Preclinical Pharmacology Studies Research Articles

Brain metastatic outgrowth and osimertinib resistance are potentiated by RhoA in EGFR-mutant lung cancer

The brain is a major sanctuary site for metastatic cancer cells that evade systemic therapies. Through pre-clinical pharmacological, biological, and molecular studies, we characterize the functional link between drug resistance and central nervous system (CNS) relapse in Epidermal Growth Factor Receptor- (EGFR-) mutant non-small cell lung cancer, which can progress in the brain when treated with the CNS-penetrant EGFR inhibitor osimertinib. Despite widespread osimertinib distribution in vivo, the brain microvascular tumor microenvironment (TME) is associated with the persistence of malignant cell sub-populations, which are poised to proliferate in the brain as osimertinib-resistant lesions over time. Cellular and molecular features of this poised state are regulated through a Ras homolog family member A (RhoA) and Serum Responsive Factor (SRF) gene expression program. RhoA potentiates the outgrowth of disseminated tumor cells on osimertinib treatment, preferentially in response to extracellular laminin and in the brain. Thus, we identify pre-existing and adaptive features of metastatic and drug-resistant cancer cells, which are enhanced by RhoA/SRF signaling and the brain TME during the evolution of osimertinib-resistant disease.

DDEL-08. NOVEL COMBINATION THERAPY USING PI3KINASE INHIBITION AND IMMUNE BASED THERAPY FOR THE TREATMENT OF GLIOBLASTOMA MULTIFORME

Abstract Hurdles to effective treatments for glioblastoma multiforme (GBM), the most aggressive form of brain cancer, are resistance to chemotherapy, radiation, and immune checkpoint inhibitors. GBMs, like other “difficult to treat” tumors, often over-utilize the phosphatidyl inositol 3 kinase (PI3K) pathway for survival, cell growth, and cell division, conferring both chemotherapy and radiation resistance. Furthermore, the PI3K pathway has been shown to modulate expression of Major Histocompatibility Complex (MHC) molecules, key components of immune recognition. Based on these observations, we examined the potential of a novel, small molecule PI3K inhibitor GCT.Glio.1 (NPT520-337), which was designed to cross the blood brain barrier, to increase the sensitivity of GBM to immune based therapies and radiation. We hypothesized that targeting PI3K would result in growth arrest and increase cell surface expression of MHC molecules and the PD-L1 checkpoint target. Treatment of GL261 mouse and human U251 GBM cell lines with GCT.Glio.1 caused growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1 and MHC class II, and synergized with radiation to cause growth arrest and cell death. Based on these results we designed and performed in vivo animal studies, implanting luciferase-flagged GL261 into the caudate nucleus of C57BL6 mice. We treated the animals orally with GCT.Glio.1 followed 24 hours later by anti-PD1 therapy in repeating cycles. The treatments significantly delayed progression of the tumor when compared to no treatment or either treatment alone, with several animals exhibiting complete regression as measured by MRI and bioluminescence. In conclusion, these results indicate that GCT.Glio.1 may sensitize GBM to immune checkpoint inhibitor and/or radiation therapies. Based on our encouraging pre-clinical data and early results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using this combination therapy.

The preclinical pharmacological study of a novel intravenous anesthetic, ET-26 hydrochloride, in aged rats.

