Estradiol benzoate as sterilant

Abstract

The invention relates to estradiol benzoate as a sterilant. Methods, compositions, and systems for delivering estradiol benzoate to an animal to sterilize the animal are presented. The physiologically acceptable oil or other carrier in the capsule is used to deliver estradiol benzoate. Estradiol benzoate is delivered intraperitoneally, intramuscularly or subcutaneously to a companion animal or livestock animal.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of July 30, 2019, application number 201980089378.4, and invention name “Estradiol benzoate as an infertility agent”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 772,878, filed on November 29, 2018, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to the technical field of making animals sterile. Background Art

[0005] Sterilization, also known as neutering / spaying (or induced infertility), refers to rendering an animal (male or female) incapable of reproduction. Sterilization involves the partial or complete removal of reproductive organs or rendering them nonfunctional. Over time, sterilization has come to play an important role in companion and food animals for a variety of purposes. In companion animals such as dogs and cats, sterilization of animals is primarily for population control, mitigating overpopulation of pets and the subsequent costs associated with caring for these animals.

[0006] In food animals such as cattle and pigs, sterilization is mainly performed on males by castration as a means of reducing testosterone production. The presence of testosterone in food animals causes an unpleasant, pungent odor when processing and cooking their meat, especially meat from pigs. Castration can also produce a higher grade of meat, i.e. leaner meat with a higher intramuscular fat content, also known as marbling, especially in beef. In addition, castration reduces aggressiveness, especially in male animals, thereby reducing potential physical harm to other livestock or to human handlers.

[0007] Currently, sterilization of both companion animals and food animals is primarily accomplished through surgical methods: physical removal of the male testicles and removal of the female ovaries and uterus. However, there are multiple problems with using surgical sterilization methods. For example, surgical sterilization can be painful for the animal, can be expensive for the animal's owner, and can lead to secondary complications, such as infection. Such complications can result in additional treatment costs and an increased risk of further injury or death to the animal.

[0008] In some male food animals, such as bulls, castration is achieved by non-surgical methods, such as using an elastomeric ring to block or otherwise disrupt the blood supply to the testicles, resulting in the death of testicular tissue. Typically, both surgical and non-surgical castration of livestock is performed without anesthesia or other pain management, which inevitably causes great discomfort / pain to the animal.

[0009] For these reasons, there is a need for non-invasive, humane methods for sterilizing animals. Summary of the invention

[0010] Described herein is the use of estradiol benzoate (EB) as a novel method for rendering animals sterile.

[0011] One embodiment provides a method for inducing infertility in an animal by administering an effective amount of estradiol benzoate (EB) to the animal to make the animal permanently infertile / sterilized. In one embodiment, EB is administered together with a carrier (e.g., oil). In another embodiment, EB is formulated into a sustained release formulation. In one embodiment, administration is a one-time single dose. In some embodiments, the composition can be administered intraperitoneally, intramuscularly, or subcutaneously. In one embodiment, the animal is a companion animal. In another embodiment, the animal is a livestock animal. In one embodiment, EB is administered before puberty. Also provided herein is the use of EB to induce infertility.

[0012] Another embodiment provides a system for delivering a sterilant to an animal, the system comprising: a capsule comprising estradiol benzoate and a delivery device for delivering the capsule to the animal. In one embodiment, the capsule is formulated to release EB within a predetermined time. In one embodiment, the delivery device is an injector comprising a main body and a force member. In another embodiment, EB is administered intraperitoneally, intramuscularly or subcutaneously. In one embodiment, the animal is a companion animal. In another embodiment, the animal is a livestock animal.

[0013] This summary is intended to provide an overview of the subject matter of the present disclosure. It is not intended to provide an exclusive or exhaustive description of the present invention. The detailed description is included to provide additional information about the present disclosure.

[0014] abbreviation

[0015] EB Estradiol Benzoate

[0016] E2 estradiol

[0017] Anteroventral Periventricular nucleus

[0018] ARC Arcuate nucleus

[0019] POA Preoptic area

[0020] KISS1 Kisspeptin protein

[0021] Kiss1 kisspeptin gene or mRNA

[0022] LHβ Luteinizing hormone subunit β

[0023] GH growth hormone

[0024] CASA Computer Assisted Semen Analysis BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings generally illustrate the multiple embodiments discussed in this document by way of example and not by way of limitation. In the accompanying drawings that are not necessarily drawn to scale, the same reference numerals may describe similar components in different views. The same reference numerals with different letter suffixes may represent different instances of similar components.

[0026] Figure 1A to B Depict serum and hypothalamic E2 levels after implantation of EB300 on the day of birth in rat pups (EB300 is a silicone rubber capsule containing 300 micrograms of EB; implantation is defined herein as a method of placing capsules subcutaneously in animals). EB300 was implanted on the day of birth, and E2 concentrations were measured in serum from postnatal day 0 (PND 0) to PND 30 (A) and measured in the hypothalamus from PND 0 to PND 10. Serum E2 levels were maintained at high levels until PND 10, and significantly decreased by PND 21, while hypothalamic E2 levels were reduced to basal levels by PND 10. It should also be noted that the E2 measurement units between serum (pg / ml) and hypothalamus (mg / ml) are different. The number of animals used for serum and hypothalamic E2 measurements at each time point was 3 to 8 and 3 to 7 for each assay point. Data are from mixed numbers of males and females. Fig. 2 shows data from males and females, respectively.

[0027] Figure 2A to BDepicted are serum and hypothalamic E2 levels after EB300 implantation. EB300 was implanted on the day of birth, and E2 concentrations were measured in serum from PND 0 to PND 30 (A) and in the hypothalamus from PND 0 to PND 10. The number of animals used for serum and hypothalamic E2 measurements at each time point was 3 to 8 and 3 to 7 for each assay point, respectively.

[0028] Figure 3A to B KISS expression in the hypothalamic ARC is shown. (A) Hypothalamus of 2.5 month old rats stained with anti-KISS1 antibody. Low and high magnification images obtained from the ARC of the hypothalamus are shown. (B) Hypothalamus of 6 to 7 month old rats stained with anti-KISS1 antibody. High magnification images obtained from the ARC are shown. Note the lower KISS immunoreactivity in the EB300 hypothalamus.

[0029] Figure 4A , Figure 4B , Figure 4C , Figure 4D , Figure 4E , Figure 4F , Figure 4G , Figure 4H , Fig. 4I , Figure 4J and Figure 4K Depicted are the KISS1 expression patterns of control (untreated) and EB300-implanted rats and the identification of cells and genes expressed in the hypothalamus at PND 29. KISS1 is expressed in the AVPV and ARC regions ( Figure 4A KISS1 expression was examined in the ARC region by anti-KISS1 antibody, KISS1 protein (white dots in the image) Figure 4A The plane of tissue section is marked by red dashed line). Compared with the control group, KISS1 expression in the hypothalamus of EB300 was significantly reduced ( Figure 4B ; Less white signal in EB300). To quantitatively measure KISS1 expression between the control and EB300 groups, hypothalamic regions (including the AVPV and ARC regions) were dissected and examined by single-cell RNA sequencing (scRNAseq) ( Figure 4C In females, from a total of 4,049 and 4,223 cells from the control and EB300 groups, respectively ( Figure 4E ) identified 10 different cell types (cell clusters) ( Figure 4D From the hypothalamic cell clusters, a Kiss1 expression cluster (cluster-9) was identified ( Figure 4F), and the number of Kiss1 expressing cells and the expression level of Kiss1 were compared between the control group and the EB300 group. In cluster-9, 307 and 194 cells were found in the control group and the EB300 group, respectively. 68 and 30 Kiss1 positive cells were identified from each group, respectively. This result indicates that the total number of cluster-9 and Kiss1 expressing cells in the EB300 hypothalamus were both decreased compared with the control group ( Figure 4G In males, ten distinct clusters were identified ( Figure 4H ), where a total of 4,674 and 4,229 cells were examined in the control and EB300 groups, respectively ( Fig. 4I From the hypothalamic cell clusters, a Kiss1 expression cluster (cluster-8) was identified ( Figure 4J ). The number of Kiss1 expressing cells and the expression level of Kiss1 were compared between the untreated group and the EB300 group. In cluster-8, 178 and 83 cells were found in the control group and the EB300 group, respectively. 65 and 37 Kiss1 positive cells were identified from each group, respectively. This finding suggests that the total number of cluster-8 and Kiss1 expressing cells was decreased in the EB300 hypothalamus compared with the control group ( Figure 4K ).

