Meta-analysis of on-the-road experimental studies of hypnotics: effects of time after intake, dose, and half-life.

BACKGROUND: The use of hypnotics is prevalent in the general population. Though these drugs have been shown to be effective, their residual effects may cause significant impairment to the user's driving ability. The objective of this meta-analysis is to determine whether there is a residual effect on driving and better evaluate the safety of hypnotics. METHOD: Randomized double-blind placebo-controlled studies were selected that employed a commonly used and valid driving measure to determine the user's driving ability the day after drug administration. The primary outcome measure for the driving task in all included studies was the Standard Deviation of Lateral Position (SDLP). Fixed effects model meta-analyses were performed. RESULTS: Fourteen studies, published from 1984 to 2013 (295 subjects), were included in this meta-analysis. Overall, significant impairment was found when morning testing (i.e., 10-11 h after initiating sleep) was compared to afternoon testing (i.e., 16-17 h after initiating sleep; P = .0001). Twice the standard dose also showed significant impairment (P = .0001) relative to the standard dose. The time of the test, morning versus afternoon, also had an impact on individual drugs. Middle of the night administration (MOTN) of zolpidem and zopiclone caused significant impairment the following morning, though no such impairment was seen with zaleplon. Finally, half-life was also assessed (short: <6 h, intermediate: 6-12 h, long: >12 h) and both intermediate- and long-acting drugs caused significant impairment the morning after bedtime administration, whereas short acting hypnotics did not. CONCLUSIONS: These analyses indicate that the half-life, dose of the hypnotic, as well as time between treatment and driving, as measured by SDLP, all significantly impact the ability to drive a car after taking hypnotic drugs.

Estrogen induces apoptosis in a rat prostatic adenocarcinoma: association with an increased expression of TGF-beta 1 and its type-I and type-II receptors.

Rats transplanted with the androgen-sensitive Dunning R3327 PAP prostatic adenocarcinoma were castrated and treated with either estrogen or vehicle alone for short periods (4 hr, 12 hr, 24 hr) and for 6 weeks. In these tumors the expression of TGF-beta 1, TGF-beta type-I and type-II receptors (TGF-beta RI, TGF-beta RII) was examined by immunohistochemistry. Apoptotic cells were identified by in situ nick and labelling (TUNEL). Tumor growth was retarded by castration and even more by additive estrogen treatment. The epithelium of the untreated tumors stained weakly for TGF-beta 1 and TGF-beta RI, but TGF-beta RII was not detected. Castration induced moderate TGF-beta 1 immunoreactivity in a major part of the glandular epithelium after 24 hr. After 12 hr already, castration plus estrogen resulted in an intense staining for TGF-beta 1 in the basal epithelial cells, some of which also showed an apoptotic appearance. The percentage of cells having stained positive for TGF-beta 1 was significantly higher in the estrogen-treated groups than in the castrated group after 12 hr, and its elevated TGF-beta 1 level remained at 6 weeks. Notably, the increased immunoexpression of TGF-beta 1 occurred before the onset of induction of apoptosis. In parallel with the upregulation of TGF-beta 1 after castration, the expression of its receptors. TGF-beta RI and RII, was induced and was further enhanced by the additive estrogen treatment. The number of intensely stained TGF-beta 1 tumor cells showed a strong correlation with the number of apoptotic tumor cells identified by TUNEL in the whole material. Furthermore, TGF-beta 1 immunoreactivity co-localized with the presence of apoptotic cells in the estrogen-treated tumors at 6 weeks after castration.

Estramustine-binding protein (EMBP) content in four different cell lines and its correlation to estramustine induced metaphase arrest.

