Mechanism-Based QSAR Modeling of Skin Sensitization.

Many chemicals can induce skin sensitization, and there is a pressing need for non-animal methods to give a quantitative indication of potency. Using two large published data sets of skin sensitizers, we have allocated each sensitizing chemical to one of 10 mechanistic categories and then developed good QSAR models for the seven categories that have a sufficient number of chemicals to allow modeling. Both internal and external validation checks showed that each model had good predictivity.

Structure-based modelling in reproductive toxicology: (Q)SARs for the placental barrier.

The replacement of animal testing for endpoints such as reproductive toxicity is a long-term goal. This study describes the possibilities of using simple (quantitative) structure-activity relationships ((Q)SARs) to predict whether a molecule may cross the placental membrane. The concept is straightforward, if a molecule is not able to cross the placental barrier, then it will not be a reproductive toxicant. Such a model could be placed at the start of any integrated testing strategy. To develop these models the literature was reviewed to obtain data relating to the transfer of molecules across the placenta. A reasonable number of data were obtained and are suitable for the modelling of the ability of a molecule to cross the placenta. Clearance or transfer indices data were sought due to their ability to eliminate inter-placental variation by standardising drug clearance to the reference compound antipyrine. Modelling of the permeability data indicates that (Q)SARs with reasonable statistical fit can be developed for the ability of molecules to cross the placental barrier membrane. Analysis of the models indicates that molecular size, hydrophobicity and hydrogen-bonding ability are molecular properties that may govern the ability of a molecule to cross the placental barrier.

Structure-based methods for the prediction of the dominant P450 enzyme in human drug biotransformation: consideration of CYP3A4, CYP2C9, CYP2D6.

Metabolic drug-drug interactions are receiving more and more attention from the in silico community. Early prediction of such interactions would not only improve drug safety but also contribute to make drug design more predictable and rational. The aim of this study was to build a simple and interpretable model for the determination of the P450 enzyme predominantly responsible for a drug's metabolism. The P450 enzymes taken into consideration were CYP3A4, CYP2D6 and CYP2C9. Physico-chemical descriptors and structural descriptors for 96 currently marketed drugs were submitted to statistical analysis using the formal inference-based recursive modelling (FIRM) method, a form of recursive partitioning. Generally accepted knowledge on metabolism by these enzymes was also used to construct a hierarchical decision tree. Robust methods of variable selection using recursive partitioning were utilised. The descriptive ability of the resulting hierarchical model is very satisfactory, with 94% of the compounds correctly classified.

QSAR study of some anti-hyperglycaemic sulphonylurea drugs.

Sulphonylureas are widely used anti-hyperglycaemic drugs for the treatment of type 2 diabetes. The only published quantitative structure-activity relationship (QSAR) models for sulphonylurea drugs have been found to be questionable, for a number of reasons. We have re-analysed the human anti-hyperglycaemic potencies, acute mouse intraperitoneal toxicities (LD50) and plasma protein-binding abilities of the 15 drugs using multiple linear regression and obtained good QSAR models for each endpoint. The obtained QSARs all comply well with the Organisation for Economic Co-operation and Development (OECD) Guidelines for the Validation of (Q)SARs. We could not carry out external validation of our models for acute toxicity and plasma protein-binding because of the very small datasets available.

Phenobarbitone interaction with oral contraceptive steroids in the rabbit and rat.

1 The effect of phenobarbitone on the single dose pharmacokinetics of the synthetic steroids, ethinyloestradiol (EE2) and norethisterone, has been studied in the rabbit and rat. 2 EE2 is subject to an extensive first pass effect (96%). The plasma clearance of EE2 approaches total hepatic blood flow. It is suggested that a secondary peak in EE2 plasma concentration time curves at 5 h is due to enterohepatic recycling. Phenobarbitone had no effect on plasma EE2 concentrations following intravenous administration and produced a variable decrease after oral administration. 3 In phenobarbitone-treated rabbits, following intravenous administration of norethisterone there was no significant change in the area under the curve (AUC) compared to controls. In contrast, following oral administration of norethisterone to treated rabbits, the AUC was 20% and the peak plasma concentration 17% of that in controls. 4 The data in rabbits are consistent with drugs which are highly extracted by the liver. 5 In rats, phenobarbitone had no effect on plasma norethisterone concentrations following intravenous or hepatic portal (bolus) administration, but caused a decrease in systemic availability after both infusion into the portal vein (over a period of 5 min) and oral administration. 6 It is concluded that the rate of delivery of norethisterone to the liver is important in determining whether or not enzyme induction will cause an increased first pass effect. 7 Phenobarbitone caused an increase in conjugation of norethisterone in the gastrointestinal tract of rats.