BackgroundET-26 hydrochloride (ET-26HCl) is a novel analogue of etomidate approved for clinical trials. However, all results from recent studies were accomplished in young adult animals. The objective of this study was to evaluate the efficacy and safety of ET-26HCl in aged rats.MethodsAged Sprague-Dawley rats were randomly divided into three groups (three males and three females in each group) were given dose of two-fold of median effective dose (ED50) of ET-26HCl, etomidate and propofol: the measurements of loss of the righting reflex (LORR) and cardiovascular and respiratory function after injection at the two-fold dose of the median effective dose were used for evaluation of effectiveness and safety, and the modified adrenocorticotropic hormone-stimulation experiment was used to evaluate the inhibition effect of the drugs on the synthesis of adrenal cortical hormones.ResultsThere was no significant difference in the onset time among propofol, etomidate and ET-26HCl. The duration of propofol (850.5 ± 77.4 s) was significantly longer than that caused by etomidate (489.8 ± 77.0 s, p = 0.007) and ET-26HCl (347.3 ± 49.0 s, p = 0.0004). No significant difference was observed in the time to stand and normal activity among drugs. A total of 66.7% of rats in the ET-26HCl group were evaluated to have mild hematuria. Then, etomidate and ET-26HCl had a milder blood pressure inhibition effect than propofol. Apnea was observed in all rats administered propofol and the duration for this side effect was 45.0 ± 9.0 s. For etomidate and ET-26HCl, no apnea was observed. No other clinical signs of side-effect were observed, and no rats died. No significant difference was observed in corticosterone concentrations between ET-26HCl and solvent group. However, rats administered etomidate had lower corticosterone concentrations than those administered ET-26HCl at 15, 30, and 60 min.ConclusionsOur results indicate ET-26HCl in aged rats is an effective sedative-hypnotic with stable myocardial and respiratory performance and also have mild adrenocortical suppression. Thus, these findings increase the potential for the clinical use of ET-26HCl in the elderly population.

Pharmacological Potential of Flavonoids against Neurotropic Viruses.

Flavonoids are a group of natural compounds that have been described in the literature as having anti-inflammatory, antioxidant, and neuroprotective compounds. Although they are considered versatile molecules, little has been discussed about their antiviral activities for neurotropic viruses. Hence, the present study aimed to investigate the pharmacological potential of flavonoids in the face of viruses that can affect the central nervous system (CNS). We carried out research from 2011 to 2021 using the Pubmed platform. The following were excluded: articles not in the English language, letters to editors, review articles and papers that did not include any experimental or clinical tests, and papers that showed antiviral activities against viruses that do not infect human beings. The inclusion criteria were in silico predictions and preclinical pharmacological studies, in vitro, in vivo and ex vivo, and clinical studies with flavonoids, flavonoid fractions and extracts that were active against neurotropic viruses. The search resulted in 205 articles that were sorted per virus type and discussed, considering the most cited antiviral activities. Our investigation shows the latest relevant data about flavonoids that have presented a wide range of actions against viruses that affect the CNS, mainly influenza, hepatitis C and others, such as the coronavirus, enterovirus, and arbovirus. Considering that these molecules present well-known anti-inflammatory and neuroprotective activities, using flavonoids that have demonstrated both neuroprotective and antiviral effects could be viewed as an alternative for therapy in the course of CNS infections.

Pharmacological effects and therapeutic potential of natural compounds in neuropsychiatric disorders: An update.

Neuropsychiatric diseases are a group of disorders that cause significant morbidity and disability. The symptoms of psychiatric disorders include anxiety, depression, eating disorders, autism spectrum disorders (ASD), attention-deficit/hyperactivity disorder, and conduct disorder. Various medicinal plants are frequently used as therapeutics in traditional medicine in different parts of the world. Nowadays, using medicinal plants as an alternative medication has been considered due to their biological safety. Despite the wide range of medications, many patients are unable to tolerate the side effects and eventually lose their response. By considering the therapeutic advantages of medicinal plants in the case of side effects, patients may prefer to use them instead of chemical drugs. Today, the use of medicinal plants in traditional medicine is diverse and increasing, and these plants are a precious heritage for humanity. Investigation about traditional medicine continues, and several studies have indicated the basic pharmacology and clinical efficacy of herbal medicine. In this article, we discuss five of the most important and common psychiatric illnesses investigated in various studies along with conventional therapies and their pharmacological therapies. For this comprehensive review, data were obtained from electronic databases such as MedLine/PubMed, Science Direct, Web of Science, EMBASE, DynaMed Plus, ScienceDirect, and TRIP database. Preclinical pharmacology studies have confirmed that some bioactive compounds may have beneficial therapeutic effects in some common psychiatric disorders. The mechanisms of action of the analyzed biocompounds are presented in detail. The bioactive compounds analyzed in this review are promising phytochemicals for adjuvant and complementary drug candidates in the pharmacotherapy of neuropsychiatric diseases. Although comparative studies have been carefully reviewed in the preclinical pharmacology field, no clinical studies have been found to confirm the efficacy of herbal medicines compared to FDA-approved medicines for the treatment of mental disorders. Therefore, future clinical studies are needed to accelerate the potential use of natural compounds in the management of these diseases.

