Safety Assessment of Metarrestin in Dogs: A Clinical Candidate Targeting a Subnuclear Structure Unique to Metastatic Cancer Cells.

Abstract

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 hrs (overall median T_max) across all doses with a mean t_1/2 over 168 hours of 55.5 hours. Dose dependent increase in systemic exposure (C_max and AUC_last) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female C_max = 82.5 ng/mL and AUC_last = 2521 hr*ng/mL, on Day 27.

1. Introduction

Tumor metastasis is a major contributor to overall cancer-related mortality (Steeg, 2016). Pancreatic cancer, known to metastasize early, is the fourth leading cause of cancer related mortality in the United States (Ma and Jemal, 2013) and is expected to increase to the second leading cause of death within the next decade (Rahib et al., 2014). The 5-year survival rate for all stages combined is between 7–9%, lowest when compared to other cancers. More than half of all pancreatic cancer patients present with metastatic disease, which carries a 5-year survival rate of 2–3% (Rahib et al., 2014; Siegel et al., 2019). The current National Comprehensive Cancer Network and European Society for Medical Oncology guidelines recommend combination chemotherapy with nab-Paclitaxel (Abraxene)/Gemcitabine (nab-P/G) or FOLFIRINOX as the preferred first-line treatments for patients with metastatic pancreatic cancer who have good performance status (Ducreux et al., 2015). However, there are currently no treatments available which specifically target metastasis.

Metarrestin was discovered from a high content drug screen of a > 140,000 compound library (Frankowski et al., 2010; Norton et al., 2009a). It disassembles the perinucleolar compartment (PNCs) at submicromolar concentrations in solid organ cancer cell lines, suppresses metastasis formation, and extends survival in preclinical cancer models including xenotransplantation models (Frankowski et al., 2018). PNC is a small subnuclear organelle located adjacent to the nucleolus (Norton et al., 2009b; Pollock and Huang, 2009; Slusarczyk et al., 2010). The PNC is enriched with RNA polymerase III transcripts, small nuclear RNAs, and RNA binding proteins (Slusarczyk et al., 2010; Wang et al., 2003). PNCs are detected in metastatic solid tumors and transformed cell lines including breast, prostate, pancreatic and colorectal cancers, but absent in normal cells including embryonic stem cells (Kamath et al., 2005; Norton et al., 2008; Wen et al., 2013). In other studies of clinical samples from patients with breast, colon, ovarian, and hepatocellular cancer PNC incidence is both a strong predictive and prognostic marker of overall patients’ clinical outcome (Kamath et al., 2005; Liu et al., 2019; Norton et al., 2008; Slusarczyk et al., 2010) suggesting that PNCs are a putative drug target for metastatic cancers including pancreatic cancer. Although the exact function of PNCs remains unclear, studies have linked possible roles of the PNC to RNA metabolism (Even et al., 2016; Norton and Huang, 2013; Politz et al., 2013; Pollock et al., 2011), therefore, inhibition of PNCs could limit cancer cell growth and migration by inhibition of ribosomal biogenesis and dysregulation of ribosome function. Considering metarrestin’s promising preclinical activity and novel mechanism of action, we performed in-vitro and in-vivo nonclinical safety studies in dogs to support its clinical evaluation for metastatic pancreatic cancer.

2. Materials and methods

2.1. Test and Vehicle Control Articles

The test article, metarrestin [ML246; trans-4-(7-benzyl-4-imino-5,6-diphenyl-4,7-dihydro-3H- pyrrolo[2,3-D]pyrimidin-3-yl) cyclohexanol], was obtained from Suven Life Sciences, Hyderabad, India. The control articles were Labrasol ^® ALF (Gattefossé, Saint-Priest/Lyon, France) and caprylic acid (MP Biomedicals, Solon, OH), which when prepared as 80% (v/v) Labrasol ^® ALF / 20% (v/v) caprylic acid constituted the vehicle control article. Dosing formulations of the test article (20 mg/mL bulk concentration) were prepared in a vehicle of 80% (v/v) Labrasol® ALF/20% (v/v) caprylic acid. The test article and vehicle in liquid formulation was administered orally as capsule(s) as this is the intended route of administration in humans.

2.2. Test System

Dogs:

Purebred beagle dogs were purchased from Ridglan Farms (Mt. Horeb, WI) with body weights ranged from 7.78 kg to 9.94 kg for males and from 6.44 kg to 8.38 kg for females. Certified Global 21% Protein Dog Diet #2021C [obtained from the Madison, WI, facility of Teklad Laboratory Animal Diets, Envigo (Huntingdon, UK)] was made available to each animal for a minimum of four hours daily. Food was provided approximately two hours after dosing and Water was provided ad libitum via an automatic watering system. All in-vivo study procedures followed applicable animal welfare acts and were approved by the Institutional Animal Care and Use Committee (IACUC). All the studies were conducted in compliance with the U.S. Food and Drug Administration Good Laboratory Practice (GLP) Regulations (Code of Federal Regulations Title 21 Part 58).

