Antalarmin (calculated molecular weight 378.56) was synthesized by Ash Stevens, Inc. (Detroit, MI), and was supplied by the National Institute of Mental Health (Bethesda, MD). Antalarmin lot numbers PB-V-205 (purity 99.53%) and BM-03-38 (99.76%) were used for these studies. The chemical structure of Antalarmin is shown in .
2.2. Animal welfare
Prior to the initiation of in vivo
experimentation, study protocols involving experimental animals were reviewed and approved by the IIT Research Institute Animal Care and Use Committee. Studies were performed in a laboratory facility that is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Program elements involving animal care and use were performed in compliance with United States Department of Agriculture regulations and the Guide for the Care and Use of Laboratory Animals
(National Research Council, 1996
2.3. Salmonella reverse mutation assay
reverse mutation assay was conducted in accordance with the methods described by Maron and Ames (1983)
. Five Salmonella typhimurium
tester strains were used in this assay; 4 tester strains (TA 98, TA 100, TA 1535 and TA 1537) detect histidine reversion at G-C sites, and one tester strain (TA 102) provides enhanced detection of oxidizing mutagens and hydrazines. All tester strains were obtained from Moltox (Boone, NC), and were evaluated both in the presence and absence of a rat liver enzyme metabolic activation system (Aroclor 1254-induced rat liver S9 fraction [Moltox]).
A 25 mg/ml stock solution of Antalarmin was prepared in dimethyl sulfoxide (DMSO) immediately prior to each assay. Based on the results of a cytotoxicity range-finding assessment, Antalarmin was tested for mutagenicity in triplicate cultures (± S9) at doses of 5, 50, 150, 625 and 1250 μg/plate; triplicate cultures exposed to vehicle only and to appropriate positive control mutagens were included in each assay. Positive control mutagens for studies including S9 were 2-aminoanthracene (TA 98, TA 100, TA 1535 and TA 1537); 2-aminofluorene (TA 98 and TA 100); and danthron (TA 102). Positive control mutagens for studies without S9 were daunomycin (TA 98); methylmethanesulfonate (TA 100); cumene hydroperoxide (TA 102); sodium azide (TA 1535); and ICR-191 (TA 1537).
The number of revertant colonies in each plate was determined using an automatic colony counter. Criteria for a valid assay included: a spontaneous revertant frequency within the historical range from this laboratory; a strain-specific positive response to a positive control mutagen; a minimum of 1 × 107 cfu/plate; no cytotoxicity in cultures incubated with at least five concentrations of Antalarmin; and a phenotypically normal background bacterial lawn (in comparison to vehicle control cultures). The test material was considered mutagenic if at least one non-toxic concentration induced a reproducible two or more-fold increase in the number of revertant colonies per plate (compared to vehicle control), and/or a reproducible dose-response trend was identified in one or more tester strains.
2.4. Mouse lymphoma [L5178Y TK(+/−)] assay
The L5178Y TK(+/−) (clone 3.7.2C) cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in log phase growth by serial subculturing. To reduce the frequency of spontaneous TK(−/−) mutants, pre-existing TK(−/−) mutants were removed from cell cultures by exposure to thymidine, hypoxanthine, methotrexate, and glutamine (THMG) for approximately 24 h; this agent combination selects against the TK(−/−) phenotype.
A stock solution of Antalarmin (25 mg/ml) was prepared in DMSO immediately prior to each assay. For metabolic activation of promutagens, Aroclor 1254-induced rat liver S9 fraction (Moltox) was used. Methyl methanesulfonate served as the positive control article for incubations without S9, and benzo[α]pyrene was used as the positive control article for incubations in which +S9 was included.
Based on cytotoxicity observed in a dose range-finding assay, Antalarmin concentrations of 0.9, 1.9, 3.9, 7.8, 15.6, 31.3, 62.5 and 125 μg/ml were tested in the definitive mutation assay without S9. Antalarmin concentrations of 1.9, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 μg/ml were tested in the definitive mutation assay in the presence of S9. Exposure times in the definitive mutation assays were 24 h (without S9) and 4 h (with S9).
At 12–15 days after exposure to Antalarmin, vehicle only, or positive control mutagens, the number of large and small mutant colonies in each plate were counted using a BIOTRAN II automatic colony counter (New Brunswick Scientific, Edison, NJ). The cloning efficiency, relative cloning efficiency, relative total growth, and mutation frequency were calculated for each plate. A positive mutagenic response was defined as a statistically significant increase in the number of small and/or large colonies in one or more groups treated with Antalarmin versus vehicle control, and/or a dose-related increase in the number of colonies versus vehicle control. A valid assay also required a vehicle control response within the laboratory historical range and a statistically significant (p ≤ 0.05) elevation of the number of colonies in cultures exposed to the appropriate positive control.
