Nonclinical safety evaluation of 2-Deoxy-D-Glucose following twice-daily oral administration for 28 days in beagle dogs

Abstract

2-Deoxy-D-Glucose (2-DG) has anticonvulsant and antiseizure effects in rodent models and is in the development phase for novel antiseizure treatment. To evaluate potential toxicity, Beagle dogs (five/sex/group) were orally gavaged with either vehicle (deionized water) or 2-DG (5, 30, and 90 mg/kg BID) for 28 days followed by a 14-day recovery period. The safety endpoints evaluated were mortality, clinical observations, body temperature, respiratory assessment, body weights, food consumption, ophthalmic examinations, electrocardiograph (ECG), blood pressure, cardiac biomarker (NT-proBNP) and pathology. Toxicokinetic analysis was conducted after the first dose on days 1 and 28. The dose formulation analysis confirmed that 2-DG concentrations were within 93–107% of the target concentrations. There were no 2-DG associated effects observed in mortality, clinical signs, body temperature, respiratory parameters, body weights, food consumption, ophthalmic examination, ECG, blood pressure and NT-proBNP. There was an increase (~1.7x) in aspartate transaminase on day 29, while histopathological evaluation revealed hepatic cytoplasmic alterations in 2 of 6 dogs on day 29 and only in 1 of 4 dogs on day 43 at 90 mg/kg BID. These changes were considered non-adverse because of minimal severity, reversibility trend after recovery period and no correlative increase in alanine transaminase. Toxicokinetic evaluation revealed dose dependent increases in C_max of ~7.8, 39.5, and 114 μg/mL, and AUCs of 12.2, 70.8, and 202 hr*μg/mL at 5, 30, and 90 mg/kg, respectively with T_max of ~0.5–0.9 hr and T_1/2 of ~3.8–5.4 hr. In conclusion, 90 mg/kg BID of 2-DG was considered as the No Observed Adverse Effect Level following 28-day administration in dogs.

1. Introduction

There is a compelling need for new, more effective treatments for epilepsy, which affects 0.5–1% of the population¹. About one-third of patients treated for epilepsy with standard-of-care medications continue to experience frequent seizures and are regarded as intractable². The glucose analog 2-Deoxy-D-Glucose (2-DG) is a reversible glycolytic inhibitor and has anticonvulsant and antiepileptic properties in seizure animal models³. During seizures, a higher metabolic demand in neural circuits increases 2-DG uptake into brain. Subsequently, 2-DG is phosphorylated to 2-DG-6P. However, it is unable to get metabolized by glucose-6P isomerase (GPI) that inhibits further step of glycolysis⁴. The antiseizure action of glycolytic inhibition is novel compared to all current antiseizure medications² and supports the potential use of 2-DG as a novel therapeutic intervention for convulsion and epilepsy.

Minor et al⁵ conducted dietary toxicity studies of synthetic 2-DG up to 2 year of dosing duration at doses ranging from 20 to 300 mg/kg (0.04%−0.6% 2-DG in the diet) in F344 and brown Norway rats, that identified cardiac toxicity. Minor et al⁵ also showed 2-DG associated mortality, pheochromocytoma in the adrenal medulla, reduction in food intake and body weight gain in male F344 rats. Furthermore, Terse et al⁶ showed that 2-DG induced cardiac toxicity (myocardial vacuolar degeneration) was associated with elevated levels of NT-proBNP and BNP (NT-proBNP being more sensitive) in F344 rats. However, it might be possible that cardiac toxicity could be limited to rats, which have increased metabolic demand. In the cardiomyocytes, the abnormal glucose metabolism is associated with pathological cardiac hypertrophy⁷.

To evaluate 2-DG in a clinical setting, it is required to provide toxicity results from rodent and non-rodent animal models⁸. As a non-rodent species, we conducted toxicity and toxicokinetic study of 2-DG gavaged daily for 28 days followed by a 14-day recovery period in Beagle dogs to support clinical trials for the treatment of convulsion and epilepsy. Furthermore, this study also determined species concordance for cardiac toxicity in rats to large animal species, Beagle dogs since larger animal findings are generally more translatable to humans. As a requirement of investigational new drug (IND) application, it was conducted following the guidance from the United States FDA Goold Laboratory Practice (GLP) (Code of Federal Regulations, Title 21 Part 58).

