Adjudin - A Male Contraceptive with Other Biological Activities

Abstract

Background

Adjudin has been explored as a male contraceptive for the last 15 years since its initial synthesis in the late 1990s. More than 50 papers have been published and listed in PubMed in which its mechanism that induces exfoliation of germ cells from the seminiferous epithelium, such as its effects on actin microfilaments at the apical ES (ectoplasmic specialization, a testis-specific actin-rich anchoring junction) has been delineated.

Objective

Recent studies have demonstrated that, besides its activity to induce germ cell exfoliation from the seminiferous epithelium to cause reversible infertility in male rodents, adjudin possesses other biological activities, which include anti-cancer, anti-inflammation in the brain, and anti-ototoxicity induced by gentamicin in rodents. Results of these findings likely spark the interest of investigators to explore other medical use of this and other indazole-based compounds, possibly mediated by the signaling pathway(s) in the mitochondria of mammalian cells following treatment with adjudin. In this review, we carefully evaluate these recent findings.

Methods

Papers published and listed at www.pubmed.org and patents pertinent to adjudin and its related compounds were searched. Findings were reviewed and critically evaluated, and summarized herein.

Results

Adjudin is a novel compound that possesses anti-spermatogenetic activity. Furthermore, it possesses anti-cancer, anti-inflammation, anti-neurodegeneration, and anti-ototoxicity activities based on studies using different in vitro and in vivo models. Conclusion: Studies on adjudin should be expanded to better understand its biological activities so that it can become a useful drug for treatment of other ailments besides serving as a male contraceptive.

INTRODUCTION

Adjudin, 1-(2, 4-dichlorobenzyl)-1H-indazole-3-carbohydrazide, formerly called AF-2364 Fig. (1), is an analog of lonidamine. Lonidamine and related compounds such as tolnidamine [1] belong to a class of indazole-based compounds synthesized in the 1970s as part of the effort of developing innovative anti-cancer drugs, which were later found to have potent antispermatogenic activity [2, 3]. Unlike other anti-cancer drugs which are mostly anti-mitotic by targeting actively dividing tumor cells, lonidamine and its related compounds are not anti-mitotic, non-cytotoxic, and have no effects on cellular nucleic acids or protein synthesis [4–6]. However, lonidamine and its related compounds (e.g., tolnidamine) are mitochondrial hexokinase inhibitors (also known as glycolytic inhibitors), by impairing the energy metabolism of neoplastic cells [7–9], impairing mitochondrial function leading to mitochondria-mediated apoptosis in tumor cells [6, 10–13]. Specifically, lonidamine binds to adenine nucleotide translocator (ANT) in mitochondria, triggering mitochondrial membrane permeabilization (MMP) that leads to cancer cell apoptosis [6, 11–13]. Lonidamine also blocks lactate transport by inhibiting lactate efflux in cancer cells, and such lactate accumulation leads to intracellular acidification, thereby reducing cell proliferation, migration and invasion, and promoting apoptotic cell death in tumor cells [14–17]. It is noted that lonidamine also induces Sertoli cell injury in the testis, thereby inducing germ cell exfoliation, when administered by oral gavage in rodents [18]. In an effort to capture the anti-spermatogenic effects of lonidamine based on its indazole ring, we had synthesized a panel of analogs and derivatives of lonidamine and other indazole-based compounds to identify potential male contraceptives without the toxicity of lonidamine [19]. Adjudin was selected from more than two-dozen lonidamine analogs and derivatives that were synthesized in the 1990s. The screening was based on an in vivo assay developed in our laboratory [19] to select an indazole-based compound that could disrupt spermatid adhesion in the mammalian testis, which could serve as a potential non-hormonal and reversible male contraceptive. Since other germ cell types, in particular undifferentiated spermatogonia are not perturbed following treatment of adult male rats with adjudin [20], the adjudin-induced infertility is transient, this is because undifferentiated spermatogonia can repopulate the germ cell-depleted seminiferous epithelium in tubules via spermatogenesis, making adjudin a reversible male contraceptive. This assay was developed based on the observation that the level of testin, a signaling/structural molecule at the Sertoli-spermatid interface which is highly expressed in the testis [21], was transiently up-regulated, usually within 4–6 hr after treatment, when the adhesion of spermatids in the seminiferous epithelium was disrupted [19]. It was noted that if the testin level remained elevated well past -6 hr, the chemical entity usually associated with acute toxicity [19, 22–24]. Adjudin was selected based on this assay because it induced a drastic but transient up-regulation on the expression of testin in the testis following treatment, usually peaked within a day vs. control rats and returned to the basal level rapidly based on Northern blot and/or immunoblot analysis [19, 24], consistent with subsequent gene profiling analysis [25]. Herein, we focus our discussion on adjudin, however, we also provide a brief account on lonidamine and other structurally related compound since findings from these other chemical entities are helpful to better understand adjudin. Also, we do not intend to provide detailed molecular mechanistic insights on the other biological activities of adjudin but a brief summary of these recent findings since the intention of this brief review is to provide the latest update regarding some ongoing studies in the field. Furthermore, additional studies are needed to better explore the mechanistic insights pertinent to other biological activities of adjudin besides its anti-fertility effects in male rodents.

