Methods for screening or monitoring the risk of cardiovascular disease relating to sex steroid compound or composition intake and methods for screening sex steroid compound

A method for screening for negative side effects of a sex steroid compound or composition in a subject, by carrying out an assay on the subject or on a sample derived from the subject determining whether an increase of the compound or composition on the level of an acute phase reactant or a metabolic derivative thereof has occurred since applying the compound or composition to the subject, the acute phase reactant being selected from the group consisting of positive Acute Phase Reactants (ARPs) with the exclusion of ceruloplasmin and coagulation/thrombosis associated factors. An increase in the level of the acute phase reactant being indicative of negative side effects. A sex steroid compound or composition characterized by a lower increase in APR level than a third generation oral contraceptive, as determined in a manner according to the invention is also provided; the compound or composition not being a second generation oral contraceptive.

EXAMPLE I Thirty-nine apparently healthy female subjects aged 20 to 40 years who had regular menstrual cycles (21 to 35 days) and not using OC or being pregnant the previous three month were enrolled in this study. None of the routine hematologic and serum-chemical screening and urine analysis showed abnormal results. Informed consent was obtained from all subjects and the study was approved by the Committee on Medical Ethics of the University of Leiden. Fasting blood samples were taken between 8.00 and 10.00 AM during the 17th and 25th day of the pretreatment cycle and between the 18th and 21 st day of the 3rd, 6th, and 12th cycle on OC. The volunteers were randomized (open label) to the use of two different OC preparations with an equal dose of ethinylestradiol (30 ug), but different type and dose of progesteron, being either 75 ug gestodene (GTD-EE) or 150 ug desogestrel (DSG-EE) C-reactive protein was measured with an enzyme-immuno-assay, using horseradish peroxidase labelled polyclonal antibodies against CRP (Dako, Copenhagen, Denmark) as catching and tagging antibody in a manner known per se. CRP standard serum (Behringwerke, Marburg, Germany) was used to standardize the results. The detailed procedure was as follows. CRP Assay Plasma CRP levels were measured by an enzyme immunoassay (ELISA) . The microtitration plates were coated with 100 &mgr;l Rabbit-anti-human CRP (DAKO, Denmark) in coating buffer (1:8000) and incubated overnight at 4° C. The wells were washed three times with 0.1% PBS-Tween-20 and ImM Na 2 EDTA. The plasma samples diluted 1:1000 (Hamilton, MicroLab 1000) in PBS-Tween containing 3% polyethylene glycol 6000 were added in duplicate to the coated wells. The plates were incubated for two hours at room temperature, the wells were rewashed three times and 100 &mgr;l peroxidase-conjugated rabbit-anti-human CRP was added for one hour at room temperature. After washing three times 100 &mgr;l TMB solution was added. The colour reaction was stopped with 4 M sulphuric acid and the extinction was measured by reading at 450 nm in a Titertek multiscan photometer. The standard line (0.08-21.5 ng/ml) was made of CRP standard serum (Behring, 7.6 mg/dl) For further details reference is made to Chapter 32 of J. A. Gevers Leuven et al in the book Fibrinolysis in Disease, Molecular and Hemovascular Aspects of Fibrinolysis edited by Pia Glas-Greenwalt, for what was analysed further and the used methods; From the sixty-nine from the Gevers Leuven study we only studied 39 for CRP. 1 Observations: CRP-values Pre- cy- treatment 3rd 6th 12th cle GTD- EE Number 19 19 18 18 Median 0.260 1.170&num; 0.750 2.075 IR 0.13-0.73 0.83-2.04 0.488-1.538 0.98-2.483 DSG- EE Number 20 20 18 19 Median 0.190 0.785&num; 0.740* 1.310&num; IR 0.113-0.560 0.353-1.422 0.435-2.093 0.550-2.030 IP &equals; interquartile range: Wilcoxon Matched-Pairs signed-ranks test compared to preatreatment, *&equals; p < 0.05; &num;&equals; p < 0.005. It was concluded that the use of both Ocs induced an increase in CRP. The increase lasted at least for 6 cycles. The increment in CRP from pretreatment to the third cycle showed a trend of being larger for GTD-EE (p&equals;0.063). 
