Veterinary Clinical Studies Research Articles

O104 | Experiences with test permits and GMP requirement for veterinary clinical studies

O104 | Experiences with test permits and <scp>GMP</scp> requirement for veterinary clinical studies

Perspectives on implementing veterinary clinical studies committees.

To address the limitations of traditional IACUC review of clinical research studies involving client-owned animals, the AVMA issued a policy describing the use of a veterinary clinical studies committee (VCSC), analogous to an institutional review board, as a way to ensure the adequate review and oversight of such studies. While IACUC composition, review, approval processes, and responsibilities are well established, uniform guidance for VCSCs is not readily available and not included in the guidance for IACUCs. In this manuscript we describe suggested best practices for scientific and ethical review of veterinary clinical research studies, regardless of the specific research setting. This resource complements the AVMA policy mentioned above by providing additional thoughts on aspects of VCSCs, including considerations necessary for the adequate review and oversight of clinical research studies using client-owned animals by VCSCs or IACUCs.

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Viscum album (L) extracts in cancer treatment: a systematic review of in vitro and in vivo studies

Background: Several reviews about phytotherapy and homeopathy have been published in the last years, including Viscum album (VA.L). VA is a parasite plant whose extract has anti-cancer proprieties and is used alone or in combination with conventional chemotherapy. &#x0D; Methods: We performed a systematic review about the in vivo and in vitro models described in the literature, including veterinary clinical trials. The literature was consulted from Pubmed database.&#x0D; Results: There are several kinds of pharmaceutical preparations about VA and their active principles used in experimental studies, lectin being frequently studied (alone or as an extract compound). More than 50% of available literature about VA is related to the lectin effects. On the other hand, the effects of viscotoxins are less studied. Among the in vivo experimental studies about VA and its compounds, the B16 murine melanoma is the most used model, followed by Ehrlich, Walker and Dalton tumors. The results point to the apoptotic effects, metastasis control and tumor regression. Some veterinary clinical studies about the use of VA in the treatment of sarcoid, fibrosarcoma and neuroblastoma are quoted in literature too, with interesting results. Considering the in vitro models, our review revealed that NALM6 leukemia cells, B16 melanoma and NC1-H460 lung carcinoma were the most studied tumor models, apoptosis signals being the most important findings. Only one study verified immunoglobulin and interleukin production. All consulted papers were related to phytotherapy preparations only.&#x0D; Conclusions: Although the literature about the anti-cancer activity of VA extract and its lectins is enough, there is a marked lack of information about viscotoxin activities and about the effects of homeopathic preparations of this plant on animal tumors and on in vitro cultivated tumor cells.

Isolation and identification of exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells

BackgroundExosomes, internal proteins, lipids, and nucleic acids coated by phospholipid bilayer membranes, are one type of small extracellular vesicles, which can mediate cell-cell communication. In recent years, exosomes have gained considerable scientific interest due to their widely applied prospect in the diagnosis and therapeutics of human and animal diseases. In this study, we describe for the first time a feasible method designed to isolate and characterize exosomes from feline plasma, urine and adipose-derived mesenchymal stem cells.ResultsExosomes from feline plasma, urine and adipose-derived mesenchymal stem cells were successfully isolated by differential centrifugation. Quantification and sizing of exosomes were assessed by transmission electron microscopy, flow nano analysis and western blotting. Detected particles showed the normal size (30–100 nm) and morphology described for exosomes, as well as presence of the transmembrane protein (TSG101, CD9, CD63, and CD81) known as exosomal marker.ConclusionsThe results suggest that differential centrifugation is a feasible method for isolation of exosomes from different types of feline samples. Moreover, these exosomes can be used to further diagnosis and therapeutics in veterinary pre-clinical and clinical studies.

Recommendations for Ethical Review of Veterinary Clinical Trials.

Ethical review of both human and animal research is critical to ensuring that studies are conducted with due regard to the welfare and safety of enrolled subjects and to the integrity of the data. However, differences exist in laws, policies, and best practices between human and animal studies. Ethical review is required for most human studies. While the laws and standards are clear for humans and for laboratory animals, the laws and standards for clinical research for client-owned animals are not as well-defined. Here, we discuss gaps in ethical review of clinical animal research in the United States of America and propose expanded functions for veterinary clinical studies committees as a solution.

