Colon-specific Prodrug Research Articles

Synthesis and In Vitro Properties of Dexamethasone 21-Sulfate Sodium as a Colon-Specific Prodrug of Dexamethasone

ABSTRACTWe synthesized dexamethasone 21-sulfate sodium (DS) as a colon-specific prodrug of dexamethasone and investigated its properties. Introduction of a sulfate group to dexamethasone lowered the apparent partition coefficient from 52.5 to 0.27 in 1-octanol/pH 6.8 phosphate buffer at 37°C. DS was stable on incubation with buffer solutions of varied pH or with the upper intestinal contents of rats at 37°C for 24 h. On incubation with the cecal contents, DS was hydrolyzed by producing dexamethasone over 80% of the dose at 10 h. When DS was incubated with the cecal contents collected from trinitrobenzenesulfonic acid (TNBS)-induced colitic rats, the degree of prodrug hydrolysis and production of dexamethasone amounted to 70% of healthy rats. In comparison with prednisolone, hydrocortisone, and cortisone, dexamethasone was stable against bioinactivation by the cecal contents, a desirable property for the development of a colon-specific prodrug. These results demonstrated that DS might be delivered specifically to the colon as an intact form to produce dexamethasone in high yield, suggesting DS as a potential colon-specific prodrug of dexamethasone.

Synthesis and in vitro properties of prednisolone 21-sulfate sodium as a colon-specific prodrug of prednisolone.

Colon-specific delivery of glucocorticoids is highly desirable for the efficient treatment of inflammatory bowel disease. We synthesized prednisolone 21-sulfate sodium (PDS) as a colon-specific prodrug of prednisolone (PD) and investigated its properties using rats as test animals. We expected that introduction of sulfate ester as a sodium salt might increase the hydrophilicity and restrict the absorption in the GI tract. If PDS is stable and nonabsorbable in the upper intestine, it will be delivered to the colon as an intact form, where it hydrolyze by the sulfatase to release PD. Compared with PD, the solubility of PDS increased and the apparent partition coefficient decreased greatly. PDS was stable on incubation with pH 1.2 and 6.8 buffer solutions and with the contents of the stomach and small intestine. On incubation with the cecal contents, PDS decreased to 9.6% of the dose in 10 h producing PD. The amount of PD increased to give a maximum 54% of the dose and decreased. As a control, when PD was incubated with the cecal contents, it decreased to 29% of the dose in 8 h, which implied that reduction of PD proceeded under such conditions. These results suggested that hydrolysis of PDS took place to produce and accumulate PD, which decreased by reduction as the incubation period extended. Our results suggested that PDS can be a promising colon-specific prodrug of PD, and sulfate ester group might serve as a potential colon-specific promoiety, especially for the drugs which are resistant to reduction in the colon.

Synthesis and properties of 5-aminosalicyl-taurine as a colon-specific prodrug of 5-aminosalicylic acid.

5-Aminosalicylic acid (5-ASA) is an active ingredient of therapeutic agents used for Crohn's disease and ulcerative colitis. Because it is absorbed rapidly and extensively in the upper intestine, delivery of the agent specifically to the colon is necessary. We selected taurine as a colon-specific promoiety and designed 5-aminosalicyltaurine (5-ASA-Tau) as a new colon-specific prodrug of 5-aminosalicylic acid (5-ASA). It was expected that introduction of taurine would restrict the absorption of the prodrug and show additive effect to the anti-inflammatory action of 5-ASA after hydrolysis. 5-ASA-Tau was prepared in good yield by a simple synthetic route. The apparent partition coefficient of 5-ASA-Tau in 1-octanol/pH 6.8 phosphate buffer or CHCl3/pH 6.8 phosphate buffer was 0.10 or 0.18, respectively, at 37 degrees C. To determine the chemical and biochemical stability in the upper intestinal environment, 5-ASA-Tau was incubated in pH 1.2 and 6.8 buffer solutions, and with the homogenates of tissue and contents of stomach or small intestine of rats at 37 degrees C. 5-ASA was not detected from any of the incubation medium with no change in the concentration of 5-ASA-Tau. On incubation of 5-ASA-Tau with the cecal and colonic contents of rats, the fraction of the dose released as 5-ASA was 45% and 20%, respectively, in 8 h. Considering low partition coefficient and stability in the upper intestine, 5-ASA-Tau might be nonabsorbable and stable in the upper intestine. After oral administration, it would be delivered to the colon in intact form and release 5-ASA and taurine. These results suggested 5-ASA-Tau as a promising colon-specific prodrug of 5-ASA.

