Method of treating endometriosis

Abstract

The invention provides a molecule that inhibits or prevents an interaction between a Src family kinase and an androgen or estradiol receptor, for use in preventing or treating a non-cancerous condition in which an activity of AR and/or ER is a contributory factor in a subject, or for use in preventing or treating a cancerous condition in which an activity of AR and/or ER is a contributory factor in a subject who wishes to preserve fertility, or for use in preventing or treating a gynecological condition in which an activity of AR and/or ER is a contributory factor in a subject. Preferably, the molecule comprises or consists of the structure: B.sub.j[(Pro).sub.n-X.sub.r-His-Pro-His-Ala-Arg-lle-Lys].sub.m-R.sub.p, or B.sub.j[lys-ile-arg-ala-his-pro-his-x.sub.r-(pro).sub.n].sub.m-R.sub.p, or a derivative or fragment thereof, wherein B is a first chemical moiety, j is 0 or 1, n is an integer from 1-10, X is any amino acid, r is an integer from 0 to 2, m is an integer from 1 to 3, R is a second chemical moiety, p is 0 or 1, and [lys-ile-arg-ala-his-pro-his-x.sub.r-(pro).sub.n] is the retro-inverso peptide of [(Pro).sub.n-X.sub.r-His-Pro-His-Ala-Arg-Ile-Lys].

Claims

1. A method of treating endometriosis in a subject in which it is desired to preserve fertility, in which an interaction between a Src family kinase and an androgen receptor (AR) and/or estradiol receptor (ER) is a contributory factor to endometriosis, the method comprising, administering to the subject an effective amount of a molecule that inhibits an interaction between the Src family kinase and the AR and/or the ER, wherein the molecule comprises the structure: Bj-Pro-Pro-Pro-His-Pro-His-Ala-Arg-Ile-Lys-Rp (SEQ ID NO: 21), wherein B is a first chemical moiety that is an acetyl group, j is 0 or 1, R is a second chemical moiety that is an NH2 group, and p is 0 or 1, wherein said inhibition occurs without a contraceptive effect in the subject.

Description

(1) The invention will now be described with the aid of the following figures and examples.

(2) FIG. 1: Mouse weight (g) in dose ranging study.

(3) FIG. 2: Lesion growth following treatment (mm2) in dose ranging study.

(4) FIG. 3: Homologues of PPPHPHARIK (SEQ ID NO: 21) in a selection of species.

EXAMPLE 1: THE PEPTIDE AC-PPPHPHARIK-NH2 (SEQ ID NO: 21) EXHIBITS THERAPEUTIC EFFICACY AGAINST ENDOMETRIOSIS WITHOUT AFFECTING FERTILITY

(5) Summary

(6) Two studies have been carried out which demonstrate the efficacy of the peptide Ac-PPPHPHARIK-NH2 (SEQ ID NO: 21) (also defined in Tables 1-4 as ValiRx1) in treating endometriosis without reducing fertility.

(7) Results

(8) In a first study, a total of 4 treatment and 5 control animals were generated in a novel in-vivo model of endometriosis (Research Horizons, University of Cambridge, 2009 Issue 8, GB 0715635.9) K-rasV12/Ah-Cre transgenic mice were crossed with Rosa26R mice to generate KrasV12+/?/Ah-Cre+/?/ROSA26R-LacZ+/? transgenic mice. The F1 offspring were inbred to generate F2 K-rasV12+/?/Ah-Cre+/+/ROSA26R-LacZ+/+ transgenic mice. The presence of the transgenes was determined by PCR using gene specific primers for K-ras, Cre and Rosa26R-LacZ. Tissue was collected from donor animals previously treated with hormones etc. and was divided between wild-type animals that were then injected with drug or vehicle for 21 days.

(9) From a total of four treated animals, three showed complete absence of lesion following treatment with one non-responder. In the control groups totaling 5 animals, one failed to develop a lesion.