Fig. (1).

Structural formulae of adjudin, lonidamine and other related indazole-based compounds.

ADJUDIN IS A POTENTIAL MALE CONTRACEPTIVE

As noted above, adjudin has been explored to serve as a male contraceptive since its synthesis in the early 1980s [19, 26, 27]. Studies have shown that adjudin exerts its effects by perturbing the apical ectoplasmic specialization (apical ES) function at the Sertoli-spermatid interface, a testis-specific actin-rich anchoring junction [22, 28], altering the function of adhesion protein complexes at the site, causing exfoliation of germ cells, initially elongating/elongated spermatids, to be followed by round spermatids and spermatocytes, but not spermatogonia [20, 26, 28]. During the epithelial cycle, the integrity of apical ES is notably supported by an array of actin microfilament bundles that lie perpendicular to the Sertoli cell plasma membrane, which are sandwiched in-between the Sertoli cell plasma membrane and the cisternae of endoplasmic reticulum in the Sertoli cell [29, 30]. In this context, it is of interest to note that a similar structural feature, namely the array of actin filament bundles, is not found in the apposing step 8–19 spermatid at the apical ES. Due to this array of bundles of actin microfilaments at the ES, study has shown that apical ES confers stronger adhesion vs. desmosome [31], the intermediate filament-based anchoring junction in the skin known to confer one of the strongest adhesion junctions [32, 33]. However, in order for spermatids to be transported across the adluminal compartment of the seminiferous epithelium during the epithelial cycle so that fully developed spermatids (i.e., spermatozoa) can be lined up near the tubule lumen to prepare for their eventual release at spermiation, these actin microfilament bundles must be rapidly re-organized via conversion between a “bundled” and “unbundled/branched” configuration to support spermatid transport until at stage VIII when spermiation takes place [30, 34]. Studies have shown this can be achieved via spatiotemporal expression of actin barbed end capping and bundling protein Eps8, and other actin bundling proteins such as palladin and ezrin vs. branched actin polymerization proteins such as the Arp2/3 (actin related protein 2/3) complex at the apical ES during the epithelial cycle [30, 35–37]. As such, actin bundles can be rapidly converted between a bundled and branched/unbundled configuration to confer plasticity to the apical ES to facilitate spermatid transport. Recent reports have shown that adjudin exerts its effects by altering the spatiotemporal expression of actin binding proteins, such as Eps8 and Arp3 [38, 39], at the apical ES, thereby actin microfilament bundles fail to be re-organized from “bundled” to “unbundled/branched” configuration and vice versa in response to stages of the epithelial cycle to support spermatid transport and adhesion. This thus causes exfoliation of spermatids, inducing infertility in male rats [28, 40] Fig. (2). Changes in the spatiotemporal expression of Arp3, Eps8 and other actin binding/regulatory proteins can possibly be regulated by p-FAK-Tyr³⁹⁷ and p-FAK-Tyr⁴⁰⁷, since studies have shown that these signaling molecules regulate actin microfilament organization at the ES based on studies in vitro and in vivo, and these proteins also display restrictive spatiotemporal expression at the apical ES during the epithelial cycle [41, 42]. It is noted that adjudin received a US6001865 patent granted on December, 1999. However, there are many advances in the field regarding the contraceptive use of other analogs of lonidmaine and indazole-based derivatives such as H2-gamendazole as well as other medical use such as for treatment of multiple diseases including cancer, chronic and inflammatory disorders and others (see Table 1). In this section, we have briefly reviewed the latest updates on the likely mechanism of action of adjudin in the testis to perturb spermatogenesis, leading to transient male infertility. In the sections that follow, we also review other biological activities of adjudin illustrating the added health benefits of adjudin. Perhaps, these other biological activities of adjudin should be better explored in future studies. We first provide a brief update on the anti-cancer characteristics of lonidamine in the next section since adjudin is a close cousin of lonidamine.