Synthesis and Evaluation of N-acyl-2-(5-Fluorouracil-1-yl)-d,l-Glycine as a Colon-Specific Prodrug of 5-Fluorouracil

N-nicotinyl-2-(5-fluorouracil-1-yl)-D,L-glycine (NFG) and N-isonicotinyl-2-(5-fluorouracil-1-yl)-D,L-glycine (INFG) were synthesized as colon-specific prodrugs of 5-fluorouracil (5-FU). As N-aromatic acyl amides of glycine, they are expected to be stable in the upper intestine and delivered to the colon as an intact form if they are nonabsorbable. Microbial hydrolysis of amide bond in the colon will give 2-(5-fluorouracil)-D,L-glycine, which releases 5-FU by spontaneous decomposition. NFG and INFG were soluble in water and stable in pH 1.2 and 7.4 buffer solutions. The apparent partition coefficient of NFG or INFG in 1-octanol/pH 7.4 phosphate buffer solution at 37 degrees was 0.025 or 0.024, respectively. On incubation with cecal contents of rats, conversion of NFG or INFG proceeded only 9 or 5% in 8 h, respectively, producing 5-FU and a metabolite. The metabolite formation was inhibited in the presence of diazouracil, a dihydrouracil dehydrogenase inhibitor. The HPLC retention time of the metabolite from the incubation of 5-FU, NFG, or INFG with cecal contents was identical to dihydro-5-fluorouracil (dihydro-5FU). When N-nicotinyl-2-hydroxy-D,L-glycine methyl ester (NHGM) was incubated with the cecal contents, the extent of amide bond hydrolysis was 85% in 24 h. The result suggested that steric hindrance imposed by 5-FU at 2-position of glycine retarded the hydrolysis of the amide bond in NFG or INFG and suppressed the prodrug conversion.

Colon-Specific Prodrugs of 5-Aminosalicylic Acid: Synthesis and In Vitro/In Vivo Properties of Acidic Amino Acid Derivatives of 5-Aminosalicylic Acid

5-aminosalicyl-L-aspartic acid (5-ASA-Asp) and 5-aminosalicyl-L-glutamic acid (5-ASA-Glu) were synthesized and their properties as colon-specific prodrugs of 5-aminosalicylic acid (5-ASA) were investigated employing rats as test animals. Incubation of 5-ASA-Asp and 5-ASA-Glu with the homogenates of tissue and contents of stomach or small intestine released no 5-ASA, indicating that they were stable in this condition. Incubation of 5-ASA-Asp with the cecal contents released 5-ASA 37%, whereas 5-ASA-Glu released only 8% of the dose in 16 h. Plasma concentration of 5-ASA-Asp after intravenous administration decreased rapidly and became undetectable in 60 min. No 5-ASA was detected in the blood, which indicated 5-ASA-Asp was stable in the plasma. After oral administration of 5-ASA-Asp, concentration of 5-ASA, its metabolite N-acetyl-5-ASA, and 5-ASA-Asp in the plasma, feces, and urine was determined. In the plasma, 5-ASA-Asp was not detected and the concentration of 5-ASA or N-acetyl-5-ASA was very low. About 33% of the administered dose was recovered as 5-ASA and N-acetyl-5-ASA and 43% as 5-ASA-Asp from feces, and 20% as 5-ASA and N-acetyl-5-ASA and 1% as 5-ASA-Asp from urine in 24 h. These results suggested that most of 5-ASA-Asp was delivered to the large intestine and about half of the administered dose was activated to liberate 5-ASA. After oral administration of free 5-ASA, fecal recovery was only 7% of the dose in 24 h and more than 80% was recovered from urine. Comparing 5-ASA-Asp and free 5-ASA, the amount of 5-ASA available in the large intestine was much larger, while the amount of 5-ASA in urine, which might be related to the systemic toxicity of 5-ASA, was much lower by the administration of 5-ASA-Asp than free 5-ASA.