Artemisia spp.: An Update on Its Chemical Composition, Pharmacological and Toxicological Profiles.

Artemisia plants are traditional and ethnopharmacologically used to treat several diseases and in addition in food, spices, and beverages. The genus is widely distributed in all continents except the Antarctica, and traditional medicine has been used as antimalarial, antioxidant, anticancer, antinociceptive, anti-inflammatory, and antiviral agents. This review is aimed at systematizing scientific data on the geographical distribution, chemical composition, and pharmacological and toxicological profiles of the Artemisia genus. Data from the literature on Artemisia plants were taken using electronic databases such as PubMed/MEDLINE, Scopus, and Web of Science. Selected papers for this updated study included data about phytochemicals, preclinical pharmacological experimental studies with molecular mechanisms included, clinical studies, and toxicological and safety data. In addition, ancient texts and books were consulted. The essential oils and phytochemicals of the Artemisia genus have reported important biological activities, among them the artemisinin, a sesquiterpene lactone, with antimalarial activity. Artemisia absinthium L. is one of the most famous Artemisia spp. due to its use in the production of the absinthe drink which is restricted in most countries because of neurotoxicity. The analyzed studies confirmed that Artemisia plants have many traditional and pharmacological applications. However, scientific data are limited to clinical and toxicological research. Therefore, further research is needed on these aspects to understand the full therapeutic potential and molecular pharmacological mechanisms of this medicinal species.

Endothelial cell expression of mutant Map2k1 causes vascular malformations in mice.

Extracranial arteriovenous malformation (AVM) is a congenital vascular anomaly causing disfigurement, bleeding, ulceration, and pain. Most lesions are associated with somatic MAP2K1 activating mutations in endothelial cells (ECs). The purpose of this study was to determine if EC expression of mutant activated MAP2K1 is sufficient to produce vascular malformations in mice. We generated mice with a ROSA26 allele containing a lox-stop-lox gene trap (GT), Map2k1 cDNA with an activating p.K57N missense mutation, an internal ribosomal entry site, and green fluorescent protein cDNA (R26GT-Map2k1-GFP). We expressed mutant MAP2K1 and GFP in ECs of fetal and newborn mice using Tg-Cdh5Cre or Tg-Cdh5CreER alleles. Tg-Cdh5Cre+/-;R26GT-Map2k1-GFP/+ animals that express mutant MAP2K1 in ECs in utero developed diffuse vascular abnormalities and died by embryonic (E) day 16.5. Tg-Cdh5CreER+/-;R26GT-Map2k1-GFP/+ animals in which mutant MAP2K1 expression was induced in ECs by tamoxifen at postnatal (P) day 1 developed vascular malformations in the brain, ear, and intestines by P23. The lesions consisted of abnormal networks of blood vessels containing recombined and non-recombined ECs. In conclusion, expression of MAP2K1 p.K57N is sufficient to cause vascular malformations in mice. This model can be used to study the malformation process and for pre-clinical pharmacologic studies.

A review for discovering bioactive minor saponins and biotransformative metabolites in Panax quinquefolius L.

Panax quinquefolius L. has attracted extensive attention worldwide because of its prominent pharmacological properties on type 2 diabetes, cancers, central nervous system, and cardiovascular diseases. Ginsenosides are active phytochemicals of P. quinquefolius, which can be classified as propanaxdiol (PPD)-type, propanaxtriol (PPT)-type, oleanane-type, and ocotillol-type oligo-glycosides depending on the skeleton of aglycone. Recently, advanced analytical and isolated methods including ultra-performance liquid chromatography tandem with mass detector, preparative high-performance liquid chromatography, and high speed counter-current chromatography have been used to isolate and identify minor components in P. quinquefolius, which accelerates the clarification of the material basis. However, the poor bioavailability and undetermined bio-metabolism of most saponins have greatly hindered both the development of medicines and the identification of their real active constituents. Thus, it is essential to consider the bio-metabolism of constituents before and after absorption. In this review, we described the structures of minor ginsenosides in P. quinquefolius, including naturally occurring protype compounds and their in vivo metabolites. The preclinical and clinical pharmacological studies of the ginsenosides in the past few years were also summarized. The review will promote the reacquaint of minor saponins on the growing appreciation of their biological role in P. quinquefolius.

