Abstract
SCH 206272, an antagonist of neurokinin receptors 1, 2, and 3, was administered orally by gavage for 1 month to 8- to 10-month-old dogs at doses of 0, 15, 30, or 60 mg/kg, and to 6-week-old rats at doses of 0, 30, 100, or 300 mg/kg. The most important changes occurred in the reproductive tract of the dogs at all doses. Absolute and relative group mean organ weights for the testes, prostate gland, epididymides, ovaries, and uterus were 33–86% lower than concurrent controls in groups receiving SCH 206272. Organ weight changes were not dose-related. Microscopic changes that correlated with the organ weight changes occurred in all groups receiving SCH 206272. For males, they included minimal to severe atrophy of the testes, epididymides, and prostate gland. In addition, the epididymides exhibited severe oligospermia or aspermia, minimal epithelial apoptosis and mild epithelial vacuolation. In female dogs, the ovaries and uteri appeared immature. Microscopic changes were similar in incidence and severity in dogs receiving 30 or 60 mg/kg, but were slightly less in dogs receiving 15 mg/kg. In contrast, similar findings were not observed in the reproductive tract of male or female rats, despite overlapping systemic, hypothalamic, and pituitary gland concentrations of SCH 206272.
Introduction
Neurokinins (NKs), also called tachykinins, are a family of small peptides that share the common C-terminal sequence Phe-X-Gly-Leu-MetNH2 (Maggi, 1995). In mammals, 3 primary members of this family have been described known as Substance P (SP), Neurokinin A (NKA), and Neurokinin B (NKB). Several subspecies, including the N-terminally elongated forms of NKA, Neuropeptide K and Neuropeptide gamma, have also been described (Maggi, 1995; Joos et al., 2000; Patacchini and Maggi, 2001). Neurokinins are produced by a subpopulation of sensory nerves (Von Sprecher et al., 1998). In addition, certain immune cell types synthesize and possibly release neurokinins during inflammation, thus representing a non-neuronal source of neurokinins in inflamed tissues. (De Giorgio et al., 1998; Lai et al., 1998; Joos et al., 2000). The SP and NKA precursor proteins are encoded by the same gene, called preprotachykinin 1, while a separate gene, preprotachykinin 2, encodes the precursor of NKB (Von Sprecher et al., 1998).
Three G-protein coupled receptors for NKs have been cloned and are termed NK1 (SP-preferring), NK2 (NKA-preferring) and NK3 (NKB-preferring) (Maggi, 1995). NK1 and NK2 receptors are distributed throughout the central and peripheral nervous system (Maggi, 1995; Von Sprecher et al., 1998; Patacchini and Maggi, 2001). NK3 receptors were previously thought to be confined to the central nervous system, but more recently have been found in peripheral tissues as well, including at sites within the reproductive tract (Patacchini et al., 1995; Blondeau et al., 2002; Crane et al., 2002).
Neurokinins have been shown to be involved either directly or indirectly in a variety of biological processes including mast cell degranulation, vasomotor function, smooth muscle contraction/relaxation, salivary gland secretion, airway mucus secretion, airway contraction, inflammatory cell recruitment and activation, control of gonadotropic hormone secretion, emesis and transmission of nociceptive responses (reviewed in Quartara and Maggi, 1998; Debeljuk and Lasagna, 1999; Rupniak and Kramer, 1999; Campos and Calixto, 2000; Joos et al., 2000). Based on the biology of the NKs, it has been suggested that antagonists of NKs may have use in treating a wide range of diseases, including inflammatory conditions, pain, emesis, cancer, affective disorders, and schizophrenia (Van Schoor et al., 1998; Von Sprecher et al., 1998; Spina et al., 1998; Rupniak and Kramer, 1999; Campos and Calixto, 2000; Saban et al., 2000; Stout et al., 2001).
SCH 206272 is a potent nonpeptide, orally active antagonist of NK receptors 1, 2, and 3. It inhibits binding at human NK1, NK2, and NK3 receptors (K i = 1.3, 0.4 and 0.3 nM, respectively), and antagonizes [Ca2+] i mobilization in Chinese hamster ovary (CHO) cells expressing the cloned human NK1, NK2, or NK3 receptors (Anthes et al., 2002). The principal metabolite of SCH 206272 is SCH 209119, which exhibits similar pharmacologic activity and high binding affinity (K i = 0.4, 0.2, and 0.2 nM, respectively), to NK1, NK2, and NK3 receptors as the parent compound (Unpublished data, Schering Plough Research Institute, 1997). As part of the preclinical development of SCH 206272, the toxicity of orally administered SCH 206272 was evaluated in Beagle dogs and Sprague–Dawley rats in 1-month toxicity studies.
