Design Of Preclinical Studies Research Articles

⍺-Synuclein Structural Diversity and the Cellular Environment in ⍺-Synuclein Transmission Models and Humans.

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are termed synucleinopathies, disorders that are characterized by the intracellular aggregation of the protein ɑ-synuclein. The cellular tropism of synuclein pathology in these syndromes is notably distinct since in the Lewy disorders, PD and DLB, ɑSyn forms aggregates in neurons whereas in MSA ɑSyn forms aggregates in oligodendrocytes. Studies examining ɑSyn pathology in experimental models and in human brain have now identified fibrillar ɑSyn with unique but distinct molecular signatures, suggesting that the structure of these ɑSyn fibrils might be closely tied to their cellular ontogeny. In contrast to the native structural heterogeneity of ɑSyn in vitro, the conformational landscape of fibrillar ɑSyn in human brain and in vivo transmission models appears to be remarkably uniform. Here, we review the studies by which we propose a hypothesis that the cellular host environment might be in part responsible for how ɑSyn filaments assemble into phenotype-specific strains. We postulate that the maturation of ɑSyn strains develops as a function of their in vivo transmission routes and cell-specific risk factors. The impact of the cellular environment on the structural diversity of ɑSyn might have important implications for the design of preclinical studies and their use for the development of ɑSyn-based biomarkers and therapeutic strategies. By combining phenotype-specific fibrils and relevant synucleinopathy transmission models, preclinical models might more closely reflect unique disease phenotypes.

Identifying applications of virtual reality to benefit the stroke translational pipeline.

As a leading cause of mortality and morbidity, stroke and its management have been studied extensively. Despite numerous pre-clinical studies identifying therapeutic targets, development of effective, specific pharmacotherapeutics remain limited. One significant limitation is a break in the translational pipeline - promising pre-clinical results have not always proven replicable in the clinic. Recent developments in virtual reality technology might help generate a better understanding of injury and recovery across the whole research pipeline in search of optimal stroke management. Here, we review the technologies that can be applied both clinically and pre-clinically to stroke research. We discuss how virtual reality technology is used to quantify clinical outcomes in other neurological conditions that have potential to be applied in stroke research. We also review current uses in stroke rehabilitation and suggest how immersive programmes would better facilitate the quantification of stroke injury severity and patient recovery comparable to pre-clinical study design. By generating continuous, standardised and quantifiable data from injury onset to rehabilitation, we propose that by paralleling pre-clinical outcomes, we can apply a better reverse-translational strategy and apply this understanding to animal studies. We hypothesise this combination of translational research strategies may improve the reliability of pre-clinical research outcomes and culminate in real-life translation of stroke management regimens and medications.

Targeting histone deacetylases in testicular germ cell tumours: an encouraging treatment approach for the future†.

Epidrugs, specifically histone deacetylase inhibitors (HDACi), have been increasingly used in preclinical studies for the treatment of testicular germ cell tumours (TGCTs). SINHCAF was recently described as a potential oncogene in TGCTs located on chromosome 12p, the hallmark of type II (malignant) TGCTs. Thefindings contribute to the field by further supporting the efficacy of HDACi in the treatment of TGCTs, promoting the design of more preclinical studies and providing the motivation for future implementation of clinical studies with these compounds. © 2022 The Pathological Society of Great Britain and Ireland.

18F-FES radiation dosimetry preliminary estimates for preclinical studies based on voxelized phantom

Small animals, such as mice, are used in radiopharmaceutical biodistribution studies and innumerous others preclinical investigations involving ionizing radiation. Longitudinal preclinical studies with five or more image procedures, involving radiopharmaceuticals injection and/or X-radiation, are not uncommon. However, a suitable dosimetric evaluation is not always available and, sometimes, absorbed doses in animal organs or tissues and their influence in experimental results were not appropriately taken into account. Accurate calculation of absorbed doses in mice organs are needed to evaluate potential radiobiological effects that may interfere with in vivo experiments. In this work, we perform a preliminary 16α-[18F]-fluoro-17β-estradiol (18F-FES) radiation dosimetry estimates for female mice. The obtained animal dosimetric results can be useful for evaluating animal doses during the design of longitudinal preclinical studies.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples.Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions.Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively.Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Age-dependent and modality-specific changes in the phenotypic markers Nav1.8, ASIC3, P2X3 and TRPM8 in male rat primary sensory neurons during healthy aging

