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The first-in-patient (FIP) starting dose for oncology agents should be reasonably safe and provide potential therapeutic benefit to the patient. For late-stage oncology patients, this dose is often based on the ICH S9 guidance, which was developed primarily based on experience with cytotoxic chemotherapeutic agents using the rodent STD10 or non-rodent HNSTD and an appropriate safety factor. With the increase in molecularly targeted chemotherapeutics, it is prudent to re-evaluate how the FIP dose is derived to ensure that the appropriate balance between risk and therapeutic benefit to the patient is achieved. Blinded data on 92 small molecule oncology compounds from 12 pharmaceutical companies who are members of the IQ DruSafe consortium were gathered to investigate if a NOAEL-based starting dose without a safety factor would have been tolerated in the FIP trial and if so, estimating how many dose escalation cohorts could have been reduced. Our analysis suggests that the NOAEL-based alternative starting dose would have been tolerated in most cases evaluated, with an anticipated mean reduction of 2.3 cohorts. Of the 12 cases where the alternative approach resulted in a starting dose that would have exceeded the MTD/RP2D, none of the nonclinical toxicities in these cases were considered irreversible and would be monitorable in all but one instance. Most non-tolerated cases were within two–threefold of the MTD/RP2D, with the clinical AEs considered manageable and mitigated by dose de-escalation. No one method of FIP dose calculation will likely be appropriate for all oncology small molecules and starting dose selection should be performed using a case-by-case approach. However, the NOAEL-based method that does not utilize a safety factor should be considered when appropriate to minimize the number of patients exposed to sub-therapeutic doses of an investigational oncology agent and accelerating development to RP2D. [ABSTRACT FROM AUTHOR]

Purpose: Latanoprost is used for the treatment of an increased intraocular pressure (IOP) to prevent the progression of glaucoma. Since the lack of compliance with topical ocular dosing may compromise efficacy, alternate methods of delivery are being sought. A 9-month study was conducted to assess the safety and tolerability of latanoprost-containing subconjunctivally implanted devices. Methods: Dutch-belted rabbits were implanted subconjunctivally with up to 5 placebo or drug-loaded devices containing from 50 to 180 μg of latanoprost per device. Study assessment consisted of irritation scoring, clinical signs, ophthalmic exams, IOP, electroretinography (ERG), ocular histology of cohorts at 3 and 9 months postimplantation, and systemic exposure to latanoprost acid. Results: The implants were well tolerated, with minimal-to-mild clinical and microscopic ocular findings attributable to either the placebo or drug-loaded devices. Mild conjunctival congestion persisted through week 13 of the study and tended to correlate with the number of devices and presence of drug. Ophthalmic examinations revealed no effects beyond conjunctival surface hyperemia. No effects on the IOP, corneal thickness, or ERG parameters were observed. The lack of changes in the IOP was expected due to the known lack of the IOP-lowering effects in rabbits from latanoprost. Microscopically, implants at the 3-month necropsy were associated with subconjunctival tissue cavities (containing the implants), fibrous encapsulation, and an infiltrate of lymphocytes and macrophages, sometimes as multinucleate cells, into the subconjunctival implant cavity. The drug-containing implants were often associated with inflammatory cell infiltrates, including heterophils (neutrophils), within the implant subconjunctival cavities and adjacent to the implant sites. At the 9-month necropsy, heterophils were no longer common among the inflammatory cell infiltrates; macrophages and lymphocytes persisted; most of the biodegradable implants were fragmented and disintegrating; and fibrovascular proliferation was present within implant luminal remnants. None of the findings were considered adverse. Systemic exposures were above the limit of quantification (0.1 ng/mL) for up to 96 h in the higher-dose groups, consistent with the initial burst phase of compound release. Conclusion: Overall, the study supports the safety of the latanoprost-containing subconjunctival device as a means of extended delivery of the antiglaucoma medication. Latanoprost-containing subconjunctival implants were well tolerated by Dutch-belted rabbits for up to 9 months. Such devices may improve patient compliance and serve as a means of extended delivery of antiglaucoma medications. [ABSTRACT FROM AUTHOR]

