Colonic homogenates

Peptidases such as aminopeptidases and diamino-peptidases are at lower concentrations in the colon than in the small intestine. However, it has been shown that prolyl endoprotease and collagenase activities are five to six times higher in the colon than in the small intestine. TRH is more readily hydrolyzed to deaminated TRH in colonic homogenates compared with small intestine and rectum. Therefore, peptide drugs that are substrates for prolyl endoprotease and collagenase will likely be degraded in the colon and may not be suitable for colon delivery. 

Wolf and his associates [109, 110] studied the effect of vitamin A on mucus synthesis by investigating the effect of vitamin A on mucopolysaccharide synthesis by colon segments and colon homogenates of vitamin deficient rats. The incorporation of in the mucus polysaccharides of colon segments of vitamin deficient rats is about half of that observed in colon segments obtained from rats fed a diet containing vitamin A. The rate of incorporation into the deficient mucosa was restored to normal by adding vitamin A to the incubation medium. 

The Caco-2 cell line was isolated from a human colon carcinoma, and has been characterized as one of the best in vitro models of intestinal epithelium. Indeed, in contrast to other intestinal cell lines, Caco-2 cells are able to constitute a homogenous monolayer and to spontaneously differentiate into polarized cells, highly similar to human mature enterocytes, after approximately 2 weeks of culture. Furthermore, the Caco-2 cells present microvillosities at the apical side and have a high transmembrane resistivity, which confirms the fact that the cells are confluent and link to one another via gap junctions. Finally, they can absorb different compounds, express many enzymes involved in intestinal metabolic pathways (Pinto et al. 1983, Musto et al. 1995, Salvini et al. 2002), and give reproducible in vitro results consistent with results obtained in in vivo studies (Artursson and Karlsson 1991). 

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

The same strategy was applied in colon-specific delivery of two corticosteroids used to treat inflammatory bowel disease [20][21], Indeed, budeso-nide /3-D-glucuronide and dexamethasone /3-D-glucuronide underwent ready hydrolysis in the luminal contents of rat colon and caecum. Rat mucosal homogenates were less active, and hydrolysis in human fecal samples was quite low. Based on these and other studies, the prodrugs were found to be suitable candidates for delivery of corticosteroids to the large intestine. 

Colon was excised, irrigated free of feces, homogenized and chromatographed on chymotrypsin sepharose (18). 

Figure 1 - Chymotrypsin-sepharose elution profile of colon wall homogenate 1 hour after i.g. administration of Arrow (+)...

Thiol methyltransferase has been detected in erythrocytes, lymphocytes, lungs, cecal, and colonic mucosae. The nature and number of thiol methyltransferases is not clear at the present time. A cytosolic enzyme and a microsomal enzyme have been reported, with the microsomal enzyme being dissociated from membrane relatively easily. The microsomal enzyme in rat liver has been purified to homogeneity. The enzyme is a 28,000-dalton monomer with an isoelectric point of 6.2. A wide variety of xenobiotic thiols are methylated, but cysteine and glutathione are not substrates. S-Methylation is an important component in the thiomethyl shunt. Thiomethyl conjugates are metabolized to the methylsulfoxides by oxidation (see Chapter 10) and reenter the mercapturic acid pathway as substrates for glutathione S-transferase. 

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