[0030] Figure 5A to B Describe the histology of pituitary and the expression level of LH and GH. (A) Luteinizing hormone β subunit (LH β) and growth hormone (GH) of male pituitary in 5 to 6 months of age are examined by histology (H&E staining) and immunohistochemistry. Representative images of tissues from control group and EB300 group are shown. (B) LH β and GH of male pituitary in 5 to 6 months of age are examined by histology and immunohistochemistry. Representative images of tissues from control group and EB300 group are shown.

[0031] Figure 6A to B Serum LH concentrations before and after KISS-10 challenge are shown. Serum levels of LH were measured 30 minutes after intraperitoneal injection of 50 nmole / animal of KISS-1 at 2 to 2.5 months of age. (A) Measurements in males. Number of animals used: control group (n=6) and EB300 group (n=3). (B) Measurements in females. Number of animals used: control group (n=4) and EB300 group (n=2). Asterisks indicate significant (p<.05) differences from KISS-1 injection. "a" and "b" indicate significant (p<.05) differences between all groups before KISS-1 injection.

[0032] Figure 7A, Figure 7B and Figure 7CNeonatal gonadal development in males is depicted. Testes at PND 1, 3, and 10 were stained with H&E (A) or TUNEL (B) and DDX4 (Dead-box helicase-4) (C). Representative images of testes from four animals per group are shown.

[0033] Figures 8A, 8B, 8C and 8D depict neonatal gonadal development in females. Ovaries at PND 1, 3 and 10 were stained with H&E (A) or TUNEL (B), DDX4 (C) and FOXL2 (D). Representative images of ovaries from four animals per group are shown.

[0034] Fig. 9 Ovaries, uterus, and pituitary gland at 5.5 months are depicted. Images were acquired immediately after tissue collection or after H&E staining.

[0035] Fig.10 Ovaries, uteri, and pituitary glands at 2.5 and 7 months are depicted. Images were obtained immediately after tissue collection or after H&E staining. Images are from seven-month-old rats.

[0036] Fig.11 Sperm count and motility (five to six months) are shown. Sperm concentration and motility were measured by CASA at 5 to 6 months of age. Four animals were used for the control group and three for the EB300 group. Data are mean ± SEM, * represents p≤0.05 when compared with the control group.

[0037] Fig. 12A and Fig. 12B Male gonads, epididymis and accessory glands are depicted. Gonads, epididymis and accessory glands were collected at 2.5 months of age, and their morphology and histology (A) were examined, and then immunohistochemistry (B) was performed using antibodies and DDX4 for StAR (Steroidogenic AcuteRegulatory Protein, steroidogenic acute regulatory protein). Arrows represent interstitial cells. White arrows represent germ cells. Black arrows represent apoptotic germ cells by TUNEL staining (which allows identification of dying cells). Representative images of tissues from four animals in each group are shown.

[0038] Fig.13 Depicted are the gonads, epididymis, and accessory glands of males collected at 5 to 6 months of age.

[0039] Fig.14 Depicted are the gonads, epididymis, and accessory glands of a male collected at 6 to 7 months of age.

[0040] Fig.15Shown are bar graphs depicting epididymal length, testicular weight, and accessory sexual organs / body weight ratio (6 to 7 months; n=8, 3).

[0041] Fig.16 Bar graphs depicting mating behavior exhibited by males are shown. Four-month-old male rats were mated with ovariectomized female rats (mount) and sexual behavior was measured during a 30-minute period. (A) Latency to first mating during the experimental time (seconds). (B) Total number of male matings during the experimental time. Number of rats, control group = 5, EB300 group = 3. The figure shows mean ± SEM, * indicates p≤0.05 when compared to the control group.

[0042] Fig.17 Bar graphs depicting serum T and E2 levels in young adult and mature animals are shown. Serum testosterone concentrations were measured in males at 2 to 3 months and 6 to 7 months of age. Serum estradiol concentrations were measured in females at 2 and 6 months of age. Note the difference in the Y-axis scale of serum estradiol levels in females. Mean ± SEM is shown; * indicates p≤0.05 when compared to the control group. Serum estradiol concentrations were measured at 2 and 6 months of age.

[0043] Fig.18 Bar graphs depicting serum T and E2 levels in young adult animals are shown. Serum testosterone concentrations were measured at 2 to 3 months and 6 to 7 months in females. Serum estradiol concentrations were measured at 2 months and 6 to 7 months in males. Mean ± SEM is shown; * indicates p≤0.05 when compared to the control group. Serum estradiol concentrations were measured at 2 and 6 months of age.

[0044] Figure 19A to B Changes in body weight over time are depicted. Body weights of males (A) and females (B) were measured at PND 0, 6, 14, 21, 28, 42, 60, 90, 120, 150 and 180. Data are mean ± SEM. (n = 3 to 7 for each time point).

[0045] Fig. 20 Organ weights of males are shown. Femur (weight / length), brain (weight), pituitary (weight), liver (weight), spleen (weight), kidney (weight) and heart (weight) were each divided by body weight to investigate the impaired growth of basic organs. Error bars represent SEM; * represents a significant difference from the control (Student t test, p<0.05). (6 to 7 months; n=7, 8).

[0046] Fig.21Organ weights of females are shown. Femur (weight / length), brain (weight), pituitary (weight), liver (weight), spleen (weight), kidney (weight) and heart (weight) were each divided by body weight to investigate the impaired growth of basic organs. Error bars represent SEM. * indicates significant difference from control (Student t test, p<0.05). (6 to 7 months; n=7, 8).

[0047] Fig. 22 Representative kidney and liver histology of males (5-6 months) are shown.

[0048] Fig.23 A representative mammary gland of a female is shown. The fourth mammary gland was dissected from a female rat and subjected to whole-mount histological analysis. The tissue was fixed in Carnoy's solution and stained with Carmine alum solution and then washed with acid ethanol. Arrows indicate mammary gland branch points. Arrows indicate mammary gland buds.

[0049] Fig.24 The effective time window for males (testes) is depicted. Male rat pups were implanted with EB300 at the indicated ages and their gonads were dissected and examined at 3 months of age.

[0050] Fig.25 The effective time window for males (reproductive tract) is depicted. Male rat pups were implanted with EB300 at the indicated ages and their gonads were dissected and examined at 3 months of age.

[0051] Fig.26 The effective time window for females is depicted. Female rat pups were implanted with EB300 at the indicated ages and their gonads were dissected and examined at 3 months of age.

[0052] Fig. 27 is a cross-sectional side view of an exemplary injector device for use in a chemosterilant delivery system (eg, subcutaneous delivery to puppies / neonatal dogs).

[0053] Fig.28 The effect of EB125 on gonadal development in hamsters is shown. Empty capsules (control) or 125 micrograms of EB contained in silicone rubber capsules (EB125) were implanted into 21-day-old hamsters. Reproductive tissues were collected from hamsters at PND 30. DETAILED DESCRIPTION

[0054] The following detailed description includes an introduction to the accompanying drawings, which form a part of the detailed description. The accompanying drawings show, by way of example, some specific embodiments in which the present invention may be implemented. These embodiments, also referred to herein as "embodiments", are described in sufficient detail to enable those skilled in the art to implement the present invention. Without departing from the scope of the present invention, the exemplary embodiments may be combined, additional embodiments may be used, or structural and logical changes may be made. Although the disclosed subject matter will be described in conjunction with the enumerated claims, it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter. Therefore, the following detailed description should not be regarded as limiting, and the scope of the present invention is defined by the appended claims and their equivalents.

[0055] In this document, unless otherwise stated, nouns without quantifiers refer to one or more; the term "or / or" is used to refer to a non-exclusive "or / or". In addition, the words or terms used herein and not otherwise defined are for descriptive purposes only and not for limiting purposes. In addition, all publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. If there is an inconsistency between the usage of this document and those documents incorporated by reference, the usage in the incorporated references should be considered to supplement the usage of this document; for irreconcilable inconsistencies, the usage in this document shall prevail.

[0056] References in the specification to "one embodiment," "an embodiment," "an exemplary embodiment," etc. indicate that the described embodiment may include a particular feature, structure, or characteristic, but not every embodiment may necessarily include the particular feature, structure, or characteristic. Furthermore, such phrases do not necessarily refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in conjunction with an embodiment, it is considered to be within the knowledge of those skilled in the art to affect such feature, structure, or characteristic in conjunction with another embodiment, whether or not explicitly described.

[0057] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as limits of the range, but also all individual values ​​or sub-ranges encompassed within the range, as if each value and sub-range were explicitly listed. For example, a concentration range of "about 0.1% to about 5%" should be interpreted as including not only the explicitly listed concentrations of about 0.1 wt.% to about 5 wt.%, but also individual concentrations (e.g., 1%, 2%, 3% and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, and 3.3% to 4.4%) within the specified range.

