Next step in the development of mesoprogestins: the preclinical profile of EC313.

Introduction: The pharmacological target for progesterone, different progestins, and Selective Progesterone Receptor Modulators (SPRMs) is the nuclear progesterone receptor (PR). EC313 is a new member of a subgroup of SPRMs, mesoprogestins, which combine especially PR- agonistic and PR-antagonistic activities in one molecule. Methods: The suitable in vivo-model for the differentiation of SPRMs from the subgroup of mesoprogestins is the estrogen-primed juvenile rabbit endometrium assay (McPhail Assay). Remarkably, in contrast to other well-known SPRMs with no agonistic effects in this test, EC313 shows clear partial PR-agonistic effects that are higher than that of the well-known mesoprogestin Asoprisnil which already demonstrated remarkable clinical effectiveness for the treatment of uterine fibroids and endometriosis. The findings from the guinea pig studies presented here can be the impetus for further preclinical development of EC313. This model shows the same features for the termination of pregnancy by antiprogestins such as Mifepristone and Ulipristal acetate (UPA) in humans. Moreover, it is possible to distinguish between progestational and anti-progestational activities in the same experiment. Results: The EC313 treatment reveals PR dominance in the genital tract and inhibits unopposed estrogenic effects. In very high doses (30.0 mg/animal/day subcutaneously (s.c.)) given twice on pregnancy days 43 and 44, no premature labor was induced (in contrast to UPA, dosed at 10.0 and 30. mg/animal/day s.c.). The anti-ovulatory activity of EC313 exceeds that of Ulipristal acetate or Mifepristone. EC313 binds to the steroid receptors in vitro with a similar affinity as the natural ligand progesterone. At the glucocorticoid receptor (GR) EC313 acts as a weak inhibitor. Minor activities at the human androgen receptor (AR) and mineralocorticoid receptor (MR) are considered negligible. No binding to the estradiol receptor was detected. In contrast to some in vitro-receptor findings, estrogenic, anti-estrogenic, androgenic, anti-androgenic, glucocorticoid, and anti-glucocorticoid actions were absent in vivo. The tissue selectivity of EC313 was demonstrated previously by reducing the growth and proliferation of uterine fibroids in animal models (lowest effective dosage 0.1 mg/kg/day s.c.).. As shown in this article, the anti-fibroid activity of EC313 was confirmed with a 10 times lower dosage (0.01 mg/kg/day s.c.). It was also shown that EC313 reduces the growth of endometriotic lesions in a human xenograft immune-deficient (NOD-SCID) mice model with a comparatively very low dosage range. In the aforementioned EC313 activity model, UPA was tested as the reference compound, the clinical effectiveness of which has already been demonstrated. Discussion: For an explanation of these findings, the possibility is discussed that the mixed agonistic/antagonistic feature of EC313 is tissue target-specific based on its super-additive synergism characteristic for active bifunctional agents. In conclusion, the specific pharmacodynamic profile of this compound opens the possibility for the development of a drug with a distinct pharmaco-endocrinological profile against uterine fibroids, endometriosis, and other PR-dependent gynecological diseases.

Treatment of cat allergy with T-cell reactive peptides.

We induced in allergic humans the counterpart of murine experimental T-cell tolerance. T-cell lines from cat-allergic humans were used to map T-cell epitopes for the principal allergen of cat dander, Fel d 1. Two peptides of 27 amino acids each were synthesized to contain the dominant epitopes (ALLERVAX CAT). After a safety trial, we carried out a blinded study of the dose required for efficacy. We randomly divided 95 cat-sensitive patients into placebo, 7.5 micrograms, 75 micrograms, and 750 micrograms groups. Patients received a subcutaneous injection weekly for 4 wk. Before and after treatment, patients were exposed in a room inhabited by live cats and scored by nose and lung symptoms. Baseline nasal and lung scores (+/-SEM) were 6.2 +/- 0.56 and 5.4 +/- 0.73 in the 750 micrograms group; 7.8 +/- 0.53 and 4.7 +/- 0.68 in the placebo group. Six weeks after treatment, scores adjusted for baseline differences were reduced in the 750 micrograms group: -2.3 +/- 4.9 and -2.3 +/- 0.59 compared with -0.84 +/- 0.50 and -0.85 +/- 0.62 in the placebo group. The 75 micrograms group showed intermediate effects and the 7.5 micrograms group no effect. Linear trend analysis indicated a significant dose response effect: p = 0.05 for nose and 0.03 for lung symptoms. Allergic side effects occurred an hour or more after the first 750 micrograms dose in 16 of 24 patients but required little or no treatment with one exception. T-cell reactive treatment peptides safely improved allergic responses to cats.

Rates of thiol-disulfide interchange reactions involving proteins and kinetic measurements of thiol pKa values.

Brønsted coefficients have been determined for the rates of thiol-disulfide interchange between low molecular weight thiols and the disulfide groups of four native or modified proteins: DNase (beta nuc congruent to 0.36), lysozyme (beta nuc congruent to 0.55), adenylate kinase(SSCH3)2 (beta nuc congruent to 0.65), and papain(SSCH3) (beta nuc congruent to 0.45). These values are similar to those observed for reductions of oxidized glutathione and Ellman's reagent by a similar set of thiols (beta nuc congruent to 0.5). Glutathione is anomalously slow in reduction of certain protein disulfide groups: although this effect may in part reflect steric hinderance to attack by the relatively large glutathione molecule at disulfides shielded by protein tertiary structure, other (presently undefined) factors appear also to be important, at least in the case of DNase. The rates of reduction of several disulfide derivatives of papain(SSR) by DTT were determined. These data provide estimates of the Brønsted coefficient for the "central" thiol in thiol-disulfide interchange: these estimates fall in the range beta c congruent to -0.25 to -0.43. Rates of reduction of protein disulfide moieties were analyzed by using a Brønsted equation developed previously [Szajewski, R. P., & Whitesides, G. M. (1980) J. Am. Chem. Soc. 102, 2011] to yield pKa values for the participating thiol moieties: in particular, for papain, pKa(Cys-25) = 8.4 at pH 9 and pKa (Cys-25) = 4.1 at pH 6. The thiols of the structurally essential cysteine group of lysozyme seem to have pKa congruent to 11. The advantages and disadvantages of this method for estimating thiol pKa values are discussed.