Development and application of an automated solution stability assay for drug discovery.

Screening of solution stability provides an early alert on potential liabilities of drug candidates so that strategies can be developed to overcome the challenges. A fully automated solution stability assay has been developed to accelerate traditional manual operation. The assay uses the advanced capabilities of a high-performance liquid chromatography instrument that is present in many pharmaceutical research laboratories. The samples are prepared automatically by a temperature-controlled autosampler. The samples are delivered to the stability matrices, mixed, incubated, and injected at selected time points during the reaction time course. This automated process occurs without operator intervention, thus allowing 96 experiments to be run with 0.5 h of a scientist's time compared to 8 h for the same study when performed manually. Automation not only eliminates the manual operation but also improves accuracy and throughput. The assay protocol has been optimized to achieve homogenous mixing and eliminate carryover. The assay is robust, flexible, and high throughput. It can be used to study stability for a large number of samples under multiple incubation conditions and has a wide range of applications in drug discovery and development, such as screening compound stability in biological assay media, obtaining a stability-pH profile, surveying compound stability in physiological fluids, and performing development forced degradation and excipient compatibility.

Laxative poisoning: toxicological analysis of bisacodyl and its metabolite in urine, serum, and stool.

An 11-year-old boy was hospitalized because of severe diarrhea (over 5 L/day). The child survived; however, the diarrhea continued while he was given intravenous fluids and his electrolyte balance was closely supervised. Based on observation of the patient and his family in the hospital, surreptitious administration of a poison by his mother was suspected, and toxicological analysis was carried out on stools from the boy and on medicine administered to him by his mother. Bisacodyl, a cathartic with a direct effect on the colon, was detected in the medicine, and a metabolite of bisacodyl was present in the stool. We devised a sensitive and reliable method to quantitate the bisacodyl metabolite in urine and serum. The sample was incubated with beta-glucuronidase at 37 degrees C for 2 h; bisacodyl metabolite was extracted with tert-butyl methyl ether, then derivatized by methylation, and subjected to gas chromatography-mass spectrometry. Bisphenol A was used as an internal standard. The calibration curve was linear in the concentration range from 2 to 1000 ng/0.2 mL, and the lower limits of detection were 1 ng/0.2 mL for the urine and 2 ng/0.2 mL for the serum. As concentrations of bisacodyl metabolite in the urine and serum of the patient were clearly defined, perhaps such investigations are warranted before extensive clinical therapy is prescribed.

Maternal drug treatment and human milk banking.

We studied prospectively during one year temporary drug use by mothers donating breast milk to assess the problem of drug treatment of donors. Sixty-four of the 284 mothers (22.5%) had to abstain from donating due to medication. The indication was infection in 50/56 treatments (89.3%). Antimicrobial agents were prescribed 44/52 times (84.6%). The channelling of milk from mothers in early phases of lactation to premature and newborn infants was identified as a special risk situation, if mothers on medication are not excluded. The limited number of such donors leads to use of milk unpooled or pooled to small volumes with increased risk for adverse effects to babies as a consequence. We recommend a wash-out period of 5 half-lives of the drug after the last ingested dose. For the majority of drugs in this study, with some important exceptions, a wash-out period of 1 day was sufficient.

Fluorescence resonance energy transfer between the nucleotide binding site and Cys-10 in G-actin and F-actin.

Intramonomer fluorescence resonance energy transfer between the donor epsilon-ATP bound to the nucleotide site and the acceptor N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) or 4-dimethylaminophenyl-azophenyl-4'-maleimide bound to Cys-10 in G-actin was measured. The donor-acceptor distance was calculated to be about 40 A. The intermonomer energy transfer in F-actin occurring between epsilon-ADP and DABMI was also measured. The radial coordinate of Cys-10 was calculated to be 25 A based on the helical symmetry of F-actin and the recently calculated radial coordinate of the nucleotide binding site in F-actin i.e. 25 A (Miki, M., Hambly, B. and dos Remedios, C.G. (1986) Biochim. Biophys. Acta 871, 137-141). (The assumption has been made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Corresponding distances separating the donor nucleotide in one monomer from acceptors on Cys-10 in the first and second nearest neighbours in F-actin are 39-40 A and 41-43 A.