A CTSA One Health Alliance guidance on institutional review of veterinary clinical studies

Harmonized institutional processes and reviewer training are vital to maintain integrity and ethical rigor of the veterinary clinical research pipeline and are a prerequisite to future work that might establish centralized or single-site ethical and regulatory review to ease initiation of multi-center studies. Funded by a CTSA One Health Alliance (COHA) pilot award, a diverse working group of veterinary clinicians and institutional representatives was convened in February 2020 to develop a guidance document detailing broadly agreed upon practices for ethical review and approval of veterinary clinical studies conducted in the United States.The working group defined key areas of need for consensus, developed a set of associated guidelines, and circulated these for review by COHA’s fifteen member institutions. Six focus areas were identified by the working group and included vital items of protocol review, composition of the review committee, post-approval monitoring and adverse event reporting, consideration of special circumstances such as satellite sites and the use of healthy veterinary subjects in research, and the informed consent process.This document outlines a broadly agreed-upon framework through which to approach vital items associated with veterinary clinical study protocol review and approval. These approaches represent current best practice in the review and approval of veterinary clinical studies, and can serve as a guidance for veterinary clinician-scientists and regulatory experts, to ensure robust and ethically conducted studies that can contribute to the advancement of both animal and human health.

The Importance of the Tumor Microenvironment and Hypoxia in Delivering a Precision Medicine Approach to Veterinary Oncology.

Treating individual patients on the basis of specific factors, such as biomarkers, molecular signatures, phenotypes, environment, and lifestyle is what differentiates the precision medicine initiative from standard treatment regimens. Although precision medicine can be applied to almost any branch of medicine, it is perhaps most easily applied to the field of oncology. Cancer is a heterogeneous disease, meaning that even though patients may be histologically diagnosed with the same cancer type, their tumors may have different molecular characteristics, genetic mutations or tumor microenvironments that can influence prognosis or treatment response. In this review, we describe what methods are currently available to clinicians that allow them to monitor key tumor microenvironmental parameters in a way that could be used to achieve precision medicine for cancer patients. We further describe exciting novel research involving the use of implantable medical devices for precision medicine, including those developed for mapping tumor microenvironment parameters (e.g., O2, pH, and cancer biomarkers), delivering local drug treatments, assessing treatment responses, and monitoring for recurrence and metastasis. Although these research studies have predominantly focused on and were tailored to humans, the results and concepts are equally applicable to veterinary patients. While veterinary clinical studies that have adopted a precision medicine approach are still in their infancy, there have been some exciting success stories. These have included the development of a receptor tyrosine kinase inhibitor for canine mast cell tumors and the production of a PCR assay to monitor the chemotherapeutic response of canine high-grade B-cell lymphomas. Although precision medicine is an exciting area of research, it currently has failed to gain significant translation into human and veterinary healthcare practices. In order to begin to address this issue, there is increasing awareness that cross-disciplinary approaches involving human and veterinary clinicians, engineers and chemists may be needed to help advance precision medicine toward its full integration into human and veterinary clinical practices.

Myofascial Pain Syndrome, myofascial trigger points and trigger points in Veterinary Medicine

Myofascial Pain Syndrome (MPS) a is a painful condition related to myofascial trigger points (TP) in skeletal muscle. The aim of this study was to perform a literature review on the etiology, pathophysiology, diagnosis and treatment of MPS in veterinary medicine. The research sites used for this purpose were: Scientific Electronic Library, PubMed and Medline. The inclusion criteria for the papers were: must be written in English; published between 1990 and 2018; include the following keywords: myofascial pain syndrome veterinary/dog/canine/cat/feline/horse/equine, trigger points veterinary/dog/canine/cat/feline/horse/equine. Through the review, it was observed that veterinary clinical studies are scarce, and the articles found lack information such as MPS description, incidence and specific treatment techniques in dogs and horses, and no studies in cats were found.

The Use of Peripheral Blood-Derived Stem Cells for Cartilage Repair and Regeneration In Vivo: A Review.

