Confirming genes influencing risk to cleft lip with/without cleft palate in a case-parent trio study.

A collection of 1,108 case-parent trios ascertained through an isolated, nonsyndromic cleft lip with or without cleft palate (CL/P) was used to replicate the findings from a genome-wide association study (GWAS) conducted by Beaty et al. (Nat Genet 42:525-529, 2010), where four different genes/regions were identified as influencing risk to CL/P. Tagging SNPs for 33 different genes were genotyped (1,269 SNPs). All four of the genes originally identified as showing genome-wide significance (IRF6, ABCA4 and MAF, plus the 8q24 region) were confirmed in this independent sample of trios (who were primarily of European and Southeast Asian ancestry). In addition, eight genes classified as 'second tier' hits in the original study (PAX7, THADA, COL8A1/FILIP1L, DCAF4L2, GADD45G, NTN1, RBFOX3 and FOXE1) showed evidence of linkage and association in this replication sample. Meta-analysis between the original GWAS trios and these replication trios showed PAX7, COL8A1/FILIP1L and NTN1 achieved genome-wide significance. Tests for gene-environment interaction between these 33 genes and maternal smoking found evidence for interaction with two additional genes: GRID2 and ELAVL2 among European mothers (who had a higher rate of smoking than Asian mothers). Formal tests for gene-gene interaction (epistasis) failed to show evidence of statistical interaction in any simple fashion. This study confirms that many different genes influence risk to CL/P.

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium.

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone-deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha-methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.

Glomerulonephritis induced in the rabbit by antiendothelial antibodies.

The effects of interaction between endothelial angiotensin converting enzyme (ACE) and goat anti-rabbit ACE (GtARbACE) antibodies were studied in rabbit glomeruli. By immunofluorescence ACE was not detectable in normal glomeruli. However, when kidneys were perfused with GtARbACE antibodies glomerular bound IgG was seven times higher than that of non-immune IgG and granular deposits of goat IgG were found on the endothelium of glomeruli and arteries. Rabbits injected intravenous for 4 d with GtARbACE antibodies showed on day 1 granular deposits of goat IgG on the glomerular endothelium; from day 3 to 24 there was gradual development of subepithelial deposits of goat IgG, rabbit IgG and C3. When GtARbACE antibodies were similarly injected into proteinuric rabbits there was formation of subepithelial granular deposits of goat IgG and ACE. The results document that a glomerular endothelial antigen is redistributed in vivo by a specific ligand, an event associated with formation of immune deposits. Furthermore, if the glomerular permeability is artificially increased, immune complexes shed from nonglomerular endothelia into the circulation can contribute to form subepithelial immune deposits.

The neonatal preventable harm index: a high reliability tool.

OBJECTIVE: The aim of this study is to identify, quantify and disseminate a novel set of safety indicators for monitoring the occurrence of preventable harm in the neonatal intensive care unit (NICU). STUDY DESIGN: Literature review and experiences in an academic, level IV NICU identified prevalent, preventable safety events: hospital-acquired infections (catheter-associated bloodstream infection, ventilator-associated pneumonia), unscheduled extubations, intravenous infiltrates requiring intervention, first week readmissions, serious adverse drug events and miscellaneous events (unanticipated harm or serious near misses). Negative binominal regression evaluated the event incidence trends. RESULTS: Of 226 preventable harm events occurring between March 2013 and January 2015, the most common were unscheduled extubations (98; 2/100 ventilator days) and intravenous infiltrates (62; 2.7/100 admissions). No trends were detected (rate ratio: 0.99; confidence limits: 0.96 to 1.01; P=0.38). CONCLUSION: The Neonatal Preventable Harm Index represents a novel and transparent means to monitor serious safety events and direct harm prevention strategies in the NICU.

Pharmacokinetic Evaluation of a Drug Transporter Cocktail Consisting of Digoxin, Furosemide, Metformin, and Rosuvastatin.