In the preceding studies, species-specific reproductive toxicity was observed. Preclinical studies evaluating the toxicity of pharmaceutical compounds and xenobiotics frequently include comparisons between rats and dogs, and species-specific differences in toxicity are often observed. However, the explanation for the differences, other than those related to interspecies differences in the pharmacokinetics of the test article (Christian, 1996), are not always easily determined. In this paper, an attempt was made to explore the species differences in toxicity by considering both the likely pharmacologic mechanism of the toxicity seen in dogs and possible pharmacokinetic differences between rats and dogs.
Materials and Methods
Animals and Husbandry
Eight- to 10-month-old purpose-bred Beagle dogs (Schering Plough Research Institute, Lafayette, NJ), weighing 7.7 to 13.3 kg (males) or 5.6 to 9.2 kg (females) at the start of dosing, were randomly assigned to 4 groups of 4 animals per sex. Dogs were housed individually in stainless-steel cages in environmentally controlled rooms (temperature 64–84°F, relative humidity 30–70%, 12-hour light/12-hour dark photoperiod). Dogs were offered Purina Mills, Inc., Certified Canine Diet #5007 daily, and provided tap water ad libitum. Because pilot studies demonstrated that SCH 206272 caused emesis in the dog that could be reduced by feeding prior to dosing, food was presented for approximately 1 hour before daily dosing was initiated.
Six-week-old rats [Crl: CD (SD) BR VAF/PLUS, Charles River Laboratories, Portage, MI], weighing 185–252 g (males) and 157–211 g (females) at the start of dosing, were randomly assigned to 4 groups of 10 animals per sex for the toxicity portion of the study. An additional 9 rats per sex were assigned to each group for plasma analysis of SCH 206272. Rats were housed individually in stainless-steel cages in environmentally controlled rooms (Temperature 64–79°F, relative humidity 30–70%, 12-hour light/12-hour dark photoperiod). Rats were offered Purina Mills, Inc., Certified Rodent Chow #5002 weekly, and provided tap water ad libitum.
Both the dog and rat toxicology studies were conducted in facilities accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC), and in compliance with the guidelines published in the National Research Council “Guide for the Care and Use of Laboratory Animals,” (National Academy of Sciences, 1996) and the Animal Welfare Act (Public Law 91–579).
Test Article and Administration
SCH 206272, supplied as a tartrate salt (Schering-Plough Research Institute), was dissolved in sterile water for injection at concentrations required to provide a solution with a total dose volume of 3 mL/kg for both species and all groups (the concentrations discussed throughout this report are based on the amount of SCH 206272 free base). The concentration of SCH 206272 varied from 5 mg/mL to 100 mg/mL, and was shown to be stable under the conditions used in these studies. Animals were dosed daily for 28 days by gavage at doses of 15, 30, or 60 mg/kg in the dog study, and 30, 100, or 300 mg/kg in the rat study. The doses for each species were selected based on previous pharmacokinetic study data as well as pharmacologic studies demonstrating NK receptor antagonist-mediated functions (Anthes et al., 2002). Control animals received 3 mL/kg of the sterile water vehicle orally by gavage.
Clinical Examinations
Animals were examined after each day’s dosing was completed, or as clinical signs warranted. Body weights were recorded on Day 1 prior to dosing, weekly thereafter, and additionally (in the rats only), on Day 16. Food consumption was recorded daily (dogs), or weekly (rats). Ophthalmoscopic examinations were conducted pretest and during week 4 for both species. Electrocardiograms and blood pressure measurements were conducted in dogs twice pretest, and approximately 4 hours postdose during Weeks 2 and 4. Blood sampling for hematology, coagulation assays, and serum chemistry parameters in the rat were conducted on Days 9 (high-dose group only), 15 (no coagulation assays), and 29; and, in the dog, twice pretest and on Days 15 and 28. Urinalysis and urine chemistry parameters were evaluated in the rat during Weeks 2 (high-dose group only), 3 and 5, and, in the dog, twice pretest and during Weeks 3 and 4.
