Preclinical Efficacy Studies Research Articles

Cissus quadrangularis Linn exerts dose-dependent biphasic effects: osteogenic and anti-proliferative, through modulating ROS, cell cycle and Runx2 gene expression in primary rat osteoblasts.

This report highlights phytoconstituents present in Cissus quadrangularis (CQ) extract and examines biphasic (proliferative and anti-proliferative) effects of its extract on bone cell proliferation, differentiation, mineralization, ROS generation, cell cycle progression and Runx2 gene expression in primary rat osteoblasts. Phytoconstituents were identified using gas chromatography-mass spectroscopy (GC-MS). Osteoblasts were exposed to different concentrations (10-100 μg/ml) of CQ extract and cell proliferation and cell differentiation were investigated at different periods of time. Subsequently, intracellular ROS intensity, apoptosis and matrix mineralization of osteoblasts were evaluated. We performed flow cytometry for DNA content and real-time PCR for Runx2 gene expression analysis. CQ extract's approximately 40 bioactive compounds of fatty acids, hydrocarbons, vitamins and steroidal derivatives were identified. Osteoblasts exposed to varying concentrations of extract exhibited biphasic variation in cell proliferation and differentiation as a function of dose and time. Moreover, lower concentrations (10-50 μg/ml) of extract slightly reduced ROS intensity, although they enhanced matrix mineralization, DNA content in S phase of the cell cycle, and levels of Runx2 expression. However, higher concentrations (75-100 μg/ml) considerably induced the ROS intensity and nuclear condensation in osteoblasts, while it reduced mineralization level, proportion of cells in S phase and Runx2 level of the osteogenic gene. These findings suggest that CQ extract revealed concentration-dependent biphasic effects, which would contribute notably to future assessment of pre-clinical efficacy and safety studies.

Understanding paratyphoid infection: study protocol for the development of a human model of Salmonella enterica serovar Paratyphi A challenge in healthy adult volunteers

IntroductionThis study will develop the first human challenge model of paratyphoid infection which may then be taken forward to evaluate paratyphoid vaccine candidates. Salmonella Paratyphi A is believed to cause...

Preclinical efficacy studies using HuMax-Axl-ADC, a novel antibody-drug conjugate targeting Axl-expressing solid cancers.

3066 Background: HuMax-Axl-ADC is an antibody-drug conjugate (ADC) that specifically targets Axl-expressing tumor cells. Aberrant expression of Axl, a receptor tyrosine kinase, has been described i...

Repeated Low-Dose Influenza Virus Infection Causes Severe Disease in Mice: a Model for Vaccine Evaluation.

Influenza infection causes severe disease and death in humans. In traditional vaccine research and development, a single high-dose virus challenge of animals is used to evaluate vaccine efficacy. This type of challenge model may have limitations. In the present study, we developed a novel challenge model by infecting mice repeatedly in short intervals with low doses of influenza A virus. Our results show that compared to a single high-dose infection, mice that received repeated low-dose challenges showed earlier morbidity and mortality and more severe disease. They developed higher vial loads, more severe lung pathology, and greater inflammatory responses and generated only limited influenza A virus-specific B and T cell responses. A commercial trivalent influenza vaccine protected mice against a single high and lethal dose of influenza A virus but was ineffective against repeated low-dose virus challenges. Overall, our data show that the repeated low-dose influenza A virus infection mouse model is more stringent and may thus be more suitable to select for highly efficacious influenza vaccines. Influenza epidemics and pandemics pose serious threats to public health. Animal models are crucial for evaluating the efficacy of influenza vaccines. Traditional models based on a single high-dose virus challenge may have limitations. Here, we describe a new mouse model based on repeated low-dose influenza A virus challenges given within a short period. Repeated low-dose challenges caused more severe disease in mice, associated with higher viral loads and increased lung inflammation and reduced influenza A virus-specific B and T cell responses. A commercial influenza vaccine that was shown to protect mice from high-dose challenge was ineffective against repeated low-dose challenges. Overall, our results show that the low-dose repeated-challenge model is more stringent and may therefore be better suited for preclinical vaccine efficacy studies.

Therapeutic targeting of acute lung injury and acute respiratory distress syndrome.