This article reports the clinical investigation of a probe drug cocktail containing substrates of key drug transporters. Single oral doses of 0.25 mg digoxin (P-gp), 5 mg furosemide (OAT1 and OAT3), 500 mg metformin (OCT2, MATE1, and MATE2-K), and 10 mg rosuvastatin (OATP1B1, OATP1B3, and BCRP) were administered separately or as a cocktail in a randomized six-period crossover trial in 24 healthy male volunteers. As a cocktail, relative bioavailabilities of digoxin and metformin and furosemide AUC0-tz were similar to separate dosing. However, when administered as a cocktail the Cmax of furosemide was 19.1% lower and the Cmax and AUC0-tz of rosuvastatin were 38.6% and 43.4% higher, respectively. In addition, the effects of increased doses of metformin or furosemide on the cocktail were investigated in 11 and 12 subjects, respectively. The cocktail explored in this trial has the potential to be used for the in vivo screening of transporter-mediated drug-drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.

Regulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells by glucocorticoids: evidence for nongenomic as well as genomic mechanisms.

We have investigated the nongenomic as well as the genomic effects of glucocorticoids on phosphate (Pi) uptake in primary rabbit renal proximal tubule cells (PTCs) and have defined the involved signaling pathways. In the present study, cortisol-BSA (cortisol-BSA) (>10(-9) M, 30 min) was found to inhibit Pi uptake in a time- and concentration-dependent manner. However, progesterone-BSA (P(4)-BSA), 17ss-estradiol-BSA (E(2)-BSA), testosterone-BSA (T(4)-BSA), aldosterone, P(4), E(2), and T(4) (10(-9) M, 1 h) had no effect on Pi uptake. In addition, cortisol-BSA (10(-9) M) did not affect either Na(+) uptake or alpha-methylglucopyranoside (alpha-MG) uptake. The cortisol-BSA-induced inhibition of Pi uptake was associated with a decrease in the V(max) for Pi uptake, rather than the K(m). The inhibitory effect of cortisol-BSA was not blocked either by actinomycin D (an inhibitor of transcription), cycloheximide (an inhibitor of translation), or classical glucocorticoid receptor antagonists (RU 486 or P(4)). The cortisol-BSA-induced inhibition of Pi uptake was blocked by two phospholipase C (PLC) inhibitors (neomycin or U73122), and two protein kinase C (PKC) inhibitors (staurosporine or bisindolylmaleimide I) but not by two adenylate cyclase/protein kinase A inhibitors [SQ 22536 (an adenylate cyclase inhibitor) or myristoylated protein kinase A inhibitor amide 14-22]. Furthermore, cortisol-BSA promoted the translocation of PKC from the cytosolic fraction to the membrane fraction, while having no effect on the activity of adenylate cyclase. Our observations may thus be interpreted as indicating that cortisol does indeed inhibit renal Pi uptake via a nongenomic mechanism, which involves the PLC/PKC pathway.

Increased prevalence of the Taq I A1 allele of the dopamine receptor gene (DRD2) in obesity with comorbid substance use disorder: a preliminary report.

