The gastrointestinal absorption of liposomally entrapped insulin in normal rats [proceedings].

The use of liposomes as carriers of therapeutic agents has been extensively reviewed (Tyrrell et al., 1976). We (Patel & Ryman, 1976) have shown that encapsulation within liposomes may be an effective way of administering insulin orally, by which route it is normally destroyed in the gastrointestinal tract. When liposomally entrapped insulin was given orally to diabetic rats, there was a significant decrease in blood glucose, whereas the same amount of free insulin had no effect on the blood glucose (Patel & Ryman, 1976). Thus liposomes may be protecting the degradation of the protein by proteolytic digestion and possibly facilitating its absorption in the gastrointestinal tract. However, the hypoglycaemic effect was not observed when liposomally entrapped insulin was administered orally to normal rats, contrary to the results of Dapergolas & Gregoriadis (1976). Two possibilities were considered to explain our failure (Patel & Ryman, 1976) to observe the hypoglycaemic effect in the normal rat. The effective absorption of the liposomally entrapped protein may possibly be dependent on the diabetic state of the animal. It is known that an increase in intestinal brush-border-enzyme activities occurs in the mucosa of diabetic rats (Caspay et al., 1972), and an increase in intestinal growth and the transport of hexose sugars (Schedl & Wilson, 1971) and amino acids (Younoszai & Schedl, 1972) has also been reported and could possibly explain our observations with liposomally entrapped insulin in diabetic rats. The second possibility is that the amount of intact insulin absorbed may not be sufficient to produce any significant change in the blood glucose in normal rats as it may be broken down by the liver. As much as 40-50 % of insulin absorbed from the intestine is destroyed by the liver in a single transhepatic circulation (Mortimore & Tietze, 1959; Madison et al., 1959; Blackard &Nelson, 1970). In diabetic rats the absorbed insulin may not be destroyed so readily by liver, because the activity of the hepatic enzyme primarily responsible for the degradation of insulin, namely glutathione-insulin transhydrogenase (EC 1.8.4.2) is reported to be decreased in diabetic rats (Cudworth &Barber, 1975). To investigate the above possibilities, we treated normal rats with indol-3-ylacetic acid (3mmol/kg administered intraperitoneally), a compound known to inhibit insulin degradation (Mirsky et al., 1956), and immediately afterwards 6i.u. of liposomally entrapped insulin was administered orally. The liposome preparation used was as described earlier (Patel & Ryman, 1976) with the small alteration that phosphate buffer was replaced by 5Om~-Tris/NaCl buffer, pH 7.4. Monocomponent pig insulin used was a gift from Dr. W. R. Buckett (Novo Industries, Copenhagen, Denmark). The results in Table I show that 6i.u. of liposomally entrapped insulin given orally to normal rats led, perhaps surprisingly, to an apparent increase in blood glucose (123% of initial value), but the same preparation given orally to indolylacetic acidtreated normal rats markedly decreased the blood glucose (54% of initial value). This decrease is greater than that observed with indolylacetic acid followed by oral feeding of control liposomes (i.e. liposomes without insulin). These results indicate that liposomally entrapped insulin is absorbed in the gastrointestinal tract of normal rats, but the absorbed insulin requires protection against its degradation (which is probably carried out by the liver) to produce a hypoglycaemic effect in such rats. This protection is provided by indolylacetic acid, probably by inhibiting hepatic glutathione-insulin transhydrogenase activity. We could not observe any decrease in blood glucose in normal rats given 2.5-5 i.u. of insulin entrapped in dipalmitoyl phosphatidylcholine/cholesterol liposomes as used by Dapergolas & Gregoriadis (1976). During these studies, we have observed that not all preparations of liposomally entrapped insulin produce a hypoglycaemic effect in either diabetic or indolylacetic acid-treated normal rats. At present we suspect that insulin might be denatured in