It is known that estramustine (EM) accumulates in cells at the G2/M-phase and causes metaphase arrest of various cell types. The inhibitory effect is mediated by interaction with microtubule-associated proteins (MAPs) and/or tubulin. Estramustine-binding protein (EMBP) is a secretory protein which has been found in a number of different tumor cells and has been shown to faciliate the uptake of EM into cells. In this study the efficacy of EM in arresting cells at metaphase was studied, using four different human cell lines; the prostatic cancer cell line DU 145, the breast cancer cell line MDA 231, the colon cancer cell line Colon 320, and the urinary bladder cancer cell line RT4. The cells were incubated with EM at a concentration of 10 micrograms/ml for 24 hours. The data reveal an increase in metaphase arrests in the DU 145 and in Colon 320 cell lines. Both of these cell lines were found to contain high amounts of EMBP using a dot-blot assay. The other two cell lines, MDA 231 and RT4 had undetectable intracellular amounts of the protein and exhibited a low increase in metaphase arrests. The cell lines were analysed regarding S-phase fraction with flow-cytometry (FCM) to exclude the growth rate of the cells as a limiting factor. The results from the FCM confirmed the cytogenic analysis, that is a higher percentage of cells were in the G2/M phase in both the DU 145 and Colon 320 cell line compared to MDA 231 and RT4. EM causes mitotic arrest in those cell lines that contain detectable amounts of EMBP.

Deoxythymidine kinase in the staging of prostatic adenocarcinoma.

The role of prostate-specific antigen in the management of prostatic adenocarcinoma is still not fully ascertained. Its place in the monitoring of patients who have undergone radical treatment is without question but its role in the primary assessment of a lesion is a point of continuous discussion. This study reports the analysis of prostate-specific antigen (PSA) in 92 patients with different stages of prostatic adenocarcinoma prior to treatment; in the case of the localized lesions, this was based to a great extent on the findings at lymphadenectomy. Apart from PSA analysis, deoxythymidine kinase (dTK) analyses were also performed in an attempt to discover whether the latter could provide additional information about the tumor load in the different patient categories, viz. those with lymph node involvement (group 1), those with lymph node involvement but without distant metastases (group 2), and those with disseminated disease (group 3). The median PSA and dTK values in groups 1-3 were 6.5 micrograms/L and 2.7 U/microliter, 16 micrograms/L and 2.6 U/microL, and 90 micrograms/L and 7.8 U/microL, respectively. If the two analyses were used concomitantly, they could differentiate true localized disease from metastatic in approximately 92% of cases. The combination should prove of value in the primary assessment of a patient with a newly diagnosed prostatic adenocarcinoma.

Elevated expression of estramustine binding protein (EMBP) in prostatic intraepithelial neoplasia (PIN) compared with malignant and benign prostatic epithelia.

The expression of estramustine-binding protein (EMBP) was studied immunohistochemically in whole-mount prostate sections. Specimens were taken from the prostates of 15 patients who had undergone total prostatectomy due to localized (TOd-T2 NO MO) prostatic cancer (PC). Almost all the examined whole-mount sections displayed areas with prostatic intraepithelial neoplasia (PIN). PIN is regarded as the main precursor of invasive PC. High- and low-grade PIN expressed EMBP. The average positively stained areas accounted for averages of 69.2% and 48.7%, respectively. High-grade PIN contained the highest EMBP levels of all the investigated (benign and malignant) epithelia, followed by moderately differentiated PC. With regard to areas with PC, the highest levels of EMBP expression (61.3%) were observed in moderately differentiated PC; poorly differentiated PC came second. Of all the examined epithelia, EMBP levels were lowest in well-differentiated PC (25.8%). Normal prostatic epithelia and hyperplasia were characterized by low EMBP expression, although somewhat higher than well-differentiated PC. A moderate expression (45%) was observed in the seminal vesicles. According to these results, EMBP was expressed mainly in the diseased peripheral zone (PZ), where PIN and prostatic cancer have their highest prevalence.

Estramustine potentiates the radiation effect in human prostate tumor transplant in nude mice.

