Intrauterine administration of CDB-2914 (Ulipristal) suppresses the endometrium of rhesus macaques.

BACKGROUND: Ulipristal (UPA; CDB-2914) is a progesterone receptor modulator with contraceptive potential. To test its effects when delivered by an intrauterine system (IUS), we prepared control and UPA-filled IUS and evaluated their effects in rhesus macaques. STUDY DESIGN: Short lengths of Silastic tubing either empty (n=3) or containing UPA (n=5) were inserted into the uteri of 8 ovariectomized macaques. Animals were cycled by sequential treatment with estradiol and progesterone. After 3.5 cycles, the uterus was removed. RESULTS: During treatment, animals with an empty IUS menstruated for a mean total of 11.66+/-0.88 days, while UPA-IUS treated animals bled for only 1+/-0.45 days. Indices of endometrial proliferation were significantly reduced by UPA-IUS treatment. The UPA exposed endometria were atrophied with some glandular cysts while the blank controls displayed a proliferative morphology without cysts. Androgen receptors were more intensely stained in the glands of the UPA-IUS treated endometria than in the blank-IUS treated controls. CONCLUSIONS: In rhesus macaques, a UPA-IUS induced endometrial atrophy and amenorrhea. The work provides proof of principle that an IUS can deliver effective intrauterine concentrations of Ulipristal.

An initial pharmacokinetic study with a Metered Dose Transdermal Systemfor delivery of the progestogen Nestorone as a possible future contraceptive.

BACKGROUND: Transdermal delivery of steroids is gaining popularity for contraception and hormone replacement therapy. This study aimed to test metered spray delivery of a precise dosage of Nestorone (NES) progestogen as a possible transdermal progestogen-only contraceptive. STUDY DESIGN: Six healthy postmenopausal volunteers, not recently using any hormonal therapies, comprise the sample for this study. Each subject was studied on two occasions with multiple blood sampling for assay of NES over a 24-h period: on the first occasion, after a single dosage of 3 x 90 microL NES sprays using a specially devised, precisely metered delivery device; on the second occasion, following the fifth in a series of five daily transdermal dosages of 3 x 90 microL of NES spray. Conventional pharmacokinetic parameters were calculated. NES was assayed in serum using a specific radioimmunoassay. RESULTS: Mean serum levels of NES peaked at around 20 h following dosing, and levels plateaued at 285-290 pmol/L after 4-5 days of daily spray application. All subjects achieved satisfactory serum levels, although substantial intersubject variation was noted. The apparent elimination half-life of NES after the last dose on Day 5 was 26.8 h. No unexpected adverse events were encountered. CONCLUSION: This early pharmacokinetic trial of a new transdermal steroid delivery system has demonstrated the feasibility of achieving serum levels of NES sufficient to block ovulation and potentially provide effective contraception.

Liver enzyme monitoring in patients treated with troglitazone.

CONTEXT: Soon after initial marketing in March 1997, troglitazone, the first thiazolidinedione antidiabetic agent, was found to cause life-threatening acute liver failure. The drug was removed from the market in March 2000. OBJECTIVE: To evaluate the effect of US Food and Drug Administration (FDA) risk management efforts, including repeated labeling changes and "Dear Healthcare Professional" letters, on periodic liver enzyme monitoring of patients taking troglitazone. DESIGN, SETTING, AND PARTICIPANTS: Claims data from a large, multistate managed care organization were used to establish 4 cohorts of patients (N = 7603) with at least 90 days of health plan enrollment before first troglitazone prescription during 4 consecutive periods spanning April 1997 to September 1999 and representing 4 progressively stringent liver monitoring recommendations. MAIN OUTCOME MEASURES: Percentage of eligible troglitazone users in each cohort with baseline, monthly (for up to 6 months of continuous use), and complete (baseline and monthly) enzyme monitoring, based on computerized records of laboratory claims. RESULTS: Baseline testing increased from 15% before any FDA monitoring recommendations (cohort 1) to 44.6% following 4 separate FDA interventions (cohort 4; P<.001). In cohort 4, 33.4% of users had follow-up testing after 1 month of therapy, falling to 13% after 5 months of continuous use. In all cohorts, less than 5% received all recommended liver enzyme tests by the third month of continuous use. CONCLUSIONS: The FDA risk management efforts did not achieve meaningful or sustained improvement in liver enzyme testing. Evaluation of the impact of regulatory actions is needed before such actions can be regarded as effective or sufficient.

