Adeno-associated virus (AAV) vectors are widely used for gene therapy as they deliver therapeutic genes with minimal toxicity. However, pre-existing anti-AAV immunity in a large percentage of humans poses a challenge to their efficacy and safety; only patients with low or no AAV-specific antibodies are currently eligible for systemically administered gene therapies. The impact of pre-existing anti-AAV total antibodies (TAbs) following local delivery is less characterized. This study explored the impact of pre-existing anti-AAV9 TAbs on biodistribution and safety of an AAV9 tool vector administered intrathecally to cynomolgus monkeys. Although high-serum AAV9 titers did not affect central nervous system vector biodistribution, it reduced distribution to peripheral tissues, except the spleen in which vector genome copies increased. Animals with high pre-existing antibodies had rapid and transient systemic interferon response, faster therapy-emergent antibody increases in the cerebrospinal fluid (CSF), and higher cellular responses in the spleen but no major safety concerns. Our findings suggest that using serum AAV titers as an eligibility criterion for intrathecal AAV-based gene therapy could exclude patients who might benefit from treatment. Stronger anti-vector immune responses in animals with high anti-AAV9 TAb titers suggest the need to closely monitor treatment-emergent immune responses and potential consequences for highly seropositive patients.

Administration of AAV-based gene therapies into the intra-cerebrospinal fl uid (CSF) compartments via routes such as lumbar puncture (LP) has been implemented as an alternative to intravenous dosing to target the CNS regions. This route enables lower doses, decreases systemic toxicity, and circumvents intravascular pre-existing anti-AAV antibodies. In this study, AAV9-GFP vectors were administered via LP to juvenile cynomolgus macaques with and without pre-existing serum antiAAV9 antibodies at a 5.0 x 1013 vector genomes per mL (vg/mL) dose and examined for 28 days. CNS and peripheral tissues were surveyed for vector genome, mRNA, and protein expression. Histopathology, clinical pathology, and humoral immune response to the viral capsid and transgene were also assessed. In addition, serum and CSF samples were analyzed to examine 276 proteomic markers curated to evaluate neural injury, organ damage, and inflammatory response. This study reveals no noticeable difference in AAV9-mediated gene transfer in the CNS tissues in the two groups; however, differences were observed for endpoints such as liver enzyme activities, histopathology, and levels of protein markers in the serum and CSF. These fi ndings provide a view into vector transduction effi ciency and safety following LP-delivered AAV9 to juvenile cynomolgus macaques with and without pre-existing antiAAV9 antibodies.

The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

Members of the ATP-dependent SWI/SNF chromatin remodeling complexes are among the most frequently mutated genes in cancer, suggesting their dysregulation plays a critical role. The synthetic lethality between SWI/SNF catalytic subunits BRM/SMARCA2 and BRG1/SMARCA4 has instigated great interest in targeting BRM. Here we have performed a critical and in-depth investigation of novel dual inhibitors (BRM011 and BRM014) of BRM and BRG1 in order to validate their utility as chemical probes of SWI/SNF catalytic function, while obtaining insights into the therapeutic potential of SWI/SNF inhibition. In corroboration of on-target activity, we discovered compound resistant mutations through pooled screening of BRM variants in BRG1 -mut cancer cells. Strikingly, genome-wide transcriptional and chromatin profiling (ATAC-Seq) provided further evidence of pharmacological perturbation of SWI/SNF chromatin remodeling as BRM011 treatment induced specific changes in chromatin accessibility and gene expression similar to genetic depletion of BRM. Finally, these compounds have the capacity to inhibit the growth of tumor-xenografts, yielding important insights into the feasibility of developing BRM/BRG1 ATPase inhibitors for the treatment of BRG1 -mut lung cancers. Overall, our studies not only establish the feasibility of inhibiting SWI/SNF catalytic function, providing a framework for SWI/SNF therapeutic targeting, but have also yielded successful elucidation of small-molecule inhibitors that will be of importance in probing SWI/SNF function in various disease contexts.