HEK-293 cells:

hERG potassium channels are expressed in the human embryonic kidney cell line HEK293 (ATCC, Manassas, VA) that lacks endogenous I_Kr. HEK-293 cells were stably transfected with hERG cDNA. Stable transfectants have been selected by co-expression with the G418 resistance gene incorporated into the expression plasmid. Selection pressure was maintained by including G418 in the culture medium. Cells were cultured in a 50:50 mix of Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 500 ug/mL G418.

2.3. Dog 28 day (14-doses) toxicity and toxicokinetics study.

Forty Dogs were divided into four dose groups and were administered every other day for 28 days with vehicle, 0.25, 0.75 or 1.50 mg metarrestin/kg body weight (Table 1) via oral route. Two dogs/sex/group were observed for 28 days following dosing phase for recovery. The doses were selected based on the range finding study. Assessment of toxicity was based on mortality/moribundity, clinical observations, body weight, food consumption, body temperature, ophthalmic examination, and clinical (during pre-test and on days 14, 28 and 57) and anatomic (on day 29 terminal sacrifice in dosing phase and on day 57 recovery phase) pathology.

Table 1:

Dog GLP study design.

Group	Dose Level (mg/kg/ dose)	Dose Concentration (mg/mL)	Capsule Volume (mL/kg)	Number of Dogsa,b,c
				M		F
				Main Study	Recovery	Main Study	Recovery
1	Vehicle (0)	0	0.0750	3	2	3	2
2	Metarrestin (0.25)	20	0.0125	3	2	3	2
3	Metarrestin (0.75)	20	0.0375	3	2	3	2
4	Metarrestin (1.50)	20	0.0750	3	2	3	2

^aMain study animals (3/sex/group) were sacrificed on Day 29 and the remaining recovery animals (2/sex/group) were sacrificed on Day 57 after a 29-day recovery period.

^bEach dog got one capsule.

^cEvery other day for 28 days (14 doses) was the dosing frequency.

ECG tracings were obtained from all dogs during pre-test, Study Week 4 (approximately 2 hours after dosing), and the last week of the recovery period. Analysis included heart rate and rhythm, amplitude of the P wave and QRS complex, and duration of the P wave, QRS and QT intervals. Blood pressure measurements using a tail cuff were performed at the same time as the ECGs. Pulmonary function measurements were collected during pre-test and at approximately 2 hours after dosing during Weeks 1 and 4. Pulmonary parameters (respiratory rate and signs) were measured qualitatively.

Blood samples for determination of plasma metarrestin levels were obtained as described below:

Group 1: On Study Days 1 and 27, blood was drawn from three dogs/sex (Main Study) approximately two hours after dosing.

Groups 2–4: On Study Days 1 and 27, blood was drawn from three dogs/sex (Main Study) at seven time points: before dosing and approximately 0.5, 2, 4, 8, 24 and 48 hours after dosing. Additionally, blood was drawn from two dogs/sex (Recovery) at approximately 96 and 168 hours post-dose (relative to Day 27 dosing; Study Days 31 and 34, respectively).

Plasma concentration-time profiles of metarrestin in individual dogs at scheduled sampling times were analyzed by non-compartmental toxicokinetic methods. Toxicokinetic (TK) modeling was performed using Phoenix WinNonlin software, Version 6.3 (Certara, Princeton, NJ). Dogs were fasted overnight prior to blood collection (except during pre-test). On study day 29 (main study dogs) and study day 57 (recovery dogs), urine samples were collected directly from the urinary bladder at necropsy. Animals were fasted overnight prior to necropsy and were anesthetized with sodium pentobarbital and euthanized by exsanguination. Weights of the adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, and thyroids were collected at necropsy, and all gross lesions and approximately 50 tissues were collected from each animal and fixed in 10% neutral buffered formalin with the exceptions of the eyes (with optic nerves), which were fixed with Davidson’s solution; the testes and epididymides, which were fixed with modified Davidson’s solution; and the bone marrow smear, which was fixed in methanol (Bourdi et al., 2018; Johnson et al., 2011). All tissues collected from dogs in the high dose and control groups in the Main Study were processed by routine histologic methods, cut at 5um, stained with hematoxylin and eosin, and evaluated by a board-certified veterinary pathologist. Target tissues from Main Study dogs in the mid and low dose groups and from Recovery dogs were evaluated.