BackgroundPeripheral blood (PB) is a potential source of chondrogenic progenitor cells that can be used for cartilage repair and regeneration. However, the cell types, isolation and implantation methods, seeding dosage, ultimate therapeutic effect, and in vivo safety remain unclear.MethodsPubMed, Embase, and the Web of Science databases were systematically searched for relevant reports published from January 1990 to December 2019. Original articles that used PB as a source of stem cells to repair cartilage in vivo were selected for analysis.ResultsA total of 18 studies were included. Eight human studies used autologous nonculture-expanded PB-derived stem cells (PBSCs) as seed cells with the blood cell separation isolation method, and 10 animal studies used autologous, allogenic or xenogeneic culture-expanded PB-derived mesenchymal stem cells (PB-MSCs), or nonculture-expanded PBSCs as seed cells. Four human and three animal studies surgically implanted cells, while the remaining studies implanted cells by single or repeated intra-articular injections. 121 of 130 patients (in 8 human clinical studies), and 230 of 278 animals (in 6 veterinary clinical studies) using PBSCs for cartilage repair achieved significant clinical improvement. All reviewed articles indicated that using PB as a source of seed cells enhances cartilage repair in vivo without serious adverse events.ConclusionAutologous nonculture-expanded PBSCs are currently the most commonly used cells among all stem cell types derived from PB. Allogeneic, autologous, and xenogeneic PB-MSCs are more widely used in animal studies and are potential seed cell types for future applications. Improving the mobilization and purification technology, and shortening the culture cycle of culture-expanded PB-MSCs will obviously promote the researchers' interest. The use of PBSCs for cartilage repair and regeneration in vivo are safe. PBSCs considerably warrant further investigations due to their superiority and safety in clinical settings and positive effects despite limited evidence in humans.

The AVMA Animal Health Studies Database

The AVMA Animal Health Studies Database (AAHSD, www.avma.org/findvetstudies) was established as a tool for investigators conducting prospective veterinary clinical studies. Investigators may use the database to list studies recruiting for patients after creation of log-in credentials, while searches of the database listings may be made freely by anyone through the public search side. Although the main intent of the AAHSD is to increase public awareness of veterinary clinical studies and enhance recruitment of suitable animal patients into the studies, the database does have other intended purposes. Specifically, the AAHSD was modeled after the NIH-NLM site for medical trials (Clinicaltrials.gov) and functions like a trial registry and results database. Study listings remain permanently recorded in the AAHSD such that the AAHSD serves as a running archive of veterinary clinical studies conducted, informing investigators of studies performed both presently and in the past as well as providing an opportunity for investigators to make public the outcomes and conclusions of studies that have been completed.

Advances in Bovine Digital Diseases

A full week was devoted to discussion, sometimes heated, of bovine digital problems at a meeting organized by the British Cattle Veterinary Association in Liverpool, England, July 16-20, 1990. The first and last days, confined to members of the international study group on diseases of the ruminant digit (sixth meeting) involved twenty papers and considerable discussion on recent observations and studies. The three intervening days' program was a forum for an "Update in Cattle Lameness" (US $20 or £12 sterling post free from Dr. R. D. Murray, Department of Veterinary Clinical Studies, University of Liverpool Field Station, "Leahurst", Neaton, S. Wirral, Cheshire L64 7TE, England) attended by over 200 veterinarians and others, including large, predominantly European and Japanese contingents. This study was conceived in response to NCA's resolution to encourage biological and pharmaceutical manufacturers to provide tissue reaction data on all animal health products and to encourage development of acceptable alternative routes of administration to the problematic IM method.

Ketoconazole-conjugated ZnO nanoparticles based semi-solid formulation and study their impacts on skin disease.

In this study, the ketoconazole-conjugated zinc oxide (ZnO) nanoparticles were prepared in a single-step approach using dextrose as an intermediate compound. The physical parameters confirmed the drug conjugation with ZnO and their size was around 70-75 nm. The drug loading and in vivo drug release studies indicated that the -CHO group from the dextrose increase the drug loading up to 65% and their release kinetics were also studied. The anti-fungal studies indicated that the prepared nanoparticles exhibit strong anti-fungal activity and the minimum concentration needed is 10 mg/ml. The nanoparticles loaded semi-solid gel was prepared using carbopol, methylparaben, propyl paraben and propylene glycol. The in vitro penetration of the ketoconazole-conjugated nanoparticles was studied using the skin. The results indicated that the semi-solid gel preparations influenced the penetration and also favoured the accumulation into the skin membrane. The veterinary clinical studies indicated that the prepared gel is highly suitable for treatment of Malassezia.

