Assessment of Epithelial Lining Fluid Partitioning of Systemically Administered Monoclonal Antibodies in Rats.

For systemically administered monoclonal antibodies (mAbs) with pharmacological targets in the epithelial lining fluid (ELF), information on the partitioning of mAb between plasma and ELF is instrumental for dose predictions. Bronchoalveolar lavage (BAL) combined with measurements of urea as indicator of sample dilution is often used to estimate ELF concentrations of a drug. However, unbalanced extraction of mAb and urea could potentially lead to a systematic bias in the back-calculated ELF concentration. In the present study 0.5, 1, or 4 mL phosphate-buffered saline was instilled to lungs of rats to obtain lavage samples after systemic dosing of mAb and tool small molecule (n≥4/group). Furthermore, extraction of urea, mAb and the small molecule was assessed by repeatedly lavaging the lung (n = 4). There was no statistically significant difference in the calculated partitioning into ELF between the evaluated instillation volumes. Repeated BAL demonstrated that urea and the small molecule were extracted from other sources than the ELF. In contrast, there was limited to none in-flow of mAb into the lavage fluid. The unbalanced extraction of urea and mAb could theoretically result in underestimated ELF concentrations and the calculated partitioning of 0.17±0.062 might therefore constitute a lower boundary for the true partitioning.

Possible Extraction of Drugs from Lung Tissue During Broncho-alveolar Lavage Suggest Uncertainty in the Procedure's Utility for Quantitative Assessment of Airway Drug Exposure.

Following inhaled dosing, broncho-alveolar lavage (BAL) is often used for sampling epithelial lining fluid (ELF) to determine drug concentration in the lungs. This study aimed to explore the technique's suitability. Urea is typically used to estimate the dilution factor between the BAL fluid and physiological ELF, since it readily permeates through all fluids in the body. As representatives of permeable small molecule drugs with high, medium and low tissue distribution properties, propranolol, diazepam, indomethacin and AZD4721 were infused intravenously to steady state to ensure equal unbound drug concentrations throughout the body. The results showed that propranolol had higher unbound concentrations in the ELF compared to the plasma whilst this was not the case for the other compounds. Experiments with different BAL volumes and repeated lavaging indicated that the amount of drug extracted is very sensitive to experimental procedure. In addition, the results show that the unbound concentrations in ELF compared to plasma differs dependent on molecule class and tissue distribution properties. Overall data suggests that lavaging can remove drug from lung tissue in addition to ELF and highlights significant uncertainty in the robustness of the procedure for determining ELF drug concentrations.

Enteric neurones modulate the colonic permeability response to luminal bile acids in rat colon in vivo.

BACKGROUND: The mechanisms behind microscopic colitis and exacerbations of ulcerative colitis are incompletely understood. It seems highly likely that both luminal antigens and bile are involved. The aim of this study was to test the hypothesis that bile acids increase colonic mucosal permeability by activating enteric neurones. METHOD: The effect of 4 mM deoxycholic acid (DCA) on the appearance rate of intravenously administered (3)H-mannitol and (14)C-urea into the lumen of the proximal and distal rat colon was measured in vivo and expressed as clearance. The nerve blocking agents atropine and hexamethonium were given intravenously, and lidocaine was applied onto the serosal surface of the colon, before and after DCA exposure RESULTS: DCA markedly increased clearance of the permeability probes into the lumen in both colonic segments and also the ratio of mannitol/urea clearance, particularly in the distal colon. Pretreatment with atropine, hexamethonium, and lidocaine significantly inhibited the increase in clearance by approximately 65-80% but did not affect the clearance ratio. In the distal colon, the inhibitory effect of lidocaine was not statistically significant. Also, administration of atropine and hexamethonium after DCA exposure significantly inhibited the DCA effect on clearance of the probes. CONCLUSION: The results suggest that in vivo, the permeability increase induced by a moderate concentration of bile acid is to a large extent mediated by a neural mechanism involving muscarinic and nicotinic receptors. This mechanism may be a link between the central nervous system and colonic mucosal barrier function, and may be a new target for treatment.

Inhibition of nitric oxide synthesis potentiates the colonic permeability increase triggered by luminal bile acids.