Plasma Drug Analysis
Plasma concentrations of SCH 206272 and its major metabolite SCH 209119 were determined at 0, 1, 2, 4, 6, 8, 12, and 24 hours after initial dosing and on Day 28 for both species. Blood samples were obtained in heparinized capillary tubes by retro-orbital bleeding from rats assigned to the plasma analysis portion of the study and by jugular venipuncture from all dogs. Plasma samples were analyzed for SCH 206272 and its major metabolite, SCH 209119, using a validated liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) assay with a limit of quantitation of 1 ng/mL for both analytes.
Pathology
Dogs were administered a barbiturate overdose, exsanguinated and necropsied on Day 29. Following an overnight fast, rats were anesthetized with C02, exsanguinated and necropsied on Day 29. Organ weights from a standard panel of organs were collected for both species. All major organs were fixed by immersion in 10% neutral-buffered formalin, with the exception of the eyes, which were collected and fixed in 3% glutaraldehyde and then transferred to neutral buffered 10% formalin. Tissues were routinely processed and hematoxylin and eosin-stained slides were prepared.
Investigative Study
Microscopic changes, suggestive of hormonal-based toxicity, were observed in the reproductive organs of dogs in the 1-month study. Similar changes did not occur in rats. A follow-up investigative study was performed to determine whether differences observed in reproductive toxicity between dogs and rats were related to differences in plasma and/or central nervous system (hypothalamus and pituitary gland), concentrations of the test article between the two species. Dogs and rats were administered [14C]-SCH 206272 at doses that provided approximately equivalent plasma concentrations of SCH 206272 in the 2 species as follows.
Two 9–10-month-old male dogs were dosed with 23 mg/kg of [14C]-SCH 206272 (approximately 50–100 μCi/kg), at a dose volume of 3 ml/kg in water. Similarly, 20 6–8-week-old nonfasted male rats (2 groups, 10 rats each) were dosed with 230 mg/kg of [14C]-SCH 206272 (approximately 50–100 μCi/kg), at a dose volume of 3 mL/kg in water. For both species, blood was collected at the anticipated Tmax as determined from previous studies (3 hours for dogs and 8 hours for rats), and the animals were terminated as above. For both species, the brain was removed, and the hypothalamus and pituitary gland were dissected, weighed, and homogenized in distilled water to give 25% homogenate (w/v). Homogenized tissues were stored frozen at −20°C pending analysis. The tissues were evaluated individually for the dogs, while tissues were pooled for each of the 2 groups of rats. Plasma and homogenates of hypothalamus and pituitary gland were analyzed from both species for total radioactivity by oxygen combustion (or solubilization), followed by liquid scintillation counting. A second aliquot was analyzed for SCH 206272 and SCH 209119 by a validated LC/MS/MS assay.
Results
Dog Toxicity Study
There was an increased incidence of emesis and soft feces in dogs receiving 60 mg/kg of SCH 206272, compared to the other dose groups, which correlated with lower food consumption. There were no test article-related changes observed during the general veterinary, ophthalmoscopic, electrocardiographic, blood pressure, and hematologic evaluations. Serum aspartate aminotransferase, alanine aminotransferase, and urea nitrogen values were minimally higher for both sexes and serum cholesterol concentrations were minimally higher for females in the 60 mg/kg group on Days 15 and 28 as compared to pretest values and concurrent control values (data not shown).
The mean absolute and relative (compared to body weight and brain weight) organ weights were lower than the concurrent control weights in all groups receiving SCH 206272 for the testes (↓ 60–79%), prostate gland (↓ 60–69%), epididymides (↓ 33–43%), ovaries (↓ 47–61%) and uterus (↓ 64–82%). Organ weight changes in the reproductive tract were similar at all doses (See Table 1). There were no SCH 206272-related macroscopic findings reported for dogs. SCH 206272-related microscopic changes occurred in the reproductive tract of both sexes in all dose groups.
On histopathologic examination, all the control male dogs were sexually mature with normal spermatogenesis evident in the testes and epididymides. The size of the uterus in control females varied between animals, but most were of sufficient size to indicate that the dogs were mature or approaching maturity. In addition, all control females had developing follicles in their ovaries. Histopathologic findings in SCH 206272-administered female dogs were limited to the appearance of small ovaries, which lacked developing follicles or corpora lutea. Aggregates of immature oocytes, with no evidence of degenerative change, were present in apparently normal distribution in the subcapsular region of the ovaries (Figures 1a and 1b). Uteri exhibited normal histologic appearance except that they were thin-walled, suggestive of immaturity (Figures 1c and 1d). All females exhibited ovarian and uterine immaturity/atrophy except 1 female/group from the 15 and 60 mg/kg groups.