In order to investigate the prevalence of the Taq I A1 allele of the dopamine receptor gene (DRD2) in obesity with and without comorbid substance use disorder, a total of 40 patients, from an outpatient neuropsychiatric clinic in Princeton, New Jersey, were genotyped for presence or absence of the Taq I DRD2 A1 allele. The primary inclusion criterion for 40 obese subjects was a body mass index (BMI) equal to or over 25 (uncharacterized); 11 obese subjects had severe substance use disorder; 20 controls had a BMI below 25; and, 33 substance use disorder (less severe) patients had a BMI below 25. The data were statistically compared with three different sets of controls divided into three separate groups (Group I, n = 20; Group II, n = 286; Group III, n = 714). They differed according to screening criteria (drug, alcohol, nicotine abuse/dependence, BMI below 25 and other related behaviours including parental history of alcoholism or drug abuse and DSM IV, Axis I and Axis II diagnoses). Groups II and III were population controls derived from the literature. The prevalence of the Taq I A1D2 dopamine receptor (DRD2) alleles was determined in 40 Caucasian obese females and males. In this sample with a mean BMI of 32.35 +/- 1.02, the A1 allele of the DRD2 gene was present in 52.5% of these obese subjects. Furthermore, we found that in the 23 obese subjects possessing comorbid substance use disorder, the prevalence of the DRD2 A1 allele significantly increased compared to the 17 obese subjects without comorbid substance use disorder. The DRD2 A1 allele was present in 73.9% of the obese subjects with comorbid substance use disorder compared to 23.5% in obese subjects without comorbid substance use disorder. Moreover, when we assessed severity of substance usage (alcoholism, cocaine dependence, etc.) increasing severity of drug use increased the prevalence of the Taq I DRD2 A1 allele; where 66.67% (8/12) of less severe probands possessed the A1 allele compared to 82% (9/11) of the most severe cases. Linear trend analyses showed that increasing use of drugs was positively and significantly associated with A1 allelic classification (p < 0.00001). These preliminary data suggest that the presence of the DRD2 A1 allele confirms increased risk not only for obesity, but also for other related addictive behaviours (previously referred to as the Reward Deficiency Syndrome) and that a BMI over 25 by itself (without characterization of macroselection or comorbid substance use disorders) is not a sufficient criterion for association with the DRD2 A1 allele.

Colloidal silica-coated tissue culture dishes for primary cell cultures: growth of rabbit renal proximal tubule cells.

The use of colloidal silica as a substratum for primary cultures of differentiated cells has significant advantages over classic tissue culture polystyrene. In this report, the growth and the level of expression of differentiated function of primary rabbit renal proximal tubule (RPT) cell cultures on colloidal silica is examined, using hormonally defined serum-free medium. Primary RPT cells grew to confluence more rapidly on colloidal silica than on tissue culture polystyrene (TC+). Moreover, following three passages, the RPT cells increased in number threefold more than parallel cultures on TC+. The morphology of primary RPT cells on colloidal silica were found by means of transmission electron microscopy to possess a polarized morphology with a brush border, and differentiated markers were retained even after passaging, including the Na+/glucose cotransport system and Glut 7.

Response to missile attacks on civilian targets in patients with panic disorder.

BACKGROUND: The complex interaction that exists between biological and cognitive factors determines the reaction of panic-disorder patients to stressors. The current study was conducted to systematically assess the behavioral effects of a real, life-threatening event on panic-disorder patients. METHOD: Sixty-five panic-disorder patients completed structured telephone interviews during the first 4 weeks of the Persian Gulf War. Evaluation included frequency of panic attacks, anxiety levels, and function levels both during and between air raid alarms. RESULTS: The findings indicate that panic-disorder patients, despite high levels of anxiety, did not demonstrate an increased frequency of panic attacks during the Persian Gulf War. In addition, the majority of patients reported good-to-high levels of functioning during the crisis in both everyday and alarm-related functioning. Grouping of subjects according to proximity to risk or current antipanic treatment did not produce significant differences in the frequency of panic attacks or levels of anxiety. CONCLUSION: The findings suggest that vulnerability of patients with panic disorder to a "panic-stricken" response does not increase during real-life stressors. The lack of increased frequency of panic attacks observed under these circumstances provides additional support for the opinion that panic and fear are two distinct entities.

Treatment of choledocholithiasis with oral chenodeoxycholic acid.

The aim of this study was to conduct a controlled trial of oral chenodeoxycholic acid in the management of radiolucent choledocholithiasis. Thirteen patients were randomized in double-blind fashion to receive either 750 mg/day of chenodeoxycholic acid (CDCA) or a placebo. After 4 months, those who had the placebo were administered CDCA; those who had received CDCA and showed a 25% or more decrease in the size of stones (evaluated blindly) received CDCA for an additional 4 months. Five of the 13 patients did not complete the study; four (one initially placebo and three CDCA) because acute biliary symptoms mandated operative intervention and one (initially placebo, then CDCA) because of asymptomatic elevations of the serum transaminase levels. Patients who were withdrawn from the study had significantly larger stones (P less than 0.02) (mean largest diameter, 11.4 mm +/- 1.6 SEM) than those who completed the study (6.5 +/- 0.5). Of the eight patients who completed the study, two of the three who received CDCA initially for 6 to 8 months experienced complete disappearance of stones; all five patients who took the placebo failed to show dissolution, and one of these subsequently had dissolution of stones after 8 months of CDCA. Biliary lipid analyses during treatment showed bile unsaturated with respect to cholesterol in the three patients whose stones dissolved with CDCA therapy. In conclusion, a patient with partial dissolution of stones and unsaturated bile after 4 months of CDCA probably will have complete dissolution of stones after 6 to 8 months of CDCA.