Fig. (2).

The biological effects of adjudin including its anti-fertility, anti-cancer, and neuroprotective and hearing-protective activities.

Table 1.

Some Recent Patents (2013 - Present) on lonidamine, Lonidamine, Adjudin and Related Indazole-Based Compounds Concerning their Medical Use^*.

Patent Number (Date of Approval)	Patent Title	Inventor(s)	Summary of Invention	Reference
US8980934 (03/17/2015)	Kinase inhibitors and method of treating cancer with same	Pauls, H.W., Laufer, R., Li, S.W., Ng, G.	An indazole-based compound or its analogs that shares the structure of lonidamine, which acts as a protein kinase inhibitor for cancer treatment including the pharmaceutical compositions and methods of use	[101]
US8377958 (02/19/2013)	Lonidamine analogues for fertility management	Georg, I.G., Tash, J.S., Chakrasali, R., Jakkaraj, S.R., Roby, K.	Lonidamine analogues in particular H2-gamendazole can be used as a reversible or irreversible male contraceptive. These compounds also affect female ovarian function, and reducing female progesterone serum level	[102]
US8791094 (07/29/2014)	Treatment of prostate cancer	Morrison, J.P., Stein, C.A., Casebier, D.S.	Methods of making, pharmaceutical composition, and methods of using indazole-based compounds that are inhibitors of cytochrome C17α-hydroxylase/C17,20-lyase to treat androgen receptor mediated diseases or conditions in particular prostate cancer	[103]
US8748633 (06/10/2014)	Selective androgen receptor modulators (SARMS) and uses thereof	Zhi, L.	Indazole-based compounds that bear similar structure as of lonidamine which also bind to androgen receptors and/or modulate activity of androgen receptors. Also included are methods of making and using such compounds	[104]
US8673948 (03/18/2014)	Chemical compounds	Turnbull, P.S., Cadilla, R., Larkin, A., Zhou, H., Stewart, E.L., Stetson, K., McDougald, D.L., Randhawa, A.S., Ray, J.A.	The methods for the making and use of non-steroidal indazole-based compounds as modulators of androgen receptor	[105]
US9056857 (06/16/2015)	Methods for treating or preventing cancer and neurodegenerative diseases	Zhu, L., Li, Y.	Methods of treating or preventing a neurodegenerative disease by treating a subject having a neurodegenerative disease based on an indazole-based compound	[106]
US9000028 (04/07/2015)	Indole and indazole compounds as an inhibitor of cellular necrosis	Kim, S.H., Kim, H.J., Koo, S.Y., Chung, C.W., Lee, S.B., Park, H.S., Yoon, S.H., Kwak, H.S., Seo, D.O., Park, E.	Using pharmaceutically acceptable salts or isomers of indole or indazole compounds for the prevention or treatment of cellular necrosis and necrosis-associated diseases by inhibiting cellular necrosis	[107]
US9090613 (07/28/2015) and US8987298 (03/24/2015)	Indazole inhibitors of the Wnt signal pathway and therapeutic uses thereof	Hood, J., Wallace, M., Sunil, K.K.C.	Use of an indazole compound or analogs of indazole compounds in the treatment of disorders, such as cancer, abnormal cellular proliferation, angiogenesis, Alzheimer’s disease and osteoarthritis, characterized by (i) the activation of and/or (ii) modulation of cellular events of Wnt signal pathway, as well as genetic diseases and neurological conditions/disorders/diseases due to mutations in one or more components of Wnt signal pathway or dysregulation of the Wnt signal pathway	[108, 109]
US8987275 (03/24/2015)	Indazole compounds and their uses	Gray, N., Zhou, W., Deng, X.	Therapeutic use of indazole-based compounds including compositions comprising an effect amount of such a compound, and methods for treating and preventing multiple diseases	[110]
NZ596071 (12/20/2013)	Oxazole substituted indazoles as PI3-kinase inhibitors	Mitchell, C.J., Keeling, S.E., Parr, N.J., Jones, P.S., Hamblin, J.N., Le, J.	Oxazole substituted indazole-based compounds served as PI3-kinase inhibitors for the treatment of diseases mediated by inappropriate PI3-kinase activity, such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and others	[111]
US8362031 (01/29/2013)	Lonidamine analogues and treatment of polycystic kidney disease	Georg, I.G., Tash, J.S., Chakrasali, R., Jakkaraj, S.R., Calvet, J.P.	Lonidamine derivatives are effective compounds to treat, inhibit, and/or prevent polycystic kidney disease (PKD). These compounds exert their effects by inhibiting ErbB2, Src, Raf-1, B-Raf, MEK, Cdk4 and NKCC1 or a combination of these biomolecules and/or kinases	[112]
US8927579 (01/06/2015)	Male contraceptive	Amobi, N.M.I., Smith, C.	A series of indazole-based compounds for reduction or prevention of the production of sperm and the transmission of viral agents transmitted in seminal fluid	[113]
WO2015044072 (04/02/2015)	Indol and indazol derivatives	Ballard, T.M., Groebke, Z.K., Pinard, E., Ryckmans, T., Schaffhauser, H.	The use of indole and indazole derivatives for the treatment of prophylaxis of Alzheimer’s disease, cognitive impairment, schizophrenia, pain or sleep disorders	[114]
US9056129 (06/16/2015)	Precision-guided nanoparticle systems for drug delivery	Hanson, R.N., Amiji, M., Weissig, V.	A method preparing multifunctionalized nanoparticles (applicable to lonidamine or indazole-based compounds) so that drugs in the nanoparticles can be delivered to a desired cellular target to exert their intended effects for treatment of diseases such as neoplastic, infectious and chronic diseases	[115]

