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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

Surgical sterilization is the mainstay of dog and cat population control, but its use is still often limited by the costs and effort involved, especially in developing countries. An ideal non-surgical sterilant that is safe, effective, permanent, administered as a single injection and capable of being manufactured inexpensively could have a significant impact on the world-wide dog and cat overpopulation problem. One approach towards developing such an agent is the targeting of pituitary gonadotrophic cells with cytotoxic agents using gonadotropin-releasing hormone (GnRH). GnRH is a peptide that binds to high-affinity receptors selectively expressed on gonadotrophs and some types of cancers. Both small molecules and proteins have been conjugated to GnRH analogues to generate targeted cytotoxic and imaging agents. Although most of these efforts have focused on development of human cancer therapeutics, available reproductive studies in rats and dogs suggest that current compounds do not have sufficient therapeutic windows for complete gonadotroph ablation, in part owing to poor stability of peptide targeting sequences. The only reported longer-term study of gonadotroph ablation in dogs reported suppression of serum testosterone for 8 months, but endocrine function then recovered, raising important questions about the mechanism of reproductive suppression and its recovery. Although studies to date suggest that this is a potentially attractive approach to non-surgical sterilization, ideal agents are yet to be developed, and important mechanistic questions remain to be answered.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

Surgical sterilization is the mainstay of dog and cat population control. Although gonadectomy is effective on an individual basis, it requires anaesthesia, medical equipment, a trained veterinarian, recovery time and post-surgical observation. The associated cost can be a barrier for many owners, especially in developing countries. Further, the effort involved is difficult to scale for larger populations of feral animals or those entering shelters, and, all too often, euthanasia is the resulting alternative. An ideal non-surgical sterilant would be safe, effective, permanent, administered as a single injection and capable of being manufactured inexpensively. Such an agent could be an important new tool for companion animal population control, and may also have additional applications in wildlife management and agriculture.

One approach that has been explored is targeted ablation of pituitary gonadotrophic cells. These highly specialized cells are critical for reproductive function in mammals and are the central player of the hypothalamic-pituitary-gonadal reproductive endocrine axis (Fig. 1). They are responsible for the biosynthesis and secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that are necessary for gametogenesis and gonadal steroid biosynthesis in both male and female mammals (Millar 2004). Transgenic mice whose gonadotrophs have been ablated by cell-specific expression of diphtheria toxin are infertile (Kendall et al. 1991). Further, all other pituitary cell types and overall phenotype appear to be normal in these animals. Therefore, if one could design efficacious chemical or biological cytotoxic agents that are highly specific for gonadotrophs, they would be expected to induce sterility without non-reproductive side effects. Efforts in this direction are the subject of this review.

image

Figure 1.  The hypothalamic-pituitary gonadal axis. In the hypothalamus, kisspeptin/NKB neurons enervate GnRH-secreting neurons in the median eminence (ME). Kisspeptin/NKB signalling regulates the pulsatile secretion of GnRH, which in turn stimulates pituitary release of the gonadotropins, luteinizing hormone and follicle-stimulating hormone that stimulate steroidogenesis and gametogenesis in the gonads.

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Background: GnRH and the GnRH Receptor

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

Secretion of LH and FSH by pituitary gonadotrophs is stimulated by the hypothalamic peptide, gonadotropin-releasing hormone (GnRH) (Matsuo et al. 1971; Burgus et al. 1972). GnRH binds to the GnRH receptor (GnRHR), a member of the G protein-coupled receptor family (Millar 2004). Activation of GnRHR by GnRH initially results in activation of Gq, activation of phospholipase C and production of the second messengers diacylglycerol and inositol phosphates (Conn 1986; Hsieh and Martin 1992). These early responses in turn stimulate LH/FSH secretion and activation of downstream signalling pathways that stimulate gonadotropin biosynthesis. Whereas pulsatile administration mimicking physiologic secretion of GnRH is stimulatory, continuous administration of GnRH or its agonist analogues results in profound down regulation of the gonadotroph and temporary loss of gonadal function (Belchetz et al. 1978).

