Assessment of ixekizumab, an interleukin-17A monoclonal antibody, for potential effects on reproduction and development, including immune system function, in cynomolgus monkeys.

The reproductive and developmental toxicity of ixekizumab, a selective inhibitor of interleukin-17A (IL-17A), was assessed in the following studies in cynomolgus monkeys: fertility (3-month dosing), embryo-fetal development (EFD; dosing from gestation day (GD) 20 through 139), and pre-postnatal development (PPND; dosing from GD 20 through parturition). Because IL-17A has functional roles in innate and humoral immunity, immune system modulation was evaluated in the EFD and PPND studies; immunological evaluations in infants comprised peripheral blood immunophenotyping, Natural Killer cell cytolytic activity, and T-cell-dependent antibody (IgG and IgM) primary and secondary responses to antigen challenge. Ixekizumab exposure was sustained during the dosing periods in most adult monkeys. Fetal exposure at Cesarean section (GD 140-142; EFD study) was 18-25% of maternal exposure and ixekizumab was present in infants for up to 29 weeks postpartum. There were no adverse effects attributed to ixekizumab in any study. Importantly, immune system development and maturation were unaffected.

Hyperplastic changes within the leptomeninges of the rat and monkey in response to chronic intracerebroventricular infusion of nerve growth factor.

Recombinant human nerve growth factor (rhNGF) was delivered for up to 6 months by continuous intracerebroventricular (i.c.v.) infusion to CD (Sprague-Dawley derived) rats and cynomolgus monkeys. Rats (n = 15/sex/group) received doses of 0 (vehicle), 6, 60, or 300 ng/day; monkeys (n = 5/sex/group) received 0, 0.6, 6, or 60 microg/day of rhNGF. Animals tolerated i.c.v. infusion with no behavioral signs attributable to rhNGF. Body weight was transiently decreased in female rats at the highest dose. At the completion of dosing, histological examination in both species revealed an increase in the thickness of the leptomeninges along the ventral and lateral surfaces of the hindbrain and extending over the dorsal aspect of the spinal cord. The change was present to varying degrees at all doses of rhNGF and tended to be more severe at higher doses. At the light microscopic level, the leptomeninges contained layers of well-differentiated, spindle-shaped cells and a plexus of axonal fibers. Cells were immunoreactive for S-100 protein and were associated with an accumulation of Type IV collagen, suggesting Schwann cell origin. Electron microscopy revealed numerous fine caliber axons ensheathed by the presumptive Schwann cells, with myelination of individual axonal segments. These findings suggest that chronic i.c.v. delivery of rhNGF has stimulated axonal sprouting and secondary hyperplasia of Schwann or Schwann-like support cells within the pia-arachnoid.

Comparison of ketorolac tromethamine with other injectable nonsteroidal anti-inflammatory drugs for pain-on-injection and muscle damage in the rat.

The local tolerance of ketorolac tromethamine (Toradol, Syntex) was compared with that of four other injectable nonsteroidal anti-inflammatory drugs (NSAIDs) (diclofenac sodium, piroxicam, ketoprofen, and metamizol magnesium) in the rat paw-lick/muscle irritation assay as described previously. All drugs were tested at concentrations approved for clinical use. After subplantar (footpad) injection, ketorolac produced virtually no pain-on-injection as assessed by the number of paw-lick/lift responses during a 15 min observation period. The other NSAIDs produced slight to moderate paw-lick/lift responses. Redness and swelling at the injection site were less severe for ketorolac than for the other NSAIDs. After intramuscular (i.m.) injection, all of the NSAIDs produced some degree of muscle damage, as assessed histopathologically 24 h after injection. The lesions, consisting primarily of muscle degeneration, were less severe for ketorolac than for the other NSAIDs. Ketorolac and metamizol produced the smallest elevations in serum creatine kinase, as measured 2 h after i.m. dosing, not significantly different from isotonic saline. Overall, ketorolac was better tolerated in the assay than the other injectable NSAIDs, thereby suggesting the possibility of improved local tolerance on clinical use.

Activation of B virus (Herpesvirus simiae) in chronically immunosuppressed cynomolgus monkeys.

Three of 14 cynomolgus monkeys given the highest dose of an immunosuppressive drug in a 6-month toxicology study developed B virus (Herpesvirus simiae) oral lesions after 3 months of dosing. This necessitated early removal of all high-dose monkeys from the study due to concerns related to B virus. The incidence and severity of parasitic (Oesphagostomum sp.) lesions of the large intestine were also increased in high-dose animals. Both B virus and Oesophagostomum are enzootic in macaques, and the lesions caused by them were considered secondary to chronic immunosuppression caused by the highest dose of the test compound. Evidence of immunosuppression included decreased lymphocyte counts (B-cells; CD2 and CD8 T-cells), histopathologic evidence of lymphoid suppression, and serum-induced inhibition of lymphocyte mitogen responses. Pathogenesis of the B virus was apparently associated with both activation of latent virus as well as transmission of active virus. Approaches for virologic monitoring of primates and for ensuring optimal safety for primate handlers are discussed.

