Emma L. McConnell

Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments.

To use rodent models effectively in in‐vivo investigations on oral drug and vaccine delivery, the conditions in the gastrointestinal tract must be understood. Some fundamental information is currently unavailable or incomplete. We have investigated the pH, water content and lymphoid tissue distribution along the gastrointestinal tract, as well as the stomach volume, as these were critical to our investigations on pH‐responsive drug delivery and colonic vaccination. The observed values were compared with those in man as an indication of the validity of the rodent model. The mouse stomach pH was 3.0 (fed) and 4.0 (fasted), and the corresponding values in the rat were 3.2 (fed) and 3.9 (fasted). The mean intestinal pH was lower than that in man (< pH 5.2 in the mouse; < pH 6.6 in the rat). This brings into question the use of rodents in investigations on enteric‐coated drug carriers targeted to the large intestine/distal gut. The water content in the gastrointestinal tract in the fed and fasted mouse was 0.98 ± 0.4 and 0.81 ± 1.3 mL, respectively, and in the fed and fasted rat was 7.8 ± 1.5 and 3.2 ± 1.8 mL. When normalized for body weight, there was more water per kg body weight in the gastrointestinal tracts of the mouse and rat, than in man. The stomach capacity was found to be approximately 0.4 and 3.4 mL for mice and rats, respectively. The low fluid volume and stomach capacity have implications for the testing of solid dosage forms in these animal models. Substantial amounts of lymphoid tissue analagous to small intestinal Peyers patches were measured in the rat and mouse colon, showing the feasibility of colonic vaccination, a route which might prove to have different applications to the more commonly studied oral vaccines. The existence of lymphoid tissue in the mouse and rat caecum has also been reported.

An in vivo comparison of intestinal pH and bacteria as physiological trigger mechanisms for colonic targeting in man

Targeting the colon for site-specific oral delivery can exploit one of two main physiological triggers; the intestinal pH changes or the increase in bacterial numbers in the distal gut. This study aimed to assess how these triggers compared in vivo to determine which concept provides better colon-specific release. Pellets were prepared using theophylline (model drug) and coated with methacrylic acid/methylmethcrylate co-polymer (Eudragit S [a pH-responsive polymer which dissolves above pH 7]) or amylose/ethylcellulose (a polysaccharide/polymeric mixture which is partially digested by colonic bacteria). The immediate release (uncoated) and the two sets of modified release (coated) pellets were administered to eight healthy fasted volunteers in a three-way crossover study. Drug levels were measured in the plasma, and the transit of the modified release pellets was followed by gamma scintigraphy. The immediate release pellets had T(max) values ranging from 0.5-2 h and bioavailability (AUC) ranging from 24.8-50.7 mcg h/ml. The pH-responsive pellets released drug in seven out of eight subjects. In those subjects in whom drug release occurred, the pellets had variable in vivo performance (T(max) ranging from 5-9 h; AUC 8.8-55.0 mcg h/ml) and drug release started in the small intestine for these pellets. The bacterially-triggered pellets (T(max) 8-10 h; AUC 16.5-47.9 mcg h/ml) were colon-specific; drug was detected in the blood only when the pellets reached the colon and release was more sustained than the pH system. The use of the bacterially-triggered delivery concept provided improved colonic delivery over the pH approach.

Assessment of gastrointestinal pH, fluid and lymphoid tissue in the guinea pig, rabbit and pig, and implications for their use in drug development

Laboratory animals are often used in drug delivery and research. However, basic information about their gastrointestinal pH, fluid volume, and lymphoid tissue is not completely known. We have investigated these post-mortem in healthy guinea pigs, rabbits and pigs, to assess their suitability for pre-clinical studies by comparing the results with reported human literature. The mean gastric pH (fed ad libitum) was 2.9 and 4.4 in guinea pig and pig, respectively. In contrast, a very low pH (1.6) was recorded in the rabbits. The small intestinal pH was found in the range of 6.4-7.4 in the guinea pigs and rabbits, whereas lower pH (6.1-6.7) was recorded in the pig, which may have consequences for ionisable or pH responsive systems when tested in pig. A relatively lower pH than in the small intestine was found in the caecum (6.0-6.4) and colon (6.1-6.6) of the guinea pig, rabbit and the pig. The water content in the gastrointestinal tract of guinea pig, rabbit and pig was 51g, 153g and 1546g, respectively. When normalized to the body weight, the guinea pig, had larger amounts of water compared to the rabbit and the pig (guinea pig>rabbit>pig); in contrast, a reverse order was found when normalized to per unit length of the gut (guinea pig<rabbit<pig). The lymphoid tissue distribution (lymphoid follicles, Peyers patches and long strips) along the length of the gut in these animals is presented; in particular, an abundance of lymphoid tissue was found in pigs stomach, small intestine and caecum, and rabbits appendix. Their ample presence indicated the potential utility of these animal species in oral and colonic vaccination. These differences in the gastrointestinal parameters of the guinea pig, rabbit and pig reiterates the crucial importance of correctly selecting animal models for pre-clinical studies.