Findings in males included atrophy of the seminiferous tubules of the testes, atrophy, epithelial vacuolation, and aspermia/oligospermia of the epididymides and atrophy of the prostate gland. Most males were affected and most findings were present in all dose groups, except epithelial vacuolation of the epididymides that occurred only in mid- and high-dose group males.
Microscopically, atrophied testes were shrunken and slightly irregular in shape with condensation of the connective tissue in the mediastinum testis and septae related to collapse of the seminiferous tubules. Tubules had decreased luminal diameter due to both a loss of spermatogenic epithelial layers and to apparently reduced production of fluid by Sertoli cells.
There were variable numbers of degenerating or necrotic germ cells (Figure 2). Giant multinucleated spermatids were common, and sloughed cells and karyorrhectic debris were present within the remaining epithelial layers, in the tubular lumina and within the rete testis. Specific stages of spermatogenesis could not be identified because of the severity of the changes. Germ cells that were present appeared to be spermatogonia or spermatocytes. The normal stages of developing round and elongate spermatids were not observed. Some tubules were lined primarily by Sertoli cells and lacked any recognizable germ cells (Figure 3). Leydig cells were prominent and occasionally exhibited discrete vacuoles within the cytoplasm (Figure 4). Leydig cells appeared to occur in normal numbers throughout the interstitium, but had increased visibility relative to the decreased surface area of seminiferous tubules. Atrophy of the epididymides was characterized by apoptosis and/or single cell necrosis and vacuolation of the epithelium throughout the tissue, as well as by reduced height of the cuboidal epithelium and reduced luminal diameter (Figure 5).
Spermatozoa were reduced in number or absent in the tubular lumina in all sections of the gland (head, body, and tail), and necrotic cellular debris, consistent with the observed sloughing of testicular germ cells, was frequently present. Atrophic prostate glands exhibited similar histopathologic changes of reduced height of the epithelium with minimal eosinophilic secretory product and reduced luminal diameter. The interstitial connective tissue in the prostate appeared comparatively more dense due to collapse or failure of development of the glandular components of the tissue (Figure 6). All SCH 206272-administered male dogs exhibited histopathologic changes of their reproductive organs, with the exception of 1 dog receiving 15 mg/kg.
Rat Toxicity Study
Four rats/sex administered the 300 mg/kg dose and 2 males administered the 100 mg/kg dose were found dead during the first week in the study. Their mortality was attributed to aspiration of the highly concentrated and irritating test article during the gavage procedure. As a consequence of the early mortality, the remaining 300 mg/kg group rats from the toxicity portion of the study were sacrificed on study Day 8. A change in the gavage procedure was implemented that reduced the risk of aspiration for the remaining study animals. Surviving rats assigned to the 300 mg/kg group for toxicokinetic analysis were maintained until the scheduled sacrifice on Day 28. Clinical observations for the study were limited to evidence of respiratory tract irritation, including nasal and ocular discharge and labored breathing that affected mainly the high-dose group animals. Group mean body weights were significantly lower in rats of both sexes receiving 300 mg/kg, and in males receiving 100 mg/kg at every interval beginning on Day 8 (data not shown).
Lower food consumption occurred in all groups, with the incidence and severity generally dose-related. The effects on food consumption and body weights were attributed, at least in part, to the respiratory effects related to aspiration in a few rats. There were no test article-related changes observed during the ophthalmoscopic examinations, and no SCH 206272-related changes in clinical pathology parameters were seen in groups receiving 30 or 100 mg/kg. High-dose group rats had sporadic increases in WBC counts (mainly neutrophilia), that were attributed to inflammatory changes in the respiratory tract related to compound aspiration. At necropsy, there were no macroscopic findings in the reproductive organs and there were no SCH 206272-related changes in the reproductive organ weights, as compared to concurrent controls, in any dose group. There were no SCH 206272-related microscopic findings in the reproductive organs of rats of either sex.
Plasma Drug Analysis
Dogs
Plasma concentrations [both Cmax and AUC (0–24 hr)] of SCH 206272 and its major metabolite, SCH 209119, increased in a dose-related manner and were gender-independent. For both sexes, absorption was variable, with Tmax values of individual animals ranging from 1–8 hours on Day 1 and 1–12 hours on Day 28. SCH 206272 and SCH 209119 accumulated in the plasma in all groups (group mean R values from 1.57 to 2.03 for SCH 206272 and 1.31 to 3.54 for SCH 209119). Systemic exposure of SCH 209119, based on AUCs, ranged from 6% to 16% of that seen with SCH 206272.