For thc establishment of a high throughput screening system using primary cell cultures, investigation of elucidatcd toxicities to assess the correlation between in vitro and in vii,o hepatotoxicity is necessary in the safety eva]uation of thc compound. [n the prcvious study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hcpatocytes culture screening system, we conducted a singlc-dose in vivo study with relatively high dose of hcpatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochernistry, Wc also carried out a proteomics analysis for estimating the reliable and scnsitive biomarkers. Histopathologica]ly, cumpound-spccific hepatotoxicity was detected at 24 hr after administration in all compounds cxcept amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative strcss-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealcd several protein biomarkcrs related to oxidative stress and mitochondrial mctabolism-regulation, which had been previoushy dctected by proteomics analysis in in vitro screening system. Oxidative stress-related protcins were considered ag uscful biomarkers of hepatotoxicity; since they were detected by immunohistoehemistry and proteomics analysis prior to appearancc of compound-specifie histopathologica] changes detected by light microscopy. Considering the relevance of in vitro system to in i,i],o system from the aspect of new biomarkers re]ated to the toxicogenomiesftoxicoproteomics, in vitro primary ce[[ culture system would be sut/ficient to detect hepatotoxicity in the ear[y gtage of drug diseover}r.

The effects of repeated complement activation in cynomolgus monkeys after chronic antisense oligonucleotide (ASO) treatment were evaluated by using ISIS 104838, a representative 2'-O-methoxyethyl (2'-MOE) modified ASO. The treatment was up to 9 months with a total weekly dose of 30 mg/kg, given either as daily [4.3 mg/kg/day, subcutaneous (s.c.) injection] or once weekly [30 mg/kg, either as s.c. injection or 30-min intravenous (i.v.) infusion]. Acute elevations of complement split products (Bb and C3a) and a transient decrease in C3 occurred after the first dose and were drug plasma concentration dependent. However, with repeated complement activation after chronic ASO treatment, there were progressive increases in basal (predose) levels of Bb and C3a, and a sustained C3 reduction in all treated groups. There was also a progressive increase in C3d-bound circulating immune complex (CIC) that was considered secondary to the C3 depletion. Evidence of vascular inflammation was observed, mostly in the liver, kidney, and heart, and correlated with severe C3 depletion and increases in plasma IgG and IgM. Vascular inflammation was accompanied by increased C3 and IgM immunereactivity in the affected vasculatures and endothelial activation markers in serum. In summary, repeated complement activations in monkeys lead to a sustained decrease in circulating C3 over time. The concomitantly increased inflammatory signals and decreased CIC clearance due to impairment of complement function may lead to vascular inflammation after chronic ASO treatment in monkeys. However, based on the known sensitivity of monkeys to ASO-induced complement activation, these findings have limited relevance to humans.

The purpose of this article is to describe a case of drug-induced cutaneous toxicity observed in cynomolgus monkeys and to introduce approaches attempted in order to elucidate mechanisms. The test article was a small molecule with a ubiquitously distributed target, especially in rapidly dividing cells, and which modulated cell cycle regulation. After 7 consecutive days of oral dosing, animals developed multifocal skin lesions. The lesions were characterized clinically by vesicles and scabs formation and were distributed mainly in thin-skinned areas of the body including the cheek, chest, abdomen, and inner limbs. Microscopically, the lesions were confirmed as epidermal vesicle formation and ulceration. Immunohistochemical staining revealed that the levels within the epidermis where separation (vesicle formation) occurred were not consistent. The differential diagnoses for vesicular skin lesions and our efforts to elucidate the mechanism of toxicity using in-house database searches and immunohistochemistry are discussed. To the best of our knowledge, similar cutaneous toxicity has not been reported previously, although there are reports of other types of cutaneous toxicities. Understanding the mechanisms of the toxicity is very important when assessing human relevancy during drug development. Our investigative approach can be utilized when unusual skin toxicities are observed in the future.

