Investigation of Correlation Among Safety Biomarkers in Serum,Histopathological Examination,and Toxicogenomics

Abstract

This article addresses the issue of miscorrelation between hepatic injury biomarkers and histopathological findings in the drug development context. Our studies indicate that the use of toxicogenomics can aid in the drug development decision-making process associated with such miscorrelated data. BLZ945 was developed as a Colony-Stimulating Factor 1 Receptor (CSF-1R) inhibitor. Treatment of BLZ945 in rats and monkeys increased serum alanine aminotransferase (ALT) and aspartate transaminase (AST). However, liver hypertrophy was the only histopathological liver finding in rats, and there was no change in the livers of monkeys. Longer treatment of BLZ945 in rats for 6 weeks caused up to 6-fold elevation of ALT, yet hepatocyte necrosis was not detected microscopically. Toxicogenomic profiling of liver samples demonstrated that the genes associated with early response to liver injury, apoptosis/necrosis, inflammation, oxidative stress, and metabolic enzymes were upregulated. Studies are ongoing to evaluate the mechanisms underlying BL945-induced ALT and AST elevations.

Introduction

Drug-induced hepatic injury has a considerable impact on public health. A survey done by the Acute Liver Failure Study Group demonstrated that in 17 US hospitals, prescribed drugs accounted for more than 50% of acute liver failure cases. ¹ Drug-related hepatotoxicity is still the most frequent reason for drug development termination and has been responsible for the costly withdrawal of several approved drugs.

In standard clinical practice, drug-related liver injury is usually detected by serum chemistry profiles since liver biopsy specimens are often not available. In the clinical setting, serum alanine aminotransferase (ALT) levels of greater than twice the upper limit of normal (ULN) are considered indicative of liver injury. ^2,3 In humans, the half-life of ALT in the blood is approximately 47 hours. ⁴ This long half-life and ALT’s predominant liver expression offer diagnostic value during and after initial liver insults. Although ALT is also present in other tissues, it has been shown that in mice, rats, and dogs, hepatic ALT activity ranges from 3 to 10 times higher than ALT activity in other tissues. ⁵ Monkeys, however, show less ALT organ specificity, as there is a comparable amount of ALT activity in heart muscle and liver tissue on a per-gram basis. ⁵ Aspartate transaminase (AST) is similar to ALT in that it is another enzyme associated with hepatocytes. Unlike hepatic ALT, however, increases in serum AST are not as organ specific, with skeletal muscle, heart muscle, and liver having the most AST activity per gram of tissue. ⁵ In preclinical safety evaluation studies, integrated evaluation of serum ALT/AST levels and liver histopathology has been crucial for the identification of drug-induced liver injury as an indicator of potential liability for hepatotoxicity in humans. Although positive correlations between the degree of liver necrosis and the serum ALT and AST levels have been demonstrated in many animal studies, discrepancies between clinical chemistry findings and liver histopathology have nonetheless been observed. ⁶ Travlos et al ⁶ reviewed 61 toxicity studies conducted for the National Toxicology Program, and found that 44% to 60% of the studies reporting treatment-related increases in ALT activity had no concurrent morphological changes in the liver. Unfortunately, there was no follow-up investigation to determine whether the compounds in those studies that reported increased ALT activities were in fact hepatotoxicants. Increased ALT levels without accompanying liver morphological changes make it difficult to determine whether the drug candidate in question actually causes hepatotoxicity. The general guidelines for the interpretation of indicators for liver toxicity in preclinical studies recommend that increases in ALT that correlate with detrimental histopathological changes should be considered adverse. ⁷ However, it does not provide guidance regarding the interpretation of increased ALT activity in the absence of concurrent histopathological changes. The American Society for Veterinary Clinical Pathology proposed that increases in serum ALT levels in the range of 2 to 4 times or higher than controls should raise concern as an indicator of potential hepatotoxicity unless a clear alternative explanation is found.

Other potential serum biomarkers for hepatotoxicity have been evaluated and developed largely by industrial efforts. ⁸ The new potential biomarkers include ALT isoforms (ALT1 and ALT2), glutamate dehydrogenase (GLDH), glutathione S-transferase-α (GST-α), serum F protein, arginase I, regucalcin, malate dehydrogenase (MDH), purine nucleoside phosphorlyase (PNP), and paraoxonase-1 (PON1). However, none of these biomarkers has been well-validated and routinely used to detect hepatotoxicity in preclinical or clinical settings. Among these potential new biomarkers, GST-α and GLDH have been shown to likely add value to interpreting increased or decreased ALT activity. One area of active research focuses on using ALT1 and ALT2 to differentiate hepatic or nonhepatic sources of ALT activity. Some studies have indicated that ALT1 might be used as liver biomarker and ALT2 for skeletal muscle and heart due to the different tissue localizations of these 2 isoforms. ^8,9 Conflicting studies, however, demonstrate that serum ALT1 and ALT2 activities might not be so easily discriminated. Yang et al ¹⁰ have examined the tissue expression of ALT1 and ALT2 at the mRNA and protein levels and have shown that ALT1 mRNA and protein were widely distributed in intestine, liver, fat tissues, colon, muscle, and heart, whereas ALT2 was more restricted, mainly in liver, muscle, brain, and white adipose tissue. Furthermore, these authors ¹⁰ have also demonstrated that under carbon tetrachloride- or acetaminophen-induced liver injury, both serum ALT1 and ALT2 were significantly elevated and correlated with ALT activity. Jadaho et al ¹¹ have shown that in fatty livers of obese mice, ALT2 gene expression is induced, but ALT1 remains the same. Furthermore, total hepatic ALT activity is elevated significantly. These authors concluded that ALT2 may be responsible for the observed increased ALT activity, and proposed using ALT2 as a biomarker of hepatic steatosis. Various issues also exist for other potential biomarkers. For example, while serum F protein is produced across mammalian species (and would thus be a useful biomarker), the correlation between elevated serum F protein levels and liver morphological changes has not been established in animal models. ⁸ Another example is arginase I. ⁸ This potential biomarker is highly liver-specific. However, a preclinical arginase I assay is not commercially available for an extensive validation. ⁸ Newer biomarkers such as MDH, PNP, and PON1 are under development, ⁸ and whether they eventually will be proven to add value to ALT activity interpretation remains to be determined. Collectively, in the practical world of preclinical and clinical safety evaluation, ALT and AST are still recognized as the gold standard biomarkers driving diagnosis of hepatotoxicity.