[0058] As used herein, the term "about" allows for a certain degree of variability in values ​​or ranges - for example, within 10%, within 5%, within 1%, within 0.5%, within 0.1%, within 0.05%, within 0.01%, within 0.005%, or within 0.001% of a specified value or a specified range limit - and includes the exact specified value or range.

[0059] As used herein, a "subject" is an animal. Animals include mammals, including companion animals, such as dogs or cats; farm animals, such as horses, cows, or pigs; laboratory animals, such as mice or rats; and non-human primates and humans. The subject also includes birds.

[0060] The term "infertility" or "sterility" refers to the state of being unable to reproduce or to produce offspring. Infertility can occur in males or females or both.

[0061] As used herein, "effective amount" means an amount sufficient to induce infertility (i.e., to render an animal infertile). An effective amount can be administered in one or more administrations. In some embodiments, an effective amount of EB can be achieved in combination with another drug, compound, or pharmaceutical composition. In other embodiments, an effective amount of EB can be achieved independently of the use of another drug, compound, or pharmaceutical composition.

[0062] As used herein, the term "eSpay" refers to any embodiment of the compositions and / or methods using EB as described herein.

[0063] As used herein, the term "carrier," "pharmaceutically acceptable carrier," or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of EB as a composition (ie, a pharmaceutical composition).

[0064] Compositions and methods

[0065] Compositions comprising estradiol benzoate (EB) and methods of using estradiol benzoate as an infertility agent / inducer for animal infertility are described herein. Other compounds that can be used in the method include, but are not limited to, estradiol, estradiol dipropionate, estradiol valerate, estradiol cypionate (e.g., chemical compounds that release estradiol or function similarly to estradiol in vivo in any form can be used). EB has the following structure:

[0066]

[0067] EB concentration / amount

[0068] The infertility agent (inducing infertility) composition comprises an amount of EB that induces infertility. The effective amount of EB that induces infertility may depend on, for example, the route of administration, the age of the animal and its size (weight). Therefore, the technician can titrate the dose and modify the route of administration of EB to obtain the best effect for a particular animal. A typical dose of EB may be from about 0.01 mg / kg to about 100 mg / kg or more. In other embodiments, the dose of EB may be from 0.1 mg / kg to about 100 mg / kg; or from 1 mg / kg to about 100 mg / kg; or from 5 mg / kg to about 100 mg / kg. For example, in rats and hamsters, 300, 100 or 30 μg may be administered to induce infertility. In larger animals, similar doses will be effective.

[0069] Application time

[0070] The composition comprising EB is administered prior to puberty (before reaching sexual maturity / being able to reproduce) to induce infertility. The composition may be administered days, weeks, months, or even years after birth, as long as the composition is administered before the animal reaches puberty. Administration of EB effectively inhibits / blocks the maturation of sex organs / gonads.

[0071] Route of administration

[0072] The administration routes of the compositions provided herein are according to known methods such as injection (intraperitoneal, intramuscular, subcutaneous) and nasal (inhalation). In one embodiment, EB is administered in a one-time single dose to sterilize the animal. In other embodiments, multiple EB administrations may be performed to produce sterilization.

[0073] Composition

[0074] In one embodiment, the EB composition for injectable administration can be in the form of an oily suspension, including oil, such as vegetable oil (e.g., corn oil), cottonseed oil, peanut oil and / or sesame oil. As an alternative to oil or in addition to oil, other carriers or fillers can be used. Carriers / fillers can include lactose, sucrose, starch powder, cellulose alkanoate, cellulose alkyl esters, talcum, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, gum arabic, sodium alginate, polyvinyl pyrrolidone and / or polyvinyl alcohol. These suspensoids can be prepared according to the method for dispersing and suspending components available in the art.

[0075] In another embodiment, the above composition can be encapsulated for administration. In one embodiment, a capsule can be formed by a silicone tube with a plug at each end to contain a mixture of, for example, EB and oil. The capsule can be placed in the subject's body, for example, by injection (described further below). The EB composition described herein can be formulated for immediate release or in a delayed release formulation (e.g., sustained release). For example, EB can be prepared together with a carrier that protects EB from rapid release, such as a controlled release formulation.

[0076] Many methods for preparing controlled release / slow release formulations are known to those skilled in the art. For example, the technology for preparing a variety of sustained or controlled delivery modes is also known to those skilled in the art, such as liposome carriers, polymers (e.g., ethylene vinyl acetate, polyanhydrides, silicones, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic acid polyglycolic acid copolymers (PLG)), microparticles, nanoparticles or porous beads, and depot injections. For example, referring to PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556, 1983), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15; 167-277, 1981; Langer et al., Chem. Tech. 12: 98-105, 1982), ethylene vinyl acetate (Langer et al., supra) or poly-D (-) -3-hydroxybutyric acid (EP 133,988). Sustained release compositions also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692, 1985; EP 36,676; EP 88,046; EP 143,949.

[0077] In some embodiments, compositions have different release rates (for example, controlled release or immediate release). Immediate release refers to that EB releases immediately after administration. In another embodiment, immediate release occurs when there is EB dissolution within 1 to 20 minutes after administration. Dissolution can be all EB or less than all EB (for example, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, 99.9% or 99.99%). In another embodiment, immediate release causes complete dissolution or less than complete dissolution in about one hour after administration.

[0078] Controlled release, slow release or sustained release refers to the release of an active ingredient (e.g., EB) from a composition or dosage form, wherein the active ingredient is released over an extended period of time. In one embodiment, controlled release causes EB to dissolve within 20 to 180 minutes after administration. In another embodiment, controlled release causes dissolution within hours to days or weeks. In another embodiment, EB is released within a period of several weeks (including about 15 to 20 days).

[0079] In another embodiment, the composition is formulated for inhalation; for example, EB can be formulated as a dry powder. The inhalation solution can also be formulated with a propellant for aerosol delivery. In another example, the inhalation solution can be atomized.

[0080] One embodiment provides a kit for producing a single-dose administration unit. The kit may include single-chamber and multi-chamber pre-filled syringes containing EB and instructions for use (sterilizing the animal).

[0081] Delivery System Implementation

[0082] The present disclosure also describes systems and methods for delivering a chemosterilant composition (also referred to as a "chemosterilant delivery system") to an animal to initiate chemical sterilization of the animal. In one embodiment, the sterilant is EB.

[0083] Fig. 27 A conceptual view of an exemplary chemical sterilant delivery system 10 (hereinafter referred to as "sterility system 10" or simply "system 10") is shown. The system 10 example includes an injector device 12 (hereinafter also referred to as "injector 12"), which is configured to deliver a sterilant container body 14 (e.g., a capsule) to an animal 2 (in the form of a sterilant container body 14). Fig. 271, which may be a male or female puppy / newborn dog) on ​​or in. Those skilled in the art will appreciate that the animal 2 may be male or female and may be another type of livestock, such as a piglet or lamb, or may be a companion animal, such as a dog or cat. The sterilant container body 14 contains a specific dose of a chemosterilant (e.g., EB) and is configured to release the chemosterilant into or on the animal 2 after the sterilant container body 14 has been delivered to the animal 2 by the injector 12. In one example, the sterilant container body 14 is generally in the shape of a capsule, such as a cylindrical or substantially cylindrical body, so that it can be incorporated into or on the animal 2 by the injector 12, as described in more detail below. For this reason, the sterilant container body 14 will also be referred to as a "sterile delivery capsule 14," "delivery capsule 14," or simply "capsule 14." Those skilled in the art will appreciate that the sterilant delivery body 14 does not need to have a cylindrical or substantially cylindrical shape that is generally considered a "capsule."

[0084] In one example, the injector 12 includes a body 16, a force member 20 (eg, a plunger), and in some examples, an insertion structure 18 and / or one or more gripping members 22. Fig. 27 In the example shown, the injector 12 is constructed similar to a medical syringe, so that in some examples the injector 12 will be referred to as a "syringe 12", the body 16 will be referred to as a "barrel 16", and the force member 20 will be referred to as a "piston 20". In one example, the body 16 at least partially surrounds a chamber 24, and a portion of the force member 20 is located in the chamber 24. In one example, the force member 20 can slide along the chamber 24 to change the internal volume experienced by the gas or fluid in the chamber 24, thereby changing the pressure of the gas or fluid in the chamber 24, and thus changing the driving force applied by the gas or fluid in the chamber 24.