There is no Food and Drug Administration-approved treatment for acute respiratory distress syndrome (ARDS), in spite of the relatively large number of patients with the diagnosis. In this report, we provide an overview of preclinical studies and a description of completed and future clinical trials in humans with ARDS. Preclinical studies dealing with acute lung injury have suggested roles for complement and complement receptors, as well as the evolving role of histones, but details of these pathways are inadequately understood. Anti-inflammatory interventions have not been convincingly effective. Various cell growth factors are being considered for clinical study. Interventions to block complement activation or its products are under consideration. Stem cell therapies have shown efficacy in preclinical studies, which have motivated phase I/II trials in humans with ARDS.

438. Oncolytic Immunotherapy for Treatment of Breast Cancer, Including Triple-Negative Breast Cancer

Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes associated with specifical biological behavior and different clinical outcomes. Triple-negative breast cancer (TNBC) is defined as estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and lacking overexpression of human epidermal growth factor receptor 2 (HER2). Among all the breast cancer subtypes, TNBC (10-15% of breast cancers) is associated with a worse prognosis.Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Cyclophosphamide (CP) is a chemotherapy drug that works by slowing or stopping cell growth. At higher doses, CP is associated with increased cytotoxicity and immunosuppression, while at low continuous dosage it shows immunostimulatory and antiangiogenic properties. Therefore, the combination of oncolytic immunovirotherapy with low-dose CP is an appealing approach.We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and animal models, in combination with low-dose CP or its main active metabolite 4-hydroxycyclophosphamide (4-HP-CP). Furthermore, we summarized the breast cancer-specific human data from the Advanced Therapy Access Program (ATAP).Low-dose CP effectively increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC xenograft mouse model. In ATAP, treatments appeared safe and well-tolerated. Fourteen out of sixteen breast cancer patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, four had stable disease, and nine had progressive disease. One patient was alive at 941 days after treatment.Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy. This preliminary data supports continuing the clinical development of oncolytic adenoviruses for treatment of breast cancer, including TNBC.

32. Safety Studies Towards a Combined Cell and Gene Therapy To Treat Critical Limb Ischemia

Over 240 clinical trials are in progress worldwide using mesenchymal stem cells (MSCs). Some critical advantages of MSCs include simple isolation and expansion, and ease for allogeneic applications due to their immune evading properties. MSCs produce many paracrine signals that promote tissue regeneration, though at sub-therapeutic levels. We have therefore developed a product that combines cell and gene therapy, by engineering MSCs to overexpress vascular endothelial growth factor (VEGF). Here we show our preclinical data that not only confirms the efficacy of our product (MSC-VEGF), but addresses multiple safety aspects. Here, MSCs are transduced with a GMP-grade lentiviral vector to obtain an average of one copy per cell (ranging from 0.8 to 1.3), minimizing occurrence of insertional mutagenesis. The secretion of VEGF is optimized by using a previously clinically evaluated constitutive promoter (MNDU3) and an enhancer element acting in cis (WPRE). Safety studies at this level of secretion (approx. 80pg/ml VEGF secreted per 1000 cells per 24 hours) did not cause edema or hemangiomas in immune deficient NOD/SCID IL-2R-gnull/null (NSG) mice. The genomic stability of MSC-VEGF was addressed by karyotyping analysis, where no abnormalities were found in MSCs transduced with increasing viral loads, and by PCR to evaluate potential genomic rearrangement of the insert. To rule out tumorigenicity of MSC-VEGF, cells transduced with increased viral load were in injected into NSG mice. In contrast to 3 distinct positive controls used, no tumors were detected in over 50 mice treated with MSC-VEGF up to 6 months after injection. Additional safety studies included confirmation by flow cytometry of homogeneity of the transduction level of MSC-VEGF, absence of replication competent lentivirus, freedom of endotoxin, mycoplasma and absence VSV-G viral envelope coding plasmid. Luciferase-expressing MSC-VEGF consistently decreased to undetectable levels by 28 days after injection and PCR confirmed the absence of human DNA 6 months after injection. Pilot efficacy studies using MSC-VEGF in an immune deficient mouse model of hind limb ischemia (HLI) have been completed. MSC-VEGF were injected IM the day after HLI surgery into the ischemic limbs of NSG mice and blood flow was monitored weekly, demonstrating that MSC-VEGF treatment leads to faster revascularization than control treatment. Furthermore, we show that a small percentage of injected MSC-VEGF take a pericyte position on blood vessels. While future assays are intended as IND-enabling safety studies, these pre-clinical safety and efficacy studies are directed towards our planned Phase I clinical trial.