Group sequential test strategies for superiority and non-inferiority hypotheses in active controlled clinical trials.

In a group sequential active controlled clinical trial, the study hypothesis may be a superiority hypothesis that an experimental treatment is more effective than the active control therapy or a non-inferiority hypothesis that the treatment is no worse than the active control within some non-inferiority range. When it is necessary to plan for testing the superiority and the non-inferiority hypotheses, we propose an adaptive group sequential closed test strategy by which the sample size is planned for testing superiority and is to be increased for showing non-inferiority given that it is deemed more plausible than superiority based on the observed sample path during the course of the trial. The proposed adaptive test strategy is valid in terms of having the type I error probability maintained at the targeted alpha level for both superiority and non-inferiority. It has power advantage or sample size saving over the traditional group sequential test designed for testing either superiority only or non-inferiority only.

Nestorone: a progestin with a unique pharmacological profile.

Nestorone(R) (Nestorone 16-methylene-17alpha-acetoxy-19-norpregn-4-ene-3,20-dione), formerly referred to as ST 1435, is a potent progestin when given parenterally via sustained release formulations. The pharmacological profile of Nestorone was compared with that of levonorgestrel and 3-keto-desogestrel by steroid receptor binding studies and by in vivo bioassays in rats and rabbits. 3-Keto-desogestrel showed the highest binding affinity to progesterone receptors (PR) followed by Nestorone, levonorgestrel, and progesterone. The binding affinity of Nestorone to androgen receptors (AR) was 500- to 600-fold less than that of testosterone. However, both levonorgestrel and 3-keto-desogestrel showed significant binding (40 to 70% of testosterone) to AR. None of the progestins bound to estrogen receptors (ER). The progestational activity of Nestorone, levonorgestrel, and progesterone was compared using McPhail index in immature rabbits and pregnancy maintenance and ovulation inhibition tests in rats after subcutaneous (s.c.) administration. In all three tests, Nestorone was the most potent progestin. The progestational activity of Nestorone was also compared after oral and s.c. administration in rabbits. The potency of Nestorone was over 100-fold higher upon s.c. administration than via the oral route. The androgenic activity of progestins, based on the stimulation of ventral prostate (androgenic target) and levator ani (anabolic target) growth in castrated immature rats, showed good correlation with their binding affinity to AR. Nestorone showed no androgenic or anabolic activity. Nestorone did not bind to sex hormone binding globulin (SHBG), whereas both levonorgestrel and 3-keto-desogestrel showed significant binding to SHBG. The estrogenic/antiestrogenic activity of Nestorone was investigated in immature ovariectomized rats. In contrast to estradiol and levonorgestrel, Nestorone showed no uterotropic activity in ovariectomized rats. Despite significant binding to glucocorticoid receptors (GR), Nestorone showed no glucocorticoid activity in vivo. It is concluded that a strong progestational activity, combined with lack of androgenic, estrogenic, and glucocorticoid-like activities, confer special advantages to Nestorone for use in contraception and hormone replacement therapy.

Evaluation of in vitro reporter gene induction assays for use in a rapid prescreen for compound selection to test specificity in the Tg.AC mouse short-term carcinogenicity assay.

