IE46481B1 - Improvements in or relating to broparestrol based therapeutic compositions - Google Patents

Abstract

The therapeutic composition contains trans-broparestrol as active principle. This composition may be used, in particular, in the treatment of disorders linked to a hormonal excess.

Description

This invention relates to a new therapeutic composition containing, as active ingredient, the trans Broparestrol isomer.

Brcparestrol or l-bromo-2-ui—etnyl-phenyl)-1,2-d.iphenylethylene, obtained by chemical synthesis (N.P. Buu Ηοΐ, Bull.

Soc. Chim. Fr., 1946, 17) is a mixture of both the cis and trans isomers.

Each of said isomers was isolated ir. a state of high purity by fractional crystallization.

The structures of both the .cis and trans components of 10 Broparestrol were ascertained by analysis of the nuclear magnetic resonance (NMR) spectra and confirmed by radiocrystallcgraphy (J.M. Fornies-Marcuina, C. Courseilie, B. Busetta & M.(Hospital, i Crystal Structure Communication, 1972, J.» 261-264). $ Said physical-chemical investigations demonstrated that the trans isomer of Broparestrol may be represented as follows: CH- CH- trans-isomer Broparestrol, i.e., the mixture of the cis- and transisomers, which contains about 40% cis-isomer and ahout 60% trans20 isomer, is already used for therapeutic purposes due to its low oestrogenic properties. Under the tradename Longestrol, it is used at an average daily dosage of three 25 mg tablets, typically for the treatment of mammary disorders and to control metastases of the neoplasms of the endocrinogenital system.

Applicant has discovered that the two Broparestrol isomers possess in fact different specific properries and that the - 2 46481 .tlGllS. Broparestrol isomer constitutes a new therapeutically useful active ingredient which, rlua to its properties, is found to differ from the cis Eroparestrol isomer and from the heretofore therapeutically used mixture of both the cis and trans isomers.

Particularly, Applicant has found that,while all said materials exhibit an action of competitive dualism type in their actin with respect to oestrogens, i.e., a potentiation of the effects of low oestrogen dosages and an antagonism with respect to high oestrogen dosages, the modulating effect of the trans Broparestrol isomer is found to differ from that of the cis isomer and also from that of the mixture of the cis and trans isomers, because tha antagonistic effect is highly predominant in the case of the trans isomer whereas the synergistic effect is predominant in the case of the cis isomer.

Thus, this invention relates to a therapeutic composition having, in particular, a modulating or regulating effect on the effects of oestrogens, which contains the trans Eroparestrol isomer as active ingredient and is substantially free of the cis-isctner, 2C togetner with a pharmaceutically acceptable carrier.

The therapeutic composition of this invention may be formulated in a form suitable for oral or sub-cutaneous administration Pharmaceutical formulations suitable for oral administration .include capsules, tablets, and suspensions. The capsules and tablets may typically contain 10-300 mg of the trans Broparestrol isomer, and preferably 10-50 mg.

Pharmaceutical formulations suitable for sub-cutanecus administration include injectable solutions in an acceptable solvent such as olive oil. Each ampoule may typically contain 3-200 mg of the trans Broparestrol isomer, and preferably 3-50 mg The trans Broparestrol isomer may be administered to human patients at a daily dosage of 0.3-10 mg/kg by the oral route and at a daily dosage of 0.05-3 mg/kg by the sub-cutaneous route.

The trans Broparestrol isomer is predominantly applicable in the'treatment of female patients in the following cases : - in the treatment of disorders related to a hormonal excess and typically : - in the treatment of hormone-dependent mammary cancers, in the treatment of menopausal and post-menopausal disorders, - to correct the effects of oral contraceptives consisting of oestrogen-progestational agent combinations, - also as contraceptive agent due to its antifertilizing action, and in male patients : - in the treatment of hormone-dependent cancers of the prostate.

When applied to counteract the secondary effects of oral contraceptives, the trans Broparestrol isomer may be administered to female- patients either independently from the oral contraceptive, or in combination with the latter.

Therefore,, this invention includes also within its scope combinations of the trans Broparestrol isomer with oral contraceptives, which combinations exhibit the primary effects of oral contraceptives without their objectionable side-effects.