Intestinal handling of bisacodyl and picosulphate by everted sacs of the rat jejunum and stripped colon.

Bisacodyl (BIS) is the acetic acid di-ester of the laxative diphenol 2-(4,4'-dihydroxydiphenyl)methyl-pyridine. A HPLC-method which permits the simultaneous determination of BIS and its monodesacetylated (MONO) as well as totally desacetylated (DES) form, has been used to study the intestinal handling of BIS (20 nmol/ml), when the compound was incubated for 60 min. at the mucosal side of the preparations specified. In the jejunal mucosal fluid, BIS disappeared completely in short time, and there was a nearly equivalent rise in DES. MONO was transitory present. Hydrolysis was also rapid in mucosal fluid which had been in contact with jejunal sacs for 30 sec., but BIS was stable in blank incubations. Hydrolysis of BIS was slower by colonic than by jejunal sacs, and all three molecular forms were present during incubation. It seemed still slower in mucosal fluid which had been in contact with colonic sacs for 5 min. BIS just as DES accumulated in the jejunal and colonic serosal fluid mainly as conjugates (greater than 95%), and DES was in all cases the only unconjugated metabolite present. Drug accumulation in jejunal serosal fluid was the same whether BIS or DES was added. However, more drug seemed to accumulate on the serosal side of colonic sacs when incubated with BIS instead of DES. In similar experiments with picosulphate, which is the sulphuric acid di-ester analogue of BIS, free DES was not detected in the mucosal fluid during incubation. The amounts of laxative accumulating in the serosal fluid were less than 1/10 of those observed with BIS.

Uptake, conjugation and transport of laxative diphenols by everted sacs of the rat jejunum and stripped colon.

Phenolphthalein (PHEN), desacetylbisacodyl (DES) and oxyphenisatin (OXY) were incubated with everted sacs of the rat jejunum and stripped descending colon; the mucosal and serosal fluid were analysed with respect to free and conjugated diphenol by means of HPLC. Conjugates were measured as the amount of free diphenol in completely hydrolyzed samples less the amount before hydrolysis. A study with double-sided administration of PHEN revealed that diphenol uptake from and conjugate output to both sides followed a rectilinear course for 15-90 min. A standard incubation time of 60 min. was chosen for the subsequent experiments, in which the diphenols were administered at the mucosal side at a low and a high concentration. Diphenol uptake, i.e. the amount of free diphenol administered less the amount recovered at the mucosal side, varied in an order (PHEN greater than DES greater than OXY) which seems to be inversely related to the order of water solubility of the compounds. Tissue accumulation and conjugate output relative to uptake varied with the dose, and from one compound to another. At low initial concentration (20 nmol/ml), the compounds were transferred to the jejunal and colonic serosal fluid almost entirely as conjugates (greater than or equal to 95%); the transfer rates followed, qualitatively, the same order as above. In jejunum, more conjugates were released to the mucosal than to the serosal side; in colon the distribution was reversed. Increasing the dose to 100 nmol/ml caused a corresponding increase in uptake, but relative output decreased and tissue accumulation increased; thus demonstrating capacity limitation. With PHEN, the ratio of conjugated:free diphenol on the serosal side remained essentially unchanged; with DES in particular, but also with OXY the ratio decreased. These findings may be interpreted to mean that in case of PHEN capacity limitation is linked to conjugate efflux, while DES and OXY may be poor substrates for glucuronide formation as well. Experiments with serosal side administration like the double-sided PHEN experiments verified the dissimilar conjugate distribution in jejunal and colonic sacs; the phenomenon is to some extent discussed in the text. Identity tests gave evidence that the conjugates were mainly monoglucuronides.

Biliary excretion of bisacodyl and picosulphate in man: studies in gallstone patients after biliary tract surgery.