Under ICH guidelines, short-term carcinogenicity assays such as the Tg.AC assay are allowed alternatives for one species in the 2-year rodent bioassay. The Tg.AC transgenic mouse, which carries the v-Ha-ras oncogene under control of the zeta-globin promoter, develops skin papillomas in response to dermal application of carcinogens and tumor promoters. The appropriate specificity of the Tg.AC model for testing pharmaceuticals has not been systematically evaluated. The selection of candidate test compounds among noncarcinogenic pharmaceuticals would be aided by a high-throughput in vitro prescreen correlative of activity in the in vivo Tg.AC assay. Here we describe the development of a prescreen based on correct response to 24 compounds tested previously in Tg.AC mice. The in vitro prescreens, chosen to reflect molecular pathways possibly involved in Tg.AC papilloma formation, consisted of a zeta-globin promoter-luciferase construct stably expressed in K562 cells (Zeta-Luc) and three of the stress-response element-chloramphenicol acetyltransferase (CAT) fusion constructs stably expressed in HepG2 cells that are part of the CAT-Tox (L)iver assay. The stress response elements chosen were the c-fos promoter, the gadd153 promoter, and p53 response element repeats. Of the four assays, the gadd153-CAT assay showed the strongest concordance with activity in the Tg.AC assay, correctly classifying 78% of Tg.AC positive and 83% of Tg.AC negative compounds. The correlation was further improved by adding the Zeta-Luc assay as a second-stage screen. These cell-based assays will be used in a novel approach to selecting candidate compounds that challenge the specificity of the Tg.AC assay toward pharmaceuticals.

Assessment of similarity between dissolution profiles.

In vitro dissolution equivalence has been assessed through profile comparison between the test and reference formulations for postapproval changes. Functions of the absolute mean difference and average squared mean differences were two of the often-used criteria to evaluate distance or similarity between general profiles. The two functions and their method of moment estimators are studied in this paper as an application to similarity assessment of two dissolution profile. Due to the complexity of the distributions of these estimators, the confidence intervals obtained from the bootstrap method were used for testing the hypothesis of dissolution similarity. The size and power of two procedures are examined with a simulation study. A numerical example is used to illustrate the application of the procedures.

Chronic toxicity and reversibility of antifertility effect of immunization against gonadotropin-releasing hormone in male rats and rabbits.

The chronic systemic toxicity of immunization with gonadotropin-releasing hormone, conjugated to tetanus toxoid (GnRH-TT), was investigated in male rats and rabbits in order to start Phase I clinical trials. Groups of rats and rabbits were immunized with GnRH-TT dissolved in aqueous adjuvant. The antigen was administered at weeks 0, 4, and 8, followed by boosters to maintain high antibody titers. At termination (8-9 months after first immunization), twenty rats and ten rabbits exhibiting the highest mean anti-GnRH titers and all the controls were selected for complete toxicological evaluation. In the rat study, a castrated control group was included for comparison with the immunized group. The hematological and serum chemistry parameters of immunized rats and rabbits were not affected in a significant manner. Most of the changes in serum chemistry of immunized rats were also found in castrated rats, indicating that the changes are most likely due to the withdrawal of androgenic support. The weights of the testes, epididymides, and sex accessory glands were lower in all immunized animals. There was significant atrophy of the germinal epithelium, which, however, sustained a population of Sertoli cells, spermatogonia, and pachytene spermatocytes. Other morphological changes in the prostate, seminal vesicles, pituitary, and mammary gland reflected the effect of androgen withdrawal. The decrease in the weight of liver, kidney, and heart seen in the immunized rats was also present in castrated rats and was not associated with any histopathological changes. The reversibility of immunization-induced infertility was investigated by mating the rats with normal females. Four months after the start of immunization, 9 out of 10 immunized rats were infertile whereas by nine months, all rats had regained fertility. Thus, it is concluded that immunization with GnRH-TT had no systemic toxicological effects in the adult male rats and rabbits for the period studied. The results also indicated that the GnRH-TT immunization had an antifertility effect in male rats. Fertility was restored following cessation of immunization and decline in anti-GnRH antibody titers.

In vitro dissolution profile comparison--statistics and analysis of the similarity factor, f2.

PURPOSE: To describe the properties of the similarity factor (f2) as a measure for assessing the similarity of two dissolution profiles. Discuss the statistical properties of the estimate based on sample means. METHODS: The f2 metrics and the decision rule is evaluated using examples of dissolution profiles. The confidence interval is calculated using bootstrapping method. The bias of the estimate using sample mean dissolution is evaluated. RESULTS: 1. f2 values were found to be sensitive to number of sample points, after the dissolution plateau has been reached. 2. The statistical evaluation of f2 could be made using 90% confidence interval approach. 3. The statistical distribution of f2 metrics could be simulated using 'Bootstrap' method. A relatively robust distribution could be obtained after more than 500 'Bootstraps'. 4. A statistical 'bias correction' was found to reduce the bias. CONCLUSIONS: The similarity factor f2 is a simple measure for the comparison of two dissolution profiles. But the commonly used similarity factor estimate f2 is a biased and conservative estimate of f2. The bootstrap approach is a useful tool to simulate the confidence interval.