The effects during healthy aging of the tetrodotoxin-resistant voltage-gated sodium channel 1.8 (Nav1.8), the acid-sensing ion channel-3 (ASIC3), the purinergic-receptor 2X3 (P2X3) and transient receptor potential of melastatin-8 (TRPM8) on responses to non-noxious stimuli are poorly understood. These effects will influence the transferability to geriatric subjects of findings obtained using young animals. To evaluate the involvement of these functional markers in mechanical and cold sensitivity to non-noxious stimuli and their underlying mechanisms, we used a combination of immunohistochemistry and quantitation of immunostaining in sub-populations of neurons of the dorsal root ganglia (DRG), behavioral tests, pharmacological interventions and Western-blot in healthy male Wistar rats from 3 to 24months of age. We found significantly decreased sensitivity to mechanical and cold stimuli in geriatric rats. These behavioural alterations occurred simultaneously with differing changes in the expression of Nav1.8, ASIC3, P2X3 and TRPM8 in the DRG at different ages. Using pharmacological blockade in vivo we demonstrated the involvement of ASIC3 and P2X3 in normal mechanosensation and of Nav1.8 and ASIC3 in cold sensitivity. Geriatric rats also exhibited reductions in the number of A-like large neurons and in the proportion of peptidergic to non-peptidergic neurons. The changes in normal sensory physiology in geriatric rats we report here strongly support the inclusion of aged rodents as an important group in the design of pre-clinical studies evaluating pain treatments.

Evaluation of therapeutic strategies targeting BCAA catabolism using a systems pharmacology model.

Background: Abnormal branched-chained amino acids (BCAA) accumulation in cardiomyocytes is associated with cardiac remodeling in heart failure. Administration of branched-chain α-keto acid dehydrogenase (BCKD) kinase inhibitor BT2 has been shown to reduce cardiac BCAA levels and demonstrated positive effects on cardiac function in a preclinical setting. The current study is focused on evaluating the impact of BT2 on the systemic and cardiac levels of BCAA and their metabolites as well as activities of BCAA catabolic enzymes using a quantitative systems pharmacology model. Methods: The model is composed of an ordinary differential equation system characterizing BCAA consumption with food, disposal in the proteins, reversible branched-chain-amino-acid aminotransferase (BCAT)-mediated transamination to branched-chain keto-acids (BCKA), followed by BCKD-mediated oxidation. Activity of BCKD is regulated by the balance of BCKDK and protein phosphatase 2Cm (PP2Cm) activities, affected by BT2 treatment. Cardiac BCAA levels are assumed to directly affect left ventricular ejection fraction (LVEF). Biochemical characteristics of the enzymes are taken from the public domains, while plasma and cardiac BCAA and BCKA levels in BT2 treated mice are used to inform the model parameters. Results: The model provides adequate reproduction of the experimental data and predicts synchronous BCAA responses in the systemic and cardiac space, dictated by rapid BCAA equilibration between the tissues. The model-based simulations indicate maximum possible effect of BT2 treatment on BCAA reduction to be 40% corresponding to 12% increase in LVEF. Model sensitivity analysis demonstrates strong impact of BCKDK and PP2Cm activities as well as total BCKD and co-substrate levels (glutamate, ketoglutarate and ATP) on BCAA and BCKA levels. Conclusion: Model based simulations confirms using of plasma measurements as a marker of cardiac BCAA changes under BCKDK inhibition. The proposed model can be used for optimization of preclinical study design for novel compounds targeting BCAA catabolism.

Understanding the Tissue Specificity of ZIKV Infection in Various Animal Models for Vaccine Development.