Preclinical safety and tolerability of a repeatedly administered human leishmaniasis DNA vaccine.

The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T-cell epitope-enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naive mice and rats, complemented by the demonstration of tolerability in Leishmania-infected mice. Biodistribution and persistence were examined following single and repeated intradermal (i.d.) administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. Although vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after i.d. administration to naive, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania-infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naive and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine.

Are adaptive randomised trials or non-randomised studies the best way to address the Ebola outbreak in west Africa?

The Ebola outbreak that has devastated parts of west Africa represents an unprecedented challenge for research and ethics. Estimates from the past three decades emphasise that the present effort to contain the epidemic in the three most affected countries (Guinea, Liberia, and Sierra Leone) has been insufficient, with more than 24 900 cases and about 10 300 deaths, as of March 25, 2015. Faced with such an exceptional event and the urgent response it demands, the use of randomised controlled trials (RCT) for Ebola-related research might be both unethical and infeasible and that potential interventions should be assessed in non-randomised studies on the basis of compassionate use. However, non-randomised studies might not yield valid conclusions, leading to large residual uncertainty about how to interpret the results, and can also waste scarce intervention-related resources, making them profoundly unethical. Scientifically sound and rigorous study designs, such as adaptive RCTs, could provide the best way to reduce the time needed to develop new interventions and to obtain valid results on their efficacy and safety while preserving the application of ethical precepts. We present an overview of clinical studies registered at present at the four main international trial registries and provide a simulation on how adaptive RCTs can behave in this context, when mortality varies simultaneously in either the control or the experimental group.

Treatment of melanoma with a serotype 5/3 chimeric oncolytic adenovirus coding for GM-CSF: Results in vitro, in rodents and in humans.

Metastatic melanoma is refractory to irradiation and chemotherapy, but amenable to immunological approaches such as immune-checkpoint-inhibiting antibodies or adoptive cell therapies. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules. Therefore, oncolytic immuno-virotherapy of malignant melanoma is an appealing approach, which was recently validated by a positive phase 3 trial. We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a panel of melanoma cell lines and animal models, and summarized the melanoma-specific human data from the Advanced Therapy Access Program (ATAP). The virus effectively eradicated human melanoma cells in vitro and subcutaneous SK-MEL-28 melanoma xenografts in nude mice when combined with low-dose cyclophosphamide. Furthermore, virally-expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated the differentiation of human monocytes into macrophages. In contrast to human cells, RPMI 1846 hamster melanoma cells exhibited no response to oncolytic viruses and the chimeric 5/3 fiber failed to increase the efficacy of transduction, suggesting limited utility of the hamster model in the context of viruses with this capsid. In ATAP, treatments appeared safe and well-tolerated. Four out of nine melanoma patients treated were evaluable for possible therapy benefit with modified RECIST criteria: one patient had minor response, two had stable disease, and one had progressive disease. Two patients were alive at 559 and 2,149 days after treatment. Ad5/3-D24-GMCSF showed promising efficacy in preclinical studies and possible antitumor activity in melanoma patients refractory to other forms of therapy. This data supports continuing the clinical development of oncolytic adenoviruses for treatment of malignant melanoma.

