Harold W. Nolen

In Vitro Evaluation of Dexamethasone-β-D-Glucuronide for Colon-Specific Drug Delivery

Dexamethasone-β-D-glucuronide is a potential prodrug for colonic delivery of the antiinflammatory corticosteroid dexamethasone. Previous studies [T. R. Tozer et al., Pharm. Res. 8:445–454 (1991)] indicated that a glucoside prodrug of dexamethasone was susceptible to hydrolysis in the upper gastrointestinal tract. Resistance of dexamethasone-β-D-glucuronide to hydrolysis in the upper gastrointestinal tract was therefore assessed. Conventional, germfree, and colitic rats were used to examine enzyme levels along the gastrointestinal tract to compare the stability of two model substrates (p-nitrophenyl-β-D-glucoside and -β-D-glucuronide) and to evaluate the prodrug dexamethasone-β-D-glucuronide. Hydrolytic activity was examined in the luminal contents, mucosa, and underlying muscle/connective tissues in all three types of rats. Enzymatic activity (β-D-glucosidase and β-D-glucuronidase) was greatest in the lumen of cecum and colon of conventional rats. In contrast, germ-free rats exhibited relatively high levels of β-D-glucosidase activity (about 80% of total activity in the conventional rats) in the proximal small intestine (PSI) and the distal small intestine (DSI). Rats with induced colitis (acetic acid) showed reduced levels of luminal β-D-glucuronidase activity in the large intestine; however, β-D-glucosidase activity was relatively unchanged relative to that of the conventional rat. Mucosal β-D-glucuronidase activity was significantly lower in the colitic rats compared with that in the conventional animals. Despite reduced luminal levels of β-D-glucuronidase activity in the colitic rats, there was still a sharp gradient of activity between the small and the large intestines. Permeability of the glucoside and glucuronide prodrugs of dexamethasone through a monolayer of Caco-2 cells was relatively low compared to that of dexamethasone. The results indicate that dexamethasone-β-D-glucuronide should be relatively stable and poorly absorbed in the upper gastrointestinal tract. Once the compound reaches the large intestine, it should be hydrolyzed to dexamethasone and glucuronic acid. Specificity of colonic delivery in humans should be even greater due to lower levels of β-D-glucuronidase activity in the small intestine compared with that in the laboratory rat.

Colonic delivery of dexamethasone from a prodrug accelerates healing of colitis in rats without adrenal suppression

BACKGROUND/AIMS Dexamethasone-beta-D-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohns colitis. The aim of this study was to evaluate colonic delivery and efficacy of this prodrug in the rat. METHODS Distribution of dexamethasone in luminal contents and tissues of the gastrointestinal tract and in plasma was measured after oral administration of dexamethasone-beta-D-glucuronide or free dexamethasone. Efficacy of the prodrug and free drug was tested in an acetic acid-induced rat colitis model. Healing of induced colitis was assessed by measuring net intestinal fluid absorption, colonic surface area of ulceration, histology, and myeloperoxidase activity. Glucocorticosteroid toxicity was evaluated with serum corticosterone and plasma adrenocorticotropic hormone levels. RESULTS The drug delivery index (a measure of relative targeting efficiency) was 6.7 and 8.6 in the cecal and colonic mucosa, respectively. The prodrug was significantly more potent than free drug in improving net colonic fluid absorption while significantly reducing surface area of ulceration and histological grade in colitic rats. Treatment with free dexamethasone significantly reduced serum corticosterone levels to subnormal levels, and treatment with the prodrug maintained serum corticosterone and plasma adrenocorticotropic hormone levels near control levels. CONCLUSIONS The prodrug dexamethasone-beta-D-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.

The measurement of urinary oxalic acid by derivatization coupled with liquid chromatography

Abstract A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o -phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C 8 column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.

Percutaneous penetration of methyl phosphonate antisense oligonucleotides

Abstract A series of antisense methyl phosphonate oligonucleotides (MPOs) were evaluated in vitro for skin penetration and retention using either hairless mouse or human cadaver skin. Several skin penetration enhancers were used to promote uptake of MPOs ranging in molecular weight from 1834 to 5500. In general, as the molecular weight of the MPOs increased, the penetration rate decreased. Tape stripping experiments with both hairless mouse skin and human cadaver skin indicated that the stratum corneum is the primary barrier to penetration. Comparison of a 14-mer MPO and the same MPO modified by the introduction of one negative charge (phosphate linkage) per molecule reduced the skin permeability by about 10-fold. The amount of MPO retained in the dermis at the end of the 24 h permeation experiments indicated that most of the compounds were able to reach a nominal target concentration of 1.0 μM. The largest MPO tested (18-mer, Mol. Wt 5500) gave the least amount of material penetrating through human cadaver skin, but depending on the vehicle, was retained in nearly sufficient amounts in the target tissues (dermis). Thus, the 18-mer represents a logical candidate for further evaluation due to the potential for delivery into the target tissue with limited systemic exposure.

