Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

In Vitro Study on the Effect of Maraviroc or Dolutegravir on Bilirubin to Albumin Binding.

We performed an in vitro evaluation of the effect of maraviroc or dolutegravir on bilirubin to albumin binding. At typical treatment and low albumin concentrations, maraviroc had no impact, while dolutegravir affected bilirubin to albumin binding to an equivalent extent as sulfisoxazole. However in vivo, neither is likely to significantly impact bilirubin to albumin binding because of their low concentrations relative to albumin.

Simultaneous determination of N(1)-acetyl sulfisoxazole and its metabolites, and relative bioavailability compare to sulfisoxazole in rats.

N(1)-acetyl sulfisoxazole (N1AS), a dihydropteroate synthase inhibitor is known to be biotransformed primarily to sulfisoxazole, partly to N(4)-acetyl sulfisoxazole (N4AS), and likely also to diacetyl sulfisoxazole (DAS) and other compounds. Although its clinical use has been limited due to urolithiasis, some countries still use the drug in combination with trimethoprim in cattle. A liquid chromatographic method using ultraviolet detection was developed for the simultaneous determination of four substances for the first time. Four analytes and sulfamethoxazole (IS) were separated on a reversed-phase column with gradient elution of a mobile phase. Because DAS and N1AS in plasma were changed very quickly into N4AS and sulfisoxazole, respectively, and esterase inhibitors (sodium fluoride and eserine) could not prevent the transformation, sulfisoxazole and N4AS were monitored in rat plasma following a single oral administration of N1AS and sulfisoxazole in five rats. The relative bioavailability of N1AS to sulfisoxazole was about two, indicating that a half-dose of N1AS would be equivalent to a dose of sulfisoxazole to achieve the same systemic exposure to sulfisoxazole.

The effect of pH and triethanolamine on sulfisoxazole complexation with hydroxypropyl-beta-cyclodextrin.

A novel complexation of sulfisoxazole with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was studied. Two systems were used: binary complexes prepared with HP-beta-CD and multicomponent system (HP-beta-CD and the basic compound triethanolamine (TEA)). Inclusion complex formation in aqueous solutions and in solid state were investigated by the solubility method, thermal analysis (differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)), Fourier-transform infrared spectroscopy (FT-IR) and dissolution studies. The solid complexes of sulfisoxazole were prepared by freeze-drying the homogeneous concentrated aqueous solutions in molar ratios of sulfisoxazole:HP-beta-CD 1:1 and 1:2, and sulfisoxazole:TEA:HP-beta-CD 1:1:2. FT-IR and thermal analysis showed differences among sulfisoxazole:HP-beta-CD and sulfisoxazole:TEA:HP-beta-CD and their corresponding physical mixtures and individual components. The HP-beta-CD solubilization of sulfisoxazole could be improved by ionization of the drug molecule through pH adjustments. However, larger improvements of the HP-beta-CD solubilization are obtained when multicomponent systems are used, allowing to reduce the amount of CD necessary to prepare the target formulation.

Comparison of the binding characteristics of serum albumins from various animal species.

In order to develop an animal model to study the bilirubin displacing effect of various drugs, we compared the bilirubin binding ability of human, pig, dog, rabbit, hamster, rat, guinea pig, and cat albumin. These albumins were used also to study the binding of monoacetyldiaminodiphenyl sulfone (MADDS). Using human, rat, guinea pig, and rabbit albumin, we studied the effect of sulfisoxazole, ceftriaxone, and tin protoporphyrin on bilirubin binding. Our results demonstrate that each animal albumin has different binding characteristics for the various chemicals tested. This variable must be considered before using an animal as a model for studying factors influencing bilirubin deposition in the brain.

Pharmacokinetic analysis of concentration data of drugs with irregular absorption profiles using multi-fraction absorption models.

