50 +/- 10; C, 180 +/- 7.0; NP, 225 +/- 15) resulted in marked differences (P < 0.05) in water flux (mul.min(-1).cm(-2): NE, 0.1 +/- 0.1; C, 1.6 +/- 0.4; NP, 1.0 +/- 0.2) and in mannitol MTC (x10(3) cm/min: NE, 0.9 +/- 0.3; C, 3.8 +/- 0.3; NP, 3.6 +/- 0.6). Concentration profiles and calculated capillary permeability and tissue diffusivity were significantly different among the groups. These results demonstrate a direct correlation of mass transfer, diffusion, capillary permeability, and water flux with peritoneal vascular density and validate a method by which mechanistic changes in transport may be measured.

Brain hypothermia treatment (BHT) is an intensive care characterized by simultaneous managements of various vital signs, such as intracranial temperature (ICT) and pressure (ICP), of the severe neuropatient. Medical treatments including therapeutic ambient cooling and diuresis are separately carried out based on the experience of the medical staff involved in the clinical management of various pathophysiological processes, such as thermodynamics, hemodynamics and pharmacokinetics. However, no special attention has been paid to the interactions among these subsystems in therapeutic hypothermia because of the lack of theoretical knowledge. Therefore, quantitative analyses using an integrated model of various physiological processes and their interactions are of pressing need. In the present paper, we propose a general compartmental model to describe the pathophysiological processes of the three aforementioned dynamics, on account of the dynamical analogy of temperature, pressure and concentration. The model is verified by the agreement of model-based simulation results with clinical evidence. Based on responses of the integrated model to various stimuli, a transfer function matrix is identified to linearly approximate the characteristic interrelationships between medical treatments (ambient cooling and diuresis) and the vital signs (ICT and ICP). Then a controller that decouples ambient cooling and diuresis is proposed for efficient management of ICT and ICP, enhancement of hypothermic decompression and reduction of diuretic dosage. Decoupling control simulation indicates that ICT and ICP of the integrated model, representing a patient under BHT, can be simultaneously regulated by a single PID controller for ambient cooling and another for diuresis. The proposed decoupler effectively establishes hypothermic decompression, reduces the dosage of diuretic and improves ICP management. Theoretical analyses of the integrated model and decoupling control of ICT and ICP provide insights into the intensive care of various pathophysiological processes in patients undergoing BHT.

Aim: Widespread use of mannitol to reduce brain edema and lower elevated ICP in brain tumor patients continues to be afflicted by the so-called rebound phenomenon. Leakage of mannitol into the brain parenchyma through an altered BBB and secondary reversal of osmotic gradient is considered the major cause of rebound . This has only been demonstrated experimentally in animals. As a contribution to this issue we decided to research the possible passage of mannitol into the brain after administration to 21 brain tumor patients., Methods: Mannitol (18% solution; 1 g/kg) was administered as a bolus to patients (ten had malignant glioma, seven brain metastases and four meningioma) about 30 minutes before craniotomy. During resection, a sample of the surrounding edematous white matter was taken at the same time as a 10 ml venous blood sample. Mannitol concentrations were measured in plasma and white matter by a modified version of the enzyme assay of Blonquist et al., Results: In most glioma patients, mannitol concentrations in white matter were 2 to 6 times higher than in plasma (mean 3.5 times). In meningioma and metastases patients plasma concentrations of mannitol were higher than white matter concentrations except in three cases with infiltration by neoplastic cells., Conclusions: The results of our study show that even after a single bolus, mannitol may leak through the altered BBB near gliomas, reversing the initial plasma-to-blood osmotic gradient, aggravating peritumoral edema and promoting rebound of ICP.

Determining the time of brain death is one of the critical issues in forensic examinations. Few authors have attempted to determine the time of brain death using pharmacokinetic approaches. We investigated cerebral concentrations of mannitol of which a single dose (1 g/kg) was administered in the course of brain death. The inflation of an epidural balloon was adopted as a rodent model of brain death. Brain death was determined using ordinary tests. Specimens were collected 4 h after brain death. Brain water content was higher in brain dead (BD) groups than those in control groups. Cerebral concentrations of mannitol in the BD group were significantly higher than those in the control group (P<0.01). In all areas of brain the concentration was the highest at the time when mannitol was administered during balloon inflation. Interhemispheric difference in the cerebrum was observed, followed by balloon inflation (P<0.05). Significant differences were observed in the average concentration of administered mannitol before and after brain death in the contralateral hemisphere (P<0.01) and in the brainstem (P<0.01). As the concentrations of mannitol in the brain are affected by cerebral trauma and brain death follows, mannitol can be used to determine the time of brain death at forensic examinations.

This review examines the available data on the use of osmotic agents in patients with head injury and ischemic stroke, summarizes the physiological effects of osmotic agents, and presents the leading hypotheses regarding the mechanism by which they reduce ICP. Finally, it addresses the validity of the following commonly held beliefs: mannitol accumulates in injured brain; mannitol shrinks only normal brain and can increase midline shift; osmolality can be used to monitor mannitol administration; mannitol should be not be administered if osmolality is >320 mOsm; and hypertonic saline is equally effective as mannitol.