^*This Table contains information on patents pertinent to lonidamine, adjudin, and other indazole-based and related compounds, and also concerning their use for male contraception, and for treatment of cancers, inflammatory and chronic diseases, neurodegenerative diseases and kidney disorders. This information also supports recent findings on adjudin (and lonidamine) in particular other potential health benefits besides adjudin’s male contraceptive activity as discussed in this review.

LONIDAMINE, A DERIVATIVE OF INDAZOLE-CARBOXYLIC ACID AND AN ANALOG OF ADJUDIN, IS AN ANTI-CANCER DRUG

Lonidamine, 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid shown in Fig. (1), is an anti-cancer drug using for different types of cancer including brain tumors, breast cancer, ovarian and lung cancer, including late stage cancer [6, 15, 43, 44]. Studies have shown that other similar analogs and/or derivatives of lonidamine also possess anti-cancer activities as noted in Table 1. Unlike many anticancer drugs, lonidamine, however, is not anti-mitotic [6, 8, 45, 46]. It was also shown to possess potent antispermatogenic activity by causing Sertoli cell injury due to the presence of the indazole ring in its chemical structure [4] as noted in Fig. (1). Lonidamine is an inhibitor of mitochondrial hexokinase 2, the predominant isoform of hexokinase found in cancer cells that is capable of phosphorylating hexoses by acting as a glycolytic inhibitor [7–9]. Thus, lonidamine effectively blocks the formation of glucose-6-phosphate from glucose - the initial step of glycolysis in mitochondria. This thus disrupts energy metabolism in cancer cells since mitochondrial hexokinase 2, especially in cells in advanced neoplasms, support cell growth by enhancing aerobic glycolysis [47], which is currently considered to be one of the major mechanisms of action of lonidamine. In this context, it is of interest to note that in normal mammalian cells, hexokinase I is predominantly used for glucose metabolism, and hexokinase 2 is restricted to rapidly dividing cells with condensed mitochondria such as those found in cancer cells in particular after they are sensitized by radiation [6, 8, 45, 48, 49]. Lonidamine also disrupts lactate transport, causing lactate accumulation in tumor cells, this thus leads to intracellular acidification which, in turn, induces tumor cell death [6, 14, 49]. Interestingly, lonidamine does not affect respiration, mitochondrial electron transport and glycolysis in Sertoli cells [50], perhaps due to the absence of condensed mitochondria in Sertoli cells. Lonidamine does disrupt the Sertoli cell cytoskeleton, affecting Sertoli cell actin microfilaments, intermediate filaments, and microtubules, and a disruption of these cytoskeletons thus leads to germ cell exfoliation as a result of Sertoli cell injury [18].