[0085] In one example, the insertion structure 18 (optionally) includes a structure through which the delivery capsule 14 will pass to provide placement of the delivery capsule 14 at a specific location in or on the animal 2. In one example, the insertion structure 18 includes a narrow structure with a small cross-sectional area, which allows the insertion structure 18 to be used to pierce one or more epidermal layers of the animal 2 so that the delivery capsule 14 can be injected therein. For this reason, the insertion structure 18 may also be referred to as a "hypodermic injection needle 18" or simply "needle 18" hereinafter. For example, the needle 18 may include a cannula 26, which extends from a proximal end connected to the barrel 16 and a distal end configured for insertion into the animal 2. The distal end of the cannula 26 may include a relatively sharp tip that can pierce one or more epidermal layers of the target animal 2. The cavity 30 passes through the cannula 26 from the proximal end to the distal end. In one example, the delivery capsule 14 can be positioned in the cavity 30, where the delivery capsule 14 can be driven out of the cannula 26. In one example, the lumen 30 is in fluid communication with the syringe chamber 24 such that the force member 20 , when moved forward, can drive the delivery capsule 14 through the lumen 30 and out the distal tip with sufficient force for injection into the target animal 2 .

[0086] The force member 20 includes a movable structure that, when moved from a first position to a second position, can cause the delivery capsule 14 to move out of the injector 12 so that the delivery capsule 14 can be delivered into or onto the animal 2. In an example where the injector 12 is a syringe-like device, the body 16 can be formed into a cylindrical tubular configuration, so that the body 16 can also be referred to herein as a "barrel 16." The force member 20 can include a piston (e.g., a structure having a cross-sectional shape and size substantially the same as the cross-sectional shape of the barrel 16) that fits tightly within the cylindrical barrel 16, so that when the force member 20 moves axially along the barrel 16, the gas (e.g., air) located within the chamber 24 is compressed, and the increased pressure acts as a driving force to drive the delivery capsule 14 through the insertion structure 18. In another example, the force member 20 includes a push structure 28 connected to or as part of the piston 20, wherein the push structure 28 engages the delivery capsule 14 and pushes the delivery capsule 14 out of the injector 12. For example, if the injector 12 includes an insertion structure 18, the push structure 28 pushes the delivery capsule 14 through the lumen 30 of the insertion structure and out of the distal tip of the cannula 26 for insertion into the target animal. In one example, one or more gripping members 22 are disposed on the body 16 of the injector 12 and allow the user to hold and stabilize the injector 12 while actuating the force member 20 to drive the delivery capsule 14 into the target animal 2.

[0087] In one example, the delivery capsule 14 is sized and shaped to fit within the lumen 30 of the insertion structure 18. In one example, the delivery capsule 14 is sized and shaped for intramuscular, intraperitoneal, or subcutaneous injection into the animal 2.

[0088] Found Fig. 27 The infertility system 10 shown in the invention effectively provides chemical-based sterilization for mammals. For example, in an experiment in which an infertility delivery capsule 14 containing an infertility composition 34 containing estradiol benzoate was subcutaneously injected into immature male rats (described in more detail in the following examples and as described above), it was found that the infertility composition 34 effectively inhibited the production of neuronal kisspeptin in the hypothalamus, testicles (e.g., the site of testosterone and sperm production) and seminal vesicles and prostate (e.g., the auxiliary male gonads that produce semen components) of the brain. Male rats injected with the delivery capsule 14 containing EB were prevented from experiencing puberty and became permanently infertile. Specifically, the testicles of male rats did not produce testosterone or sperm. When the infertility delivery capsule 14 with the infertility composition 34 containing estradiol benzoate was injected into immature female rats, it was found that estradiol benzoate inhibited the female reproductive system, preventing the female from experiencing the menstrual cycle and preventing the production of eggs.

[0089] The subject rats also did not have any apparent negative health effects as a result of injection of estradiol benzoate via the sterilant delivery capsule 14. Examination of the animals' immune system, liver, and kidney function revealed no clinical pathology in either male or female subject rats. Furthermore, after testing more than 200 animals with the delivery capsule 14 containing estradiol benzoate, no subject animals died or exhibited disease during the six-month testing period.

[0090] The development and function of the reproductive system are consistent in all mammalian species. All mammals use the same reproductive hormones, which vary little between species. For example, reproductive hormones extracted from one species, such as estrogen, progesterone, kisspeptin, luteinizing hormone (LH) and follicle stimulating hormone (FSH), have the same or substantially similar biological effects in all or nearly all other mammalian species. Due to the high level of conservation, estradiol benzoate infertility composition 34 (and other estradiol-type compositions) will effectively make all mammals and birds infertile.

[0091] Example

[0092] Various embodiments may be better understood by reference to the following examples which are provided by way of illustration.The present disclosure is not limited to the examples given herein.

[0093] Example 1

[0094] The eSpay implant induces permanent sterility in males and females.

[0095] introduction

[0096] In mammals, kisspeptin initiates puberty by directly stimulating the release of gonadotropin-releasing hormone (GnRH), also known as gonadoliberin. Thus, kisspeptin promotes the correct timing of puberty and normal gonadal development. It has been found that knocking out the Kiss1 gene responsible for encoding kisspeptin or its receptor GPR54 leads to infertility in both male and female mice (wherein mice are used as model organisms). Kispeptin is primarily expressed in two regions of the hypothalamus, the preoptic area (POA) and the arcuate nucleus (ARC). Although kisspeptin neurons in the POA are thought to play a key role in the luteinizing hormone surge in females, arcuate nucleus kisspeptin neurons mediate the negative feedback effect of steroids on GnRH release in both male and female adults. Although kisspeptin in pre-pubertal mammals is necessary for the onset of puberty, the contribution of a specific group, whether derived from the arcuate nucleus or the preoptic area, remains unclear.

[0097] The development of kisspeptin neurons and the expression of kisspeptin may be different between POA and ARC. In the preoptic area, Kiss1-expressing cells appear between 8 and 10 days postnatal day (PND) in laboratory animals (mice and rats) of both sexes. Sexual dimorphism is manifested on the 10th to 12th day after birth, with females showing more kisspeptin cells than males in the preoptic area. In contrast, kisspeptin in the arcuate nucleus is expressed during embryonic development. In both sexes, messenger RNA and protein expression appear at about 14.5 days of gestational age, increase at 18.5 days of gestational age, and decrease just before birth. Arcuate Kiss1 messenger RNA is detected on day 0 after birth (as fast as 0 to 4 hours). Unlike the preoptic area, arcuate Kiss1 is usually expressed in the first ten days of rodent life. Then expression decreases during the third week after birth, but begins to show improvement during puberty.

[0098] Compared further to the expression in the preoptic area, arcuate kisspeptin expression is not highly sexually dimorphic in adults, compatible with the function of mediating negative feedback that occurs in both sexes. It has been found that arcuate kisspeptin expression appears to increase in both sexes at about the onset of early puberty. For example, in female rats, both Kiss1 messenger RNA and neuronal fiber density increase before the onset of puberty, with increases first occurring in the arcuate nucleus and later in the preoptic area. This suggests that kisspeptin in the arcuate nucleus may be the cause of the onset of puberty.

[0099] Sex steroids appear to be required for the proper development and organization of kisspeptin neurons and for Kiss1 expression. In kisspeptin cell-specific estrogen receptor α knockout mice, the number of kisspeptin fibers in the hypothalamus is reduced. This is also true when the hormone is depleted by gonadectomy in developing rodents. Estrogen and testosterone inhibit Kiss1 expression in the arcuate nucleus but increase Kiss1 expression in the preoptic area. Kisspeptin neurons have been found to colocalize with estrogen receptors; moreover, prior to puberty, estrogen signaling inhibits kisspeptin in the arcuate nucleus, which interrupts activation of the hypothalamic-pituitary-gonadal (HPG) axis, thereby preventing the early onset of puberty.

[0100] Furthermore, exposure of females to estrogens during postnatal days 0 to 10 resulted in permanent suppression of kisspeptin expression in the arcuate nucleus. These rats reportedly did not exhibit luteinizing hormone secretion or estrous cycles. Although GnRH was produced, its release appeared to be disturbed. The number of Kiss1-expressing cells and kisspeptin-immunoreactive cells observed was significantly reduced. However, it is unclear whether estrogen exposure affects kisspeptin neuron proliferation and differentiation or the transcription and expression of the Kiss1 gene.

[0101] Materials and methods

[0102] Animals. Male and female Sprague-Dawley rats of 3 months old were purchased from Charles River Laboratory and used as breeders. If not used for experimental purposes, newborns were designated as continuous breeders. When breeders reached breeding age (6 weeks of age for females and 10 weeks of age for males), breeding pairs were set in separate cages for continuous breeding until the pups of the desired number were produced or the breeders reached one year old. Pregnancy was represented by the changes in the weight and abdominal protuberance of female breeders. At birth (postnatal day or PND 0), the pups were distinguished in sex and assigned to specific experiments. When the pups reached 21 days of age, they were weaned.