In this study, we have investigated the combined effect of estramustine treatment and external beam radiation on human prostatic cancer tumor cells (DU 145) transplanted in nude mice. The treatment was given according to two different schedules. In the first treatment regimen, estramustine was administered intraperitoneally (i.p.) intermittently for 20 days. The radiation therapy, which was started on day 9, was given with 6 Gy fractions during an 11-day-long period to a total dose of 36 Gy. The combination treatment (estramustine + radiation) resulted in a significant tumor growth retardation as compared to the control group. This pronounced effect was seen neither with radiation alone nor with estramustine alone. In order to further extend the radiation treatment time, a second therapy regimen was employed. In this part of the study, estramustine was administered i.p. intermittently for 26 days. The radiation therapy, which was started on day 6, was given with 4 Gy fractions during a 21-day-long period to a total dose of 40 Gy. Under these conditions, a significant tumor growth retardation was disclosed, when comparing the combination treatment (estramustine + radiation) with radiation alone. The tumors were analyzed for content of necrosis and proliferative activity. The largest proportion of necrosis was seen in the combination (estramustine + radiation) treatment group. Also, the tumors from this group expressed a decreased proliferative activity. The data indicate that estramustine acts as a radiosensitizing agent in human prostatic cancer cells in vivo. The radiosensitizing properties of the drug encourage further studies with respect to clinical application.

Lack of the latent transforming growth factor beta binding protein in malignant, but not benign prostatic tissue.

Transforming growth factor beta (TGF-beta) is a family of proteins which act as a potent growth inhibitor for most cell types including epithelial cells. TGF-beta is synthesized as latent high molecular weight complexes, composed of TGF-beta, the NH2-terminal part of the TGF-beta precursor and the third molecule, the latent TGF-beta binding protein (LTBP). We here ascertain that TGF-beta is expressed in human prostatic cancer tissue as well as in cystectomized prostatic tissue and in materials from transurethral resections with benign prostatic hyperplasia, analyzed by immunohistochemistry. TGF-beta is observed in both epithelial cells and stromal cells. No significant correlation was obtained between TGF-beta expression in tumor cells and their degree of differentiation. However, analysis by immunohistochemistry using antibodies against LTBP revealed that specimens from histopathologically verified human prostatic cancer are mostly negative for this molecule, although it is expressed in cystectomized prostatic and benign prostatic hyperplasia tissues. These results indicate that in cystectomized prostatic and benign prostatic hyperplasia tissues, TGF-beta may be produced in a complex associated with LTBP; whereas in prostatic carcinoma, TGF-beta is produced without associating with LTBP. The biological significance of the production of TGF-beta in relation to LTBP and the possible association with prognosis are discussed.

Radiation sensitization by estramustine studies on cultured human prostatic cancer cells.

In low-stage prostatic carcinoma, local cure can be obtained with radiation therapy alone, while in locally advanced disease the chances for cure are less. In this study, we have addressed the question of whether estramustine (EM), the main cytostatic metabolite of estramustine phosphate (Estracyt), may act as a radiosensitizing agent. This drug accumulates in prostatic cancer and has also been shown to arrest cancer cells at metaphase both in vitro and in vivo. The human prostatic cancer cell line DU 145 was grown as cultures monolayer and incubated with EM in concentrations varying from 1 to 20 micrograms/ml. External beam irradiation was performed with doses ranging from 0 to 8 Gy using gamma rays from a 60Co source. Clonogenic cell survival (CS) was used to analyse the radiation sensitizing effect of EM. The radiation dose modifying factor (DMF) at the survival level 0.1 was found to be 0.77 in the presence of EM (5 micrograms/ml), i.e., 23% sensitization was obtained. When irradiating cells at the standard fraction dose of 2 Gy in the absence of EM, 22% of the cells lost their clonogenic ability. In presence of EM (5 micrograms/ml), 2 Gy caused 40% of the cells to lose their clonogenic ability. Thus a radiation sensitizing effect of EM was established in the CS assay. It was also of interest to determine if the radiosensitizing effect of EM could be confirmed in a rapid assay. The rapid fluorescence assay was used to observe early damage of the cells. Results showed that by 2 days after exposure to irradiation a weak tendency towards sensitization was seen, while a clear sensitization was obtained after 4 days. This indicates that the rapid assay might be developed to a predictive assay for detection of the response of primary prostate tumor cells to the radiation sensitizing effect of EM.

The expression of estramustine-binding protein in the human prostatic cancer cell line DU 145 is not androgen dependent.