2.4. Dog cardiovascular and respiratory safety pharmacology study

Four male and four female, purebred beagle dogs from telemetry dog colony were used for this study. Dosing sequence assignments were determined using a balanced Latin-Square crossover design to maximize the number of dosing combinations possible. Each dose was followed by a washout period lasting at least seven days. In this non-terminal study, the dogs were subjected to cardiovascular and respiratory function measurements. The cardiovascular measurements were taken on study days 1, 8, 15 and 22; and respiratory function measurements were taken on study days 29, 36, 43 and 50 (Table 2). Surgically implanted telemetry devices (DSI; St. Paul, MN) continuously monitored the following parameters approximately twenty-four hours prior to each dose and until at least forty-eight hours after each dosing event: body temperature; heart rate; systolic, diastolic, and mean blood pressure; and electrocardiogram (ECG) data (in milliseconds; PR, RR, QTc, and QRS intervals). ECG intervals (PR, RR, QRS, and QTc) were measured on the ECG waveform tracings at hourly intervals from 24 hours pre-dose to 48 hours post-dose using DSI Physiostat ECG Analysis software. During respiratory measurements, animals were placed in restraining slings and a pneumotachograph, which was attached to a mask positioned over each animal’s muzzle, measured the following respiratory parameters: respiratory rate (breathing frequency), tidal volume, and minute volume (calculated). Respiratory measurements were taken for five minutes prior to each dose and approximately 1, 2, 3 and 4 hours after each dose. Assessment of general health of the dogs was based on mortality/moribundity, clinical observations, body weight, body temperature and clinical pathology.

Table 2:

Dog safety pharmacology study.

Animals	Study Day a, b
	1 and 29	8 and 36	15 and 43	22 and 50
Male 1 and Female 1	Low Dose	High Dose	Mid Dose	Control
Male 2 and Female 2	Mid Dose	Low Dose	Control	High Dose
Male 3 and Female 3	Control	Mid Dose	High Dose	Low Dose
Male 4 and Female 4	High Dose	Control	Low Dose	Mid Dose

^aCardiovascular measurements were collected on Days 1, 8, 15 and 22; respiratory function measurements were collected on Days 29, 36, 43 and 50.

^bControl and Low, Mid and High dose = 0 (empty capsule), 0.25, 0.75 and 1.50 mg/kg, respectively.

2.5. hERG assay

Metarrestin was evaluated in-vitro for inhibitory e□ects on the hERG (human ether-à-go-go-related gene) channel current (a surrogate for I_Kr, the rapidly activating delayed rectifier cardiac potassium current) (Redfern et al., 2003). The concentration-response relationship of the effect of metarrestin (0.03, 0.1, 0.3, 3 and 30 μM) on the hERG potassium channel current was evaluated at near-physiological temperature in stably transfected HEK-293 cells that express the hERG gene. This assay was conducted according to the study-specific procedure of the test facility and available literature (Kirsch et al., 2004).

2.6. Data evaluation and statistical analysis

2.6.1. Dog 28 day (14-doses) toxicity study.

Descriptive statistics (mean and standard deviation) were calculated and analyzed for statistical significance for body weight/body weight change, food consumption, clinical pathology [clinical chemistry, hematology, coagulation and select urinalysis parameters (refractive index, specific gravity and pH)], and organ weight/organ-to-body weight ratio data using the ToxData® system. Blood pressure and respiratory rate were analyzed for statistical significance using SigmaPlot software (version 13.0; Systat Software, Inc.; Chicago, IL). Recovery phase data were not statistically analyzed due to the small number of animals (2/sex/group).

For all analyses, if the data set was normally distributed and of equal variance, statistical comparisons were conducted using a one-way analysis of variance (ANOVA), with post hoc comparisons made (if necessary) using Dunnett’s test. If normality and/or equal variance failed for a data set, statistical comparisons were conducted using nonparametric Kruskal-Wallis ANOVA, with post hoc comparisons made (if necessary) using Dunn’s test. Incidence data (e.g., clinical signs, physical examinations) were evaluated using Chi-square analysis and/or Fisher’s Exact test. A minimum significance level of p<0.05 was used for the statistical comparisons in this study.

2.6.2. Cardiovascular and respiratory safety pharmacology study

Body weight/body weight change data and adjusted body temperature, heart rate, blood pressure (systolic, diastolic, mean), electrocardiogram (PR, RR, QTc, QRS), and respiratory function (respiratory rate, tidal volume, minute volume) data were analyzed for statistical significance using analysis of variance (ANOVA), with post hoc Dunnett’s test comparisons as necessary with SYSTAT software (version 10.2; Systat Software Inc.; Chicago, IL) or SigmaPlot® software (version 13.0; Systat Software, Inc.). A minimum significance level of p ≤ 0.05 was used for the statistical comparisons in this study.

For each analyzed endpoint (except body weight/body weight change), data collected during the corresponding time interval of the 24-hour period prior to dosing was used as a baseline value. (For respiratory function parameters, a single baseline value was collected for each animal.) Data collected within the 48-hour (cardiovascular) or 4-hour (respiratory) period following dosing were averaged per hour, and these post-dosing hourly averages were “baseline-adjusted” by calculating the percent change from the baseline average [percent change = (unadjusted post-dose value - baseline {i.e., average} value) ÷ baseline {i.e., average} value * 100]. The adjusted values for each endpoint at each time interval were statistically analyzed using group-to-group comparisons.