Since its discovery, the roles of GnRH and its receptor in mammalian reproductive physiology have been well established. High-level expression of the GnRHR gene is restricted to pituitary gonadotrophic cells, GnRH-secreting neurons of the hypothalamus and peripheral reproductive tissue (ovary, uterus, prostate and breast) (Cheng and Leung, 2005). Neither radioligand binding assays nor RT-PCR could detect receptor in other tissues such as liver, kidney, spleen, heart or hematopoietic stem cells (Grundker et al. 2002). Expression of this and other neuropeptide receptors is now well established in a variety of human cancers (Heasley, 2001; Petit et al., 2001; Dyba et al., 2004).

However, the exclusively reproductive role of GnRH and GnRHR in normal physiology remains controversial, and some groups have reported effects of GnRH on cardiac myocytes and other tissues (Skinner et al. 2009; Dong et al. 2011). This is an important controversy to resolve, because if GnRHR-expressing cells are critical for non-reproductive functions, then cytotoxic agents targeted to GnRHR could have an intrinsically poor safety window. Important data in this regard are provided by GRIC knock-in mice that co-express Cre recombinase with the GnRHR gene (Wen et al. 2008, 2010, 2011). When these mice are crossed with mice carrying a ROSA26-YFP reporter (incorporating a floxed terminator preventing YFP expression), the Cre recombinase removes the terminator and allows YFP expression. In these mice, 99.9% of gonadotropin-containing cells are labelled with YFP, indicating that the GnRHR gene is expressed in essentially all gonadotrophs (Wen et al. 2008). Furthermore, YFP expression is found in brain and testis, but not in kidney, liver, adrenal, ovary, uterus, spleen or heart (Wen et al. 2010). When the GRIC mice are crossed to ROSA26-DTA mice (carrying a floxed terminator prior to the diphtheria toxin A chain), diphtheria toxin is expressed in GnRHR gene-expressing cells, resulting in cell death (Wen et al. 2010). These mice are severely hypogonadal and 94.4% of the LH-expressing cells and 68.3% of the FSH-expressing cells in the pituitary are ablated. Offspring carry the transgene sequences with Mendelian frequencies and are viable, indicating that ablation of GnRHR-expressing cells is not lethal. Other than the altered reproductive system, the only other apparent phenotype is a reduced body weight of males, together with reduction in size of heart, kidney and liver. Therefore, while detailed histopathology of these animals was not performed to rule out more subtle impacts of extrapituitary GnRHR-expressing cell ablation, these data suggest that gonadotroph ablation appears to be possible without major non-reproductive side effects.

GnRH is a linear decapeptide amide with the sequence, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Matsuo et al. 1971; Burgus et al. 1972). The structural and conformational requirements for peptide analogues to maintain receptor binding and activity have been well established (Struthers et al. 1990; Millar 2004). The post-translational modifications to form pyroglutamic acid (pGlu) and carboxy-amide are required for biological activity. Endogenous GnRH is highly labile to proteolysis in plasma and its half-life in vivo is only a few minutes (Dupont et al. 1974). To overcome this, soon after the discovery of GnRH, a large number of analogues were synthesized and tested. Replacement of Gly6 with D-amino acids improved both potency and stability. Replacement of Gly10 with ethyl-amide also increased enzymatic stability and, when combined with the D-Leu6 substitution, resulted in the drug leuprolide (Lupron™; Abbott, Abbott Park, Illinois, USA). Gonadotroph downregulation by leuprolide and related agonist analogues has become a mainstay of hormone deprivation therapy for treatment of prostate and breast cancer (Conn and Crowley 1991; Engel and Schally 2007). Substitutions of His2 in the peptide result in antagonists which bind to the GnRH but fail to stimulate signalling (Vale et al. 1972). When combined with extensive additional modifications, these antagonist analogues also have been developed into approved human therapeutics for control of reproductive function and reproductive hormone levels (Huirne and Lambalk 2001; Herbst 2003). For both agonist and antagonist GnRH peptides, the bioactive conformation required for receptor binding involves a β-turn at position 6–7 and that the two ends of the peptide are in proximity and engaged in specific receptor interactions (Struthers et al. 1990; Millar 2004). More recently, small molecule GnRH antagonists have been developed that lack the peptide backbone structure, are highly stable and are orally bioavailable (Betz et al. 2008). The most advanced of these agents, elagolix (Struthers et al. 2009) has completed a series of Phase II clinical trials for the treatment of endometriosis, an oestrogen-dependent disease of women. Despite extensive efforts in the development of GnRHR modulators, all of these agents share a common feature that their effect is lost and reproductive function is restored once administration of the drug is discontinued.

