Research articleMelphalan, alone or conjugated to an FSH-β peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo
Introduction
The most common method of sterilizing male animals relies on removal of the testes, which requires skilled personnel and can cause significant postprocedural pain [1]. Removal of the testes also deprives the animal of testosterone, which may lead to health and behavioral problems [2], and increase the risk of prostate cancer [3], [4], [5] or osteoporosis [2], [6] later in life. An alternative approach to male sterilization is the injection of a solution of calcium chloride [7] or zinc gluconate [8] directly into the testes. These injections are very effective from a contraceptive standpoint; however, severe injection site reactions requiring surgery can occur in up to 4% of recipients [9], with others experiencing scrotal ulceration or dermatitis [10]. Therefore, there is a need for novel methods of permanent sterilization for male animals. Ideally, such a method would be inexpensive and administered with a single injection.
Several investigators have examined the potential of gonadotoxicants for the purposes of male sterilization. For example, ketoconazole [11], alpha-chlorohydrin [12], and emblein [13] have been tested in animals. However, the suppression of spermatogenesis was incomplete, and systemic toxicity was limited [14]. We sought to test a novel approach to male sterilization using the potent gonadotoxicant melphalan. Melphalan, an alkylating agent, is widely used in the treatment of lymphomas and multiple myeloma in both humans and animals. Infertility is frequently seen after administration of melphalan [15], [16], and melphalan is fairly unique among gonadotoxicants in that it kills both spermatogonial stem cells and dividing germ cells in vivo [17]. Melphalan has been used extensively in animals for the treatment of malignancies [18], [19], and its pharmacokinetics and marrow toxicity are well described [20], [21]. Despite its widespread use in animals, the effect of melphalan on spermatogenesis in animals has not been well studied. We sought to: (1) determine if melphalan could be used to induce sterility, and (2) investigate if we could facilitate melphalan effect and minimize any toxicity by targeting the melphalan specifically to the testes via conjugation to peptides derived from the β-chain of human FSH-β.
FSH is a protein hormone produced in the anterior pituitary. FSH binds to the FSH receptor (FSHr) on Sertoli cells within the seminiferous tubules of the testes [22], stimulating them to nurture the developing germ cells. The FSHα chain is not required for binding to the FSHr, but the FSHβ chain specifically binds to the FSHr with high affinity and is required for receptor activation [23]. Because of its high expression in the gonads and very low levels of expression in other tissues, FSHr provides an elegant targeting mechanism for drug delivery to the gonads. Indeed, FSHβ has been shown to be an effective gonad-specific drug delivery vehicle for experimental forms of reversible contraception in mice. In one such study, the contraceptive efficacy of adjudin was increased 10,000-fold by conjugation to FSHβ [24]. In another example, FSHβ conjugated to a peptide that interrupted the integrity of the blood–testes barrier caused significant loss of germ cells and a decrease in fertility [25].
Use of the entire FSHβ molecule for targeting, however, is impractical for widespread use because of the expense of either the chemical synthesis or recombinant production of the entire FSHβ chain, which is 112 amino acids in length. Fortunately, FSHβ-derived peptides that bind to the FSHr with high affinity have been described [26], [27], [28], [29], [30]. The most attractive candidates for drug delivery are peptides based on the amino acids in positions 33 to 53 and 81 to 95 of FSHβ [27], [28]. Each of these peptides sequences has good in vitro FSHreceptor-binding inhibition and demonstrated in vivo efficacy. Furthermore, there is good recognition of these conserved peptides by the FSHr across species [27], [31]. Peptides with terminal modifications and substitutions of serine for cysteine increase FSHr binding, as measured by competitive inhibition studies [26], [30]. These peptides have been used to target drugs to the gonadal tissue. Both peptides specifically targeted ovarian cancer cells with nanoparticles [32], [33]. Therefore, we hypothesized that the conjugation of FSHβ-derived peptides to the gonadotoxicant melphalan would allow for sterilization of male animals while minimizing the side effects induced by high-dose melphalan administered alone. We tested melphalan and FSHβ-melphalan conjugates in vitro and in vivo for their ability to kill mouse testicular cells and thereby induce sterility.
Section snippets
Melphalan and FSHβ-melphalan conjugate preparation
Melphalan was obtained commercially (Sigma, St. Louis, MO, USA). Two different FSHβ-derived peptide sequence residues β33 to 53 and β81 to 95 were employed as the carriers for melphalan. The melphalan was conjugated to either the amino or carboxy terminus of each peptide, with or without the presence of terminal modifications and addition or deletion of terminal amino acids. The molecular weights and FSHβ-amino acid composition of the 16 conjugates tested are listed in Table 1. FSHβ-melphalan
In vitro cytotoxicity testing of melphalan and FSHβ-melphalan conjugates
The results of the cytotoxicity testing of melphalan and the FSHβ-melphalan conjugates (Table 1), as demonstrated by the release of LDH over time, are summarized in Figure 1. Good cytotoxicity was observed when the N-terminal acetate was included with the β33 to 53 peptide C-terminal conjugate F. The β33 to 53 peptide lacking an N-terminal acetate was less effective (C, D) than conjugate F. There was also less cytotoxicity when the melphalan was placed on the N-terminus of the β33 to 53 peptide
Discussion
Herein, we report on our efforts to develop a method of permanently sterilizing male animals by the use of a gonadotoxicant, melphalan, alone and conjugated to a peptide derived from FSHβ intended to specifically target the melphalan to the testes. Our extensive in vitro work resulted in the identification of a conjugate consisting of a 20-amino acid peptide and melphalan (conjugate F) that exhibited significant cytotoxicity in vitro that appeared to be more specific for testicular cells as
Acknowledgments
The authors would like to thank Erin Pagel MA for assistance with the sperm analysis and David W. Amory MD, PhD for thoughtful review of the article. This work was funded by the University of Washington Royalty Research Fund, and in part, by the Eunice Kennedy Shriver National Institute of Child Health and Human Development through cooperative agreement U54 HD-42454 as part of the Cooperative Contraceptive Research Centers Program and funds from Focused Scientific, Inc. as well as Grants 1 U01
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