Embryo implantation

Abstract

The present invention relates to methods of and compositions comprising cytokines for, improving the success rate of embryo implantation and the success rate of pregnancy rates in females, by providing an immunopermissive uterine environment prior to insemination or implantation of embryos. The methods of the present invention are used to make the uterus more receptive or less hostile to, for example, transferred embryos, sperm or other allografted tissue.

Claims

1. A composition consisting essentially of a recombinant eotaxin and a recombinant RANTES, wherein the composition releases the recombinant eotaxin and the recombinant RANTES in situ in a vagina within a physiological concentration range as corresponding eotaxin and RANTES found in seminal fluid of a fertile male.

2. The composition of claim 1, wherein the composition is shaped in the form of a vaginal insert or vaginal delivery system.

3. The vaginal insert or vaginal delivery system according to claim 2, wherein the recombinant eotaxin and the recombinant RANTES are active ingredients and are released in situ within the physiological concentration range as corresponding eotaxin and RANTES found in seminal fluid of the fertile male.

4. The vaginal insert or vaginal delivery system according to claim 2, wherein the vaginal insert or vaginal delivery system is a vaginal capsule, vaginal gel, vaginal tablet, vaginal powder, vaginal solution, vaginal pessary, vaginal cup, vaginal sponge, vaginal aerosol, vaginal foam or vaginal spray.

5. The vaginal insert or vaginal delivery system according to claim 2, further including an adjuvant, excipient or carrier.

6. A rodent pessary for transmucosal vaginal delivery comprising the composition according to claim 1.

7. The rodent pessary according to claim 6 for promoting uterine receptivity in a rodent.

8. The rodent pessary according to claim 6, wherein the rodent is a mouse or a rat.

9. A kit comprising: (i) a vaginal delivery vehicle comprising the composition of claim 1; and (ii) an apparatus for inserting said vaginal delivery vehicle into the vagina of a recipient female.

10. The composition of claim 1, wherein the recombinant eotaxin and the recombinant RANTES are the active ingredients.

11. A composition consisting essentially of a recombinant eotaxin, a recombinant RANTES, and one or more recombinant cytokines selected from the group consisting of TNF-, TGF-, IL-12, MCP-1, MIP, IL-17, IL-9, GM-CSF and combinations thereof, wherein the composition releases the recombinant eotaxin, the recombinant RANTES, and the one or more recombinant cytokines in situ in a vagina within a physiological concentration range as corresponding eotaxin, RANTES, and cytokines selected from the group consisting of TNF-, TGF-, IL-12, MCP-1, MIP, IL-17, IL-9 and GM-CSF found in seminal fluid of a fertile male.

12. The composition according to claim 11, wherein the IL-12 is IL-12 (p40) or IL-12(p70).

13. The composition according to claim 11, wherein the MIP is MIP-1a or MIP-1 b.

14. A method of reducing maternal alloreactivity against seminal fluid, sperm, or an embryo, the method comprising: (i) exposing a vaginal mucosa to a composition consisting essentially of a recombinant eotaxin and a recombinant RANTES, wherein the composition releases the recombinant eotaxin and the recombinant RANTES in situ in a vagina within a physiological concentration range as corresponding eotaxin and RANTES found in seminal fluid of a fertile male; (ii) introducing at least one vaginal delivery vehicle comprising the composition consisting essentially of the recombinant eotaxin and the recombinant RANTES into the vagina of a female, wherein the recombinant eotaxin and the recombinant RANTES are active ingredients; (iii) allowing a sufficient period of time to elapse to allow the active ingredients of the vaginal delivery vehicle to be released and to penetrate into the vagina and be absorbed into a uterus; and (iv) inseminating the female by either mating with a male or by artificial insemination, donor gametes or introducing an embryo into the uterus for implantation.

15. The method according to claim 14, wherein steps (iii) and (iv) are performed simultaneously or in close chronological sequence.

16. A method of improving pregnancy rate or outcome in a female prior to insemination or implantation of an embryo, the method comprising: (i) exposing a vaginal mucosa to a composition consisting essentially of a recombinant eotaxin and a recombinant RANTES, wherein the composition releases the recombinant eotaxin and the recombinant RANTES in situ in a vagina within a physiological concentration range as corresponding eotaxin and RANTES found in seminal fluid of a fertile male; (ii) introducing at least one vaginal delivery vehicle comprising the composition consisting essentially of the recombinant eotaxin and the recombinant RANTES into the vagina of the female, wherein the recombinant eotaxin and the recombinant RANTES are active ingredients; (iii) allowing a sufficient period of time to elapse to allow the active ingredients of the vaginal delivery vehicle to be released and to penetrate into the vagina and be absorbed into a uterus; and (iv) inseminating the female by either mating with a male or by artificial insemination, donor gametes or introducing an embryo into the uterus for implantation.

