No surgery required: the future of feline sterilization

Abstract

Overview:

For many years, researchers have been studying reproduction of cats and dogs, including approaches to non-surgical sterilization, but scant funding has been available for this work. Recognizing the need to fund research and to attract researchers from the biomedical community to apply their expertise to this area, the Michelson Prize & Grants (MPG) in Reproductive Biology program was founded. Since 2009, it has funded 34 research projects in seven countries toward discovery of a safe single-administration lifetime non-surgical sterilant in male and female cats and dogs.

Goal:

The goal of the MPG program is the reduction or elimination of the approximately 2.7 million deaths of healthy shelter cats and dogs in the US every year. The successful product is expected to be a single-dose injectable product approved by the US Food and Drug Administration as a veterinary prescription item. The most optimistic prediction is that such a product will reach the hands of practicing veterinarians within the next decade.

Areas of research:

Active research is in progress using approaches such as immunocontraception with a single-administration vaccine against gonadotropin releasing hormone (GnRH). Long-term therapy with GnRH agonists such as deslorelin administered in controlled-release devices is also being studied. Other scientists are targeting cells in the brain or gonads with cytotoxins, such as are used in cancer chemotherapy. Gene therapy expressing proteins that suppress reproduction and gene silencing of peptides essential to reproduction are further avenues of research. Findings are available at www.michelsonprizeandgrants.org/michelson-grants/research-findings.

Introduction

For over 30 years, researchers have investigated non-surgical technologies that might be used to suppress fertility in cats and dogs. When women began using oral contraceptives composed of progestogens and/or estrogens, various similar oral drugs for dogs were tried briefly, but were found to have deleterious side effects. In cats, progestational drugs suppress male and female reproduction but also lead to type 2 diabetes, mammary nodules and, in females, increased risk of pyometra. These sex steroid therapeutic approaches cannot provide permanent sterility without repeated dosing, and so cannot be considered a true alternative to castration for males and ovariohysterectomy for females.

Immunocontraception has been tested in cats and dogs using vaccines against zona pellucida proteins and gonadotropin-releasing hormone (GnRH). These methods showed some promise, but they require multiple boosters. Hormone implants with the GnRH agonist deslorelin (Suprelorin^®; Virbac) became available about 6 years ago in Europe. However, they also have the limitation of only lasting a year and being labeled for use only in male dogs. All of these approaches are short-term solutions and particularly unsuited to use for feral or shelter cats and dogs, where permanent sterility is the goal.

Surgical ovariohysterectomy (or ovariectomy) and castration remain the gold standard for inducing permanent sterility in cats and dogs. Surgical sterilization not only prevents generation of offspring, it also prevents undesirable behaviors associated with estrogen secretion in the female and testosterone secretion in the male that can be barriers to successful pet ownership. Although surgical sterilization is associated with undesirable sequelae in some pets (obesity, urinary incontinence, joint disease and some cancers), it also prevents significant undesirable outcomes that occur in intact pets. These include a high incidence of mammary and testicular cancer, prostatic hypertrophy, undesired mating and pregnancy, pyometra and pregnancy disorders such as dystocia.

An estimated 2.7 million healthy dogs and cats are euthanized in US shelters alone every year. ¹ While surgical sterilization has had a significant impact in reducing the numbers of shelter animal deaths, it requires substantial and expensive infrastructure and community commitment. There are not enough veterinary surgeons or adequate financial resources to make surgery available to all homeless pets. (For a comprehensive review of the various methods of cat and dog non-surgical contraception used over the years, see the Alliance for Contraception in Cats & Dogs’ 2013 report.) ²

The Michelson Prize & Grants program

Prior to 2008, very little competitive research funding from federal or foundation sources was available to the scientific community to investigate technologies for safe and effective contraception in cats and dogs. In 2008, the Los Angeles billionaire orthopedic surgeon, Gary Michelson, MD, established Found Animals Foundation to reduce or eliminate shelter euthanasia of cats and dogs in the United States. He funded a $25 million Michelson Prize in Reproductive Biology and committed $50 million for Research Grants in Reproductive Biology (www.michelsonprizeandgrants.org).

Both the prize and grants are directed toward discovery of a safe low-cost single-dose non-surgical sterilant for male and female cats and dogs that lasts for their 10–20 year lifespan. The sterilant also must ablate the presence or action of sex steroids (thereby preventing sexual behaviors as well as mating), have a pathway to US Food and Drug Administration (FDA) regulatory approval as a veterinary prescription product, and be amenable to delivery in a field setting; an injectable product (SC, IM or IV) is preferred. While the Foundation’s mission is directed toward reducing euthanasia of shelter pets, it anticipates that a successful product also will be made available domestically and internationally to groups managing feral and community-owned populations of cats and dogs.