Zika virus (ZIKV) is an arthropod-borne virus that belongs to the Flavivirus genus and is principally transmitted by Aedes aegypti mosquitoes. ZIKV infection often causes no or only mild symptoms, but it can also trigger severe consequences, including microcephaly in infants and Guillain-Barré syndrome, uveitis, and neurologic manifestations in adults. There is no ZIKV vaccine or treatment currently approved for clinical use. The primary target of ZIKV infection has been recognized as the maternal placenta, with vertical transmission to the fetal brain. However, ZIKV can also spread to multiple tissues in adults, including the sexual organs, eyes, lymph nodes, and brain. Since numerous studies have indicated that there are slightly different tissue-specific pathologies in each animal model of ZIKV, the distinct ZIKV tropism of a given animal model must be understood to enable effective vaccine development. Here, we comprehensively discussed the tissue specificity of ZIKV reported in each animal model depending on the genetic background and route of administration. This review should facilitate the selection of appropriate animal models when studying the fundamental pathogenesis of ZIKV infection, thereby supporting the design of optimal preclinical and clinical studies for the development of vaccines and therapeutics.

Topical ophthalmic administration: Can a drug instilled onto the ocular surface exert an effect at the back of the eye?

Topical ophthalmic instillation is an appealing strategy to deliver drugs to the back of the eye to treat retinal diseases such as neovascular age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, and glaucomatous optic neuropathy. It has several advantages such as being non-invasive and user-friendly, e.g., allowing self-administration. However, the main obstacle has been how to achieve therapeutic drug concentrations in the retina due to the eye’s protective mechanisms, flows, and barriers. Less than 4% of the instilled drug dose enters the anterior chamber, and much less is expected to reach the posterior segment. It is crucial to understand a drug’s topical pharmacokinetics in humans and how one can extrapolate data from rabbits to humans. In this review, the available data on the retina and vitreous drug concentrations from pharmacokinetics studies conducted in human patients and rabbits have been compiled, together with the critical physiological factors to be considered for this route of administration. Improvements in the design of preclinical studies are suggested to increase their translatability to the treatment of human patients. Finally, the current status of clinical trials with topical ophthalmic formulations intended to treat the back of the eye is depicted. At present, no topical ophthalmic formulations to treat neovascular age-related macular degeneration or other retinal neurodegenerative illnesses have reached the market.

β-Cell Cre Expression and Reduced Ins1 Gene Dosage Protect Mice From Type 1 Diabetes.

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in preclinical models. In the type 1 diabetes (T1D) research field, multiple lines of nonobese diabetic (NOD) mice have been engineered to express Cre recombinase in pancreatic β cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background have high β-cell specificity with no reported off-target effects. We explored whether NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in T1D disease modeling. We studied wild-type (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knockin in Ins1Neo/Cre mice were not additive to the Cre knockin alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at age 12 weeks. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has important implications for the experimental design and interpretation of preclinical T1D studies using β-cell-selective Cre in NOD mice.

Orthotopic and heterotopic triple negative breast cancer preclinical murine models: A tumor microenvironment comparative

Breast cancer is the most frequent type of cancer worldwide and triple negative breast cancer is a particularly aggressive subtype. Novel therapies for the treatment of cancer patients focus on the remodeling of the tumor microenvironment (TME). Orthotopic and heterotopic syngeneic mice are the most common model used to study the TME in preclinical research. Despite this, there are no published studies that address the differences between orthotopic and heterotopic murine breast cancer models at the TME level. In this report we compared proliferation, immune cell infiltrates, extracellular matrix, vascular density, and response to chemotherapy between the mammary fat pad orthotopic model, and the air pouch heterotopic model. Our study shows that the orthotopic tumors form more metastasis, however, the heterotopic tumors grow larger, have a higher FOXP3 cell infiltrate, and resemble more accurately the breast cancer TME. Our findings show that both models are very similar, there are however some differences that should be considered in the experimental design of preclinical studies.