Rat paw-lick/muscle irritation model for evaluating parenteral formulations for pain-on-injection and muscle damage.

A two-phase assay was developed in the rat to evaluate parenteral formulations intended for intramuscular administration for the induction of both acute pain-on-injection and delayed pain/discomfort at the injection site (secondary to muscle damage). Phase 1 of the assay assessed pain-on-injection using a modified version of the previously published rat paw-lick assay. Adult male CD rats (10/group) were given subplantar (footpad) injections of 0.1 ml and then observed for 15 min for paw-lick responses. To increase assay sensitivity, responses more subtle than paw licks (ie., paw lifts) were scored, and injection-site clinical signs were recorded 6, 24, and 48 hr after injection. Phase 2 of the assay assessed myotoxic potential, using the same rats after a 1-week recovery period. The rats were injected intramuscularly in the anterior thigh with 0.2 ml, bled from the orbital sinus at 2, 6, and 24 hr for analysis of serum creatine kinase (CK), and then necropsied at 24 hr to prepare tissue sections of the injection site for microscopic examination. A series of cephalosporin-type antibiotics produced pain-on-injection and muscle damage consistent with reported clinical experience (cefazolin less than cephalothin less than cefoxitin). Several nonantibiotic parenteral formulations (diazepam, digoxin, phenytoin, and lidocaine) tested in the paw-lick/muscle irritation model also produced responses that correlated with the clinic, i.e., virtually no acute pain but moderate to marked muscle damage. The results indicate that the two-phase rat paw-lick/muscle irritation model is effective in evaluating parenteral formulations for clinical acceptability, and that both phases of the assay are necessary to optimize predictability of the assay for human clinical experience.

The use of multiple endpoints to define the mechanism of action of reproductive toxicants and germ cell mutagens.

Although a variety of endpoints are routinely assessed in reproductive toxicity studies, the inclusion of additional (and potentially more sensitive) endpoints may increase our ability to detect adverse effects on male or female reproduction and also provide information pertaining to the mechanism of action of the reproductive toxicant. Methyl chloride (MeCl) is a well characterized reproductive toxicant in the male rat, and can serve as a model to illustrate the importance of using multiple endpoints to determine the biological basis of chemically induced toxicity in the reproductive system. Exposure of male rats to MeCl results in bilateral testicular degeneration and epididymal inflammation and sperm granuloma formation. Females bred to these males in a dominant lethal assay exhibit elevated rates of postimplantation embryonic death during the first 2 weeks after treatment and increased preimplantation embryonic loss during weeks 2 to 8 post-exposure. Since the chemical is known to be a direct-acting mutagen in vitro and a kidney carcinogen in vivo, the increased embryo death rate observed might reasonably be considered good evidence that MeCl is a direct-acting germ cell mutagen. However, subsequent investigations revealed that MeCl-induced preimplantation loss was a result of cytotoxic rather than genotoxic effects on sperm, with a significant decrease in the count of motile sperm of normal morphology in exposed males during weeks 2 to 8 after treatment. In fact, examination of the fertilization rate during these weeks using a system of embryo recovery and culture revealed that the entire elevated rate of preimplantation loss detected in the dominant lethal assay was the result of failure of fertilization; it had no genetic component at all. Postimplantation death is considered a more reliable indicator of dominant lethality than is preimplantation loss. In the MeCl dominant lethal assay, such increased postimplantation loss was detected only when the fertilizing sperm had been present at the site of MeCl-induced acute inflammation in the cauda epididymis. Inflammatory cells, such as those in the MeCl-exposed epididymis, are known to produce a variety of genetic lesions in the DNA of neighboring cells. Therefore, male rats were concurrently exposed to MeCl and treated with an anti-inflammatory agent (BW755C) to inhibit the epididymal inflammation.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of testicular versus epididymal toxicity in the induction of cytotoxic damage in Fischer-344 rat sperm by methyl chloride.

