Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome linked to biallelic inactivation of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH).Individuals with HLRCC are at risk to develop cutaneous and uterine leiomyomas and an aggressive form of kidney cancer. Pseudohypoxic drive-the aberrant activation of cellular hypoxia response pathways despite normal oxygen tension-is considered to be a likely mechanism underlying the etiology of this tumor. Pseudohypoxia requires the oxygen-independent stabilization of the ␣ subunit of the hypoxia-inducible transcription factor (HIF-1␣). Under normoxic conditions, proline hydroxylation of HIF-1␣ permits VHL recognition and subsequent targeting for proteasomal degradation. Here, we demonstrate that inactivating mutations of FH in an HLRCC-derived cell line result in glucose-mediated generation of cellular reactive oxygen species (ROS) and ROS-dependent HIF-1␣ stabilization. Additionally, we demonstrate that stable knockdown of FH in immortalized renal epithelial cells results in ROS-dependent HIF-1␣ stabilization. These data reveal that the obligate glycolytic switch present in HLRCC is critical to HIF stabilization via ROS generation.Patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) harbor germ line mutations of the FH gene, which encodes the tricarboxylic acid cycle enzyme fumarate hydratase, and affected individuals are at risk for the development of leiomyomas of the skin and uterus (fibroids) as well as kidney cancer (11,25,37). Genetic analysis of tumor samples indicates that FH acts as a tumor suppressor gene (37). The renal tumors that develop in HLRCC patients are notable for their aggressiveness, and effective systemic therapies are lacking at this time. Hence, identification of the molecular mechanisms that underlie the pathogenesis of this disease is needed to facilitate the development of targeted therapeutic strategies. Moreover, such studies may provide further insight into the role of mitochondrial metabolism in both normal and aberrant cellular physiology.FH catalyzes the enzymatic step of the tricarboxylic (TCA) cycle that hydrates fumarate to form malate. Proposed mechanisms for HLRCC tumor formation include apoptotic resistance, oxidative stress, and pseudohypoxic drive (10). Of these, most reports to date support a role for pseudohypoxic drive, based specifically on studies of hypoxia-inducible transcription factor 1␣ (HIF-1␣) expression. Pseudohypoxia is defined as the aberrant activation of hypoxia response pathways under normal oxygen conditions. HIF-1␣ expression is elevated both in HLRCC tumor specimens and in normoxic cells in which FH expression has been transiently suppressed with small interfering RNA (siRNA) (16). HIF-2␣ expression is also elevated in HLRCC tumor samples, although to a lesser extent than is HIF-1␣. In addition, there is clear evidence of upregulated transcription of HIF target genes in HLRCC tumor samples and in FH siRNA-treated cells (16,30). Fur...

Prednisolone 21-sulfate sodium (PDS) was synthesized as a colon-specific pro-drug of prednisolone with the expectation that it would be stable and non-absorbable in the upper intestine and release prednisolone by the action of sulfatase once it was delivered to the colon. In-vitro/in-vivo properties were investigated using rats as test animals. PDS was chemically stable at pH 1.2, 4.5, 6.8 and 8.0, and the apparent partition coefficient was 0.11 in 1-octanol/pH 6.8 buffer solution at 37 degrees C. PDS was stable on incubation with the contents of the stomach or small intestine. When PDS (0.1 mg equiv. of prednisolone) was incubated with the caecal contents (0.05 g), prednisolone was produced to a maximum 54% of the dose in 6 h and decreased thereafter, which suggested that reduction of the A ring took place in addition to the hydrolysis by sulfatase. After oral administration of PDS, a small portion of prednisolone was recovered from the cecal contents but not from the small intestine. Neither PDS nor prednisolone was detected in the plasma, suggesting that absorption of PDS is limited. The data demonstrate that the sulfate ester can serve as a novel colon-specific pro-moiety by limiting the absorption of the pro-drug in the upper intestine and releasing the active compound by the action of microbial sulfatase in the colon.

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.

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.

We 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 degrees C. DS was stable on incubation with buffer solutions of varied pH or with the upper intestinal contents of rats at 37 degrees 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.