Abstract 3574: A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Despite the therapeutic success of novel small molecules (e.g. ibrutinib, idealisib and venetoclax), patients frequently experience disease relapse due to the persistence of minimal residual disease (MRD). Targeting the residual CLL cells by immunotherapy could further improve the depth and persistence of CLL remission. Here, we report on the preclinical development and clinical implementation of a phase I trial evaluating safety, immunogenicity and efficacy in terms of MRD response of a personalized multi-peptide vaccine adjuvanted with the novel toll-like receptor (TLR) 1/2 agonist XS15 emulsified in Montanide࣪ ISA51 VG (Rammensee et al. JIC 2019) in CLL patient receiving Bruton-Tyrosin-Kinase (BTK)-inhibitor based regimes (NCT04688385). So far, broad application of peptide vaccines in cancer patients is hampered by time- and cost-intensive design and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. To improve on these drawbacks, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics (Nelde et al., Front Immunol, 2021). For the trial reported here, comparative mass spectrometry-based immunopeptidome analyses of 61 CLL patient samples and a dataset of benign tissues samples (n = 351) enabled the identification of high-frequent non-mutated CLL-associated antigens for the most common HLA allotypes (HLA-A*02, -A*24, and -B*07). These antigens were proven to be recognized as T cell epitopes by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, thereby enabling the selection of a 12-peptides panel comprising 9 HLA-class I (3 for each HLA allotype) and 3 HLA class II restricted high frequent CLL-exclusive T cell epitopes as warehouse for the personalized composition of study vaccine cocktails. Our meanwhile ongoing phase I trial recruits CLL patients eligible for treatment with BTK inhibitors. For those with matching HLA allotypes, a personalized peptide-cocktail is formulated comprising 6 CLL-associated peptides (3 HLA class I and 3 HLA class II peptides), selected from our warehouse based on individual immunopeptidome analysis, and 2 highly immunogenic HLA class II control peptides, one viral (human adenovirus C) and one tumor associated (Baculoviral IAP repeat-containing protein 5). Three doses of the vaccine are administered in a 4-weekly interval, after reduction of CLL-load by BTK inhibitors. So far, 10 CLL patients were included in the trial. The first vaccinations will be applied in November and December 2021 with first immunogenicity data expected in March 2022 and to be reported at the meeting. Citation Format: Maddalena Marconato, Annika Nelde, Yacine Maringer, Marcel Wacker, Marion Richter, Monika Denk, Helmut Salih, Jonas Heitmann, Juliane Sarah Walz. A Phase I trial of personalized multi-peptide vaccination in combination with the TLR1/2 ligand XS15 in CLL patients under Bruton-Tyrosin-Kinase-inhibitor based regimes - Preclinical development and study design [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3574.

Preclinical nerve conduction: Nerve battery options for primate studies

Electrophysiological neurodiagnostic tests of nerve conduction (NC) are key assays included in preclinical safety and toxicology programs to assess the peripheral neuropathy (PN) liability of a new drug. Despite their increased use, standardization of nerve conduction studies (NCS) is lacking in the preclinical space, with limited regulatory guidelines stipulating type and number of nerves or minimum combinations appropriate for each stage of drug development or indication. Detection of subtle peripheral toxicities depends on choosing appropriate nerve targets for testing, especially when functional changes remain above the lower limit of normal values. To support robust preclinical toxicology study designs, the current short communication provides options and recommendations for selecting peripheral nerves for clinically translatable nerve conduction batteries applicable to toxicology and gene therapy, with a focus on primate models. A comprehensive compilation of accessible nerve locations is offered including lower and upper extremity motor nerves, and sensory nerves with origin at multiple DRG levels. Rankings of technique difficulty and repeatability across serial collections are presented for each assay informed by serial nerve conduction from 500 adult primates. The goal of this communication is to support the standardization and preclinical implementation of this important assay.