Useful oral contraceptives include combinations of an oestrogen such as Ethinyl oestradiol or Mestranol and of a progestational drug such as Norgestrel, Norethisterone acetate, Quingestanol acetate and Lynoestrenol.

Results of pharmacological investigations which demonstrate the valuable properties of the trans Broparestrol isomer, particularly a3 compared to the cis Broparestrol isomer and to ths jnixture of the cis and trans Bropare/.trcl isomers aregivenbelow 1 - Comparative uterotroohic action a) Material_and_Method d -· Procedure Use was made of the Lauson test in 2550 Sprague Dawley impuberal female rats (21 days, 40 g + 2 g). The animals were distributed into 85 groups of 30 animals each and were adminis10 tered the test material or its carrier (1 reference group for each test) for three consecutive days.

The animals were sacrificed on the fourth day. After ether anesthesia and bleeding, the uteri were dissected and weighed ina fresh condition.

The test materials were dissolved in virgin olive oil and administered orally at a . uniform volume of 0.2 ml/20 g.

The materials used were Ethinyl oestradiol, trans Broparestrol, cis Broparestrol and a 60 trans/40 cis Broparestrol mixture.

I?·· Expres3ion_of_the_results The uterotrophic action is evaluated by the percent weight variation of the uterus of the treated animals with respect to that of the reference animals.

The effects are represented by an action (with respect 25 to the maximum effect of the Ethinyl oestradiol reference) vs/dosage (as moles/kg) diagram.

The affinities and intrinsic activities were then calculated.

In this respect, it should be remembered that when a drug or agonist A becomes attached to a receptor R, thus forming a complex RA - 5 46481 EA and when:· Eft = the effect of agonist A at a dosage of E^ = the maximum biological effect obtainable = the maximal effect obtainable with agonist A +_3· A/ defined as being the value /— S A defined by the AM = 0( ratio and intrinsic Affinity is activity is At one-half the effect = τ , K. = , eam 2 A pi>50 is the co-logarithm of the agonist concentration which ID produces 50% of the maximum effect. b) Results 1) Action of the individual products per se The results obtained are given in Table I (as percent weight variation of the uterus/vs the maximum effect of Ethinyl oestra15 diol Ea/Em) and illustrated in Pig.l.

TABLE 1 Uterotrophic action of the compounds per se Dosage «7 ..A -6 -5 -A “3 -2 -1 -7 (g/kg) 10 ' 10 2 5x10 5x10 10 10 ' 10 10 10 χ 5x10 *· Ethinyl oestradiol 0.55 27.6 64 85.5 96.3 loo — Cis-Rropa- restrol - - - - 7.9 9.9 32 60 83.3 85.6 Bropares- trol - - - - 3.4 17.4 36.7 59.5 59 66 Trans-Bro- parestrol - - *- o.b 20.5 28.6 35.2 39.4 35 Both the cis and trans isomers and their admixture have an uterotrophic action and, thus, behave like oestrogenomimetic - 6 46481 drugs (Fig.l).However, their agonistic actions — characterized by the intrinsic activity — are markedly differentiated and follow the reverse law of their affinities, as apparent from following Table II which gives both the affinity and the intrinsic activity for each material.

TABLE II Product Uptake Agonistic action Affinity (pD2) Intrinsic activity ¢( Ethinyl oestradiol 8.20 1.00 trans-Broparestrol 6.80 0.375 Broparestrol 5.55 0.690 cis-Broparestrol 4.95 0.790 Ethinyl oestradiol is a total agonist which exhibits the strongest affinity and maximum intrinsic activity? cis-Broparestrol is a partial agonist which exhibits a very strong intrinsic activity, but the lowest affinity. Thus, it ie very weakly taken up, but it is capable of exhibiting a good oestrogenic action at high dosages.

Broparestrol (mixture of cis- and trans-isomers) is the 2D partial agonist which exhibits both average affinity and average intrinsic activity.