During the development of a selective colony-stimulating factor 1 receptor (CSF-1R) inhibitor, BLZ945, more than 4-fold increases in serum ALT levels without concurrent hepatocyte necrosis were observed in subacute toxicology studies. Long-term studies (eg 13-week study) with continued dosing might show clear hepatocyte necrosis under the microscope. However, practically speaking, it would take enormous effort and resources to undertake such studies for each drug candidate, and it is thus not a sound drug development strategy. In addition, the guiding principle of international guidelines on drug development is to reduce, refine, and replace the use of animals, whenever possible. But perhaps most important in the context of this report is the ever increasing use of emerging “-omics” technologies that have been integrated into preclinical studies to better assess drug safety by allowing investigation of the cellular and molecular events underlying adverse drug reactions. The signature genes for hepatotoxicity and liver hypertrophy have been well established. Therefore, we applied toxicogenomic profiling in order to further our mechanism-based investigation. We hope that this article will help to fill in the gap between the use of biomarkers in preclinical hepatotoxicity studies and the lack of correlative histopathological findings associated with these studies that one frequently comes across during pharmaceutical drug development. It is our hope that this report strengthens the argument that toxicogenomics can prove useful in determining hepatoxicity risk during drug development.

Materials and Methods

Chemicals

BLZ945 is 4-[2-((1R, 2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid methylamide. It was formulated in 0.5% (w/v) methyl cellulose (MC), Type 400 cPs, aqueous solution. Aberrant activation of osteoclasts due to bone metastasis causes osteolysis, skeletal-related events, and severe pain in patients with cancer. Macrophage-CSF (M-CSF) signaling through its receptor CSF-1R in the monocytic lineage is essential for osteoclastogenesis, providing an opportunity to inhibit this pathway and suppress tumor-induced osteolysis. BLZ945 is a selective and ATP competitive CSF-1R kinase inhibitor. BLZ945 potently inhibits CSF-1R (IC50 1.2 nmol/L) as determined by an in vitro kinase assay with recombinant CSF-1R kinase domain. It reduces the tyrosine-phosphorylated levels of CSF-1R in cells (EC₅₀ pCSF-1R 58 nmol/L), and exerts a significant antiproliferative effect on the M-CSF-dependent cell line MNFS-60 (EC₅₀ 71 nmol/L).

Animals

All procedures were performed under approved animal study protocols in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. Wistar Hannover rats (8 weeks old) were obtained from Charles River Laboratories (Raleigh, North Carolina). Cynomolgus monkeys (Macaca fascicularis; 2-4 years old) were obtained from Covance (Denver, Pennsylvania).

Studies in Rats and Monkeys to Evaluate Toxicity of BLZ945

In the rat study, BLZ945 was administered orally to 4 groups of Wistar Hannover rats at doses of 0, 10, 50, or 150 mg/kg per day for 4 weeks (10/sex per group with additional 6/sex in the control and high-dose groups for recovery). The dosing volume was 5 mL/kg. At the end of the dosing period, 6 rats/sex from the control and high-dose groups were placed on recovery for 4 weeks. In the monkey study, BLZ945 was administered orally via gavage to 4 groups of cynomolgus monkeys at doses of 0, 10, 30, and 100 mg/kg per day for 4 weeks (3/sex per group with additional 2/sex in the control and high-dose groups for recovery). The dosing volume was 5 mL/kg. Two animals per sex from the control and high-dose groups were maintained on study for a 4-week recovery period. In both studies, clinical signs, body weight, serum chemistry, and hematology evaluations were performed. Complete necropsies including macroscopic assessments and organ weight determinations were performed at the end of dosing and recovery periods. Tissues were processed and stained with hematoxylin and eosin for histopathology evaluation. Liver samples were also collected for genomic expression profiling. In the monkey study, additional liver samples were processed for immunohistochemical staining for CD68, a marker for macrophages, and examined to determine the presence of macrophages. In both studies, blood samples were also collected on day 1 after a single dose and on day 28 after for toxicokinetic analysis.

Study to Investigate Kinetics of Serum ALT and AST Elevation Upon Treatment of BLZ945

Wistar rats were cannulated with jugular and femoral indwelling catheters, and the patency was maintained by infusion of sterile dextrose 50% with heparin 100 units/L and vancomycin 1 mg/mL. The first group (n = 12 males) was used as the regression group, and received BLZ945 daily at 150 mg/kg per day. Once a 3-fold increase in serum ALT was determined at 2 consecutive blood samplings, dosing was stopped. In all, 8 out of the 12 rats were euthanized, and the liver samples were taken for histopathology examination. The other 4 rats were put on recovery, and the regression of elevated serum ALT and AST was studied. The change of serum AST was also evaluated. The second group (n = 12 males) was used as the progression group, and received BLZ945 daily at 150 mg/kg per day for 6 weeks or until serum ALT elevation ≥10-fold at 2 consecutive measurements. Then the dosing was stopped. There was a control group for each BLZ945-treatment group (n = 8). Control animals received vehicle (0.5% MC). Blood samples were collected periodically for the evaluation of ALT and AST. At necropsy, the liver samples were collected for histopathological examination. Due to the findings in the muscle from the above-mentioned 4-week monkey study, serum creatine kinase (CK) was evaluated. Several muscle samples including diaphragm, gastrocnemius, and soleus were also collected for histopathological examination of potential muscle damage. During the course of the study, several BLZ945-treated animals were found dead or were sacrificed moribund with catherization-related septicemia likely the cause. At each time point of ALT and AST determinations, blood samples were collected from at least 4 animals.

RNA Extraction and Purification

Total RNA was isolated from each frozen liver using TRIzol Reagent (Invitrogen, Carlsbad, California) and purified on an affinity resin using RNeasy kit (Qiagen, Valencia, California) according to the manufacturer’s instructions. The concentration of RNA samples was determined by the UV absorbance at 260 nm (A _260nm), and the RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California). RNA was stored at −80°C until analysis.

Expression Array

Expression experiments were conducted using the Affymetrix GeneChip Rat Genome 230 2.0 Array and GeneChip HG-U133 Plus 2 Array for monkey (Affymetrix). The total RNA from each sample was converted into fragmented-labeled cRNA following the manufacturer’s recommendations (Affymetrix, 1-cycle amplification). Briefly, 10 μg of the fragmented-labeled cRNA was hybridized for approximately 16 hours at 45°C to an expression probe array. The array was washed and stained twice with streptavidin-phycoerythrin (Molecular Probes) using the GeneChip Fluidics Workstation 450 (Affymetrix). The array was then scanned using a solid-state laser scanner (GeneArray Scanner 3000, Affymetrix). Expression data were processed by the MAS5 program (Affymetrix) using a target intensity of 150.

Genomic Data Analysis

Control animals from the main study and from the recovery period were grouped together, as well as male and female animals from each dose group. In order to identify meaningful information on molecular pathways and processes, and increase the reproducibility and the robustness of the interpretation of gene expression profiles, the Gene Set Enrichment Analysis (GSEA) was used. ¹² The leading edge analysis was based on mean raw data expression of >80 in any of the treatment conditions and minimum fold change of 1.3. The a priori–defined sets of genes for GSEA were on their existing link to a biological process based on the literature. The enrichment score ¹² (referred herein as score) of established gene signatures was calculated as the geometric mean of a group divided by the median of the baseline. Additionally, any expression values lower than 50 were set to a base value of 50.