An Investigation into the Digestion of Chitosan (Noncrosslinked and Crosslinked) by Human Colonic Bacteria

Chitosan salts are being investigated as materials for bacterially triggered colonic drug delivery, via the oral route, based on the assumption that they will be degraded by the enzymes produced by the human colonic bacteria. The actual susceptibility of chitosan to these enzymes is, however, unclear. The digestion of chitosan films (noncrosslinked, and crosslinked with glutaraldehyde or tripolyphosphate) by human colonic bacteria (using human faecal material) was therefore investigated, and in addition, their digestion by pancreatic enzymes (of porcine origin) was assessed. Noncrosslinked chitosan films were digested by both pancreatic and colonic enzymes within 4 h, while glutaraldehyde crosslinked chitosan films were resistant to both enzyme systems. In contrast, tripolyphosphate crosslinked chitosan films resisted pancreatic digestion, but were susceptible to faecal digestion over the same 4 h time period. As expected, lowering crosslinker concentration and increasing incubation time (to 18 h) allowed greater digestion. The difference between the crosslinkers is attributed to the mechanism of crosslinking, and the associated degree of film swelling in an aqueous environment. Swelling studies in acidic conditions suggest that only glutaraldehyde or higher concentrations of tripolyphosphate would be able to prevent film dissolution in gastric conditions.

Colonic treatments and targets: issues and opportunities

The colon provides a plethora of therapeutic opportunities. There are multiple disease targets, drug molecules, and colon-specific delivery systems to be explored. Clinical studies highlight the potential for systemic delivery via the colon, and the emerging data on the levels of cell membrane transporters and metabolic enzymes along the gut could prove advantageous for this. Often efflux transporters and metabolic enzyme levels are lower in the colon, suggesting a potential for improved bioavailability of drug substrates at this site. The locoregional distribution of multiple metabolic enzymes (including cytochromes), efflux transporters (including P-glycoprotein and breast cancer resistance proteins), and influx transporters (including the solute carrier family) along the intestine is summarized. Local delivery to the colonic mucosa remains a valuable therapeutic option. New therapies that target inflammatory mediators could improve the treatment of inflammatory bowel disease, and old and new anticancer molecules could, when delivered topically, prove to be beneficial adjuncts to the current systemic or surgical treatments. New issues such as pharmacogenomics, chronotherapeutics, and the delivery of prebiotics and probiotics are also discussed in this review. Targeting drugs to the colon utilizes various strategies, each with their advantages and flaws. The most promising systems are considered in the light of the physiological data which influence their in vivo behavior.

Colonic antigen administration induces significantly higher humoral levels of colonic and vaginal IgA, and serum IgG compared to oral administration.

It was hypothesised that different immune responses would be obtained following oral and colonic antigen administration, due to the significant differences in the immune environments of the colon and that of the small intestine. Antigen administration to the mouse colon (via the rectum) was found to generate different profiles of immune responses compared to oral administration (by gavage). Serum IgG and IgA levels in faecal and colonic extracts and in the vaginal wash were significantly higher following colonic administration of soluble (plus cholera toxin B subunit adjuvant) or encapsulated (in microspheres) antigen while smaller differences were seen in the small intestinal IgA levels. This reflects the compartmentalisation within the common mucosal immune system and suggests that the colon may be an appropriate vaccination target for diseases of the colon, and for sexually and vertically transmitted diseases. Antigen was also administered rectally and intramuscularly as controls. Colonic administration was superior to rectal administration, possibly due to the greater amounts of lymphoid tissue in the colon, although the immune response profiles were similar.