Rats
Plasma concentrations of SCH 206272 and its major metabolite, SCH 209119, increased in a dose-related manner. Absorption of SCH 206272 was slow and gender-dependent, with Tmax values of 4–8 hours in males and 8–12 hours in females. Systemic exposure [AUC (0–24 hours)] was less than dose proportional in female rats, and greater than dose proportional in male rats. Systemic exposure to SCH 206272 was greater in females than males with the inter-gender difference decreasing as the dose increased. For example, systemic exposure in females was greater than in males by approximately 10-fold at the 30 mg/kg dose, by approximately 5-fold at the 100 mg/kg dose, and by approximately 3-fold at the 300 mg/kg dose.
Systemic exposure of SCH 209119 was less than 2% of that seen with SCH 206272. Both SCH 206272 and SCH 209119 accumulated in the plasma in male and female rats receiving 30 or 300 mg/kg and females receiving 100 mg/kg (group mean R values from 1.25 to 3.38 for SCH 206272 and 1.77 to 5.60 for SCH 209119). Accumulation was not observed in male rats receiving 100 mg/kg (group mean R value = 1.00).
Species Comparison
In comparing the species, the toxicokinetic data for dogs was considered for both sexes combined, while the data for rats was considered for each sex separately due to gender dependent differences in plasma concentrations (Cmax and AUC), (Figure 7). The systemic exposures to SCH 206272, [AUC (0–24 hours)] were similar in dogs receiving 15 mg/kg and female rats receiving 30 mg/kg (20,000–35,000 ng-hr/mL) or male rats receiving 100 mg/kg (20,000–30,000 ng-hr/mL). With regard to SCH 209119, dogs achieved higher exposure levels than rats at all doses. Systemic exposures were most similar in dogs receiving 15 mg/kg and female rats receiving 300 mg/kg (2500–3400 ng-hr/mL). Male dogs receiving the lowest dose of SCH 206272 still had exposure to SCH 209119 that was approximately 50% higher than that seen in the rat at the highest dose of 300 mg/kg.
Investigative SCH 206272 Distribution Study
The mean concentration of SCH 206272 was higher in the rat pituitary gland compared to the dog, while the mean concentration in the rat hypothalamus was slightly lower than that in the dog (Table 2). These concentrations were 23–36-fold higher than in the systemic circulation. The major metabolite, SCH 209119, could not be detected in the pituitary gland or hypothalamus of either species.
Discussion
Administration of SCH 206272 to rats for 28 days produced no changes in organ weights or microscopic appearance of the reproductive organs of either sex. Administration of SCH 206272 to Beagle dogs for 28 days resulted in lower absolute and relative group mean organ weights for the testes, prostate gland, epididymides, ovaries, and uterus. Lower organ weights correlated with the microscopic finding of atrophy in males and atrophy or hypoplasia of the reproductive organs in females. The severity of the organ weight changes was generally comparable for all dose groups, although the microscopic findings were less frequent or severe in the group receiving 15 mg/kg.
The pathogenesis of male reproductive tract toxicity in laboratory animals is complex (see reviews in Russell et al., 1981; Creasy, 2001). Toxic changes can occur as a direct insult targeting specific cell types in the testes, such as Sertoli cells (Boekelheide, 1993), spermatogonia (Meistrich, 1984) or Leydig cells (Bartlett et al., 1986). Hormonal deficiencies or imbalances can also produce changes in the testes that mimic local toxicants, whether they result from effects on testosterone production by Leydig cells (Bartlett et al., 1986) or from changes in the hormonal regulatory system at other sites. Potential hormone-related mechanisms (reviewed in Creasy, 1999; Chapin and Williams, 1989), include interference with androgen or gonadotropin receptor binding, alterations in circulating gonadotropin concentrations related to decreased secretion from the hypothalamus or pituitary gland or to changes in the metabolism or clearance of any of the hormones involved in the endocrinology of reproduction.
If testicular toxicity is identified at an early stage, careful examination of the microscopic pattern of changes may be used to help elicit the pathogenetic mechanism (Russell et al. 1990; Creasy, 2001; Lanning et al., 2002). Unfortunately, the dogs in this study had sufficiently prolonged exposure to the test article to result in severe atrophy. Because atrophy is the end stage consequence of many different mechanisms (Russell et al., 1981), the microscopic appearance of the testes was not useful in identifying the pathogenesis of the changes seen. However, the lower mean weights and reduced secretory activity of the prostate is suggestive of reduced androgen status.