Carboxymethyl Chitosan Oligosaccharide Holds Promise for Treatment of Stenosis Crohn's Disease.

Crohn's disease (CD) is a chronic intestinal disturbance mediated by mucosal immune hyperactivity that is often associated with the formation of stenosis. No reliable solution to stenosis CD exists so far. Therefore, we generated carboxymethyl chitosan oligosaccharide (CMCOS) as a new promising therapy and investigate its efficacy in an improved rat CD model. CMCOS was synthesized by enzymatic hydrolysis, and its biosafety was evaluated in vivo. The rat model of stenosis CD was optimized by an orthogonal experiment of 75 or 100 mg/kg trinitrobenzenesulfonic acid (TNBS) in a 50 or 75% ethanol enema. The therapeutic efficacy of CMCOS on the rat model of stenosis CD was investigated and compared with the commercial drug 5-aminosalicylic acid over a 28 day period of disease progression. The rat model of stenosis CD was well established by intracolonic administration of 75 mg/kg TNBS in 75% ethanol. CMCOS significantly alleviated CD symptoms morphologically, hematologically, and pathologically, promoting functional recovery of intestinal epithelium in a dose-dependent manner. CMCOS reduced infiltrations of inflammatory cells by regulating the IL-17A/PPAR-γ pathway and reduced fibro-proliferation and fibro-degeneration of the colon tissue by downregulating the TGF-β1/WT1 pathway. 75 mg/kg TNBS in a 75% ethanol enema induces a rat model of stenosis CD suitable for preclinical pathology and pharmacological studies. The safety, antifibrosis, and functional repair performance of CMCOS make it a promising candidate for the treatment of stenosis CD.

A Review on Clinical and Preclinical Pharmacological Studies in Muskmelon Fruit

Muskmelons are extensively cultivated throughout India particularly in the hot and dry North-Western are as belongs to family Cucurbitaceae. Muskmelon, also referred to as ‘sweet melon,’ got its name from its strong musky smell. It is a yellow-coloured fruit with tempting sweetness and a pleasant aroma. Generally, muskmelon fruit is eaten fresh or made salad or as a dessert. Seed kernels are used in soups and stews as a thickening, emulsifying, fat binding and flavouring agent. Muskmelon has rich calories, carbohydrate, protein, dietary fibre, sodium, vitamin A, folic acid, niacin, Vitamin C it is also nutrient enriched with good quantities of calcium, magnesium, potassium and carotenoids, does not have fat, cholesterol or saturated fat and have high content of polyphenol antioxidants which enhance the immune function and cardiovascular system. Polyphenols regulates nitric oxide that assists in prevention of heart attacks and also strengthens blood vessels. It can be used for weight loss purposes. During summer, it prevents dehydration of body and replenishes water content and electrolytes in the body. Musk melon is scientifically proven for its useful medicinal values such as Analgesic and Anti-inflammatory Activity, Anti-oxidant and Anti-Vitiligo Effect, Anti-ulcer activity, Anti-cancer activity, Hepato-protective activity, Diuretic effect, Anti-diabetic activity, Antibacterial activity, Antifungal activity. In this review, the effort has been made to focus on the pharmacological activities of the Muskmelon.