By contrast, all germ cells, unlike Sertoli cells, in particular post-meiotic spermatids, except spermatogonial stem cells, spermatogonia, and preleptotene spermatocytes, contain condensed mitochondria [51]. Thus the structural features of mitochondria in post-meiotic spermatids are similar to tumor cells. Unlike somatic or neoplastic cells, germ cells, such as spermatocytes and spermatids, do not utilize glucose and/or hexose; instead, they rely on lactate generated by Sertoli cells to serve as their energy source [52, 53]. Interestingly, in order to accommodate the physiological needs of germ cells, Sertoli cells also metabolize glucose preferably to lactate instead of pyruvate to enter the Krebs cycle to maximize the production of ATPs [54, 55]. In short, Sertoli cells are similar to cancer cells as noted in their energy management strategy by producing lactate from glucose [56]. Germ cells obtain lactate from Sertoli cells in the testis via their integral membrane lactate transporter MCT2 (monocarboxylate transporter 2). Once lactate enters a germ cell, it will be converted to pyruvate by a germ cell-specific lactate dehydrogenase (LDH) called LDHX, also known as LDHC4. Pyruvate then enters the Krebs cycle to produce ATPs to sustain germ cell development [53]. The effects of lonidamine on germ cells, in particular late spermatocytes and post-meiotic spermatids, are most obvious within the condensed mitochondrion, since lonidamine was shown to induce mitochondrial membrane swelling and also mitochondrial degeneration [57]. A significant reduction in oxygen consumption and a remarkable decline in ATP production by germ cells, but not by liver cells, are also evident after lonidamine exposure [57]. Other changes include the vacuolization of round spermatid nuclei and the formation to giant multinucleated round spermatids following lonidamine treatment have also been reported [57].

As noted above, Sertoli cells prefer to metabolize glucose to pyruvate, and pyruvate to lactate, instead of having pyruvate to enter the Krebs cycle to produce ATPs more effectively, almost analogous to tumor cells [53, 58]. In fact, the production of lactate via anaerobic glycolysis in Sertoli cells is significantly higher than that of normal differentiated or tumor cells such as Ehrlich ascites tumor cells [50]. In the testis, Sertoli cell-produced lactate is pumped out via the monocarboxylate transporter 1 (MCT1) and uptaken by meiotic and post-meiotic germ cells via the MCT2 [53]. Tumor cells generate energy by metabolizing glucose via anaerobic glycolysis even when oxygen is present in the environment [59, 60], which essentially yields two pyruvate molecules and two adenosine triphosphates (ATP) from a single glucose molecule; pyruvate is then metabolized to lactate. This phenomenon, the hallmark of tumor cell metabolism, is known as the Warburg effect [61, 62], and it explains how tumor cells maintain their active mitotic activity when there is a shortage of oxygen (i.e., hypoxia) because they are usually located away from the microvasculature during initial tumorigenesis [63]. Although Sertoli cells have access to oxygen from the microvasculature in the testis, they also produce pyruvate and lactate via anaerobic glycolysis, but supply lactate to developing germ cells [53], instead of putting pyruvate through the Krebs cycle that requires oxygen consumption. As each Sertoli cell requires to nurture ~30–50 developing germ cells at different stages of development [64], germ cell nourishment is no small task. Besides serving as the substrate for the production of ATP through mitochondrial respiration via the Krebs cycle, studies have shown that lactate also regulates the reactive oxygen species (ROS) level in germ cells [65], sperm motility, protein tyrosine phosphorylation during capacitation, and fertilization [66].