Based on lower reproductive organ weights, both sexes were considered to be affected by SCH 206272; however, the assessment of toxicity in the female dogs was more difficult. Both the cyclic nature of the female estrus cycle and the young peripubertal age of the dogs, could have contributed to the immature appearance of the ovaries and uterus in individual animals. Although there is limited data on reproductive toxicants in female dogs, there have been a few reports in rats of compounds that produced microscopic lesions in the ovary involving degenerative changes and/or loss of primordial follicles, with subsequent reduced fertility (Mattison et al., 1983; Flaws et al., 1994; Hoyer and Sipes, 1996). No specific degenerative changes were observed in the ovaries of female dogs in this study, and the study was not designed to evaluate fertility. The microscopic appearance of the ovaries was suggestive of a deficiency of hormonal stimulation.
Primordial follicles were evident in microscopic sections, but the various stages of developing follicles and/or corpora lutea that would be expected in mature females were not present. While it is not clear if SCH 206272 administration produced effects on organ weights and histopathologic appearance of reproductive tissues by blocking the normal process of maturation in immature females or by causing atrophy of formerly mature tissues, the latter is more likely. The dogs were randomly assigned to the different dose groups. At the end of the 1-month study, all the controls appeared mature, while all but 2 of the treated dogs displayed changes suggestive of gonadotropin deficiency.
SCH 206272-related reproductive toxicity in dogs was suspected of being the result of interference in gonadotropin production or release, primarily at the level of the hypothalamus or pituitary gland. The observations that both sexes showed organ weight changes, and that atrophy was evident in all the reproductive tissues, supported a pathogenesis involving gonadotropic hormones rather than a local toxicity of the reproductive organs. Most cell-specific gonadal toxicants do not target the testes and ovaries equally, nor do they result in generalized atrophic changes in the secondary sex organs (Russell et al., 1981; Creasy, 2001).
Neurokinins are known to play a number of roles in mammalian reproduction, including modulation of smooth muscle contractility of the uterus of rats (Moodley et al., 1999), and humans (Patak et al., 2003), the influencing of sexual behavioral responses in rats (Dornan et al., 1993), and the hormone-dependent support of reproductive organ development and maintenance in mice and rats (Pintado et al., 2003). The latter effect has been attributed to the role of NKs as paracrine effectors involved in modulating reproductive hormonal interactions.
There is extensive anatomic evidence supporting a role of neurokinins in reproduction physiology that includes studies, using in situ hybridization and/or radioimmunoassays, that show the distribution of neurokinins in the CNS of the rat (Tsuruo et al., 1987; Harlan et al, 1989), monkey (Ronnekleiv et al., 1984) and human (Covenas et al., 2003). Neurokinins in the CNS are produced by small populations of cells, representing 5% to 7% of neurons, localized to crucial regions such as the median eminence and arcuate hypothalamic nucleus, septal-preoptic area and periventricular hypothalamus (Makara et al., 1986; Debeljuk and Lasagna, 1999; Mantyh, 2002). These sites are in close proximity and are believed to communicate with the neurons producing gonadotropic releasing hormone (GnRH), (Kalra and Kalra, 1983, Tsuruo et al., 1987), which in turn secret into the portal system of blood vessels that supplies the pituitary gland.
The density of this neuronal population in rats varies during the estrous cycle and lactation, demonstrating that the hypothalamic levels of neurokinins are interrelated with the concentration of circulating sex steroids (Tsuruo et al., 1987). In addition, SP-positive immunoreactive nerve fibers have been identified in the anterior pituitary of monkeys (Ju and Liu, 1989), dogs (Ju and Liu, 1990; Ju and Zhang, 1990), rats (Larsen, 1992; Ju and Liu, 1993), and humans (Liu, 2004), in close contact with secretory cells. The latter observation has led researchers to believe that neurokinins may also act directly on the pituitary gland via a neurologic mechanism, as well as by influencing hypothalamic-releasing hormones.
Literature describing neurokinin reasearch in dogs is sparse and mainly limited to studies on anti-emetic mechanisms. Therefore, most of the information we have on the likely physiologic role of NKs in reproduction are derived from studies in rats. Vijayan and McCann (1979) first showed that administration of SP into the third ventricle of rats, presumably acting directly on the hypothalamus, stimulated the release of rat luteinizing hormone (LH). Similarly, intraventricular injection of anti-SP serum resulted in a decrease in plasma LH and FSH (Arisawa et al., 1990).