(10) A second study was designed to evaluate the effect of the peptide on reproduction in healthy mice and efficacy against autografted lesions derived from one uteri re-implanted in the same animal (Becker et al, AM J Pathol 178 (4): 1782-91).

(11) Estrous Cycling

(12) 8-10 week old, female nulliparous C57BL6 mice were acclimatised for 1 week. A group of 10 animals were treated with 10 ng of peptide injected subcutaneously 3 days prior to experimentation with a group of 10 controls dosed with vehicle alone. Daily vaginal smears were taken for a period of 10 days. Normal smears were obtained in all cases

(13) Mating (Female Treatment Group)

(14) 8-10 week old, female nulliparous C57BL6 mice were acclimatised for 1 week. A group of 10 female animals were dosed with 10 ng of peptide injected subcutaneously 3 days prior to experimentation with a control group of 10 animals treated with vehicle alone.

(15) 5 male C57BL6 animals were introduced and the females checked daily for mucous plug (before 9 a.m.). When mucous plug was recorded the females were evaluated for:

(16) a) Length of possible pregnancy (days)

(17) b) Number of offspring

(18) c) Anomalies in offspring

(19) All animals delivered normal litters (with the exception of one of the dosed group)

(20) Mating (Male Treatment Group)

(21) 8-10 week old, nulliparous C57BL6 mice were acclimatised for 1 week. A group of 5 male animals were dosed with 10 ng of peptide injected subcutaneously 3 days prior to experimentation with a control group of 5 animals dosed with vehicle alone (10 ng daily SC). 10 non-treated female C57BL6 animals were introduced to each group and the females checked daily for mucous plug (before 9 a.m.). When mucous plug was recorded the females were evaluated for:

(22) a) Length of possible pregnancy (days)

(23) b) Number of offspring

(24) c) Anomalies in offspring

(25) As above all animals delivered normal litters

(26) Mating (2 Generation from Treated Females)

(27) Offspring from dosed females were mated and checked for fertility to determine possible inheritable effects. Animals were evaluated as above following plugging with no abnormalities noted.

(28) Dose Ranging Prevention Study

(29) 8-10 week old, female nulliparous C57BL6 mice were each transplanted with 6? plugs of uterine tissue from donor animals after one week of acclimatisation. Three groups of 5 animals began immediate daily dosing with 1 ng, 10 ng or 100 ng of peptide injected subcutaneously with the ARP peptide and a control group of 5 animals treated with vehicle alone. Lesion growth following treatment in the dosed group was significantly reduced compared to the control group (see FIG. 2).

(30) The model resulted in excellent lesion establishment (97.67%-100%, Table 4). A reduction in lesion burden (6.02 mm.sup.2, 5.53 mm.sup.2 and 4.52 mm.sup.2 vs. 7.18 mm.sup.2, Table 2) and growth (6.23 mm.sup.2, 5.72 mm.sup.2 and 4.52 vs 7.18 mm.sup.2 Table 3).

(31) With regard to dose, there is an option to synchronize estrous cycle by single injection of estrogen 2-3 days prior to surgery.

(32) The data obtained from the experiments described above are illustrated in FIGS. 1 and 2 and Tables 1-4.

(33) TABLE-US-00001 TABLE 1 Mouse weight following treatment with Valirx1 or control without treatment Mouse # Treatment Approach Application Dose Frequency Weight Average Weight STD SEM 1 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 25 2 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 27 3 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 23 4 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 27 5 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 24 25.2 1.79 0.8 1 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 30 2 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 30 3 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 23 4 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 30 5 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 24 27.4 3.58 1.6 1 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 25 2 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 27 3 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 22 4 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 34 5 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 34 28.4 5.41 2.4207 1 Control Prevention s.c. injection n/a Daily 28 2 Control Prevention s.c. injection n/a Daily 27 3 Control Prevention s.c. injection n/a Daily 30 4 Control Prevention s.c. injection n/a Daily 25 5 Control Prevention s.c. injection n/a Daily 28 27.6 1.82 0.8124