Preparation of prednisolone-appended α-, β- and γ-cyclodextrins: Substitution at secondary hydroxyl groups and in vitro hydrolysis behavior

The carboxyl group of prednisolone 21-hemisuccinate was conjugated to one of the hydroxyl groups of α-, β-, and γ-cyclodextrins using a coupling agent, carbonyldiimidazole. The direct coupling produced prednisolone-appended cyclodextrin conjugates in which the drug is selectively introduced at one of the secondary hydroxyl groups of cyclodextrins through an ester linkage. The aqueous solubility (> 50% w/v at 25°C) of these conjugates was much higher than those of prednisolone and its 21-hemisuccinate. Prednisolone was slowly released from the conjugate: the percents of prednisolone and its hemisuccinates released from the α-, β-, and γ-cyclodextrin conjugates were 49, 57, and 85%, respectively, for 24-h. The release pathway is proposed to be via two fast acyl migrations between the 2- and 3-hydroxyl groups of cyclodextrins and between the 21- and 17-hydroxyl groups of prednisolone. The slow release of prednisolone from the ester conjugates was in sharp contrast to the fast release of the prednisolone amide conjugate reported previously. Because of relatively slow and/or site-specific release properties, the present prednisolone-appended cyclodextrin conjugates may be of value as an orally administered delayed-release and/or colon-specific prodrug. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:493–503, 2001

Synthesis and Properties of Dextran–Nalidixic Acid Ester as a Colon-Specific Prodrug of Nalidixic Acid

Dextran–nalidixic acid ester (dextran-NA) with a varied degree of substitution (DS) was synthesized as a colon-specific prodrug of nalidixic acid (NA). Solubility in water (mg/ml) of dextran-NA with DS (mg NA/100 mg dextran-NA) of 7, 19, or 32 was 57.57 (equivalent to 4.00 mg NA/ml), 0.53 (equivalent to 0.10 mg NA/ml), or 0.03 (equivalent to 0.01 mg NA/ml), respectively, and that for NA was 0.03 at 25°C. To ensure the chemical stability of dextran-NA at conditions similar to those of the stomach and small intestine, dextran-NA was placed in a solution of pH 1.2 hydrochloric acid buffer or pH 6.8 phosphate buffer and incubated at 37°C; no NA was detected during the 6 h of the incubation period, which indicated that dextran-NA might be chemically stable during the transit through the gastrointestinal tract. Degree of depolymerization (%) by dextranase determined by the 2,4-dinitrosalicylic acid (DNS) method at 37°C for dextran-NA with DS of 7, 19, or 32 was 81, 68, or 8, respectively, in 8 h, and that for dextran was 91. When dextran-NA (equivalent to 50 μg of NA) with a DS of 7 or 17 was incubated with cecal contents (100 mg) of rats at 37°C, the extent of NA released in 24 h was 41% or 32% of the dose, respectively. NA was not liberated from the incubation of dextran-NA with the homogenate of tissue and contents of the small intestine.

Chemoprevention of colon cancer carcinogenesis by balsalazide: inhibition of azoxymethane-induced aberrant crypt formation in the rat colon and intestinal tumor formation in the B6-Min/+ mouse.

Non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin have been shown to suppress colon carcinogenesis and in some cases reduce the size of colorectal polyps. Balsalazide disodium (BSZ) is a colon-specific prodrug of the salicylate, 5-aminosalicylic acid. The aim of the present study was to test the chemopreventive activity of BSZ in two established animal models of colon tumorigenesis, azoxymethane-induced aberrant crypt formation in the rat and intestinal tumor formation in the B6-Min/+ mouse. Aberrant crypt foci (ACF) were induced in Fischer 344 rats via 2 subcutaneous injections of azoxymethane (20 mg/kg). BSZ was supplied in the drinking water for 8 weeks and ACF quantitated. B6-Min/+ mice were treated from 55 days of age for 90 days and intestinal tumors scored for number, size and location. BSZ treatment of AOM-injected rats reduced ACF formation in a dose-dependent manner by 60% with the greatest effect observed on ACF with 4 or more crypts. In B6-Min/+ mice a dose-dependent reduction of intestinal tumor number was observed which reached 80% in the distal small intestine and colon. A preliminary mechanistic study in cultured human colon cancer cells showed that both BSZ and 5-ASA inhibited colon cancer cell proliferation in vitro. However, 5-ASA but not BSZ produced changes consistent with the induction of apoptosis. BSZ produces a dose-dependent chemopreventive effect on colon carcinogenesis. A possible mechanism is consistent with the inhibition of cellular proliferation and the induction of apoptosis.