Clinical use of plasma lactate concentration. Part 1: Physiology, pathophysiology, and measurement.

To review the current literature with respect to the physiology, pathophysiology, and measurement of lactate. Data were sourced from veterinary and human clinical trials, retrospective studies, experimental studies, and review articles. Articles were retrieved without date restrictions and were sourced primarily via PubMed, Scopus, and CAB Abstracts as well as by manual selection. Lactate is an important energy storage molecule, the production of which preserves cellular energy production and mitigates the acidosis from ATP hydrolysis. Although the most common cause of hyperlactatemia is inadequate tissue oxygen delivery, hyperlactatemia can, and does occur in the face of apparently adequate oxygen supply. At a cellular level, the pathogenesis of hyperlactatemia varies widely depending on the underlying cause. Microcirculatory dysfunction, mitochondrial dysfunction, and epinephrine-mediated stimulation of Na+ -K+ -ATPase pumps are likely important contributors to hyperlactatemia in critically ill patients. Ultimately, hyperlactatemia is a marker of altered cellular bioenergetics. The etiology of hyperlactatemia is complex and multifactorial. Understanding the relevant pathophysiology is helpful when characterizing hyperlactatemia in clinical patients.

Simple and Fast Gas-chromatography Mass Spectrometry Assay to Assess Delta 9-Tetrahydrocannabinol and Cannabidiol in Dogs Treated with Medical Cannabis for Canine Epilepsy.

To date, an increasing number of pet owners, especially in the USA, are using cannabis-derived products containing generally delta 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) to help their animals' health. Unfortunately, studies on the clinical use of cannabinoids in veterinary medicine are still limited, and the application of analytical methodologies for the determination of cannabinoids in animal (especially dog) biological matrices such as plasma, is still missing. A reliable, fast, accurate, simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the quantification of THC and CBD in plasma samples of eight dogs under therapeutic treatment for epilepsy and receiving oral administration of medical cannabis (Bediol). The method was linear for both the analytes under investigation with coefficients of determination (r2) of at least 0.99. Absolute analytical recovery (mean ± SD) ranged from 80.6 ± 6.2% for THC and 81.7 ± 4.3% for CBD. The matrix effect showed less than 10% analytical suppression due to endogenous substances for both the analytes. The intra-assay and inter-assay precision values ranged from 4.9% to 12.7%, and from 5.2% to 8.7% respectively. The intra-assay and inter-assay accuracy values ranged from 2.3% to 9.6% and from 3.4% to 13.0%, respectively. The validated method was successfully applied to real samples; moreover, to assess the potential of the method applicability and robustness in future veterinary clinical studies on cannabinoids therapy, we attempted to follow the kinetic of THC and CBD in the plasma of two dogs under therapy at different times after Bediol administration.

Recommendations for designing and conducting veterinary clinical pathology biologic variation studies

The recent creation of a veterinary clinical pathology biologic variation website has highlighted the need to provide recommendations for future studies of biologic variation in animals in order to help standardize and improve the quality of published information and to facilitate review and selection of publications as standard references. The following recommendations are provided in the format and order commonly found in veterinary publications. A checklist is provided to aid in planning, implementing, and evaluating veterinary studies on biologic variation (AppendixS1). These recommendations provide a valuable resource for clinicians, laboratorians, and researchers interested in conducting studies of biologic variation and in determining the quality of studies of biologic variation in veterinary laboratory testing.

Defining the primary research question in veterinary clinical studies.

A thoughtful, clearly defined research question should be the foundation of any clinical trial or research study. The research question helps determine key study methods, and defining a specific research question helps avoid problems with inadequate sample size, inappropriate design, or multiple statistical comparisons. Rationales and strategies for formulating research questions and using them to define study protocols are discussed, with a focus on application in clinical trials.

Evaluation of Clinical Metrology Instrument in Dogs with Osteoarthritis.

