Altasciences at the ACT 40th Annual Meeting
November 17–20, 2019
Altasciences is a proud sponsor of the 40th Annual American College of Toxicology 2019 Meeting. Our team will be available to meet with you to discuss your safety assessment needs for both small molecules and biologics.
Whether you have a stand-alone study or a full IND program, we have the scientific and technical expertise to support your project and to deliver the highest quality results, on time and on budget.
The Altasciences team is delighted to present at ACT. Join us at our exhibitor-hosted session and at the wine and cheese poster session.
Monday, November 18th | 5:00 p.m.–6:30 p.m.
Strategies for Screening Nonhuman Primates in Gene Therapy Studies
Presented by Tina Rogers, PhD, Vice President, Preclinical Sciences
Sunday, November 17, 12:00 p.m.-12:55 p.m. in the Grand Sonoran H room.
As gene therapy treatments become more prevalent, properly pre-screening nonhuman primates (NHPs) for single nucleotide polymorphisms (SNPs) and anti-adeno-associated virus (AAV) antibodies becomes increasingly important. While SNPs can be rare, incidence rates of AAV-neutralizing antibodies in NHP colonies can be widely varied, based on several factors, and seroconversion can also be a concern. This review evaluates potential prescreening strategies to ensure quality nonclinical studies.
Development of an IL-31-Induced Pruritus Model in Cynomolgus Monkeys
Presented by Julie Forget, Director of Safety Assessment
Itching and scratching has evolved as an important protection mechanism against various threats to the skin. Pruritus is a severe itching condition most commonly associated with skin disorders, such as atopic dermatitis or psoriasis. Interleukin-31 (IL-31) is a protein produced by activated T cells in mammals and has been shown to induce a profound pruritogenic response. In order to evaluate the efficacy of therapies intended to treat pruritic skin diseases, an IL-31 pruritus cynomolgus monkey model has been established.
To develop the pruritus model, cynomolgus monkeys were administered subcutaneous (SC), intradermal (ID), or intravenous (IV) injection(s) of cIL-31, ranging from 0.3 to 24 µg/kg. Pharmacological activity was monitored based on the number of scratching and self-grooming events over a 24-hour period.
Intradermal injections of cIL-31 in cynomolgus monkeys at doses of 6, 12, 19.5 and 24 µg/kg resulted in a consistent and robust systemic pharmacological response, as evidenced by a 3- to 6-fold increase from baseline. The pharmacological response was more pronounced between 0.5 and 1.5 hours post-dose and appeared to restore nearly to baseline levels by 24 hours post-dose.
The IV route elicited a comparable scratching response to the highest ID levels tested but with smaller dose levels (0.25 to 10 µg/kg); SC provided the least useful data.
Based on these observations, ID or IV injections of cIL-31 in cynomolgus monkey resulted in a consistent and robust model for future research and development of treatment strategies for atopic dermatitis and many other pruritic skin diseases.
Effect of Housing on Stress-Related Parameters in NHPs
Presented by Tina Rogers, PhD, Vice President, Preclinical Sciences
In preclinical studies, it is important to reduce animal stress, both for animal welfare reasons and to minimize the impact on toxicologic parameters. Effects of stress on in-life parameters (body weight, food consumption), clinical pathology (white blood cell and differential counts), organ weights (endocrine, immune, reproductive) and histopathology (lymphoid, GI tract) are well documented. Myostatin (MSTN), a member of the TGF-β superfamily, is a negative regulator of muscle deposition and has been shown to increase in response to psychological stress in mice; increased MSTN correlated directly with decreased muscle mass. Plasma levels of MSTN were measured in 15 young adult male cynomolgus monkeys during a two-week period of housing in standard four packs (pairs and trios) and after transfer into gang housing enclosures (three animals per cage) that meet or exceed EU standards. Average MSTN levels in standard housing ranged from approximately 3,900 to 7,100 pg/mL, and in gang housing from 1,700 to 5,000 pg/mL. On an individual animal basis, average MSTN levels decreased by 10-32%, suggesting potential stress reduction in the gang housing setting. The significance of this change in MSTN levels is unknown. Potential correlation with other stress related parameters, including body weights, clinical pathology, and behavioral evaluations, was assessed. The specific benefits of gang vs. standard housing were examined to better understand the manifestations of stress in a laboratory environment that are relevant to toxicology study outcomes.
Use Of Thromboelastography (TEG) in Preclinical Studies.
Presented by Narine Lalayeva Study Director, Preclinical Services
Preclinical drug development focused on coagulation will typically include frequent analyses of hematology and coagulation. In addition, thromboelastography (TEG) is useful to monitor clot strength, fibrinolysis, and helping to diagnose platelet dysfunction and hypercoagulability. TEG must be initiated shortly following blood collection, thus it is essentially a point-of-care technique. Methods: In practice, whole blood is collected in sodium citrate and an aliquot of citrated blood is added to a vial containing Kaolin. The Kaolin mixture is then pipetted into a disposable TEG cup and analyzed on a calibrated machine. The resulting TEG tracing provides data on the rate of clot formation (alpha-angle), time to achieve a certain clot strength (K), time to initial fibrin formation (R), overall stability of the clot (MA), and decrease in stability of the clot at 30 or 60 min (LY30 or LY60). Results: TEG analysis has been utilized in pre-clinical studies focused on bleeding disorders and platelet dysfunction in multiple large animal models and correlated well with conventional hematology data. For example, a TEG tracing illustrating long R and K times, and low alpha-angle and MA suggestive of thrombocytopenia directly correlated with decreased platelet counts. The long R time suggestive of a deficiency in clotting factors was compared to measured levels of individual coagulation factors. Data indicates that coagulation factors VIII, XI, XII, and XIII were markedly altered during the critical point of thrombocytopenia. These data support the continued use of multiple approaches to evaluate the coagulation cascade to provide the most meaningful interpretation.