The pharmacokinetics of norethisterone in the rabbit and rat after systemic and oral administration: effect of phenobarbitone [proceedings].

PIP: The pharmacokinetics of norethisterone in the rabbit and the rat after systemic and oral administration and the effect of phenobarbitone on pharmicokinetic parameters is presented. Norethisterone was given by iv and oral routes (85 mcg/kg) in the rabbit and by portal administration in the rat. Blood samples were collected over 24 hours in the rabbit and over 2 hours in the rat. Norethisterone was measured in plasma by radioimmunoassay. The plasma concentration-time curve was resolved into values for "fast disposition" half-life, "slow disposition" half-life, and the area under the curve (AUC). In the rabbit, AUC after oral administration was 53% of iv administration, while in the rat the AUC after portal administration was 32% of that after iv administration. In the rabbit phenobarbitone was without effect on plasma norethisterone concentrations after iv administration but significantly reduced the plasma concentration after oral administration. In the rat phenobarbitone had little effect on plasma norethisterone concentrations despite evidence of enzyme induction.^ieng

The temporal relationship between the secretion of luteinizing hormone and testosterone in man.

It has been suggested recently that testosterone secretion by the human testis may be controlled by factors other than luteinizing hormone (LH). In order to re-examine this hypothesis, plasma LH and testosterone concentrations were determined throughout the day in eight studies. A new method of data analysis revealed that the levels of the two hormones were closely related, but that the testicular response to LH was sluggish. These results explain some inconsistencies in the literature. It was demonstrated that average values for LH varied throughout the day, with a morning maximum and an evening minimum. It was also shown that injections of LH releasing hormone in man resulted in an increase in plasma testosterone above control levels. These results are consistent with the concept that LH controls the major changes in testosterone secretion in men. They do not exclude, however, the possible existence of other factors which might affect the peripheral concentration of testosterone, such as changes in testicular blood flow.

Interindividual variation and drug interactions with hormonal steroid contraceptives.

Large interindividual differences occur in the plasma concentration of contraceptive steroids. With the present tendency to decrease the dose of progestagen and oestrogen any factor which reduces the bioavailability of the lower dose preparations becomes very important. The possibility that other drugs and environmental chemicals may interact with contraceptive steroids is currently being investigated. Clinical reports suggest that the most important interacting drugs are the antituberculosis agent rifampicin, anticonvulsants and antibiotics. In the case of the first two, evidence is available suggesting that microsomal enzyme induction, either in the liver or the gut wall, is the operative mechanism. Antibiotics interfere with the pharmacokinetics of contraceptive steroids by interfering with their enterohepatic circulation in animal species: whether this is operative in man is still unclear. Other environmental and constitutional factors such as smoking, variations in diet, and concurrent disease may alter the disposition of contraceptive steroids and affect response accordingly. Additional studies are needed to estimate the significance of such interactions.

PIP: There has been little study on the clinical pharmacology of OCs (oral contraceptives). Studies have shown that large interindividual differences occur in the plasma concentration of contraceptive steroids, indicating a difference in metabolism. As estrogen and progestogen content of OCs are being continually lowered, any substance which reduces the bioavailability of the steroids within OCs may actually lower contraceptive efficacy also. The literature is studied for mention of drug and other environmental chemical interactions with OCs. 3 main groups of drugs have been shown to react adversely with OCs: 1) antituberculous drugs, especially rifampicin; 2) anticonvulsant drugs; and 3) antibiotics. Studies on all 3 of these types of drugs are cited along with advice to patients concerning each category of drug. For the 1st 2 types of drugs, microsomal enzyme induction in either the liver or the gut wall is the operative mechanism. Antibiotics interfere with enterohepatic circulation in animal species. It is believed that smoking and diet may also affect the efficacy of OCs. Probably only individuals with low plasma concentrations of the contraceptive steroids are at serious risk from drug and environmental interactions.