Ephedrine in treatment of urinary incontinence.

Patients with urinary incontinence caused by various urologic disorders were evaluated and treated with oral ephedrine sulfate. The drug was found to improve continence in those patients with mild degrees of wetting due to urethral dysfunction regardless of cause. The failures occurred in severely damaged posterior urethras or denervated bladders and urogenital diaphragms.

Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line.

The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the stabilized dipeptides D-Glu-Ala and D-Asp-Ala as pro-moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding D-Glu-Ala and D-Asp-Ala model prodrugs' affinity for hPepT1 in Caco-2 cells. A second aim was to investigate the transepithelial transport and hydrolysis parameters for D-Asp(BnO)-Ala and D-Glu(BnO)-Ala across Caco-2 cell monolayers. In the present study, all investigated D-Asp-Ala and D-Glu-Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used to establish a QSAR of our benzyl alcohol modified model prodrugs, aided at elucidating the observed differences in model prodrug affinity for hPepT1; additionally, these data suggest that the hydrophobicity of the side-chain model drug is the major determinant in the compounds affinity for hPepT1. Transepithelial transport studies performed using Caco-2 cells of D-Asp(BnO)-Ala and D-Glu(BnO)-Ala showed that the K(m) for transepithelial transport was not significantly different for the two compounds. The maximal transport rate of the carrier-mediated flux component does not differ between the two model prodrugs either. The transepithelial transport of D-Asp(BnO)-Ala and D-Glu(BnO)-Ala follows simple kinetics, and the release of benzyl alcohol is pH-dependent, but unaffected by 1 mM of the esterase inhibitor Paraoxon in 80% human plasma and Caco-2 cell homogenate.

A double-blind crossover comparison of clomipramine and desipramine in the treatment of panic disorder.

OBJECTIVE: To compare the efficacy of clomipramine hydrochloride (CMI), a serotonin reuptake inhibitor with the noradrenergic tricyclic antidepressant agent, and desipramine hydrochloride (DMI) for patients with panic disorder (PD). METHOD: Following a 2-week, single-blind placebo washout phase, 17 PD outpatients completed a 16-week, double-blind, crossover comparison of CMI and DMI. Key outcome measures included panic attacks frequency, the NIMH Global Scales for Anxiety, Depression and Impairment, Hamilton Anxiety Scale (Psychic and Somatic Subscales), Zung Anxiety Inventory (Raw and Index Subscales) and the Spielberger State Anxiety Scale. RESULTS: Both CMI and DMI led to significant improvement from baseline placebo state in panic attacks frequency and behavioral ratings (p<0.001). CMI led to a greater reduction in the frequency of panic attacks (p=0.028) and was superior to DMI on ratings of anxiety: NIMH Global Anxiety, Zung Anxiety Scale (Raw and Index) and the Spielberger Anxiety Scale. No difference was found between the drugs on the NIMH Global Impairment Scale and the Hamilton Somatic and Psychic Scales. CONCLUSION: Both drugs appeared to have significant therapeutic effects in patients with PD, but CMI appeared to be more effective. The effectiveness of the serotonergic drug suggests that the role of the serotonergic system in the pathogenesis of PD should be further explored.

Realism of confidence in obsessive-compulsive checkers.