The influx of resources for research with the goal of better understanding reproductive control in cats and dogs has attracted wide interest from scientists in the global biomedical research community, with grant applications from around the world and from disciplines such as reproductive biology, gene therapy, neuroendocrinology, immunology, oncology, bioengineering, medicinal chemistry and many more.

Applications for Michelson grant funding are accepted after approval of a two-page Letter of Intent, and they are reviewed by a 20-member Scientific Advisory Board (www.michelsonprizeandgrants.org/about/scientific-advisory-board) that meets three times per year to review and recommend approval of applications.

Research in progress

Found Animals Foundation has received 338 Letters of Intent and 124 full applications for research funding since 2008, of which 34 proposals have been funded. Proposals funded to date encumber approximately $14.2 million of the $50 million available.

Research in projects funded has fallen into four broad approaches:

• Immunocontraception;

• High-dose/long-term GnRH agonists;

• Targeted delivery of cytotoxins; and

• Gene silencing/gene therapy.

Immunocontraception

Immunocontraception uses the immune response of the animal to block reproduction. It works by vaccinating cats or dogs with an antigen that is important in maintaining normal reproduction, such as GnRH. Because this antigen is a ‘self’ antigen, various ingredients are added to the vaccine to help make the antigen more immunogenic. Vaccines to GnRH and zona pellucida proteins (porcine origin, PZP) have been developed successfully for use in wild deer and wild horses ³ (GonaCon™, ZonaStat-H™), but both require boosters to remain effective. GnRH vaccines have proven to be successful for long-term suppression of fertility in cats and dogs.

GnRH is a promising target for immunocontraception, as this decapeptide is the same in male and female dogs and cats, and is at the top of the reproductive cascade from the brain to the gonads that regulates reproduction. Vaccines against GnRH have been reported to suppress reproduction safely in cats ⁴ and dogs, ⁵ but a drawback is that booster vaccinations are necessary at intervals to maintain suppression.

New research areas in immunocontraception involve the use of different antigens and different ways to present those antigens to the immune system. Antigens being tested in vaccines, not before investigated, include unique antigens on sperm, GnRH receptor proteins, GnRH DNA or specific recombinant zona pellucida proteins. Various methods for presentation or delivery of familiar antigens, such as GnRH, include some type of delivery vehicle or matrix that will allow for longer-term exposure. Some of these approaches use viral vectors or ‘smart’ delivery devices that can expose dogs or cats to antigen over a lifetime, without booster injections. ⁶

High-dose/long-term GnRH agonists

GnRH agonists act by binding to the GnRH receptors on gonadotroph cells of the anterior pituitary, causing an initial stimulation of these cells (to secrete luteinizing hormone [LH] and follicle-stimulating hormone [FSH]); in males this produces an increase in testosterone, and in females it may elicit an estrous cycle. Native GnRH delivery in normal reproduction is episodic and pulsatile; constant delivery is hypothesized to suppress reproduction by eliminating the pulsatility of effect. After about 10 days, the GnRH receptors in the gonadotrophs are downregulated, and this results in a complete shutdown of the reproductive system in both males and females (called ‘medical castration’ in human medicine).

For many years, GnRH agonist drugs have been used in humans to suppress secretion of reproductive hormones. GnRH agonists (eg, leuprolide) are used to treat such human conditions as precocious puberty and are formulated as subcutaneous implants that can release the drug for as long as 1 year. Similarly, in the past 10 years, a GnRH agonist called deslorelin has been formulated in implants that release the drug over 6 or 12 months (Suprelorin) and have been approved in Australia, New Zealand and Europe for suppression of fertility in male dogs.

Several grantees are exploring how GnRH agonists might be used for induction of sterility in cats and dogs. For example, if the length of time an implant could deliver a drug could be increased to over 5 years, it may be that this is long enough in some populations, perhaps feral cats, to essentially cause lifetime sterility. This type of delivery might be achieved using some kind of novel implant formulation, or perhaps a medical device that could hold sufficient drug for lifetime administration (Microchips Biotech, www.mchips.com, is a company developing such a device for human contraception). Although it sounds far-fetched that a device might deliver a drug for the lifetime of an animal, because only very low levels of GnRH agonists are needed to maintain reproduction suppression, this might be possible.