GnRH-Drug Conjugates

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

The ectopic expression of GnRH receptors on ovarian, breast and endometrial cancer cells (Fekete et al. 1989; Srkalovic et al. 1998; Grundker et al. 2002; Schally and Nagy 2004; Engel et al. 2007; Wilkinson et al. 2008) prompted efforts to use GnRH analogues for targeting cytotoxic agents as a novel form of cancer therapy. It was previously known that GnRH analogues with D-amino acids at position 6 could undergo substitution with a wide variety of chemical groups, including large groups such as proteins and colloidal gold (Jennes et al. 1983). These results suggest that this region of the peptide reaches into solvent while other portions of the peptide are responsible for receptor interactions (Struthers et al. 1990; Millar 2004). Schally et al. inferred from these structure activity relationships that a variety of small molecule cytotoxic groups could be attached to both agonist and antagonist analogues of GnRH peptides with maintenance of GnRHR binding activity (Janaky et al. 1992). Short-term treatment (24 h) of primary pituitary cell cultures with several of these analogues showed selective loss of LH content compared to other pituitary hormones growth hormone (GH) and prolactin (PRL), consistent with a selective effect on gonadotrophs (Rekasi et al. 1993). Additional optimization of the linker between DLys6-GnRH and doxorubicin or 2-pyrrolinodoxorubicin led to the identification of AN-152 and AN-207, respectively (Janaky et al. 1992; Nagy et al. 1993; Rekasi et al. 1993). Doxorubicin is a potent anthracycline antibiotic used in cancer chemotherapy that acts by intercalating DNA, and pyrrolinodoxorubicin is a more potent analogue. Both conjugates retain cytotoxic activity similar to free toxin as well as high-affinity GnRHR binding (Nagy et al. 1996).

A wide range of other groups have been conjugated to position 6 of GnRH analogues, including ferritin and colloidal gold (Jennes et al. 1983), methotrexate (Nagy et al. 1993), camptothecin (Dharap et al. 2003), anthraquinones (Lev-Goldman et al. 2006), iron oxide nanoparticles (Kumar et al. 2007), siRNA nanoparticles (Kim et al. 2008) and radiotherapeutic chelates (Guo et al. 2011a,b). Most studies with these analogues focused on their use in tumour models, and will not be summarized here. However, the effects of AN-207 on the reproductive endocrine axis in rats were examined in some detail (Kovacs et al. 1997, 2002). Regularly cycling female rats treated with a single IV injection of 150 nmol/kg AN-207 experienced interrupted reproductive cycling for 4–7 days, although 8/10 rats returned to normal cycling by 6 days following treatment. Ex vivo cultures of pituitaries isolated from these rats 1 week after treatment showed a selective 63% loss of GnRH-stimulated LH release from pituitary cells, but LH content remained at 73% of vehicle treated. Full gonadotroph function had recovered by 2 weeks. GH and thyroid-stimulating hormone (TSH) content in treated pituitaries was unchanged, indicating that effects were specific for gonadotrophs. In a subsequent study, male and female rats were treated with 175 nmol/kg (0.29 mg/kg; the maximum tolerated dose) resulting in a 39–51% decrease in pituitary GnRHR mRNA levels after 5 h, that returned to normal 1 week later (Kovacs et al. 2002). These data indicate that AN-207 at the doses tested was unable to kill the majority of gonadotrophs, but rather appeared to result in an acute injury to the gland.