To further pinpoint the site of NK action in the CNS, in vitro studies have been designed using isolated organ preparations. When SP was added to preparations derived from normal rats of perifused hypothalamus and pituitary gland in sequence, there resulted a statistically significant increase in LH in the efflux, as well as an increase in GnRH from perifused hypothalamus alone (Ohtsuka et al., 1987). These findings support the theory that NKs, or at least SP, have a stimulatory effect on gonadotropin production at the level of the hypothalamus that is mediated in part or fully by GnRH and can be antagonized by NK receptor inhibition.
Although the preceding paradigm appears to offer a likely explanation for the reproductive toxicity of SCH 206272 in dogs, it does not explain the lack of toxicity in the rat study with this compound. Unfortunately, the extensive body of literature investigating the physiology of NK-hormonal interactions in rats presents conflicting information and conclusions. Some researchers have suggested that the predominant effect of NK administration or antagonism in rats occurs not at the hypothalamus, but at the level of the pituitary gland (Duval et al., 1996a, 1996b).
However, different results, involving either stimulation or inhibition of LH secretion by the pituitary gland, have been achieved in rat studies by adjusting the route of NK administration, whether systemic, intraventricular or by use of in vitro isolated pituitary gland preparations (Vijayan and McCann, 1979; Arisawa et al., 1990; Battmann et al., 1991; Duval et al., 1996a, 1998). In addition, conflicting effects of NK administration on LH secretion have been reported as a consequence of manipulating the gonadal steroid environment of both male and female rats in different test systems (DePalatis et al., 1985; Kalra et al, 1992; Duval et al., 1998). Researchers have theorized that the variable consequences of NK administration or antagonism reported in different rat studies may depend, at least in part, on “priming” of the hypothalamus and pituitary gland by up- or down-regulation of receptors for NKs, gonadal steroid hormones, and GnRH (Duval et al., 1998; Kerdelhue et al., 2000). The one conclusion on which there is agreement is that a delicate balance exists for these chemical mediators, and that additional research is needed to characterize the functional role of NKs in mammalian reproduction.
In recent years, a number of selective NK agonists and/or antagonists have been developed as potential therapeutic agents and there is considerable literature describing their pharmacology (reviewed in Patacchini and Maggi, 2004; Emonds-Alt, 2004; Mazzone and Canning, 2004; Rumsey and Kerns, 2004). However, there are no previous reports of reproductive toxicity in dogs or other species associated with any of these agents. Because dogs are not commonly chosen to assess reproductive toxicity, the lack of toxic findings in the literature may simply reflect the fact that the effects on dogs have not been investigated with these compounds.
In the present studies, an effort was made to compare the plasma and tissue (pituitary gland and hypothalamus) concentrations of SCH 206272 and its main metabolite in rats and dogs to see if differences in drug exposure could explain the differences in toxicity. It is noteworthy that dogs absorbed SCH 206272 more efficiently than rats. Dogs achieved higher plasma levels of SCH 206272 at lower administered doses than rats of either sex. Dogs also achieved relatively higher levels of SCH 209119, the main active metabolite, with individual dogs achieving exposure to SCH 209119 up to 6–16% of the parent compound. Mean plasma levels of the metabolite in rats were less than 2% of the SCH 206272 concentration in all cases. These differences, however, are not believed to be sufficient to explain the differences in toxicity.
Dogs of both sexes exhibited reproductive toxicity at the lowest administered dose of 15 mg/kg. The AUCs of SCH 206272 achieved by dogs at 15 mg/kg overlapped those achieved by female rats at 30 mg/kg and male rats at 100 mg/kg, and the AUCs of SCH 209119 achieved by dogs at 15 mg/kg overlapped those of female rats at 300 mg/kg. Since no rats exhibited histopathologic findings in their reproductive organs, it is unlikely that toxicokinetic differences were responsible for the discrepancy. It is not clear if the higher concentrations of SCH 209119 in a few dogs may have contributed to the reproductive toxicity. However, it is not likely that the metabolite was wholly responsible for the findings in dogs, because both parent and metabolite have similar NK antagonistic activity and binding affinities, and the concentration of metabolite in the plasma of dogs was still small compared to the concentration of the parent drug.