Abstract: Attrition due to safety reasons remains a serious problem for the pharmaceutical industry. This has prompted efforts to develop early predictive in vitro screens that can assist in selecting compounds with a more desirable safety profile early on in the drug discovery process. Here we examined the relationship between physicochemical properties, such as partition coefficient (clogP), topological polar surface area (TPSA), acid dissociation constant (pK a), and in vitro mechanistic endpoints generated using a high content imaging approach. We demonstrate in our initial analysis that compounds with clogP>2 and pK a >5.5 flagged more endpoints than compounds with clogP⩽2 and pK a ⩽5.5. In contrast, TPSA did not stand on its own in predicting cytotoxicity. When this knowledge was applied to eight different mechanistic cytotoxicity endpoints (cell loss, apoptosis, ER stress, DNA fragmentation, mitochondrial potential, nuclear size, neutral lipids/steatosis and lysosomal mass), we found that compounds with such properties preferentially flagged in the lysosomal endpoint. We also saw a slight enrichment of such compounds in the endpoints cell loss, DNA fragmentation and nuclear size. We demonstrate that lysosomal compound accumulation is a potential contributor to cell death and possibly organ toxicity. [Copyright &y& Elsevier]

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11β-hydroxysteroid dehydrogenase 1 were induced while actin α2 and procollagen VIII α1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases. [Copyright &y& Elsevier]

Summary This study addresses the contribution of an Sp1 response element in the proximal promoter of the transglutaminase 1 gene to transcription in normal epidermis and in a case of lamellar ichthyosis lacking transglutaminase 1 activity. The latter exhibited an Sp1 promoter mutation previously hypothesized to suppress transcription. In this study, several experiments indicated that the native Sp1 response element was functional, but it had only a small influence on transcription, and the previously observed mutation had no effect. These experiments involved mobility shift assays and transfections of promoter constructs in which the Sp1 site was mutated or lacking altogether. In addition the proximal 1.6 kb of the promoter from the affected individual was as active in transfections as the promoter from unaffected individuals. A search for sequence alterations in mRNA transcribed in keratinocytes from the patient revealed a novel single base mutation in codon 661 of the transglutaminase coding region predicted to result in premature termination of protein translation. The presence of this mutation in parental genomic DNA was confirmed by restriction digestion. Thus the lamellar ichthyosis phenotype in this case is likely attributable to a novel non-sense mutation in the coding region leading to reduced transglutaminase 1 mRNA levels rather than mutation of the Sp1 site. [ABSTRACT FROM AUTHOR]

The retina is a highly structured tissue that is formed by layers containing 7 different cell types. The photoreceptor cell is a specialized type of neuron in the retina that is capable of absorbing and converting light into electrophysiological signals. There is a constant renewal process for photoreceptors consisting of intermittent shedding of the distal tips of the photosensitive outer segment and subsequent phagocytosis (uptake, degradation and recycling) by retinal pigmented epithelial (RPE) cells. This rebuilding process is essential for vision and the survival of photoreceptors and RPE cells. Drugs with a basic moiety have the potential to accumulate in the lysosome and impair its functions including the phagocytosis process, which could hinder clearance of outer segments and ultimately induce retinopathy. To determine the prevalence of this cellular mechanism in retinal toxicity, a collection of proprietary compounds associated with retinal toxicity were subjected to a battery of in vitro tests using the human adult retinal pigmented epithelium cell line, ARPE-19. The tests included a phagocytosis assay, and lysosomal and autophagosomal staining. The compounds that induced retinopathy clustered in the basic and lipophilic region, which drives lysosomal sequestration. This accumulation coincided with phagocytosis inhibition and an increase in autophagosome staining, suggesting a blockage of the membrane trafficking process. A correlation between the physicochemical properties and in vitro lysosomal pathway effects was established. These data reveal the importance of physicochemical properties of compounds and lysosome accumulation as a potential mechanism for drug-induced retinopathy and demonstrate the usefulness of in vitro screening in predicting this liability. [ABSTRACT FROM AUTHOR]