This chapter contains sections titled: General Introduction Introduction of in vitro Screening Tests Correlations between in vitro Systems and in vivo Systems Application of Toxicopanomics Technologies to in vitro Safety Evaluation Systems Introduction of Evaluation Systems Using in vitro Hepatotoxicity Tests in the Early Phase of Drug Discovery Future Prospects References

We investigated the overall protein expression profiles in the in vivo hepatotoxicity of rats induced by four well-recognized hepatotoxicants. Acetaminophen (APAP), amiodarone (AMD), tetracycline (TC) and carbon tetrachloride (CTC) were administered to male rats by gavages and the liver at 24 hr post-dosing was applied to the proteomic experiment. Blood biochemistry and histopathology were examined to identify specific changes related to the compounds given. Protein expression in the liver was investigated by 2-dimensional gel electrophoresis (2DE), and spots showing a significantly different expression in treated versus control group were excised from gels and identified by Q-Tof mass spectrometer. They were well characterized based on their functions related to the mechanisms of toxicity of the compounds. Among them, we focused on the 8 proteins that were affected by all 4 compounds examined. Proteins related to oxidative stress response such as carbonic anhydrase III (CA3) and 60kDa heat shock protein (HSP60), and energy metabolism such as adenylate kinase 4 (AK4) were found. Moreover, hierarchical clustering analysis using 2D-gel spots information revealed the possibility to differentiate the groups based on their toxicity levels such as severity of liver damage. These results suggested that assessing the effects of hepatotoxicants on protein expression is worth trying to screen candidate compounds at the developmental stage of drugs.

For the establishment of a high throughput screening system using primary cell cultures, investigation of elucidated toxicities to assess the correlation between in vitro and in vivo hepatotoxicity is necessary in the safety evaluation of the compound. In the previous study, we reported the usability of rat primary cultured hepatocytes for establishment of high throughput screening system. To confirm the reliability of rat primary hepatocytes culture screening system, we conducted a single-dose in vivo study with relatively high dose of hepatotoxicant in rats using 4 reference compounds (acetaminophen, amiodarone, tetracycline, carbon tetrachloride), and investigated histopathological changes and expression of oxidative stress-related proteins by immunohistochemistry. We also carried out a proteomics analysis for estimating the reliable and sensitive biomarkers. Histopathologically, compound-specific hepatotoxicity was detected at 24 hr after administration in all compounds except amiodarone, which is known to induce phospholipidosis. Immunohistochemically, oxidative stress-related proteins were increased within 6 hr after administration in all treated groups. Proteomics analysis revealed several protein biomarkers related to oxidative stress and mitochondrial metabolism-regulation, which had been previously detected by proteomics analysis in in vitro screening system. Oxidative stress-related proteins were considered as useful biomarkers of hepatotoxicity; since they were detected by immunohistochemistry and proteomics analysis prior to appearance of compound-specific histopathological changes detected by light microscopy. Considering the relevance of in vitro system to in vivo system from the aspect of new biomarkers related to the toxicogenomics/toxicoproteomics, in vitro primary cell culture system would be sufficient to detect hepatotoxicity in the early stage of drug discovery.

The present study was undertaken to estimate the usefulness of genomic approaches to predict hepatotoxicity. Male rats were treated with acetaminophen (APAP), carbon tetrachloride (CCL), amiodarone (AD) or tetracycline (TC) at toxic doses. Their livers were extracted 6 or 24 hr after the dosings and were used for subsequent examinations. At 6 hr there were no histological changes noted in any of the groups except for the CCL group, but at 24 hr, such changes were noted in all but the AD group. Regarding genomic analysis, we performed hierarchical cluster analysis using S-plus software. The individual microarray data were clearly classified into 5 treatment-related clusters at 24 hr as well as at 6 hr, even though no morphological changes were noted at 6 hr. In the gene expression analysis using GeneSpring, transcription factor and oxidative stress- and lipid metabolism-related genes were markedly affected in all treatment groups at both time points when compared with the corresponding control values. Finally, we investigated gene networks in the above-affected genes by using Ingenuity Pathway Analysis software. Down-regulation of lipid metabolism-related genes regulated by SREBP1 was observed in all treatment groups at both time points, and up-regulation of oxidative stress-related genes regulated by Nrf2 was observed in the APAP and CCL treatment groups. From the above findings, for the application of genomic approaches to predict hepatotoxicity, we considered that cluster analysis for classification and early prediction of hepatotoxicity and network analysis for investigation of toxicological biomarkers would be useful.