Statistical Analyses Used in Animal Studies

An analysis of variance (ANOVA) was used to correlate the effects of treatment (all except genomic data). The ANOVA was followed by a Bartlett’s test for homogeneity of variances. If the variances were homogeneous, Dunnett’s t test was employed to determine the statistical significance between control and treated groups. If the variances were not homogeneous, a modified t test was used to determine which groups are statistically different from the controls. For organ weight data, an automated program Pathdata System (PDS Pathology Data Systems Ltd, Basel, Switzerland) was used to decide whether parametric or nonparametric group comparisons should be made. This program uses Kolmogorov’s test to examine the normality of the data and Bartlett’s test to examine the homogeneity of variances. Accordingly, either Dunnett’s test or Student’s t -test for parametric group comparisons or Dunn’s test or Wilcoxon’s test (U test) for nonparametric group comparisons were used.

Results

Changes in Serum Biomarkers and Liver Histology in 4-Week Rat Study

Oral administration of BLZ945 at doses of 0, 10, 50, or 150 mg/kg per day for 4 weeks was reasonably well tolerated in rats. No mortality was seen at any dose levels, and animals gained weight. For the discussion purpose, only the serum chemistry and histopathological data related to liver and muscle injury are included here. Dose-dependent increases in group mean serum ALT activities relative to concurrent controls were observed ranging approximately from 2- to 6-fold (Table 1). Serum AST activity was also increased 2- to 4-fold in a dose-dependent manner. Other serum biomarkers of hepatobiliary or hepatic synthetic function including alkaline phosphatase, total bilirubin, total protein, albumin, triglycerides, cholesterol, glucose, urea, activated partial thromboplastin time, and prothrombin time were not altered when compared to those in the control animals. Increased mean liver weights (absolute and relative to body and brain) were observed in the female rats only at the high dose compared with controls (32% absolute weight increase). Microscopic examination indicated diffuse or panlobular hepatocellular hypertrophy of minimal to slight severity in male rats at doses of 50 and 150 mg/kg per day. This change was not observed in BLZ945-treated females. Under the light microscope, the cytoplasm of the hypertrophic hepatocytes had an eosinophilic, ground glass appearance, suggesting an increase in the number of ribosomes and enzyme induction. No increased CK or morphological evidence of skeletal muscle damage was observed in rats in this study. Following single and multiple doses of BLZ945, the exposure of BLZ945 increased with increasing dose in a roughly proportional manner irrespective of gender (Table 2 ). Following multiple administration of BLZ945 for 28 days, no significant increase in exposure was observed when comparing to the exposure on day 1 for the low and mid-doses. However, an increase of exposure was observed on day 28 for the high dose when comparing to that of day 1.

Table 1.

Serum ALT and AST Changes in the Rats After Treatment of BLZ945 for 4 Weeks ^a

	ALT (U/L)		AST (U/L)		Liver Weight		Liver Histopathology b
	Male	Female	Male	Female	Male	Female	Male	Female
0 mg/kg	37 ± 6.5	37 ± 6.1	76 ± 11.8	79 ± 6.9	—	—	—	—
10 mg/kg	68 ± 7.3	68 ± 13.2	141 ± 11.7	150 ± 20.2	—	—	—	—
50 mg/kg	135 ± 25.5	137 ± 22.5	220 ± 19.2	245 ± 25.3	—	—	Hypertrophy 4 (1.5)	—
150 mg/kg	212 ± 29.8	173 ± 26.6	267 ± 24.4	264 ± 50.4	—	↑~32%	Hypertrophy 2 (1.0)	—

Abbreviations: ALT, alanine aminotransferase; AST, aspartate transaminase.

^a Recovery data were not shown. After a 4-week recovery period, serum ALT and AST levels in the BLZ934-treated rats were similar to the controls. Increased liver weight and liver hypertrophy were not observed in the BLZ945-treated rats after the 4-week recovery period.

^b Under liver histopathology, the number outside ( ) represented the number of animals that had the finding, and the number inside ( ) represented average severity grade: 1, minimal; 2, slight; and 3, moderate.

Table 2.

Toxicokinetic Data of BLZ945 After Single and Multiple Doses of BLZ945 in Rats and Monkeys

	Dose (mg/kg)	AUC (ng × Hours/mL)	Cmax (ng/mL)	Tmax (Hours)
Rat
Day 1	10	85 550	9240	0.75
	50	480 000	37 250	2.00
	150	798 000	55 150	0.75
Day 28	10	100 950	11 750	1.00
	50	546 500	54 750	1.00
	150	1 510 000	87 450	0.75
Monkey
Day 1	10	52 600	7405	1.34
	30	137 250	20 750	1.00
	100	362 000	38 150	1.50
Day 28	10	73 800	6500	2.50
	30	282 500	25 750	1.50
	100	672 500	46 150	1.00

Changes in Serum Biomarkers and Liver Histology in 4-Week Monkey Study

Treatment of BLZ945 at doses of 0, 10, 30, or 100 mg/kg per day for 4 weeks was reasonably well tolerated in monkeys. At 30 and 100 mg/kg, emesis (with feed or apparent compound) or diarrhea was observed, but weight gain was unaffected and no mortality occurred. The most prominent changes in clinical pathology parameters were the increases in the serum activities of ALT, AST, and CK (Table 3 ). Time- and dose-dependent increases in serum ALT and AST were observed at all doses. The average group mean increase relative to baseline values of serum ALT was 3- to 8-fold, and 3- to 13-fold for AST. At the doses of 30 and 100 mg/kg, the magnitudes of the ALT and AST elevations were similar. Glutathione S-transferase-α is considered an early marker of posttraumatic hepatic injury, and was added in the monkey study. There was no elevation in serum GST-α upon BLZ945 treatment. Biomarkers for billiary excretion, including alkaline phosphatase and total bilirubin, were not increased compared to the control levels. Biomarkers that indicate hepatobilliary function, including total protein, albumin, triglycerides, cholesterol, glucose, urea, activated partial thromboplastin time, and prothrombin time, were also not elevated compared to the control levels. There were mild to marked increases in serum total CK activity. Evaluation of CK isoenzymes indicated that these increases were due almost solely to increases in CK-MM isoenzyme activity, indicating a skeletal muscle origin of the increased CK activity. Morphological injury of skeletal muscle was not observed in the specimens examined under the light microscope. Although the serum AST elevation tended to be higher than that of ALT, the average AST:ALT ratio was less 1.5-fold. Other changes included a trend of increases in serum bilirubin concentrations (still within normal historical range of 0.3-0.8 mg/dL) at the high dose, and minimally increased serum globulin concentrations. Increased liver weights (absolute and relative to brain and body weights) were observed in females at a dose of 100 mg/kg per day. The increase in the mean absolute liver weight was 41% compared to the controls. This appeared to be a reversible change as it was not observed after the 4-week recovery period. Liver histopathological examination did not indicate any evidence of necrosis, hypertrophy, or any other cellular abnormality. There was moderate BLZ945-related reduction (up to 56%) in CD68-positive macrophages in the liver at doses of ≥10 mg/kg per day. CD68-positive macrophages were randomly distributed in the sinusoidal spaces and the reduction was distributed throughout the hepatic lobule. Following 4 weeks of recovery, the number of CD68-positive macrophages was comparable between the high dose and control animals of both sexes, indicating a complete recovery. Following single and multiple administrations of BLZ945, the exposure of BLZ945 increased with increasing dose in a roughly proportional manner irrespective of the gender (Table 2). Following multiple administrations of BLZ945, an increase of the exposure was observed on day 28 when comparing to the exposure on day 1.