Exposure of male Fischer-344 (F-344) rats to methyl chloride (MeCl) results in testicular and epididymal toxicity and the induction of both pre- and postimplantation embryonic loss; the preimplantation loss is caused by cytotoxic damage to sperm that leads to failure of fertilization (Toxicol Appl Pharmacol 1986; 86:124-130). The present study examined whether the cytotoxicity of MeCl to sperm is due to the testicular or epididymal toxicity of MeCl. Groups of 18 males were exposed to 3000 ppm MeCl 6 h/day for 5 days, with and without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C; 10 mg/kg, i.p. 1 h pre- and postexposure); BW755C was used to inhibit the epididymal toxicity of MeCl. Control groups were untreated or injected as described above with BW755C. Six males from each group were killed weekly for 3 weeks. Toxic effects of MeCl on the testis were demonstrated by decreased relative organ weight (week 3), testicular histopathology (weeks 1-3) and decreased daily sperm production (weeks 1-3); these effects were not prevented by BW755C. In both the MeCl and the MeCl + BW755C treatment groups, tubules devoid of sperm were observed in regions 4 and 5 of the epididymis at week 2, and in regions 6A and 6B at week 3. Sperm were present in the vas deferens of both groups at week 3 in decreased numbers and had decreased motility and more frequent morphologic abnormalities compared to untreated controls. In conjunction with known epididymal transit times for F-344 rat sperm, these data indicate that the induction of preimplantation loss by MeCl at weeks 2 and 3 postexposure is likely to result from cytotoxic effects on sperm located in the testes at the time of exposure.

Role of epididymal inflammation in the induction of dominant lethal mutations in Fischer 344 rat sperm by methyl chloride.

This study assessed the possible relationship between methyl chloride (MeCl)-induced epididymal inflammation and the formation of dominant lethal mutations in sperm of Fischer 344 rats. Groups of 40 males were exposed to MeCl (3000 ppm 6 hr/day for 5 days), with or without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C; 10 mg/kg, i.p. 1 hr pre- and postexposure); BW 755C was shown previously to inhibit MeCl-induced epididymal inflammation. Control groups (n = 20) were either untreated, injected as described above with BW 755C, or injected on the afternoon of day 5 with triethylenemelamine (0.2 mg/kg), a known dominant lethal mutagen. Each male was caged with one female weekly for 3 weeks; 12-18 days after mating, females were killed to assess dominant lethal parameters. In females bred to MeCl-exposed males, significant increases were observed in postimplantation loss at postexposure week 1 (0.84 dead implants per female vs. 0.29 in untreated controls) and in dead implants/total implants at both week 1 (0.10 vs. 0.04 control) and week 2 (0.24 vs. 0.06 control). These increases were not observed in females bred to males treated with BW 755C during MeCl exposure. Coadministration of BW 755C to males along with MeCl also reduced the percentage of mated females with two or more postimplantation losses from 31% to 8% (week 1) and 30% to 12% (week 2). Therefore, the dominant lethal mutations induced by MeCl appear to be a consequence of its induction of inflammation in the epididymis. These data demonstrate the potential genotoxicity of inflammatory processes in vivo.

Inhibition of the acute toxicity of methyl chloride in male B6C3F1 mice by glutathione depletion.

Previous data have demonstrated that methyl chloride (MeCl) is toxic to B6C3F1 mice under both acute and chronic exposure conditions, and that conjugation of MeCl with glutathione (GSH) is a key step in the metabolism of MeCl. This study examined the role of GSH in mediating the acute toxicity of MeCl to liver, kidney, and brain of male B6C3F1 mice. The lethal effects of a single 6-hr inhalation exposure of B6C3F1 males to 2500 ppm MeCl were completely prevented by pretreatment with the GSH synthesis inhibitor, L-buthionine-S,R-sulfoximine (4 mmol L-BSO/kg, ip 1.5 hr prior to MeCl exposure). GSH levels (measured as nonprotein sulfhydryl) in liver and kidney were depleted to 19 and 25% of control values, respectively, at the start of the exposure; the ratio of dead/exposed mice during the 18-hr postexposure declined from 14/15 mice to 0/10. Also, the LC50 for MeCl increased from 2200 to 3200 ppm in male mice pretreated with BSO. The hepatic toxicity of MeCl was detected by increased alanine aminotransferase (ALT) activities in serum 18 hr after a 6-hr exposure to 1500 ppm MeCl (2147 +/- 1327 IU/liter vs 46 +/- 6 in controls). Liver toxicity was inhibited when B6C3F1 males were depleted of GSH prior to MeCl exposure by BSO pretreatment (43 +/- 2), fasting (100 +/- 47), or injection of diethyl maleate (42 +/- 16). The effects of GSH depletion on MeCl toxicity to brain and kidney were determined in B6C3F1 males exposed to 1500 ppm MeCl 6 hr/day, 5 days/week for 2 weeks, with and without daily pretreatment with 2 mmol L-BSO/kg. This dose of BSO depleted hepatic and renal GSH by 28 and 60%, respectively, at the start of MeCl exposure. BSO-pretreated mice were protected from the central nervous system toxicity of MeCl, as assessed by microscopic examination of the granule cell layer of the cerebellum. BSO pretreatment also inhibited the renal toxicity of MeCl as measured by incorporation of [3H]thymidine ([3H]TdR) into renal DNA, an indicator of cell regeneration after cortical necrosis. [3H]TdR incorporation was 105 +/- 10,337 +/- 40, and 60 +/- 15 dpm/microgram DNA in nonexposed controls, MeCl, and MeCl + BSO treatment groups, respectively. These results indicate that GSH is an important component in the toxicity of MeCl to multiple organ systems in B6C3F1 mice. Reaction of MeCl with GSH appears to constitute a mechanism of toxication, contrary to the role usually proposed for GSH in detoxifying xenobiotics.