17. The method according to claim 16, wherein steps (iii) and (iv) are performed simultaneously or in close chronological sequence.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the Bayesian mathematical modelling of cytokine networks in mouse seminal plasma. The nodes (cytokines) are colour-coded according to the conditional probability of corresponding mediator relative concentrations being high (green), low (red) or medium (white) concentration given the state(s) of their parent nodes (the bar charts adjacent to each node reflect underlying conditional probabilities of categorization into one of the three concentration bins from low on the left to high on the right). The normalized concentration (low or high) determines the intensity of the node colour. Edges (causal connecting lines between nodes) represent causal directed interactions between nodes. These cytokines will interact with the maternal reproductive tract to induce immunopermissiveness to paternal antigens.

(3) FIG. 2 shows the Bayesian mathematical modelling of cytokine networks in rat seminal plasma (details discussed above). Very high confidence level edges are coloured in red, based upon the confidence analysis of the Bayesian result (based on occurrence in >90% bootstrapping iterations).

(4) FIG. 3 shows a bar chart of the percentage of successful deliveries following induction of pseudopregnancy by mating with a vasectomised male or a pessary (n=14 per group) of full mix, mix 1, mix 2 and mix 3.

DETAILED DESCRIPTION

(5) Reference herein to embryo is intended to include a blastula, blastocyst, fertilized ovum or an organism in its early stages of development, especially before it has reached a distinctively recognizable form that is to be implanted into a female recipient. The terms are used interchangeably.

(6) Reference herein to an improved pregnancy rate is intended to include a positive pregnancy outcome or improved perinatal survival or general viability following artificial insemination with processed semen or natural insemination or following transplantation of fresh or frozen or otherwise preserved embryos. The term pregnancy as used hereinafter is to be interpreted as encompassing a pregnancy resulting from natural or artificial insemination or following transplantation of a fresh or frozen or otherwise preserved embryo(s) and gametes.

(7) Reference herein to an intra-uterine device is intended to include any pessary-based, gel-based, solution-based, emulsion-based, powder-based or aerosol-based delivery system that is capable of delivering the compositions of the present invention into the vagina so as to permit the compositions of the present invention to have a pharmacological effect on the uterine environment.

(8) Reference herein to a pessary is intended as a means of delivery of the pharmaceutical substances of the present invention so that they are easily absorbed through the mucosal surfaces of the vagina, or intended to have action in the locality, for example against inflammation, or on the uterus.

(9) Pharmaceutical ingredient or excipient means a pharmacologically inactive pharmaceutically acceptable compound added to a mucoadhesive composition of the invention. The ingredient or excipient does not have any pharmacological properties.

(10) Rapid delivery means initial immediate rapid release and delivery of the components from the composition. The rapid delivery is typically followed by a time-dependent reduction in release of the components from the composition or device and delivery of the drug to the plasma/uterine wall tissues (or gastrointestinal tract, where appropriate).

(11) Controlled delivery means a release wherein the active agent is released from the material in a predesigned manner. The release of the active agent may be constant over a long period, it may be cyclic over a long period, or it may be triggered by the environment or other external events.

(12) Continuous delivery means continuous and uninterrupted release of the components from the formulation or device and delivering such components in a continuous manner. Continuous delivery may be preceded by the rapid delivery.

(13) Pulsed delivery means a release and delivery of the components in intermittent intervals. Such pulsed delivery may be provided, for example, by formulating the composition in individual layers interspaced with inactive layers of dissolvable coatings or by using different pharmaceutical ingredients.

(14) Seminal Fluid Cytokine Analysis

(15) Sexually mature CD1 male mice (n=20) and Wistar rats (n=20) were sacrificed and seminal fluid collected from the seminal glands post mortem, a post mortem approach was chosen to avoid collecting samples by electroejaculation since semen quality is variable by this method, and because the samples coagulate rapidly, making analysis problematic. Seminal vesicle sampling is ideal as the fluid (rather than that of the accessory glands) contains the maternal tract immunomodulatory factors investigated and because coagulating gland secretions can more easily be avoided.

(16) Seminal fluid samples were weighed individually, suspended in phosphate buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA), and weighed again. By inference to standard weight:volume ratio of murine seminal fluid, it was possible to determine the original volume isolated and the dilution factor introduced by the PBS. This step was necessary because the fluid is too viscous to be pipetted accurately. Samples were spun and the supernatant frozen at 80 C. until analysed simultaneously for the following 23 cytokines: interleukin (IL)-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-, keratinocyte-derived chemokine (KC), monocytes chemotactic protein (MCP)-1, macrophage inhibitory protein (MIP)-1, MIP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and tumour necrosis factor (TNF)-. This was achieved by custom 23-plex fluid-phase immunoassay kits run on a Luminex-100 equipped with StarStation software. Serum diluent was used in all cases to avoid false positive/negatives and dilution adjusted to 1:1 in order to maximise sensitivity to baseline levels. Similar analysis was performed on rat seminal fluid.