Table 3.

Serum ALT and AST Changes in the Monkeys After Treatment of BLZ945 for 4 Weeks

	Pretreatment		Week 2		Week 4		End of Recovery a
	Male	Female	Male	Female	Male	Female	Male	Female
ALT (U/L)
10 mg/kg	50 ± 11.3	60 ± 13.7	110 ± 57	139 ± 22.7	134 ± 89.5	200 ± 16	—	—
30 mg/kg	41 ± 9.3	39 ± 6.4	249 ± 18.4	193 ± 73.2	336 ± 43.5	271 ± 81.4	—	—
100 mg/kg	59 ± 30.2	54 ± 25.3	257 ± 126.2	237 ± 60.5	284.8 ± 109.8	267 ± 124	38 ± 9.2	49 ± 8.5
AST (U/L)
10 mg/kg	50 ± 3.8	44 ± 10.4	126 ± 54.7	145 ± 32.2	145 ± 93	219 ± 53.3	—	—
30 mg/kg	33 ± 5.5	32 ± 5.1	349 ± 104.6	316 ± 189.2	477 ± 175.2	404 ± 127.2	—	—
100 mg/kg	48 ± 18.1	33 ± 2.7	343 ± 157.6	294 ± 83.4	348.2 ± 125.8	373 ± 120.1	38 ± 7.8	33 ± 6.4
CK (U/L)
10 mg/kg	215 ± 37.6	238 ± 79.7	1021 ± 5887	1083 ± 276.1	1545 ± 1219	2252 ± 693.7	—	—
30 mg/kg	191 ± 18.8	180 ± 25.1	6736 ± 2174.2	4495 ± 3468.4	11552 ± 2953	7811 ± 2639.6	—	—
100 mg/kg	285 ± 203.4	194 ± 47.3	4081 ± 1586.2	3542 ± 824.0	11080 ± 6009.4	13275 ± 5741.4	158 ± 31.8	192 ± 43.8

Abbreviations: ALT, alanine aminotransferase; AST, aspartate transaminase; CK, creatine kinase.

^a At doses of 10 and 30 mg/kg, there were no recovery animals; therefore, the recovery data are not available.

Study to Investigate Kinetics of Serum ALT and AST Elevation in Rats

Daily administration of BLZ945 at the dose of 150 mg/kg resulted in time-dependent, mild to moderate increases in serum ALT and AST activities when compared to both the pretest values and those from the concurrent controls. The changes are shown in Figure 1 . In the BLZ945-treated rats, serum ALT elevation progressed slowly, and did not reach a 3-fold increase until after about 10 days of daily treatment. In the regression group, BLZ945 was withdrawn on day 15. Serum ALT activities continued to increase up to day 17, and then began to decrease, likely reflecting degradation half-life rather than a delay in cellular release. By study day 24, serum ALT activity was similar to the activities measured both from the pretest period and from the concurrent control. In the progression group, animals were continuously dosed for 6 weeks, and serum ALT elevation reached up to approximately 5-fold compared to controls. The pattern of serum AST elevation was similar to that of the ALT, and the magnitude of AST was about 4-fold by the end of the 6-week treatment. Under the light microscope, BLZ945-related changes included hepatocellular hypertrophy, especially in animals that were terminated after 6-week treatment. Sections of the skeletal muscle and diaphragm exhibited occasional single fiber degeneration/regeneration, and along with the lack of meaningful changes in serum CK activity suggested that muscle injury was not involved in the increased ALT and AST activities. During the course of the study, a total of 9 rats (3 rats in the regression group, and 6 rats in the progression group) were found dead or sacrificed moribund between days 8 and 35. These early deaths were likely owing to cannulation-related septicemia, as pyelitis/pyelonephritis and/or bronchopneumonia containing bacterial colonies were observed in 6 out of the 9 rats. Due to expected infection-related mortality, 12 rats were used in each group. Even after the death of some animals, at each time point of ALT and AST determinations, blood samples were collected from at least 4 animals. Therefore, the mortality did not significantly affect study validity or interpretation.

Figure 1.

Onset, progression, and regression of serum alanine aminotransferase (ALT) activity upon treatment of BLZ945.

Effect of BLZ945 on mRNA Expression of ALT and AST

In the 4-week studies in the BLA945-treated rats and monkeys, increases in mRNA levels of hepatic ALT and AST were not detected by microarray analysis. Therefore, it does not appear that the elevation of ALT and AST activities in serum was due to the increased intracellular levels of ALT and AST expression of the relevant mRNA following the BLZ945 treatment.

Transcriptional Upregulation Upon Treatment of BLZ945 From Rat Liver

The administration of BLZ945 led to a dose-dependent upregulation of early response genes in the liver, such as EGR-1, Fos, Igfbp-1, and Zfp36 (Table 4 ). The mRNA coding for proinflammatory proteins, such as interferon regulatory factor 9, interferon-induced GTPase, STAT1, and Cxcl9, were upregulated in a dose-dependent manner, indicating a possible activation of the innate immune response. A dose-dependent increase in the mRNA levels of P450 oxidoreductase (Por), NAD(P)H dehydrogenase, quinone 1 (Nqo1), and epoxide hydrolase 2 (Ephx2) was observed. Moreover, an upregulation of antioxidant-related genes vanin1 and peroxiredoxin 6 was observed. An increase of apoptosis-/necrosis-related genes was also observed, in particular genes coding for a death-associated protein and a variety of proteasome subunits. Administration of BLZ945 to rats also led to a dose-dependent upregulation of multiple genes involved in the xenobiotic metabolism, such as CYP1A1, CYP4A10, flavin monooxygenase, GST, and UDP glycosyltransferase, which may correlate with the increase in liver weight. Finally, upregulation of genes related to fatty acid oxidation such as malic enzyme 1 (ME1), stearoyl-coenzyme A desaturase 1 (Scd-1), and fatty acid synthase (Fasn) was observed.