Abstract LB136: Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme

Abstract The purpose of this study was to test the effects of a novel small molecule inhibitor of phosphatidylinositol-3 kinase (PI3K) for the treatment of glioblastoma multiforme (GBM). The key hurdles to effective treatment for GBM, the “most deadly” form of brain cancer, are resistance to chemotherapy, to radiation, and to immune check point inhibitor therapy. The overarching goal of our experiments has been to determine if a well-characterized small molecule inhibitor of PI3K, GCT.Glio.1, would improve treatment outcomes for GBM patients. The PI3K pathway has been shown to confer both chemotherapy and radiation resistance in GBM. GCT.Glio.1 (NPT520-337), a small molecule compound known to cross the blood brain barrier (BBB), was developed to target PI3K to treat GBM. GBMs, like other “difficult to treat” tumors, often over-express and actively utilize the PI3K pathway for survival, cell growth, and cell division. We used this small molecule inhibitor to determine how targeting the PI3K pathway impacts treatment resistance in GBM. We hypothesized that targeting PI3K would result in growth arrest, increase cell surface expression of a key immune check-point inhibitor target PD-L1, and would sensitize the GBM to treatment with radiation. Treatment of GL261 mouse GBM and U251 human GBM cell lines with GCT.Glio.1 resulted in profound growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1, and synergized with radiation to cause growth arrest and subsequent cell death. In conclusion, these results indicate that GCT.Glio.1 may be a strong candidate for treatment of GBM, and suggest that GCT.Glio.1 is a candidate for rationally designed combinations with currently available radiation and immune checkpoint inhibitor therapies. Currently, the animal studies using GCT.Glio.1 in the GL261 mouse model of GBM have been initiated. Based on our promising pre-clinical data and early encouraging results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using GCT.Glio.1 for GBM patients. Citation Format: Ekokobe Fonkem, M. Karen Newell-Rogers, Richard Tobin, Debbie Healey, Mita Das, Sara Bowen, Chad Quarles, John Clark. Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme [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 LB136.

Abstract 5524: AK127, a novel monoclonal antibody (mAb) targeting T cell immunoreceptor (TIGIT)

Abstract Introduction: Immune checkpoint blocking antibodies achieved great clinical success, yet a large fraction of cancer patients receive minimum benefit from current immunotherapies targeted PD-1 and CTLA-4. As a new multifunctional immune checkpoint molecule, TIGIT holds promise to become an important cancer immune therapy target. AK127 is a novel humanized immunoglobulin (Ig) G1 monoclonal antibody (mAb) targeting T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT). AK127 binds to TIGIT specifically and blocks the interaction of TIGIT with its ligands, thus relieves immune suppression and promotes anti-tumor immune response. Method: The effect of AK127 on secretion of IL-2 in mixed culture of Jurkat-TIGIT and HT1080-aCD3scFv cells (ELISA) was determined in cellular assays. Binding kinetics of AK127 to C1q, FcγR were measured by Fortebio. ADCC, ADCP and CDC activities were determined in cellular assays. In preclinical in-vivo pharmacology studies, synergistic anti-tumor activity of AK127 and a bi-specific anti-PD1/CTLA4 antibody Cadonilimab (AK104) was investigated in BALB/c-hPD-1/hTIGIT transgenic mice with CT26.WT tumor model. Result: AK127 blocked the immune inhibitory signal mediated by TIGIT, resulting in enhanced IL-2 secretion. AK127 exhibited binding to FcγIa, FcγRIIIa and C1q, and eliciting ADCC, ADCP and CDC. In animal models, both AK104 (0.5 mg/kg) and AK127 (4 mg/kg) significantly inhibited tumor growth. The combination of AK127 and AK104 produced a significantly enhanced anti-tumor effect than either monotherapy, demonstrating the synergistic anti-tumor activity of AK127 combined with AK104. Conclusion: AK127 binds to human TIGIT with high affinity, competitively blocks the binding of TIGIT with its ligand CD155 and CD112, relieves immune suppression in both cellular and animal model systems, and promotes anti-tumor immune response. Citation Format: Zhaoliang Huang, Xinghua Pang, Tingting Zhong, Chunshan Jin, Na Chen, Dennis Xia, Peng Zhang, Max Wang, Michelle Xia, Baiyong Li. AK127, a novel monoclonal antibody (mAb) targeting T cell immunoreceptor (TIGIT) [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 5524.

