In vivo evaluation of the third generation biodegradable stent: a novel approach to avoiding the forgotten stent syndrome.

PURPOSE: Ureteral stents are prone to irritation, encrustation and infection, and they require additional procedures for removal. Furthermore, indwelling polymer stents are often forgotten with devastating consequences to the patient. We describe the degradation time, and physiological and histological responses elicited by a novel biodegradable ureteral stent in a porcine model. MATERIALS AND METHODS: A total of 16 female Yorkshire pigs were used in the study. Ten biodegradable Uriprene™ stents and 6 biostable Polaris™ stents were cystoscopically inserted unilaterally in 2 groups of animals. Excretory urogram, and blood and urine tests were performed on different days until day 28. Biostable stents were removed on day 21. On day 28 all pigs underwent necropsy for microscopic and histological evaluation. RESULTS: Nine of the 10 biodegradable stents (90%) degraded completely by 4 weeks, while 1 pig had 3 fragments smaller than 1.5 cm in the bladder. Excretory urogram showed equivalent drainage and significantly less hydronephrosis in biodegradable stented kidneys. Blood and urine parameters were similar in the 2 groups. A transient increase in serum creatinine on day 7 in 40% of the pigs with a degradable stent resolved by day 10. There were significantly fewer abnormal histological findings in the degradable stent group. We evaluated drainage characteristics in an unobstructed ureter and results may not be representative of what develops in obstructed ureters. CONCLUSIONS: The third generation biodegradable stent is a safe, effective alternative to conventional polymer stents, resulting in equivalent drainage and less hydronephrosis.

Investigation of drug delivery by iontophoresis in a surgical wound utilizing microdialysis.

PURPOSE: This study investigated the penetration of lidocaine around and through a sutured incision following the application of iontophoretic and passive patches in the CD Hairless rat. MATERIALS AND METHODS: Concentrations in localized areas (suture, dermis, subcutaneous, and vascular) were determined using microdialysis sampling followed by analysis using liquid chromatography with UV detection. RESULTS: Iontophoresis significantly enhanced the dermal penetration of lidocaine. In an intact skin model, dermal concentrations were 40 times greater following iontophoretic delivery compared to passive delivery. In a sutured incision model, iontophoresis enhanced localized concentrations in the dermis, suture, and subcutaneous regions by 6-, 15-, and 20-fold, respectively. Iontophoretic delivery to a region containing a sutured incision was focused to the incision resulting in a greater increase in the suture concentration and in the subcutaneous region directly below the incision. CONCLUSIONS: The four microdialysis probe design was successful in the determination of localized drug penetration in a sutured incision model. Iontophoresis enhanced skin penetration and allowed for site specific delivery when applied to a sutured incision.

Indole-3-carbinol, but not its major digestive product 3,3'-diindolylmethane, induces reversible hepatocyte hypertrophy and cytochromes P450.

Indole-3-carbinol (I-3-C) and 3,3'-diindolylmethane (DIM) have been shown to reduce the incidence and multiplicity of cancers in laboratory animal models. Based on the observation that I-3-C induced hepatocyte hypertrophy when administered orally for 13 weeks to rats, a treatment and recovery study was undertaken to test the hypothesis that the induction of hepatocyte hypertrophy and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses. Additionally, we directly compared the effects of I-3-C to those of its principle metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and 50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day) and then one-half of the animals were not treated for an additional 28 days. Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2, 2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28 days significantly increased liver weights and CYP enzyme activities. The effects in males were more pronounced and persistent after recovery than the effects in females. The increased organ weights returned to control values after treatment. Conversely, DIM did not alter liver weights and had no effect on CYP activities after the 28-day treatment. Some changes in CYP activities were measured after the DIM recovery period but the magnitudes of the changes were considered biologically insignificant. The results show that I-3-C, but not DIM, induces reversible adaptive responses in the liver.

Evaluation of hydrogen ion concentrations in prostates from rats and dogs using fluorescent confocal microscopy.

The knowledge of intracellular spatial distribution of pH in prostates in animal models reflective of human prostate may have implications for drug development upon pH dependent drug delivery and activity. Freshly dissected prostate tissues (in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes and viewed using confocal microscopy. Images were initially taken in tissues perfused with RPMI-1640 medium. Calibration in situ was performed with high potassium buffers of known pH containing nigericin. Acetoxymethyl ester carboxy-SNARF-1 was visible in epithelial cells (but not stroma) in rat and dog prostates. The pH of lysosomes in prostate epithelial cells was 5.2 as determined by fluorescence of Lyso Sensor Green DND-189. A method of in situ confirmation of tissue viability was developed by a secondary loading and visualization of the BCECF fluorescent dye. Besides the direct measurement of the pH in rat and dog tissues (pH approximately 7.0), a method of pH measurement in prostate tissue (rather than in cell culture) was developed.

