Effects of Enzymatic Inhibition and Increased Paracellular Shunting on Transport of Vasopressin Analogues in the Rat ANNA-LENAUNGELL", ANNIKEANDREASSON*,KERSTI LUNDIN', AND LENA UlTER* Received April 29, 1991, from the 'Department of Dru Delivery Research, Pharmaceutical R 8 0, Astra Hdssle AB, S-437 83 Mdlndal, Sweden, and the *Departmentof Zoophys/olcgy, Unlversrfy of 8dteborg, Box 250 59, S-400 37 Gbteborg, Sweden. Accepted for publication August 26, 1991. Abstract 0 The Ussing chamber technique was used as an oral absorption model for studies of the relative effects of the inhibition of enzymaticdegradation and increased paracellular route on the transport of the poorly absorbed vasopressin analogues lysine vasopressin (LVP) and desmopressin (DDAVP). The rates of transport of LVP or DDAVP at 250 pM across ileum and colon segments were studied in the absence and in the presence of protease inhibitors (aprotinin and bestatin) and cytochalasin-B.During the different treatments, the rates of degradation of the peptides were also studied. Detectable amounts of LVP could only be measured on the serosal side of the intestinal segment in the presence of protease inhibitors or cytochaiasin-B. The treatment with cytochalasin-Bincreased the rates of transport of both peptides severalfold, and the effect was reversible. We suggest that the Ussing chamber technique can be used to evaluate the reasons for low transport rates across intestinal membranes. The results also show that, apart from enzymatic degradation, the vasopressin analogues LVP and DDAVP have additional permeation problems; therefore, it may be necessary to increase the paracellular route to increase the absorption of these peptides.
the electrical parameters. Compared with perfusion or in vivo studies, the Ussing chamber is less time consuming and requires fewer animals. In addition, the mechanism by which the substance crosses the epithelial barrier can be studied without the further complexity of the endothelial layer, as in perfusion studies, or influence from the body circulation, as in in vivo studies. The further advantage of using excised intestinal segments instead of cultured epithelial layers is that the influence of anatomy (crypt-villus axis) and morphology (heterogeneous membrane) on the transport rate of a drug may also be studied. The objectives of the present study were to evaluate the importance of decreased proteolytic activity and increased paracellular route on the rate of transport through the intestinal epithelia, by the use of the Ussing chamber technique as a tool for studying drug absorption, of the vasopressin analogues LVP and desmopressin (DDAVP).
Experimental Section For many peptides to enter the circulation from the gastrointestinal tract, several barriers have to be dealt with. First, many peptides are degraded by enzymes within the lumen of the intestinal tract and therefore do not reach the epithelial membrane. Second, the mucus and the aqueous diffusion layer that lies external to the microvillus membrane can be difficult to penetrate for hydrophobic or ionic molecules. In the third barrier, the epithelial membrane itself, the peptide can be degraded during its permeation or encounter physical difficulties to permeation. This last physiological barrier seems to be the most difficult one to deal with in the delivery of peptide drugs. Two routes exist for permeation of the epithelial barrier, transcellular and paracellular. During transcellular passage, the peptide can be exposed to enzymes within the cells. The peptide is, however, not always protected against degradation by paracellular transport because of the high proteolytic activity in the brush-border membrane region.1 Additionally, it is not known if any proteolytic activity exists on the basolateral membrane facing the intercellular spaces. However, inhibition of proteolytic activity can increase the absorption of peptides, as has been reported for insulin, arginine vasopressin, and lysine vasopressin (LVP).24 However, the question arises whether an increased paracellular route would permit higher transport rates, despite ongoing metabolic degradation, than would decreased proteolytic activity. Among a vast number of in vitro models for the prediction of drug absorption,&11 those that describe transport across isolated intestinal segments9 or cultured epithelial cell layers" offer several advantages. The diffusion-cell technique (i.e., Ussing chambe199 allows measurement of intestinal (or epithelial) viability and integrity simultaneously during studies of drug transport across the membrane by monitoring 640I Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 7992