Translatability of Nonhuman Primate Cytokine Data to In-Life Parameters in Nonclinical Toxicology Studies
Presented by Christopher Do, PhD Scientist, Analytical Biology
Cytokines are important immunoregulatory proteins that have gained focus in safety assessment. Interpreting cytokine data comes with challenges due to the variable nature of their stimuli and responses. Contributing factors to the variability in cytokine expression include species specific reactions, individual variations, dose-response relationships, and unanticipated immunotoxicity. Therefore, evaluating cytokine measurements in conjunction with additional parameters such as clinical observations, and clinical pathology data, can be used to provide more definitive assessments in nonclinical safety studies. In several case studies, cytokines were evaluated for a presence of a dose response relationship. Multiplex platforms such as Luminex or MSD® were used in determining cytokine levels in non-human primates. In multiple studies, increases in IL-6, a pro-inflammatory cytokine, and MCP-1, a monocyte chemoattractant were consistently correlated with a rash, diarrhea, lethargy, or hunched posture. More specifically, increases in both analytes ranged from undetectable (at predose) to over 20,000 pg/mL (after dosing). In other instances, elevated IL-2 (>2,000 pg/mL) or IL-12 (>350 pg/mL) correlated with bruising, injury, or abnormal feces which were not necessarily considered test article-related. In cases of test article-related effects, animals becoming moribund also had elevated TNF-α and IL-6 (>20,000 pg/mL for both). Therefore, when interpreting cytokine variations for assessment of potential toxicity, other measurements such as clinical observations, and clinical pathology parameters should also be considered in addition to the test article-related effects.
Detection of Peripheral Blood Foxp3+ Regulatory T Cell Population in a Preclinical Setting
Presented by Milan Patel Scientist, Flow Cytometry
Research focused on T regulatory (Treg) cells has seen an increase for immunotherapy treatments designed to treat various malignancies. For instance, studies in humans and animal models have shown that by suppressing tumor immunity, Tregs can prevent the host immune system from detecting and controlling tumor growth. As a result, interest in targeted depletion of Treg cells has also increased in preclinical and clinical settings. However
,it is difficult to quantify and characterize the naturally occurring Treg population characterized as CD3+CD4+ CD25+Foxp3+ using flow cytometry in peripheral blood. This is due to difficulties involved in Foxp3 staining and relatively rare presence of Treg cells in peripheral blood. To alleviate these concerns, we developed a method that can reproducibly detect a large number of Treg cells in 250ul of whole peripheral blood of cynomolgus monkeys; this allows effective evaluation of therapies designed to characterize or deplete Treg cells. In animals analyzed using our developed method, the Foxp3+ Treg population ranged from 2.1% to as low as 0.2% of total lymphocyte population. Nonetheless,our method was reliably able to detect up to 21,500 Foxp3+ Treg cells in peripheral blood. The results were confirmed using an extracellular method of detecting Treg population characterized as CD3+CD4+CD25+127low. Our method provides a reproducible way of detecting Foxp3+ Treg cell population in cynomolgus monkeys and presents a unique opportunity of studying modulation of the Treg cell population in vivo.
Repeated Ultrasound-Guided Liver Biopsies in Non-human Primates
Presented by Steve Mason, Executive Vice President of Operations
Despite several improvements in serology, the need to collect a sample directly from an organ remains a valuable option in nonclinical research. The liver biopsy is an invasive procedure with potential risk for complications. Nonetheless, the procedure has been refined to become a rapid, precise, and safe collection in non-human primates (NHPs), with a relatively short recovery period. In order to mitigate the risk associated with liver biopsies, a 2D ultrasound was used to identify major structures in and around the liver, to allow for the safe insertion of the biopsy needle (14 or 16G), and to monitor any potential bleeding post retraction of the needle. Ultrasound-guided non-terminal single liver collections have been successfully performed on 150 NHPs. Repeated non-terminal liver collections were performed on 541 NHPs with 2 or 3 biopsies (4 to 7 days apart) and on 237 NHPs with 6 to 9 monthly collections. Minor complications such as prolonged recovery from sedation, second incision needed, or difficulty penetrating the hepatic capsule were noted for a handful of animals without affecting the health of the animals. A monthly frequency has been shown to be successful for long-term studies requiring up to 9 liver biopsies per animal and no less than 4 to 7 days between occasions for up to 3 biopsies per animal. Based on these observations, ultrasound-guided liver biopsies have been proven to be minimally invasive, safe, and well tolerated in NHPs for non-terminal repeated collections.