Nonlinear regression analysis of plasma drug concentration data with irregular or stepwise absorption profiles was studied using multi-fraction absorption models in which drugs in the gastrointestinal tract were assumed to be divided into several fractions each with its respective lag time and absorption rate constant. Plasma allopurinol concentration data, with two-phase absorption profiles in dogs after oral administration, were found to be satisfactorily fitted using a two-fraction absorption model. Plasma sulfisoxazole concentration data in humans were also successfully analyzed using a two-fraction absorption model. Plasma and urinary concentrations of pindolol in humans after oral administration of sustained-release preparations were fitted to two- or three-fraction absorption models. The pharmacokinetic absorption behavior of a sustained-release preparation of diltiazem hydrochloride was studied using a multi-fraction absorption model. Pharmacokinetic parameters derived from these models and those from the discontinuous absorption model were compared.

Pharmacokinetics of sulfisoxazole in renal transplant patients.

We studied the elimination of sulfisoxazole in eight renal transplant patients. The patients received sulfisoxazole prophylactically for urinary tract infection commencing 7 days postoperatively. The renal elimination of sulfisoxazole (unbound renal clearance) was decreased in this patient population and was highly correlated with creatinine clearance. The unbound metabolic clearance and apparent unbound formation clearance of N4-acetyl sulfisoxazole did not differ from values found in healthy volunteers. The protein binding was marginally lower in this patient population than in healthy subjects after a single dose. The reduced binding was compatible with a reduced albumin concentration. In contrast to the situation for healthy subjects, the binding of sulfisoxazole decreased upon multiple dosing. This is probably due to a relatively higher sulfisoxazole and N4-acetyl sulfisoxazole-to-albumin ratio in this patient population than in healthy subjects. No complications of sulfisoxazole therapy were seen, although in three subjects concentration of the N4-acetyl sulfisoxazole in urine exceeded its theoretical solubility on a few occasions.

Segmental difference and effect of glucose on drug exsorption across the small intestine of rats.

An exsorption technique which can assess the transport of a drug from blood to the intestinal lumen was used to study the effects of glucose and to determine the presence of segmental differences in drug transport across the rat small intestinal membrane following intravenous drug administration. The drugs used in the study were un-ionized (sulfanilamide) or ionized (metoclopramide and sulfisoxazole) at the physiological pH (6.5) of rat small intestine. An intestinal segment of 20 cm from the ligament of Treitz to the distal part was used as the jejunum and the same length from the ileocecal junction to the proximal part was regarded as the ileal segment. Isotonic NaCl or glucose was used as the intraluminal perfusate. In the case of sulfanilamide, the exsorption rate remained almost constant during the perfusion of NaCl or glucose and no segmental differences in the exsorption rates were observed. For the weakly basic drug (metoclopramide) the exsorption rate in the jejunum was significantly decreased by glucose. Conversely, for the weakly acidic drug (sulfisoxazole), the exsorption rate in the jejunum was increased during glucose perfusion. In contrast to the effects observed in the jejunum, glucose did not have any significant effect on the exsorption rate of drugs studied in the ileum. These observations might be explained by the intraluminal pH in the respective segments and changes in the pH mediated by glucose perfusion.

Binding and disposition of sulfisoxazole in alcoholic cirrhosis.

The disposition of sulfisoxazole was studied in six male patients hospitalized with biopsy-proven Laennec's alcoholic cirrhosis and six normal, healthy volunteers. Four of the patients were restudied at a time their liver disease had improved clinically. On the average, compared to the normal group, the metabolism of sulfisoxazole (clearance values with respect to unbound concentration values) appears to be unaltered for the cirrhotic patients. For patients with alcoholic cirrhosis having normal renal function for age and weight, the renal elimination of sulfisoxazole was normal. For those subjects with decreased renal function, the renal clearance of sulfisoxazole appeared to be disproportionately decreased, as evidenced by a lower-than-normal sulfisoxazole renal clearance-to-creatinine clearance values. The apparent steady-state volume of distribution of unbound sulfisoxazole was not altered in cirrhotic patients when compared to normal subjects, while the apparent volume of distribution of total drug increased by more than what could be expected from protein binding changes alone. The elimination rate constant did not differ from values found in normals. These data suggest that the total binding capacity of sulfisoxazole in cirrhotic patients is not different from that of normal subjects.

Use of PKM-MULTI program for fitting discontinuous absorption profiles using a microcomputer.