Multipoint dissolution specification and acceptance sampling rule based on profile modeling and principal component analysis.

In dissolution testing, multiple dissolution measurements at specific time points are needed in quality control when the compliance of the product requires controlled dissolution throughout the time course. The dissolution specification based on general multivariate confidence region was proposed by Chen and Tsong (8). This paper presents two alternative procedures when the dissolution profile consists of important measurements at more than 4 time points. In the first procedure, when the dissolution profile can be described by a physical curve through modeling, the dissolution specification is developed based on the confidence region of the parameters of the physical curve. In the second procedure, the principal components (PCS) as the linear combinations of the dissolution measurements are identified and dissolution specification is set based by the confidence intervals of the values of principal components. In both approaches the specification can be set at lower dimensions than the general multivariate confidence region approach. A single-stage acceptance rule can be used in both approaches by first projecting the dissolution values of each tablet in the new testing batch onto the determined parameters axes (through modeling in modeling approach and through projection on the selected PCS in principal component approach). Then check if the projections of the new tablet fall within the specifications. Finally, count the number of tablets that fall outside the specification limits and reject the batch if the proportion of out-of-specification tablet is high and accept the lot for release if the proportion is low.

Pharmacokinetics of 7 alpha-methyl-19-nortestosterone in men and cynomolgus monkeys.

Testosterone and its esters are widely used for androgen replacement therapy. In the prostate, testosterone ins 5 alpha-reduced to dihydrotestosterone (DHT), which leads to an amplification of its stimulatory activity in this and other tissues that have significant 5 alpha-reductase activity. While this amplification is essential during fetal development, it has potentially undesirable consequences during adult life. 7 alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that does not undergo 5 alpha reduction and is therefore being investigated for long-term clinical use because it is expected to be less stimulatory to the prostate. Since we anticipate using MENT acetate (MENT Ac) rather than MENT as the form of this androgen in humans, the bioavailability of MENT following the administration of MENT and MENT Ac was investigated in cynomolgus monkeys. Equimolar concentrations of MENT or MENT Ac were administered as a continuous subcutaneous infusion via Alzet osmotic pumps. Serum MENT levels were measured by radioimmunoassay (RIA) in blood samples collected daily for 4 days during steady state. The serum MENT levels were not significantly different in the two groups (11.3 +/- 1.6 vs. 13.1 +/- 1.2 nmol/L). This suggested that MENT Ac was rapidly converted to MENT in circulation. The hydrolysis of MENT Ac to MENT was confirmed by the in vitro incubation of MENT Ac with blood or plasma and the demonstration of MENT in products following separation by high-performance liquid chromatography (HPLC). Following the demonstration of the safety of MENT Ac in subchronic toxicity studies in rats and rabbits, a pharmacokinetic study was performed in men. In normal men, a single intravenous bolus of 500 micrograms of MENT led to peak serum MENT levels at 3 minutes after dosing (when the first samples were collected), followed by an exponential decline, reaching undetectable levels by 180 minutes. The average terminal half-life and the metabolic clearance rate (MCR) were calculated to be 40 minutes and 2,360 L/day, respectively. The results of the pharmacokinetic studies show that in both men and monkeys, the MCR of MENT is much faster than the values reported for testosterone. The faster MCR can be attributed, in part, to the finding that, in contrast to testosterone, MENT showed no binding to sex hormone binding globulin (SHBG).

Pharmacokinetics and pharmacodynamics of 7alpha-methyl-19-nortestosterone after intramuscular administration in healthy men.