3. Results

3.1. Dog 28 day (14- Doses) GLP study

All 40 dogs survived the 28-day dosing period. Treatment-related clinical observations seen in dogs at the 0.75 and/or 1.50 mg/kg dose levels consisted of hypoactivity, shaking/shivering, thinness, irritability, vomiting, salivation, discolored inguinal fur, abnormal gait, tremors, intermittent seizure-like activity, and ataxia. The seizure-like activity was seen in 2 out of 10 male dogs at the 0.75 mg/kg dose level and in 4 out of 10 male and 2 out of 10 female dogs at the 1.50 mg/kg dose level. This seizure-like activity was intermittent, limited to occur on one day only, with the exception of one of the female dogs at the 1.50 mg/kg dose level, in which they occurred on three separate days during the dosing period. In addition, one male dog in the 1.50 mg/kg dose group was prostrate on Day 21. Hypoactivity, shaking/shivering, and abnormal gait were observed in one male dog at the 0.25 mg/kg dose level. All dogs were normal at the end of the 29-day recovery period. No treatment-related effect on body temperature was seen in either sex at any dose level. Mean body weight gain was statistically significantly decreased in both males and females at the 1.50 mg/kg dose level during the first week of dosing (Table 3). In males, mean body weight was decreased 5% compared to the control group, while mean body weight gain was decreased 94% compared to the control group. Females lost 6% of body weight compared to the control group. At the end of the 28-day treatment period, mean weekly body weight gain was statistically significantly decreased (84%) in the Group 4 (1.50 mg/kg) males. Mean body weight gain was decreased 41% and 51% in the Group 2 (0.25 mg/kg) and Group 3 (0.75 mg/kg) males, respectively, while mean body weight gain was decreased 26% in the Group 3 females at the end of the 28-day treatment period. All test article-treated dogs gained weight during the 29-day recovery period. Mean food consumption was statistically significantly decreased on Day 11 in the Group 4 (1.50 mg/kg) males and on Days 7, 13 and 25 in the Group 4 females. The only statistically significant effects on organ weight were decreased absolute and relative thymus weights in the Group 4 (1.50 mg/kg) males. Administration of metarrestin resulted in the following observations: gross (small) and microscopic findings in the thymus (atrophy) at 1.50 mg/kg (Table 4); no gross findings with microscopic findings in the thymus [atrophy (females only)] at 0.75 mg/kg and no gross or microscopic findings at 0.25 mg/kg. At the end of the recovery period, there was nearly complete recovery of the thymic atrophy.

Table 3:

Males (M) and Females (F) Summary of Weekly Body Weight Change Data (kg)

Day		G 1 /M 0 mg/kg	G 2/M 0.25 mg/kg	G 3/M 0.75 mg/kg	G 4/M 1.50 mg/kg
d 1 to 8	Mean	0.72d	0.41	0.39	0.04**
	S.D.	0.310	0.112	0.464	0.161
	N	5	5	5	5
	P-Value	0.0176	0.2603	0.2117	0.0055
d 8 to 15	Mean	0.14a	0.28	−0.11	−0.09
	S.D.	0.110	0.182	0.300	0.292
	N	5	5	5	5
	P-Value	0.0503
d 15 to 22	Mean	0.42 k	0.11	0.47	0.36
	S.D.	0.125	0.488	0.730	0.647
	N P-Value	5 0.5406	5	5	5
d 22 to 28	Mean	0.06k	0.00	−0.08	−0.08
	S.D.	0.121	0.366	0.340	0.368
	N	5	5	5	5
	P-Value	0.9266
d 1 to 28	Mean	1.36d	0.80	0.67	0.22**
	S.D.	0.517	0.484	0.268	0.410
	N	5	5	5	5
	P-Value	0.0067	0.1431	0.0585	0.0020
		G 1 / F 0 mg/kg	G 2 / F 0.25 mg/kg	G 3 IF 0.75 mg/kg	G 4/ F 1.50 mg/kg
d 1 to 8	Mean	−0.03d	0.09	0.06	−0.38*
	S.D.	0.090	0.226	0.141	0.313
	N	5	5	5	5
	P-Value	0.0095	0.6999	0.8633	0.0426
d 8 to 15	Mean	0.15a	0.20	−0.14	−0.07
	S.D.	0.211	0.111	0.287	0.199
	N	5	5	5	5
	P-Value	0.0595
d 15to 22	Mean	0.30k	0.36	0.58	0.43
	S.D.	0.279	0.325	0.113	0.285
	N	5	5	5	5
	P-Value	0.2978
d 22 to 28	Mean	0.12 a	−0.03	−0.08	−0.05
	S.D.	0.167	0.181	0.221	0.473
	N	5	5	5	5
	P-Value	0.6925
d 1 to28	Mean	0.54k	0.61	0.40	−0.07
	S.D.	0.462	0.461	0.138	0.326
	N	5	5	5	5
	P-Value	0.0749

^dANOVA-DUNNETT;

^*p < 0.05;

^**p < 0.01;

^aANOVA;

^kKRUSKAL-WALLIS

Table 4:

Summary of Microscopic Findings in Thymus

Terminal day 29	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	0.25	0.75	1.50	0	0.25	0.75	1.50
No. Animals Examined	3	3	3	3	3	3	3	3
Thymus (No. Examined)	3	3	3	3	3	3	3	3
Atrophy	(0)a	(0)	(0)	(2)	(0)	(0)	(1)	(2)
Mild	0	0	0	1	0	0	0	1
Moderate	0	0	0	1	0	0	1	0
Marked	0	0	0	0	0	0	0	1
Recovery day 57	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg/day)	0	0.25	0.75	1.50	0	0.25	0.75	1.50
No. Animals Examined	2	2	2	2	2	2	2	2
Thymus (No. Examined)	2	0	0	2	2	0	2	2
Atrophy	(0)a	-	-	(0)	(0)	-	(1)	(0)
Minimal	0	-	-	0	0	-	1	0

^aNumbers in parentheses represent the number of animals with the finding.

There were no ocular or cardiovascular (ECG) treatment-related abnormalities seen in either sex at the end of the 28-day treatment period. Heart beats per minute were statistically significantly increased at Week 4 in the Group 2 (0.25 mg/kg) females; however, the increase was not considered treatment-related due to the lack of a dose response. Minimal increases upto 23% in mean systolic, diastolic and arterial blood pressure were seen in all dose groups for both sexes at Week 4 compared to the control group (Table 5 and 6). Blood pressures were still increased at the end of the recovery period (Week 8) for both sexes in all dose groups, except for the Group 4 (1.50 mg/kg) females. None of these increases were statistically significant (Table 5 and 6). These trends of increase are mostly due to animal struggle during measurement, especially in males metarrestin-treated dogs. Two female dogs data could not be collected in high dose for the same reason (Table 6 (N=3 instead of 5)). There were no treatment-related effects on respiratory rate observed in either sex during or at the end of the 28-day dosing period. There were also no treatment-related effects on any hematology, clinical chemistry, coagulation or urine parameters indicative of any systemic toxicity seen on Day 14 or 28 following metarrestin administration.

Table 5:

Summary of Blood Pressure Data- Treatment and Recovery (MALE)

Dose Group		Week 4 (Approximately 2 Hours Post-Dose)
		Blood Pressure		Arterial Pressure	Heartbeats per minute
		Systolic (mmHg)	Diastolic (mmHg)
G 1 / M 0 mg/kg	Mean	135 a	86 a	98 a	102 a
	S.D.	15.5	20.7	16.0	27.2
	N	5	5	5	5
G 2 / M 0.25 mg/kg	Mean	154	103	115	124
	S.D.	27.2	18.3	18.0	37.4
	N	5	5	5	5
G 3 / M 0.75 mg/kg	Mean	156	92	108	95
	S.D.	50.6	36.6	38.4	25.9
	N	5	5	5	5
G 4 / M 1.50 mg/kg	Mean	164	106	122	94
	S.D.	40.0	37.4	34.7	46.6
	N	5	5	5	5
Dose Group		Week 8b
		Blood Pressure		Arterial Pressure	Heartbeats per minute
		Systolic (mmHg)	Diastolic (mmHg)
G 1 / M 0 mg/kg	Mean	124	75	90	124
	S.D.	21.2	3.5	8.5	24.0
	N	2	2	2	2
G 2 / M 0.25 mg/kg	Mean	182	113	129	113
	S.D.	16.3	12.0	11.3	21.2
	N	2	2	2	2
G 3 / M 0.75 mg/kg	Mean	174	116	131	136
	S.D.	42.4	42.4	36.1	16.3
	N	2	2	2	2
G 4 / M 1.50 mg/kg	Mean	154	116	126	108
	S.D.	26.9	1.4	8.5	60.1
	N	2	2	2	2

^aANOVA

^bRecovery phase data not statistically analyzed due to the small number of animals.

Table 6:

Summary of Blood Pressure Data- Treatment and Recovery (FEMALE)

Dose Group		Week 4 (Approximately 2 Hours Post-Dose)
		Blood Pressure		Arterial Pressure	Heartbeats per minute
		Systolic (mmHg)	Diastolic (mmHg)
G 1 / F 0 mg/kg	Mean	106 a	73 a	80 a	88 a
	S.D.	29.1	17.4	19.4	23.8
	N	5	5	5	5
G 2 / F 0.25 mg/kg	Mean	129	87	97	141 *
	S.D.	26.4	27.1	27.1	13.1
	N	5	5	5	5
G 3 / F 0.75 mg/kg	Mean	117	81	89	111
	S.D.	24.6	27.6	25.7	28.3
	N	5	5	5	5
G 4 / F 1.50 mg/kg	Mean	127	76	88	108
	S.D.	62.2	26.3	34.8	6.0
	N	3	3	3	3
Dose Group		Week 8b
		Blood Pressure		Arterial Pressure	Heartbeats per minute
		Systolic (mmHg)	Diastolic (mmHg)
G 1 / F 0 mg/kg	Mean	116	87	92	98
	S.D.	50.2	46.7	46.7	4.2
	N	2	2	2	2
G 2 / F 0.25 mg/kg	Mean	145	91	100	139
	S.D.	49.5	11.3	1.4	31.8
	N	2	2	2	2
G 3 / F 0.75 mg/kg	Mean	154	89	100	119
	S.D.	14.1	4.9	9.2	5.7
	N	2	2	2	2
G 4 / F 1.50 mg/kg	Mean	112	78	83	98
	S.D.	38.9	34.6	33.2	29.0
	N	2	2	2	2

^aANOVA

^*p < 0.05 (vs. control group)

^bRecovery phase data not statistically analyzed due to the small number of animals.