[0103] Implantation of estradiol benzoate capsules. EB powder (17β-estradiol 3-benzoate, Sigma E8515) was dissolved in sesame oil (Sigma S3547) to make a solution with a concentration of 300 μg / 5 μl. EB-300 μg capsules (EB300) were made by using a silicone tube (Dow Corning 508-005) containing 5 μl of EB solution and then sealing it with a silicone sealant (Factor II, Inc. medical adhesive A-100). The capsules (referred to herein as EB300 or eSpay) were placed at room temperature for one hour to dry the sealant and then immersed in sesame oil. The size of the capsules was 8 mm long and 2.16 mm in diameter. The skin of the implantation site (on the back between the shoulders) was disinfected by applying a povidone-iodine solution. A 2 mm long transverse incision was cut on the skin with sterile surgical scissors on the back near the shoulders. A packet is formed between the skin and muscle layers by inserting the tips of blunt-tipped forceps. The capsule is inserted into the pocket and pushed sideways so that the capsule is facing away from the incision. The incision is sealed by applying surgical adhesive.

[0104] Hormone measurements. Blood was collected from the tail vein or by cardiac puncture. The blood was centrifuged at 2000×g for 10 minutes; serum was then collected and stored at −20°C until further analysis. Serum estradiol concentrations were measured by using an ELISA kit (DRG EIA4399), with a reportable range of 1.4 to 200 pg / ml. The concentration of circulating testosterone was measured using an ELISA kit (DRG EIA1559), with a reportable range of 0.083 to 16 ng / ml. Serum luteinizing hormone (LH) concentrations were measured by sandwich immunoassay at the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core using monoclonal antibodies against both bovine LH (no. 581B7) and human LH-β subunit (no. 5303: Medix Biochemica, Kauniainen, Finland) as described previously (PMID: 8462469), with a reportable range of 0.04 to 37.4 ng / mL.

[0105] Test male mating behavior. In order to study sexual behavior, male rats of 4 months old were paired with ovariectomized females induced by estrus. Estrus was induced by treatment with exogenous estradiol (50 μg / kg sc, 54 hours before the test) and progesterone (2.0 mg / kg sc, 6 hours before the test). Male sexual behavior was observed in a fresh transparent cage (56 × 35 × 31 cm) illuminated by a 40W lamp with a red filter. Each untreated male rat was placed in a test cage for five minutes. Then a receptive female was introduced and sexual behavior was observed for 30 minutes. As previously described (PMID: 17016704), the time (latency) and the number of matings to the first mating of each animal were recorded.

[0106] Sperm analysis. The cauda epididymis was removed and minced in warm (37°C) phosphate-buffered saline with fine scissors. The sperm suspension was incubated at 37°C for 10 minutes to allow the sperm to swim out of the minced epididymis. Sperm motility was then analyzed by a computer-assisted sperm analyzer (CASA; Sperm Vision II, Minitube of America, Vernon, WI, USA). At least ten microscopic fields of view covering the entire visible area of ​​the semen analysis chamber and without overlapping continuous fields of view were examined. Sperm motility was measured by the percentage of active sperm, progressively active sperm, and immotile sperm. For total sperm counts, two aliquots of semen samples were collected from each mouse and diluted in formalin at 1:200 for fixation. The number of sperm was counted using a hemocytometer, and the average sperm concentration per milliliter was calculated and reported as million sperm / ml.

[0107] Hypothalamic-pituitary responsiveness after KISS-10 injection. Peripheral blood (50 μL) was collected from the ventral tail vein of adult male and female rats aged 60 to 70 days. Immediately after blood collection, 50 nanomoles / animal of kiss-10 (Metastin 45-54, Millipore-Sigma, San Diego, CA) was injected intraperitoneally into the rats, and additional blood samples were collected 30 minutes after injection. Samples were centrifuged at 2000 × g for 10 minutes, and serum was collected and stored at -20 ° C until further analysis. Serum luteinizing hormone (LH) concentrations were measured by sandwich immunoassay at the University of Virginia Reproductive Ligand Assay and Analysis Core Research Center using monoclonal antibodies against both bovine LH (no. 581B7) and human LH-β subunit (no. 5303: Medix Biochemica, Kauniainen, Finland) as previously described (PMID: 8462469), with a reportable range of 0.04 to 37.4 ng / mL.

[0108] Fertility test. To evaluate the fertility of male rats, each male rat was housed with a breeder female for two weeks at 4 months of age. The male fertility percentage (number of males producing litters / total number of males × 100) and litter size (number of pups per litter) were recorded. To evaluate the fertility of female rats, each female rat was housed with a breeder male for two weeks at 3 months of age. The female fertility percentage (number of females producing litters / total number of females × 100) and litter size (number of pups per litter) were recorded.

[0109] Histopathology.At the respective sampling age of rats, rats were euthanized by CO2 asphyxiation followed by bilateral thoracotomy. All or part of the organs (brain, pituitary, gonads, reproductive tract, heart, liver, kidney, spleen, mammary gland and femur) were dissected out and immediately fixed in freshly prepared 4% paraformaldehyde for 24 hours, then cleaned in PBS (phosphate buffered saline pH 7.4), and then stored in 70% ethanol at 4 ° C until use. The fixed tissue was processed by an automatic tissue processor (Miles Scientific Tissue Tek VIP 2000) and embedded in paraffin blocks. The embedded tissue was cut into 7 μm thick slices using a microtome (Leica Biocut Model 3011) and mounted on a positively charged glass slide (type). After dewaxing, the tissue was subjected to hematoxylin and eosin staining or immunohistochemistry (IHC) for a variety of biomarkers. Antigen retrieval for IHC was done using citrate buffer (pH 6.0) and microwaved at 10% power for 15 min. Endogenous peroxidase activity was blocked using 3% H2O2 for 20 min, and slides were blocked with 5% host serum for 1 h before incubation with primary antibodies overnight at 4°C. The following primary antibodies were used: kisspeptin (rabbit-anti-kisspeptin 1:250, AB9754-Millipore-Sigma), DDX4 / MVH (rabbit-anti-DDX4 / MVH 1:2,000, AB13840 Abcam), LHβ (rabbit-anti-LHβ 1:100, National Hormone & Peptide Program), StAR (rabbit-anti-StAR 1:2,000, SC-25806 Santa Cruz), GH (rabbit-anti-GH 1:100, National Hormone & Peptide Program), Cyp17A1 (rabbit-anti-bovine Cyp17A1 1:5,000, kindly provided by Dr. Alan Conley, UC Davis). Peroxidase-conjugated donkey or goat anti-rabbit secondary antibodies were used at a concentration of 1:200 and detected using a DAB kit (VectorLabs). Whole-mount mammary gland preparation and analysis were done as described previously (10.21769 / BioProtoc.2915).Apoptosis assays were performed using the In Situ Apoptosis Detection Kit S7100 (Millipore) stained with DAB chromogen (diaminobenzidine) and counterstained with hematoxylin according to the manufacturer's instructions.

[0110] Blood biochemistry. Blood was collected by cardiac puncture and immediately divided into two samples: (a) For complete blood count (CBC), blood was mixed with EDTA in a hematological tube (BD Microtainer 365967) and stored at 4°C before analysis within 24 hours. (b) For serum biochemistry profile, blood was centrifuged at 2000×g for 10 minutes; the separated serum was then collected and stored at −20°C until analysis. Sample analysis was performed by the Veterinary Diagnostic Laboratory at the University of Illinois at Urbana-Champaign.

[0111] Single cell RNA sequencing. In order to study the differential gene expression in the hypothalamic region between vehicle and EB300, the hypothalamus was obtained from two rats in each group, and the single cell suspensions from the same group were combined for single cell RNA sequencing (scRNAseq). M199 culture medium was used to remove blood by perfusion after euthanasia. The brain was quickly dissected and transferred to ice-cold M199 culture medium containing 10% FBS. The hypothalamic region was dissected using curved fine forceps. Hypothalamic tissue was separated into single cells using a neural tissue separation kit-postnatal neurons (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer's instructions. The survival rate in all samples was determined by trypan blue exclusion>85%. The cell suspension was filtered using a 40 μm filter, and the separated cells were precipitated and resuspended in PBS containing 1% bovine serum albumin (bovine serum albumin) (BSA; Sigma-Aldrich, St.Louis, MO). Cell suspensions (approximately 3000 cells) of each sample were loaded onto a 10× Chromium instrument (10X Genomics, Pleasanton, CA) according to the manufacturer's protocol. Single-cell RNA-Seq libraries were prepared using the Chromium Single Cell 3′ v2 Kit (10X Genomics) according to the manufacturer's protocol. An initial step performed emulsification, in which individual cells were separated into droplets together with gel beads coated with unique primers, unique molecular identifiers (UMIs), and poly (dT) sequences with 10× cell barcodes. Reverse transcription reactions were performed to generate barcoded full-length cDNA, followed by breaking the emulsion using a restorer and cleaning the cDNA with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Large amounts of cDNA were amplified using a GeneAmp PCR System 9700 (Applied Biosystems) with a 96-well Gold SampleBlock Module. Amplified cDNA products were cleaned with the SPRI Selection Kit (Beckman Coulter). Indexed sequencing libraries were constructed using reagents from the Chromium Single Cell 3′ v2 kit following these steps: (1) fragmentation, end repair, and A-tailing; (2) size selection with SPRI selection; (3) adapter ligation; (4) post-ligation cleanup with SPRI selection; and (5) sample indexing PCR and cleanup with SPRI selection beads.Library quantification and quality assessment were performed using the Qubit fluorescence assay (Invitrogen) with the dsDNA HS (High Sensitivity) assay kit and the Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (Agilent Genomics). Index libraries were pooled equimolarly and sequenced on an Illumina HiSeq4000.