2. Materials and Methods

2.1. Formulation preparation

2-DG with purity 98.47% (Lot # SAI1206A01) was manufactured at Norac Pharma, Azusa, CA. In each week of the dosing period, 2-DG formulations (5, 30, and 90 mg/mL) were made by mixing 2-DG into deionized water. These formulations were protected from light. The formulation analyses were performed using validation method. Dose formulation concentrations were analyzed using high performance liquid chromatography with ultraviolet (HPLC/UV). The stability was also determined for 5 and 90 mg/mL dose formulation concentrations to establish stability for 15 days.

2.2. Test system

Forty Beagle dogs (20 male, 9.5–13.3kg; 20 female, 6.9–8.7 kg) with an age range of 9 to 11 months were received from Covance Research Products, Inc (Cumberland, VA). The dogs were fed Purina Lab Diet 5007 once daily, a freshwater supply was provided ad libitum. This study procedure followed applicable animal welfare acts and was approved by the Institutional Animal Care and Use Committee (IACUC). This study was conducted in compliance with the US FDA GLP Regulations (Code of Federal Regulations Title 21 Part 58).

2.3. Group assignment and dose administration

Prior to day 1, dogs were assigned to groups using the computer program, Provantis (INSTEM, Staffordshire, UK), which ensures similar group mean body weights by sex. All dogs were orally gavaged twice daily with either vehicle control (deionized water) or 2-DG. BID dosing was used because of the short half-life (0.6–5.4h) of 2-DG, and to mimic clinical BID dosing regimen. Group 1 dogs received vehicle control only, while groups 2, 3 and 4 received 2-DG formulations at doses 5, 30, and 90 mg/kg BID, respectively, for 28 days followed by a 14-day recovery (Table 1). The doses for this study were selected based on the 14-day dose range finding (DRF) study in the dogs where 100 mg/kg BID was a well-tolerated-dose, and 250 mg/kg BID was maximum tolerated dose (MTD) based on body weight loss and clinical signs. Daily two doses (BID) were administered at a target of 6 hours ± 10 minutes apart except on toxicokinetics (TK) blood collection days 1 and 28 (the second dose was administered after the 12-hour blood collection). On Days 1 and 28, the 12-h interval was used to obtain a full TK profile, whereas, on the remaining days, the 6-h interval was used to avoid dosing during nighttime for practical reasons.

Table 1.

Study design.

Groupa	Target single 2-DG concentration (mg/mL)	Target total daily 2-DG dose (mg/kg) BID	Number of dogs
			Corec (M+F)	Recoveryd (M+F)
1-Vehicle Controlb	0	0	6 (3+3)	4 (2+2)
2-Low Dose	5	5	6 (3+3)	4 (2+2)
3-Mid Dose	30	30	6 (3+3)	4 (2+2)
4-High Dose	90	90	6 (3+3)	4 (2+2)

^aAll groups were dosed for 28 days. Blood samples were also collected from study dogs for TK analysis on day 1 and day 28 following the first dose administration.

^bVehicle control was deionized water.

^cCore male (M) and female (F) dogs were humanely sacrificed on day 29 for necropsy.

^dRecovery male (M) and female (F) dogs were humanely sacrificed on day 43 for necropsy.

2.4. Clinical observation, body weights and food consumption

For all animals, the cage-side clinical observations were conducted at least twice daily for the duration of treatment and once daily during the recovery period. In each group, individual animal body weights were recorded weekly from day 1 onwards. Food consumption for all animals was recorded daily for the duration of the study.