Lonidamine is most effective as an antineoplastic drug when tumor cells are sensitized by radiation, which generates more condensed mitochondria, and in combination with other chemotherapeutic drugs, such as paclitaxel, which damages the plasma membrane and condensed mitochondrial membrane in tumor cells, causing mitochondrial degeneration and tumor cell death [6, 48, 67]. Adjudin has also been shown to possess anticancer activity in a mouse model as well as tumor cells cultured in vitro. For example, adjudin has been reported to reduce lung and prostate tumor cell growth in vivo after A549 (adenocarcinomia human alveolar basal epithelial cells) or PC3 (human prostate adenocarcinoma cells) cells were subcutaneously injected into adult nude mice [68]. Furthermore, adjudin has also been shown to possess anti-inflammatory activity in a brain model [69], indicating that it might alleviate symptoms associating with neurodegenerative diseases. However, these results must be interpreted with caution because outcomes from mouse models do not reliably predict outcomes in humans. Furthermore, most types of cancer and neurodegeneration have a strong environmental and/or behavioral underpinning, and they usually occur in aged individuals. Most mouse models that study cancer or neuro-degeneration do not mimic these features in particular humans. Below is a more detailed discussion on the other biological activities of adjudin.

It is also noted that when used at identical doses, adjudin is more potent than lonidamine and less toxic based on results of acute and subchronic toxicity tests conducted by licensed toxicologists [70], even though they share a common chemical structure of indazole. Comparison between adjudin and lonidamine perhaps are similar to salicyclic acid and aspirin. Salicyclic acid is a highly toxic active ingredient of willow bark leaves, but aspirin is acetylated salicyclic acid prepared in 1897 by Felix Hoffman in 1897 at Bayer AG, less toxic, and aspirin subsequently used to treat pain, fever, and inflammation, and remains one of the most widely used medicines with multiple biological activities [71–73]. Different lonidamine analogs currently under development by others (e.g., gamendazole) [74, 75] and our laboratory (e.g., adjudin) [22, 28] may prove to be viable non-hormonal male contraceptives with other added health benefits. In the following sections, we summarize relevant findings on adjudin in particular its other biological activities besides its anti-fertility activity as recently reviewed [27, 28].

BIOLOGICAL ACTIVITIES OF ADJUDIN IN ADDITION TO ITS ANTI-FERTILITY EFFECTS IN THE TESTIS

Anti-Cancer Activity

Since adjudin is an analog of the anticancer drug lonidamine, it is not surprising that it possesses anticancer activity. Studies in vitro using human cancer cell lines including SGC-7901 cells (human gastric adenocarcinoma cells), MDA-MB-231 cells (human breast adenocarcinoma cells), Smmc-7721 cells (human hepatoma cells), and MIA PaCa-2 cells (human pancreatic adenocarcinoma cells) have shown that adjudin is capable of inhibiting cell proliferation dose-dependently with an IC₅₀ of 58, 13.8, 72.3 and 52.7μM, respectively, following treatment of 24hr [68]. Furthermore, the potency of adjudin in blocking cancer cell proliferation is ~2–3 fold better than lonidamine when the IC₅₀ of adjudin vs. lonidamine are compared in A549 cells (human lung adenocarcinoma cells) and PC3 (human prostate cancer cells) cells: 63.1 and 93 μM for adjudin vs. 200 and 120μM for lonidamine, respectively [68]. Adjudin also induced cell apoptosis of A549 and PC3 cancer cells via the caspase 3-dependent apoptotic pathway, perturbing mitochondrial function, thereby reducing intracellular ATP levels in these cancer cells [68] noted in Fig. (2). Adjudin apparently exerts its anti-cancer effects by triggering mitochondrial dysfunction via a loss of mitochondrial membrane potential, serving as a hexokinase 2 inhibitor by reducing cellular ATP level [68]. Adjudin also induces cancer cell apoptosis via the caspase-3-dependent pathway [68]. More important, the anti-cancer activity of adjudin noted in vitro has also been shown in studies in vivo. For instance, when adult nude mice were inoculated subcutaneously with A549 cells and PC3 cells to induce tumor up to a size of ~6-mm in diameter in 2-wk, an administration of adjudin via i.p. at 100mg/kg b.w. (note: to obtain effective contraceptive dose in male rats, two weekly dose of 50mg/kg b.w. is required [22]) with ~6 doses in 2-wk, the tumor size was shrunk considerably, by as much as ~40–50% in ~10 days following adjudin treatment [68]. These findings thus illustrate the added health benefits of adjudin when used as a male contraceptive, in which adjudin may be able to suppress cancer growth and/or cancer development in men taking adjudin as a contraceptive. Nonetheless, the mechanism(s) by which adjudin disrupts cancer metabolism and induces cancer apoptosis must be better delineated in future studies.