Malnutrition and absence of exogenous luminal nutrients in the gastrointestinal tract affect intestinal permeability (IP) leading to an increased penetration of substances that passively cross intestinal epithelium via intercellular pathways. We hypothesised that an increase in IP could occur in patients with anorexia nervosa because of their prolonged fasting and chronic malnutrition. Therefore, we assessed IP in 14 drug-free anorexic women and 19 drug-free age-matched healthy women by means of the lactulose/mannitol (LA/MA) test. To this purpose, after an overnight fast, subjects ingested an oral solution containing 5 g lactulose and 2 g mannitol in 100 ml water. Urine specimens were collected immediately before and 30, 60, 120, 180, 240 and 300 min after the ingestion of the sugar solution. Urinary lactulose and mannitol were determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection. We found that IP, as expressed by the 5-h LA/MA excretion ratio, was significantly decreased in anorexic women because of a lower urinary recovery of lactulose. Moreover, in patients, the time course of lactulose excretion significantly differs from healthy controls. These results do not confirm our hypothesis of increased IP in anorexia nervosa. Since IP reflects the anatomo-functional status of intestinal mucosa, the present findings support the idea that changes in the anatomo-physiology of intestinal mucosa occur in anorexia nervosa.

Butyrate enemas have been demonstrated to ameliorate inflammation in ulcerative colitis. The mechanism of this protective effect of butyrate is not known, and this study examines the effect of butyrate on epithelial function, inducible heat shock protein 70 (HSP70) expression, and NF-kappaB activation in experimental colitis. Colitis was induced in rats by oral dextran sulfate sodium (DSS) and by butyrate or saline enemas. Mucosal barrier function was assessed by electrical resistance and [14C]mannitol permeability. HSP70 production was determined by [35S]methionine labeling, Western blot analysis, and immunohistochemistry. Activation of heat shock factors (HSFs) and NF-kappaB was evaluated by EMSA. Butyrate showed a significant protection against the decrease in cell viability, increase in mucosal permeability, and polymorphonuclear neutrophil infiltration seen in DSS colitis. Butyrate inhibited HSP70 expression in DSS colitis and also inhibited the activation of HSF and NF-kappaB. Thus butyrate enema was found to be cytoprotective in DSS colitis, an effect partly mediated by suppressing activation of HSP70 and NF-kappaB.

Sonophoresis at a frequency of 20 kHz has been shown to enhance transdermal drug delivery, a phenomenon referred to as low-frequency sonophoresis. This study provides an investigation of the dependence of low-frequency sonophoresis on various ultrasound parameters, including the distance of the horn from the skin, intensity, and frequency. We performed in vitro experiments with full thickness pig skin to measure enhancements of skin conductivity and drug permeability. Ultrasound was applied to pretreat the skin using a sonicator operating at a frequency of either 20 or 40 kHz. We also measured pitting of aluminum foil to measure cavitation, which is the principal mechanism of low-frequency sonophoresis. The skin conductivity enhancement was found to be inversely proportional to the distance of the horn from the skin. As the intensity increased, skin conductivity enhancement also increased up to a certain threshold, and then dropped off. The intensities (I(max)) at which maximum enhancement occur are about 14 W/cm2 for 20 kHz and 17 W/cm2 for 40 kHz. These findings may be useful in optimizing low-frequency sonophoresis. Overall, the dependence of transport on ultrasound parameters is similar to that of aluminum foil pitting. These results support the role of cavitation in low-frequency sonophoresis.

Human tumor xenografts established in athymic rat brains were used to determine the feasibility of intravascular delivery of tumor suppressor genes to brain tumors. Both tumor size and number were compared to characterize the effect of tumor burden on tumor transduction efficacy by a control LacZ-containing adenoviral vector. Experiments with tumors grown in vivo for either 3, 5, or 7 days demonstrated that 5-day-old tumors provided the best target for vector infection and transgene expression by this mode of administration. Intra-arterial mannitol facilitated transduction efficiency. Tumor burden did not seem to affect transduction, while tumor location appeared to be an important factor. Based on these results, intra-arterial infusion of a p53-containing adenoviral vector was carried out and resulted in significant retardation of brain tumor growth 3 days after administration. Effects at longer time points were not as significant. These findings indicate that intra-arterial administration of adenoviral vectors containing p53 is efficient and can result in changes in tumor size, but that long-term control of tumor growth may require multiple adenoviral treatments.

The application of low-frequency ultrasound enhances drug transport through the skin, a phenomenon referred to as low-frequency sonophoresis. This enhancement is mediated through cavitation, the formation and collapse of gaseous bubbles. We hypothesized that the efficacy of low-frequency sonophoresis can be significantly enhanced by provision of nuclei for cavitation. In this study, we used two porous resins, Diaion HP20 and Diaion HP2MG (2MG), as cavitation nuclei. We measured the effect of these resins on cavitation using pitting of aluminum foil. 2MG showed a higher efficacy in enhancing cavitation compared with Diaion HP20. 2MG was also effective in enhancing transdermal mannitol transport. These results confirm that the addition of cavitation nuclei such as porous resins further increases the effect of low-frequency ultrasound on skin permeability., (Copyright 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.)

A method for simultaneous determination of glycerol and mannitol in various human tissues was devised and for this we used high-performance liquid chromatography (HPLC). Specimens were homogenized in a mixture of chloroform and methanol, phosphate buffer (pH 7.0) and pentaerythritol (IS) solution. After centrifugation, an aliquot of the aqueous layer was evaporated to dryness and derivatized with p-nitrobenzoyl chloride at 50 degrees C for 1h, then applied to HPLC with analytical conditions of: column, CAPCELL PAK C18 MG (250 mm x 3.0 mm i.d., 5 microm, Shiseido Co. Ltd., Tokyo, Japan); column temperature, 1-2 degrees C; mobile phase, 75% acetonitrile-distilled water containing 0.05% trifluoroacetic acid, 0.05% heptafluoro-n-butyric acid and 0.1% triethylamine; flow rate, 0.5 ml/min; wavelength, 260 nm. Calibration curves for both substances were linear in concentration ranges from 1 to 500 microg/0.1g and correlation coefficients exceeded 0.99. The relative standard deviation (R.S.D.) of the method was evaluated at concentrations of 10 and 100 microg/0.1g, and ranged from 0.84 to 10.6%. Using this method, we determined the regional distribution levels of glycerol and mannitol in various tissues from an autopsied brain dead man.