An investigation of the effects of phenobarbitone on the pharmacokinetics of norethindrone in the rat using liver perfusion and everted gut sacs.

Previous in vivo studies have suggested that phenobarbitone increases the first pass clearance of norethindrone in the rat by induction of enzymes both in the gut wall and liver. In the present study phenobarbitone caused an increase in both the production of highly polar ether-extractable metabolites and the conjugation of the steroid as it crossed the wall of the everted gut sac preparation. In addition, there was a marked increase in the uptake of norethindrone into the liver followed by increased phase I metabolism in the isolated perfused liver. As expected for a highly cleared drug, enzyme induction had no measurable effect on the terminal half-life of norethindrone in the perfused liver preparation.

Analysis of intracytoplasmic sperm injection procedures related to delayed insemination and ejaculated, epididymal and testicular spermatozoa.

In all, 1210 treatment cycles were divided into three categories for retrospective analysis according to the period of delay between oocyte retrieval (occurring at a fixed time after human chorionic gonadotrophin) and intracytoplasmic sperm injection (ICSI) of <3 h, 3-5 h, >5 h (referred to as 'delayed ICSI'). Three stages from oocyte to the birth of a live baby were identified for statistical analysis, (i) fertilization (2PN zygotes), (ii) cleavage of 2PN zygotes, (iii) transferred embryo to live birth. Stages 1, 2 and 3 were analysed statistically for the three time periods. Chi-square analysis showed no significant effect of delayed ICSI on fertilization (chi(2) = 3.615, P = 0.65), and embryo transfer to birth (chi(2) = 1.840, P = 0.399). The effect on cleavage was significant (chi(2) = 9.625, P = 0.008). However, shorter incubation times produced results which were better than the traditional longer ones. The success rate at the cleavage stage was so high that the marginal advantage had very little effect on the overall process. This study of a substantial patient sample establishes that ICSI on a peri-ovulatory oocyte (<3 h after oocyte retrieval) does not compromise outcome parameters, and that longer periods of incubation (>5 h) do not offer a statistically significant advantage.

The pharmacokinetics of tritiated ethinylestradiol in the rabbit.

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life (t1/2) of 5.5 min and the second, apparently terminal exponential was also rapid (t1/2-69 min). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterophepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administration of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.

A comparison of the fatty acid profiles of subcutaneous fat from women with breast cancer, benign breast disease and normal controls.

Using gas chromatography, the fatty acid profile has been measured in subcutaneous fat obtained from women undergoing biopsy for benign and malignant breast conditions. There was a similar proportion of saturated and unsaturated fatty acids in both types or fat, suggesting that long-term differences in fatty acid constituents of diet are unrelated to risk of breast cancer.

An investigation of the pharmacokinetics of ethynylestradiol in women using radioimmunoassay.

A radioimmunoassay for ethynylestradiol (EE) which is applicable to plasma samples obtained from women, who have taken a combination type oral contraceptive, has been developed and fully validated. Plasma concentration of EE rise to a peak of 128 pg/ml following the oral administration of 50 microgram EE. Following the intravenous administration of the same dose of EE, plasma concentrations of the steroid declined biexponentially, the two half-lives being 0.83 and 6.75 hours. Comparison of the results of the intravenous and oral administration of the steroid suggested that its oral bioavailability is 42%. Although EE thus has a lower bioavailability than norethindrone, the pharmacokinetics of the two steroids, as reflected by half-lives, plasma clearance and volume of distribution, are very similar. The occurrence of a secondary peak in plasma at around 12 hours after dosing gave strong evidence that EE undergoes enterohepatic circulation in women; an event that may have considerable clinical significance.

