Melphalan, alone or conjugated to an FSH-β peptide, kills murine testicular cells in vitro and transiently suppresses murine spermatogenesis in vivo

Abstract

New approaches to sterilizing male animals are needed to control captive and wild animal populations. We sought to develop a non-surgical method of permanent sterilization for male animals by administering the gonadotoxicant melphalan conjugated to peptides derived from the β-chain of follicle stimulating hormone (FSHβ). We hypothesized that conjugating melphalan to FSHβ peptides would magnify the gonadotoxic effects of melphalan while minimizing systemic toxicity. The ability of conjugates of melphalan and FSHβ peptides to kill murine testicular cells was first tested in vitro in a three-dimensional testicular cell co-culture system. In this system, melphalan caused considerable cell death as measured both by increases in LDH concentrations in the culture supernatant and direct visualization of the cultures. Of the conjugates tested, melphalan conjugated to a 20 amino acid peptide derived from human FSHβ consisting of amino acids 33-53 (FSHβ (33-53)-melphalan) was very potent, with cell cytotoxicity and LDH release roughly one-half that of melphalan. The effects of melphalan and FSHβ (33-53)-melphalan on spermatogenesis were then tested in vivo in mature C56Bl/6 male mice. Four weeks after intraperitoneal injection, all mice treated with either FSHβ (33-53)-melphalan or melphalan had ~75% reductions in testicular spermatid counts compared with control animals. Testicular histology revealed significant reduction in mature spermatids and spermatocytes in most tubules. However, 12 weeks after the injection, testicular spermatid counts and histology were similar to controls, except in one animal receiving FSHβ (33-53)-melphalan that had no apparent spermatogenesis. We conclude that melphalan and FSHβ (33-53)-melphalan are potent gonadotoxicants in male mice resulting in marked suppression of spermatogenesis 4 weeks after a single intraperitoneal injection. However, this effect is transient in most mice as spermatogenesis is similar to control animals 12 weeks after drug administration. Melphalan or FSHβ (33-53)-melphalan may be useful for the temporary control of fertility in male animals, but additional research will be needed to develop a single dose method of permanent sterilization for male animals.

Alkylating agents‐induced gonadotoxicity in prepubertal males: Insights on the clinical and preclinical front

“…Finally, gonadotoxic effects of melphalan in males when administered during puberty have only been explored in preclinical studies where it was shown to impair the viability of germ cells. 141,150 Nevertheless, it was reported to be gonadotoxic at doses above 140 mg/m 2 . 13 Similarly, Thio-TEPA.…”

“…No studies with patients treated during prepuberty/adolescence Germ cells: Reduction in viability. 150 Increase in oxidative stress; decrease in sperm numbers and motility. 141 Note:…”

Effective Delivery of Male Contraceptives Behind the Blood-Testis Barrier (BTB) – Lesson from Adjudin

“…Also, it requires parenteral administration, posing an acceptability obstacle, unless this mutant-adjudin conjugate can overcome the proteolysis of FSH polypeptide in the GI tract when the conjugate is orally administered, such as using the approach developed for oral delivery of insulin [86], including the use of nanoparticles containing absorption enhancers ( e.g., surfactants, zonula occludens toxins) and proteolytic enzyme inhibitors ( e.g., bacitracin, aprotinin, soybean trypsin inhibitor, polymer-inhibitor conjugates) [86–88]. A recent study had also used this approach to conjugate a permanent male contraceptive melphalan (also a cytotoxic and gonado-toxic nitrogen mustard alkylating agent that kills murine testicular cells) to FSH-ß peptide to be used to target melphalan to the testis for chemical sterilization in wild-life animals and also pets such as cats and dogs [89]. …”

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: An approach to non-surgical sterilization

“…Specifically, our aim was to harness the mouse model to develop targeting vectors for two specialized cell populations; the Sertoli cells that nurse differentiating male germ cells and form the protective blood-testis-barrier [ 16 ], and the androgen producing Leydig cells, that reside within the interstitial space [ 17 , 18 ]. Our approach builds on previous evidence that peptides have utility for selectively delivering cytotoxic agents to the gonadotrophic cells of the pituitary [ 19 , 20 ] or the Sertoli cells of the testes [ 21 , 22 ], whereupon they transiently disrupt male fertility. To extend this response to achieve a state of permanent sterility, we elected to target cytotoxic agents to the Sertoli and Leydig cell populations concurrently, with the ultimate goal of ablating a sufficient number of these cells to permanently disrupt spermatogenesis.…”