(34) TABLE-US-00002 TABLE 2 Lesion Burden following treatment with various doses of Valirx1 or control without treatment BUR- DEN P1 P2 P3 P4 P5 P6 Ave Mean StDev SEM Ttest 1 ValiRx1 Pre- s.c. 1 ng/ Daily 15.1 5.7 8.0 3.8 0.2 6.2 6.5 ven- injec- mouse tion tion 2 ValiRx1 Pre- s.c. 1 ng/ Daily 10.2 10.6 16.6 3.8 0.5 9.4 8.5 ven- injec- mouse tion tion 3 ValiRx1 Pre- s.c. 1 ng/ Daily 1.8 0.2 0.1 0.0 3.5 1.1 1.1 ven- injec- mouse tion tion 4 ValiRx1 Pre- s.c. 1 ng/ Daily 1.1 3.1 2.5 0.2 1.1 3.5 1.9 ven- injec- mouse tion tion 5 ValiRx1 Pre- s.c. 1 ng/ Daily 4.2 16.7 15.2 17.0 11.2 8.0 12.1 6.02 5.69 1.04 ven- injec- mouse tion tion 1 ValiRx1 Pre- s.c. 10 ng/ Daily 1.0 3.5 10.9 1.0 1.0 0.2 2.9 ven- injec- mouse tion tion 2 ValiRx1 Pre- s.c. 10 ng/ Daily 7.3 10.0 4.5 6.7 7.1 4.5 6.7 ven- injec- mouse tion tion 3 ValiRx1 Pre- s.c. 10 ng/ Daily 12.6 7.0 9.6 5.4 21.1 12.1 11.3 ven- injec- mouse tion tion 4 ValiRx1 Pre- s.c. 10 ng/ Daily 3.1 3.5 3.5 3.0 3.5 3.1 3.3 ven- injec- mouse tion tion 5 ValiRx1 Pre- s.c. 10 ng/ Daily 1.1 2.5 3.5 0.0 4.0 9.7 3.5 5.53 4.63 0.85 ven- injec- mouse tion tion 1 ValiRx1 Pre- s.c. 100 ng/ Daily 7.0 18.5 11.0 8.4 12.6 13.2 11.8 ven- injec- mouse tion tion 2 ValiRx1 Pre- s.c. 100 ng/ Daily 4.5 2.8 2.3 0.2 1.0 0.3 1.8 ven- injec- mouse tion tion 3 ValiRx1 Pre- s.c. 100 ng/ Daily 2.8 2.3 2.3 3.8 7.5 0.3 3.2 ven- injec- mouse tion tion 4 ValiRx1 Pre- s.c. 100 ng/ Daily 2.0 3.1 3.5 2.5 3.8 2.8 3.0 ven- injec- mouse tion tion 5 ValiRx1 Pre- s.c. 100 ng/ Daily 1.5 3.5 4.9 1.8 2.0 3.5 2.9 4.52 4.31 0.79 ven- injec- mouse tion tion 1 Control Pre- s.c. n/a Daily 0.2 4.9 6.6 9.3 9.6 10.2 6.8 ven- injec- tion tion 2 Control Pre- s.c. n/a Daily 7.5 6.6 9.1 7.1 9.8 7.5 7.9 ven- injec- tion tion 3 Control Pre- s.c. n/a Daily 2.5 3.8 3.8 7.0 6.2 1.8 4.2 ven- injec- tion tion 4 Control Pre- s.c. n/a Daily 4.2 4.9 5.3 9.6 8.5 4.9 6.2 ven- injec- tion tion 5 Control Pre- s.c. n/a Daily 7.0 15.7 16.7 0.5 18.4 6.2 10.8 7.18 4.26 0.78 0.37 0.15 0.01908652 ven- injec- tion tion