[0112] Analysis of scRNA-seq data. First, single-cell expression was analyzed using the Cell Ranger single-cell software suite (v2.1.1) for quality control, sample demultiplexing, barcoding, and single-cell 3' gene counting. Sequencing reads were aligned to the UCSC rn6 rat transcriptome using the Cell Ranger suite with default parameters. Samples were merged using the cell-ranger aggregation function with default parameters. A total of 3,634 and 3,339 single cells from the control and EB300 groups, respectively, were analyzed. The average raw reads / cell were 45,201 and 48,751, respectively. The median genes / cell were 1,496 and 1,429, respectively. Further analysis was performed in R (v3.5) using the cellrangerR kit (v2.2.0), Seurat (v2.0), and Monocle packages (v2.8.0). The gene-cell-barcode matrix of the sample was log-transformed and filtered based on the number of genes detected / cell (any cell with less than 300 genes or more than 5,000 genes / cell was filtered out), as were cells with more than 6% mitochondrial UMI counts and more than 50% ribosomal UMI counts. Gene expression was regressed based on the number of UMI counts and the percentage of mitochondrial genes. Only genes detected in at least three cells were included. The cells were then scaled to a total of 1e4 molecules. A total of 6,964 cells were retained for statistical analysis. To reduce the dimensionality of the data, 2,505 variable genes were selected based on gene expression and dispersion (expression cutoff = 0, and dispersion cutoff = 0.5). PCA was run on the normalized gene-barcode matrix. The Barnes-hut approximation to t-SNE was then performed on the first 12 principal components to visualize the cells in two-dimensional space. The first 11 principal components were used for t-SNE projection and cluster analysis using the Elbow Plot method. Clusters were identified using the "FindClusters" function in Seurat with a resolution parameter of 0.6. This graph-based clustering method relies on a clustering algorithm based on shared nearest neighbor (SNN) modular optimization. Unique cluster-specific genes were identified by running the Seurat "FindAllMarkers" function using the MAST framework. A total of 17 clusters were identified from tSNE, and five marker genes in each cluster were identified using the "FindMarkers" function. tSNE plots, histograms, scatter plots, and violin plots were drawn using Seurat.

[0113] statistics

[0114] Overall data analysis was performed using statistical software (SPSS22). The normal distribution of continuous data was tested by the Shapiro-Wilk test. After confirming the homogeneity of variance, all normally distributed continuous data were analyzed with parametric tests and Tukey's HSD post hoc tests. All non-normally distributed continuous data were analyzed by nonparametric tests (Kruskal Wallis ANOVA). Similar analyses were performed on ordered data. The data are graphically represented as the mean and standard error of the mean. For all analyses, the alpha value was set to 0.05. In order to draw conclusions from scRNAseq, unique cluster-specific genes were identified by running the Seurat "FindAllMarkers" function using the MAST framework. A total of 17 clusters were identified from tSNE; the top five marker genes in each cluster were identified using the "FindMarkers" function. Differential gene expression between control and EB300 was tested by the default "bimod" likelihood ratio test in Seurat (cutoff, p_val_adj<0.05). tSNE plots, histograms, scatter plots, and violin plots were drawn using Seurat.

[0115] result

[0116] Controlled delivery of 17β-estradiol 3-benzoate by eSpay to neonatal rats irreversibly inhibits hypothalamic kisspeptin expression and germ cell development in both males and females.

[0117] Manufacturing of the eSpay (or alternatively known as the EB300).

[0118] Silicone capsules comprising 17β-estradiol 3-benzoate (EB, Sigma E8515) are used as devices for increasing blood 17β-estradiol (E2) concentration to super physiological levels. Once implanted subcutaneously, silicone capsules (called eSpay) with EB release EB for a prolonged period of time. EB is selected instead of E2 because E2 is rapidly removed by liver metabolism, and EB is slowly hydrolyzed to E2, providing a continuous high blood E2 level, which lasts for about three weeks of extended time period. After a large number of studies, it was determined that 300 μg of EB implanted subcutaneously will effectively induce infertility. Therefore, EB (EB300 in this article) comprising 300 μg of EB is prepared by dissolving 300 μg of EB in 5 μl of sesame oil (Sigma S3547), and inserted into a silicone tube (Dow Corning 508-005), which is then sealed with a silicone sealant (Factor II, Inc. medical adhesive A-100). Capsules are kept at room temperature until use. EB300 capsule is about 8mm long and about 2.16mm in diameter. The skin of the implantation site (back between shoulders) is disinfected by applying povidone-iodine solution. A 2mm long transverse incision is cut on the skin with sterile surgical scissors. A bag is formed between the skin and the muscle layer by inserting the tip of blunt-headed forceps. The capsule is inserted into the bag and pushed sideways so that it is away from the incision and the incision is sealed by applying surgical adhesive.

[0119] The efficacy of EB300 capsules as a means of increasing E2 levels in their recipients was tested after implantation of EB300 capsules on the day of birth (postnatal day 0, PND 0). Serum and hypothalamus were collected at PND 1, 2, 3, 4, 7, 10, 21, and 30, and E2 concentrations were measured. Serum E2 concentrations increased and remained at a stable level for 10 days ( Figure 1A to B ) and gradually decreased over time, but in the EB-treated group it remained at a slightly higher level than the untreated control group until PND 30 (Figure 1A). E2 measured from the hypothalamus (calculated as 1 mg tissue volume equals 1 mL) showed a similar pattern of increase, but the concentration was 100 times higher than that in the serum, and then dropped sharply by PND 4 and returned to near basal levels by PND 7 (Figure 1B). The same hormone profile pattern was observed in males and females ( Figure 2A to B ). These results indicate that EB300 implants induce sustained high levels of E2 in the circulating blood for up to about 30 days. In addition, the selective increase in hypothalamic E2 levels relative to serum levels over the first 4 days and the subsequent sharp drop in hypothalamic E2 levels when serum E2 levels are still high suggest that the hypothalamus may have a unique regulatory system for the temporary accumulation and removal of E2.

[0120] Effects of EB300 implants on kisspeptin expression in the hypothalamus.

[0121] In both male and female, in the early adulthood when 2.5 months old, neonates were exposed to estrogen and significantly reduced arcuate nucleus (ARC) kisspeptin immunoreactivity (Fig. 3A), and similarly, lower immunoreactivity (Fig. 3B) was observed in the EB300 group when 6 to 7 months old. In general, in young rats and old rats, the kisspeptin immunoreactivity of males was lower than that of females, but EB reduced the kisspeptin (KISS, the protein product of Kiss1 gene) immunoreactivity in both sexes. This result shows that the EB300 implant in the neonate reduces KISS expression, and the effect lasts for a long time. As rats reach puberty when 2 months old, and KISS is the key major hormone to establish fertility, the reduced KISS affects the fertility of rats receiving EB300 implants.

[0122] Effects of EB300 implants on hypothalamic gene expression. Figures 4A to 4K Depicted is the adult hypothalamus, where KISS1 is expressed in the AVPV and ARC regions ( Figure 4A ).