The study examined whether obsessive-compulsive (OC) checkers have reduced confidence in their knowledge. OC checkers were compared with panic disorder (PD) patients and nonpatient controls using a calibration-of-knowledge procedure. Participants completed a general knowledge questionnaire, rated their confidence in each answer, and estimated the total number of correct answers. These responses were converted to 2 measures of confidence relative to performance--over/underconfidence and over/underestimation. OC checkers had lower scores than nonpatients did on both measures, whereas the PD patients did not differ from either group. For the OC checkers, relative confidence was inversely related to the severity of obsessions. The authors speculate that confidence may depend on a confirmation bias in testing hypotheses and that the reduced confidence in OC checkers may reflect a disconfirmation bias in this population.

Specific inhibition of breast cancer cells by antisense poly-DNP-oligoribonucleotides and targeted apoptosis.

Two membrane-permeable and RNase-resistant antisense poly-2'-O-(2,4-dinitrophenyl)-oligoribonucleotides (poly-DNP-RNAs) have been synthesized as inhibitors of human breast cancer, with nucleotide sequences complementary to the genes of RIalpha subunit of protein kinase A (RIalpha/PKA) and erbB-2, respectively. Both compounds inhibit the proliferation of SK-Br-3 breast cancer cells in culture above the concentration of 10 microg/ml, but have no effect on nontumorigenic MCF-10A breast cells. These antisense inhibitors also block the cell colony formation in methylcellulose medium, whereas the control poly-DNP-RNA with either random or sense sequence has no effect. RT-PCR data show that the antisense inhibition decreases the concentration of the mRNA. TdT-mediated dUTP nick-end labeling (TUNEL) fluorescence assay indicates that the targeted antisense inhibition by poly-DNP-RNAs leads to apoptosis of SK-Br-3 cells but does not affect nontumorigenic MCF-10A cells. The control poly-DNP-RNAs with random or sense nucleotide sequence are completely inactive.

Primary kidney cells.

Hormonally defined serum-free media have been developed for growth and functional studies with kidney epithelial cell cultures. Not only can several established kidney epithelial cell lines (MDCK and LLC-PK1) be grown in a serum-free environment (1,2), but in addition, primary cultures of kidney epithelial cells can also be grown serum free (1-4). Investigations with primary kidney cell cultures are advantageous for several reasons. First of all, kidney cells can be grown in vitro from the animal of choice. Thus, the results of tissue culture studies can be more closely correlated with animal studies. Secondly, new tissue culture systems can be developed that more closely resemble kidney cells in vivo than those obtained with established kidney cell lines.

Pantomography vs mandibular series for the detection of mandibular fractures.

OBJECTIVE: The two primary radiographic techniques used for the evaluation of mandible injury are a pantomographic series (PS) and the standard four-view mandibular series (MS). Despite a tenuous foundation, there is apparent bias in favor of PS compared with MS. Many emergency departments do not have ready access to the specialized equipment necessary to perform a pantomographic study. The hypothesis of this study was that a high-quality standard MS is as sensitive and specific as a PS in the detection of mandibular fractures. METHODS: This was a prospective, blinded study of 54 patients presenting with acute mandibular injury comparing MS and PS. The study design used two board-certified emergency physicians and a single staff radiologist who read a series of MS and PS films in a randomized fashion without access to clinical information or identifying patient data. The absolute number of fractures present was determined by a neuroradiologist with access to both MS and PS simultaneously as well as pertinent clinical information. RESULTS: Thirty patients had 47 mandibular fractures. The sensitivity for fracture detection for each physician was 0.85, 0.77, and 0.89 with MS and 0.79, 0.74, and 0.83 with PS (p > or = 0.51, p > or = 1.00, and p > or = 0.51, respectively, McNemar's binomial test). The specificity for fracture detection for each physician was 0.88, 0.92, and 0.96 for MS and 0.96, 1.00, and 0.92 for PS (p > 0.625, p > 0.50, and p = 1.00, respectively, McNemar's binomial test). CONCLUSIONS: A standard mandibular series is as sensitive and specific as pantomography in the detection of mandibular fractures.

Primary cultures of rabbit renal proximal tubule cells: II. Selected phase I and phase II metabolic capacities.