Changes in epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone in surgically and chemically induced rat models of osteoarthritis

Purpose: Several experimental animal models have been developed for human osteoarthritis (OA) and used to study the preclinical efficacy of disease and symptom modifying OA drug candidates. The preclinical efficacy has been determined by various microscopic scoring systems and joint pain assessments. The histopathology initiative of Osteoarthritis Research Society International (OARSI) has presented recommendations for OA assessment in order to standardize preclinical efficacy studies. Recommendations for rat samples include the histological analysis of cartilage degeneration and extracellular matrix (ECM) loss, osteophytes, calcified cartilage and subchondral bone, synovium, joint capsule, and growth plate. As subchondral bone has been identified to play an important role in the development and progression of OA, treatment effects on bone tissue have been studied even in more detailed. In this study, we performed a systemic characterization of epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone together with the assessment of knee joint discomfort and pain and degenerative changes in articular cartilage and synovium in four rat OA models. Methods: The study was conducted using male Lewis rats (body weight range 330-380 g). Unilateral OA was induced in their knee joints by applying the following models: 1) intra-articular injection of monoiodoacetate (MIA) at the dose of 1 mg, 2) medial meniscal tear (MMT) combined with medial collateral ligament transection (MCLT), 3) anterior cruciate ligament transection (ACLT) combined with partial medial meniscectomy (pMMx), 4) ACLT. Body weight and OA symptoms were followed in each model during the study. Knee joint discomfort and pain were used as the symptoms of OA. Knee joint discomfort was analyzed as static weight bearing using Incapacitance Tester and knee joint pain as static mechanical allodynia using von Frey monofilaments. Knee joints were harvested at two different time points in each model as follows: in the MIA model at 2 and 4 weeks, in the MMT + MCLT model at 3 and 6 weeks, in the ACLT + pMMx model at 4 and 8 weeks, and in the ACLT model at 5 and 10 weeks. The structure of epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone were analyzed in coronal sections of medial tibial plateau followed by the histological OA assessment as recommended by the OARSI histopathology initiative. This experimental protocol was approved by National Animal Experiment Board, Regional State Administrative Agency for Southern Finland, Hämeenlinna, Finland. Results: Knee joint discomfort was observed in operated hind limbs as decreased static weight bearing during the first week of the study. Knee joint pain was identified in operated and MIA-injected hind limbs as decreased paw withdrawal threshold during the first week and at the end of in-life phase of each model. In the rat MIA model, this knee joint pain was associated with mild synovial inflammation at 2 weeks, cartilage degeneration and ECM loss in superficial layer at 2 weeks and exacerbating down to intermediate layer at 4 weeks, and with the reduction in the amount of epiphyseal and subchondral bone. In the rat MMT + MCLT model, the knee joint pain was observed together with synovial inflammation, cartilage degeneration and the loss of ECM from superficial layer down to tidemark at 3 and 6 weeks, large osteophytes, and with the increased amount of epiphyseal and subchondral bone. In the rat ACLT + pMMx model, the pain of knee joint was identified in association with synovial inflammation, cartilage degeneration and ECM loss from superficial layer down to tidemark at 4 and 8 weeks, and with large osteophytes. In the rat ACLT model, the knee joint pain was associated with mild synovial inflammation, cartilage degeneration and the loss of ECM in superficial layer at 5 weeks and exacerbating down to deep layer at 10 weeks, osteophytes, and with the reduction in the amount of subchondral bone. Conclusions: This study characterized changes in the structure of epiphyseal bone, subchondral bone plate and epiphyseal trabecular bone together with degenerative changes in articular cartilage and synovium, and knee joint discomfort and pain in four rat OA models. The amount of bone decreased in the rat OA models exhibiting from mild to moderate degenerative changes in articular cartilage, whereas the amount of bone remained unchanged or increased in the rat OA models exhibiting from moderate to severe degenerative cartilage changes. These results can be used to design studies for testing the preclinical treatment effects of OA drug candidates on articular cartilage, synovium, epiphyseal bone, and knee joint discomfort and pain.