Solutiont-+All transport studies were carried out in a Krebsbicarbonate buffer (KBR)with the following composition (KIM): NaCl (108.0); KC1 (4.7);Na2HP04 (1.8); KH,P04 (0.4);NaHCO, (15); MgSO, (1.2); CaCl, (1.25); D-glucose (11.5); and the additional respiratory substrates sodium tglutamate (4.9), disodium fumarate (5.4), and sodium pyruvate (4.9). The buffer solution was gassed with CO,:O, (595) to a constant pH of 7.4.The drugs were dissolved in the KBR and preheated to 37 "C before being added to the transport chambers. DDAVP and LVP were gifts from Fening AB, (Malmo, Sweden). Aprotinin, bestatin, and cytochalasin-B were purchased from Sigma Chemical Company (St. Louis, MO). Segment Preparation-Female Sprague-Dawley rats weighing 250-300 g were used in this study and were received at least 1week prior to use. The rats had free access to food and water prior to the time of sacrifice to allow intestinal sheets to survive in the transport chamber longer than the time required for the experiments. After an animal was sacrificed with C02, the whole intestine was removed and washed with cold KBR, put in a beaker with KBR solution on ice, and continuously gassed with 02:COz(95:5). The distal part of the ileum (5 cm proximal to the ileo-cecal junction) and the descending colon (2 cm proximal to the rectum) were then opened along the mesenteric border. Care was taken to avoid the Peyer's patches. For the ileum and the colon segments, the serosa and both the serosa and the muscularis externa, respectively, were removed by using blunt dissection. During the preparation, the tissues were submerged in KBR solution (10 "C), which was continuously gassed. Each segment was then mounted on a segment holder connected to the transport chamber (modified Ussing chamber) consisting of two 10-mL poly(methy1 methacrylate) (Lucite) chambers with rotors for effective stirring of the test solutions. The exposed segment area was 1 cm', and all tissues were allowed to equilibrate in the transport chamber before the start of the experiments (usually 30-40m i d . The equilibration period was followed by monitoring the electrical parameters of potential difference (PD) and short-circuit current (SCC). Electrical Measurements-The electrical parameters PD and SCC were recorded simultaneously during the experiments with agar-Ringer bridges connected through 3 M KCl to either calomel electrodes (positioned not further than 5 mm from the tissue surface) 0022-3549/92/0700-0640$02.50/0 0 7992, American Pharmaceutical Association
for measurement of PD or to silver electrodes (positioned at the far end of the chamber) for measurement of SCC. The electrodes were connected to an amplifier with a voltage-clamp facility. SCC was recorded by passing a brief period of alternating current through the tissue to reduce the PD to zero at appropriate time intervals. The electrical setup was calibrated for the resistance in the KBR solution and for differences in electrodes prior to each experiment. The resistance of the tissue segments was calculated from the PD and SCC values according to Ohm's law. Any tissue registering
the electrical parameters. Compared with perfusion or in vivo studies, the Ussing chamber is less time consuming and requires fewer animals. In addition, the mechanism by which the substance crosses the epithelial barrier can be studied without the further complexity of the endothelial layer, as in perfusion studies, or influence from the body circulation, as in in vivo studies. The further advantage of using excised intestinal segments instead of cultured epithelial layers is that the influence of anatomy (crypt-villus axis) and morphology (heterogeneous membrane) on the transport rate of a drug may also be studied. The objectives of the present study were to evaluate the importance of decreased proteolytic activity and increased paracellular route on the rate of transport through the intestinal epithelia, by the use of the Ussing chamber technique as a tool for studying drug absorption, of the vasopressin analogues LVP and desmopressin (DDAVP).