It would appear likely that the therapeutic window may be limited by the poor stability of these conjugates. In mouse serum, the closely related AN-152 (which differs only in the structure of the doxorubicin toxin) has a half-life of only 19 min owing to enzymatic cleavage (Nagy et al. 2000). One would expect a similar half-life of AN-207 in rats, suggesting that the vast majority of conjugate is cleaved to non-specific toxin within the first hour following IV administration. It remains to be seen whether more complete gonadotroph ablation could be achieved with conjugates either employing more potent toxins or more stable targeting moieties.

GnRH-Protein Toxin Conjugates

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

GnRH peptides also have been used to target protein toxins to gonadotrophs. The best characterized of these are reported in a series of studies using GnRH analogues chemically conjugated to pokeweed antiviral protein (PAP) (Yang et al. 2003; Qi et al. 2004). Pokeweed antiviral protein is a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana with potent cytotoxic activity once it reaches the cytoplasm of a cell. The initial conjugate, [DLys6]GnRH-PAP, retained high GnRH receptor binding affinity (KI = 3 nm) and was selectively cytotoxic for GnRH receptor-expressing cells, although not particularly potent (EC50 = 300 nm in a clonogenic assay) (Yang et al. 2003). Subsequently, the carboxy-terminal Pro9-N ethylamide used in leuprolide was incorporated into the more potent and stable [DLys6, Pro9-NEt]GnRH for targeting of PAP (Qi et al. 2004). This conjugate showed improved cytotoxic potency in vitro (EC50 = 2 nm) but surprisingly was not used in subsequent in vivo studies.

Treatment of adult male dogs with [DLys6]GnRH-PAP (0.15 mg/kg infused IV over 36 h) resulted in suppression of serum LH and T for approximately 5 months (Sabeur et al. 2003) without impact on serum cortisol or thyroxin, suggesting selective impairment of gonadotroph function. However, during the period of effect on serum reproductive hormone levels, LH response to GnRH challenge was maintained, and testosterone levels did not achieve castrate levels (T < 0.5 ng/ml), indicating that the gonadotroph population, while impaired, was not ablated.

Subsequent studies in peripubertal dogs (Ball et al. 2006) showed that a higher-dose bolus (0.25 mg/kg SQ) had little effect on serum LH or T, but a second bolus 4 months later resulted in castrate T levels and reduced but clearly detectable LH levels. Although good suppression of the HPG axis was achieved, residual LH secretion indicates that complete gonadotroph ablation was not achieved.

Surprisingly, these suppressed animals then began to recover T levels 10 months following the second injection of conjugate. Although the mechanism of this recovery was not investigated, there appear to be two possible explanations. First, studies in mice suggest that a significant portion of the adult pituitary cell population is derived from adult stem cells (Gleiberman et al. 2008; Castinetti et al. 2011). It is possible that adult stem cells in these peripubertal dogs regenerated a sufficient population of gonadotrophs to restore testosterone biosynthesis. Alternatively, because ribosomal inactivating proteins are highly antigenic (Pastan et al. 2007; Onda et al. 2008), perhaps the conjugate acted as an effective immunogen for GnRH, and that immunoneutralization of endogenous GnRH contributed to efficacy. This is in part supported by the observation that two injections of the PAP conjugate were required for maximum inhibition of testosterone, analogous to many immunization protocols. It has previously been observed that administration of a GnRH-based vaccine to dogs results in inhibition of the reproductive endocrine axis, but that antibody titres decline and hormone levels return to pre-immunization levels after 3 months (Ladd et al. 1994). However, pituitary histology and anti-GnRH antibody levels were not reported in this study; therefore, the mechanistic basis of both loss and recovery of testosterone levels is unknown.