The brain and CSF distribution of the HIV reverse transcriptase inhibitor, 2'3'-dideoxycytidine (ddC), was investigated by the in situ brain perfusion and isolated incubated choroid plexus methods in the guinea pig. Multiple-time brain perfusions indicated that the distribution of [3H]ddC to the brain and CSF was low and the unidirectional rate constant (K(in)) for the brain uptake of this nucleoside analogue (0.52 +/- 0.10 microL/min/g) was not significantly different to that for the vascular marker, [14C]mannitol (0.44 +/- 0.09 microL/min/g). The influence of unlabelled ddC, six organic anion transport inhibitors and 3'-azido 3'-deoxythymidine (AZT) on the CNS uptake of [3H]ddC was examined in situ and in vitro. ddC, probenecid and 2,4-dichlorophenoxyacetic acid altered the distribution of [3H]ddC into the brain and choroid plexuses, indicating that the limited distribution of [3H]ddC was a result of an organic anion efflux transporter, in addition to the low lipophilicity of this drug (octanol-saline partition coefficient, 0.047 +/- 0.001). The CNS distribution was also sensitive to p-aminohippurate and deltorphin II, but not digoxin, suggesting the involvement of organic anion transporters (OAT1/OAT3-like) and organic anion transporting polypeptides (OATP1/OATPA-like). AZT did not effect the accumulation of [3H]ddC, indicating that when these nucleoside analogues are used in anti-HIV combination therapy, the CNS distribution of ddC is unchanged.

Five TMC polymers with different degrees of quaternisation (12-59%) were synthesised and administered together with [14C]-mannitol in the nasal route of rats at a pH of 6.20 and 7.40, respectively. All the TMC polymers increased the nasal absorption of [14C]-mannitol significantly at pH 6.20, but only TMC polymers with higher degrees of quaternisation (>36%) were able to increase the absorption of this hydrophilic model compound at pH 7.40. The absorption of [14C]-mannitol at pH 7.40 increased with an increase in the degree of quaternisation of TMC until a maximum absorption value was reached with TMC with a degree of quaternisation of 48%. The absorption of [14C]-mannitol did not increase further, even when TMC with a higher degree of quaternisation (59%) was used. This can probably be explained by steric effects caused by the attached methyl groups and changes in the flexibility of the TMC molecules with an increase in the degree of quaternisation above an optimum value for absorption enhancement in a neutral environment. It was concluded that the degree of quaternisation of TMC plays an important role in the absorption enhancement properties of this polymer across nasal epithelia in a neutral environment.

1. The barrier function of colonic epithelia is challenged by apoptotic loss of enterocytes. In monolayers of human colonic HT-29/B6 cells, apoptosis induced by camptothecin was assessed by poly-(ADP-ribose)-polymerase (PARP) cleavage, histone ELISA and DNA-specific fluorochrome staining (with 4',6'-diamidino-2'-phenylindoladihydrochloride (DAPI)). Epithelial barrier function was studied in Ussing chambers by measuring transepithelial conductivity and unidirectional tracer fluxes. The ion permeability associated with single cell apoptoses was investigated with the conductance scanning technique. 2. The spontaneous rate of apoptotic cells was 3.5 +/- 0.3 % with an overall epithelial conductivity of 3.2 +/- 0.1 mS cm(-2). Camptothecin induced a time- and dose-dependent increase of apoptosis and permeability. With 20 microg ml(-1) of camptothecin for 48 h, apoptosis increased 4.1-fold to 14.3 +/- 1.5 % and the conductivity doubled to 6.4 +/- 1.0 mS cm(-2). 3. While 3H-mannitol flux increased 3.8-fold and 3H-lactulose flux increased 2.6-fold, the flux of 3H-polyethylene glycol 4000 remained unchanged. Hence, the higher permeability was limited to molecules < 4000 Da. 4. The local epithelial conductivity was higher at the sites of apoptosis than in non-apoptotic areas. With camptothecin the leaks associated with apoptosis became more numerous and more conductive, while in non-apoptotic areas the conductivity remained at control level. Hence, the camptothecin-induced increase in epithelial conductivity reflected the opening of apoptotic leaks and thus the results described, for the first time, epithelial permeability as a function of apoptosis only. 5. The conductivity of apoptotic leaks contributed 5.5 % to the epithelial conductivity of controls and 60 % to the conductivity of monolayers treated with 20 microg ml(-1) of camptothecin. Thus apoptosis increased the contribution of paracellular pathways to the overall epithelial permeability. Under control conditions the paracellular conductivity (G(para)) was smaller than the transcellular (G(trans)), but with 12 % apoptosis, G(para) exceeded G(trans). By definition, the epithelium became 'leaky'.