7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that is resistant to 5alpha-reductases and therefore less prone to over-stimulate the prostate. It is a good candidate for implant administration in long-term androgen replacement therapy for hypogonadal men or as part of a male contraceptive system. To investigate the pharmacokinetics of MENT after i.m. administration, single i.m. injections of 2, 4 or 8 mg of micronized MENT were given in aqueous suspension to 18 healthy men in two clinics. Blood was sampled frequently for 8 h and 1, 2, 3, 4 and 9 days after the injections. Serum MENT concentrations were determined by radioimmunoassay. Peak MENT concentrations were dose-dependent and were reached about 1-2 h after the injections. Doubling the dose of MENT resulted in an increase of 60% in peak serum MENT concentrations. The mean +/- SE clearance rate was 1790 +/- 140 l/day. The antigonadotrophic activity of MENT was investigated by giving six consecutive daily i.m. injections of 1, 2 or 4 mg of MENT to 24 healthy men in two clinics. Blood was sampled before each injection and up to 24 days after the last injection. Serum testosterone and gonadotrophin concentrations (determined by radioimmunoassay and fluoroimmunoassay respectively) decreased in a dose-dependent and statistically significant manner. The highest dose caused a 74% fall in testosterone, a 70% fall in luteinizing hormone, and a 57% fall in follicle stimulating hormone concentrations. MENT injections did not cause any side-effects. The results show that MENT is a potent antigonadotrophic agent in men.

Synthesis and progestational activity of 16-methylene-17 alpha-hydroxy-19-norpregn-4-ene-3,20-dione and its derivatives.

16-Methylene-17 alpha-hydroxy-19-norpregn-4-ene-3,20-dione 1 and its 17 alpha-acylated derivatives were synthesized. The length of the 17 alpha-side-chain ranges from C2-C6. As anticipated, compound 1 did not show any progestational activity or receptor binding activity; whereas, the acylated compounds, especially the butyrate, showed remarkable ability to bind to progesterone receptors. These compounds also showed progestational activity in an in vitro T47D cell culture assay in which progestins increase alkaline phosphatase activity and in an in vivo ovulation inhibition assay. All of the compounds synthesized were without estrogenic activities. The results showed that acylation of 16-methylene-17 alpha-hydroxy-19-norprogesterone can increase progestational activity. The progestational activities of these compounds varied with the 17 alpha-side chain.

Multiple-time-point dissolution specifications for a sampling acceptance plan.

In dissolution testing, multiple dissolution measurements at specific time points are used to obtain the dissolution characteristics for most extended-release and some immediate-release drug products. This paper presents a general procedure for defining specifications by a multivariate confidence region or by simultaneous confidence limits on the dissolution values of individual time points. The confidence regions and simultaneous confidence limits were estimated using two approaches: the first approach assumed a multivariate normal distribution on the multiple dissolution values and the second approach used the bootstrap resampling method. The multivariate confidence region was constructed using the Hotelling's T2 statistic (equivalently, Mahalanobis distance D2), and the simultaneous confidence limits were based on the Mahalanobis statistic as well as on the Bonterroni adjustment. The Mahalanobis simultaneous confidence limits specification criteria have the overall false out-of-specification rate too low in both parametric and bootstrap approaches.

In vitro dissolution profile comparison and IVIVR. Carbamazepine case.

Dissolution data for the immediate or modified release drug products are usually collected as percent dissolved at multiple time points. Once an in-vitro/in-vivo relationship is established on a drug product, the dissolution profile becomes meaningful and important. In that context, if a firm desires to modify its formulation on which the in-vitro/in-vivo association has been established, a meaningful insight into the pharmacokinetics may be obtained by comparing the dissolution profiles of the two lots. In this presentation, we demonstrated a model dependent dissolution profile comparison approach using example of carbamazepine tablet dissolution data. Once a mathematical function was selected to describe the dissolution data coming from various standard lots, a similarity region could be constructed using the model parameter variances. To compare the test and reference lot dissolution profiles, a statistical distance was calculated between the mean parameters. A confidence region generated around the normalized mean statistical distance could then be compared with the similarity region to assess the similarity or dissimilarity of the dissolution profiles.

In-vitro dissolution profile comparison: statistics and analysis, model dependent approach.