The programs for the pharmacokinetic models with discontinuous absorption, written in BASIC, were connected to MULTI, which had been proposed for nonlinear least squares for a microcomputer by Yamaoka et al. Using this program (designated as PKM-MULTI), the same data sets previously calculated by HFCM, FITSI2 and NONLIN were fitted and the finally obtained estimates were in close agreement with those obtained previously. The plasma data of nalidixic acid were also fitted to the pharmacokinetic model with discontinuous absorption, which were suggested to be more valid compared with one compartment model with continuous absorption.

Abnormal serum protein binding of acidic drugs in diabetes mellitus.

Nonenzymatic glycosylation of proteins is common in diabetes mellitus and glycosylation of serum albumin in this condition has been described. To evaluate whether glycosylation of albumin affects acidic drug binding, sulfisoxazole and diazepam binding was examined in samples of normal serum incubated with glucose and in samples of serum from 42 patients with diabetes mellitus. Incubation of normal serum with 20mM glucose for several days resulted in progressive glycosylation of proteins, with decreased binding of sulfisoxazole (100 micrograms/ml) but not of diazepam (3 micrograms/ml). Free fractions of sulfisoxazole and diazepam were higher in serum from patients with diabetes. The percentage of free sulfisoxazole in serum from 10 normal subjects was 5.1% +/- 0.2%, whereas it was 16.0% +/- 1.3% in serum from 42 patients with diabetes with varying degrees of carbohydrate control. The percentage of free diazepam in plasma was 2.6% +/- 0.1% in the normal group and 3.6% +/- 0.4% in patients with diabetes. Decreased serum albumin levels, increased levels of free fatty acids, and glycosylation of plasma proteins seem to play a role in the defective acidic drug binding in diabetes. Elevated free fatty acid levels explain the abnormal binding of diazepam and the increased free fraction of sulfisoxazole is directly related to glycosylation of plasma proteins.

Influence of protein binding and metabolic interconversion on the disposition of sulfisoxazole and its N4-acetyl metabolite by the isolated perfused rat kidney.

The renal clearances of sulfisoxazole (SX) and N4-acetylsulfisoxazole (NSX) were studied in the isolated perfused rat kidney (IPK). Studies were conducted with conventional bovine serum albumin perfusates as well as with dextran perfusates to assess the influence of perfusate protein binding on the disposition of these compounds by the IPK. The results presented herein indicate that the disposition of sulfisoxazole by the IPK involves both metabolism and excretion. The metabolism of SX to NSX is reversible and is influenced by protein binding since metabolism increased with increased free fraction (Ff). The excretion of SX and NSX reflects a complex interaction of filtration, secretion, and reabsorption. A comparison of clearance values between kidneys perfused with bovine serum albumin perfusate (Ff 0.1) and dextran perfusate (Ff 1.0) suggests that tubular secretion of SX is a function of total (unbound plus bound) rather than free (unbound) drug in the perfusate.

Pharmacokinetics of sulfisoxazole in young and elderly subjects.

The pharmacokinetics of sulfisoxazole was studied in 6 elderly (age 63-75 years) and 7 young (age 22-37 years) healthy nonsmoking volunteers following the oral administration of 2 g of Gantrisin. The plasma levels of sulfisoxazole obtained in the postabsorption phase were higher in the elderly subjects. There was no significant variation between the two groups of volunteers in the absorption rate constant, Cmax, bioavailability, the fraction of the dose of sulfisoxazole excreted unchanged, the area under the plasma curve of the N4-acetyl conjugate formed, and in the apparent volume of distribution of the drug. The tmax value and plasma half-life of sulfisoxazole were significantly longer, and the total body and renal clearances of the drug decreased in the elderly subjects. Diminished renal function as estimated by the creatinine clearance and urinary flow may explain the slower elimination of sulfisoxazole in the elderly subjects. Therefore, advancing age should be considered as a factor in the adjustment of sulfisoxazole dosage.

Use of metzler's NONLIN program for fitting discontinuous absorption profiles.