High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.

Predictive biomarkers of testicular toxicity are needed for an efficient development of drugs. The purpose of the present study was to obtain further insight into the toxicity mechanisms of various male reproductive toxicants and to detect genomic biomarkers for rapid screening of testicular toxicity. Four reproductive toxicants, 2,5-hexanedione (Sertoli cells toxicant), ethylene glycol monomethyl ether (EGME; spermatocytes toxicant), cyclophosphamide (spermatogonia toxicant) and sulfasalazine, were orally administered to male rats once. Six hours after the single dosing, gene expression in the testes was monitored by cDNA microarray and real-time RT-PCR and the testes were histopathologically examined. No histopathological abnormality was detected except for slight degeneration of spermatocytes in the EGME-treated testes. cDNA microarray analysis revealed differential gene expression profiles, and it was possible based on the profiles to characterize the action of the compounds in the testes. Interestingly, 3 spermatogenesis-related genes -- heat shock protein 70-2, insulin growth factor binding protein 3 and glutathione S transferase pi -- were affected by all the compounds. The above changes of gene expression were detectable within a short period after the dosing prior to the appearance of obvious pathological changes. These data suggest that cDNA microarray is a useful technique for evaluation of primary testicular toxicity. Furthermore, we propose the above 3 spermatogenesis-related genes as potential biomarkers of testicular toxicity.

We investigated the effects of three hepatotoxicants, acetaminophen (APAP), amiodarone (AD) and tetracycline (TC), on protein expression in primary cultured rat hepatocytes with toxicoproteomic approach, which is two-dimensional gel electrophoresis (2DE) and mass spectrometry. The objectives of this study were to search for alternative toxicity biomarkers which could be detected with high sensitivity prior to the appearance of morphological changes or alterations of analytical conventional biomarkers. The related proteins in the process of cell degeneration/necrosis such as cell death, lipid metabolism and lipid/carbohydrate metabolism were mainly affected under exposure to APAP, AD and TC, respectively. Among the differentially expressed proteins, several oxidative stress-related proteins were clearly identified after 24-hr exposure, even though they were not affected for 6-hr exposure. They were glutathione peroxidase (GPX) as a down-regulated protein as well as peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as up-regulated proteins, which are known to serve as antioxidative enzymes in cells. These findings suggested that the focused proteins, GPX and PRXs, could be utilized as biomarkers of hepatotoxicity, and they were useful for setting high throughput screening methods to assess hepatotoxicity in the early stage of drug discovery.

The utilization of safety biomarkers to predict the possibility of compound-related toxicity provides several advantages for drug discovery and development, especially at an early stage. The objectives of this study were to investigate the effects of male reproductive toxicants on protein expression profiles in the rat testes and to identify potential biomarker candidates. Four well-known reproductive toxicants, ethylene glycol monomethyl ether (EGME), cyclophosphamide (CP), sulfasalazine (SASP) and 2,5-hexanedione (2,5-HD), were administered to male rats in a single dose, and protein expression profiles were investigated after 24 hr by two-dimensional gel electrophoresis (2DE). Histopathological examination of the testes and serum concentration analysis were also performed. From the results of the comparison of 2D-gels among different doses of a compound and among compounds, 52, 20, 24 and 111 spots were nominated as differentially expressed spots with EGME, CP, SASP and 2,5-HD treatments, respectively. Several spermatogenesis-involved proteins were identified, including glutathione S-transferase (GST), testis-specific heat shock protein 70-2 (HSP70-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphatidylethanolamine-binding protein (PEBP). Some of them were altered by more than one compound. In summary, remarkable histopathological findings were observed only in the EGME high-dose group, and most of the protein changes were detected before histopathological changes occurred. Therefore, the proteins identified in this study could potentially serve as biomarkers to evaluate male reproductive toxicity at an early stage of drug discovery and development.