After oral administration of metarrestin to female and male dogs, overall median T_max across all doses, sexes and study days was 3 hours (2 to 48 hours). There was no difference in median T_max between females and males, nor was there a consistent relationship to dose. Median T_max on Day 1 was 4 hours (2 to 48 hours) compared to 2 hours (2 to 4 hours) on Day 27 (Figure 1 and 2). On Day 27, mean t_1/2 over 168 hours was estimated to be 55.5 hours. Mean t_1/2 was similar between females and males and did not vary over all doses. When considering over all doses and both study days, there was either no difference or a slight difference in systemic exposure between females and males (Table 7). The ratio of female/male C_max was 0.91. AUC_last was slightly higher in females than males, with a ratio of 1.08. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. The accumulation ratios of both C_max and AUC_last were similar, as were the accumulation ratios of females and males. Systemic exposure to metarrestin (as C_max and AUC_last) increased as dose increased over a range of 0.25 to 1.5 mg/kg on both Days 1 and 27 (Figure 3 and 4). From 0.25 to 0.75 mg/kg, the increase was approximately proportional to dose. From 0.75 to 1.5 mg/kg, the increase was generally greater than proportional to dose.

Figure 1:

Mean Plasma Concentration vs. Time to 168 hr beginning Day 27 after Oral Administration of Metarrestin at Doses from 0.25 to 1.5 mg/kg to Female Dogs

Figure 2:

Mean Plasma Concentration vs. Time to 168 hr beginning Day 27 after Oral Administration of Metarrestin at Doses from 0.25 to 1.5 mg/kg to Male Dogs

Table 7:

Dose Proportionality of Mean C_max and AUC_last from 0.25 to 1.5 mg/kg in Female and Male Dogs on Days 1 and 27 after Oral Administration of Metarrestin

Day	Sex	Dose (mg/kg)	Fold-increase	Cmax ng/mL	Fold-increase	AUClast hr*ng/mL	Fold-increase
1	Female	0.25	1.00	33.7	1.00	1259	1.00
		0.75	3.00	81.9	2.43	2822	2.24
		1.5	6.00	228	6.76	7831	6.22
	Male	0.25	1.00	37.2	1.00	1068	1.00
		0.75	3.00	102	2.76	2919	2.73
		1.5	6.00	278	7.49	7176	6.72
28	Female	0.25	1.00	78.2	1.00	2778	1.00
		0.75	3.00	220	2.82	6998	2.52
		1.5	6.00	544	6.96	18407	6.63
	Male	0.25	1.00	87.4	1.00	2264	1.00
		0.75	3.00	289	3.31	9077	4.01
		1.5	6.00	429	4.91	14630	6.46

Figure 3:

C_max and AUC_last vs. Dose on Day 1 after Oral Administration of Metarrestin at Doses of 0.25, 0.75 and 1.5 mg/kg to Female and Male Dogs

Figure 4:

C_max and AUC_last vs. Dose on Day 27 after Oral Administration of Metarrestin at Doses of 0.25, 0.75 and 1.5 mg/kg to Female and Male Dogs

Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female C_max = 82.5 ng/mL and AUC_last = 2521 hr*ng/mL, on Day 27.

3.2. Cardiovascular and Respiratory Safety Pharmacology Study of Metarrestin in Telemetered Dogs

The administration of metarrestin at dose levels of 0, 0.25, 0.75 and 1.50 mg/kg in beagle dogs did not result in any clinical signs of toxicity or any treatment-related effects on body weight/body weight change, body temperature, arterial blood pressure (Figure 5), heart rate (Figure 6), corrected QT, PR, RR, or QRS intervals, respiratory function parameters (respiratory rate, tidal volume, or minute volume), or clinical pathology parameters (clinical chemistry, hematology, or coagulation). In males there is a non-significant increase in mean blood pressure at the high dose at >20 hr post dose.