Anti-Neuroinflammatory Activity

Microglia cells are resident macrophages in the central nervous system (CNS) which play an important role in brain function including innate immunity, brain development, repair and also etiology of CNS disorders such as Alzheimer’s disease [76–79]. Studies have also shown that activation of microglia that leads to chronic inflammation is closely related to neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, dementia and multiple sclerosis [80–83]. Indazole-based compounds, such as bindarit, and benzydamine, some structurally related compounds to lonidamine and adjudin, have also been shown to possess anti-inflammatory activities, acting as non-steroidal anti-inflammatory drugs (NSAID) to relief some pathological conditions [84–91]. Using a murine BV2 microglia cell line that mimics primary microglia and is widely used to study microglial biology in Alzheimer’s disease [92], adjudin was shown to inhibit LPS (lipopolysaccharide)-induced microglia activation [69], the hallmark of microglia-mediated inflammation in the brain. For instance, adjudin considerably inhibited LPS-mediated IL-6 release and also the expression of IL-6, IL-1β, and TNFα in BV2 microglia cells [69]. More important, using a permanent middle cerebral artery occlusion (pMCAO) in vivo mouse model that closely mimics focal cerebral ischemia [93] by reducing the cerebral blood flow with ~10% of normal mouse to induce ischemia, adjudin was found to significantly attenuate ischemia-induced microglia activation [69]. For instance, it was noted that after pMCAO, the expression of CD11b (also known as integrin αM chain), an indicator of microglia activation in various CNS diseases [80], was significantly up-regulated when frozen sections of pMCAO-induced ischemic cerebral cortex and striatum vs. controls (sham operated+vehicle control, and sham operated + adjudin treated rats) were examined by immunofluorescence analysis using an anti-CD11b antibody, and adjudin treatment (50mg/kg b.w. by i.p.) 2hr before ischemia reduced CD11b expression [69]. Furthermore, this anti-inflammatory activity in microglia cells is mediated by inhibiting NF-κB activation and ERK MAPK (but not p38 or JNK) phosphorylation [69]. Adjudin also reduced ischemia-induced brain edema [69]. More important, adjudin treated rats displayed behavioral improvement at 72hr after ischemia [69]. Subsequent studies have shown that adjudin likely exerts its effects by preventing blood-brain barrier disruption after ischemia, and also by reducing the expression and the release of TNFα, IL-1β and IL-6 in the brain based on the use of a tMCAO (transient MCAO) model [94]. Collectively, these findings illustrate the anti-neuroinflammatory activity of adjudin in the CNS during ischemia-mediated brain damage, suggesting neuroprotective function of adjudin as noted in Fig. (2).

Otoprotective Activity in Cochlea Hair Cells

In mammals, hair cells in the cochlea of the ear convert sound signals into electrical impulses [95, 96]. However, degeneration of hair cells due to aging, acoustic trauma and exposure to aminoglycoside antibiotics (e.g., gentamicin is known to cause ototoxicity in ~5–10% of the patients when given intravenously or during peritoneal dialysis in humans) are generally irreversible [97, 98]. However, a recent study using rat cochlear explant cultures and cochlear primary cell cultures subjected to gentamicin-induced ototoxicity, adjudin has shown to offer otoprotection by up-regulating mitochondrial Sirt3, a member of the Sirtuin family protein known to regulate reactive oxygen species (ROS) production [99]. In short, adjudin protects hair cells from gentamicin-mediated apoptosis [99]. Treatment of hair cells with adjudin also induces a surge in mitochondrial Sirt3 expression, concomitant with a significant reduction in ROS level [99], illustrating adjudin exerts its otoprotection via the mitochondrial Sirt3-ROS pathway Fig. (2). These findings are consistent with an earlier report illustrating that Sirt3 mediates reduction of oxidative damage by reducing ROS production in the inner ear, and promoting hair cell survival, thereby preventing age-related hearing loss following caloric restriction using a Sirt3^-/− mouse model [100]. The otoprotective effects of adjudin on cochlea hair cells are further confirmed by overexpressing Sirt3 using lentivirus (lenti-Sirt3) in cochlear explant cultures, illustrating that Sirt3 overexpression in cochleae indeed can rescue gentamicin-induced hair cell loss, analogous to adjudin treatment [99]. Furthermore, using an in vivo gentamicin injury mouse model combined with hearing function tests including Auditory Brainstem Response (ABR) and Compound Action Potential (CAP) tests, adjudin was shown to have significant otoprotective effects on gentamicin-induced hearing loss in mice [99]. Adjudin was also shown to reduce hair cell loss from cochleae in gentamicin-treated mice in this mouse model, likely by blocking cell apoptosis induced by gentamicin [99]. In short, adjudin is an otoprotective drug.