2.5. In vivo rat bone marrow micronucleus assay
Male and female CD rats [Crl:CD®
(SD)IGSBR] were received at approximately 6 weeks of age from virus-free colonies maintained at Charles River Laboratories (Kingston, NY); animal husbandry was as described below in section 2.6. Bone marrow micronucleus assays were performed on six rats/sex/group using the same Antalarmin dosing formulations and dose levels (0 [vehicle], 30, 100, and 300 mg/kg/day) used in the subchronic toxicity bioassay. Antalarmin or vehicle was administered orally (by daily gavage) to rats for 14 consecutive days; cyclophosphamide (30 mg/kg) served as the positive control article, and was administered via intravenous injection to the positive control group once on study day 14. All study animals were euthanized by CO2
overdose on study day 15. The femur was excised and the bone marrow was flushed using fetal bovine serum (FBS). Bone marrow cells were suspended in FBS and centrifuged. The supernatant was removed except for a small volume (0.1–0.5 ml) and the cells were resuspended in this volume. Three wedge smears of each suspension were made on glass slides, fixed in methanol, and air dried before staining with acridine orange (Hayashi et al., 1983
). Slides were coded and analyzed by a reviewer who was unaware of the group identity of any slide. At least 2000 polychromatic erythrocytes (PCE) per animal were screened for the presence of micronuclei using a fluorescent microscope and filters for fluorescein isothiocyanate. In order to stabilize the variance, the micronucleus frequency per 2000 PCEs was transformed by adding one to each count and then taking the log of the adjusted number. The transformed micronucleated PCE data were then analyzed by comparing Antalarmin dose groups versus the vehicle control group using a paired t
A positive response was defined as a statistically significant increase in the number of micronucleated cells in one or more groups treated with Antalarmin versus vehicle control and/or a dose-related increase in the number of micronucleated PCE versus vehicle control. A valid assay also required that the vehicle control response fall within the historical range for this laboratory, and a statistically significant (p ≤ 0.05) increase in the number of micronucleated cells in the positive control group (versus vehicle controls).
2.6. Subchronic oral toxicity study in rats
Male and female CD rats [Crl:CD®(SD)IGSBR] were received at approximately 5 to 6 weeks of age from virus-free colonies maintained at Charles River Laboratories (Portage, MI). Rats were housed individually in suspended stainless steel cages in a temperature-controlled room maintained on a 12 h light/dark cycle, and were held in quarantine for two weeks prior to the initiation of drug administration. With the exception of scheduled fasting periods, rats were allowed free access to Certified Rodent Diet 5002 (PMI Nutrition International, Inc., Brentwood, MO). City of Chicago drinking water was supplied to rats ad libitum using an automatic watering system.
After release from quarantine, animals were assigned to experimental groups using a computer-based randomization procedure that blocks for body weights. Groups of 20 rats/sex received daily oral (gavage) exposure to Antalarmin at doses of 30, 100, or 300 mg/kg/day (180, 600, and 1800 mg/m2/day, respectively) for 90 days, or to vehicle only (0.5% [w/v] aqueous carboxymethylcellulose/0.2% [w/v] Tween 80) for the same period. Antalarmin dose levels used in the 90-day toxicity study were selected on the basis of a 14-day range-finding toxicity study. In that study, administration of Antalarmin at 500 or 1000 mg/kg/day for 14 days induced mortality, whereas no limiting toxicity was observed in rats receiving Antalarmin at doses of ≤ 250 mg/kg/day.
Throughout the study, rats were observed a minimum of twice daily to monitor their general health status; detailed clinical examinations and measurements of body weight and food consumption were performed once weekly. To identify possible neurotoxicity, functional observational battery (FOB) evaluations were conducted on 5 rats per sex per group during quarantine (pre-test), during weeks 4, 8 and 12, and during the last week of the recovery period. FOB evaluations in rats included home cage observation, handheld observation, audition (click) reflex response, body temperature, open field (mobility/gait), tail pinch, pupil response, vision, hindlimb extension, catalepsy, grip strength (forelimb and hindlimb), aerial righting reflex, and foot splay. Indirect funduscopic ophthalmic examinations were performed on all study animals during the quarantine period (pre-test) and during the final week of the treatment period.