Another approach being investigated is the possibility that giving high doses of GnRH agonists early in life might significantly delay or even prevent puberty. One grantee demonstrated a significant delay in the onset of puberty (to 42–91 weeks of age) in male and female kittens given a 1.6 mg deslorelin acetate implant within 24 h of birth; control kittens demonstrated onset of puberty at 15.5 ± 1.7 weeks. ⁷

Targeted delivery of cytotoxins

Instead of surgically removing reproductive organs such as the uterus, ovaries or testes, some scientists are exploring whether it might be possible to specifically target and kill cells that are vital to maintain reproduction. Would it be possible to create a drug that, when injected, could go directly to a specific subset of cells, such as the GnRH neurons in the brain or primordial follicle cells in the ovary, and kill those cells without damaging other tissues?

In order for this approach to work, three things are required: a method for targeting a particular subtype of cell; a potent toxin that can kill cells when delivered by the targeting mechanism; and, finally, a way to get the drug–toxin conjugate to the specific cells. This approach is used to kill cancer cells in human medicine. For example, a prostate cancer therapy has been developed that uses antibodies to a protein specific to many prostate cancer cells and conjugates this antibody to a potent toxin. Once the antibody has bound to the prostate cancer cell surface, the toxin is delivered and kills only the cancer cells. ⁸

A number of grants using this type of approach have been funded by the MPG program. Researchers are targeting neurons in the brain that secrete GnRH with the understanding that, if these neurons are killed, the entire reproductive cascade is shut down in both male and female cats and dogs. The challenge with this approach is getting treatments through the blood–brain barrier. Despite this obstacle, these neurons are an appealing target. Another brain target is the population of gonadotrophs in the anterior pituitary. Gonadotrophs are located outside of the blood–brain barrier, but the challenge here is that stem cells of the pituitary may be able to replenish gonadotroph populations killed with toxins. ⁹

Another exciting target are the stem cells in the gonads – the cells that differentiate into ovarian follicles and sperm. If those cells could be specifically destroyed, then cats or dogs could be made sterile. This may be easier in females born with a finite population of follicles than in male animals that continuously make sperm. Targeting of the exact cell population to be killed is important to this approach as well; male and female gonadal cells will likely need different methods.

Several grants have been awarded to study these approaches. Grantees are targeting GnRH neurons with IV administration of the peptide kisspeptin conjugated to the toxin saporin; kisspeptin binds to receptors on GnRH neurons. Others are linking GnRH analogs with toxins that are expected to bind to, and kill, gonadotrophs. Scientists targeting gonadal stem cells are searching for homing peptides that can specifically deliver an agent to those cells, and seeking agents that can be internalized into cells and destroy them.

Gene silencing/gene therapy

Some of the most cutting-edge technologies in biomedical sciences are gene therapy and gene silencing. It has been known for many years that it is possible to genetically engineer viruses to be vectors to deliver genes to cells. Early days of gene therapy focused on developing therapies for genetic diseases, where a child might be born with, for example, hemophilia because they lacked the gene for a specific clotting factor. Gene therapies were designed to deliver these genes to the child’s cells, so the cells could actually produce the clotting factors – in essence, providing the gene that the child lacked. Such an approach sounds simple, but it took many years for these therapies to be developed and made safe enough for clinical use. Gene therapy for hemophilia in dogs has resulted in dogs with genetic clotting defects having normal clotting function restored for 8 or more years. ¹⁰

How could this approach work for fertility suppression? Suppose we could identify a specific protein that could suppress reproduction and then deliver that protein for a lifetime using a viral vector? Instead of the gene restoring function, it could potentially interfere with function. In order to make this approach possible, we need to identify a protein or toxin that suppresses reproduction, make a viral vector that carries the gene for that substance into cells in a cat or dog, and get that protein expressed, either systemically or locally, such as within the ovaries or testes.

Some progress has been made exploring this approach to suppressing reproduction. Scientists are studying the feasibility of inserting genes that cause over-expression of antibodies to GnRH. They are also studying the feasibility of inserting genes that cause over-expression of gonadotropin-inhibitory hormone (GnIH) or Mullerian inhibiting substance (which regulates primordial follicle recruitment in adult females and testosterone production in adult males) in both cats and dogs.

In human medicine, a number of companies are using the technology of gene silencing to ‘turn off’ specific genes to regulate various physiological functions. Gene silencing is a biological phenomenon that has fairly recently been discovered and involves the realization that small RNA fragments are used by cells to regulate gene expression. If synthetic small interfering RNA (siRNA) can be synthesized and delivered to the body as a drug, it could theoretically be designed to specifically shut off expression of certain genes. Companies such as Alnylam (www.alnylam.com) are developing small molecule drugs for amyloidosis and porphyria using this mechanism of action. But can this approach be used for suppression of reproductive genes?