Male rats were exposed to the parent drug at concentrations comparable to those in dogs, while female rats in the 300 mg/kg group were exposed to much higher concentrations than dogs, that would be expected to produce toxicity if rats had been susceptible to it. In addition, the investigative study that assessed the concentration of parent and metabolite in the likely target tissues of hypothalamus and pituitary revealed comparable exposure of SCH 206272 and negliglible exposure of the metabolite for both species. Thus, the difference in toxic effects between the species is likely to be related to physiologic differences intrinsic to rats and dogs.
Investigators have reported interspecies differences in a number of nonreproductive pharmacologic responses to NK receptor antagonists, and, in some cases, have identified mechanisms to explain them. Some differences in response to NK antagonists have been shown to be the result of species-specific differences in homologues of the 3 NK receptors (Maggi, 1995). For example, at least 2 subtypes of the NK1 receptor are known, the “classical” and “septide” types, which have different distribution in various species. Bioassays assessing the NK receptor-blocking ability of different antagonists showed variable potency among species, based on preference for the receptor subtype (Patacchini and Maggi, 2004). Heterogeneity of NK2 and NK3 receptors is also likely to exist (reviewed in Maggi, 1995). Pharmacologic studies, using specific NK receptor antagonists, have also demonstrated vastly different binding affinities of the different compounds for the tissues of different species, possibly related to heterogeneity of the NK receptors.
For example, RP67580, an NK1 antagonist, was found to be 10–20-fold more potent in blocking NK1 receptors in rats as compared to those in guinea pigs or humans. In contrast, CP96345 was 30–100-fold more potent in humans, guinea pigs, and dogs than in rats or mice (Patacchini and Maggi, 2004). Differences in the pharmacologic action of these drugs between species have been linked to discrete point mutations on the human and rat NK1 receptors that affect binding affinity (Fong et al., 1992).
Although the data in the present studies suggests that the likely pathogenesis of SCH 206272-related effects on the reproductive system of dogs is through impaired LH secretion, we can not rule out the possibility of a direct gonadal effect as well. Neurokinins, particularly SP, have been identified in Leydig cells of rodents (Angelova et al., 1991) and humans (Schulze et al., 1987) and in the ovaries of rats (Dees et al., 1985). Although the role of NKs in the ovary has not been greatly studied, a number of researchers have demonstrated that NKs, including SP and NKA, inhibit the production or release of testosterone by the Leydig cells (Debeljuk and Lasaga, 1999), while increasing the secretory activity of Sertoli Cells (Rao et al., 1995).
These findings have led to the theory that NKs might be involved in the modulation of metabolic processes in the testes that are required for spermatogenesis (Delbejuk and Lasaga, 1999). Neurokinins may also be involved in controlling the blood supply to the gonads. Recent reports have shown that SP is present in the nerve fibers of the tunica albuginea of the dog testes (Tamura et al., 1997). These SP immunopositive nerve fibers penetrate the testicular parenchyma and run along blood vessels. A similar observation was made in the ovaries of female rats, where SP was shown by immunofluorescence to be localized in the nerve fibers closely associated with the theca externa of antral follicles, as well as being in the interstitial tissues and within the advential layer of small arterioles (Dees et al., 1985).
However, none of these findings, whether involving vascular or metabolic mechanisms, can be clearly related to the observed toxicity of SCH 206272. The degenerative/atrophic changes in the reproductive organs of dogs were inconsistent with the histopathologic appearance of ischemia produced by hypoperfusion. In addition, SCH 206272, as an NK receptor antagonist, would have been expected to produce opposite effects to those described (inhibition of testosterone production/release) for NKs, rather than similar effects. As with the studies involving the role of NKs in hypothalamic-pituitary interactions, there is a need for further research to understand the local effects of these chemical mediators.
In summary, SCH 206272 administration resulted in reproductive toxicity in dogs but not rats. This difference in sensitivity could not be explained by a species-specific difference in SCH 206272 and metabolite exposures in the plasma and in the tissues (hypothalamus and pituitary gland) that were considered to be the most likely targets for pharmacologic action. The suspected mechanism of toxicity may be the result of SCH 206272-related suppression in production or release of GnRH and/or LH, subsequently resulting in reproductive organ atrophy, although a secondary local effect of NK antagonism on reproductive organs cannot be ruled out. The reason for the discrepancy in the reproductive toxicity between the two species could not be determined. Based on the presented findings, additional studies were recommended to investigate the hormonal effects of SCH 206272 administration in dogs.
Footnotes
Acknowledgements
The authors would like to acknowledge the assistance of K. B. Lee in preparing the photographic images.