PIP: A radioimmunoassay for ethinyl estradiol (EE) was developed and fully evaluated. It is applicable to plasma samples obtained after administration of oral contraceptives containing norethindrone or norgestrel in addition to EE. In addition, the bioavailability of EE, by comparison of its pharmacokinetics after intravenous and oral ingestion, was determined. Plasma concentrations of EE rose to a peak of 128 pg/ml after oral doses of 50 mcg of EE. After the intravenous administration of 50 mcg of EE, plasma levels of the steroid declined biexponentially; respective half-lives for the 2 routes were .83 and 6.75 hours. By comparing these route-of-administration results, the bioavailability of EE was determined to be 42%. EE thus has a lower bioavailability than norethindrone, yet the pharmacokinetics of the 2 steroids, including half-lives, plasma clearance, and volume of distribution, were similar. A secondary peak occurred in plasma at about 12 hours after dosing, giving strong evidence that EE undergoes enterohepatic circulation in women, an event of considerable clinical significance. The most distinctive feature of the radioimmunoassay used in this study is the use of partition between methanol/water and petroleum ether to remove excess fats from the initial ether extract. The inter- and intraassay precision of this newly developed method were within the range that is normally expected of such an assay.

The pharmacokinetics of a large (3 mg) oral dose of ethynylestradiol in women.

Plasma levels of ethynylestradiol (EE), EE sulphate (EES), EE glucuronide (EEG) and prolactin were measured in women up to 72 h following the oral administration of 3 mg of EE. The decline in plasma EE levels showed a sharp discontinuity at 10 h which is assumed to be due to the beginning of enterohepatic circulation (EHC). After this event, an apparently terminal monoexponential decline was eventually established with a half-life of 13.1 h. As a result of EHC, the volume of distribution was increased by 60% to 6.0 1.Kg-1 but it does not appear that any significant accumulation of EE would occur during multiple dosing. Plasma levels of EES were, on average, 22.5 times greater than those of EE and this circulating metabolite may act as a reservoir of EE. No circulating EEG could be detected. Comparing these results with those previously obtained with 50 microgram EE, there was no evidence of dose dependency in the pharmacokinetics of this steroid. Plasma prolactin levels were markedly enhanced by this single dose of oestrogen.

PIP: To test the effects of Progynon M, a postcoital contraceptive used in Tubingen where these studies were carried out, 6 female volunteers, aged 20-30 years, with no previous use of this or other hormonal medication and in good health, were dosed with 3 mg of EE (ethinyl estradiol) on Day 25 of menstrual cycle. Blood samples were removed hourly for 4 hours, and then at 4, 6, 8, 10, 12, 16, 24, and 48 hours after dosing. Plasma levels of EE, EE sulfate, EE glucuronide, and prolactin were measured. EE levels declined and showed sharp discontinuity at 10 hours postdosing, possibly due to the initiation of enterohepatic circulation (EHC). Once the EHC had begun, a monoexponential decline was established for EE with a half-life of about 13.1 hours. Because of EHC, the volume of distribution was increased by 60%; it is theorized, however, that multiple dosing with EE would not result in accumulation of the steroid. EE sulfate levels in plasma were 22.5 times greater than EE, leading to the postulation that EE sulfate is the reservoir for EE. EE glucuronide was not detected. The results reported for this study were compared with previously reported results of metabolite action of EE, and like an earler trial of 50 mcg of EE, there was no evidence of dose dependency in the pharmacokinetics of EE. Plasma prolactin levels jumped after EE dosing.

The effect of rifampicin on the pharmacokinetics of ethynylestradiol in women.

A single dose of Minovlar (50 microgram ethynylestradiol (EE) and 1 mg norethindrone acetate) was given to seven women during treatment with rifampicin (450-600 mg/day) and again one month after stopping rifampicin. Blood samples were taken at intervals over a 24-hour period. The area under the plasma EE versus time curve increased significantly on stopping rifampicin from 1014 +/- 317 pg/ml x h (mean +/- SE) to 1747 +/- 218 pg/ml x h (p less than 0.01). The terminal plasma half-life increased from 2.9 +/- 0.8 h to 6.3 +/- 1.4 h (p less than 0.005). The sex hormone binding globulin (S.H.B.G.) capacity was also reduced from 213.4 +/- 11.5 nmoles to 129.0 +/- 7.7 nmoles/1 after stopping rifampicin. In one patient starting rifampicin who was taking Minovlar as a long-term oral contraceptive, a fall in the plasma EE concentration was associated with a rise in the plasma follicle stimulating hormone concentration. These effects of rifampicin on EE pharmacokinetics are consistent with induction of the microsomal enzymes that metabolise EE.