2.5. Body temperature, respiratory measurements and ophthalmic examinations

For all animals, body temperatures were measured using a thermometer on day −3 (baseline), and days 1 and 28 at a target of 1h ± 15 min after the second dose administration. Manual respiratory rates were collected as a part of clinical observation once from all animals on day 23 or 24 at a target of 1h ± 15 min after the first dose administration. Ophthalmic examinations were performed on all animals prior to group assignment (pre-study) and on day 26 or 27. The pupils of the animals were dilated by the instillation of Tropicamide ophthalmic solution (1%) into each eye before the examination. Each ophthalmic examination included an examination of the adnexal structures (conjunctiva, eyelids, and eyelashes), the anterior segment of the eye (cornea, sclera, iris, pupil, lens, aqueous humor, and anterior chamber), and the posterior segment of the eye (the vitreous humor, retina, and optic disc). A Zeiss hand slit lamp HSO 10 was utilized for all direct ophthalmic examinations, while a Keeler all-pupil indirect ophthalmoscope with a volk 30 diopter double aspheric lens was utilized for all indirect ophthalmic examinations.

2.6. Blood pressure, ECG, and cardiac biomarker evaluations

Blood pressure and ECG tracings were examined once for all study animals on day 23 at a target of 1h ± 15 min after the first dose administration. Blood pressures were determined non-invasively by a Cardell vital signs monitor. ECG waveform tracings of leads I, II, III, aVR, aVL, aVF, and V10 were collected by a Hewlett Packard PageWriter Xli Cardiograph for morphologic evaluation. Leads I, aVf, and V10 were collected for rhythm evaluation. ECG intervals (RR, PR, QRS, and QT) were measured on the tracings. QTc intervals were calculated according to Fridericia’s formula⁹: QTc = QT/(RR)^1/3.

For cardiac biomarker evaluation, blood samples were collected on day −1 (baseline) and prior to dose administration on Days 2, 15, 29, and 43 for the plasma analysis of NT-proBNP (N-Terminal pro-B-type Natriuretic Peptide). NT-proBNP has 76 amino acids, which is a part of prehormone (proBNP)¹⁰. NT-proBNP is the gold standard biomarker for heart failure in humans¹¹. The enzyme-linked immunosorbent assay (ELISA) was conducted with the commercially available kit (Kamiya biomedical company; Catalog # KT-23770), and plates were read by the μQuant Plate Reader. The data were then transferred to Softmax Pro 5.2, Rev C for the generation of reference curves and sample analysis.

2.7. Toxicokinetics (TK)

Blood was collected from the jugular vein for the 2-DG plasma concentration analysis on day 1 and day 28 following the first dose administration of the BID dosing regimen. TK specimen collections were at pre-dose (0min) and post-dose (10min, 20min, and 1, 2, 4, 8 and 12h). The second dose of the BID dosing regimen was administered after 12h sample collection. After centrifugation, plasma samples were separated, processed by protein precipitation, and analyzed with a validation method of liquid chromatography with tandem mass spectrometry (LC-MS). 2-DG concentrations were calculated using peak area ratio [analyte peak area/internal standard (IS) peak area] and a regression line constructed from the concentrations and peak area ratios of the calibration standards. TK analysis was performed using the non-compartmental analysis (NCA) module in the WinNonlin software package (version 6.3, Pharsight corporation, Mountain View, CA). The evaluated TK parameters were C_max, T_max, elimination half-life (T_1/2), apparent clearance, and AUC (to AUC_last and AUC_∞).

2.8. Clinical pathology

Blood was collected from the jugular vein of all animals to assess hematology, coagulation, and serum chemistry on Days −1 (baseline), 2, 15, 29, and 43 (non-fasted). To evaluate urine parameters, urine was also collected via free catch or from the cage pan on days 28 and 29.

2.9. Necropsy

Dogs (three/sex/group) were weighed and humanely sacrificed before necropsy on day 29. Following a 14-day recovery period, the remaining dogs (two/sex/group) were similarly necropsied on day 43. Organ weights were weighed, and absolute weight, organ to body weight, and organ to brain weight ratio were estimated.