Synthesis and evaluation of 5-aminosalicyl-glycine as a potential colon-specific prodrug of 5-aminosalicylic acid.

As a new colon-specific prodrug of 5-aminosalicylic acid (5-ASA), 5-aminosalicyl-glycine (5-ASA-Gly) was prepared by a simple synthetic route in good yield. Apparent partition coefficients of 5-ASA-Gly were lower than those of 5-ASA, which determined in CHCl3/pH 6.8 buffer or n-octanol/pH 6.8 buffer system. Stability of 5-ASA-Gly by peptidases was investigated by incubation of 5-ASA-Gly with the homogenates of tissue and contents of stomach, proximal small intestine or distal small intestine of rats at 37 degrees C. 5-ASA was not detected, indicating that the prodrug was stable in the upper intestine. The amount of 5-ASA liberated from incubation of the prodrug in cecal or colonic contents of rats was about 65% or 27% in 8 hrs, respectively, which indicated that the prodrug activation took place more readily in the rat cecum whose bacterial counts are high like human colon. Results from in vitro experiments suggested 5-ASA-Gly as a promising candidate of a colon-specific prodrug of 5-ASA.

Synthesis and properties of dextran-5-aminosalicylic acid ester as a potential colon-specific prodrug of 5-aminosalicylic acid.

Dextran-5-aminosalicylic acid ester (dextran-5-ASA) was synthesized as a colon-specific prodrug of 5-aminosalicylic acid (5-ASA) which is active against inflammatory bowel diseases. Chemical stability of dextran-5-ASA in the bath of pH 1.2 or 6.8 was investigated at 37 degrees C for 6 hrs, and 5-ASA was not released on such conditions. Depolymerization (%) of dextran-5-ASA by dextranase with the degree of substitution (DS) of 18, 23, or 30 was 92, 62 or 45 in 8 hrs respectively, but was not affected by the MW of dextran (9,000, 40,600, 80,200 or 580,000). Distribution of 5-ASA in dextran, determined by gel filtration chromatography, appeared to be relatively uniform. Incubation of dextran-5-ASA (DS 18) in cecal contents of rats released 20% (28 g) and 35% (49 g) of 5-ASA in 8 hrs and 24 hrs, respectively, but no 5-ASA was liberated from small intestinal contents.

A novel colon-specific steroid prodrug enhances sodium chloride absorption in rat colitis.

A recently synthesized novel colon-specific dexamethasone prodrug, dexamethasone-beta-D-glucuronide, delivers efficacious amounts of dexamethasone to the colon with limited adrenal suppressive effects. During experimentally induced colitis in rats, the dexamethasone prodrug is significantly more potent than free dexamethasone in improving colonic fluid and electrolyte absorptive injury. The present studies examined whether the improvement in colonic absorption seen with the prodrug occurred as a consequence of alterations in sodium and chloride epithelial transport. The efficacy of the dexamethasone prodrug and free dexamethasone were tested in an acetic acid-induced rat model of colitis. Healing of the induced colitis was assessed by measuring net colonic fluid absorption and surface area ulceration. Transmural unidirectional fluxes of 22Na and 36Cl across sheets of colonic mucosa were measured in Ussing chambers. Treatment of colitis with the prodrug delivered a sixfold higher concentration of dexamethasone to the colon than did treatment with the free drug. The prodrug accelerated healing of colitis by returning in vivo colonic fluid absorption to normal and virtually eliminated colonic macroscopic ulceration, whereas the free drug did not. In vitro transmural fluxes demonstrated that, in addition to repair of mucosal integrity, the prodrug enhanced electroneutral sodium chloride absorption over and above that seen in control animals or after treatment with the free drug. Both the prodrug and the free drug limited theophylline-mediated net chloride and sodium secretion, an effect that would be consistent with the antidiarrheal effect induced by these drugs in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Background/Aims: Dexamethasone-β- d-glucuronide, a colon-specific prodrug of dexamethasone, may be useful in the treatment of ulcerative colitis and Crohn's 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-β- 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-β- d-glucuronide delivers efficacious amounts of dexamethasone to the large intestine from lower doses than free dexamethasone.