Example 1

(17) Table 2 below shows a variety of cytokines analyzed and measured in mouse seminal fluid. Eotaxin and RANTES appear to be the predominant cytokines present, with levels above 500 pg/ml. IL-9, TNF- and MIP-1a had levels above 100 pg/ml whereas several cytokines such as G-CSF and IFN- had levels between 50-100 pg/ml and several others such as IL-13 and TGF- had levels below 50 pg/ml.

(18) TABLE-US-00002 TABLE 2 Mouse Mean SEM IL-1 8.19 1.96 IL-1 87.48 9.04 IL-2 3.03 0.49 IL-3 0.35 0.04 IL-4 0.11 0.01 IL-5 0.56 0.07 IL-6 3.63 0.44 IL-9 135.14 33.47 IL-10 19.95 3.36 IL-12 p40 5.25 0.53 IL-12 p70 10.91 1.08 IL-13 20.64 1.86 IL-17 5.10 0.90 Eotaxin 857.22 73.85 G-CSF 45.03 3.33 GM-CSF 4.16 0.39 IFN- 46.38 3.95 KC 37.17 3.56 MCP-1 30.23 2.65 MIP-1 114.32 8.31 MIP-1 6.68 1.36 RANTES 618.62 84.17 TNF- 102.27 9.11 TGF- 27.63 6.54

(19) Table 3 below shows a variety of cytokines analyzed and measured in rat seminal fluid. RANTES appears to be the predominant cytokine present. Of the cytokines analyzed only RANTES and GRO/KC had levels above 200 pg/ml. IL-10 and IL-6 had levels above 100 pg/ml, whereas several cytokines such as MCP-1 had levels between 50-100 pg/ml and several others such as IL-17 had levels below 50 pg/ml.

(20) TABLE-US-00003 TABLE 3 Rat Mean SEM IL-1 3.28 0.97 IL-1 20.41 0.84 IL-2 29.11 3.40 IL-4 20.17 1.13 IL-5 9.58 0.95 IL-6 149.17 1.13 IL-9 54.56 0.84 IL-10 114.89 1.45 IL-12 p70 55.14 4.31 IL-13 8.29 1.35 IL-17 15.80 1.11 IL-18 6.66 0.89 TNF- 2.27 0.16 IFN- 2.93 0.39 Eotaxin 34.84 1.45 GCSF 1.51 0.07 GMCSF 40.53 2.10 MCP-1 61.56 2.21 LEPTIN 43.69 2.61 MIP-1 0.13 0.02 IP-10 4.24 0.34 GRO/KC 228.00 2.10 RANTES 287.31 2.21 VEGF 0.00 0.00 TGF- 0.00 0.00

Example 2

(21) Eotaxin and RANTES appear to be the predominant cytokines each being present in an amount of more than 500 pg/ml (see Tables 1 and 2). The cytokines IL-1, IL-6, IL-10, IL-12 (p40), IL-12 (p70), GM-CSF and MIP-1 were present at levels below 20 pg/ml and cytokines IL-1, IL-9, 1L-13, G-CSF, TNF-, MCP-1, KC, MIP-1 and IFN- were present at levels above 20 and below 150 pg/ml.

(22) Based on these analyses, a solution of cell culture-tested recombinant mouse cytokines was made up in PBS using recombinant cytokines at the concentrations found in seminal fluid (Table 3). This was stored at 80 C. until required for imbibing the pessary.

(23) TABLE-US-00004 TABLE 4 Cytokine Concentrations in utero in a Mouse Pessary Preparation once solubilised. Pessary solution Cytokine concentration (pg/ml) IL-1 8.19 IL-1 87.48 IL-6 3.63 IL-9 135.14 IL-10 19.95 IL-12 (p40) 5.25 IL-12 (p70) 10.91 IL-13 20.64 G-CSF 45.03 GM-CSF 4.16 TNF- 102.27 MCP-1 30.23 RANTES 618.62 Eotaxin 857.22 KC 37.17 MIP-1 114.32 MIP-1 6.68 IFN- 46.38

(24) Pessaries also include an amount of TGF-1 as this is important in eliciting uterine receptivity.