All tissues were collected and preserved in 10% neutral-buffered formalin (NBF), with the exceptions of testes, which were placed in Bouin’s fixative and subsequently transferred to 70% ethanol. The eyes with optic nerve were fixed in Davidson’s fixative and subsequently transferred to 10% NBF. Bone marrow smears were fixed in methanol. All fixed tissues from control and high-dose groups (day 29) and gross lesions from all groups were processed to slides with staining of hematoxylin and eosin. Liver tissues from day 29 (low and mid-dose groups) and day 43 (all groups) necropsied animals were also collected for histopathology. Microscopic findings in histopathology were graded as per the following scale: minimal (Grade 1), mild (Grade 2), moderate (Grade 3) and marked (Grade 4). These grades were used to calculate the average severity of microscopic findings (Table 3).

Table 3.

Stability on day 0 and day 15.

Prepared formulation concentration (mg/mL)	Determined concentration on day 0 (mg/mL)	Average determined concentration on day 15 (mg/mL)	Average RE* of Day 0
5	5.07	4.88 ± 0.02	−3.7
90	87.8	87.2 ± 0.6	−0.7

^*RE: Relative Error

2.10. Statistical analysis

To evaluate the 2-DG associated effect, the parametric or nonparametric analysis of variance (ANOVA) was used to analyze quantitative in-life data, which was collected using the Provantis system. To determine the significant differences among the group means, the ANOVA F-test was used for the analysis of parametric data, which was normally distributed and homogenous among groups. If this test was significant, then Dunnett’s test was used to determine differences between the control and each of the comparison groups. The Shapiro-Wilk’s test and Levene’s test determined normality and homogeneity of variances, respectively, for all data. Kruskal-Wallis test was used to determine the significant differences among the group means for normally distributed and/or non-homogenous nonparametric data. If this test was significant, then the Wilcoxon test was used to determine differences between the control and each of the comparison groups, while the Bonferroni-Holm method was used to correct for multiple comparisons.

3. Results

3.1. Dose formulation analysis and stability

As per Table 2, the formulation concentrations were within 92.7 to 107% of the target concentrations of 2-DG (within ±10% acceptance criteria) for formulation prepared in each week of the dosing period. For 2-DG formulations of 5 and 90 mg/mL, stability was established for up to 15 days at room temperature (Table 3).

Table 2.

Formulation analysis.

Week of dosing	Target concentration (mg/mL)	Average determined concentration (mg/mL)	Average relative error (RE)
1	0	BLOQa	NAb
	5	5.07 ± 0.03	1.3
	30	29.5 ± 0.3	−1.6
	90	87.8 ± 0.3	−2.4
2	0	BLOQ	NA
	5	5.35 ± 0.05	7.0
	30	31.9 ± 0.1	6.3
	90	94.8 ± 0.3	5.4
3	0	BLOQ	NA
	5	4.64 ± 0.11	−7.3
	30	28.3 ± 1.0	−5.6
	90	84.5 ± 0.5	−6.1
4	0	BLOQ	NA
	5	4.66 ± 0.18	−6.7
	30	28.5 ± 0.1	−5.0
	90	86.0 ± 1.0	−4.4

^aBLOQ: Below the limit of quantitation.

^bNA: Not Applicable.

3.2. Clinical observation, body weights and food consumption

Mortality was not observed. 2-DG administered dogs had intermittent yellow, soft feces or diarrhea in a dose-dependent manner in 3, 6 and 9 dogs in the 5, 30 and 90 mg/kg BID, respectively. Two control dogs were also noted with similar intermittent abnormal feces. Since there was no indication of dehydration or changes in body weights/food consumption in any animals, these intermittent abnormal feces are not considered adverse effects.

3.3. Body temperature, respiratory measurements and ophthalmic examinations

There were no 2-DG associated changes in body temperature, respiratory measurements and ophthalmic observations.

3.4. Blood pressure, ECG, and cardiac biomarker evaluations

There were no 2-DG associated findings in blood pressure, ECG and biomarker (NT-proBNP levels).