In both studies, arthralgia, pyrexia and injection site reactions were observed, although it was not discussed if these were dose limiting. As a result of the conjugation method, approximately 25–35% of the final preparation consisted of unconjugated PAP, which could be expected to contribute to non-specific toxicity. Similar to the doxorubicin conjugates, the clearance of intact [DLys6]GnRH-PAP in sheep is very rapid (t1/2  15’) (Yang et al. 2006), suggesting that significant amounts of untargeted toxin also may be formed in vivo. Once again, these data suggest that the ability to explore higher doses may be limited by non-specific toxicity possibly due to generation of free toxin from peptide conjugates.

There also are reports in the literature of chimeras between GnRH-like sequences and peptide or protein toxins. In one, a GnRH-like sequence (MEHWSYWLRPG) was genetically fused to a Pseudomonas exotoxin domain and expressed in bacteria (Nechushtan et al. 1997). Although the resulting protein showed cytotoxic activity in adenocarcinoma cells, there were no data presented to demonstrate that GnRH receptor binding was maintained. Rather, it is clear from the known structure activity relationships of GnRH peptides that the recombinant protein should be unable to bind GnRH receptors, because the amino terminal pyroglutamic acid is missing (and proceeded by a methionine), and the large carboxy-terminal extension is not consistent with the binding mode of GnRH (Struthers et al. 1990). Similarly, in a second approach, the GnRH sequence was placed at the amino-terminus of a chimeric peptide where the carboxy-terminal region was the lytic peptide, hecate (Leuschner et al. 2003a,b). Again, this design violates known GnRH receptor binding requirements by placing a large substitution on the carboxy terminus of the peptide which is required for receptor binding. Although some cytotoxic activity data were reported, no data were presented to demonstrate maintenance of receptor binding. Unfortunately, for both these constructs it is unlikely that the reported biological responses were GnRHR mediated.

Future Directions

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References

The results with GnRH-Dox and GnRH-PAP conjugates demonstrate that gonadotrophic cells can be selectively targeted using GnRH-toxin conjugates. However, at the doses tested, gonadotroph function was only partially impaired, indicating that at least a portion of the gonadotroph population remained. Only a very narrow range of doses was explored in these studies, because higher doses that may have exhibited greater anti-gonadotroph efficacy were precluded by systemic toxicities. For both the GnRH-Dox and GnRH-PAP conjugates, it would appear likely that the use of relatively unstable [DLys6]-GnRH-targeting sequences and the resulting short half-life of the targeting moiety in vivo could cause a high relative exposure to non-specific toxins. Therefore, it is important to determine if more stable analogues for targeting can improve the therapeutic window of these agents and allow complete ablation of the gonadotroph population.

Another important unanswered question is raised by the recovery of serum T levels after a sustained period (approximately 8 months) of castrate T levels following GnRH-PAP treatment (Ball et al. 2006). Immunohistochemistry to directly evaluate gonadotroph populations in the pituitary during the period of gonadal suppression and after recovery would be important to shed light on the underlying mechanisms. It will be critical to understand if the gonadotroph population can regenerate from a population of adult stem cells that do not express GnRH receptors. If so, then the prospects of GnRH-targeted gonadotroph ablation providing permanent sterilization would appear unlikely. However, it is also possible that the gonadotroph population was merely injured in these animals, and eventually recovered function. Alternatively, the residual differentiated gonadotrophs apparently present in these animals may have proliferative capacity. In these scenarios, more complete ablation of the gonadotroph population may result in more prolonged or permanent sterility. Regardless, understanding the underlying mechanisms resulting in gonadal suppression and recovery with these agents would be highly important to guide the future efforts of the field.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Background: GnRH and the GnRH Receptor
  5. GnRH-Drug Conjugates
  6. GnRH-Protein Toxin Conjugates
  7. Future Directions
  8. Acknowledgements
  9. Conflicts of interest
  10. References