Aim To determine if there is an association between the use of latanoprost ophthalmic solution and malignant melanoma and to assess the evidence of a plausible biological mechanism. Methods Two safety databases were reviewed: one representing all latanoprost (n=24) and fixedcombination latanoprost/timolol (n=16) clinical trials conducted from November 1992 through November 2007 and a global safety database of all spontaneous non-trial-related clinical reports spanning 13 and 9 years for latanoprost and for latanoprost/timolol, respectively. A systematic PubMed search for studies evaluating potential mechanisms was conducted. Results Amongst 12 880 latanoprost-treated subjects in clinical trials, no reported cases of ocular melanoma and three cases of cutaneous melanoma were identified. Of 19 940 cases recorded in the global safety database, 22 reports of ocular/cutaneous neoplasms were identified. Of these neoplasms, 11 were ocular and six were cutaneous melanomas. Possible association with latanoprost use could not be excluded in three ocular and one periorbital report. In vitro and in vivo data were consistent with a mechanism whereby the increased iris pigmentation results from stimulation of melanin synthesis by induction of tyrosinase transcription without increasing mitotic activity. Conclusion There is no evidence at present that establishes a link between latanoprost use and either ocular or cutaneous melanoma. [ABSTRACT FROM AUTHOR]

The article describes lesions in preclinical studies produced by a pan-Cyclin Dependent Kinase (CDK) inhibitor (AG-12986) not previously reported. This class of compounds is currently an important component of the approach of targeted therapy against cancer. The Center for Disease Control has reported cancer as the second leading cause of mortality in the U.S.; it is expected to become the leading cause of death in this decade. Therapy against cancer continues its evolution, relying heavily on traditional DNA damaging agents or blockers of DNA synthesis. Many targeted therapies against cancer are focusing on one of the foundations of tumorigenesis, the cell cycle. Studies have shown that abnormal expression or function of molecules involved in the cell cycle are the origin of a variety of malignant neoplasms. Targeting the cell cycle controlling molecules is one of the most dynamic approaches for anticancer treatment.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside lysosomes (lysosomotropic). How cells respond to the lysosomal stress associated with lysosomotropism is not well characterized. Our goal is to assess the lysosomal changes and identify the signaling pathways that involve in the lysosomal changes. Eight chemically diverse lysosomotropic drugs from different therapeutic areas were subjected to the evaluation using the human adult retinal pigmented epithelium cell line, ARPE-19. All lysosomotropic drugs tested triggered lysosomal activation demonstrated by increased lysosotracker red (LTR) and lysosensor green staining, increased cathepsin activity, and increased LAMP2 staining. However, tested lysosomotropic drugs also prompted lysosomal dysfunction exemplified by intracellular and extracellular substrate accumulation including phospholipid, SQSTM1/p62, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) and opsin. Lysosomal activation observed was likely attributed to lysosomal dysfunction, leading to compensatory responses including nuclear translocation of transcriptional factors TFEB, TFE3 and MITF. The adaptive changes are protective to the cells under lysosomal stress. Mechanistic studies implicate calcium and mTORC1 modulation involvement in the adaptive changes. These results indicate that lysosomotropic compounds could evoke a compensatory lysosomal biogenic response but with the ultimate consequence of lysosomal functional impairment. This work also highlights a pathway of response to lysosomal stress and evidences the role of TFEB, TFE3 and MITF in the stress response. [ABSTRACT FROM AUTHOR]

Purpose: PF-06653157 is a bifunctional antagonist monoclonal antibody (mAb) that targets human VEGF-A ligand and PDGF-Rβ. With the advent of PF-06653157 as an angiogenesis inhibitor and potential treatment for angiogenesis deregulation diseases, a relevant toxicology species is needed for toxicity and efficacy studies. Investigative studies were conducted to validate the mAb dual antagonist properties in a human system and determine its cross-reactive pharmacology in nonhuman cells.Methods: Sequence alignment was used to determine percent sequence identity of VEGF and PDGF receptors and ligands; qualitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the presence of PDGF-Rβ on cells of interest. The functional activity of PF-06653157 antibody was assessed in human, dog, porcine, rabbit, rat, mouse, and cynomolgus monkey cells treated with VEGF and PDGF ligands through cell proliferation assays and western blot analysis of AKT and p44/p42 (ERK1/2) protein phosphorylation and enzyme-linked immunosorbent assay.Results: PF-06653157 attenuated phosphorylation of AKT and p44/p42 proteins in human and cynomolgus monkey cells. The antibody did not attenuate AKT nor p44/p42 phosphorylation in any other species tested. PDGFR signaling could not be activated with human PDGF ligand in the porcine cells, so PF-06653157 activity in porcine remains inconclusive.Conclusion: The PF-06653157 mAb cross-reacts with cynomolgus monkey cells in a similar manner to human cells. Therefore, cynomolgus monkeys are considered the appropriate species for efficacy and regulatory toxicology studies in PF-06653157 development. [ABSTRACT FROM AUTHOR]