3.5. Toxicokinetics

The TK results after single dose administration are shown in Table 4 and Figure 1. Briefly on day 1, T_max was between 0.47 to 1.03 hr for all dose groups. Group mean apparent clearance values ranged between 349 to 471 mL/hr/kg for all dose groups. These results indicated no dose or gender-dependent effect on T_max and apparent clearance. Group mean T_1/2 values were ranging between 2.04 to 3.16 hr for all dose groups with the exception of 0.611 hr for the 5 mg/kg males. The shorter T_1/2 for males in 5 mg/kg groups was due to T_last, which was only 4 hr. Systemic exposure of 2-DG was evaluated using C_max and AUC values. C_max values were 8.55, 51.2 and 127 μg/mL, while AUC_∞ values were 14.6, 83.6 and 224 hr*μg/mL for 5, 30, 90 mg/kg, respectively (Table 4).

Table 4.

Toxicokinetics (TK) in dogs after first 2-DG administration on day 1 and day 28.

Gender	Dosea (mg/kg)	Cmax (μg/mL)	Tmax (hr)	T1/2 (hr)	Apparent Clearance (mL/hr/kg)	AUClast (hr*μg/mL)	AUC∞ (hr*μg/mL)
Day 1
Male	5	7.84	0.47	0.611	440	10.2	11.7
	30	51.2	0.87	2.61	370	82.9	83.6
	90	127	0.60	2.59	405	223	224
Female	5	8.55	1.03	2.04	349	12.6	14.6
	30	48.8	0.60	3.16	410	75.2	75.9
	90	117	0.73	2.76	471	197	199
Day 28
Male	5	6.98	0.47	5.41	446	10.7	11.3
	30	39.5	0.47	4.89	463	64.6	67.7
	90	82.3	0.87	3.81	557	162	167
Female	5	7.77	0.87	4.59	397	12.2	12.7
	30	36.2	0.87	4.41	417	70.8	73.3
	90	114	0.87	3.89	443	202	208

^aTK blood collections were done after first 2-DG dose administration of BID dosing regimen.

Figure 1.

Toxicokinetic after the first 2-DG dose administration on day 1 and day 28.

On Day 28, T_max ranged between 0.47 to 0.87 hr for all animals. Group mean apparent clearance values ranged from 398 to 557 mL/hr/kg, while group mean T_1/2 values ranged from 3.81 to 5.41 for the three dose groups. There were no apparent dose or sex effects on T_max, T_1/2 or apparent clearance. C_max values were 7.77, 39.5 and 114 μg/mL, while AUC_∞ values were 12.7, 73.3 and 208 hr*μg/mL for 5, 30, 90 mg/kg, respectively (Table 4).

When comparing day 1 and day 28 TK parameters, the mean T_max was relatively unchanged. In addition, group mean clearance values were all within a range of 349–557 mL/hr/kg. However, group mean T_1/2 values were increased on Day 28 for all groups. Systemic exposure (C_max and AUC_∞) remained relatively similar (within 2-fold) from day 1 to day 28. The plasma TK parameters (especially C_max and AUC_∞) increased in a dose-dependent-manner although it was not consistently a dose proportional manner for day 1 from 5–30 mg/kg (male). In addition, there was no gender difference in the measured TK parameters.

3.6. Clinical pathology

There were no 2-DG associated effects on coagulation, hematology and urinalysis parameters. In addition, 2-DG related serum chemistry changes were limited to only aspartate aminotransferase (AST). AST was mildly increased on days 15 and 29 in the 90 mg/kg BID (1.1x to 1.7x). However, changes in AST levels were reversed on day 43.

3.7. Necropsy

On day 29, there were no significant differences in group mean terminal body weights, absolute organ weights, and organ-to-terminal body weight ratios in all animals of 2-DG treated groups compared to the control group. Although, spleen to brain weight ratio significantly increased in 90 mg/kg BID females as compared to control group females, similar increases in absolute spleen weight and spleen-to-terminal body weight ratios were not observed in 90 mg/kg BID females. Since, spleen to brain weight ratio was significantly higher only in females of 90 mg/kg BID with no correlative histopathological changes in any gender, these changes in weight ratio are considered as non-adverse. The heart-to-brain weight ratio at 30 mg/kg/day in males was significantly decreased as compared to the control males. However, the decrease in the ratio for males in other 2-DG treated groups was not observed. In addition, the absolute and relative heart weights of female groups were not decreased significantly. Since this change in heart-to-brain weight ratio was only observed at 30 mg/kg BID males (indicating no dose-dependent response) with no correlative histopathological changes in any other 2-DG treated groups, the decrease in heart-to-brain weight ratio of the males was considered as not associated with the 2-DG treatment.