Blood samples for clinical chemistry, hematology, and coagulation evaluations were collected from fasted rats designated for necropsy at the end of the treatment and recovery periods (days 91 and 121, respectively), and from non-fasted rats during study weeks 5 and 9. Clinical pathology assays were performed using automated instruments (Synchron CX5 Clinical Chemistry Analyzer [Beckman Instruments, Brea, CA]; Advia System 120 Hematology Analyzer [Bayer Corp., Tarrytown, NY]; MLA Electra 900 Automatic Coagulation Timer [Hemoliance, Raritan, NJ]). Overnight urine samples were collected from fasted rats on days 91 and 121, and were analyzed by dipstick and microscopy.
On study day 91, 15 rats/sex/group were euthanized by CO2 overdose and received a complete gross necropsy. All gross lesions plus approximately 45 tissues per rat were collected and fixed in 10% neutral buffered formalin. Remaining rats in each group (designated as recovery animals) were held for an additional 30 days without further treatment, and were euthanized and necropsied (as above) on study day 121. Histologic processing and histopathologic evaluations were performed on all tissues collected at the end of the dosing period from all rats in the high dose and vehicle control groups. Histopathologic evaluation of tissues collected from rats in the low and middle dose groups (euthanized on day 91) and from all rats in the recovery groups (euthanized on day 121) were limited to gross lesions and identified target tissues.
Continuous in vivo data from the rat toxicology study were compared by analysis of variance (ANOVA), followed by post-hoc analysis using Dunnett’s test for comparisons of multiple treatment groups to a single control group. Comparisons of incidence data were performed using by X2 analysis or Fischer’s exact test. A minimum significance level of p ≤ 0.05 was used in all comparisons.
2.7. Subchronic oral toxicity study in dogs
Male and female purebred beagle dogs were received at approximately 6 to 7 months of age from Covance Research Products (Kalamazoo, MI), and were held in quarantine for three weeks prior to randomization into experimental groups. Dogs were housed individually in stainless steel cages in a temperature-controlled room maintained on a 12 h light/dark cycle. Dogs were provided with 400 g of Certified Canine Diet 5007 (PMI Nutrition International, Inc.) for a minimum of 2 h each day, and were permitted free access to City of Chicago drinking water supplied via an automatic watering system. Each dog received a supervised daily exercise period outside of its cage.
After release from quarantine, dogs were assigned to experimental groups using a computerized randomization program that blocks for body weight. Groups of 5 dogs/sex received daily oral (capsule) exposure to Antalarmin at doses of 4, 8 and 16 mg/kg/day (80, 160, and 320 mg/m2/day, respectively) for a minimum of 90 days, or to empty capsules only for the same period. Antalarmin dose levels used in the 90-day toxicity study were selected on the basis of a 14-day range-finding toxicity study. In that range-finding study, administration of Antalarmin at doses of ≥ 50 mg/kg/day was associated with repeated emesis, dose-related decreases in body weight and food consumption, duodenal inflammation, and dose-related bone marrow suppression; these doses were considered to be above the maximum tolerated dose for repeat-dose administration of Antalarmin in dogs. In the range-finding study in dogs, administration of Antalarmin at 10 mg/kg/day induced decreases in white blood cell and reticulocyte counts, lymphoid depletion, and minimal hepatic changes, but did not induce emesis or other clinical evidence of toxicity.
Throughout the study, dogs were observed a minimum of twice daily to monitor their general health status; detailed clinical examinations and measurements of body weight and food consumption were performed once weekly. To identify possible neurotoxic effects of Antalarmin, FOB evaluations were conducted on all dogs once during quarantine (pre-test), during weeks 4, 8 and 12, and during the last week of the recovery period. FOB evaluations in dogs included mental status, gait and posture, righting reflex (with and without sight placing responses), papillary reflex, core body temperature, hopping test, wheelbarrow test, hindlimb and forelimb flexor reflexes, perineal reflex, patellar reflex and menace reflex. Indirect funduscopic ophthalmic examinations were performed on all study animals during the quarantine period (pre-test), during the final week of the treatment period, and during the final week of the recovery period. Electrocardiograms (ECGs) were obtained from all dogs during the quarantine period (pre-test), the final week of the treatment period, and the final week of the recovery period; ECGs were evaluated for heart rate and rhythm, amplitude of the P wave and QRS complex, and duration of the P wave, PR, QRS and QT intervals.