Zonula occludens toxin (Zot), a protein elaborated from Vibrio cholerae, has been shown to be capable of reversibly opening tight junctions between intestinal cells The objective of this study was to examine the effect of Zot on the flux of various molecules across Caco-2 cell monolayers. In addition, the transport of a series of anticonvulsants, the enaminones was also evaluated in the presence of Zot. The flux of [(14)C]mannitol, [(14)C]inulin and various enaminones across Caco-2 cell monolayers (n=6) was examined after pre-incubation for 1h with Zot (0 or 4000ng/ml) or phosphate-buffered saline (PBS). At the end of the incubation period, the flux of radiolabeled compounds or enaminones (1x10(-4)M) was assessed over a 2-h period. In addition, dose-response studies with Zot (0, 1000, 2000 or 4000ng/ml) were performed using mannitol. The flux of both mannitol and inulin significantly increased (P<0.05) in the presence of Zot. The transport of the enaminones with Zot ranged from 9.42 to 26.83x10(-5)cm/s vs. 4.68 to 13.83x10(-5)cm/s without Zot. Zot significantly increased the transport of all agents tested. This suggests that the co-administration of drugs with Zot may be a useful delivery strategy to increase the intestinal permeability and hence oral absorption of poorly bioavailable agents.

1. The rabbit Na(+)-glucose cotransporter rbSGLT1 and its carboxy-terminal part, C5, which contains transmembrane helices 10-14 of SGLT1 and functions as a low affinity glucose uniporter, were expressed as individual proteins in Xenopus oocytes. Transport of 55 microM urea, ethylene glycol, mannitol and alpha-methyl-D-glucopyranoside (alphaMDG) by control oocytes and by oocytes expressing SGLT1 and C5 was studied by uptake measurements of the 14C-labelled substrates. 2. There was a 5- to 6-fold increase in urea transport mediated by C5, compared with control oocytes. Similar to SGLT1, the C5-urea uptake was cation independent, linear in time and with increasing urea concentration, and blocked with the same sensitivity by the inhibitor phloretin (K(i) approximately 1 mM). Like SGLT1 in choline buffer, the C5-mediated uptake was insensitive to phlorizin. 3. Mannitol was transported by C5 but not by SGLT1 or control oocytes. 4. The activation energy (E(a)) for urea transport through C5 was low (5 +/- 3 kcal mol(-1)) compared with that of non-injected oocytes (16 +/- 0.5 kcal mol(-1)) and comparable with the E(a) of passive urea or water transport through intact SGLT1. 5. The urea influx through C5 increased in the presence of alphaMDG, but not in the presence of the same concentration of mannitol. 6. We conclude that the five carboxy-terminal transmembrane helices of SGLT1 form a channel for the permeation of small molecules such as urea and water.

Skeletal muscle glucose uptake requires delivery of glucose to the sarcolemma, transport across the sarcolemma, and the irreversible phosphorylation of glucose by hexokinase (HK) inside the cell. Here, a novel method was used in the conscious rat to address the roles of these three steps in controlling the rate of glucose uptake in soleus, a muscle comprised of type I fibers, and two muscles comprised of type II fibers. Experiments were performed on conscious rats under basal conditions or during hyperinsulinemic euglycemic clamps. Rats received primed, constant infusions of 3-O-methyl-[3H]glucose (3-O-MG) and [1-14C]mannitol. Total muscle glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-MG concentration, which distributes based on the transsarcolemmal glucose gradient (TSGG), were used to calculate glucose concentrations at the inner and outer sarcolemmal surfaces ([G](im) and [G](om), respectively) in muscle. Muscle glucose uptake was much lower in muscle comprised of type II fibers than in soleus under both basal and insulin-stimulated conditions. Under all conditions, the TSGG in type II muscle exceeded that in soleus, indicating that glucose transport plays a more important role to limit glucose uptake in type II muscle. Although hyperinsulinemia increased [G](im) in soleus, indicating that phosphorylation was a limiting factor, type II muscle was limited primarily by glucose delivery and glucose transport. In conclusion, the relative importance of glucose delivery, transport, and phosphorylation in controlling the rate of insulin-stimulated muscle glucose uptake varies between muscle fiber types, with glucose delivery and transport being the primary limiting factors in type II muscle.

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprotein, gp60, was studied in cultured type II alveolar epithelial cells obtained from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 microg ml(-1)) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunostained for caveolin-1; however, lysosomes did not stain for caveolin-1. Filipin depleted the epithelial cells of the caveolin-1-positive, albumin-transporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-gp60 antibody produced loss of cell-surface gp60 and abolished endocytic albumin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetric rat lung preparation perfused at 37 degrees C. (125)I-labelled albumin was instilled into distal airspaces of lungs, and the resulting (125)I-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1)) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10(5) counts (instilled ml)(-1)) with an IC(50) value of 0.34 g (100 ml)(-1). 8. Filipin blocked the displacement-sensitive component of (125)I-labelled albumin transport, but had no effect on the transport of the paracellular tracer (3)[H]mannitol. 9. Displacement-sensitive (125)I-labelled albumin transport had a significantly greater Q(10) (27-37 degrees C) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive (125)I-labelled albumin transalveolar transport in intact rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system, the percentage of instilled (125)I-labelled albumin counts remaining in lung tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled albumin. Approximately 25 % of instilled (125)I-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pathway. This component was blocked by filipin.