PIP: 7 female patients between ages 18-42 who were receiving treatment with rifampicin (450-600 mg daily) for tuberculosis were given a single dose of Minovlar (50 mg ethynlestradiol (EE) and 1 mg norethindrone acetate) and given a dose 1 month after stopping rifampicin. Blood samples were taken at intervals over a 24-hour period. The area under the plasma EE versus time curve increased significantly on stopping rifampicin from 1014 + - 317 pg-ml x h (mean + - SE) to 1747 + - 218 pg-ml x h (p 0.01). The terminal plasma half-life increased from 2.9 + - 0.8 h to 6.3 + - 1.4 h (p 0.005). The sex hormone binding globulin capacity was reduced from 213.4 + - 11.5 nmoles to 129.0 + - 7.7 nmoles-1 after stopping rifampicin. In 1 patient starting rifampicin who was taking Minovlar as a long-term contraceptive, a decline in the plasma EE concentration was associated with an increase in the plasma follicle stimulating hormone concentration. Women taking oral contraceptive steroids and rifampicin at the same time are likely to suffer a decrease in the effectiveness of the OC. It is suggested that it is unwise for women taking rifampicin to use OCs as the sole method of contraception.

The pharmacokinetics of levonorgestrel and ethynylestradiol in women - studies with Ovran and Ovranette.

A radioimmunoassay for levonorgestrel (Ng), which is applicable to plasma samples obtained from women who have taken a combination type oral contraceptive, has been developed and fully validated. Plasma concentrations of Ng rise to a peak of 3.6 ng/ml after an oral dose of 150 microgram and to 5.0 ng/ml after a 250 microgram dose. Following the intravenous administration of Ng, plasma concentrations of the steroid decline bi-exponentially with mean half-lives of 0.76 and 11.55 hours. Comparison of the results of the intravenous and oral administration of the steroid show a mean systemic bioavailability of 89% after the 150 microgram dose and 99% after a 250 microgram dose. This difference was not statistically significant. The values for volume of distribution were approximately half and the values for plasma clearance three times less than that previously reported for norethindrone. The plasma ethynylestradiol levels were also measured following administration of 30 microgram both intravenously and orally. The bioavailability and kinetics were similar to that previously reported for a 50 microgram dose. The peak values after oral dosing were 103 pg/ml and were reached by 1.0 hour in all subjects.

Studies on the enzymatic cleavage of norethisterone oenanthate.

Norethisterone oenanthate (NET OEN) was extensively hydrolysed in rabbit, rat and guinea pig plasma (99.9%, 76.1% and 46.0% hydrolysed in 90 min, respectively). In contrast, there was negligible hydrolysis (less than 2.5%) in dog, goat and human plasma. Rabbit liver, kidney, gut wall, stomach, heart and muscle showed marked hydrolytic activity in vitro, but there was little hydrolysis of NET OEN by either human muscle or fat. It is proposed that following intramuscular administration of NET OEN to rabbits, the ester is rapidly hydrolysed at the injection site and in the circulation whereas in humans the liver is the main site of hydrolysis since neither muscle nor plasma cause significant breakdown of the ester. The rabbit is not a good model for predicting the pharmacokinetics of NET OEN in humans.

The interaction of phenobarbital and other anticonvulsants with oral contraceptive steroid therapy.