Preclinical toxicity of a geldanamycin analog, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), in rats and dogs: potential clinical relevance.

PURPOSE: 17-DMAG is a hydrophilic derivative of the molecular chaperone inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG; NSC-330507), which is currently being evaluated for the treatment of cancer in clinical trials. 17-DMAG offers a potential advantage over 17-AAG because its aqueous solubility eliminates the need for complicated formulations that are currently used for administration of 17-AAG. In addition, 17-DMAG undergoes only limited metabolism compared to 17-AAG. The present results are from preclinical toxicity studies evaluating 17-DMAG in rats and dogs. METHODS: Doses of 0, 2.4, 12 and 24 mg/m2 per day were administered to rats, while dogs received doses of 0, 8 or 16 mg/m2 per day. In both species, 17-DMAG was administered i.v. (slow bolus for rats; 1-h infusion for dogs) daily for 5 days. An additional cohort of dogs received 16 mg/m2 per day orally for 5 days. Clinical observations were noted, and standard hematology and clinical chemistry parameters were monitored. Selected tissues were evaluated microscopically for drug-related lesions. Tissue and plasma 17-DMAG concentrations were measured by HPLC/MS at selected time-points on days 1 and 5. RESULTS: Daily i.v. administration of 17-DMAG at doses of 24 mg/m2 per day in rats or 16 mg/m2 per day in dogs produced lethality on day 6, approximately 24 h following the last dose. Body weight loss was common in rats and dogs. Drug-related gastrointestinal, bone marrow and hepatic toxicities were also common in rats and dogs. Dogs also exhibited signs of renal and gallbladder toxicity. Plasma concentrations of 17-DMAG increased proportionately with dose in rats and disproportionately with dose in dogs. In rat tissues, however, only fourfold to sixfold increases in 17-DMAG concentrations were observed with a tenfold increase in dose. The highest concentrations of 17-DMAG were found in the liver of rats, with progressively lower concentrations in the spleen, lung, kidney and plasma. Regardless of the route of administration, higher drug concentrations were present in plasma (rat and dog) and tissue (rat) samples obtained on day 5 compared to those obtained on day 1. Although plasma concentrations decreased with time, 17-DMAG was still detected in dog plasma for at least 24 h after drug administration. CONCLUSIONS: With the recent approval of 17-DMAG for clinical use, the data generated from these preclinical studies will provide guidance to clinicians as they administer this drug to their patients. The MTD of 17-DMAG was 12 mg/m2 per day in rats and 8 mg/m2 per day in dogs; therefore, the recommended starting dose for phase I trial is 1.3 mg/m2 per day for 5 days. Gastrointestinal and bone marrow toxicity were dose-limiting in rats, and gastrointestinal, renal, gallbladder and bone marrow toxicity were dose-limiting in dogs. All adverse effects were fully reversible in surviving animals after treatment was complete.

Toxicogenomics of resveratrol in rat liver.

Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the modulation of liver genes at the high dose of resveratrol may implicate the potential toxicity observed.

The effects of tetrathiomolybdate (TTM, NSC-714598) and copper supplementation on fertility and early embryonic development in rats.

Based on its ability to chelate copper, TTM is being studied as an antiangiogenic agent for cancer therapy. The purpose of this study was to evaluate the toxicity of TTM and the protection of copper supplementation on the reproductive capability of male and female CD rats. Doses of 0, 1, 4, and 12 mg/kg/day with copper supplementation (110 mg/kg of diet) were given by gavage. There were no effects on the estrous cycle or reproductive indices, or maternal toxicity in any female dose group. Male rats given 12 mg/kg/day showed significant decreases in body weight gains and food consumption, and anemia. Serum ceruloplasmin levels were dose-dependently decreased in all male dose groups. Reduced epididymal weights, sperm counts, and sperm motility, sperm morphologic abnormalities and histopathologic changes in testis and epididymis occurred only at 12 mg/kg/day. Dietary copper supplementation prevented the adverse sperm effects produced by 12 mg/kg/day of TTM.

Resveratrol-associated renal toxicity.