Abstract 2743: Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal models

Abstract Successful oncology drug development hinges on proper use of preclinical xenograft and allograft mouse models that faithfully reflect histopathology and genomic features of patient tumors and exhibit clinically similar drug response. For in vivo pharmacology studies, tumor-bearing mice are treated by drugs and efficacy is monitored by continuingly measuring tumor volumes over weeks or even months, resulting in mouse-specific tumor growth data. In our previous study, we used linear mixed model (LMM) to fit tumor growth data and demonstrated its superiority to simple readouts, such as tumor growth inhibition (TGI), in discovering biomarkers and exploring mechanism of action for drugs. Here we extended the study to more parametric statistical models on an expanded dataset with over 100 thousand tumor growth curves. Six parametric models (exponential, exponential square, Gompertz, Logistic, Monomolecular, and von Bertalanffy) were fit to tumor growth curves within a single-treatment group, by the least square method after variance stabilization transformation. Best models were selected by Akaike information criterion (AIC)-based confidence set. In the Irinotecan case study, logarithmic transformation of tumor volume converts exponential or exponential square models to mixed-effects linear models that integrate random effect of mouse difference within a treatment group, and are used to identify predictive biomarkers for drug efficacy. We compared three variance stabilization transformations and found that log transformation of tumor volume is appropriate for majority of tumor growth curves. In log transformed data, exponential or exponential square model can satisfactorily fit nearly all tumor growth curves, as 96.1% of best model confidence sets contain exponential or exponential square model, followed by von Bertalanffy (92.5%), Gompertz (92.1%), Logistic (90.5%), and Monomolecular (58.9%). The percentage of studies, in which all treatment groups can be best fit by exponential or exponential square model, is 91.3% (82.4% for von Bertalanffy, 81.4% for Gompertz, 79.1% for Logistic, 37.3% for Monomolecular). Thus, exponential or exponential square model can be applied to most in vivo mouse studies, and should be preferred for their mathematical simplicity and interpretability. We further demonstrated the utility of the modeling method on a 16-PDX-model study for Irinotecan to identify biological processes and pathways related to its mechanism of action. Citation Format: Huajun Zhou, Binchen Mao, Sheng Guo. Tumor growth modeling and its applications in preclinical pharmacology studies to improve translatability of animal models [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 2743.

0756 Preclinical Pharmacology of Solriamfetol: Potential Mechanisms for Wake Promotion

Abstract Introduction Solriamfetol is a wake-promoting agent (WPA) approved for the treatment of excessive daytime sleepiness associated with narcolepsy and obstructive sleep apnea. The wake-promoting mechanism of solriamfetol may result from dopamine and norepinephrine reuptake inhibition. Preclinical pharmacology studies were conducted to further elucidate the molecular targets activated by solriamfetol and compare them to that of known WPAs and traditional stimulants. Methods In vitro binding and functional studies were conducted in a panel of cell lines or membrane preparations expressing transmembrane receptors and monoamine transporters to measure the activity of solriamfetol, comparator WPAs, and traditional stimulants. Electrophysiology studies were conducted in slice preparations from mouse ventral tegmental area (VTA). Studies to measure locomotor activity and wake-promoting effects were conducted in mice. Results In vitro functional studies showed agonist activity of solriamfetol at human, mouse, and rat TAAR1 receptors. hTAAR1 EC50 values (10–16 µM) were within the clinically observed therapeutic solriamfetol plasma concentration range and overlapped with the observed DAT/NET inhibitory potencies of solriamfetol in vitro. TAAR1 agonist activity was unique to solriamfetol; neither the WPA modafinil nor the DAT/NET inhibitor bupropion had TAAR1 agonist activity. Solriamfetol (1–10 µM) dose-dependently inhibited the firing frequency of dopaminergic VTA neurons in mouse brain slices, similar to known TAAR1 agonists; however, these effects were inhibited by a D2 antagonist, suggesting a DAT-mediated effect. Unlike traditional stimulants, solriamfetol did not increase locomotor activity in naive mice, but inhibited the increase in locomotor activity in DAT knockout mice. Conclusion Preclinical studies have identified agonist activity at the TAAR1 receptor and, possibly, lower potency agonist activity at 5-HT1A receptors as potential pharmacological targets for solriamfetol, in addition to its activity as a DAT/NET inhibitor. Given the current understanding of TAAR1 agonists as modulators of monoamine transmission with potential wake-promoting effects in multiple preclinical species, agonist activity at the hTAAR1 receptor may represent an additional pharmacological target underlying the wake-promoting effects for solriamfetol, in addition to its DNRI activity. Support (If Any) Jazz Pharmaceuticals

Development and technical validation of an ultrasound nebulizer to deliver intraperitoneal pressurized aerosols in a rat colon cancer peritoneal metastases model