Application of ultrasound enhances transdermal transport of drugs (sonophoresis). The enhancement may result from enhanced diffusion due to ultrasound-induced skin alteration and/or from forced convection. To understand the relative roles played by these two mechanisms in low-frequency sonophoresis (LFS, 20 kHz), a theory describing the transdermal transport of hydrophilic permeants in both the absence and the presence of ultrasound was developed using fundamental equations of membrane transport, hindered-transport theory, and electrochemistry principles. With mannitol as the model permeant, the role of convection in LFS was evaluated experimentally with two commonly used in vitro skin models- human cadaver heat-stripped skin (HSS) and pig full-thickness skin (FTS). Our results suggest that convection plays an important role during LFS of HSS, whereas its effect is negligible when FTS is utilized. The theory developed was utilized to characterize the transport pathways of hydrophilic permeants during both passive diffusion and LFS with mannitol and sucrose as two probe molecules. Our results show that the porous pathway theory can adequately describe the transdermal transport of hydrophilic permeants in both the presence and the absence of ultrasound. Ultrasound alters the skin porous pathways by two mechanisms: (1) enlarging the skin effective pore radii, or (2) creating more pores and/or making the pores less tortuous. During passive diffusion, both HSS and FTS exhibit the same skin effective pore radii (r = 28 +/- 13 A). In contrast, during LFS, r within HSS is greatly enlarged (r > 125 A), whereas r within FTS does not change significantly (23 +/- 10 A). The observed different roles of convection during LFS across HSS and FTS can be attributed to the different degrees of structural alteration that these two types of skin undergo during LFS., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.)

Furosemide (frusemide) is a weakly acidic diuretic drug. Its absorption is poor and variable, in part due to its restricted sites of absorption, mainly the stomach. The narrow absorption window of this drug can be explained by pH partition theory. The purpose of this study was to investigate the feasibility of widening the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract with officially used additives. Methacrylate copolymer (Eudragit L100-55), hydroxypropylmethylcellulose phthalate (HP-55) and hydroxypropylmethylcellulose acetate succinate (AS-MF) were selected as additives. The pH of suspensions of these additives was about 4, and the pH was adjusted to about 6-7 by the addition of NaOH. The Eudragit L100-55 suspension was found to be the most resistant to NaOH titration. When Eudragit L100-55 was used in an in-situ ileal loop experiment in rats, the pH of the intestinal contents was significantly reduced, from 7.9+/-0.1 to 5.7+/-0.1, and the plasma concentration of furosemide 15 min after administration was about 3 times higher than that in controls, 1.81+/- 0.42microg mL(-1) vs 0.63+/-0.08 microg mL(-1). However, the plasma concentration of [14C] mannitol was not changed by the co-administration of Eudragit L100-55. Furthermore, the AUC of furosemide was significantly increased by a factor of about 1.6 relative to that in controls by the co-administration of Eudragit L100-55, to 21.4+/-4.0 microg h mL(-1) from 13.3+/- 3.9 microg h mL(-1), and the gastrointestinal pH in the midgut and ileum was significantly reduced, with most of the furosemide remaining in these segments at 2 h following the oral administration of furosemide with Eudragit L100-55 to rats. These findings clearly demonstrate that the addition of Eudragit L100-55 can increase the absorption of furosemide in distal portions of the gastrointestinal tract. In conclusion, it is feasible to widen the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract by the co-administration of Eudragit L100-55.

Our knowledge of the epithelial permeability of different sections of the colon as well as of the surface and crypt epithelium is patchy and contradictory. Therefore, movement of radiolabelled urea, mannitol and Cr-EDTA between the lumen and the plasma of rats was studied, and expressed as clearance. In experiments studying movement from the lumen to the plasma, only the clearance of urea was significant. In experiments on the movement from plasma to the lumen, all three permeability probes exhibited significant clearance in the proximal colon, while in the distal colon the clearance of Cr-EDTA was not significant and the other clearance values were lower than in the proximal colon. Thus, the two methods are proposed to mainly reflect the permeability of two different parts of the epithelium, i.e. the surface and the crypt epithelium. Furthermore, it is proposed that the rat surface epithelium only allows passage of hydrophilic substances smaller than monosaccharides [radius below 0.35 nm (3.5 A] while the crypt epithelium, particularly in the proximal colon, is a heteroporous membrane of higher permeability containing pores corresponding to radii of >3.5-4.0 nm (35-40 A) and 0.4-0.5 nm (4-5 A). Moreover, the results indicate that in vivo luminal fluid solution has no access to the crypt epithelium, a conclusion strengthened by the observation that Evans-blue-labelled albumin and FITC-dextran 4000 do not seem to reach the crypt lumina.

Background & Aims: Release of neurotensin (NT) from intestines is markedly stimulated by ingested fat, and NT may facilitate lipid digestion and absorption through various actions that are not fully understood. Our recent finding that NT stimulates hepatic output of bile acids only when bile delivery to the intestine is maintained has led us to investigate the effects of NT on bile acid absorption in the rat small intestine., Methods: We measured the effects of intravenous infusion of NT (3-10 pmol x kg(-1) x min(-1)) on biliary recovery of (3)H-taurocholate ((3)H-TC) and (3)H-cholate administered into proximal and distal intestines or into isolated intestinal segments in situ in biliary fistula rats. To further understand the underlying mechanisms involved, the effects of NT on intestinal absorption of (3)H-D-glucose, (3)H-leucine, (14)C-antipyrine, and (51)Cr-EDTA were investigated by monitoring the absorption of radioactivity into superior mesenteric venous blood., Results: Infusion of NT, at doses that caused near physiologic increases in blood NT levels, increased biliary recovery of (3)H-TC from the jejunum (3.4-fold) and ileum (1.7-fold), but did not enhance absorption of (3)H-cholate. NT also facilitated transcellular uptake of (3)H-glucose and (3)H-leucine and increased paracellular uptake to (51)Cr-EDTA and (3)H-mannitol, but did not alter the absorption rate for (14)C-antipyrine., Conclusions: These results indicate that NT can exert a facilitative effect on intestinal bile acid absorption and return to liver. This effect of NT may involve increases in paracellular absorption and carrier-mediated transport by mechanisms not yet identified.