Inhibition of methyl chloride toxicity in male F-344 rats by the anti-inflammatory agent BW755C.

This study examined the effectiveness of the cyclooxygenase/lipoxygenase inhibitor 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C) in preventing the toxicity induced in male F-344 rats by methyl chloride (MeCl). BW755C (10 mg/kg ip, 1 hr pre and postexposure) prevented both lethality (0/6 vs 8/12 in controls) and epididymal granuloma formation (0/6 vs 4/4 in controls) in rats exposed to 7500 ppm MeCl 6 hr/day for 2 days. Additional rats (n = 5 per group) were exposed to 5000 ppm MeCl 6 hr/day for 5 days, with and without BW755C treatment as described above. The rats were killed on Day 5 and tissues processed for light microscopic examination. MeCl-exposed rats showed hepatocellular cloudy swelling, degeneration of renal proximal convoluted tubules, vacuolar degeneration in the adrenal cortex, necrosis of the internal granular layer of the cerebellum, and degenerative changes in the testis and epididymis, including formation of epididymal sperm granulomas. With the exception of the adrenal, tissues examined in rats of the MeCl/BW755C treatment group showed virtually no histologic evidence of lesions. BW755C did not significantly alter metabolism of [14C]MeCl to 14CO2 or 14C in urine, nor did it affect the distribution to various organs of radioactivity derived from [14C]MeCl. Therefore, BW755C protection against MeCl toxicity did not appear to result from altered MeCl metabolism or disposition. Instead, the protection was apparently related to the pharmacologic activity of BW755C as an inhibitor of leukotriene and prostaglandin synthesis.

Resistance to cadmium-induced necrosis in testes of inbred mice: possible role of a metallothionein-like cadmium-binding protein.

Altered accumulation and subcellular disposition of testicular Cd were examined as possible explanations for the resistance to Cd-induced testicular damage observed in certain inbred strains of mice. Mouse strains susceptible (129/J) or resistant (A/J) to Cd-induced testicular necrosis were injected iv with 10 or 45 mumol CdCl2/kg, respectively. This dosing regimen compensated for decreased Cd accumulation by A/J testes and established similar concentrations of Cd in testes of both strains (approximately 4 nmol Cd/g tissue). Twenty-four hours later, 129/J testes showed marked interstitial hemorrhage and seminiferous tubule necrosis, while A/J testes showed no microscopic evidence of damage. Two hours postinjection, no histopathologic changes were detected in testes of either strain; however, A/J testes had 15% more Cd associated with the cystosol than 129/J testes, and three times more Cd bound to a 14,500 MW cytosolic protein which had gel filtration and ion-exchange chromatography properties in common with metallothionein (MT). Therefore, resistance of A/J testes to Cd does not appear to be determined solely by decreased Cd accumulation, but is associated with increased binding of testicular Cd to a MT-like protein. However, this increase is not accompanied by a proportional increase in the total Cd-binding capacity of the MT-like protein in A/J testes compared to 129/J testes.

Decreased uptake and altered subcellular disposition of testicular cadmium as possible mechanisms of resistance to cadmium-induced testicular necrosis in inbred mice.

Mechanisms responsible for resistance to Cd-induced testicular necrosis in inbred mice were investigated using strains resistant (A/J) or susceptible (129/J) to Cd-induced testicular damage. Cadmium accumulation was measured in testes of both strains 15 min, 30 min, 1 h, 2 h, 6 h, 12 h and 24 h after intravenous injection of 1 mumol 109CdCl2/kg. The subcellular disposition of Cd was determined at 15 min, 6 h and 24 h by fractionation of testicular cytosol on Sephadex G-75 Superfine. Testicular accumulation of Cd was 5-6 times less in A/J mice than in 129/J mice at all time points examined. Gel filtration revealed 4 Cd-binding peaks; in both strains testicular Cd was bound to a protein with a relative molecular mass (Mr) of 30 000 and to metallothionein (MT). The fraction of the total testicular Cd bound to the 30 000 Mr protein was similar in both strains after 15 min (13-18%) and declined rapidly to 5-7% by 6 h. A/J testes had a significantly greater fraction of the total Cd bound to MT both 15 min; 38% vs. 24% at 6 h). By 24 h both strains had approximately 43% of the total testicular Cd bound to MT. The results indicate 2 possible mechanisms of resistance to Cd-induced testicular necrosis in inbred mice: decreased testicular Cd uptake and sequestration of a greater fraction of the tissue Cd by MT.