Bisacodyl (=BIS) and picosulphate (=PICO) have been given perorally to postoperative gallstone patients, who have undergone biliary tract surgery with the insertion of an indwelling T-tube. The doses corresponded to 7.7 mg of their common free diphenol desacetylbisacodyl (=DES). The bile was sampled in hourly fraction from the external limb of the T-tube; these fractions were analysed by a modification of the HPLC method previously used to study biliary excretion in the rat. In the BIS-patients (n=8), DES in conjugated form occurred in significant concentration already in the first fractions; peak excretion values equivalent to 4-8 microgram DES/ml bile were reached in 2-5 hours. Unchanged BIS could not be detected, and the concentration levels of unconjugated DES were insignificant. The PICO-patients (n=8) on the other hand showed low DES concentrations (conjugated + free less than or equal to 0.5 microgram DES/ml) in all fractions, or low initial concentrations followed by a more or less pronounced rise in the later fractions. These results are, qualitatively, as in the rat. However, the dose fractions excreted in bile (assuming a total hepatic output of 50ml/hours) seem smaller than those to be expected from rat experiments, at least as far as BIS is concerned.

Enterohepatic circulation, urinary excretion and laxative action of some bisacodyl derivatives after intragastric administration in the rat.

Bisacodyl (BIS), the parent diphenol (DES) and its sulphuric acid di-ester (picosulphate = PICO) were given by stomach tube to fasted rats at a dose of 3.1 mumol/100 g rat. Bile was sampled in the periods 0-6, 6-12 and 12-18 hrs after drug administration, and assayed for total diphenol (= free + conjugated) by HPLC. Mean fractions (% of dose +/- S.E.M.) excreted in 5 rats per compound and period were: BIS 74.0 +/- 4.7, 51.9 +/- 7.9 and 30.8 +/- 2.5; DES 41.2 +/-4.3, 46.8 +/- 4.7 and 25.1 +/- 2.5; PICO 9.0 +/- 0.9, 26.0 +/- 5.4 and 19.6 +/- 3.1. Only minor amounts were excreted as free diphenol. Urine samples taken by bladder puncture and assayed as above furthermore showed that the renal excretion of total diphenol was insignificant compared to the amounts excreted in bile. Practically no diphenol was present in urine 0-6 hrs after the administration of PICO. In experiments with BIS and DES at 0.85 mumol/100 g, total diphenol excreted in bile during 0-6 hrs was: BIS 67.1 +/- 2.6 (n = 5); DES: 55.4 +/- 3.0 (5). - The latency time for laxative effect was studied in groups of 10 unfasted rats per compound. cumulative time response curves showed that PICO caused diarrhoea more promptly at 0.85 mumol/100 g than either BIS or DES. In most rats, this delayed action of BIS and DES persisted also at 1.7 mumol/100 g. At 3.1 mumol/100 g, however, the majority of the rats reacted as promptly to these two compounds as to PICO. These results are discussed in relation to the biliary excretion experiments, and interpreted in terms of the relative importance at the different dose levels of: 1. The enterohepatic recirculated fraction, and 2. The non-absorbed fraction, which passes directly to the large intestine. For PICO, the latter fraction is the single determinant of the effect, which is triggered when the di-ester is being hydrolyzed to active diphenol in this part of the GI-tract.

Bis-(p-hydroxyphenyl)-pyridyl-2-methane: The common laxative principle of Bisacodyl and sodium picosulfate.

After both oral and rectal administration in humans (4,4'-diacetoxy-diphenyl)-(pyridyl-2)-methane (bisacodyl, Dulcolax) and 4,4'-(2-pyridyl-methylene)-diphenol-disulfuric acid semiester disodium (sodium picosulfate, Laxoberal) are hydrolyzed to bis-(p-hydroxyphenyl)-pyridyl-2-methane (BHPM). In both cases BHPM is responsible for the laxative action. Experiments in rats and guinea pigs have shown that the hydrolysis of picosulfate, in contrast to that of bisacodyl, is attributable to the microorganisms of the intestinal flora.