Purpose: Irradiation inflicts acute injuries to the intestinal mucosa with rapid apoptosis induction and subsequent reduction in epithelial surface area. It may therefore be assumed that the intestinal barrier function is affected. The aim of this study was to compare the mucosal permeability in irradiated rectum and nonirradiated sigmoid colon from patients subjected to radiation therapy before surgical treatment for rectal cancer., Methods: Segments from sigmoid colon and rectum obtained from irradiated and nonirradiated patients were stripped from the serosa-muscle layer and mounted in Ussing diffusion chambers. The mucosa-to-serosa passage of the marker molecules 14C-mannitol, fluorescein isothiocyanate-dextran 4,400, and ovalbumin was followed for 120 minutes., Results: The permeability to the markers was size-dependent and increased linearly across time in all specimens. The passage of all markers was increased in irradiated rectum compared with nonirradiated sigmoid colon, whereas in specimens from nonirradiated patients there were no differences between rectum and sigmoid colon. Histologic signs of crypt and mucosal atrophy were found in the irradiated rectal specimens., Conclusions: Early gastrointestinal complications after radiation therapy may be the result of mucosal atrophy in addition to mucosal damage, with a loss of barrier integrity.

This report characterizes an in vivo intracerebral long-distance diffusion model using dual-probe microdialysis. Two probes 1 mm apart were implanted into the striatum of control and 6-hydroxydopamine (6-OHDA)-lesioned halothane-anaesthetized male rats. Either tritiated dopamine (500 nM 3H-DA) or mannitol (1.5 microM 3H-mannitol) was infused continuously for 5 h, while samples were collected from the other probe. Samples (10 microl) were counted by liquid scintillation. For the DA-infused rats, another 10 microL was separated with high-pressure liquid chromatography (HPLC)-electrochemical detection into individual fractions containing 3,4-dihydroxy phenylacetic acid (DOPAC) and homovanillinic acid (HVA), and counted for beta-decay. The total transfer of 3H-labelled compounds described the overall effect of cellular uptake, metabolism and clearance into the microcirculation, and was compared with that of an extracellular marker, 3H-mannitol. The migration reached steady-state levels, generating an equilibrium between delivery and removal from the extracellular space. The half-time of the steady-state values, t50%, was in all cases lower in 6-OHDA-treated rats compared with control. In addition, the t50% values of 3H-mannitol were lower than those following the 3H-dopamine infusion in both control or 6-OHDA-lesioned rats. However, it was not possible to detect any unmetabolized 3H-dopamine at the 1 mm distance. In conclusion, the dual-probe microdialysis approach proved to be a valid method to study in vivo diffusion and migration in the brain, and the intracerebral spread of compounds highly depends on the nature of the compound infused.

The ion pair skin transport of cephalexin was investigated using various counter ions and solvents. The permeability of cephalexin was enhanced by 1-alkylsulfonates (ASs) at pH 3.0 and by tetraalkylammoniums (AAs) at pH 7.0; the enhancing ratio increased with the number of carbon atoms in their alkyl chains. The corresponding effects of these additives were observed on the partitioning of cephalexin. Most of the additives did not affect the skin transport of D-mannitol and cortisone. These results suggest that the enhanced transport of cephalexin results from the ion pair formation with additives. Although ASs increased the partitioning of cephalexin above that of AAs, the transport enhancement effect of ASs was lower than AAs having the same number of carbon atoms in their alkyl chains, indicating higher diffusivity of the ion pairs with AAs in skin. Moreover, the transport enhancement by AAs increased even more when ethanol-buffer solutions were used as solvents. The conductivity measurement of dissolving solutes in donor solvents showed that the further enhancement might be caused by the increasing ion pair formation in solvents with low dielectric constants. To obtain the maximum enhancement of skin transport of zwitterionic drugs via ion pair concept, one should select a counter ion having high lipophilicity and small volume, and a solvent with suitable pH and low dielectric constant.

The objectives were: (1) to develop a more rapid, reduced serum culture system for Caco-2 monolayers, relative to the traditional 21-day, 10% fetal bovine serum (FBS) system; and (2) to determine the biopharmaceutical drug classification of an oral therapeutic agent using this new system. Caco-2 cells were grown in the six well format on polycarbonate filters, in medium containing 2% iron supplemented calf serum (sCS) and a combination of growth factors and hormones. After 4 days in culture, permeabilities of three marker compounds (metoprolol, mannitol, and taurocholate) across monolayers were determined, and compared to permeabilities from the traditional 21-day, 10% FBS system, using cells at similar passage number. Cell morphology, degree of cell differentiation, and the presence of two efflux pumps were assessed. The 2% sCS model was also used to classify the permeability of an oral therapeutic agent as high or low. No difference in permeability was observed for metoprolol transport (P = 0.38) between the two culture methods, and the values obtained were independent of passage number of the cells. Mannitol permeability was about 2-fold higher from the 2% sCS system, as compared to the 10% FBS system. Taurocholate permeability was low indicating the 2% sCS culture at 4 days lacked this particular active transporter capability. Electron micrographs of cells grown in the 2% sCS system at 4 days revealed the presence of microvilli and tight junctions. P-glycoprotein and an efflux pump for furosemide were functionally present. The 2% sCS system indicated the oral therapeutic agent as highly permeable, which agreed with the 10% FBS system. This new system provides a rapid, accurate, and economical option for passive permeability determination, and appears to be applicable to the proposed Biopharmaceutics Classification System (BCS).