Menthol-β-D-Glucuronide: A Potential Prodrug for Treatment of the Irritable Bowel Syndrome

Menthol-β-D-glucuronide is a potential prodrug for colonic delivery of the spasmolytic agent menthol. Menthol is the primary constituent of peppermint oil, which is used to treat the irritable bowel syndrome. The chemical stability of menthol-β-D-glucuromde was assessed at various pHs (1.5,4.5, 6.0 and 7.4) over a 4 to 24 h period at 37°C. The prodrug was stable, i.e., there was less than 0.1% hydrolysis of the prodrug, at pHs of 4.5, 6.0 and 7.4. At pH 1.5, the prodrug was about 20% hydrolyzed over a 4 h period suggesting the need for an enteric coating to prevent premature hydrolysis in the stomach. The stability of the prodrug was also assessed in luminal contents of the laboratory rat and in human stool samples. These studies were performed at concentrations designed to assess relative velocities of hydrolysis (i.e., substrate concentrations in excess of the Km). The prodrug was stable in luminal contents of the rat stomach, proximal small intestine, and the distal small intestine. The rate of hydrolysis of menthol-β-D-glucuronide was 6.26 ± 2.88 nmol min−l mg−l and 2.34 ± 1.22 nmol min−l mg−l in luminal contents of the rat cecum and colon, respectively. The hydrolysis rate of menthol-β-D-glucuronide was lower in human stool samples (0.52 ± 0.46 nmol min−1 mg−!). The prodrug had a measured log octanol/buffer partition coefficient of −1.61 suggesting it should be poorly absorbed from the lumen of the gastrointestinal tract. The data support the hypothesis that menthol-β-D-glucuronide is a candidate for the delivery of menthol to the large intestine under in vivo conditions.

Steady-state pharmacokinetics of corticosteroid delivery from glucuronide prodrugs in normal and colitic rats.

Ulcerative colitis and Crohns colitis are chronic intestinal diseases usually treated with various nonsteroidal antiinflammatory agents to maintain remission. Corticosteroids, while useful in acute treatment of these diseases, present side‐effects generally too serious to allow maintenance therapy. Colon‐specific drug delivery may permit use of corticosteroids for maintenance therapy if doses can be reduced while maintaining efficacy. In this study, two prodrugs (dexamethasone‐β‐D‐glucuronide (DXglrd) and budesonide‐β‐D‐glucuronide (BUDglrd)) were administered by intragastric (ig) infusion to conventional and colitic rats. In addition, dexamethasone (DX) and budesonide (BUD) were administered either ig or subcutaneously (sc) to healthy and colitic rats. Colon‐specific delivery was assessed using the drug delivery index (DDI). In conventional rats, DDIs for DXglrd ranged from about five to as high as 11 in the luminal contents relative to DX administered sc or ig. DDI values were also elevated in the mucosa of both healthy and colitic rats following ig administration of DXglrd. BUD was delivered somewhat less effectively from BUDglrd to the rat large intestine than was DX from DXglrd. The data are consistent with efficacy studies and support the conclusion that local delivery of corticosteroids to the large intestine is due, at least in part, to higher levels of drug delivery into the mucosal tissues. ©1997 John Wiley & Sons, Ltd.

Combined chromatographic—isotopic dilution analysis of fecapentaenes in human feces

Fecapentaene-12 (FP-12) and fecapentaene-14 (FP-14) are genotoxic unsaturated ether lipids produced by colonic bacteria in man. We have developed and applied to feces collections from normal volunteers direct isotopic dilution procedures using tritium-labeled (at C5) FP-12 and FP-14 for measuring these compounds. FPs were recovered from feces by solvent extraction, silica cartridge clean-up, and analytical liquid chromatography. Low levels of FP-12 and FP-14 (less than 0.1 to 2.4 micrograms/g of freeze-dried feces) were observed. Identity of chromatographic peaks was established by co-elution and by ultraviolet absorption spectra obtained via photodiode array scanning. Two unknown peaks were tentatively identified from absorption spectra as closely related compounds with increased (hexane?) or decreased (tetraene?) number of double bonds. Levels of FPs increased after incubation of feces at 37 degrees C for 96 h under anaerobic conditions and pre-FP-12 and pre-FP-14 peaks were observed, which showed identical spectra with authentic FPs. These were interpreted to be isomeric forms of the all-trans-[3H]FPs used for the isotopic dilution analysis. Total FPs (including pre-FP) yielded a range of 0.3-80 micrograms FP-12 and 2.8-44 micrograms FP-14 per g of freeze-dried feces from the study group.