In a group of 5 women on long-term anticonvulsant and oral contraceptive therapy, the plasma ethynylestradiol (EE) concentration on 50 microgram EE daily was 11.1 +/- 4.5 pg/ml. These values were at the lower end of the range found in normal women in this laboratory taking 30 microgram EE daily (6-190 pg/ml). Four women have been studied prospectively for 3 months, over 1 cycle before and 2 cycles during phenobarbital 30 mg b.i.d. therapy. Significant falls in the plasma EE concentration were seen in two women (from 104.8 +/- 13.4 to 37.7 +/- 2.0 pg/ml and from 125.6 +/- 23.8 to 34.8 +/- 6.7 pg/ml p less than 0.01) and breakthrough bleeding was seen in both women. No changes in plasma concentrations of follicle stimulating hormone, progesterone, norethindrone or norgestrel were seen. There was a significant increase in the sex hormone binding globulin capacity from 100.7 +/- 5.8 to 133.3 +/- 1.2 nmoles/1 (p less than 0.05). These changes are consistent with the known microsomal enzyme inducing effect of phenobarbital.

PIP: In a group of 5 women on long-term anticonvulsant and (OC) oral contraceptive therapy, the plasma (EE) ethinyl estradiol concentration on 50 mcg EE daily was 11.1 + or - 4.5 pg/ml. These values were at the lower end of the range found in normal women in this laboratory taking 30 mcg EE daily (6-190 pg/ml). 4 women have been studied prospectively for 3 months, over 1 cycle before and 2 cycles during phenobarbital 30 mg b.i.d. therapy. Significant falls in the plasma EE concentration were seen in 2 women (from 104.8 + or - 13.4 to 37.7 + or - 2.0 pg/ml and from 125.6 + or - 23.8 to 34.8 + or - 6.7 pg/ml, (P 0.01), and breakthrough bleeding was seen in both women. No changes in plasma concentrations of follicle stimulating hormone, progesterone, norethindrone, or norgestrel were seen. There was a significant increase in the sex hormone binding globulin capacity from 100.7 + or - 5.8 to 133.3 + or - 1.2 nmoles/l (P 0.05). These changes are consistent with the known microsomal enzyme-inducing effect of phenobarbital.

The influence of moisture content on the consolidation properties of hydroxypropylmethylcellulose K4M (HPMC 2208).

The effect of moisture content, compression speed and compression force on the compaction properties of HPMC K4M has been evaluated. As the moisture content increased from 0 to 14.9% w/w, the thickness of HPMC K4M compacts increased at constant compression force and speed. This increase in moisture content also resulted in a marked increase in the tensile strength of the tablets. At a speed of 15 mm s-1 and force of 10 kN, as the moisture content increased from 0 to 14.9% w/w, the tensile strengths increased from 1.34 to 8.54 Mpa. Equivalent tensile strengths could be obtained with less compression force as the moisture content in the polymer was increased. Increasing the compression speed generally decreased the tensile strength of HPMC K4M tablets. The dependence of tablet porosity and tensile strength on compression speeds showed that HPMC K4M is consolidated by plastic deformation. At all compression speeds, an increase in moisture content reduced the percentage elastic recovery of HPMC compacts due to greater tablet consolidation. The lowest elastic recovery (1.18%) was found for tablets made at 15 mm s-1 and 5 kN, containing 14.9% w/w moisture content.

The effect of moisture on the heckel and energy analysis of hydroxypropylmethylcellulose 2208 (HPMC K4M).

The influence of moisture content on the Heckel analysis, energy analysis and strain-rate sensitivity of hydroxyproplymethylcellulose 2208 (HPMC K4M) has been evaluated. An increase in moisture content from 0 to 14.9% w/w decreased the mean yield pressure, probably due to a plasticizing effect of moisture which reduced the resistance of particles to deformation. For each moisture content (0, 2.2, 3.8, 5.9, 9.6 and 14.9% w/w), the initial relative density and the extrapolated density from the linear portion of the Heckel plot, tended to decrease with increasing compression speed. Minor changes were observed in the initial relative density due to changes in the moisture content. The strain-rate sensitivity increased from 21.6 to 50.7% as the moisture content increased from 0 to 14.9% w/w, indicating that the plasticity of HPMC increased with increase in moisture content, whereas increase in moisture content from 0 to 14.9% w/w decreased the plastic energy. Increase in compression force or speed of compaction increased both the plastic and elastic energies. An increase in moisture content from 0 to 5.9% w/w slightly reduced the elastic energy but above 5.9% moisture content the elastic energy was unaffected by the moisture content.