[0123] KISS1 is expressed in the AVPV and ARC regions ( Figure 4A KISS1 expression was examined in the ARC region by anti-KISS1 antibody, KISS1 protein (white dots in the image) Figure 4A The plane of tissue section is marked by red dashed line). Compared with the untreated (control) group, KISS1 expression in the hypothalamus of EB300 was significantly reduced ( Figure 4B To quantitatively compare KISS1 expression between the control and EB300 groups, hypothalamic regions (including the AVPV and ARC regions) were dissected and examined by single-cell RNA sequencing (scRNAseq) ( Figure 4C In females, from a total of 4,049 and 4,223 cells from the control and EB300 groups, respectively ( Figure 4E ) identified 10 different cell types (cell clusters) ( Figure 4D From the hypothalamic cell clusters, a Kiss1 expression cluster (cluster-9) was identified ( Figure 4F ), and the number of Kiss1 expressing cells and the expression level of Kiss1 were compared between the control group and the EB300 group. In cluster-9, 307 and 194 cells were found in the control group and the EB300 group, respectively. 68 and 30 Kiss1 positive cells were identified from each group, respectively. This result indicates that the total number of cluster-9 and Kiss1 expressing cells in the EB300 hypothalamus were both decreased compared with the control group ( Figure 4G In males, ten distinct clusters were identified ( Figure 4H), where a total of 4,674 and 4,229 cells were examined in the control and EB300 groups, respectively ( Fig. 4I From the hypothalamic cell clusters, a Kiss1 expression cluster (cluster-8) was identified ( Figure 4J ). The number of Kiss1 expressing cells and the expression level of Kiss1 were compared between the control and EB300 groups. In cluster-8, 178 and 83 cells were found in the control and EB300 groups, respectively. 65 and 37 Kiss1 positive cells were identified from each group, respectively. This finding suggests that the total number of cluster-8 and Kiss1 expressing cells was decreased in the EB300 hypothalamus compared with the control group ( Figure 4K ). Taken together, these results indicate that EB treatment of newborn female and male rats reduces the number of KISS1 cells and, consequently, KISS1 protein expression in the hypothalamus.

[0124] Effects on the pituitary gland

[0125] Histopathological analysis of male and female pituitary glands was performed to observe whether the pituitary glands underwent changes after EB300 injection ( Figure 5A to B ). Paraffin sections of the pituitary obtained from the control and EB300 groups were examined histologically by H&E staining and no histological differences were found between the groups. Immunohistochemistry for LHβ (luteinizing hormone β subunit) and GH (growth hormone) did not reveal differences in LH and GH expression between the control and EB-treated groups ( Figure 5A to B ). These results suggest that the effects of EB implants may not affect growth hormone secretion or LH hormone secretion. LH secretion after KISS-10 injection.

[0126] When KISS is secreted from kisspeptin neurons, KISS binds to its cognate receptor KISS1R on GnRH neurons and stimulates GnRH secretion, which in turn stimulates LH secretion from pituitary gonadotrophs. To determine whether GnRH neurons and gonadotrophs retain their responsiveness and functionality in animals implanted with EB300, serum LH concentrations were measured before and after injection of kisspeptin-10 (KISS-10; a 10 amino acid long analog of KISS) in 2.5 month old animals ( Figure 6A to B Prior to KISS-10 injection, basal serum LH levels were low in EB-treated males and females. However, robust responses to KISS-10 were observed in both control and EB-treated males and females ( Figure 6A to B ). This finding indicates that EB300 implants do not alter the development and function of GnRH neurons or pituitary gonadotrophs, indicating the specificity of the biological effects of EB300.

[0127] Effects of EB300 on gonads.

[0128] To determine whether neonatal estradiol exposure affects gonadal development in males, testes were collected at PND 1, 3, and 10, and the seminiferous tubules, rete testis, and interstitium were stained with H&E and examined under a microscope. No obvious histopathological differences were detected between control rats and EB rats (Figure 7A). However, when TUNEL staining (which detects cells undergoing programmed cell death) was performed, many germ cells from the EB300 group showed positive signals at PND 3 and 10 days of age (Figure 7B), indicating that EB300 implants induce germ cell death in the testes. This finding was supported by reduced staining of DDX4 (germ cell marker) in the same tissue ( Figure 7C ). However, in females, TUNEL staining in the ovaries, fallopian tubes, or uterus did not find any significant differences between the control group and the EB300 group (Figure 8B). However, at PND 10, in the ovaries of EB300 rats, many germ cells gathered together without experiencing germ cell cyst rupture. When the oocytes of the control group were separated into single primordial follicles, subsequent immunostaining of DDX4 and FOXL2 (granulosa cell markers) confirmed this phenomenon in the EB300 group (Figure 8C). This result is consistent with previous reports that E2 inhibits germ cell nest rupture in neonatal ovaries (PMID: 17446182). In short, these results indicate that EB300 implants can directly affect gonadal development. A single implantation of EB300 in prepubertal rats induces complete infertility in males and females.

[0129] Fertility.

[0130] To determine whether the effects on kisspeptin neurons and gonads could lead to infertility, verified breeders were introduced into the cages of control and EB300 rats and housed with them for two weeks, and the number of fetuses and pups born by them was counted. Control rats produced fetuses, while EB-treated rats did not produce pups (Table 1).

[0131] Table 1. Fertility (male and female)

[0132]

[0133] Effects on female reproductive organs.

[0134] In females, EB300-implanted / treated animals did not show any signs of estrous cycle as determined by vaginal cytology, and their vaginal opening remained narrow and obstructed (Table 2). Morphological and histological examination of the ovaries and uteri of 5.5-month-old rats in the EB300 group were smaller and thinner, respectively, than those in the control group ( Fig. 9 and Fig.10 EB300-treated rats lacked glands in the uterus and corpora lutea in the ovaries ( Fig. 9 and Fig.10 The ovaries and uteri of young (2.5 months) and old (7 months) EB300-treated rats showed similar morphological and histological patterns ( Fig.10 ), indicating that these phenotypes persist. However, in the pituitary, no significant differences were observed ( Figure 5A to B ), indicating the organ-specific effects of EB300 implants.

[0135] Table 2. Estrous cycle patterns

[0136]

[0137] Effects on male reproductive organs.

[0138] To determine whether sperm count and motility were affected by neonatal EB300 implants, sperm quantity and quality were measured at 5 months of age. Sperm concentration (million / mL) was significantly lower in the EB-treated group compared to the control group (p=0.001) ( Fig.11 ). The percentage of motile sperm was significantly reduced in rats implanted with EB300 (p=0.003). In addition, the percentage of motile sperm was significantly reduced in rats treated with EB300 compared to control rats (p=0.02), and they had a significantly higher percentage of immotile sperm (p=0.01) ( Fig.11 ). These results suggest that neonatal EB300 implants affect sperm concentration and quality, affecting male fertility.

[0139] When examined at 2.5 months of age, all EB300 group animals had significantly smaller testes, epididymis, seminal vesicles, and prostate glands compared with controls ( Fig. 12A ). Epididymis from the EBx11 and EB300 groups had smaller tubule diameters and contained few or no germ cells ( Fig. 12A The lower expression of StAR (a marker of steroidogenic cells, Leydig cells) and DDX4 (a marker of germ cells) and the higher number of TUNEL-positive cells in EB300 animals indicated that EB300 implants resulted in lower testosterone synthesis capacity, lower sperm production, and higher levels of apoptosis ( Fig. 12B Similar effects on the testis and epididymis were evident in older animals ( Fig.13 and Fig.14 No significant differences in the pituitary were observed between control and EB animals ( Fig.13 and Fig.14 When comparing tissue weights, the testis, epididymis, and accessory glands in the EB300 group were lighter than those in the control group ( Fig.15 ).

[0140] Male sexual behavior is primarily dependent on testosterone, a hormone secreted by the interstitial cells of the testes in all vertebrate species. To assess the sexual behavior of EB-treated rats, 4-month-old males were housed with females in estrus (a reproductive stage in which females are receptive to males) and allowed to mate. The time required to first copulation (latency) and the number of copulations within 30 minutes were recorded and compared. Animals treated with EB300 had significantly longer latencies (p=0.01) and made fewer copulation attempts (p=0.02) compared to control animals. Fig.16 ), indicating that EB implants significantly reduced the mating behavior of males.

[0141] Taken together, these reproductive characteristics indicate that neonatal implantation of EB capsules significantly inhibits gonadal and accessory gland development, gametogenesis, and sexual behavior, resulting in sterility in both males and females.

[0142] In both males and females, rats implanted with silicone capsules (EB) containing estradiol benzoate produced significantly less gonadal sex steroids than controls.

[0143] Developmental defects of the reproductive system will inevitably affect the synthesis of sex steroid hormones, so the blood concentration of sex steroids was checked. Peripheral blood was collected from males of 2 months and 6 months and their serum hormone levels were measured. At 2 to 3 months of age, the serum testosterone level of the group treated with EB300 was significantly lower than that of the control group ( Fig.17 The same trend was observed when testosterone concentrations were measured at 6 to 7 months of age ( Fig.17 In females, there was no significant difference in serum E2 levels between the EB300-treated and control groups at young ages. However, E2 levels were significantly lower in animals in the EB300-treated group than in the control group ( Fig.17 On the other hand, there was no difference in testosterone levels in females between the EB300-treated and control groups, regardless of age. Interestingly, males implanted with EB300 capsules had significantly higher E2 concentrations compared to male controls ( Fig.18 ), which may be caused by increased aromatase activity in the male gonads. On the other hand, due to defective testicular and ovarian development (Figure 7A to Fig.14 ), low testosterone levels in males and low E2 levels in adult females in EB-treated animals, respectively, were expected. Developmental defects of the seminal vesicles and prostate as well as low mating behavior in EB-treated animals could be caused by these low testosterone levels, since their development is testosterone-dependent.