PURPOSE: To develop and propose a "model dependent' approach for the in-vitro dissolution profiles comparison. METHODS: Diltiazem hydrochloride tablet dissolution profiles were compared using a statistical approach based on a mathematical model. A similarity region (SR) was defined based on the intra- and inter-lot parameter variances of the final production size standard lots. Statistical distances between the test and reference lot parameter means were computed and normalized. A 90% confidence region (CR) was developed around the statistical distance. The confidence region was compared with the similarity region to assess the similarity or dis-similarity of the test and reference (REF) lot dissolution profiles. Two test lots, one with a "minor' modification (mm) the other with a "major' modification (MM), were evaluated. RESULTS: "Weibull' was selected as the "model' function. A comparison of the confidence regions around the statistical distance of "mm-REF' and "MM-REF' with the similarity region, suggested that the dissolution profiles of the "minor' modification lot were similar and that of "major' modification lot were dis-similar to the reference lot. CONCLUSIONS: A "model dependent' approach was shown to be useful for the inter-lot in-vitro dissolution profiles comparison.

Active immunization against LHRH alone or combined with LHRH-analogue treatment impedes growth of androgen-dependent prostatic carcinoma.

PROBLEM: To determine whether active immunization against LHRH can serve as treatment for androgen-dependent prostatic carcinoma. METHOD: Male rats of Copenhagen X Fisher strain, implanted with Dunning R-3327 prostatic carcinoma cells were either immunized against LHRH, treated with LHRH-antagonist, or received a combined treatment of active immunization against LHRH and LHRH-antagonist. RESULTS: Testicular histology was consistent with infertility in all treatment groups. The rate of tumor growth was inhibited by all three treatment regimens. Tumor size increased by 3.8 +/- 1.4 cm2 in the LHRH-antagonist group, 3.2 +/- 1.1 cm2 in the immunized group, and 1.0 +/- 0.4 cm2 in the combined treatment group, as compared to 8.2 +/- 2.6 cm2 in non-treated control group. CONCLUSION: LHRH-antagonist administration combined with immunization against LHRH appeared to exert a synergistic effect. This may be due to the blockade of prostatic LHRH-like receptors by the antagonist, while androgen depletion was rapidly achieved by LHRH-antagonist, and maintained by continued gonadotropin suppression caused by active immunization against LHRH once antagonist treatment had been discontinued.

Dissolution test acceptance sampling plans.

The U.S. Pharmacopeia (USP) general monograph provides a standard for dissolution compliance with the requirements as stated in the individual USP monograph for a tablet or capsule dosage form. The acceptance rules recommended by USP have important roles in the quality control process. The USP rules and their modifications are often used as an industrial lot release sampling plan, where a lot is accepted when the tablets or capsules sampled are accepted as proof of compliance with the requirement. In this paper, the operating characteristics of the USP acceptance rules are reviewed and compared to a selected modification. The operating characteristics curves show that the USP acceptance rules are sensitive to the true mean dissolution and do not reject a lot or batch that has a large percentage of tablets that dissolve with less than the dissolution specification.

Estimation of the shelf-life of drugs with mixed effects models.

This paper proposes a normal mixed effects model for stability analysis. An EM algorithm is developed to compute the maximum likelihood estimates of regression coefficients of the fixed effects and random effects, and variance components. The likelihood ratio test is used for the preliminary testing of batch-to-batch variation. An example from a marketing stability study is given to illustrate the proposed procedure.

Comparing reporting rates of adverse events between drugs with adjustment for year of marketing and secular trends in total reporting.

The Food and Drug Administration has collected spontaneous reports on adverse events (AE) from manufacturers and individuals. These data provide useful information on the safety of marketed drugs. Due to many unique characteristics of this reporting system, the information is difficult to evaluate. Incidence rates for specific adverse events and drug combinations cannot be estimated. However, reporting rates (number of reports per market share) based on prescriptions can be computed. These reporting rates provide signals of serious adverse experience that may deserve attention. When the ratio of reporting rates is used for the comparison of two drugs of the same drug class, adjustments are needed for the marketing year and secular trends of all-drug-all-AE reporting. The Mantel-Haenszel procedure is used to combine the multiyear data into one summary statistic. Application of this analysis is illustrated on reports of upper gastrointestinal bleeding, perforation, and ulcer associated with nonsteroidal anti-inflammatory drugs, as given in Rossi et al. (12).