High density mineralised protrusions from the tidemark into hyaline cartilage in human joints

High density mineralised protrusions from the tidemark into hyaline cartilage in human joints

Enhanced oral bioavailability of paclitaxel by solid dispersion granulation

The main objective of this study was to develop novel orally administrable tablets containing solid dispersion granules (SDG) of amorphous paclitaxel (PTX) prepared by fluid bed technology, and to evaluate its in vitro dissolution and in vivo pharmacokinetics (PK) in beagle dogs. The SDG were prepared using optimized composition by fluid bed technology, and characterized for solid-state properties. The release study of SDG tablet (SDG-T) in simulated gastric fluid showed a rapid release of PTX, reaching maximum dissolution within 20 min. Finally, the PK profile of SDG-T and a reference formulation Oraxol™ (oral solution formulation used in Phase I clinical study) at a dose of 60 mg orally with co-administration of P-gp inhibitor HM38101, and Taxol® at a dose of 10 mg intravenously (i.v.) was investigated in beagle dogs. The mean absolute BA% of PTX following SDG-T and Oraxol™ solution was 8.23 and 6.22% in comparison to i.v. administration of Taxol®. The relative BA% of PTX from SDG-T in comparison to Oraxol™ solution was 132.25% at a dose of 60 mg following oral administration. In conclusion, we have successfully prepared PTX tablets with solid dispersion granules (SDG) of amorphous PTX using fluid bed technology that could provide plasma PTX concentration in the range of 10–150 ng/mL for a period of 24 h following oral administration in dogs with a P-gp inhibitor. Hence, this could be a promising formulation for PTX oral delivery and could be used in our intended clinical studies following pre-clinical efficacy studies.

Safety and efficacy of VCN-01, an oncolytic adenovirus combining fiber HSG-binding domain replacement with RGD and hyaluronidase expression.

Tumor targeting upon intravenous administration and subsequent intratumoral virus dissemination are key features to improve oncolytic adenovirus therapy. VCN-01 is a novel oncolytic adenovirus that combines selective replication conditional to pRB pathway deregulation, replacement of the heparan sulfate glycosaminoglycan putative-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK for tumor targeting, and expression of hyaluronidase to degrade the extracellular matrix. In this study, we evaluate the safety and efficacy profile of this novel oncolytic adenovirus. VCN-01 replication and potency were assessed in a panel of tumor cell lines. VCN-01 tumor-selective replication was evaluated in human fibroblasts and pancreatic islets. Preclinical toxicity, biodistribution, and efficacy studies were conducted in mice and Syrian hamsters. Toxicity and biodistribution preclinical studies support the selectivity and safety of VCN-01. Antitumor activity after intravenous or intratumoral administration of the virus was observed in all tumor models tested, including melanoma and pancreatic adenocarcinoma, both in immunodeficient mice and immunocompetent hamsters. Oncolytic adenovirus VCN-01 characterized by the expression of hyaluronidase and the RGD shaft retargeting ligand shows an efficacy-toxicity prolife in mice and hamsters by intravenous and intratumoral administration that warrants clinical testing.

Preclinical examination of clofarabine in pediatric ependymoma: intratumoral concentrations insufficient to warrant further study.

Clofarabine, a deoxyadenosine analog, was an active anticancer drug in our in vitro high-throughput screening against mouse ependymoma neurospheres. To characterize the clofarabine disposition in mice for further preclinical efficacy studies, we evaluated the plasma and central nervous system disposition in a mouse model of ependymoma. A plasma pharmacokinetic study of clofarabine (45mg/kg, IP) was performed in CD1 nude mice bearing ependymoma to obtain initial plasma pharmacokinetic parameters. These estimates were used to derive D-optimal plasma sampling time points for cerebral microdialysis studies. A simulation of clofarabine pharmacokinetics in mice and pediatric patients suggested that a dosage of 30mg/kg IP in mice would give exposures comparable to that in children at a dosage of 148mg/m(2). Cerebral microdialysis was performed to study the tumor extracellular fluid (ECF) disposition of clofarabine (30mg/kg, IP) in the ependymoma cortical allografts. Plasma and tumor ECF concentration-time data were analyzed using a nonlinear mixed effects modeling approach. The median unbound fraction of clofarabine in mouse plasma was 0.79. The unbound tumor to plasma partition coefficient (K pt,uu: ratio of tumor to plasma AUCu,0-inf) of clofarabine was 0.12±0.05. The model-predicted mean tumor ECF clofarabine concentrations were below the in vitro 1-h IC50 (407ng/mL) for ependymoma neurospheres. Thus, our results show the clofarabine exposure reached in the tumor ECF was below that associated with an antitumor effect in our in vitro washout study. Therefore, clofarabine was de-prioritized as an agent to treat ependymoma, and further preclinical studies were not pursued.

Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin

A central treatment resistance mechanism in solid tumors is the maintenance of epithelial junctions between malignant cells that prevent drug penetration into the tumor. We have developed a small recombinant protein (JO-1) that triggers the transient opening of intercellular junctions and thus increases the efficacy of monoclonal antibodies and chemotherapeutic drugs without causing toxicity in mouse tumor models. Here, we provide data toward the clinical translation of an affinity-enhanced version of JO-1, which we call JO-4, in combination with PEGylated liposomal doxorubicin (PLD)/Doxil for ovarian cancer therapy. We have presented X-ray crystallography data suggesting a structural basis for the higher affinity of JO-4 to DSG2. We also confirmed JO-4 efficacy in a xenograft model with primary ovarian cancer cells showing that JO-4 can salvage Doxil therapy when given at a dose that was threefold lower than the therapeutic dose. Furthermore, we tested the safety of intravenous JO-4 alone and in combination with Doxil in Macaca fascicularis, an adequate animal model for predicting toxicity in humans. Our studies did not show critical JO-4-related toxicity or an increase of Doxil-related side effects. Our efficacy and safety data will help to support an Investigational new drug-filing for a JO-4/Doxil combination treatment.

Ultrasound-Based Measurement of Molecular Marker Concentration in Large Blood Vessels: A Feasibility Study

Ultrasound molecular imaging has demonstrated efficacy in pre-clinical studies for cancer and cardiovascular inflammation. However, these techniques often require lengthy protocols because of waiting periods or additional control microbubble injections. Moreover, they are not capable of quantifying molecular marker concentration in human tissue environments that exhibit variable attenuation and propagation path lengths. Our group recently investigated a modulated acoustic radiation force-based imaging sequence, which was found to detect targeted adhesion independent of control measurements. In the present study, this sequence was tested against various experimental parameters to determine its feasibility for quantitative measurements of molecular marker concentration. Results indicated that measurements obtained from the sequence (residual-to-saturation ratio, Rresid) were independent of acoustic pressure and attenuation (p > 0.13, n = 10) when acoustic pressures were sufficiently low. The Rresid parameter exhibited a linear relationship with measured molecular marker concentration (R2 > 0.94). Consequently, feasibility was illustrated in vitro, for quantification of molecular marker concentration in large vessels using a modulated acoustic radiation force-based sequence. Moreover, these measurements were independent of absolute acoustic reflection amplitude and used short imaging protocols (3 min) without control measurements.

Gene augmentation for X-linked retinitis pigmentosa caused by mutations in RPGR.

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is a severe and early onset form of retinal degeneration, and no treatment is currently available. Recent evidence in two clinically relevant canine models shows that adeno-associated viral (AAV)-mediated RPGR gene transfer to rods and cones can prevent disease onset and rescue photoreceptors at early- and mid-stages of degeneration. There is thus a strong incentive for conducting long-term, preclinical efficacy and safety studies, while concomitantly pursuing the detailed phenotypic characterization of XLRP disease in patients that may benefit from such corrective therapy.

Abstract 4645: Clofarabine, a potent anticancer compound with limited penetration in an orthotopic murine model of ependymoma