[0144] Animals with EB300 implants showed no pathology.

[0145] Body weight is often measured as an indicator of health status. EB300-treated and control animals were weighed from birth to 180 days of age ( Figure 19A to B ). In males, the body weight of the EBx11 and EB300 treated groups was lower than that of the control group. At 180 days of age, the body weight of the EB300 treated group was significantly lower than that of the control group, but there was no significant difference between the other experimental groups (Figure 19A). In females, the EB treated rats gained weight more rapidly than the control group rats. At 180 days of age, all EB treated groups showed significantly higher body weight than the control group (Figure 19B).

[0146] When the rats reached 6 to 7 months of age, they were euthanized and the ratios of organ length / body weight or organ weight / body weight were compared. In males, there was no difference in femur length / body weight between the control and EB300 groups ( Fig. 20 ). The brain / body weight and pituitary / body weight ratios were higher in the EB300 group, while the kidney / body weight ratio was lower in the EB300 group compared to the control group. In males, the weights of the liver, spleen, and heart remained essentially the same between the control and EB300 groups ( Fig. 20 In females, the EB300 group showed decreased organ / body weight ratios in all organs of the EB300 group ( Fig.21 ). However, these differences may be caused by effects on body weight gain; EB300 females were significantly heavier (approximately 20%) than controls ( Fig.21 In support, histological examination of the kidneys and liver revealed no pathology in any of the groups ( Fig. 22 ). Blood chemistry also showed no pathology in the kidney and liver (data not shown). Carnoy's staining of the whole mammary gland showed normal branching of the mammary gland in the EB300 group. However, in the EB300 group, shrinkage / dysplasia of the terminal buds was evident. It is believed that the low budding in the EB300 group is caused by low E2 levels ( Fig.23 ).

[0147] To investigate the effects of EB injection on hematopoiesis, blood cells, red bold cells (RBC), hemoglobin, platelets, and hemoglobin amount / erythrocyte (MCH), mean corpuscular hemoglobin concentration (MCHC) and white blood cells (WBC) were counted (Table 3). In both males and females, RBC, platelet, and WBC counts were not different from the control group, and hemoglobin concentration, MCH, and MCHC were also not different. In summary, overall histological and hematological data indicate that hematopoiesis is not affected by EB implants.

[0148]

[0149] EB300 has a wider effective time window.

[0150] Knowing that implantation of EB300 at PND 1 induced complete and irreversible infertility, we then tested whether infertility was induced when the capsules were implanted at a later time point. EB300 were implanted at PND 1, 5, 12, and 21. Unexpectedly, all implantations at these different times effectively inhibited gonadal development in males, as determined by testicular size and testicular histology along with immunohistochemistry using antibodies to Cyp17 (interstitial cell marker), DDX4, and TUNEL staining ( Fig.24 The seminal vesicles and prostate gland were also significantly smaller ( Fig.25 In females, the ovaries are small and no corpus luteum is found, indicating that ovulation has not occurred ( Fig.26 ). In conclusion, EB300 has a wide effective time window in inducing sterility in both males and females.

[0151] EB100 and EB30 were also effective in inducing sterility.

[0152] The data presented below demonstrate that EB30 (30 μg of EB) is effective in rendering both male and female animals (rats; EB implanted at PND 1 or 2) sterile.

[0153]

[0154] EB effectively induces infertility in hamsters.

[0155] The effect of EB125 implants on hamster gonadal development was determined. Empty capsules (control) or 125 micrograms of EB contained in silicone rubber capsules were implanted into 21-day-old hamsters. Reproductive tissues were collected from subjects at PND 30. Testes from hamsters implanted with EB125 were significantly smaller than those of control animals.

[0156] The above description includes reference to the accompanying drawings, which form a part of the detailed description. The accompanying drawings show some specific embodiments in which the present invention can be implemented by way of example. These embodiments are also referred to as "embodiments" in this article. Such embodiments may include elements other than those shown or described. However, the inventors also contemplate embodiments in which only those elements shown or described are provided. In addition, the inventors also contemplate embodiments using any combination or arrangement of those elements (or one or more aspects thereof) shown or described herein with respect to a specific embodiment (or one or more aspects thereof) or with respect to other embodiments (or one or more aspects thereof).

[0157] In the event of an inconsistency in usage between this document and any document incorporated by reference, the usage in this document controls.

[0158] The above description is intended to be illustrative, not restrictive. For example, the above embodiments (or one or more aspects thereof) may be used in combination with each other. After reviewing the above description, other embodiments may be used, for example, "by a person of ordinary skill in the art". An abstract is provided to allow the reader to quickly determine the nature of the technical disclosure. It should be understood that the submitted abstract is not used to interpret or limit the scope or meaning of the claims. In addition, in the above detailed description, multiple features may be combined together to streamline the disclosure. This should not be interpreted as meaning that the disclosed features that are not claimed for protection are essential to any claim. On the contrary, the subject matter of the present invention may exist in a case where there are less than all the features of a specific disclosed embodiment. Therefore, the attached claims are incorporated into the detailed description as an example or an embodiment, wherein each claim is individually an independent embodiment, and it is expected that such embodiments may be combined with each other in a variety of combinations or arrangements. The scope of the present invention should be determined with reference to the attached claims and the full scope of the equivalent schemes given by such claims.

[0159] The following content corresponds to the original claims in the parent application and is now incorporated herein as part of the specification:

[0160] 1. A method for inducing infertility in an animal, comprising administering to the animal an effective amount of estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof to render the animal infertile.

[0161] 2. The method of claim 1, wherein the estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof is administered as a composition comprising a carrier.

[0162] 3. The method of claim 2, wherein the carrier is a physiologically acceptable oil.

[0163] 4. The method of claim 1, wherein the estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof is formulated into a sustained-release preparation.

[0164] 5. The method of claim 1 , wherein the administration is a single-dose one-time administration.

[0165] 6. The method of claim 1, wherein the estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof is administered intraperitoneally, intramuscularly, or subcutaneously.

[0166] 7. The method of claim 1, wherein the animal is a companion animal.

[0167] 8. The method of claim 1, wherein the animal is a livestock animal.

[0168] 9. The method of claim 1, wherein the estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof is administered prior to puberty.

[0169] 10. A system for delivering a sterilizing agent to an animal, the system comprising:

[0170] capsules comprising estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof; and

[0171] - A delivery device for delivering the capsule to the animal.

[0172] 11. The system of claim 10, wherein the capsule is formulated to slowly release EB.

[0173] 12. The system of claim 10, wherein the delivery device is an injector for injecting the capsule into or onto the animal.

[0174] 13. A system according to item 10, wherein the estradiol, estradiol benzoate, estradiol dipropionate, estradiol valerate, estradiol cypionate, or a combination thereof is administered intraperitoneally, intramuscularly, or subcutaneously.

[0175] 14. The system of any one of items 10 to 13, wherein the animal is a companion animal.

[0176] 15. The system of claim 10, wherein the animal is a livestock animal.

Claims

1. A method for inducing permanent sterility in an animal, comprising administering to said animal an effective amount of estrogen to render said animal sterile, wherein the estrogen is formulated in a sustained release carrier that releases the estrogen over 15 to 20 days, The estrogen is administered once in a single dose within a few weeks after birth and before puberty.

2. The method of claim 1, wherein the 15 to 20 day release of estrogen results in decreased expression of KISS1 cells and KISS1 protein.

3. The method of claim 1, wherein permanent sterility is a state in which the animal is incapable of producing offspring.

4. The method of claim 3, wherein the condition comprises a lack of estrous cycles and stenosis and obstruction of the vaginal opening in the female animal.

5. The method of claim 4, wherein the condition comprises a lower sperm concentration, a lower motile sperm concentration, or fewer sexually motile sperm and reduced mating behavior in the male animal.

6. The method of claim 1, wherein the estrogen is administered intraperitoneally, intramuscularly, or subcutaneously.

7. The method of claim 1, wherein the animal is a livestock animal.

8. The method of claim 1, wherein the animal is a rodent, a cat, or a dog.

9. The method of claim 1, wherein the estrogen is estradiol benzoate (EB).

10. The method of claim 9, wherein 1-100 mg / kg of the estradiol benzoate is administered.

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