CURRENT & FUTURE DEVELOPMENTS

As briefly reviewed herein, adjudin is a male contraceptive that exerts its effects at the testis-specific Sertoli-spermatid interface known as apical ES. Adjudin alters the spatiotemporal expression of actin regulatory proteins such as actin barbed end capping and bundling protein Eps8, and branched actin inducing protein Arp3, thereby perturbing the proper organization of actin microfilament bundles at the apical ES, leading to germ cell exfoliation from the seminiferous epithelium. In this context, it is of interest to note that adjudin has been subjected to a battery of standard toxicity studies, including its acute toxicity tests performed in both mice and rats, bacterial mutation assay, mammalian erythrocyte micronucleus test and mammalian chromosome aberration test, performed by licensed toxicologists. It was noted that adjudin had passed all the standard toxicity tests [70]. However, in a 29-day subchronic toxicity tests with n = 10 males and n = 10 females, the no-observable-adverse-effect level (NOAEL) for adjudin when administered once daily by oral gavage for female rats was shown to be 50 mg/kg/day since adjudin displayed no noticeable signs of damage in organs in female rats examined by a licensed pathologist. A NOAEL for males, however, could not be determined, since at 50 mg/kg/day for 29 days, one male rat displayed signs of chronic-active portal inflammation of minimal severity, and another male rat had midzonal vacuolization of minimal severity; also 3 out of the 10 male rats displayed skeletal muscle degeneration atrophy even though there were no adverse effects on other organs such as brain, kidney and heart (see Final Report of Subchronic Toxicity Report of Adjudin in [70], and reviewed in [26–28]). These findings thus illustrate that in order to develop adjudin as a male contraceptive for humans, the efficacy and toxicity dose must be significantly widened, such as by lowing the efficacy dose of adjudin from 50mg/kg b.w., to <5mg/kg b.w. Work is now in progress to develop a better formulation [70], including the co-delivery of adjudin with another drug that can induce transient disruption of the blood-testis barrier to improve its bioavailability. These recent advances have been recently reviewed (see [26–28]) and work is in progress in our laboratory. Recent studies, however, have shown that adjudin has other added health benefits, such as anti-cancer activity that appears to be more potent than lonidamine with less toxicity as summarized in Fig. (2). Besides, adjudin also has neuroprotective activity by inhibiting neuroinflammatory response. Since neuroinflammation is one of the hallmarks of neuro-degenerating diseases, such as Alzheimer’s disease and Parkinson disease, long-term use of adjudin may have added protection to such ailments as noted in Fig. (2). A more recent report illustrating the otoprotective function of adjudin based on the use of a gentamicin-induced ototoxicity study model in vitro and in vivo further illustrates the medical benefits of adjudin as summarized in Fig. (2). It is of interest to note that recent patents also report other medical benefits of lonidamine, its relative indazole-containing compounds including their protective roles in inflammation and neurodegeneration besides their anti-cancer activities as noted in Table 1 [100–115], consistent with the observations noted for adjudin as discussed herein. Nonetheless, many open questions remain to be addressed. Does long-term low-dose use of adjudin also offer otoprotective effects due to aging? Does the in vivo otot-protective effects of adjudin based on the in vivo gentamicin model is mediated by mitochondrial Sirt3-ROS pathway analogous to the studies in vitro? Does Sirt3-ROS pathway is also involved in the anti-cancer activity or neuroprotective (i.e., anti-inflammatory activity in the CNS) activity of adjudin? The answers to some of these questions will provide the incentive to better explore the medical use of adjudin besides male contraception. With the rapid advances in the field at the technological level and the availability of different in vitro and in vivo models to study major human diseases, we will likely witness a smooth development in the next decade regarding the use of adjudin as a non-hormonal male contraceptive and also for other pathological conditions, such as for cancer treatment, and treatments for neurodegeneration and ototoxicity once the underlying molecular mechanisms and efficacy are better understood.