AIM: Experiments were performed in anaesthetized rats to clarify the role of nitric oxide (NO) in the control of colonic permeability. METHODS: Colonic luminal pressure, the transmucosal potential difference (PD) and the clearance of [3H] mannitol and [14C] urea from blood to lumen were measured. NO synthesis was blocked with Nomega-nitro-L-arginine (L-NNA) i.v. and mucosal permeability was increased by deoxycholic acid (DCA, 4 mm). The involvement of histamine in the response was studied by giving the histamine H1 receptor blocker pyrilamine. RESULTS: In proximal colon, L-NNA per se increased luminal pressure and PD but had no significant effect on clearance. DCA per se increased luminal pressure, had no significant effect on PD, but increased mannitol and urea clearance and the clearance ratio. L-NNA and pyrilamine both blocked the luminal pressure effect of DCA but L-NNA had no significant effect on the clearance response to DCA. In distal colon, L-NNA per se had no significant effect on pressure and clearance, but increased PD like in proximal colon. DCA had no significant effect on luminal pressure, but markedly reduced PD and increased both clearance and clearance ratio. In this segment, L-NNA significantly potentiated the clearance response to DCA, and further increased clearance ratio to a value not significantly different from unity (1.00 +/- 0.05). CONCLUSION: The data suggest that in vivo, moderate concentrations of bile acids increase colonic permeability in rats via a mechanism that is inhibited by NO in distal but not in proximal colon. In distal colon, NO may contribute to the maintenance of epithelial barrier function.

Involvement of enteric nerves in permeability changes due to deoxycholic acid in rat jejunum in vivo.

AIM: Stress and Clostridium difficile toxin A increase epithelial permeability in the small intestine via vagus and visceral afferents, in turn activating mucosal mast cells. Bile acids also increase epithelial permeability but it is not known if nerves or mast cells are involved in this effect in the small intestine. METHOD: In jejunum of anesthetized rats, the effects of hexamethonium and atropine on deoxycholic acid (DCA) induced fluid secretion and increase in epithelial permeability was therefore studied by determining the appearance and disappearance rates of 14C-mannitol and 51Cr-EDTA into and from a perfusion system containing 4 or 8 mm DCA and expressed as clearance. RESULTS: DCA increased net fluid transport and appearance and to a less extent disappearance rates of the probes. Hexamethonium but not atropine, chronic denervation or the NO synthase inhibitor L-NNA did significantly decrease the appearance rate and net fluid secretion. The levels of the mast cell protease II (RMCP II) in perfusate and plasma were not increased by DCA. The clearance ratio Cr-EDTA/mannitol indicates that the plasma clearance of the permeability probes is partly secondary to net fluid transport only at higher DCA concentrations. CONCLUSION: We conclude that the DCA effect on epithelial permeability is to a large part induced by intramural reflex(es) containing nicotinic receptors. The results also suggest that mast cell degranulation and NO release are not involved in the mechanism. This indicates that the nerve effect on intestinal paracellular permeability is not mediated by the mechanisms described for stress or Clostridium difficile toxin A.

Water absorption enhances the uptake of mannitol and decreases Cr-EDTA/mannitol permeability ratios in cat small intestine in situ.

BACKGROUND: Recently, we hypothesized that mannitol absorption in human intestinal permeability tests is a reflection of small intestinal water absorption and is dependent mainly on the efficiency of the countercurrent multiplier in the villi. This may affect the outcome of clinical double-sugar permeability tests. We tested the hypothesis in cats, another species with an efficient countercurrent multiplier. METHODS: The lumen-to-tissue transport of [14C]mannitol and [51Cr]EDTA was studied in in situ perfused jejunum of eight anaesthetized cats using four isotonic perfusion solutions with varying sodium and glucose content. The transport of water was monitored, and the absorption rate of the probes was calculated by their disappearance from the perfusate. RESULTS: There was a significant positive correlation between water absorption and [14C]mannitol clearance from the different perfusates (r = 0.99; P < 0.01), whereas this correlation was absent for [51Cr]EDTA clearance (r = 0.05; P = 0.95). There was also a significant negative correlation between water absorption and [51Cr]EDTA/[14C]mannitol clearance ratios (r = 0.98; P < 0.02). CONCLUSIONS: The results show a prominent effect of water absorption on mannitol uptake through pores which, also during glucose transport, exclude Cr-EDTA. The difference in water absorption from the solutions used in cat small intestine is dependent on the effectiveness of the countercurrent multiplier; we conclude that the capability of this mechanism influences mannitol absorption in vivo. Qualitatively comparable results were obtained using oral test solutions with varying NaCl and glucose concentrations in human volunteers. We propose that the functioning of the countercurrent multiplier is essential for the interpretation of double-sugar tests in clinical studies.

Effect of cholera toxin on passive transepithelial transport of 51Cr-ethylenediaminetetraacetic acid and 14C-mannitol in rat jejunum.