Of all tne partial agonists, trans-Bronarestrol is the one which exhibits the lowest oestrogenomimetic activity together with one of the highest uptake,after Ethinyl oestradiol. 2) Interaction of the different products with Ethinyl oestradiol In another series of tests, Ethinyl oestradiol was administered simultaneously with the trans Broparestrol isomer, the cis Broparestrol isomer and the mixture of both isomers (60/40), respectively.

The results obtained with the trans Broparestrol isomer, - 7 30 the mixture of 5)oth isomers (60/40) and the cis Broparestrol isomer are illustrated in Pigs. 3 and 4, respectively. On the diagrams of said Pigs. 2-4, the relative uterotrophic effect Ε /E (Effect observed/maxinum effect observed with Δ Ethinyl oestradiol) is plotted as a function of the log of the Ethinyl oestradiol dosage (mole/kg).

A modulation of the uterotrophic action of Ethinyl oestradiol (—0—) by the trans Eroparestrol isomer, the mixture of both isomers, and the cis Broparestrol isomer, administered ’ —3 —9 simultaneously at dosages of 10 g/kg (-e-—) and 10 g/kg ( Λ ) is apparent from Figs. 2, 3 and 4, respectively.

It is apparent that the three products enter into a competitive dualism at different levels with Ethinyl oestradiol. They are synergistic at low Ethinyl oestradiol dosages and potentiate the minimum effects; they are antagonistic at high Ethinyl oestradiol dosages, inducing a maximum response which is lower than that of Ethinyl oestradiol. In addition, the values noted arp reported in following Table III.

TABLE III Uterotrophic effect (percent weight variation of the uterus with respect to the controls and compared to the maximum value of Ethinyl oestradiol) Dosage g/kg ETHINYL OESTRADIOL 0 io7 10“6 2.5xl0~6 5χ1θ”6 IO5 I o i-i fS reference groups O 0 5.5 27.6 64 85.5 96.3 100 trans-Broparestrol IO-3 28-6 21.2 21.3 27.8 28.6 25.9 58.7 trans-Broparestrol io2 35.2 38.8 35.3 40.4 47.4 47.4 50.1 Broparestrol(60:40) io3 36.5 35 38.2 39 43.6 53.5 78.2 Broparestrol(60:40) 10-2 59.6 44.7 46.3 48 45.4 48.5 52.5 cis-Broparestrol io3 32 41.9 47.54 58.9 87.8 103.7 108.4 cis-Broparestrol IQ-2- 60 67.5 70.9 75.25 69.7 73.5 1 69.8 - 8 46481 First of all, ir is apparent that ail three products are partial agonists and are intermediates between a pure agonist such as Ethinyl oestradiol (high intrinsic activity) and the competitive antagonists ( as yet undiscovered antioestrogens) It is also apparent, however, that the dualist activities of the different products are highly different.

For purposes of clarity, in the case of each compound. Applicant integrated the areas limited by the dosage effect curve of Ethinyl oestradiol alone and combined. This gives an area corresponding to the synergy (1) and an area corresponding to the antagonism (2), as illustrated below.

The percentage .and the ratio of said areas permitted quantification Of the dualism.

The results of said calculations are reported in Table IV and in the diagram of Fig.5. This diagram illustrates respectively, for each product: in the left column , the ratio antagonism area/synergy area; and, in the right column, the ratio synergy area/anfcagonism area.

It is clearly apparent that the trans-Broparc-strol isomeiexhibits a predominant antagonistic effect, whereas the cisBroparestrol isomer exhibits a predominant agonistic effect. - 9 46481 CO CJ rH rH c\ rH in tn m cn co * o <0 rH in rn t- in KO rH un o • β 0 Cn rt rH r-i cu o rH +> a YY YY vo rH OJ un CJ C KO LL TABLE Y° (0 Φ U d fc Φ β Ph W Q\ 00 oj -=r I3· -=1CO tnfco ro ϋ ω\ Oto rn oj o rH n oj I I o o tn oj I I o o μ β Ό Ο Μ ti rH Ο fc -Ρ Μ 0) fc ω ctf C Ρ. co ο fc fc Η ΡΊ γΗ ο fc ω ω fc a ο fc c Πί Φ ti rH Ο fc -Ρ w Q) fc <0 ,P< WO •H fc eta - 10 This difference in the dualist action may be evaluated in the following manner. 48481 TABLE V PRODUCT DUALISM LIMIT Agonism Antagonism Ethinyl oestradiol 100 % cp,i Pure agonist cis-Broparestrol 84' % • 1875 Agonistic tendency Broparestrol 47.2,5 52.855 Pai'tial agonist-antagonist trans-Bropares trol 26.275 73.855 Antagonistic tendency Thus, the different products modulate the response curve to the oestrogens, but at different levels. Thus, the transBroparestrol isomer provides a regulation of the effects of oestrogens at relatively low levels, which permits, by substantial cutting down at the top, to prevent the detrimental effects of high oestrogen dosages, and, by slight cutting down at the bottom to retain the indispensable minimum physiological effects. Thus, the trans-isomer constitutes a much better regulator of the effects of oestrogens than the cis-isomer which induces modulation only at a high level.