Background/aimTo develop and validate a nebulizer device for anti-cancer research on pressurized intraperitoneal aerosol supply in a preclinical peritoneal metastases (PM) rat model.Material and methodsFor aerosol generation, an ultrasonic nebulizer (USN) was modified. Aerosol analyses were performed ex-vivo by laser diffraction spectrometry (LDS). Intraperitoneal (IP) 99mtechnetium sodium pertechnetate (99mTc) aerosol distribution and deposition were quantified by in-vivo single photon emission computed tomography (SPECT/CT) and compared to liquid IP instillation of equivalent volume/doses of 99mTc with and without capnoperitoneum. PM was induced by IP injection of HCT116-Luc2 human colon cancer cells in immunosuppressed RNU rats. Tumor growth was monitored by bioluminescence imaging (BLI), 18F-FDG positron emission tomography (PET) and tissues examination at necropsy.ResultsThe USN was able to establish a stable and reproducible capnoperitoneum at a pressure of 8 to 10 mmHg. LDS showed that the USN provides a polydisperse and monomodal aerosol with a volume-weighted diameter of 2.6 μm. At a CO2 flow rate of 2 L/min with an IP residence time of 3.9 s, the highest drug deposition efficiency was found to be 15 wt.-%. In comparison to liquid instillation, nebulization showed the most homogeneous IP spatial drug deposition. Compared to BLI, 18F-FDG-PET was more sensitive to detect smaller PM nodules measuring only 1–2 mm in diameter. BLI, 18F-FDG PET and necropsy analyses showed relevant PM in all animals.ConclusionsThe USN together with the PM rat model are suitable for robust and species-specific preclinical pharmacological studies regarding intraperitoneal delivery of pressurized aerosolized drugs and cancer research.

A Review on Bioanalytical Method Development and Validation by Liquid Chromatography–Tandem Mass Spectrometry

The development of sound bioanalytical method(s) is of paramount importance during the process of drug discovery and development, culminating in a marketing approval. The objective of this paper is to review the sample preparation of drug in biological matrix and to provide practical approaches for determining selectivity, specificity, limit of detection, lower limit of quantitation, linearity, range, accuracy, precision, recovery, stability, ruggedness, and robustness of liquid chromatographic methods to support pharmacokinetic (PK), toxicokinetic, bioavailability, and bioequivalence studies. Bioanalysis, employed for the quantitative determination of drugs and their metabolites in biological fluids, plays a significant role in the evaluation and interpretation of bioequivalence, PK, and toxicokinetic studies. Selective and sensitive analytical methods for quantitative evaluation of drugs and their metabolites are critical for the successful conduct of pre-clinical and/or biopharmaceutics and clinical pharmacology studies.

Preclinical Studies of a Novel Human PD-1 B-Cell Peptide Cancer Vaccine PD1-Vaxx From BALB/c Mice to Beagle Dogs and to Non-Human Primates (Cynomolgus Monkeys)

Immunotherapy with monoclonal antibodies to checkpoint inhibitors against the PD-1/PD-L1 signaling pathway is a landmark achievement in cancer therapy. Some anti-PD-1 inhibitors such as nivolumab and pembrolizumab have shown clinical success, in a percentage of patients with prolonged survival rates. However, adverse effects accompany these benefits. In this case, strategies with lower toxicity and increased specificity are urgently required. Cancer vaccines have the ability to stimulate the native immune system and in particular, an engineered B-cell epitope can elicit high-affinity polyclonal antibodies with similar efficacy to PD-1 monoclonal antibodies in murine animal models. We have previously designed and synthesized a unique B-cell vaccine, PD1-Vaxx [MVF-PD-1(92-110)], and we have tested the immunogenicity and antitumor properties in CT26 colon cancer BALB/c syngeneic mice model. This manuscript provides results from comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics, biodistribution, and safety studies. The results from these preclinical studies support the use of PD1-Vaxx in a first-in-human clinical trial in patients with non-small cell lung cancer (NSCLC). A phase I trial in patients with NSCLC has commenced.