Table 4.

Upregulation of Genes in the Rat Liver After Treatment of BLZ945 for 4 Weeks

Probe Set	Symbol	Gene Name	Controls	10 mg/kg	50 mg/kg	150 mg/kg	150 mg/kg Recovery
			Average	Fold Change
Early response genes
1368160_at	Igfbp1	Insulin-like growth factor-binding protein 1	772	1.9	1.8	2.4	1.0
1368321_at	Egr1	Early growth response 1	168	−1.3	1.6	2.3	1.1
1375043_at	Fos	FBJ osteosarcoma oncogene	21	−1.2	1.8	1.3	−1.2
1386994_at	Btg2	B-cell translocation gene 2, anti-proliferative	88	1.1	1.4	1.3	1.1
1370153_at	Gdf15	Growth differentiation factor 15	46	−1.0	1.4	1.3	−1.1
1387870_at	Zfp36	Zinc finger protein 36	290	1.0	1.2	1.3	1.1
Inflammation-specific genes
1373544_at	Cxcl9	Chemokine (C-X-C motif) ligand 9	100	1.3	1.5	1.8	1.0
1382454_at	Cxcl9	Chemokine (C-X-C motif) ligand 9	110	1.1	1.4	1.7	1.1
1387354_at	Stat1	Signal transducer and activator of transcription 1	378	1.2	1.2	1.5	−1.1
1397304_at	Igtp	Interferon γ-induced GTPase	677	1.1	1.3	1.4	−1.0
1368835_at	Stat1	Signal transducer and activator of transcription 1	466	1.1	1.2	1.4	−1.1
1372757_at	Stat1	Signal transducer and activator of transcription 1	452	1.1	1.1	1.3	−1.0
1396163_at	Igtp	Interferon γ-induced GTPase	252	1.1	1.3	1.3	1.1
1383448_at	Irf9	Interferon regulatory factor 9	211	1.2	1.3	1.2	1.0
Oxidative stress-related genes
1387109_at	Por	P450 (cytochrome) oxidoreductase	419	−1.1	1.2	1.5	1.0
1368213_at	Por	P450 (cytochrome) oxidoreductase	312	−1.1	1.1	1.5	1.0
1379361_at	Pex11a	Peroxisomal biogenesis factor 11A	268	1.1	1.3	1.5	1.0
1387599_a_at	Nqo1	NAD(P)H dehydrogenase, quinone 1	1028	1.1	1.2	1.5	1.1
1387053_at	Fmo1	Flavin containing monooxygenase 1	813	1.4	1.2	1.2	1.1
1383248_at	Fmo5	Flavin containing monooxygenase 5	2013	1.2	1.3	1.4	1.1
1369663_at	Ephx2	Epoxide hydrolase 2, cytoplasmic	693	1.0	1.2	1.3	1.1
Antioxidant-related genes
1371089_at	Gsta2	Glutathione S-transferase A3	508	1.1	1.5	3.4	1.5
1389253_at	Vnn1	Vanin 1	145	1.3	1.9	2.9	1.2
Apoptosis-/necrosis-related genes
1394082_at	Psmd14	Proteasome 26S subunit, non-ATPase, 14	330	1.1	1.1	1.3	1.1
1369992_at	Psmd1	Proteasome 26S subunit, non-ATPase, 1	333	1.0	1.1	1.3	1.1
1382385_at	Psmc6	Proteasome 26S subunit, ATPase, 6	120	1.0	1.2	1.3	1.1
1369941_at	Dap	Death-associated protein	1463	1.0	1.1	1.3	−1.0
Fatty acid oxidation-related genes
1370870_at	Me1	Malic enzyme 1, NADP(+)-dependent, cytosolic	245	−1.1	1.6	2.9	1.5
1370067_at	Me1	Malic enzyme 1, NADP(+)-dependent, cytosolic	378	−1.1	1.6	2.7	1.3
1394401_at	Elovl6	ELOVL family member 6, elongation of long-chain fatty acids	102	−1.4	1.2	2.0	1.7
1388108_at	Elovl6	ELOVL family member 6, elongation of long-chain fatty acids	304	−1.2	1.2	1.7	1.4
1367707_at	Fasn	Fatty acid synthase	698	−1.1	1.1	1.3	1.5
1367708_a_at	Scd1	Fatty acid synthase	2182	−1.2	1.1	1.2	1.4
1370893_at	Fasn	Acetyl-coenzyme A carboxylase α	846	−1.1	1.1	1.3	1.4
1388644_at	Acaca	Monoglyceride lipase	211	1.1	1.1	1.4	−1.0
1391462_at	Mgll	Emopamil binding protein-like (predicted)	312	−1.0	1.1	1.3	1.0
Xenobiotic metabolism-specific genes
1370269_at	Cyp1a1	Cytochrome P450, family 1, subfamily a, polypeptide 1	13	1.5	1.8	8.8	−1.0
1371089_at	Gsta2	Glutathione S-transferase A3	508	1.1	1.5	3.4	1.5
1370465_at	Abcb1a	ATP-binding cassette, sub-family B, member 1A	90	1.4	1.4	2.3	1.4
1369455_at	Abcg5	ATP-binding cassette, sub-family G, member 5	111	1.4	1.4	2.3	−1.4
1387022_at	Aldh1a1	Aldehyde dehydrogenase 1 family, member A1	861	−1.1	1.4	2.2	1.4
1369698_at	Abcc3	ATP-binding cassette, subfamily C, member 3	16	−1.5	−1.0	2.2	1.2
1387094_at	Slco1a4	Solute carrier organic anion transporter family, member 1a4	1060	1.0	1.1	1.9	1.1
1370464_at	Abcb1a	ATP-binding cassette, subfamily B, member 1A	99	1.1	1.4	1.9	1.1
1368121_at	Akr7a3	Aldo-keto reductase family 7, member A3	577	1.1	1.1	1.7	1.0
1387759_s_ at	Ugt1a1	UDP glycosyltransferase 1 family, polypeptide A1	1760	1.1	1.3	1.7	1.1
1387093_at	Slco1a4	Solute carrier organic anion transporter family, member 1a4	3606	−1.0	1.0	1.6	1.1
1387109_at	Por	P450 (cytochrome) oxidoreductase	419	−1.1	1.2	1.5	1.0
1370613_s_at	Ugt1a1	UDP glycosyltransferase 1 family, polypeptide A1	4411	1.1	1.2	1.5	1.1
1370583_s_at	Abcb1a	ATP-binding cassette, subfamily B, member 1B	101	1.0	1.1	1.5	1.3
1369850_at	Ugt2a1	UDP glucuronosyltransferase 2 family, polypeptide A1	120	−1.3	1.1	1.5	−1.1
1368934_at	Cyp4a10	cytochrome P450, family 4, subfamily a, polypeptide 10	2516	1.3	1.4	1.5	−1.2
1370698_at	Ugt2b1	UDP glucuronosyltransferase 2 family, polypeptide B1	2647	−1.1	1.1	1.4	1.1
1395644_at	Abca8b	ATP-binding cassette, subfamily A, member 8b-predicted	118	1.1	1.4	1.3	−1.0
1392539_at	Blvrb	Biliverdin reductase B (flavin reductase [NADPH])-predicted	405	1.1	1.1	1.4	1.0