3.8. Gross findings and histopathology

There were no 2-DG related gross findings observed in all treated groups. In histopathology, the 2-DG-related findings were confined to the liver tissue (Table 5). Microscopically, cytoplasmic alteration was present in the liver of two (one male and one female) out of six core necropsy animals receiving 90 mg/kg BID on day 29. Cytoplasmic alteration, characterized by subtle enlargement of the hepatocytes and microvesicular cytoplasmic vacuolation, was present in midzonal to centrilobular hepatocytes. This finding was also observed in one male out of four recovery (two males and two females) necropsy animals receiving 90 mg/kg BID on day 43. Cytoplasmic alteration was not seen in any females on day 43 indicating complete recovery in females.

Table 5.

Microscopic findings in the Liver of 2-DG administered dogs.

Day	Observation	Dose (mg/kg BID)
		0		5		30		90
		Male	Female	Male	Female	Male	Female	Male	Female
29	Midzonal, cytoplasmic alteration in hepatocyte	0/3a	0/3	0/3	0/3	0/3	0/3	1/3	1/3
		0	0	0	0	0	0	1c	1
43	Midzonal, cytoplasmic alteration in hepatocyte	0/2b	0/2	0/2	0/2	0/2	0/2	1/2	0/2
		0	0	0	0	0	0	1c	0

^aNumber of dogs showing microscopic findings from a total number of dogs (core) examined (n= 3) on day 29.

^bNumber of dogs showing microscopic findings from a total number of dogs (recovery) examined (n= 2) on day 43.

^cAverage severity was calculated by adding all the grades of affected animals in the group and dividing by the number affected. Microscopic findings in histopathology were graded as Grade 1: minimal, Grade 2: mild, Grade 3: moderate and Grade 4: marked.

4. Discussion

2-DG is a known glycolysis pathway inhibitor, and this property of 2-DG has been used to develop anticonvulsant and antiepileptic therapy with proven proof of concept generated using different animal models^2,12–14. In order to support 2-DG’s clinical evaluation, the current toxicity study was conducted as a part of the IND application requirement⁸. The results of this study also helped to determine species concordance for cardiac toxicity in rats^5,6 to large animal species. This study was performed to characterize the toxicity, reversibility of toxicity, dose-response, and exposure assessment to determine no observed adverse effect level (NOAEL) in non-rodent species. This information along with rodent NOAEL will be used to determine the maximum recommended starting dose (MRSD) for phase I clinical trial^8,15.