Example 3

(25) Additional formulation components of pessaries for laboratory animals was dictated principally by toxicity (in case of accidental ingestion), palatability (to dissuade ingestion) and impact on luminal pH (the bioactivity of certain cytokines is promoted by vaginal pH). The size and shape of the pessaries is largely determined by the species for which their use is intended. For example, pessaries of approximately 4 mm in diameter are particularly suitable for mice since the size has been determined as appropriate for insertion without undue discomfort and is also of a suitable size to be retained in the vaginal vestibule. Larger laboratory animals or indeed larger breeds of mice may necessitate larger pessaries. Pessaries were made from laser-etched nylon at a setting of between 5-10 Watts, use of this technique makes it possible to manipulate porosity (which facilitates loading) and overall shape and dimensions. It is envisaged that pessaries will be provided in a range of sizes and that the stalks may be snapped off from a central holding unit for use as desired and that a range of different sizes of pessaries may be provided to the user. Pessaries are prepared for use by soaking them overnight in 500 l of 100 times the concentration of cytokine solution so as to load the pessaries with the necessary active agents to guarantee a seminal fluid like final concentration in the maternal reproductive tract. A pessary head is then removed from the stalk and inserted by means a suitable device directly into the mouse vagina. The pessary is then left in the mouse vagina for a period of time and then it is either removed at the time of embryo transfer when the animals are anaesthetised, or it self-dissolves or the pessary self ejects once the active ingredients have been absorbed.

(26) It will be appreciated that the above embodiment is only one example of a means of delivering the compositions of the present invention and that the pessary may be in the form of a slow or fast melt wax type formulation and that the method of delivering the compositions of the present invention may vary from species to species. The delivery means may also be in the form of a biodegradable product and for example, in humans a vaginal sponge may be a more convenient method of delivering the compositions.

Example 4

(27) A full mix and three different combinations of reduced cytokine mixes, at physiological concentrations, were prepared as shown in the Table 5. The full mix comprised IL-12 (P40), RANTES, Eotaxin, MIP-1, TNF-, 1L13, IFN-, TGF-, IL-1, IL-6, 1L-12 (p70), MCP-1, IL-10, IL-1, IL-9, GM-CSF, KC, MIP-1 and GCSF.

(28) Mix 1 contained IL-12 (P40), RANTES, MIP-1, IL-13 and TGF-. IL-12 (P40) was included due to its role as a major network ancestral parent (given the marked number of outgoing edges) and its putative causal control of multiple downstream mediators. RANTES and MIP-1 were included as these were hub nodes (likely involved in multiple signalling integration based on the high number of incoming edges). The importance of MIP-1 signalling highlighted by the conservation of this hub node function across species where its function is likely analogous to that highlighted in the rat network (FIG. 2) IL-13 was included following a similar rationale and because it appeared the be the principal node fulfilling this function in the right hand side of the network main branch illustrated in FIG. 1. TGF- was included due to its purported potent immunomodulatory properties which temper the mating-induced inflammatory response.

(29) Mix 2 contained IL-12 (P40), RANTES, MIP-1, IL-13 and IFN-. The choice of mediators for this mix is given above with the exception of IFN- which, despite being a recognised abortifacient likely has a physiological role to play as a hub node at low levels as presented herein.

(30) Mix 3 contained RANTES, TNF- and TGF- for the reasons highlighted aboveTNF- was added given that it was the only terminal node of the network. Its likely key role is underscored by the fact that its terminal node position was consistent across both the mouse and rat, despite the difference in species and the change in some of the network nodes analysed. Its role in reproductive immunity is hotly debated but it is increasingly being recognised as a likely key player in triggering the early inflammatory response associated with this process. This selection was based on the mathematical modelling of cytokine networks in seminal plasma (see FIGS. 1 and 2). The full mix composition is based on the most extensive murine multiplex immunoassay-based analysis available for seminal plasma analysis.

(31) TABLE-US-00005 TABLE 5 The cytokine composition of the four pessaries used in this study (the checked boxes indicate inclusion in the mix) Cytokine Full Mix Mix 1 Mix 2 Mix 3 IL-12 (p40) RANTES Eotaxin MIP-1 TNF- IL-13 IFN- TGF- IL-1 IL-6 IL-12 (p70) MCP-1 IL-10 IL-1 IL-9 GM-CSF KC MIP-1 G-CSF

(32) The data shown in FIG. 3, confirms that the current nylon prototype pessary, loaded with either a full complement or reduced numbers of cytokines, can successfully induce pseudopregnancy in CaCBAxC57Bl/6 F1 mice. We found that our three mixes of reduced cytokines gave different rates of success but that one combination in particular, Mix 3, induced pseudopregnancy with a similar success rate as purchased vasectomised males (n=14).

(33) Throughout the description and claims of this specification, the words comprise and contain and variations of them mean including but not limited to, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

(34) Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

(35) The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.