Evaluation of renal injury and function biomarkers, including symmetric dimethylarginine (SDMA), in the rat passive Heymann nephritis (PHN) model

Symmetric dimethylarginine (SDMA) is a serum biomarker of excretory renal function which consistently correlates with glomerular filtration rate (GFR) across multiple species including rats, dogs, and humans. In human and veterinary clinical settings SDMA demonstrates enhanced sensitivity for detection of declining renal function as compared to other serum biomarkers, but application in preclinical study designs thus far has been limited. The purpose of this study was to determine the performance of serum SDMA in a rat passive Heyman nephritis model of glomerulopathy. In addition to SDMA other biomarkers of excretory renal function were measured including serum creatinine (sCr), blood urea nitrogen (BUN), and cystatin C along with creatinine clearance. Urinary renal biomarkers including microalbumin (μALB), clusterin (CLU), cystatin C, kidney injury marker-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin (OPN) were also measured. PHN was induced using commercial sheep anti-Fx1A serum. Tissue, serum, and urine were collected from groups of control and anti-Fx1A-treated animals for biomarker evaluation, hematology, urinalysis, serum biochemistry, and histologic examination of kidney. Over the course of a 28-day study, concentrations of the urinary biomarkers μALB, CLU, cystatin C, NGAL, KIM-1 and the serum biomarker cystatin C increased significantly in anti-Fx1A-treated rats as compared to controls but no significant increase in serum SDMA, sCr, BUN, or creatinine clearance were noted in anti-Fx1A-treated rats. Given lack of direct GFR measurement or significant change in the renal function biomarkers sCr, BUN, and creatinine clearance, it is unclear if GFR differed significantly between control and anti-Fx1A-treated rats in this study, though urinary biomarkers and histopathologic findings supported renal injury in anti-Fx1A-treated rats over the time course investigated. This study is among the first to investigate serum SDMA in a rat model relevant to preclinical safety assessment and serves to inform future experimental designs and biomarker selection when evaluation of glomerular injury is of priority.

An innovative study design with intermittent dosing to generate a GLP-regulatory package in preclinical species for long lasting molecule M5717, inhibitor of Plasmodium eukaryotic translation elongation factor 2

M5717 is a novel drug inhibiting synthesis of elongation factor 2 (PeEF2) in Plasmodium species, showing potent anti-malarial activity in preclinical studies. Traditional daily-dosing animal experiments estimating maximum safe starting dose for a first-in-human study (‘no observed adverse effect level’; NOAEL) were unsuccessful due to the long pharmacokinetic half-life of M5717, causing significant drug accumulation and high exposure. This study describes an innovative strategy to produce a GLP-certified toxicology package and estimate NOAEL for long-lasting molecules like M5717. Simulated pharmacokinetic/toxicokinetic profiles were used to design the dosing schedule for preclinical safety studies and to determine the 14-day total exposure. Animals (rats/dogs) were administered various doses of M5717 using an intermittent dosing schedule allowing partial drug elimination and alleviation of toxicity during off-treatment days to maintain a minimal parasitical concentration (MPC) of 10 ng/mL; subsequently animals were monitored for toxicity and mortality. Results showed good correlation to the modelled data used to design the dosing regimen and required MPC was reached for M5717 in study animals and could be used to calculate NOAEL. This fit-for-purpose study design allowed for maintaining clinically relevant exposure to M5717, whilst minimizing toxicity-causing compound accumulation, an aspect unaddressed by traditional NOAEL-estimating experiments. This is the first time that a compound-specific, species-specific, kinetic model-based approach to preclinical study design for regulatory toxicology studies has been described and applied to an antimalarial drug candidate with long pharmacokinetic half-life. It has potential for application to other drugs with long half-lives, supporting their clinical development.

Pharmacokinetic Variability in Pre-Clinical Studies: Sample Study with Abiraterone in Rats and Implications for Short-Term Comparative Pharmacokinetic Study Designs.