An alternative to the use of integral hybrid flow/compartmental model (HFCM) equations in fitting cases I and II discontinuous absorption profiles is presented. It is proposed that HFCM-integral equations be replaced by a system of differential equations in which sequential sets of equations describe the absorption profile from time zero to infinity. The required sets of differential equations for these two cases are presented as they apply to a two-compartment drug, potentially undergoing multiple absorption steps. It was shown that the use of the NONLIN program in the differential equation mode provides good fits for some unusually shaped absorption profiles of buformin, sulfisoxazole, and griseofulvin. The values of the parameter estimates and the sum of squared deviations, sigma SD, obtained with NONLIN were almost identical to those obtained with the FITS12 program utilizing HFCM equations. While HFCM-integral equations required less computer time, they introduced the potential for negative absorption times. This problem is avoided by use of the differential equations method.

Isoniazid, quinidine, and sulphafurazole absorption in patients with jejunum transposition 15 years earlier.

Gastric surgery has formerly been shown to affect drug absorption. The absorption of oral isoniazid (INH), quinidine, and sulphafurazole (sulfisoxazole) was therefore studied in 19 ulcer patients operated on some 15 years earlier with resection of the ventricle combined with jejunum transposition. Twelve medical inpatients served as controls. The operated patients had all experienced relief of their ulcer symptoms, and there was no evidence of clinical malabsorption. In operated patients the peak serum levels of all three drugs were reached earlier than in controls. At 1 and 6 h the INH serum levels were increased in operated patients, but the sulphafurazole serum levels were at no time different in operated and control patients. As measured by the 24-h urinary excretion, jejunum transposition did not modify the total amounts of INH or sulphafurazole absorbed. In contrast, the quinidine absorption was decreased by about 50% in operated patients, which seems to be the result of increased gastric pH in the operated patients, leading to a poor dissolution and/or precipitation of quinidine in the gastric contents. Obviously, the jejunum transposition per se may not affect the total amounts of drugs absorbed, provided the physiochemical effects resulting from, for example, pH changes due to antrectomy and/or vagotomy are taken into account.

Penetration of amoxicillin, cefaclor, erythromycin-sulfisoxazole, and trimethoprim-sulfamethoxazole into the middle ear fluid of patients with chronic serous otitis media.

Penetration into the middle ear of four antibiotics commonly used in treatment of otitis media was studied by administering a single oral dose of amoxicillin, cefaclor, erythromycin-sulfisoxazole, or trimethoprim-sulfamethoxazole to 83 children with chronic serous otitis media. The antibiotic was given 15-240 min before the removal of middle ear fluid (MEF) by ventilation tubes inserted through the tympanic membrane. At the time MEF was obtained, a sample of blood was drawn from the patient, and concentrations of antibiotic in both specimens were assayed either microbiologically by a disk diffusion method or by high-pressure liquid chromatography. Amoxicillin had the highest ratio of mean peak concentration in MEF to minimal inhibitory concentration (MIC) for the three most common pathogens of otitis media (Streptococcus pneumoniae, ampicillin-sensitive Haemophilus influenzae, and Streptococcus pyogenes), whereas trimethoprim-sulfamethoxazole had the highest ratio of mean peak concentration in MEF to MIC for ampicillin-resistant Haemophilus influenzae.

Effects of phenobarbital on the distribution pharmacokinetics and biological half-lives of model nonmicrosomal enzyme metabolizable sulfonamides in rats.

The pharmacokinetics of sulfisoxazole and sulfanilamide were studied in control rats and in rats treated for 5 days with a daily 100 mg/kg ip dose of phenobarbital. These drugs represent the organic anionic and nonionized drugs, respectively, whose nonmicrosomal enzymatic metabolisms were unstimulated by phenobarbital. Sulfisoxazole showed the characteristics of a two-compartment open model. However, its biological half-life and the apparent distribution volume of the central compartment were significantly lower and the intercompartmental transport rate constants and the urinary excretion rate constant were significantly greater, in phenobarbital treated rats than in control rats. The apparent steady-state distribution volume of sulfisoxazole was smaller in the phenobarbital treated rats at the 90% confidence level. Sulfanilamide showed characteristics of a one-compartment model in both the control and phenobarbital treated rats, but none of the pharmacokinetic parameters of the compound in the phenobarbital treated rats were significantly different from those in the control rats.