Autophagy refers to the catabolic process in eukaryotic cells that delivers cytoplasmic material to lysosomes for degradation. This highly conserved process is involved in the clearance of long-lived proteins and damaged organelles. Consequently, autophagy is important in providing nutrients to maintain cellular function under starvation, maintaining cellular homeostasis, and promoting cell survival under certain conditions. Several pathways, including mTOR, have been shown to regulate autophagy. However, the impact of lysosomal function impairment on the autophagy process has not been fully explored. Basic lipophilic compounds can accumulate in lysosomes via pH partitioning leading to perturbation of lysosomal function. Our hypothesis is that these types of compounds can disturb the autophagy process. Eleven drugs previously shown to accumulate in lysosomes were selected and evaluated for their effects on cytotoxicity and autophagy using ATP depletion and LC3 assessment, respectively. All eleven drugs induced increased staining of endogenous LC3 and exogenous GFP-LC3, even at non toxic dose levels. In addition, an increase in the abundance of SQSTM1/p62 by all tested compounds denotes that the increase in LC3 is due to autophagy perturbation rather than enhancement. Furthermore, the gene expression profile resulting from in vitro treatment with these drugs revealed the suppression of plentiful long-lived proteins, including structural cytoskeletal and associated proteins, and extracellular matrix proteins. This finding indicates a retardation of protein turnover which further supports the notion of autophagy inhibition. Interestingly, upregulation of genes containing antioxidant response elements, e.g. glutathione S transferase and NAD(P)H dehydrogenase quinone 1 was observed, suggesting activation of Nrf2 transcription factor. These gene expression changes could be related to an increase in SQSTM1/p62 resulting from autophagy deficiency. In summary, our data indicate that lysosomal accumulation due to the basic lipophilic nature of xenobiotics could be a general mechanism contributing to the perturbation of the autophagy process. [ABSTRACT FROM AUTHOR]

Purpose: Taprenepag isopropyl is an EP2 receptor agonist that is in development for the treatment of glaucoma. Iritis, photophobia, and increased corneal thickness observed in a Phase 2 clinical trial with taprenepag isopropyl were not previously observed in topical ocular toxicity studies in rabbits and dogs. In vivo studies using cynomolgus monkeys and in vitro models were used to elucidate the mechanisms underlying these ocular events. Methods: Monkeys were dosed daily for 28 days in 1 eye with taprenepag and in the other with vehicle control. Complete ophthalmic examinations were performed at baseline and weekly thereafter. Serial sections of eyes were examined histopathologically at the end of the study. Recovery after the discontinuation of taprenepag was assessed for 28 days in the monkeys in the high-dose group. In vitro studies evaluated cell viability, paracellular permeability, and cytokine induction with human corneal epithelial or endothelial cell cultures. Results: Monkeys demonstrated a dose-related incidence of iritis and increased corneal thickness that resolved within 28 days of discontinuing taprenepag. There was no evidence in vivo of taprenepag toxicity to the corneal endothelium or epithelium. Cell viability of stratified epithelial cells was primarily affected by excipients and was similar to Xalatan®. The viability of HCEC-12 cells was not affected by taprenepag at concentrations up to 100 μM. Conclusions: The lack of in vivo or in vitro endothelial cytotoxicity and the reversibility of the increase in corneal thickness and iritis in the monkey provide confidence to permit further clinical development of taprenepag. [ABSTRACT FROM AUTHOR]

The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition. [ABSTRACT FROM PUBLISHER]