One of the major concerns for all in vivo experiments is intra- and inter-subject variability, which can be a great source of inaccuracy. The aim of this study is, therefore, to estimate the ability of parallel vs. cross-over design studies in order to describe the relative pharmacokinetic performance of the studied drug formulations. We analyzed the data from a drug development program that examined the performance of innovative abiraterone acetate formulations against the identical reference product in three stages. In stages 1–3, groups A–F were dosed with the reference product once in a parallel manner. Stage 4 was performed to evaluate the intra-individual variability (IIV) by repeated administration of the reference product to the same animals. Although the geometric mean (90% CI) values of abiraterone AUClast in groups A–F were similar to the IIV group (24.36 (23.79–41.00) vs. 26.29 (20.56–47.00) mg/mL·min·g), the results generated in the isolated parallel groups provided imprecise estimates of the true AUClast values ranging from 9.62 to 44.62 mg/mL·min·g due to chance. Notably, in 4 out of 15 possible pair comparisons between the parallel groups, the confidence intervals did not include 100%, which is the true ratio for all comparisons tested after identical formulation administration to all groups. A cross-over design can significantly improve the methodology in short-term comparative pre-clinical pharmacokinetic studies, and can provide more precise and accurate results in comparison to more traditional pre-clinical study designs.

Minimum Effective Dose of DPP-4 Inhibitors for Treating Early Stages of Diabetic Retinopathy in an Experimental Model

The neurovascular unit (NVU) plays an essential role in the development of diabetic retinopathy (DR). We previously reported that the topical administration (eye drops) of sitagliptin and saxagliptin, two dipeptidyl peptidase-4 inhibitors (DPP-4i), prevents retinal neurodegeneration and vascular leakage in db/db mice. The aim of the present study is to evaluate the minimum effective dose of the topical administration of these DPP-4i. For this purpose, sitagliptin and saxagliptin were tested at different concentrations (sitagliptin: 1 mg/mL, 5 and 10 mg/mL, twice per day; saxagliptin: 1 and 10 mg/mL, once or twice per day) in db/db mice. As end points of efficacy, the hallmarks of NVU impairment were evaluated: reactive gliosis, neural apoptosis, and vascular leakage. These parameters were assessed by immunohistochemistry, cell counting, and the Evans blue method, respectively. Our results demonstrated that the minimum effective dose is 5 mg/mL twice per day for sitagliptin, and 10 mg/mL twice per day for saxagliptin. In conclusion, this study provides useful results for the design of future preclinical regulatory studies and for planning clinical trials.

Intracellular islatravir pharmacology differs between species in an in vitro model: implications for preclinical study design.

Islatravir (4'-ethynyl-2-fluoro-2'-deoxyadenosine; EFdA) is a first-in-class nucleoside reverse transcriptase translocation inhibitor (NRTTI) being investigated for HIV treatment and prevention. EFdA is intracellularly phosphorylated to EFdA-triphosphate (EFdA-tp), a competitive substrate of deoxyadenosine-triphosphate (dATP). Thus, translating safety and efficacy findings from preclinical studies relies on the assumption that EFdA's intracellular pharmacology can be extrapolated across species. We investigated how EFdA is phosphorylated across animal species commonly used for preclinical models in drug development to identify those that most closely matched humans. PBMCs were isolated from whole blood of six species (human, rhesus macaque non-human primate (rmNHP), rat, minipig, dog, and rabbit) using Ficoll separation and counted on a haemocytometer by Trypan blue staining. One million live cells were cultured in media supplemented with 10 U/mL human IL-2, 10% FBS and 1% antibiotics and treated with 0, 17, 170, and 1700 nM EFdA (n = 3 replicates per concentration). After 24 h, representative cell counts were derived from untreated control wells (as above), cells were washed in PBS, and lysed with 70:30 methanol:water. EFdA-tp and dATP concentrations were quantified by HPLC-MS/MS and normalized to the representative live cell counts for each species. When compared to human values, EFdA-tp concentrations for each EFdA treatment concentration were lower in all species (rmNHP 1.5-2.1-fold, rat 4.5-15-fold, minipig 37-71-fold, dog and rabbit >100-fold). Additionally, rmNHP and dog PBMCs exhibited significantly higher (7-10-fold; P < 0.001) dATP when compared with human PBMCs. Given intracellular pharmacology differences, these preclinical models may be a conservative estimate of EFdA's intracellular pharmacokinetics and efficacy in humans.