II - Prevention of spontaneous mammary. tumors in mice C_h/m submitted to forced reproduction by treatment J e with trans-Broparestrol - Antifertilizing effect in the same animals a) Preamble Mammary tumors may develop spontaneously in mice C^H/^ carrying the Bittner viral factor. Accelerated reproduction promotes the formation of such tumors and shortens the period of time preceding their appearance. While other typos of experimental mammary tumors induced by eaneerogenie materials are hormone-dependent - and whose formation and evolution are relatively readily controlled, -.he spontaneous tumors in mice C H/ are more difficultly controlled. Similarly, the 3 no development of such tumors appears to be essentially dependent on an increased secretion of prolactine by the pituitary gland. b) Katerial_and_method The experiment was conducted in female mice C.H/„ (ln-le).

J He Only mice bred by Applicant and closely attended to for a number of years were used. Due to suitable caretaking conditions, both where the mothers and the new-born are concerned, the animals may live under good physical conditions for more than two years. The spontaneous death rate is infinitesimal; this authorizes the undertaking of experiments of long duration.

Female mice (ln-le) were isolated from litters born at 48 hour intervals. At the age of four weeks, animals weighing between 19 and 20 g are selected. Thirty-five females are thus selected; they are each given a number and they are distributed into two groups: - Group 1: 20 animals, submitted to treatment with transBroparestrol. 50 mg of product per kg of body weight, diluted with pharmaceutical grade olive oil, are injected sub-cutaneously once a week for 2 months. On completion of the treatment, ths females are placed in cages (4 per cage) in the presence of a male and submitted to reproduction.

- Group 2: Reference group. 15 mice are given only the oily excipient, under the same conditions, during a period of time of 2 months. At the end of that time, they are placed by -fours and threes in cages in the presence of a male and submitted to reproduction.

Accelerated reproduction is conducted in the usual manner: immediately after birth, the young are removed and the females are again sui-r.iittcd to thr- males.

The animals arc weighed once a week. The number of gestations and litters, together with the formation of mammary tumors, the period of time prior to their appearance, are individually recorded for each mouse.

The total duration of the experiment is eighteen months, c) Results ei ) Prevention of mammary tumors After carrying out the experiment for 1.5 year, the development of mammary tumors is markedly lower in the group treated with trnns-Broparestrol.

In the reference group, except for a mouse sacrificed at the age of 6 months due to a poor systemic condition, and which diif not carry any tumor, all other mice exhibit mamniary tumors. Out of 20 animals preventively treated with trans-Broparestrol, there are noted only three cases of mammary tumors. One of the mice, sacrificed at the age of 18 months due to a poor systemic condition, does not carry any mammary tumor. Autopsy of the other mice, at the end of the experiment, did not disclose any tumorous lesion, either in the mammary tissue areas or in the other organs.

The number and percent mammary tumors of each of the groups are reported in Table Vl.

Whereas, in the reference group, the percent tumor® is 93% (frequent percentage for this line of mice), treatment with trans-Broparestrol reduced the percentage to 15%.

Thus, in the group treated with trans-Broparestrol, the development of tumors is markedly lower than that noted in the reference group.

VI Inhibition with trana-Bronarestrol of the development of spontaneous mammary tumors in mice Number of tumors, percent tumors, and time to the appearance 5 of tumors in the reference and treated groups.