Abstract Clofarabine, a deoxyadenosine analog, was a potent hit in our in vitro high-throughput screening against murine ependymoma neurospheres. To prioritize clofarabine for further preclinical efficacy studies, we evaluated the plasma pharmacokinetic (PK) disposition and central nervous system penetration in a murine model of ependymoma. A plasma PK study of clofarabine (45 mg/kg IP) was performed using CD1 nude mice bearing ependymoma cortical allographs (Ink4a/Arf-null + RTBDN) to obtain initial plasma PK parameters. These estimates were used to derive D-optimal plasma sampling time-points (e.g., 0.25, 2.5, and 5 hr) for cerebral microdialysis studies. Comparison of the clofarabine systemic exposure obtained from the plasma PK study and that simulated from pediatric patients using a published population PK model (Bonate, Cancer Chemotherap Pharmacol, 2011) suggested a dosage of 30 mg/kg in mice would be equivalent to a pediatric dosage of 180 mg/m2 given as a 2 hr infusion. Cerebral microdialysis was applied in CD1 nude mice bearing ependymoma cortical allographs (Ink4a/Arf-null + RTBDN), which permitted repeated in situ sampling of clofarabine tumor extracellular fluid (tECF). A microdialysis probe (BASi; 1 mm membrane) was introduced into the tumor through a cannula inserted during tumor cell implantation. After microdialysis probe equilibration, 7 mice were dosed with 30 mg/kg of clofarabine IP. In each mouse, serial plasma samples were collected at 0.25, 2.5, and 5 h post-dose, and tECF dialysate fractions were collected over 60 min intervals for up to 5 h post-dose. To measure clofarabine in both plasma and tECF, a robust, sensitive LC-MS/MS method was developed and validated. Both within-day and between-day precision (%CV) were ≤ 5.1% and accuracy ranged from 86% to 109%. A two-compartment model with absorption and tumor compartments linked to a central compartment was fitted to plasma and tECF concentration-time data using a nonlinear mixed effects modeling approach (NONMEM 7.2.0). For modeling purposes, the volume of the tECF compartment was fixed to published values. Unbound fraction of clofarabine in murine plasma was 0.82 ± 0.14. The model derived area under unbound concentration-time curve (AUCu,0-8) values for 30 and 45 mg/kg dosages were 5185 ± 550 µg/L*hr and 7677 ± 699 µg/L*hr, respectively. Clofarabine was absorbed rapidly from the peritoneal cavity with Tmax (time to reach maximum concentration) value of 0.33 ± 0.17 hr. Tumor to plasma partition coefficient (Kpt,uu: ratio of tumor to plasma AUCu,0-inf) of clofarabine was 0.12 ± 0.05. The model predicted mean tECF clofarabine concentrations were below the in vitro 1-hr IC50 (1.34 µM) for ependymoma neurospheres. In summary, we have shown the tECF clofarabine concentrations were below that required for antitumor effect in our in vitro washout studies, thus we have not pursued clofarabine for detailed efficacy studies in our preclinical pipeline. Citation Format: Yogesh T. Patel, Megan O. Jacus, Abigail D. Davis, Pradeep Vuppala, Jason D. Dapper, Burgess B. Freeman, Nidal Boulos, Stacy L. Throm, Richard J. Gilbertson, Clinton F. Stewart. Clofarabine, a potent anticancer compound with limited penetration in an orthotopic murine model of ependymoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4645. doi:10.1158/1538-7445.AM2014-4645

MiR-25 in heart failure.

Recent work by Wahlquist et al 1 showed miR-25 to reduce levels of rco/endoplasmic reticulum calcium-ATPase (SERCA) 2a during heart failure, whereas therapeutic inhibition of this microRNA (miRNA) was able to reverse pre-established heart failure in a mouse model of pressure overload. Although these data suggest that miR-25 might be a drug target for the treatment of heart failure, further in vivo analysis is required before we can consider miR-25 inhibition for clinical application. Recently, Wahlquist et al1 reported on the pathological upregulation of miR-25 during heart failure and showed that inhibition was able to block and reverse the disease in mice. Although an increase in cardiac miR-25 levels caused a decline in cardiac function, anti–miR-based inhibition of miR-25 halted established heart failure at least in part by increasing the mRNA of SERCA2a. This is intriguing because phase II clinical data from the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial has demonstrated that gene therapy to increase SERCA2a levels in humans delays the progression of chronic heart failure. This could imply that pharmacological regulation of miR-25 may regulate at least 1 clinically validated target, which may be more straightforward than current gene therapy approaches. However, miRNA biology seems to sketch a somewhat more complex picture than a miRNA functioning through 1 single-gene target. In addition, conflicting data on the cardiac functions of miR-25 highlight the need for further investigation before advancing miR-25 as a clinical target for heart failure. miRNAs are short single-stranded noncoding RNAs that modulate the expression of target proteins by annealing to complementary sequences in their target mRNAs.2 It is thought that a single vertebrate miRNA on average targets a few hundred transcripts.3,4 Because individual miRNAs often target numerous related mRNAs that encode multiple components of …