The doses (5, 30 and 90 mg/kg BID) for this GLP toxicity study were selected based on the 14-day DRF toxicity study. In the DRF study, 100 mg/kg BID was a well-tolerated-dose, and 250 mg/kg BID dose was the MTD based on decreased activity, emesis, excess salivation, decreased food consumption and body weight loss (up to −11%). In this 28-day toxicity study, dose formulations were analyzed for 2-DG concentrations and were found to be within acceptable range (±10%) of the target concentrations. Oral 2-DG administration (up to 90 mg/kg BID) did not show any mortality, clinical signs, or changes in body weights, food consumption, body temperature, respiratory measurements, ophthalmic examination, blood pressure, ECG and NT-proBNP levels. Previous dietary toxicity study in rats displayed increased mortality, pheochromocytoma, body weight gain reductions and cardiac myocyte vacuolation with elevation of autophagic flux⁵. Mortality in four males and one female has been reported after the repeated oral gavage administration of 2-DG at 375 mg/kg BID in rats⁶. The repeated daily administration of 2-DG at ≥60 mg/kg BID induced dose-dependent cardiac vacuolation in rats;^5,6, however, cardiac vacuolation was not observed with daily doses of 50 mg/kg BID⁶. In the rat study (unpublished data), the dose dependent increase in plasma level of 2-DG with no accumulation in systemic exposure was observed. We also observed a C_max of 47.5 ug/mL and AUC_∞ of 177 hr*ug/mL on day 28 at 125 mg/kg BID with microscopic changes in myocardiocytes and the significantly elevated ALT and AST values. In the previously reported in-vitro hERG study, 2-DG at 5mM (~833 μg/mL) induced 38% inhibition of hERG¹⁶. However, the source of 2-DG was Sigma¹⁶. We conducted a GLP in vitro hERG study (unpublished data) to evaluate 2-DG for hERG inhibition. 2-DG did not induce significant hERG inhibition (only 2.6% inhibition in hERG current) up to 6 mM (~1000 μg/mL) concentration. The same batch (Lot SAI1206A01) with a purity of 98.47% of 2-DG was used in both studies (in vitro hERG and in vivo dog). Also, this dog study showed systemic exposure (C_max of 114 μg/mL and AUC_∞ of 208 hr*μg/mL) at 90 mg/kg BID following 28-days of oral gavage administration with no adverse effects observed. This indicated that dogs may be less sensitive for 2-DG associated cardiac toxicity as compared to rats since toxicity in rat was observed at systemic exposure with C_max of 47.5 ug/mL and AUC_∞ of 177 hr*ug/mL in rat whereas dogs did not show toxicity at C_max of 114 μg/mL and AUC_∞ of 208 hr*μg/mL.

Furthermore, no sex or dose related effects were observed in T_max, T_1/2 or apparent clearance values. Cmax and AUC values increased in dose dependent manner from 5 to 90 mg/kg BID with no accumulation in systemic exposure after 28 days of 2-DG administration in dogs.

In the current dog study, no cardiac histopathology findings or changes in cardiac biomarker (NT-proBNP) were observed up to 90 mg/kg BID for 28 days. Compared to these dog findings, rat showed a different pattern after 2-DG administration with a significant increase in the plasma concentrations of NT-proBNP with concurrent myocardial vacuolar degeneration⁶. NT-proBNP is a potential early safety biomarker for cardiac toxicity in rats. It has been reported that the plasma levels of NT-proBNP in dogs are the only biomarkers to diagnose and monitor cardiac congestive processes¹⁷. However, 2-DG administered groups in 28-day dog study did not show an increase in NT pro-BNP concentration or cardiac histopathological changes, which indicated species differences either due to doses tested were not high enough in dog or sensitivity of dog may be lower than rat to cardiac toxicity. The lack of cardiac toxicity in dogs might be explained by less metabolic demand. Beagle dogs achieve maximum weight gain and growth by 9 months of age (https://www.pawlicy.com/blog/beagle-growth-and-weight-chart/). The energy requirement declines with age in dogs¹⁸, especially after attaining maximum growth and weight gain. Thus, the metabolic demand decreases as body mass increases¹⁹. Current study used 9 to 11-month old Beagle dogs that may have less metabolic demand since they attained maximum growth and weight. A low metabolic demand tends to reduce 2-DG uptake²⁰ to protect from its toxic effects.

The 2-DG associated changes were not observed in parameters such as coagulation, hematology and urinalysis. However, 2-DG related change was observed in the serum chemistry, which was limited to a mild increase in AST in the 90 mg/kg BID on day 29. There was no change in ALT level observed in any 2-DG administered groups. ALT upregulation is associated with liver damage or disease²¹. In the absence of an increase in ALT levels, which is a selective biomarker for hepatocyte injury, a mild increase in AST was considered non-adverse in the current study. Furthermore, the histopathological changes in the liver were also considered non-adverse as these cytoplasmic alterations were graded as minimal, no increase in ALT or only a mild increase in AST, and it demonstrated a trend towards recovery following a 2-week recovery period.

Based on these results, the No Observed Adverse Effect Level was considered to be 90 mg/kg BID after 28 days of 2-DG administration in Beagle dogs.