Reliability of differential sugar absorption tests is hampered by a lack of standardization of the content and osmolarity of the test solutions. We evaluated the effect of osmolarity of the test solution of the sugar absorption test on the 5 hour urine excretion of orally administered lactulose and mannitol. A group of 28 controls and 14 coeliacs, with villous atrophy grade II to IV, ingested a hyperosmolar sugar absorption test solution and a "low"-osmolar solution, respectively. After an overnight fast, each subject ingested hyperosmolar sugar absorption test solution (2 g mannitol, 5 g lactulose and 40 g sucrose/100 ml (around 1,560 mmol/l)). After two days, this procedure was repeated with low-osmolar solution (2 g mannitol and 5 g lactulose/100 ml (around 375 mmol/l). The influence of the sequence of the tests on the results had previously been excluded. All urine from the 5 h-period following ingestion of the test solution was collected. To calculate the low-osmolar solution ratio, samples were analysed for lactulose and mannitol concentrations by gas chromatography The sensitivity of hyperosmolar SAT solution and low-osmolar solution for the detection of mucosal abnormalities in coeliacs was 64% and 43%, respectively. In conclusion, a hyperosmolar solution discriminates better between normal and damaged mucosa of the small bowel such as villous atrophy due to a relative increase in permeability for lactulose.

Low-frequency (20 kHz) ultrasound has been shown to enhance transdermal transport of drugs, a phenomenon referred to as sonophoresis. In this paper, we report the threshold energy dose for ultrasound-induced transdermal drug transport. The threshold was determined by in vitro measurements of the dependence of sonophoretic enhancement on ultrasound parameters, including intensity, duty cycle, and exposure time. While the enhancement varies linearly with ultrasound intensity and exposure times, it is independent of the duty cycle in the range of parameters studied. The enhancement is also directly proportional to the ultrasound energy density once the threshold value is crossed. For full thickness pig skin, the threshold value is about 222 J/cm(2). The overall dependence of transport enhancement on ultrasound parameters is similar to that of cavitation measured in a model system, pitting of aluminum foil. Specifically, the extent of pitting is proportional to ultrasound intensity and exposure time and is independent of duty cycle. Furthermore, the extent of pitting is also proportional to the ultrasound energy density. The similarity between the parametric dependence of transport enhancement and cavitation is consistent with previous findings that cavitation plays the dominant role in sonophoresis.

Background: We compared symptoms and indices of fluid distribution after experimental administration of glycine and mannitol solutions, since these irrigating fluids are sometimes absorbed by the patient during genitourinary surgery., Methods: Glycine 1.5% and mannitol 3%, both with ethanol 1% added, were given by intravenous infusion at a rate of 0.5 ml/kg/min during 30 min to 12 male volunteers. Symptoms, cognitive status, hemodynamics, electrocardiogram during 24 hr, computerized tomography of the brain, bioimpedance, blood chemistry, and breath ethanol concentrations were recorded., Results: Glycine was associated with more symptoms than mannitol (P< 0.006), but the cognitive status, computerized tomography examinations, electrocardiograms, and breath ethanol concentrations did not differ between the solutions. The urinary excretion of fluid and sodium was greater after mannitol (P< 0.04), while only the glycine infusions hydrated the cells (P< 0.05). For both fluids, the intravascular and interstitial volumes were below baseline 3 hr after the experiment started (P< 0.01)., Conclusions: Glycine 1.5% had a higher tendency than mannitol 3% to cause symptoms and to accumulate in the cells., (Copyright 1999 Wiley-Liss, Inc.)

Purpose: To further characterize CYP3A4-transfected Caco-2 cells with regard to morphological, transport, and metabolic properties, and to evaluate a different Caco-2 cell strain transfected with both CYP3A4 and oxidoreductase (OR)., Methods: Transfected Caco-2 cells, Caco-2 TC7 cells, and wild-type Caco-2 cells grown onto Millicell were used. We determined the morphological characteristics of transfected cell monolayers using light and transmission electron microscope. We determined the transport and metabolic capabilities of the transfected cells, TC7 cells, and wild-type cells with a variety of drugs, nutrients, and marker compounds., Results: The transfected Caco-2 cells formed a tight monolayer with TEER values and mannitol transport similar to the untransfected parent cell strain (wild type). However, the transfected cells (grown onto Millicell) reached maturity approximately 33% faster than the wild-type cells. Permeabilities of propranolol, nifedipine, testosterone, linopirdine, mannitol, and cephalexin were similar in transfected and wild-type Caco-2 cells. On the other hand, the transfected cells of early passages were much more metabolically active, and metabolized standard CYP3A4 substrates (e.g., testosterone and nifedipine) as much as 100 times faster than untransfected cells. In addition, metabolism of standard substrates was inhibitable by ketoconazole and TAO. Using comparable data, the transfected cells metabolized testosterone the fastest, followed by linopirdine and nifedipine (approximate ratio: 10:6:2). The metabolites of standard substrates were generally preferably excreted to the apical membrane., Conclusion: The monolayers of newly transfected cells (CYP3A4 + OR) have a significantly increased level of CYP3A4 activities compared to untransfected cells. These cell monolayers also have desirable morphological and transport characteristics that are similar to untransfected cells.