Resveratrol, (3,5,4'-trihydoxystilbene) a compound found in grapes, mulberries, and peanuts, has antimycotic, antiviral, and beneficial cardiovascular and cancer preventive activities. It is being developed for several clinical indications. To evaluate the potential toxicity of resveratrol, rats were administered by gavage 0, 300, 1000, and 3000 mg trans-resveratrol per kilogram body weight per day for 4 weeks. Most of the adverse events occurred in the rats administered 3000 mg per kilogram body weight per day. These included increased clinical signs of toxicity; reduced final body weights and food consumption; elevated BUN, creatinine, alkaline phosphatase, alanine aminotransferase, total bilirubin, and albumin; reduced hemoglobin, hematocrit, and red cell counts; and increased white cell counts. Increases in kidney weights and clinically significant renal lesions, including an increased incidence and severity of nephropathy, were observed. Diffuse epithelial hyperplasia in the bladder was considered, equivocal and of limited biological significance. No histological effects on the liver were observed, despite the clinical chemistry changes and increased liver weights in the females. Effects seen in the group administered 1000 mg resveratrol per kilogram body weight per day included reduced body weight gain (females only) and elevated white blood cell count (males only). Plasma resveratrol concentrations in blood collected 1 h after dose administration during week 4 were dose related but were relatively low given the high dosage levels; conjugates were not measured. Under the conditions of this study, the no observed adverse effect level was 300 mg resveratrol per kilogram body weight per day in rats.

Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry.

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.

Pharmacokinetics and tissue distribution of orally administered lycopene in male dogs.

Consumption of lycopene, the predominant carotenoid in tomatoes and tomato products, is associated with reduced prostate cancer risk. The purpose of this study was to measure the pharmacokinetics and tissue distribution of lycopene after oral administration to male dogs. After single doses of 10, 30 and 50 mg/kg body weight (BW) lycopene to 2 dogs/dose, the mean half-life was 36 h and the plasma systemic exposure levels (AUC(0-)( infinity ), area under the curve) after the 30 and 50 mg/kg BW doses were similar. In a repeat dose study, 30 mg/(kg BW. d) administered orally to six dogs for 28 d resulted in steady-state plasma concentrations between 785 and 997 nmol/L lycopene. Apparent clearance, volume of distribution and apparent elimination half-life were 2.29 L/(h. kg), 96 L/kg and 30.5 h, respectively. Dogs were killed 1 or 5 d after the last dose and 23 tissues were collected for lycopene analysis. Lycopene concentrations were highest in liver, adrenals, spleen, lymph nodes and intestinal tissues. Liver lycopene concentrations were 66 and 91 nmol/g 1 and 5 d after cessation of treatment, respectively. Prostate lycopene concentrations were < 0.2 nmol/g both 1 and 5 d after dosing ceased (<0.4% of liver concentrations). Although 70% trans-lycopene was used in the dosing material, most of the lycopene identified in plasma and tissues was cis-lycopene.

Preclinical toxicity studies of kahalalide F, a new anticancer agent: single and multiple dosing regimens in the rat.

PURPOSE: Kahalalide F (KF) is a new anticancer agent currently in clinical trials for solid tumors, including prostate cancer. During the preclinical development of this drug, the studies reported here were conducted to determine the acute and multiple dose toxicities of KF when administered intravenously (i.v.) to rats. This dosing route is the intended route of clinical administration. METHODS: KF was administered i.v. to male and female CD rats using single- and multiple-dose (daily for 5 days) schedules. Animals were observed for clinical signs, and body weight, hematology, and clinical chemistry parameters determined. Animals were necropsied, gross observations and organ weights recorded, and numerous tissues were collected and examined microscopically. RESULTS: KF produced lethality at 375 and 450 microg/kg in males and females, respectively, and the maximum tolerated dose (MTD) was estimated to be 300 microg/kg (1800 microg/m(2)). The nervous system appeared to be a potential site of action for the production of lethality. Single-dose administration of KF at 150 and 300 microg/kg produced organ toxicity in which the kidney was the primary target. Injury to distal convoluted tubules was the most toxicologically significant lesion, and was observed on day 4. However, by day 29, resolution of renal toxicity had occurred in the 150-microg/kg group, but only partial resolution was seen at 300 microg/kg. Renal injury correlated with increased serum creatinine, BUN, and kidney weights at 300 microg/kg, indicating impairment of renal function. Subacute, necrotizing inflammation of bone marrow and peritrabecular osteocyte hyperplasia of bone were seen at 300 microg/kg on day 4, with recovery thereafter. Injury to blood vessels and surrounding tissue at the injection site were produced by KF, likely due to local cytotoxicity. In general, reversibility of toxicity was seen at 150 microg/kg but not at 300 microg/kg. When KF was administered once daily for five consecutive days at a dose of 80 microg/kg per day (400 microg/kg total dose), slightly decreased body weight gain was the primary drug-related effect. Therefore, the no-adverse-effect dose was at or near 80 microg/kg per day (480 microg/m(2) per day). CONCLUSIONS: These findings demonstrate that fractionation of a lethal or MTD dose of KF by daily administration for 5 days reduces drug-induced toxicity, and appears to be a viable option for the clinical evaluation of KF for the treatment of cancer.