Transcriptional Upregulation Upon Treatment of BLZ945 From Monkey Liver

Upregulation of acute-phase and inflammation markers such as serum amyloid A1 (SAA1) and of members of the interferon pathway was observed, indicating a minimal activation of the innate immune response (Table 5 ). An upregulation of the CYP1A1 gene was detected, however, mainly at the high dose. An upregulation of genes related to cholesterol biosynthesis was identified. Upregulation of apoptosis-/necrosis-related genes, including a variety of proteasome subunits and Caspase 10, was identified. together with an upregulation of MHC class I markers was also noted, suggesting an activation of the immune system and a possibly increased antigen processing further supported by the concurrent upregulation of TAP1 and TAPBP. TAP1 has similarity to multidrug-resistant transporters and transports antigenic peptides across the endoplasmic reticulum membrane in the preparation for MHC class I presentation. ¹³

Table 5.

Upregulation of Liver Genes in Monkeys After 4-Week Treatment of BLZ945

Probe Set	Symbol	Gene Name	Controls	10 mg/kg	30 mg/kg	100 mg/kg	100 mg/kg Recovery
			Average	Fold Change
Acute-phase-specific genes
214456_x_at	SAA1	Serum amyloid A1	365	1.3	1.8	6.8	1.1
208607_s_at	SAA1	Serum amyloid A1	46	2.5	1.8	4.1	2.3
203649_s_at	PLA2G2A	Phospholipase A2, group IIA (platelets, synovial fluid)	300	1.6	1.0	3.9	1.2
211560_s_at	ALAS2	Aminolevulinate, delta-, synthase 2	18	1.1	−1.1	2.8	−2.0
201891_s_at	B2M	β-2-Microglobulin	681	1.6	1.6	2.4	1.3
205040_at	ORM1	Orosomucoid 1	8813	1.3	1.3	2.2	1.0
205041_s_at	ORM1	Orosomucoid 1	16252	1.2	1.3	1.8	1.0
216231_s_at	B2M	β-2-Microglobulin	32025	1.2	1.3	1.6	1.0
214465_at	ORM1	Orosomucoid 1	115	1.1	1.3	1.5	−1.1
204846_at	CP	Ceruloplasmin (ferroxidase)	21997	1.3	1.4	1.5	1.1
206697_s_at	HP	Haptoglobin	5780	1.1	1.3	1.4	−1.1
Inflammation-specific genes
204415_at	IFI6	Interferon, α-inducible protein 6	146	2.4	3.2	7.8	−2.6
202269_x_at	GBP1	Guanylate-binding protein 1, interferon-inducible, 67 kDa	271	1.6	1.8	3.6	1.2
203153_at	IFIT1	Interferon-induced protein with tetratricopeptide repeats 1	235	2.7	1.7	3.6	1.6
205660_at	OASL	2′-5′-oligoadenylate synthetase-like	83	2.1	1.7	3.5	2.2
214453_s_at	IFI44	Interferon-induced protein 44	555	2.2	2.1	3.5	1.4
202411_at	IFI27	Interferon, α-inducible protein 27	539	−1.0	1.3	2.6	1.3
201315_x_at	IFITM2	Interferon-induced transmembrane protein 2 (1-8D)	964	1.6	1.7	2.6	1.2
231577_s_at	GBP1	Guanylate-binding protein 1, interferon-inducible, 67 kDa	191	1.3	1.6	2.5	−1.0
204972_at	OAS2	2′-5′-oligoadenylate synthetase 2, 69/71 kDa	72	1.5	1.3	2.3	1.2
201601_x_at	IFITM1	Interferon-induced transmembrane protein 1 (9-27)	202	1.1	1.1	2.2	−1.2
203882_at	IRF9	Interferon regulatory factor 9	329	1.5	1.4	2.0	1.1
212203_x_at	IFITM3	Interferon-induced transmembrane protein 3 (1-8U)	1241	1.6	1.4	1.9	1.2
Apoptosis-/necrosis-related genes
209040_s_at	PSMB8	Proteasome (prosome, macropain) subunit, β-type, 8	95	4.2	4.0	7.5	2.7
202659_at	PSMB10	Proteasome subunit, β-type, 10	117	1.9	1.9	4.8	−1.4
204279_at	PSMB9	Proteasome subunit, β-type, 9	187	1.2	1.2	2.2	1.2
211367_s_at	CASP1	Caspase 1, apoptosis-related cysteine peptidase	77	1.1	−2.0	1.6	1.2
201762_s_at	PSME2	Proteasome activator subunit 2 (PA28 β)	2613	1.2	1.1	1.4	−1.0
Xenobiotic metabolism-specific genes
205749_at	Cyp1a1	Cytochrome P450, family 1, subfamily a, polypeptide 1	273	1.1	1.5	4.8	−1.1
Cholesterol biosynthesis-related genes
211423_s_at	SC5DL	Sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, Saccharomyces cerevisiae)-like	1351	1.9	1.7	2.6	1.5
209146_at	SC4MOL	Sterol-C4-methyl oxidase-like	1509	1.4	1.2	2.4	1.1
221750_at	HMGCS1	3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1	223	1.3	1.2	2.1	−1.1
211162_x_at	SCD	Stearoyl-CoA desaturase (delta-9-desaturase)	454	1.2	−1.2	2.1	−1.0
205822_s_at	HMGCS1	3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1	462	1.0	1.2	2.1	1.1
216607_s_at	CYP51A1	Cytochrome P450, family 51, subfamily A, polypeptide 1	2132	1.1	1.1	1.9	1.1
MHC class I genes
211799_x_at	HLA-C	Major histocompatibility complex, class I, C	991	1.4	1.2	4.5	1.1
213932_x_at	HLA-A	Major histocompatibility complex, class I, A	1284	1.7	1.7	4.3	1.0
208729_x_at	HLA-B	Major histocompatibility complex, class I, B	2219	1.5	1.5	3.7	1.7
211911_x_at	FAM20B	Major histocompatibility complex, class I, B	2901	1.6	1.5	3.7	1.4
214459_x_at	HLA-C	Major histocompatibility complex, class I, C	2119	1.9	1.6	3.2	1.4
209140_x_at	HLA-B	Major histocompatibility complex, class I, B	5474	1.3	1.4	3.1	1.4
216526_x_at	HLA-B	Major histocompatibility complex, class I, B	2826	1.4	1.3	3.0	−1.0
204806_x_at	HLA-F	Major histocompatibility complex, class I, F	1381	1.6	1.6	3.0	1.2
208812_x_at	HLA-C	Major histocompatibility complex, class I, C	1723	1.8	1.4	3.0	1.3
215313_x_at	HLA-A	Major histocompatibility complex, class I, A	4385	1.7	1.7	2.8	1.2
221875_x_at	HLA-F	Major histocompatibility complex, class I, F	1575	1.5	1.4	2.8	1.2
217436_x_at	HLA-J	Major histocompatibility complex, class I, J	1658	1.4	1.4	2.6	1.3
211528_x_at	HLA-G	Major histocompatibility complex, class I, G	2919	1.6	1.3	2.5	1.2
211529_x_at	HLA-G	Major histocompatibility complex, class I, G	3508	1.5	1.3	2.4	1.2
217456_x_at	HLA-E	Major histocompatibility complex, class I, E	960	1.5	1.4	2.2	1.1
210514_x_at	HLA-G	Major histocompatibility complex, class I, G	731	1.3	1.3	2.2	1.1