Figure 5:

Group Mean Blood Pressure (Mean)

Figure 6:

Group Mean Heart Rate

3.3. Effect of Metarrestin on Cloned hERG Potassium Channels Expressed in Human Embryonic Kidney Cells

Metarrestin inhibited hERG current by (Mean ± SEM) 16.1 ± 0.8% at 0.03 μM (n = 3), 41.8 ± 4.2% at 0.1 μM (n = 4), 71.6 ± 2.4% at 0.3 μM (n = 4), 101.8 ± 0.8% at 3 μM (n =3) and 102.5 ± 0.4% at 30 μM (n = 3) versus 2.5 ± 0.7% (n = 3) in control (Table 8). hERG inhibition at 0.03, 0.1, 0.3, 3 and 30 μM was statistically significant (P < 0.05) when compared to vehicle control values. Nominal concentrations were used to evaluate the concentration-response relationship. As a result, the estimated IC₅₀ value could be over- or under-estimated. The positive control terfenadine inhibited hERG potassium current by (Mean ± SD; n = 2) 91.2 ± 0.5 at 60 nM. This result confirms the sensitivity of the test system to hERG inhibition. The IC₅₀ for the inhibitory effect of metarrestin on hERG potassium current was 0.13 μM (Hill coefficient = 1.2).

Table 8:

Metarrestin Summary Statistics- Mean percent hERG inhibition at each Metarrestin concentration (Mean), standard deviation (SD), standard error of the mean (SEM) and number of cells (N).

Concentration (μM)	Mean	SD	SEM	N
0	2.5%	1.1%	0.7%	3
0.03	16.1%*	1.4%	0.8%	3
0.1	41.8%*	8.4%	4.2%	4
0.3	71.6%*	4.8%	2.4%	4
3	101.8%*	1.4%	0.8%	3
30	102.5%*	0.7%	0.4%	3

^*Value is statistically different from vehicle alone.

4. Discussion

Metastasis involves complex, multistep processes (Waclaw et al., 2015; Yachida et al., 2012). Despite decades of extensive research efforts, the main mechanisms of cancer progression and metastasis are still not completely understood. Metarrestin, a small molecule selective against metastasis across different preclinical cancer, was discovered from a high content drug screen of a >140,000 compound library (Frankowski et al., 2010; Norton et al., 2009a). Metarrestin inhibits Pol I transcription, induces nucleolar segregation, reduces nucleolar volume, and disrupts PNCs, in part by interfering with eEF1A2 function, selectively in cancer but not normal cells (Frankowski et al., 2018). Metarrestin disrupted PNCs in PC3M-GFP-PTB cells with an IC₅₀ of 0.39□M without acute cytotoxicity. Previous study in mice showed no toxicities at efficacious doses ranging between 5 and 25 mg/kg (Frankowski et al., 2010; Frankowski et al., 2018). A pharmacokinetic profile of metarrestin in wild-type and tumor-bearing KPC mice revealed a high oral bioavailability, a large volume of distribution, relatively long half-life, and moderate clearance with accumulation in tumor tissues (Vilimas et al., 2018). The excellent tissue penetration of metarrestin and the monitorable toxicity support it as therapeutic drug candidate.

In this study, metarrestin was administered orally (via capsule) to beagle dogs every other day for 28 days (14 doses) at dose levels of 0.25, 0.75 and 1.50 mg/kg. The doses were selected based on previous dose range finding study. Every other day dosing schedule was chosen due to its relatively long half life (~55hr) and 28 day dosing duration was selected to provide more flexibility for clinical trial duration upto 28 day, if warranted. There was no mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting primarily of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like symptoms were seen in both sexes at the 0.75 and 1.50 mg/kg dose levels. Interestingly, no statistically significant differences were noted in a standard battery of FOB tests in the rat toxicology study (unpublished data). The mechanism behind the seizures is unknown but could likely be due to inhibition of eEF1A2 by metarrestin in the CNS. eEF1A2 is upregulated/re-expressed in cancers, including breast, pancreas, prostate, ovarian cancer, where its expression levels correlate with disease outcomes (Pinke et al., 2008; Tomlinson et al., 2005; Worst et al., 2017). Importantly, eEF1A2 is, with the exception of low level expression in neuronal cells and skeletal myocytes, not expressed in normal tissues (Murray et al., 2008). Metarrestin crosses the blood brain barrier, albeit yielding a 10- to 100-fold lower exposure levels in brain than in other tissues including tumor (Vilimas et al., 2018). Metarrestin-induced eEF1A2 inhibition is involved in the drug-induced neurological phenotype is supported by: (1) eEF1A2^−/− mice display a similar phenotype to metarrestin treatment including seizures, ataxia, and neurological wasting (Abbott et al., 2009; Davies et al., 2017), and (2) children in families with rare autosomal-recessive eEF1A2 loss-of-function germline mutations suffer from seizures, epilepsy, and neurodevelopmental delay (McLachlan et al., 2019). It is speculated that loss of eEF1A2 function affects axonal transport of neurotransmitters to the synapse (McLachlan et al., 2019). A relative imbalance of brain neurotransmitters may predispose to seizures activity. A depletion of the inhibitory neurotransmitter gama-aminobutyric acid (GABA) may trigger seizures (Olsen and Avoli, 1997). The seizure-like activity were seen in two male dogs at the 0.75 mg/kg dose level and in four male and two female dogs at the 1.50 mg/kg dose level, and were most likely neurologic in origin. They were intermittent, occurring on only one day (day 5 for one female and four males group 4; day 8 for one male group 3 and day 12 for another male group 3), with the exception of one of the female dogs at the 1.50 mg/kg dose level, in which the seizure-like activity occurred on three separate days (day 11; 17 and 23) during the dosing period. In addition, one male dog in the 1.50 mg/kg dose group was prostrate on Day 21. All dogs were normal at the end of the 29-day recovery period.