Purpose: PF-655, a synthetic 19-mer siRNA, targeting the RTP801 gene is currently in clinical trials for the treatment of wet age-related macular degeneration and diabetic macular edema. Preclinical studies have shown a dose-related suppression of RTP801 expression in rat disease models. Investigative studies were conducted with PF-655 to validate the Dutch-Belted rabbit as a biologically relevant species for gene silencing to support nonclinical ocular toxicity and continual dosing studies. Methods: Cross-species comparison and DNA sequencing was done to determine the level of homology between PF-655 and rabbit RTP801. Human (HEK 293) and rabbit (SIRC cornea) cell lines were stimulated with CoCl2 to mimic hypoxic stress (an inducer of RTP801 expression) and treated with PF-655. Taqman-polymerase chain reaction and immunoblot analysis were performed to gauge RTP801 expression in cell culture and rabbit retinas. Results: Sequence analysis showed a 1-base mismatch in the PF-655 targeting site from genomic DNA of Dutch-Belted rabbit and the SIRC cell line, a cornea cell derived from the New Zealand White rabbit. HEK and SIRC CoCl2-stressed cells induced RTP801 expression 10-20-fold above control conditions. Treatment with 20 or 100 nM PF-655 showed a decrease in gene expression, 40%-50% relative to appropriate controls. RTP801 mRNA was detectable in primary rabbit retina tissues, with cycle threshold values showing a large linear range for the assay. Conclusion: These results support our investigation into cross-species validation of gene suppression by a therapeutic siRNA designed to a human gene. The SIRC cell line was utilized as a surrogate to test the degree of RTP801 gene silencing induced by PF-655 in vitro. With a 1-base mismatch, the level of silencing in a rabbit ocular cell line was comparable to that of a human cell line. Sequence analysis and expression data confirmed the relevance of the RTP801 target gene in rabbits and the utility of this species as a relevant animal model. Additionally, our work outlines a tractable method that validates relevant larger non-rodent species for ophthalmic drug testing. [ABSTRACT FROM AUTHOR]

Abstract: Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow. [Copyright &y& Elsevier]

Abstract: Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP >2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity. [Copyright &y& Elsevier]

Abstract: Preservatives are used in ocular medications to prevent microbial contamination. The use of benzalkonium chloride (BAC), the most widely used preservative in ocular medications, has been scrutinized with a number of studies indicating its toxicity to monolayer cultures of corneal and conjunctival epithelial cells. The purpose of this study was to evaluate and compare the toxicity of BAC and other preservatives and common components of ocular formulations on monolayer and stratified air-lifted cultures of Chang conjunctival cells. Air-lifting Chang cells grown on transwell filters increased stratification as assessed by transepithelial electrical resistance and histology. Unlike monolayer cultures in which ocular medications containing BAC caused near complete loss of cell viability, stratified, air-lifted cultures were not affected by the presence of BAC in ocular medications with up to 30-min exposures. Stratification shifted the dose–response curve to the right for benzalkonium chloride, thimerosal, chlorhexidine digluconate, potassium sorbate and EDTA. These results demonstrate that stratification significantly affects cell viability of Chang conjunctival cells in response to preservatives and additives of ophthalmic preparations. [Copyright &y& Elsevier]

Prostaglandin receptor agonists have intraocular pressure—lowering effects in humans and are of interest in the treatment of glaucoma. The prostanoid receptor agonist PF-04475270 is a potent and selective agonist of the prostaglandin E2 receptor EP4. This paper characterizes the toxicity associated with topical ocular administration of PF-04475270 in beagles. Dogs were given PF-04475270 topically to the eye on a consecutive daily dosing schedule for one or four weeks followed by a one- or four-week reversal period, respectively. Clinical observations, ophthalmic, and laboratory parameters were recorded. Necropsies were conducted at the end of the dosing and recovery phases, and histologic examinations performed. Corneal neovascularization that was considered adverse was observed at doses of ≥1.0 μg/eye and was not reversed by the end of the recovery phase. Dogs dosed with ≥0.25 μg/eye developed a dose-related conjunctival hyperemia that persisted throughout the reversal period. Corneal neovascular cells stained positive with EP4 and the endothelial biomarker Factor VIII-vWF. Other histopathology findings observed at doses of ≥1.0 μg included single-cell necrosis and neutrophils in the cornea, inflammatory cell infiltrates in the iris/ciliary body, and iridal endothelial cell hypertrophy. A resolving acute to subacute inflammation in the iris/ciliary body was observed after the four-week recovery period. [ABSTRACT FROM PUBLISHER]

Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects. Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m2, respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at ≥3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose. No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at ≥6 mg/kg. AG-024322 at ≥6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at ≥6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent. The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0–24.5) of 2.11 μg h/mL. [ABSTRACT FROM AUTHOR]

Abstract: The inhibition of 11βhydroxysteroid dehydrogenase 1 (11βHSD1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is an attractive target to treat diabetes by suppressing hepatic gluconeogenesis. To test this hypothesis, we developed a novel glucocorticoid-induced diabetic KK mouse model and used 11βHSD1 antisense oligonucleotide (ASO) as an inhibitory tool. KK mice were treated with 25 or 50mg/kg/day of 11βHSD1 ASO for 28 days. On day 25, cortisone pellets were surgically implanted to induce diabetes. In the ASO-treated mice, plasma blood glucose levels were significantly reduced by up to 54%. In parallel, cortisol and other diabetes endpoints were also significantly reduced. Hepatic 11βHSD1 mRNA was suppressed by up to 84% with a concomitant respective decrease of up to 49% in the expression of PEPCK. The results suggest that inhibition of 11βHSD1 activity reduces the availability of cortisol to activate the glucocorticoid receptor, down regulates gluconeogenesis and thus reduces plasma glucose levels in cortisone-induced diabetic KK mice. [Copyright &y& Elsevier]

Cyclin-dependent kinases (cdks) play a crucial role in cell cycle regulation and are considered promising targets for cancer therapy. Intravenous administration of AG-012986, a pan-cyclin-dependent kinase inhibitor (cdk i ), resulted in unexpected retinal and peripheral nerve toxicity in mice. AG-012986 was administered daily to CD-1 or B6C3F1 mice for 5 consecutive days. Mice were euthanized 24 h after the last dose (study day 6) or after a 21-day post-dose period (study day 26). Compound related microscopic findings were seen in the sciatic nerves (axonal degeneration) of both strains and in the retina (retinal degeneration/atrophy) of CD-1 mice only after the post-dose period. Although retinal degeneration/atrophy was not detected by routine histology in mice euthanized on day 6, apoptotic retinal cells were evident at this time using TUNEL assay. To our knowledge retinal or peripheral nerve toxicity secondary to the administration of cdk i s has not been previously reported. Although the pathogenesis of these lesions is unclear, the toxicities may reflect the unique profile of cdk inhibition, off-target kinase inhibition or receptor binding, or metabolism/distribution properties of AG-012986. Multi-targeted-inhibitors may interfere with cdks and other kinases involved in a wide range of functions other than cell cycle regulation, which could result in unexpected toxicities that may hinder their clinical applications. [ABSTRACT FROM AUTHOR]

Retina is a highly compact neural structure composed of distinct layers. The retinal pigmented epithelium (RPE) is the pigmented cell layer just outside the neurosensory and serves a number of crucial metabolic functions including the phagocytosis and removal of shed photoreceptor cell outer segments. Drugs with a basic moiety can accumulate in the lysososme and disturb lysosome function including phagocytosis, which will retard the clearance of outer segments and ultimately induce retinopathy. To test this hypothesis we chose a compound that contains a basic moiety and induced retinopathy in long term in vivo studies in rats In vitro assays including viability, LC-3 staining, and phagocytosis were conducted using the human retinal pigmented epithelium cell line ARPE-19. These studies showed that this compound caused cellular vacuolation and dose dependent cytotoxicity that was related to the initial cell number and dosing volume, indicating an accumulation effect. This compound also decreased phagocytosis function of ARPE-19, and induced accumulation of LC-3, suggesting a blockage of the autophagy process. EM analysis of eyes taken from an in vivo study showed the presence of large phagolysosomes, supporting the hypothesis of impairment of lysosome function as a mechanism for retinal toxicity. Twenty three proprietary compounds for which the in vivo effects on the retina were known were tested in a phagocytosis assay to examine the prevalence of this cellular mechanism in retinal toxicity. A correlation amongst the physiochemical properties, in vitro lysosomal pathway effects, and in vivo retinal effects was established. This data reveals the importance of physiochemical properties and lysosome accumulation as a common mechanism for drug induced retinopathy and demonstrates the potential usefulness of in vitro screening in predicting this liability. [ABSTRACT FROM AUTHOR]