Blood samples for clinical chemistry, hematology and coagulation evaluations were collected from fasted dogs at pre-test, from fasted dogs designated for necropsy (during week 13 and during the final week of the recovery period), and from unfasted dogs during study weeks 4 and 9. Clinical pathology assays were performed using automated instruments (Synchron CX5 Clinical Chemistry Analyzer [Beckman Instruments]; Advia System 120 Hematology Analyzer [Bayer Corp.]; MLA Electra 900 Automatic Coagulation Timer [Hemoliance]). Urine samples were collected from fasted dogs at pre-test, week 13 and during the final week of the recovery period, and were analyzed by dipstick and microscopy.
On day 92, 3 dogs/sex/group were euthanized and necropsied with full tissue collection. The remaining 2 dogs in each group (designated as recovery animals) were held for an additional 29–30 days without further treatment, and were euthanized and necropsied on study days 120–121. At necropsy, all gross lesions and approximately 45 tissues were collected from each animal and fixed in 10% neutral buffered formalin for histopathologic evaluation. All tissues collected from all dogs were processed by routine histologic methods, stained with hematoxylin and eosin, and evaluated histopathologically.
Statistical evaluation of continuous in vivo data from the canine toxicology study was performed by ANOVA, with post-hoc analyses performed using Dunnett’s test. Incidence data were compared by X2 analysis or Fischer’s Exact Test. A minimum significance level of p ≤ 0.05 was used in all comparisons.
2.8. In vitro comparative myelotoxicity assay
In order to determine if the observed species differences in susceptibility to Antalarmin myelotoxicity occurred at the level of the myeloid progenitor cell, and to compare the relative sensitivity of rat, dog, and human myeloid progenitor cells to Antalarmin cytotoxicity, studies were performed to characterize the bone marrow toxicity of Antalarmin using the colony forming unit-granulocyte macrophage (CFU-GM) assay. Human, rat, and canine bone marrow mononuclear cells were isolated and processed in Iscove’s Modified Dulbecco’s Medium (IMDM). Human bone marrow aspirates were obtained from four healthy volunteers (Poietics, Inc., Gaithersburg, MD), and bone marrow mononuclear cells were isolated via centrifugation over a Ficoll density gradient. Rat bone marrow cells were isolated following sterile dissection of femurs from male F344 rats (Battelle Memorial Institute, Columbus, OH); femurs were placed into 50 ml sterile cell culture tubes containing 20 ml nutrient media supplemented with gentamicin (20 μg/ml final concentration). Dog bone marrow aspirates were collected sterilely from the iliac crest or femoral canal of donor beagle dogs into a 50 ml syringe containing sodium heparin and gentamicin to final concentrations of 10 IU/ml and 20 μg/ml, respectively. CFU-GM assays were performed as previously described (Erickson-Miller et al., 1997
; Parchment, 1998
; Parchment et al., 1993
), except that species-specific recombinant GM-CSF was used as the sole cytokine in each culture.
A pilot study of Antalarmin was conducted using a single marrow donor from each species and final test concentrations of 0.01, 0.10, 0.33, 1.0, 3.3 and 10 μg/ml. This pilot study was followed by the conduct of a definitive myelotoxicity study in which eight concentrations of Antalarmin (1, 3.3, 6.7, 10, 25, 33, 67, and 100 μg/ml) were evaluated using bone marrow progenitors collected from three marrow donors per species. In each study, vehicle control cultures contained 0.05% (v/v) DMSO, without added Antalarmin.
CFU-GM colonies were defined as foci of clonal cell proliferation containing at least 64 myeloid cells. All colony morphologies (focal, focal diffuse, mixed) were included, and studies performed using marrow harvested from each donor were considered to be independent experiments. For each donor, colony counts from triplicate cultures were averaged to obtain a single data point at each concentration, and the percent inhibition was determined by comparing the number of colonies in the treatment group to the number of colonies in the vehicle control cultures exposed to DMSO only (vehicle without added drug). Colony counts (normalized as a percentage of control) at each concentration were then plotted to generate the concentration-response curve. As previously reported (Erickson-Miller et al., 1997
; Parchment et al., 1994
; Pessina et al., 2003
), the endpoint of the assay for quantifying inter-species differences in susceptibility to drug toxicity is the IC90
, which is the concentration that inhibits colony formation by 90%. IC90
values were calculated from log-linear regression on concentration-response between the flanking data points on either side of the 90% inhibition level. If the concentration-response curve did not reach the 90% effect level, then the IC90
was projected from the log-linear regression line on the last two data points. In the present studies, insufficient in vitro
toxicity prevented the derivation of IC90
values in myeloid progenitor cells of rat and human origin.