BackgroundIn veterinary clinical pain studies, there is a paucity of data on test‐retest variability in Clinical Metrology Instruments (CMIs), and it is unknown whether CMIs should be administered using independent (respondents not permitted to see previous answers) or dependent (respondents shown previous answers) interviewing.ObjectivesTo compare baseline variability in CMIs designed to assess pain in dogs with osteoarthritis, and compare CMI scores using independent (InD) and dependent interviewing (DI) for the Canine Brief Pain Inventory (CBPI) and the Client‐Specific Outcome Measures (CSOM).AnimalsFifty‐one client‐owned dogs with radiographic evidence of osteoarthritis and associated pain.MethodsClinical Metrology Instruments data were collected during 2 randomized, double‐masked, placebo‐controlled, proof of principle pilot studies with parallel treatment groups. Enrolled dogs received either placebo or antinerve growth factor antibody (NV‐01).ResultsAgreement between baseline CMI scores was good (CBPI Pain P = .29, CBPI Interference P = .32, CSOM P = .036, LOAD P = .67, HCPI P = .27), being best for the LOAD (ICC = 0.89). CMI responses collected during independent and dependent interviewing were not statistically different (CBPI Pain P = .33, CBPI Interference P = .28, CSOM P = .42) and showed good agreement. Additionally, dependent interviewing resulted in increased treatment effect sizes.Conclusions and Clinical ImportanceThere is little difference between independent and dependent interviewing, however, dependent interviewing resulted in increased treatment effect sizes. By using dependent interviewing, investigators could increase clinical trial power through minimal change to study design. Further research is warranted to investigate the use of dependent interviewing.

Gene Electrotransfer of Canine Interleukin 12 into Canine Melanoma Cell Lines.

A gene electrotransfer (GET) of interleukin 12 (IL-12) had already given good results when treating tumors in human and veterinary clinical trials. So far, plasmids used in veterinary clinical studies encoded a human or a feline IL-12 and an ampicillin resistance gene, which is not recommended by the regulatory agencies to be used in clinical trials. Therefore, the aim of the current study was to construct the plasmid encoding a canine IL-12 with kanamycin antibiotic resistance gene that could be used in veterinary clinical oncology. The validation of the newly constructed plasmid was carried out on canine malignant melanoma cells, which have not been used in GET studies so far, and on human malignant melanoma cells. Canine and human malignant melanoma cell lines were transfected with plasmid encoding enhanced green fluorescence protein at different pulse parameter conditions to determine the transfection efficiency and cell survival. The IL-12 expression of the most suitable conditions for GET of the plasmid encoding canine IL-12 was determined at mRNA level by the qRT-PCR and at protein level with the ELISpot assay. The obtained results showed that the newly constructed plasmid encoding canine IL-12 had similar or even higher expression capacity than the plasmid encoding human IL-12. Therefore, it represents a promising therapeutic plasmid for further IL-12 gene therapy in clinical studies for spontaneous canine tumors. Additionally, it also meets the main regulatory agencies' (FDA and EMA) criteria.

Red blood cell storage lesion.

To summarize current understanding of the mechanisms responsible for changes occurring during red blood cell (RBC) storage, collectively known as the storage lesion, and to review the biological and clinical consequences of increasing storage time of RBCs. Human and veterinary clinical studies, experimental animal model studies, and reviews of the RBC storage lesion with no date restrictions. Experimental studies have characterized the evolution of human RBC and supernatant changes that occur during storage and form the basis for concern about the potential for harm from long-term storage of RBCs. Although 4 randomized controlled trials of varying sizes failed to find an association between RBC storage time and negative clinical outcomes, a recent meta-analysis and numerous observational clinical studies have demonstrated that transfusion of old versus fresh stored RBCs is associated with an increased risk of morbidity and mortality, particularly among trauma victims and cardiac surgery patients. Potential clinical consequences of RBC transfusion following development of the storage lesion include risk of organ dysfunction, organ failure, infections, and death. Experimental animal models have contributed to the evidence supporting adverse consequences of the RBC storage lesion. Studies on relevant RBC storage issues such as the effect of different preservative solutions and leukoreduction have been completed. Transfusion with RBCs stored for 42 days increases mortality in dogs with experimental sepsis. Storage of RBCs induces progressive biochemical, biomechanical, and immunologic changes that affect red cell viability, deformability, oxygen carrying capacity, microcirculatory flow, and recipient response. Most reports in the human and veterinary literature support the concept that there are deleterious effects of the RBC storage lesion, but additional studies with improved experimental design are needed to identify compelling reasons to modify current blood banking and transfusion practices.