Experimental Section For many peptides to enter the circulation from the gastrointestinal tract, several barriers have to be dealt with. First, many peptides are degraded by enzymes within the lumen of the intestinal tract and therefore do not reach the epithelial membrane. Second, the mucus and the aqueous diffusion layer that lies external to the microvillus membrane can be difficult to penetrate for hydrophobic or ionic molecules. In the third barrier, the epithelial membrane itself, the peptide can be degraded during its permeation or encounter physical difficulties to permeation. This last physiological barrier seems to be the most difficult one to deal with in the delivery of peptide drugs. Two routes exist for permeation of the epithelial barrier, transcellular and paracellular. During transcellular passage, the peptide can be exposed to enzymes within the cells. The peptide is, however, not always protected against degradation by paracellular transport because of the high proteolytic activity in the brush-border membrane region.1 Additionally, it is not known if any proteolytic activity exists on the basolateral membrane facing the intercellular spaces. However, inhibition of proteolytic activity can increase the absorption of peptides, as has been reported for insulin, arginine vasopressin, and lysine vasopressin (LVP).24 However, the question arises whether an increased paracellular route would permit higher transport rates, despite ongoing metabolic degradation, than would decreased proteolytic activity. Among a vast number of in vitro models for the prediction of drug absorption,&11 those that describe transport across isolated intestinal segments9 or cultured epithelial cell layers" offer several advantages. The diffusion-cell technique (i.e., Ussing chambe199 allows measurement of intestinal (or epithelial) viability and integrity simultaneously during studies of drug transport across the membrane by monitoring 640I Journal of Pharmaceutical Sciences Vol. 87, No. 7, July 7992
Solutiont-+All transport studies were carried out in a Krebsbicarbonate buffer (KBR)with the following composition (KIM): NaCl (108.0); KC1 (4.7);Na2HP04 (1.8); KH,P04 (0.4);NaHCO, (15); MgSO, (1.2); CaCl, (1.25); D-glucose (11.5); and the additional respiratory substrates sodium tglutamate (4.9), disodium fumarate (5.4), and sodium pyruvate (4.9). The buffer solution was gassed with CO,:O, (595) to a constant pH of 7.4.The drugs were dissolved in the KBR and preheated to 37 "C before being added to the transport chambers. DDAVP and LVP were gifts from Fening AB, (Malmo, Sweden). Aprotinin, bestatin, and cytochalasin-B were purchased from Sigma Chemical Company (St. Louis, MO). Segment Preparation-Female Sprague-Dawley rats weighing 250-300 g were used in this study and were received at least 1week prior to use. The rats had free access to food and water prior to the time of sacrifice to allow intestinal sheets to survive in the transport chamber longer than the time required for the experiments. After an animal was sacrificed with C02, the whole intestine was removed and washed with cold KBR, put in a beaker with KBR solution on ice, and continuously gassed with 02:COz(95:5). The distal part of the ileum (5 cm proximal to the ileo-cecal junction) and the descending colon (2 cm proximal to the rectum) were then opened along the mesenteric border. Care was taken to avoid the Peyer's patches. For the ileum and the colon segments, the serosa and both the serosa and the muscularis externa, respectively, were removed by using blunt dissection. During the preparation, the tissues were submerged in KBR solution (10 "C), which was continuously gassed. Each segment was then mounted on a segment holder connected to the transport chamber (modified Ussing chamber) consisting of two 10-mL poly(methy1 methacrylate) (Lucite) chambers with rotors for effective stirring of the test solutions. The exposed segment area was 1 cm', and all tissues were allowed to equilibrate in the transport chamber before the start of the experiments (usually 30-40m i d . The equilibration period was followed by monitoring the electrical parameters of potential difference (PD) and short-circuit current (SCC). Electrical Measurements-The electrical parameters PD and SCC were recorded simultaneously during the experiments with agar-Ringer bridges connected through 3 M KCl to either calomel electrodes (positioned not further than 5 mm from the tissue surface) 0022-3549/92/0700-0640$02.50/0 0 7992, American Pharmaceutical Association
for measurement of PD or to silver electrodes (positioned at the far end of the chamber) for measurement of SCC. The electrodes were connected to an amplifier with a voltage-clamp facility. SCC was recorded by passing a brief period of alternating current through the tissue to reduce the PD to zero at appropriate time intervals. The electrical setup was calibrated for the resistance in the KBR solution and for differences in electrodes prior to each experiment. The resistance of the tissue segments was calculated from the PD and SCC values according to Ohm's law. Any tissue registering