Microbiota-triggered colonic delivery: Robustness of the polysaccharide approach in the fed state in man

Polysaccharide-based colonic drug delivery requires that the polysaccharide in question avoids pancreatic digestion but undergoes fermentation by the colonic bacteria. Resistance of such dosage forms to pancreatic enzyme digestion is generally only tested in the fasted state, despite the higher enzymatic challenge in the fed state. Theophylline pellets coated with a polysaccharide-based (amylose) colon-specific film were administered to seven healthy volunteers (two-way crossover study, fed/fasted). The transit of the pellets through the gut was followed by gamma scintigraphy. The amount of drug released in the gut from the theophylline pellets was calculated after recovering and assaying any intact pellets in the faecal material. Of the drug released, the amount absorbed was measured using plasma profiling. Gastric empyting of pellets was delayed in the fed state, and this translated to a delayed colon arrival time. In both fed and fasted states, there was no drug release in the stomach or small intestine confirming the ability of the amylose in the coating to resist pancreatic digestion despite elevated enzyme levels in the fed state. Drug plasma levels were detected after the pellets arrived in the colon and there was a delayed Tmax in the fed state; the mean caecal arrival time in the fasted state was 5.5 ± 1.1 h and the Tmax was 9.3 ± 0.5 h, whereas in the fed state the mean caecal arrival time was 6.9 ± 2.1 h and the Tmax was 10.3 ± 0.8 h. On average, over 92% of the drug was released in the colon; the remaining was removed in faecal material. Bioavailability was similar (p>0.05) in both feeding states (26.0 ± 6.4 μg h/ml fasted and 24.4 ± 5.1 μg h/ml fed). In conclusion, feeding has no detrimental effects on the behaviour of this polysaccharide-based colonic delivery concept.

Decoupling the role of image size and calorie intake on gastric retention of swelling-based gastric retentive formulations: pre-screening in the dog model.

Gastric retention is postulated as an approach to improve bioavailability of compounds with narrow absorption windows. To elucidate the role of image size on gastric retention and pharmacokinetics, formulations with different image sizes and swelling kinetics but similar dissolution rates were designed and imaged in dogs. Diet had a clear effect, with increasing calorific intake prolonging retention in the dog model. In contrast to clinical observations, no obvious effect of image size on gastric retention was observed in the dog, with the larger gastric retentive (GR) and smaller controlled release (CR) formulations both demonstrating similar gastric emptying. Comparable pharmacokinetic profiles were observed for the two formulations, corroborating the imaging data and providing evidence of similar in vivo dissolution rates and dosage form integrity in the dog. Food, specifically meal composition, resulted in comparable enhancements in exposure in the dog and clinic due to prolonged gastric retention. However, differentiating retention based on image size in the dog was not feasible due to the smaller pyloric aperture compared to humans. This work illustrates that the dog is capable of determining the pharmacokinetic advantage of gastric retention relative to immediate release (IR) or CR formulations, however, has limited value in differentiating between CR and GR formulations.

Drug distribution in enteric microparticles

The aim of this study was to assess the distribution of three fluorescent drug or drug-like molecules in enteric microparticles. Microparticles were prepared using the pH-responsive methylmethacrylate polymer Eudragit L by an emulsion solvent evaporation process. In the process drug and polymer are dissolved in ethanol, and dispersed in a liquid paraffin external phase using sorbitan sesquioleate as stabiliser. The incorporation and distribution of riboflavin, dipyridamole and acridine orange into these microparticles were investigated using confocal laser scanning microscopy (CLSM). The influence of the physicochemical properties of the molecules (solubility in the inner phase, partition coefficient [ethanol/paraffin]) on the distribution, encapsulation efficiency and pH-responsive dissolution behaviour of the microparticles were examined. The drug that tended to partition in ethanol rather than liquid paraffin (riboflavin) was efficiently encapsulated and evenly distributed. In contrast, compounds which partitioned in favour of the liquid paraffin localised towards the surface of the microparticles and exhibited lower encapsulation efficiency (dipyridamole and acridine orange). All three sets of drug-loaded microparticles showed a limited release in acid (<10% release); drug distribution appeared to have a minimum effect on drug release. This microparticle technology has the potential to provide effective enteric drug release with a wide variety of molecules.

Drug Delivery to the Colon

The colon is a challenging target for drug delivery, as reaching the distal regions of the gastrointestinal tract presents significant physiological challenges and environmental barriers. Many approaches have been used to surmount these, with mixed success rates. Colonic delivery has historically been limited to treatment of local conditions such as inflammatory bowel disease. Latterly, efforts have also concerned delivery for treating colon cancer and for systemic delivery of selected compounds. Such approaches have concerned use of enteric coatings, sustained release systems, bacterially triggered treatments, or combinations of these. Possibilities are discussed in this chapter, along with historical experiences with systems for treating ulcerative colitis and Crohn’s disease.