Gene Downregulation Upon Treatment of BLZ945 From Rat and Monkey Liver

At the transcriptomic level, the pharmacological action of BLZ945 was reflected by a marked dose-dependent downregulation of CSF-1R and other Kupffer cell–specific genes like Fc receptor, IgG, low-affinity III, CD53, and CD68 in both rats and monkeys. This effect was reversible after 4 weeks.

Discussion

Elevated serum ALT and AST activities in the absence of histological damage in the liver can confound preclinical interpretation. Historically, increases in serum ALT activities in the range of 2 to 4 times or higher than controls raise concern for potential hepatotoxicity, unless a clear alternative explanation is found. In preclinical studies with BLZ945 in rodent models, at dose levels where efficacy or target inhibition of the pharmacodynamic markers was identified (data not shown), up to 6-fold increases in serum ALT and up to 13-fold increases in serum AST were observed in the absence of notable morphological damage in the liver. Consequently, it is predicted that therapeutic doses of BLZ945 might similarly cause serum ALT and AST elevations in humans. Therefore, it is critical to understand whether the elevations of serum ALT and AST are truly indicative of liver injury. We then investigated whether there was a benign explanation for the evaluations of serum ALT and AST activities. Our first study was to determine whether the increased ALT and AST levels could be owing to the increased expression of the ALT and AST genes. For example, glucocorticoids have been the most studied class of drugs with regard to induction of de novo ALT synthesis, and are known to cause increases in serum ALT levels in rat liver. ^{14 –16} We examined whether ALT and AST transcript levels increased following the treatment of animals with BLZ945. Hepatic ALT and AST mRNA levels in the rats and monkeys following daily dosing of BLZ945 for 4 weeks were not upregulated, indicating that there was no increase in gene transcription of ALT or AST following the treatment with BLZ945.

We then investigated whether the increased ALT and AST activities could be related to injury of nonhepatic tissues. Skeletal muscle is known to contain CK, AST, and ALT, which may be released into the bloodstream following muscle necrosis. In the 4-week rat study, serum CK level was not increased, and morphological injury of skeletal muscle was not observed in the specimens examined under the light microscope. There was no indication of skeletal muscle injury in the 4-week rat study. It can be argued that not all muscles were observed by histology. In order to further evaluate whether BLZ945 causes location-specific skeletal muscle damage, in the subsequent 6-week rat study, serum CK analysis and extensive sampling of various muscles, including diaphragm, gastrocnemius, and soleus were examined. However, again no meaningful changes in either serum CK activity or histopathological findings indicated muscle injury in rats upon 6 weeks of BLZ945 treatment, and the elevated ALT and AST could not be explained by the speculated muscle injury in the rat studies. In the 4-week monkey study, mild to marked increases in serum total CK activity were detected, and specifically in the CK isoenzyme (CK-MM), which is associated with skeletal muscle. However, morphological damage of skeletal muscle was not identified using light microscopy. Moreover, studies have shown that the AST:ALT ratio following muscle injury is ≥3 in rat, monkey and human, ^{17 –19} while in the present monkey study, the average AST:ALT ratio was <2, even in individual animals. Although a slight contribution from minimal muscle injury could not be ruled out, it does not support the theory that increased ALT was fully due to the muscle injury, due to lack of corresponding histopathological findings, and because the ratio of AST:ALT is <2. Collectively, no indication of muscle injury in the rats, and the missing concrete data for muscle injury in the monkeys, seriously called into question a possible muscular origin of the increased serum ALT and AST after the treatment of BLZ945. Therefore, a hepatic origin of the increased serum AST and ALT activities was considered more likely.

Another possibility as to the origin of the increased serum ALT and AST levels could be liver hypertrophy. Histological evidence of hepatic hypertrophy and serum chemistry markers of hepatic origin have been examined in rats and cynomolgus monkeys. ^{20 –22} In each animal species, 9 or 10 investigative drug candidates representing 5 therapeutic classes were studied. Studies in rats showed that slight elevations of serum ALT (<2-fold) were occasionally seen with slight to moderate hepatic hypertrophy. ¹⁷ Ennulate et al ²² reported that an average of 3-fold increases in serum ALT could be found under hepatic hypertrophy after reviewing multiple studies done with drug candidates in rats. An analysis of monkey data revealed that significant induction of liver microsomal enzymes did not commonly cause hepatic hypertrophy or increased serum ALT and AST. ¹⁸ Based on the literature, it is unlikely that the up to 6- and 8-fold increases of serum ALT in the BLZ945-treated rats and monkeys, respectively, are due simply to liver hypertrophy.

We next examined the possibility that increased serum ALT and AST levels could be due to decreased clearance resulting from Kupffer cell depletion. Kamimoto et al ²³ suggested that “sinusoidal liver cells” (a mixture of Kupffer and endothelial cells) were responsible for the plasma clearance of AST by endocytosis. Smit et al ^24,25 demonstrated that plasma ALT and AST were likely endocytosed by Kupffer cells rather than endothelial liver cells. However, numerous studies have shown that depletion of Kupffer cells by gadolinium chloride, allyl alcohol, and concanavalin A in a range of 40% to 90% did not cause elevations of ALT and/or AST. ^{26 –29} In the 4-week monkey study, treatment of BLZ945 caused reduction of Kupffer cells in a range of 20% to 50%, which was expected as the pharmacology effect of this compound. Therefore, the BLZ945 treatment-related elevation of ALT and AST is unlikely due to the inhibition of Kupffer cells. Furthermore, our unpublished data have shown that another compound which shares the same pharmacology of BLZ945 also caused 50% inhibition of Kupffer cells. Yet, significant ALT elevation was not observed. Thus, in this instance, it is difficult to conclude that the increased serum ALT and AST levels were due to decreased clearance as a result of decreased Kupffer cell numbers following BLZ945 treatment.