Intestinal fluid secretion, mainly derived from the crypts, induced, for example, by cholera toxin, decreases the passive transport of small hydrophilic molecules into the lumen. However, the effect of the fluid secretion on the passive absorption of these substances and thus on the permeability of the villus absorptive area is not known. Therefore, the transport rates of 51Cr-ethylenediaminetetraacetic acid (EDTA) and 14C-mannitol from lumen to plasma and from plasma to lumen were recorded in jejunal loops of anaesthetized rats during cholera toxin-induced fluid secretion in the absence and presence of glucose in the intestinal lumen and expressed as clearance (microL (min g)(-1)). The results showed that the cholera toxin induced fluid secretion and abolished the passive absorption of 51Cr-EDTA both in the absence and presence of luminal glucose during a high perfusion rate (0.5 mL min(-1)). The clearance of mannitol was also inhibited at the low perfusion rate (0.2 mL min-1) with the glucose-free perfusate but only reduced with the glucose perfusate. The results show that mechanisms activated by cholera toxin inhibit the passive absorption of inert hydrophilic substances. This is proposed to be mainly caused by a reduction in the accessibility of the villus epithelium to the luminal content. Furthermore, the secretion seems predominantly to inhibit the passive absorption at the basal parts of the villus while the absorption rate at the villus tips is better preserved. The results also show that the intestinal absorption and secretion of fluid takes place at different locations (villus and crypts, respectively).

Permeability of the proximal and distal rat colon crypt and surface epithelium to hydrophilic molecules.

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.

Permeability of the rat small intestinal epithelium along the villus-crypt axis: effects of glucose transport.

BACKGROUND & AIMS: The aim of this study was to elucidate the permeability characteristics of the epithelium along the villus-crypt axis and investigate the effect of glucose transport on these characteristics along this axis. METHODS: The disappearance rates of (14)C-mannitol and (51)Cr-EDTA or (3)H-inulin were determined as clearance (Cl(x)) from a recirculating perfusion system of the jejunal lumen in anesthetized rats. Net fluid transport was varied over a large range by exchanging mannitol with glucose in the perfusate solution and by inhibition of nervously mediated secretory processes with hexamethonium. The perfusion rate was 0.5 or 0.2 mL/min. RESULTS: Cl(Man) enhanced significantly with increasing net fluid transport (secretion 8.50+/-1.88, to absorption 16.72+/-1.75 microL x min(-1) x g(-1)) and with glucose perfusates. Cl(Cr-EDTA) was constant irrespective of net fluid transport and was reduced to insignificant values at a perfusion rate of 0.2 mL/min. Cl(In) was not different from zero. CONCLUSIONS: The absorbing apical part of the villus contains small pores (radius, <6 A) allowing passive transport via solvent drag of, e.g., monosaccharides, whereas the pores in the crypts are large (50-60 A) and inaccessible to the luminal content. The basal part of the villus contains medium-sized pores (10-15 A) through which no solvent drag occurs. Active glucose transport in the rat mainly increases the number of small pores accessible for passive transport, whereas the size of these pores seems to stay constant.

Effects of neural blocking agents on motor activity and secretion in the proximal and distal rat colon: evidence of marked segmental differences in nicotinic receptor activity.

BACKGROUND: Neuromodulation may be a new therapeutic approach in inflammatory bowel disease, but very little is known about neural control of colonic secretion in vivo. We therefore determined the effects of neural blockade on colonic motor activity and mucosal secretion in anaesthetized rats. METHODS: A proximal and a distal colonic segment were isolated in four groups of chloralose-anaesthetized rats (n = 8 in each group), and we measured luminal pressure and transmural potential difference (PD) as a marker of electrogenic chloride secretion. Recordings were made from proximal and distal segments simultaneously, which made it possible to directly compare response patterns. RESULTS: Under control conditions luminal pressure waves were associated with phasic, lumen-negative increases in PD which had a significantly greater magnitude and longer duration in the distal colon. Atropine blocked both pressure waves and PD waves in the proximal colon, but some PD waves, although of lower magnitude, remained in the distal colon. Hexamethonium abolished pressure waves in both segments and induced a marked reduction in PD in the distal but not in the proximal colon. Lidocaine also reduced PD, more so in the distal colon, and dissociated the pressure-PD linkage. CONCLUSION: In the distal but not in the proximal colon, there is a strong nicotinic, neurogenic 'tone' that maintains a high basal secretory activity. The results encourage the search for neuromodulatory agents in the treatment of colonic secretory disease.