Design, synthesis and evaluation of a series of novel long-acting dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes

Type 2 diabetes mellitus (T2DM) is one of the most popular chronic diseases around the whole world. To improve the compliance of patients, long-acting antidiabetic drugs needed to be developed. Dipeptidyl peptidase-4 (DPP-4) inhibitors are becoming increasingly important in the T2DM treatment due to the favorable properties. In the present study, a series of new substituted dihydropyrido [4′,3′:3,4] pyrazolo [1,5-a] pyrimidin-9(10H)-yl)-2-(2,5-difluorophenyl)tetrahydro-2H-pyran-3-amine were designed and synthesized as potent DPP-4 inhibitors. All compounds were characterized by 1H NMR, 13C NMR and HRMS and were evaluated in vitro. The inhibitory activity ranged from 0.43 to 12.70 μM while the inhibitory activity of positive control (omarigliptin) was 3.63 μM on DPP-4 in Caco-2 cells. Then pharmacokinetic studies were carried out in rats and compound 6c was finally selected for the further study because of its better pharmacokinetic profile. Additionally, preclinical pharmacological study of compound 6c exhibited extraordinary efficacy in vivo and good safety profile. In conclusion, compound 6c was considered as a promising DPP-4 inhibitor, which could be taken once a week or once every two weeks for the treatment of T2DM. More comprehensive researches will be carried out in the future for the further development of compound 6c.

Preclinical safety evaluation of levonadifloxacin, a novel anti-methicillin-resistant Staphyloccocus aureus benzoquinolizine fluoroquinolone by intravenous and oral administration.

Fluoroquinolone (FQ) antibacterials have drawn heightened attention from various international regulatory agencies due to their class-specific side effects. Levonadifloxacin is a novel broad spectrum benzoquinolizine FQ active against methicillin-resistant Staphyloccocus aureus (MRSA). Owing to FQ-associated safety concerns, extensive preclinical safety pharmacology (central nervous system and cardiac safety) and toxicology studies (subacute repeat-dose toxicity, genotoxicity, phototoxicity and chondrotoxicity) of levonadifloxacin were performed at relatively high doses. Intravenous (IV) and oral studies were conducted using WCK 771 (l-arginine salt of levonadifloxacin) and WCK 2349 (l-alanine ester prodrug of levonadifloxacin), respectively. Safety pharmacology studies following single dose revealed no adverse effects on central nervous system (including seizure) in mice and cardiovascular system (hERG and monkey telemetry). In repeat-dose toxicity studies, except for IV bolus dosing related effects in rat (hyperactivity, mild convulsion, polypnoea and injection site irritation) and dog (emesis and salivation), no other adverse findings limiting the dosing duration were observed. No major biochemical, haematological, gross or histopathological changes suggestive of damage to vital organs were observed in either WCK 771- or WCK 2349-treated groups. WCK 771 and WCK 2349 were found to be nongenotoxic; however, they showed weak phototoxicity that was comparable with levofloxacin. WCK 771 showed chondrotoxicity in the Beagle dog pups on repeat-dose administration; however, the severity level was lower than ofloxacin. Overall, preclinical safety studies helped establish wider safety margin for WCK 771 and WCK 2349 that supports administration of higher therapeutic doses in humans by both IV and oral routes, thereby enabling safe anti-MRSA treatment.

A novel device for serial venous blood sampling in a canine model

Diagnostic serial venous blood sampling has the potential to introduce stress-related artifactual elements into the analytical results. We have evaluated the use of a wearable automated serial blood sampling device in a canine model that obviates a need for multiple manual venipuncture. Efficacy in measuring cortisol levels during prolonged serial blood sampling is described. Automated serial blood sampling from the jugular vein was performed in two Beagle dogs. Over two separate 22-h periods, serial blood samples were withdrawn for routine cell counting and blood chemistries from each animal, whilst animal behavior along with durability of catheter placement and tolerability by the vascular system were assessed.During the study, no behavioral changes attributed to the serial blood sampling device were seen and no stress-related deviations from normal blood cell counts and cortisol levels were detected. Minor erythema was eident at the sites of catheter placement, which was consistent with the presence of an indwelling catheter. The wearable, serial blood sampling device performed well and was tolerated by the animals without stress-related changes in blood analysis or behavioral observation. This study has demonstrated the potential significant benefit of the Fluispotter® device for serial blood sampling in veterinary and clinical settings. The samples collected are free from stress-related signatures in blood chemistry otherwise common to serial blood sampling protocols requiring repeated venipuncture. This device has valuable implications for use in routine preclinical pharmacology and toxicology studies.