For induction of sterility in cats and dogs, use of siRNA drugs is not practical, since they would need to be given daily and likely by injection. If, however, these molecules could be delivered to cells using viral vectors, where the vector would incorporate into cells and deliver the gene-interfering siRNA over long periods of time, it is possible to imagine that this would be an elegant system for the induction of long-term and possibly permanent sterility.

Three steps are required of such a system in order for it to be effective: a gene to turn off needs to be defined; a viral vector to deliver the siRNA required to suppress that gene needs to be identified; and, finally, that viral vector needs to get to the cells that express the gene to be turned off. One approach that is under investigation is construction of a viral vector that targets kisspeptin, a regulatory protein that is required to elicit GnRH secretion.^11,12 This viral vector must be tailored to deliver the siRNA to the brain – a tall order, since penetrating the blood–brain barrier is difficult. However, if the appropriate hypothalamic neurons could be reached by a vector, that viral vector could cause the cell to produce a lifetime of siRNA to shut down reproduction. If this could be achieved, a single product could theoretically work in both male and female cats and dogs for a lifetime of fertility suppression.

Targeting GnRH neurons in the brain makes sense as these cells are the master regulators of reproduction, but it may be easier to target specific cells in the gonads to avoid the difficulties of getting a therapy across the blood–brain barrier. Another interesting approach is to use microRNA technology to inhibit androgen receptor expression in the testes, as androgen stimulation is required for normal testicular function including sperm production. Androgen receptor gene silencing constructs could be delivered with viral vectors to ‘turn off’ male reproduction.

Another fascinating approach is to target the small RNA molecules that have been identified as vital for ova and sperm to undergo maturation. Interfering with these small RNA molecules, which appear to be unique to gametes, might be an interesting target.^13,14 Once the right cat and dog small RNAs that regulate, for example, spermatogenesis have been identified, then antagonists to those RNAs can be designed and potentially delivered using viral vectors.

Safety and animal welfare

The goal of the supported research is to discover an effective method to sterilize male and female cats and dogs for a lifetime. In pursuing this goal, the Foundation committed to making sure that any product that might be developed using the various approaches outlined above is not only effective, but is also safe for the treated animals. Emphasis is placed on developing methods that accurately target reproduction while avoiding side effects on other organ systems.

In addition, the Foundation and MPG program are committed to the welfare of all research animals used in the grant-supported research. Grantees are required to review the Foundation and MPG program’s ‘Policy for Animals Involved in Research’ (www.michelsonprizeandgrants.org/resources/animal-welfare-policy), are required to rigorously justify numbers of animals (usually mice and rats, occasionally cats and dogs) used in projects, and must, prior to approval of any project, demonstrate that behavioral enrichment will be provided to research animals. In addition, grant applicants must provide a plan for placing research cats and dogs in adoptive homes at the end of the study where they can enjoy living as a pet.

Targeted research

One of the interesting things learned as grants have been submitted and reviewed is how very little is known about control of normal reproduction in cats and dogs. Agencies that fund biomedical research do not provide support for this work, and entities such as the United States Department of Agriculture (USDA) and pharmaceutical companies have, for the most part, stopped funding reproductive research in animals.

As part of the MPG program, significant new findings are becoming available to the research community that help shed light on basic reproductive mechanisms in domestic cats and dogs. As these findings are shared via publication in peer-reviewed journals, other researchers benefit and the knowledge of reproductive biology is spread. This, in turn, should encourage new approaches and collaborations, something we are already seeing happen among the grantees.

Key Points

• The Michelson Prize & Grants (MPG) in Reproductive Biology program supports the scientific community to advance non-surgical sterilization research for cats and dogs. The MPG Program has allocated $50 million grant funding for promising research; since 2009, it has funded over 30 projects in seven countries. A $25 million prize is available to the first entity to develop a product meeting established criteria.

• The MPG program has the goal of developing a safe low-cost single-dose lifelong non-surgical sterilant for cats and dogs of both sexes. In addition, in order to win the Prize, the solution must ablate the presence or action of sex steroids, have a pathway to US FDA regulatory approval as a veterinary prescription product, and be amenable to delivery in a field setting.

• MPG-funded grantees are investigating a wide range of approaches. Current research falls into four broad categories: immunocontraception, high-dose and/or long-term GnRH agonists, targeted delivery of cytotoxins, and gene silencing/gene therapy.