(35) TABLE-US-00003 TABLE 3 Lesion Growth following treatment with various doses of Valirx1 or control without treatment Growth P1 P2 P3 1 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 15.1 5.694 8.042 2 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 10.18 10.56 16.62 3 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1.767 0.196 0.126 4 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1.131 3.142 2.545 5 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 4.155 16.74 15.21 1 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 0.95 3.464 10.95 2 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 7.288 10.02 4.524 3 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 12.57 7.037 9.621 4 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 3.142 3.464 3.464 5 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1.131 2.545 3.464 1 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 7.037 18.47 11 2 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 4.453 2.827 2.27 3 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 2.827 2.27 2.27 4 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 2.011 3.142 3.464 5 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1.539 3.464 4.909 1 Control Prevention s.c. injection n/a Daily 0.196 4.909 6.605 2 Control Prevention s.c. injection n/a Daily 7.548 6.605 9.079 3 Control Prevention s.c. injection n/a Daily 2.545 3.801 3.801 4 Control Prevention s.c. injection n/a Daily 4.155 4.909 5.309 5 Control Prevention s.c. injection n/a Daily 7.037 15.71 16.74 P4 M1 M2 Ave Mean StDev SEM Ttest 1 3.801 0.196 6.158 6.5 2 3.801 0.503 9.425 8.5 3 3.456 1.131 1.3 4 0.196 1.131 3.464 1.9 5 16.96 11.2 8.042 12.1 6.23 5.67 1.05 1 0.95 0.95 0.196 2.9 2 6.739 7.069 4.524 6.7 3 5.372 21.11 12.06 11.3 4 2.969 3.464 3.142 3.3 5 3.958 9.66 4.2 5.72 4.59 0.85 1 8.357 12.57 13.2 11.8 2 0.196 0.95 0.283 1.8 3 3.801 7.548 0.283 3.2 4 2.545 3.801 2.835 3.0 5 1.767 2.011 3.464 2.9 4.52 4.31 0.79 1 9.346 9.621 10.18 6.8 2 7.061 9.802 7.548 7.9 3 7.037 6.158 1.767 4.2 4 9.55 8.545 4.909 6.2 5 0.503 18.38 6.158 10.8 7.18 4.26 0.78 0.47 0.019086519

(36) TABLE-US-00004 TABLE 4 Lesion establishment ESTABLISHMENT P1 P2 P3 P4 M1 M2 Ave Mean StDev SEM Ttest 1 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1 1 1 1 1 1 100 2 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1 1 1 1 1 1 100 3 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1 1 1 0 1 1 83.33 4 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1 1 1 1 1 1 100 5 ValiRx1 Prevention s.c. injection 1 ng/mouse Daily 1 1 1 1 1 1 100 96.67 7.45 1.36 1 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1 1 1 1 1 1 100 2 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1 1 1 1 1 1 100 3 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1 1 1 1 1 1 100 4 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1 1 1 1 1 1 100 5 ValiRx1 Prevention s.c. injection 10 ng/mouse Daily 1 1 1 0 1 1 83.33 96.67 7.45 1.36 1 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1 1 1 1 1 1 100 2 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1 1 1 1 1 1 100 3 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1 1 1 1 1 1 100 4 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1 1 1 1 1 1 100 5 ValiRx1 Prevention s.c. injection 100 ng/mouse Daily 1 1 1 1 1 1 100 100.00 0.00 0.00 1 Control Prevention s.c. injection n/a Daily 1 1 1 1 1 1 100 2 Control Prevention s.c. injection n/a Daily 1 1 1 1 1 1 100 3 Control Prevention s.c. injection n/a Daily 1 1 1 1 1 1 100 4 Control Prevention s.c. injection n/a Daily 1 1 1 1 1 1 100 5 Control Prevention s.c. injection n/a Daily 1 1 1 1 1 1 100 100.00 0.00 0.00