Due to the seizure like activity observed in this study it is highly recommended that precautions should be exercised during the clinical trials such as no swimming alone, no climbing on ladders or heights unsupervised, in addition to the precaution about driving. No treatment-related cardiovascular effects were observed in either males or females at the end of the 28-day treatment period. Minimal increase (upto 23%) in blood pressure was seen in all dose groups for both sexes at Week 4 compared to the control group. Since the increase in blood pressure was small in magnitude it was considered non-adverse and within biological variability. Heart beats per minute were statistically significantly increased at Week 4 in the Group 2 (0.25 mg/kg) females (Table 6); however, the increase was not considered treatment-related due to the lack of a dose response.

Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Since no gastrointestinal toxicity or emesis was observed, the changes on body weight and food consumption could be due to decrease of appetite. There were no treatment-related effects on body temperature or clinical pathology parameters (hematology, clinical chemistry, coagulation and urinalysis) seen in either sex (data not shown). The only statistically significant effects on organ weight were decreased absolute and relative thymus weights in 1.50 mg/kg males with microscopic finding of thymic atrophy in males and females at 1.50 mg/kg and in females at 0.75 mg/kg (Table 4). At the end of the recovery period, there was nearly complete recovery of the thymic atrophy. Thymic changes were not considered adverse since these could have been attributed to stress and were fully recoverable.

Safety pharmacology helps predict whether a drug is likely to cause cardiovascular, respiratory, and/or central nervous systems serious adverse effects if administered to humans (Grandi et al., 2018). The primary cardiovascular test battery includes an evaluation of proarrhythmic risk using in-vitro hERG assays and in-vivo measurements in conscious animals via telemetry. A major focus has been to evaluating drug-induced prolongations in the QT interval, a surrogate biomarker for torsades de pointes liability in humans (Grandi et al., 2018; Pollard et al., 2010). The IC₅₀ for the inhibitory effect of metarrestin on hERG potassium current was 0.13 μM nominal concentration, suggesting that metarrestin may prolong QT in the clinic. However, cardiovascular safety pharmacology study in dogs showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or on respiratory function parameters (respiratory rate, tidal volume, minute volume), diluting a risk of arrhythmogenesis. In male, there might be a minimal increase in mean blood pressure at the high dose at >20 hr post dose, which could be a concomitant with reflex bradycardia at similar timepoint and considered non-adverse. It is widely accepted that there is a good agreement between hERG liability and risk of torsadogenesis but there are many exceptions exist as well (Chouabe et al., 1998; GY et al., 2014; Redfern et al., 2003; Witchel, 2011). Moreover, there are cases of drugs that remain on sale despite carrying a QT risk but cannot be prescribed to certain patient groups (Pollard et al., 2010). Furthermore, safety margin for cardiac arrhythmogenic effect would be 3.5 fold (0.461 ng/ml safety pharmacology study mean Cmax / 0.13 ng/ml in-vitro hERG IC50).

After oral administration of metarrestin to dogs, median T_max across all doses, sexes and study days was 3 hours. Mean t_1/2 was similar between females and males and did not vary over all doses. Systemic exposure to metarrestin increased as dose increased on both Days 1 and 27 with no gender difference. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of 2.34.

5. Conclusion

The studies demonstrated the safety of metarrestin in dog with the NOAEL of 0.25 mg/kg based on clinical signs after every other day oral dosing (capsule) for 28 days with a mean of male and female C_max = 82.5 ng/mL and AUC_last = 2521 hr*ng/mL, on Day 27.

Highlights.

• Metarrestin didn’t produce mortality when tested up to 1.5 mg/kg every other day for 28 days.

• Clinical signs of toxicity were hypoactivity, salivation, tremors, ataxia and intermittent seizure-like activity.

• Metarrestin accumulated from Day 1 to Day 27 at all dose levels by an overall factor of about 2.34.

• Safety pharmacology study showed no adverse changes in cardiovascular or respiratory parameters.

• NOAEL in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days via oral capsules.

ABBREVIATIONS:

ANOVA

Analysis of variance

AUC_last

Area under the plasma concentration vs. time curve from the time of drug administration, time zero, until the last time with a quantifiable concentration

C_max

peak plasma concentration

ECG

Electrocardiogram

GLP

Good Laboratory Practice

hERG

human ether-à-go-go-related gene

HEK-293

human embryonic kidney cell

IC₅₀

half-maximal inhibitory concentration

MF

Combined male and female

NOAEL

No Observed Adverse Effect Level

PNC

perinucleolar compartment

T_max

Time of the observed maximum concentration

t1/2

Half-life: time required for a 50% decrease in drug concentration

TK

Toxicokinetics