In vitro and in vivo preclinical evaluation of a minisphere emulsion-based formulation (SmPill®) of salmon calcitonin.

Salmon calcitonin (sCT, MW 3432Da) is a benchmark molecule for an oral peptide delivery system because it is degraded and has low intestinal epithelial permeability. Four dry emulsion minisphere prototypes (SmPill®) containing sCT were co-formulated with permeation enhancers (PEs): sodium taurodeoxycholate (NaTDC), sodium caprate (C10) or coco-glucoside (CG), or with a pH acidifier, citric acid (CA). Minispheres protected sCT from thermal degradation and the released sCT retained high bioactivity, as determined by cyclic AMP generation in T47D cells. Pre-minisphere emulsions of PEs combined with sCT increased absolute bioavailability (F) compared to native sCT following rat intra-jejunal (i.j.) and intra-colonic (i.c.) loop instillations, an effect that was more pronounced in colon. Minispheres corresponding to ~2000I.U. (~390µg) sCT/kg were instilled by i.j. or i.c. instillations and hypocalcaemia resulted from all prototypes. The absolute F (i.j.) of sCT was 11.0, 4.8, and 1.4% for minispheres containing NaTDC (10µmol/kg), CG (12µmol/kg) or CA (32µmol/kg) respectively. For i.c. instillations, the largest absolute F (22% in each case) was achieved for minispheres containing either C10 (284µmol/kg) or CG (12µmol/kg), whilst the absolute F was 8.2% for minispheres loaded with CA (32µmol/kg). In terms of relative F, the best data were obtained for minispheres containing NaTDC (i.j.), a 4-fold increase over sCT solution, and also for either C10 or CG (i.c.), where there was a 3-fold increase over sCT solution. Histology of instilled intestinal loops indicated that neither the minispheres nor components thereof caused major perturbation. In conclusion, selected SmPill® minisphere formulations may have the potential to be used as oral peptide delivery systems when delivered to jejunum or colon.

Contribution of ion-pair complexation with bile salts to the transport of organic cations across LLC-PK1 cell monolayers.

PURPOSE: To examine the effect of ion-pair complexation with endogenous bile salts on the transport of organic cations (OCs) across LLC-PK1 cell monolayers. METHODS: The transport of tributylmethyl-ammonium (TBuMA) and triethylmethylammonium (TEMA) across the cell monolayer was measured in the presence of taurodeoxycholate (TDC), an endogenous organic anion that forms an ion-pair complex with TBuMA, but not with TEMA. RESULTS: Under proton gradient conditions (i.e., pH 6.0 apical/pH 7.4 basal), the above OCs exhibited similar transport charactersistics, consistent with the well-established OC/H+ antiporter, and the presence of TDC had no measurable effect on the transport of these OCs. Under pH-equilibrated conditions (i.e., pH 7.4 apical/pH 7.4 basal); however. basal to apical transport of TBuMA, not that of TEMA, was increased in the presence of TDC, probably as a result of the formation of a lipophilic ion-pair complex between TBuMA and TDC. The transport and efflux of the TBuMA-TDC complex across the apical membrane of the cell was inhibited by representative substrates of the P-glycoprotein (P-gp), indicating the involvement of P-gp in this process. The increased affinity of the ion-pair complex to P-gp is consistent with a mechanism involving increased transport. CONCLUSION: In cases where there is no proton gradient between the plasma and urine, the formation of lipophilic ion-pair complexes in the kidney with endogenous bile salts might be involved in the in vivo urinary excretion of large Mw OCs, such as TBuMA.

Repetitive short-term bile duct obstruction and relief causes reproducible and reversible bile acid regurgitation.

BACKGROUND: Long-term bile duct obstruction causes sinusoidal regurgitation of bile acids, a shift in bile acid metabolism, and alterations of liver histology. In this study we investigated the regurgitation of bile acids during short-term bile duct obstruction and its reversibility and reproducibility. In addition, the biotransformation of taurodeoxycholate and its appearance in bile and perfusate effluent were studied as well as liver histology. METHODS: Rat livers (n = 5) were perfused in vitro with 32 nmol/min/g liver taurodeoxycholate over 85 min with the bile duct being intermittently closed for 30 and 20 min, respectively. RESULTS: Within the first 5 min after bile duct obstruction bile acids started to regurgitate to the perfusate effluent amounting to approximately 15% of hepatic uptake until the end of the perfusion period. After relief of obstruction, bile flow and biliary bile acid excretion showed an overshoot phenomenon and were almost doubled compared to preobstruction. In contrast, sinusoidal bile acid regurgitation declined. The same phenomenon was observed during the second closure/opening cycle of the bile duct. Regurgitated bile acids consisted of significantly more taurodeoxycholate metabolites (approximately 70%) than did biliary bile acids (approximately 30%). Histology of liver parenchyma was preserved. CONCLUSIONS: During repetitive short-term bile duct obstruction bile acid regurgitation is reversible and reproducible. The absence of altered mechanical barriers suggests that specific pathways are involved in the regurgitation process of bile acids.

Colchicine inhibits taurodeoxycholate transport in pericentral but not in periportal hepatocytes.

Indirect evidence for a microtubule-dependent vesicular hepatocellular transport of bile acids has accumulated. Since inhibition of this transport by colchicine can be achieved only at high but not at low bile acid infusion rates we were wondering whether this transport pathway shows a hepatic zonation or not. To answer this question we perfused isolated rat livers antegradely or retrogradely, respectively, with unlabeled and labeled taurocholate or taurodeoxycholate. Inhibition of microtubule-dependent bile acid transport was aimed at co-infusion of colchicine. Periportal cells eliminated the likewise hydrophobic taurodeoxycholate as fast as the more hydrophilic taurocholate. In contrast, pericentral cells excreted taurodeoxycholate much slower than taurocholate. Colchicine did not change the biliary taurocholate excretion profile in periportal and pericentral cells. However, colchicine reduced significantly taurodeoxycholate excretion in pericentral but not in periportal cells. It is concluded that a microtubule-dependent vesicular, colchicine-sensitive transport pathway seems to be involved in the translocation of taurodeoxycholate in pericentral but not in periportal cells. Since such a vesicular bile acid transport is regarded to be much slower than transcellular transport by diffusion, this observation may explain the much slower excretion of hydrophobic bile acids like taurodeoxycholate in pericentral than in periportal cells under physiological conditions.

Hepatic uptake and intestinal absorption of bile acids in the rabbit.

The existence of transporters for bile acids (BA) in liver and intestine has been well documented, but information is still needed as to their respective transport capacity. In the present investigation, we compared the hepatic and intestinal transport rates for BA, using perfused livers and intestines. The livers and intestines were separately perfused and dose-response curves (0.25-10 mM) for tauroursodeoxycholate, taurocholate and taurodeoxycholate were obtained. The intestinal and mesenteric concentration and bile acid pattern were also evaluated in six non-fasting rabbits. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid ileal absorption showed saturation kinetics in the intestine as in the liver; the maximal uptake velocity for each bile acid in the liver was tenfold higher than the respective maximal transport velocity in the intestine; the Km values obtained in the liver were of the same order of magnitude, i.e. in the millimolar range. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid transport differences in the liver paralleled those in the intestine. Although the intestine was not homogeneously filled, the bile acid concentration in the ileal content fell into the range of the Km for the three studied bile acids, while the portal blood total bile acid concentration was inferior to the observed Kms of liver uptake. Therefore, both the hepatic and intestinal systems do not operate at their maximal transport rates at the prevailing concentrations in portal blood and luminal content, and the hepatic transport occurs at its highest efficiency (below the Km values) in physiological conditions.