 EXAMPLE II In the pretreatment samples of example I (n&equals;38) a very strong correlation (Spearman) between CRP and ceruloplasmin (r&equals;0.664, p<0.0005) was further identified in agreement with the known acute phase character of ceruloplasmin. In contrast to Ruokonen and Kaar (cited above) who regard ceruloplasmin purely as an indicator of oestrogenic activity of Ocs we chose to regard ceruloplasmin primarily as a powerful acute phase indicator. In agreement herewith a correlation of ceruloplasmin with another plasma component with an acute phase character was identified: fibrinogen (r&equals;0.3264; p&equals;0.012, n&equals;58). The change in CRP induced by OC-use (example I) was also correlated with a change in ceruloplasmin. For both Ocs or example I taken together (n&equals;39) the increment in CRP correlated with a Spearman coefficient of 0.4839 (p&equals;0.002) with an increment in ceruloplasmin after three cycles. For all three sampling moments during OC-use (example I; combining both OCs) the changes in ceruloplasmin compared to the pretreatment situation also correlated with the changes in fibrinogen (r&equals;0.3143; 0, 4386, 0, 4576 (n&equals;58). In addition the changes in von Willebrand factor and the closely related variable, Factor VIIIc. correlated with the changes in fibrinogen at all sampling moments (r&equals;0.3185 to 0.4751)(n&equals;58). The conclusion was that OCs apart from inducing changes in CRP also induce related changes in other acute phase reactants either originating from the liver (APP e.g. ceruloplasmin) or the vessel wall (APP e.g. von Willebrand factor). When using this insight to analyse in retrospect FIG. 1 of Ruokonen and Kaar (cited above) it is apparent levonorgestrel causes the ceruloplasmin concentration to decrease whereas desogestrel does not induce this effect or does so to an insufficient degree. FIG. 1 illustrates: thrombosis predominantly occurs in the first few cycles of third generation pills. Levonorgestrel decreases ceruloplasmin specifically in the first cycle lynestrenol appears to be undesirable. We feel FIG. 1 could be interpreted as a suppression of the low-grade acute phase as basically present in women by levonorgestrel. This provides a strong argument with regard to the recently discovered problem of the third generation pills in comparison to the second generation pills and our hypothesis concerning stimulatory effect on a package of acute phase reactants. 
 EXAMPLE III The effect of estrogen replacement therapy on CRP blood concentrations in 40 postmenopausal women with type 2 diabetes was studied in a randomized placebo-controlled trial. Twenty patients were treated orally with 2 mg/d of micronized 17 beta estradiol and 20 patients were treated with placebo for six weeks. Age (range: 49-68 years) and body mass index (range: 18,9-41.1 kg/m2) did not differ between the groups. Fasting blood samples were taken at baseline and after 6 weeks. For analysis of CRP, samples were available from 19 subjects from the placebo group and from 16 from the treatment group. Informed consent was obtained from all subjects and the study was approved by the Committee on Medical Ethics of the University of Leiden. C-reactive protein was measured with an enzyme-immuno-assay, using horseradish paroxidase labelled polyclonal antibodies against CRP (Dako, Copenhagen, Denmark) as catching and tagging antibody. CRP standard serum (Behringwerke, Marburg, Germany) was used to standardize the results (see detailed procedure according to example I). Observations: 2 CRP-values Pretreatment 6 weeks Placebo Number 19 19 Median 1.602.11 IR 0.98-5.09 0.80-4.99 Treatment Number 16 16 Median 4.13 6.70&num; IR 1.09-10.568 2.313-16.025 IP &equals; interquartile range; Wilcoxon Matched-Pairs signed-ranks test compared to pretreatment. &num; p &equals; 0.0027. The pretreatment values in both groups are not statistical significant different. We concluded that the use of 17-beta estradiol induced an increase in CRP. Note: It is also clear that these diabetic women already have relatively high levels of CRP, when compared to young women (see example I), healthy middle aged man (median 0.427; IR 0.144-0.776; n&equals;46) and healthy males and females 65 years and over (median 1.38: IR 0.643-2.528; n&equals;140) (not statistically tested). This however does not render the test impracticable.