Purpose: To validate our hypothesis that a bisphosphonate (BP) having a nitrogen-containing heterocyclic ring on the side chain, and with no hydroxyl on the geminal carbon would possess increased activity, and better oral bioavailability due to enhanced solubility of its calcium complexes/salts and weaker Ca chelating properties., Methods: A novel BP, 2-(2-aminopyrimidinio)ethylidene-1, 1-bisphosphonic acid betaine (ISA-13-1) was synthesized. The physicochemical properties and permeability were studied in vitro. The effects on macrophages, bone resorption (young growing rat model), and tumor-induced osteolysis (Walker carcinosarcoma) were studied in comparison to clinically used BPs., Results: The solubility of the Ca salt of ISA-13-1 was higher, and the log beta(Ca:BP) stability constant and the affinity to hydroxyapatite were lower than those of alendronate and pamidronate. ISA-13-1 exhibited effects similar to those of alendronate on bone volume, on bone osteolysis, and on macrophages, following delivery by liposomes. ISA-13-1 was shown to have 1.5-1.7 times better oral absorption than the other BPs with no deleterious effects on the tight junctions of intestinal tissue., Conclusions: The similar potency to clinically used BPs, the increased oral absorption as well as the lack of effect on tissue tight junction of ISA-13-1 warrant its further consideration as a potential drug for bone diseases.

The blood-brain barrier permeabilities of the type-A cholecystokinin receptor antagonists devazepide and A-65186 (Nalpha-3-quinolinoyl-D-Glu-N,N-dipentylamide) have been compared with those of the reference compounds iodoantipyrine, which readily penetrates the blood-brain barrier, and mannitol, which does not. Anaesthetized rats received a bolus injection into the left carotid artery of [14C]iodoantipyrine (0.25 microCi) combined with [3H]mannitol, [3H]devazepide or [3H]A-65186 (1 microCi each). Rats were decapitated 12s after injection and the brains were removed. Four samples of left cerebrum (ca 100 mg each) were solubilized overnight and 14C and 3H activity were measured. The brain-uptake index for each test compound was determined as [(3H/l4C for sample)]/[(3H/14C for injectate)] x 100, with a value of 100 representing blood-brain barrier permeability equal to that for iodoantipyrine. The brain-uptake index (mean+/-s.e.m.) was 1.6+/-0.3 for [3H]mannitol (n=5), 90.6+/-4.1 for [3H]devazepide (n=7, P<0.001 compared with mannitol) and 3.5+/-0.7 for [3H]A-65186 (n=4, P > 0.05 compared with mannitol, P < 0.001 compared with devazepide). Thus, devazepide readily penetrated the blood-brain barrier whereas A-65186 did not. It is concluded that devazepide and A-65186 are likely to be useful pharmacological tools for determining whether cholecystokinin is acting peripherally or at brain sites beyond the blood-brain barrier to produce satiety or any other function mediated by the type A cholecystokinin receptor.

Purpose: Grapefruit juice (GJ) is known to increase the oral bioavailability of many CYP3A-substrates by inhibiting intestinal phase-I metabolism. However, the magnitude of AUC increase is often insignificant and highly variable. Since we earlier suggested that CYP3A and P-glycoprotein (P-gp) form a concerted barrier to drug absorption, we investigated the role of P-gp in GJ-drug interactions., Methods: The transcellular bidirectional flux of drugs that are (i) CYP3A-and/or P-gp substrates (Vinblastine, Cyclosporine, Digoxin, Fexofenadine, Losartan) or that are (ii) primary CYP3A-substrates (Felodipine, Nifedipine) was evaluated across MDCK-MDR1 cell monolayers with or without GJ, verifying monolayer integrity at all times., Results: While both apical-to-basal (A-B) and basal-to-apical (B-A) fluxes of all CYP3A/P-gp substrates tested were increased in the presence of GJ, the resulting net efflux (B-A/A-B) was in all cases significantly greater with GJ than control (Vin, 28.0 vs. 5.1; CsA, 9.9 vs. 2.8; Dig, 22. 9 vs. 14.7, Fex, 22.3 vs. 11.1, Los, 39.6 vs. 26). In contrast, no such GJ flux effect was observed with Fel and Nif, substrates of CYP3A only (2 vs. 1.7 and 1.2 vs. 1.3)., Conclusions: GJ significantly activates P-gp-mediated efflux of drugs that are substrates of P-gp, potentially partially counteracting the CYP3A-inhibitory effects of GJ.

The blood-brain barrier is formed by the endothelial cells of the brain capillaries. Its primary characteristic is the impermeability of the capillary wall due to the presence of complex tight junctions and a low endocytic activity. Essential nutrients are delivered to the brain by selective transport mechanisms, such as glucose transporter and a variety of amino acid transporters. Although most drugs enter the brain by passive diffusion through the endothelial cells depending of their lipophilicity, degree of ionization, molecular weight, relative brain tissue and plasma bindings--some of them can use specific endogenous transporters. In these cases, binding competition on the transporter with endogenous products or nutrients can occur and limit the drug transfer. The blood-brain barrier can be a major impediment for the treatment of diseases of the central nervous system, since many drugs are unable to reach this organ at therapeutic concentrations. Various attempts have been made to overcome the limiting access of drugs to the brain: chemical modification of drugs, development of more hydrophobic analogs or linking an active compound to a specific carrier. Transient opening of the blood-brain barrier has been achieved by intracarotid infusion of hypertonic mannitol solutions or of bradykinin analogs in humans. Another way to increase or decrease brain delivery of drugs is to modulate the P-glycoprotein (P-gp) whose substrates are actively pumped out the cell into the capillary lumen. We actually dispose of many P-gp inhibitors or inducers in order to enhance the therapeutic effects of centrally acting drugs or to decrease central adverse effects of peripheric drugs.