Specific characteristics of cells vary as a function of time in culture. We have determined the stability of selected Phase I and Phase II biotransformation capacities in rabbit renal proximal tubule cells in primary culture. When grown in hormonally-defined medium, proximal tubule cells lost Phase I metabolic capacity. Cytochrome P-450 content and associated mixed-function oxidase activities present in kidney cortex microsomes were not detectable after 14 days in culture. Phase II glutathione-dependent metabolic functions were well retained in cultured cells compared with freshly isolated proximal tubules (FIPT). Cellular total glutathione content was 2.8 mug/mg protein in FIPT compared with approximately 10 mug/mg protein in stable confluent cultures. A higher total glutathione content of 20.6 mug/mg was noted in preconfluent cultures. The glutathione redox state was initially perturbed in FIPT with 37% of the total glutathione present found in its oxidized form. Tubule cells recovered to a normal ratio (6-13% of total glutathione in the oxidized form) while in culture. The glutathione S-transferase activity in 4-day-old cells in culture was reduced to 50% of the 4 U/mg protein level found in FIPT. No appreciable further decline in glutathione S-transferase activity was detected during 15 days in culture. The level of gamma-glutamyl-transpeptidase (a brush-border enzyme necessary for glutathione uptake into proximal tubule cells) declined from 1499 mU/mg protein in homogenates of FIPT to 636 mU/mg in homogenates of 8-day-old cultured cells. A further decline in activity occurred during the next 7 days in culture. In conclusion, although Phase I metabolic functions were diminished in primary cultured rabbit proximal tubule cells, Phase II metabolic functions were retained at levels comparable with FIPT and well above those found in several established kidney cell lines.

Toxicity of ifosfamide and its metabolite chloroacetaldehyde in cultured renal tubule cells.

Renal injury is a common side effect of the chemotherapeutic agent ifosfamide. Current evidence suggests that the ifosfamide metabolite chloroacetaldehyde may contribute to this nephrotoxicity. The present study examined the effects of ifosfamide and chloroacetaldehyde on rabbit proximal renal tubule cells in primary culture. The ability of the uroprotectant medication sodium 2-mercaptoethanesulfonate (mesna) to prevent chloroacetaldehyde-induced renal cell injury was also assessed. Chloroacetaldehyde (12.5-150 microM) produced dose-dependent declines in neutral red dye uptake, ATP levels, glutathione content, and cell growth. Coadministration of mesna prevented chloroacetaldehyde toxicity while pretreatment of cells with the glutathione-depleting agent buthionine sulfoximine enhanced the toxicity of chloroacetaldehyde. Ifosfamide (1000-10,000 microM) toxicity was detected only at concentrations of 4000 microM or greater. Analysis of media collected from ifosfamide-treated cell cultures revealed the presence of several ifosfamide metabolites, demonstrating that renal proximal tubule cells are capable of biotransforming this chemotherapeutic agent. This primary renal cell culture system should prove useful in studying the cause and prevention of ifosfamide nephrotoxicity.

Stability, metabolism and transport of D-Asp(OBzl)-Ala--a model prodrug with affinity for the oligopeptide transporter.

The model prodrug D-Asp(OBzl)-Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of D-Asp(OBzl)-Ala and its in vitro metabolism in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of D-Asp(OBzl)-Ala in situ using the single-pass rat perfusion model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of D-Asp(OBzl)-Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids, the half-lives were >3 h and 2.3+/-0. 03 h, respectively. Using the rat single-pass perfusion technique, the effective jejunal permeability (Peff) of D-Asp(OBzl)-Ala was determined to be high (1.29+/-0.5.10-4 cm/s). The 32 times higher Peff value found in the perfusion model compared to Caco-2 cells is most likely due to a higher functional expression of the oligopeptide transporter. Rat jejuna Peff was reduced by approximately 50% in the presence of well known oligopeptide transporter substrates, such as Gly-Sar and cephalexin. It may be that D-Asp(OBzl)-Ala is primarily absorbed intact by the rat jejunal oligopeptide transporter, since the stability in the intestinal homogenate and fluids was rather high (t1/2>2.3 h).