Toxicogenomic profiling of the liver was next used to provide a possible molecular mechanistic explanation for the dose-dependent increases in AST and ALT levels observed in the 4-week studies in rats and monkeys. Toxicogenomic profiling of the liver following BLZ945 treatment indicated that at the transcriptional level the effects of BLZ945 consisted of a dose-dependent upregulation of genes associated with early response to injury, inflammation, oxidative stress, or apoptosis/necrosis. In the rat liver, for example, upregulations of EGR-1, Fos, Zfp36, and Igfbp1 were identified. These genes are known as signature genes for liver insult in the case of acetaminophen- or carbon tetrachloride–induced liver injury. ^{30 –32} Significant increases in the concentration of reactive oxygen species (ROS) and reactive nitrogen species (RNS), and/or decreases in detoxification mechanisms, can lead to oxidative stress. In this study, a dose-dependent increase in the mRNA expression of genes encoding proteins that have the potential to generate such ROS was observed, including upregulation of P450 Por and Nqo1. Aleksunes et al ^33,34 have observed that Nqo1 was induced in the livers of acetaminophen-treated mice, and also in the livers of patients who died from acetaminophen overdose, suggesting that Nqo1 induction might be an adaptive response to acetaminophen-induced hepatotoxicity. An upregulation of the antioxidants vanin1 and peroxiredoxin 6 was also reported following the treatment with BLZ945 in rats, which could be interpreted as a response to oxidative stress. ^35,36 An increase of apoptosis-/necrosis-related genes was also observed, in particular the gene (Dap) coding for death-associated protein, and a variety of proteasome subunits following the treatment with BLZ945 in rats or monkeys. The slight increase in the transcription of proteasome genes could reflect the presence of minimal cell damage, including single-cell necrosis in such an amount that may not be detectable by histopathology. In total, these genomic changes were considered an adaptation of the liver to a hepatocellular stress.

Numerous investigators have evaluated toxicogenomic changes under drug-induced liver hypertrophy, and identified some common biochemical pathways in mediating liver hypertrophy, including stimulation of triglyceride hydrolysis, fatty acid oxidation, and cell cycle/proliferation. ^{37 –39} In none of these studies were genes reflecting early response to injury, inflammation, or apoptosis/necrosis reported under the conditions of hepatic hypertrophy. This information further confirmed our belief that liver hypertrophy was not the sole reason behind the ALT and AST elevation. Toxicogenomic analysis of the BLZ945-treated rats and monkeys also indicated the induction of genes associated with hepatic hypertrophy. There are a variety of mechanisms by which drug-induced hepatocellular hypertrophy can occur, including increases in smooth endoplasmic reticulum contents (or P450 activities), peroxisome proliferation, hypertrophy of mitochondria, or hepatocellular proliferation. We demonstrate that administration of BLZ945 to rats led to a dose-dependent upregulation of genes coding for metabolic enzymes including CYP1A1, CYP4A10, flavin monooxygenase, GST, and multiple UDP glycosyltransferase subfamilies, as well as various ATP-binding protein subfamilies (Abcb1 and Abcg5). All of these proteins are endoplasmic reticulum–resident proteins. The upregulation of these genes could be indicative of increased endoplasmic reticulum contents and/or induced metabolic enzyme activities, leading to an explanation for hepatocellular hypertrophy observed under the microscope in the rat. In the monkey study, hepatic hypertrophy was not observed, and only minimal mRNA inductions of CYP1A1 and CYP51A1 were observed. Studies have shown that hepatic hypertrophy is associated with a number of distinct gene expression profiles. ^37,38 For example, the induction of genes related to fatty acid oxidation, such as stearoyl-CoA desaturase 1 (Scd1), acetyl-CoA carboxylase α (Acaca), and fatty acid synthase (Fasn) are recognized as signature genes for hepatic hypertrophy. In our studies, Scd1, Acaca, and Fasn were indeed upregulated upon treatment with BLZ945. Collectively, the toxicogenomic data corroborated the liver hypertrophy observed in rat, and provided a possible mechanistic explanation that the cause of hypertrophy was at least partially due to the increases in smooth endoplasmic reticulum contents and/or enzyme activities. Although genes encoding cell-cycle proteins have been consistently reported in PPARα- or non-PPARα-induced liver hypertrophy, ^{37 –39} such increases in transcript levels were not observed in our study. Therefore, in our study, the hypertrophy was unlikely due to hepatocellular proliferation.

BLZ945 is a potent CSF-1R inhibitor that affects macrophage maturation. The toxicogenomic data indicated that CSF-1R transcript levels and Kupffer cell–specific genes were markedly downregulated following BLZ945 treatment in both rats and monkeys. This profile is consistent with the BLZ945-induced Kupffer cell reduction evidenced by immunohistochemistry analysis of the liver slides in the 4-week monkey study. Whether the observed hepatocellular stress is due to BLZ945-induced Kupffer cell inhibition is unknown. It has been shown ⁴⁰ that Kupffer cells may have a protective role in drug-induced acute liver injury, and the hepatoprotective role is likely mediated by cytokines and other mediators (such as IL-10, IL-6, IL-18BP, and C1q), which are important in counteracting inflammatory responses. In the rats and monkeys treated with BLZ945, depletion of Kupffer cells could decrease the levels of these protective cytokines and mediators, leading to ultimate liver injury. It is also possible that the downregulation of Kupffer cells and the hepatocyte stress are 2 independent events. Further studies, such as a time-course toxicogenomic study with some early time points, might help to explore the involvement of Kupffer cells in the hepatocyte stress upon the treatment of BLZ945.

In summary, our studies showed increased levels of ALT and AST with dose-dependent increases of the transcription of various stress genes, even in the absence of any detectable liver injury by light microscopy, might be indicative of drug-induced stress to the hepatocytes. Based on the current knowledge and available data, the development of BLZ945 was terminated due to potential liver toxicity at efficacious dose levels. In situations such as these, where the correlation between serum hepatic enzyme biomarkers and liver histopathological changes is poor, toxicogenomic profiling may assist in the interpretation of human-relevant hepatotoxic risk. In this case, elevation of serum ALT and AST levels upon BLZ945 treatment could be due to the combined effects of hepatic stress, hepatocellular hypertrophy, and reduction in Kupffer cell population, although using traditional methodology did not permit us to identify the source of ALT and AST leakage. Further investigations are being run to investigate the underlying mechanisms of elevated hepatic injury biomarkers in the absence of histopathological evidence of hepatocyte injury.