Age of the animals (months) Time of latency (days from the beginning of the treatment) Animals with mammary tumors Treated group Reference group 0/ /o % 5 103 0 0 0 0 5 110 0 0 1 6.6 6 138 0 0 1 13.3 6 145 0 0 1 20 6 152 0 0 1 26.6 7 166 0 0 2 40 8 194 0 0 1 46.6 8 202 0 0 2 60 9 215 0 0 1 66.6 9 230 0 0 2 80 12 307 1 5 1 86.6 16 432 1 10 0 86.6 18 516 1 15 1 93.3 19 (sacrifice) 547 0 1 - Total 3/20, i .e.,155( 14/15 , i.e. 9355 It should be noted that where the period of time prior to the appearance of the tumors is concerned, there is a marked difference between the two groups. In the reference group, most tumors appear in the animals at the age of 6-9 months. In the group treated with trans-Broparestrol, this period of time is extended; the first tumor- appears belatedly at the age of 12 months, and the other two at the 16th and 18th month of age. β} Action of trans-Broparestrol on reproduction After two months of treatment, trans-Broparestrol reduces markedly the reproduction in mice.

Among the 20 animals of the treated group, two females only gave birth a single time, four months after the end of the treatment. It should be noted that one of these two females (mouse n°9) developed a mammary tumor, 4 months thereafter.

The other mouse did not exhibit any tumorous lesion.

During the experiment, the males were changed three times in each of the treated cages. As a comparison, reproduction was normal in the reference group; each female gave birth several times. The number of litters per female varies between three and eleven litters, depending on the more or less early appearance of tumors in said mother-mice.

This contraceptive effect is durable; it extended to the end of the experiment, i.e., 16 months after interruption of the treatment. d) Conclusion Early administration of trans-Broparestrol (50 mg/kg/s.c./ once weekly)from the 1st to the 3rd month of age, in mice G35I0le su-bn:’-ttG'3· to forced reproduction, decreases considerably the development of tumors. Similarly, the time before the appearance of the first tumors is significantly extended in the mice treated with trans-Broparestrol.

On the other hand, a durable inhibition of reproduction was noted in ths animals treated with trans-Broparestrol.

III - Action of trans-Broparestrol on fertility a) Material_and_Method The test was conducted in adult Sprague Dawley female rats weighing about 200 + 10 g distributed into groups of 8 animals each. - 15 46481 Trans-Broparestrol is administered orally for 35 days.

The females (groups of 4 females each) arc placed in the prusenco of 2 males from the 6th day of treatment.

The females are isolated and the treatment is interrupted as soon as the females are thought to be fecundate.

The criterion of appreciation is a supranormal weight increase of the fecundate females. The weight increase of reference animals is on the average of 14 g over 12 days, and the fecundate females are isolated after about 12 days when their increase in weight is in excess of 35 g. trans-Broparestrol was administered at dosages of 10 t 2.5xlO-4 and 10~3 gAg. b) Results The results obtained are given in Table VII.

The results are expressed as percent anti-fertilizing action, trans-Broparestrol has a total anti-fertilizing action at a dosage of 10~3 gAg.

TABLE VII Dosages Percent anti-fertilizing activity -4 o 10 2.5xlO-4 25 10 J 100 IV - Toxicological investigation of trans-Broparestrol a) Acute toxicity Acute toxicity was investigated in male and female rats and in male and female mice after administration of transBroparestrol by two different routes: orally and sub-outaneously.

Whatever the route of administration, trans-Broparestrol is non-toxic.

TAELS VIII Species Sex Route Maximum dosage administrable Mortality Rat male oral 4000 mg/k<3 Nil fatale oral 4000 mgAg Nil male sub-cutaneous 2000 mg/kg Nil female sub-cutaneous 2000 mg/kg Nil Mouse male oral 4000 mg/kg Nil female oral 4000 mgAg Nil male sub-cutaneous 2000 mgAg Nil female sub-cutaneous 2000 mgAg Nil Ko symptom of intoxication is noted on administration of the compound. Behaviour remains normal; no fatal issue was noted after 14 days of observation. Autopsy does not disclose any macroscopic lesion of the principal organs; however, a decrease of the weight of the uteri is observed in the females. b) Long-term toxicity Long-term toxicity was investigated in male and female rats which were orally administered trans-Broparestrol during a period of time of three months at dosages of 1 - 5 - 10 and 20 mg/kg/day, respectively. Behavior of the animals remained normal throughout the treatment; no death occurred.