Unilateral Corneal Insult Also Alters Sensory Nerve Activity in the Contralateral Eye.

After the unilateral inflammation or nerve lesion of the ocular surface, the ipsilateral corneal sensory nerve activity is activated and sensitized, evoking ocular discomfort, irritation, and pain referred to the affected eye. Nonetheless, some patients with unilateral ocular inflammation, infection, or surgery also reported discomfort and pain in the contralateral eye. We explored the possibility that such altered sensations in the non-affected eye are due to the changes in their corneal sensory nerve activity in the contralateral, not directly affected eye. To test that hypothesis, we recorded the impulse activity of the corneal mechano- and polymodal nociceptor and cold thermoreceptor nerve terminals in both eyes of guinea pigs, subjected unilaterally to three different experimental conditions (UV-induced photokeratitis, microkeratome corneal surgery, and chronic tear deficiency caused by removal of the main lacrimal gland), and in eyes of naïve animals ex vivo. Overall, after unilateral eye damage, the corneal sensory nerve activity appeared to be also altered in the contralateral eye. Compared with the naïve guinea pigs, animals with unilateral UV-induced mild corneal inflammation, showed on both eyes an inhibition of the spontaneous and stimulus-evoked activity of cold thermoreceptors, and increased activity in nociceptors affecting both the ipsilateral and the contralateral eye. Unilateral microkeratome surgery affected the activity of nociceptors mostly, inducing sensitization in both eyes. The removal of the main lacrimal gland reduced tear volume and increased the cold thermoreceptor activity in both eyes. This is the first direct demonstration that unilateral corneal nerve lesion, especially ocular surface inflammation, functionally affects the activity of the different types of corneal sensory nerves in both the ipsilateral and contralateral eyes. The mechanisms underlying the contralateral affectation of sensory nerves remain to be determined, although available data support the involvement of neuroimmune interactions. The parallel alteration of nerve activity in contralateral eyes has two main implications: a) in the experimental design of both preclinical and clinical studies, where the contralateral eyes cannot be considered as a control; and, b) in the clinical practice, where clinicians must consider the convenience of treating both eyes of patients with unilateral ocular conditions to avoid pain and secondary undesirable effects in the fellow eye.

Methodological Approaches to Preclinical Evaluation of General Toxicity of Therapeutic Radiopharmaceuticals

Radiopharmaceuticals differ from other medicines mainly by the specific nature of their pharmacological action based on radioactivity, by absence of multiple-dose regimens, and by miniscule concentrations of the active pharmaceutical ingredient (10-9–10-12 mol/L). However, the official regulations and standards do not contain specific requirements for preclinical evaluation of radiopharmaceuticals. The aim of the study was to summarise preclinical data on general toxicity of therapeutic radiopharmaceuticals, which were obtained by the Laboratory of Preclinical and Clinical Studies of Radiopharmaceuticals of the Federal State Budgetary Institution ‘State Research Center of the Russian Federation¾Burnasyan Federal Medical Biophysical Center’ of the Federal Medical Biological Agency. Results: the authors analysed criteria for: selection of the study design based on physico-chemical nature of the active pharmaceutical ingredient; calculation of doses and selection of dosage regimens for radiopharmaceuticals with different administration routes; radiation safety for personnel performing the study. The authors used the data on three therapeutic radiopharmaceuticals to formulate methodological approaches to preclinical evaluation of their general toxic properties. The analysis of long-term experience in preclinical studies of therapeutic radiopharmaceuticals demonstrated the need for a judicious individual approach to the development of a preclinical study design. The authors proposed methodological approaches to preclinical evaluation of general toxic properties of therapeutic radiopharmaceuticals, which make it possible to adequately assess potential toxic effects and obtain meaningful study results. Conclusions: there is a need for a specific guideline for preclinical studies of therapeutic radiopharmaceuticals, which would take into account specific pharmacological action and nuclear-physical characteristics of radionuclides.