Estramustine-binding protein (EMBP) constitutes one of the major proteins in the prostatic gland, it binds estramustine and estromustine, the active metabolites of estramustine phosphate (Estracyt). Previous studies in rats have indicated that the expression of EMBP is androgen dependent, with diminishing quantities following castration and estrogen treatment as well as restored pre-castration production upon administration of androgens. In this study, we have used the human prostatic cancer cell line DU 145 transplanted in female and male nude mice. This cell line, which is sex hormone independent, gave rise to subcutaneous tumors in the rats with no difference in growth characteristics between the males and females. The expression of EMBP was analysed by radioimmunoassay, immunohistochemical and Western blot techniques. No difference was seen between the two sexes with respect to EMBP content, demonstrating that the expression of EMBP, in contrast to that reported for the normal prostate, is neither androgen- nor estrogen-dependent in tumor tissues.

Evidence for a non-estrogenic cytostatic effect of estramustine on human prostatic carcinoma cells in vivo.

Estramustine is one of the main metabolites of estramustine phosphate (EstracytR), a drug used in the treatment of advanced prostatic cancer. In the present study the hormone independent human prostatic carcinoma cell line DU 145 implanted subcutaneously in nude mice (NMRI) was used to investigate the mode of action of estramustine in vivo. Metaphase arrest was found in mice treated with estramustine intraperitoneally, 200 and 400 micrograms daily for 2 weeks, 5 days a week. A significant dose dependent decrease in the number of anaphase figures was found. A 7 to 8 fold increase in the number of abnormal metaphases, i.e., highly contracted and unaligned chromosomes, was found. Uptake and retention of 3H-estramustine was found in tumour tissue. No increase in the mitotic index or the number of abnormal metaphases was found in animals treated with polyestradiol phosphate (EstradurinR). This is the first time evidence has been presented demonstrating the anti-mitotic effect of estramustine in vivo.

Expression of Ki-67--a proliferation-associated antigen--in prostatic cancer.

The expression of the proliferation-associated antigen Ki-67 was studied in human prostatic cancer. The antigen was analyzed with an immuno-histochemical technique in TUR specimens. A correlation was seen between Ki-67 positivity and differentiation grade. All TUR specimens (15/15) with poorly differentiated carcinomas expressed the antigen. Moderately differentiated carcinomas constituted an intermediate group and slightly less than half of the cancers (12/27) were positive for the antigen. Only one of the highly differentiated carcinomas (1/12) expressed the antigen. All TUR specimens from patients with benign prostatic hyperplasia (8/8) were negative. The effect on Ki-67 positivity was also investigated in a human prostatic cancer heterotransplanted to nude mice and subjected to ionizing irradiation with or without concomitant estramustine treatment. The antigen expression was compared with that seen in tumour tissues from untreated mice and from mice treated with estramustine alone. A pronounced effect was seen in the combination treatment group with an approximately 50% reduction of the Ki-67 positive cells. The results are discussed in relation to prognosis and follow-up after radiation therapy and the possible use of estramustine in combination with radiation therapy.

Modal DNA values and estramustine-binding protein (EMBP) as prognostic markers in prostatic cancer.

In this study, we have investigated the model DNA values and the expression of estramustine-binding protein (EMBP) in formalin-fixed and paraffin-embedded TUR specimens from 76 untreated patients with prostatic cancer. In addition, specimens from 13 patients were analyzed for tumour EMBP expression only. Ploidy was measured as diploid, tetraploid and non-tetraploid aneuploid or aneuploid in the near-diploid region. All patients had been referred during 1978-1981, and were subjected to TUR due to urinary obstruction. Survival data were obtained for all patients through March 1988. Statistical analyses were performed using a Cox's regression model with respect to survival and cause specific survival and correlated to the DNA pattern and the expression of EMBP. The existence of a near-diploid aneuploid cell population as well as poor differentiation grade were both statistically significantly correlated with poor survival. Near-diploid aneuploid cell lines were seen in 9/76 (12%) of the patients and were also seen in well differentiated cancers (4/17). The expression of EMBP was most abundant in the moderately differentiated cancers. However, all prostatic cancer specimens investigated were positive for the antigen. Patients with poorly differentiated carcinomas and high EMBP expression showed a tendency towards better prognosis than those with poorly differentiated carcinomas and low EMBP expression. The present patient material was, however, too small to show a statistically significant correlation between EMBP and survival.