Hematological examination does not disclose any change in the counts and in the hemoglobin content.

As a whole, the modifications of the various biological constants are within the limits of the normal physiological variations in the controls.

Histological examination of the main organs is normal. In male rats, trans-Broparestrol has no histologically visible effect on the different viscera of male rats and also no perceivable oestrogenic effect at tho level of the endocrine glands. In female rats, the histological structure of all organs is normal, except for the ovaries in which an endocrinic effect is noted at very high dosages.

Examples of administrable formulations containing, as active ingredient, substantially pure trans-Broparestrol (i.e., substantially free from cis-Broparestrol) are given hereinunder. 1. Tablet containing 10 mg trans-Broparestrol trans-Broparestrol 0.010 Lactose 0.130 Rice starch 0.045 Pregelatinized corn starch 0.006 Sodium carhoxymethylcellulose 0.002 Talc 0.004 Magnesium stearate 0.003 2. Capsule containing 50 mg Jurans-Eronarestrol trans-Brocaresrrol Lactose Talc Magnesium stearate 0.050 g 0.243 g 0.005 g 0.002 g 3. Injectable solution containing 5% trans-Broparestrol trans-Broparestrol 0.005 g Benzyl benzoate 0.001 g Neutralized olive oil, sufficient to make 1 ml Combinations of trans-Broparestrol with oral contraceptives themselves consisting of an oestrogen-progestational drug combination, may be formulated as pills, capsules or tablets containing 0.1-5 mg of a progestational agent such as Norgestrel, Norethisterone acetate, Quingestanol acetate or Lynoestrenol, 0.01-0.5 mg of an oestrogen such as Ethinyl oestradiol or Mestranol, and 30-300 mg trans-Broparestrol (preferably 50-80 mg).

Examples of such combinations are given below. 1· Ordinary tablet Mestranol 0.075 mg Lynoestrenol 2.5 mg trans-Broparestrol 60 mg Excipient, sufficient to make one 150 mg tablet (lactose, rice starch, glycerine, talc, magnesium stearate) 2. Coated tablet Ethinyl oestradiol 0.05 mg Norgestrel 0.5 mg trans-Broparestrol 40 mg Excipient, sufficient to make one 150 mg coated tablet (lactose, corn starch, gelatin, white wax, carnauba wax, magnesium stearate, methyl paraben, propyl paraben, talc, polyvinylpyrrolidone, tricalcium phosphate) 3. Capsule trans-Broparestrol 150 mg Mestranol 0.075 mg Lynoestrenol 2.5 mg Lactose 143 mg Talc 5 mg Magnesium stearate 2 mg

Claims (9)

1. Therapeutic composition comprising trans-Broparestrol isomer as active ingredient and being substantially free of the cis-isomer, together with a pharmaceutically acceptable 5 carrier.

2. Composition as claimed in claim 1, formulated in a form suitable for oral administration.

3. Composition as claimed in claim 2, in unit dosage form, each unit dose containing 10-300 mg trans-Broparestrol isomer 10

4. Composition as claimed in claim 1, formulated in a form suitable for sub-cutaneous administration.

5. Composition as claimed in claim 4, consisting of an injectable solution in a pharmaceutically acceptable solvent.

6. Composition as claimed in claim 5, formulated as · 15 ampoules containing each 3-200 mg trans-Broparestrol isomer.

7. Composition as claimed in claim 2, wherein there is additionally present an oral contraceptive consisting of an oestrogen-progestational agent combination.

8. Composition as claimed in claim 7, comprising 0.1-5 mg 2o of a progestational agent, 0.01-0.5 mg of an oestrogen, and 30-300 mg trans-Broparestrol.

9. Composition as claimed in claim 1, substantially as described in any one of the formulation examples herein.