The Usefulness of In Vitro Assays and Animal Experiments in the Design of Chemopreventive Protocols with Beta-Carotene and Vitamin a on Tobacco Chewers

Abstract In vivo pharmacokinetics (PK) studies using mice and monkeys are the main approaches for evaluating and predicting the PK of antibodies, and there is a strong demand for methods that do not require animal experiments. In this work, we focused on quantitatively predicting the nonlinear PK of an antibody based on cell-based assays. An anti-mouse Fc gamma receptor IIB antibody was used as a model antibody. To determine the PK parameters related to nonspecific elimination in vivo, the plasma concentration profile at 100 mg/kg, at which target-specific clearance is saturated, was analyzed by a 2-compartment model. To estimate the parameters related to target-specific elimination, the Michaelis–Menten constant (Km) and the maximum elimination rate (Vmax) were determined by an uptake assay using Chinese hamster ovary (CHO) cells expressing the target receptor. Finally, the integration of all of these parameters permitted the PK to be predicted at doses ranging from 1 to 100 mg/kg regardless of whether target-specific clearance was saturated or nonsaturated. The findings presented herein show that in vitro assays using target-expressing cells are useful tools for obtaining PK parameters and predicting PK profiles and, in some cases, eliminate the need for in vivo PK studies using experimental animals.

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Antioxidant and Cytoprotective Effects of an Ethanol Extract of Acalypha wilkesiana var. macafeana from Malaysia

Natural Product Communications ◽

10.1177/1934578x1300800325 ◽

2013 ◽

Vol 8 (3) ◽

pp. 1934578X1300800

Author(s):

Wardah M. Din ◽

Jessica Chu ◽

Garry Clarke ◽

Khoo T. Jin ◽

Tracey D. Bradshaw ◽

...

Keyword(s):

Wound Healing ◽

Antioxidant Properties ◽

Ethanol Extract ◽

Beta Carotene ◽

In Vitro Assays ◽

Liver Carcinoma ◽

Single Class ◽

Traditional Medicines ◽

Acalypha Wilkesiana

In the annals of biomedical theory perhaps no single class of natural product has enjoyed more ingenious speculation than antioxidants formally aimed at counteracting oxidative insults which are involved in the pathophysiology of Alzheimer's and Parkinson's disease, cancer, amyotrophic lateral sclerosis, skin ageing and wound healing. In pursuing our study of Malaysian traditional medicines with antioxidant properties, we became interested in Acalypha wilkesiana var. macafeana hort., used traditionally to heal wounds. To examine whether Acalypha wilkesiana var. macafeana hort. could suppress oxidation an ethanol extract was tested by conventional chemical in vitro assays i.e., ferric reducing antioxidant potential assay (FRAP), DPPH scavenging assay and beta-carotene bleaching (BCB) assay. To explore whether Acalypha wilkesiana var. macafeana hort. protected cells against oxidative injuries, we exposed human hepatocellular liver carcinoma (HepG2) cells to tert-butylhydroperoxide ( t-BHP). In all the aforementioned experiments, the ethanol extracts elicited potent antioxidant and cytoprotective activities. To gain a better understanding of the phytochemical nature of the antioxidant principle involved, five fractions (F1-F5) obtained from the ethanol extract were tested using FRAP, DPPH and BCB assays. Our results provided evidence that F5 was the most active fraction with antioxidant potentials equal to 2.090 ± 0.307 μg/mL, 0.532 ± 0.041 μg/mL, 0.032 ± 0.025 μg/mL in FRAP, DPPH and BCB assay, respectively. Interestingly, F5 protected HepG2 against t-BHP oxidative insults. To further define the chemical identity of the antioxidant principle, we first performed a series of phytochemical tests, followed by liquid-chromatography and mass spectrometry (LC/MS) profiling which showed that the major compound contained in F5 was geraniin. To the best of our knowledge, this is the first report showing that the wound healing property of Acalypha wilkesiana var. macafeana hort. is mediated by a geraniin containing extract. Furthermore, our data leads us to conclude that geraniin could be used as a potential pharmaceutical and/or cosmetic topical agent.

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757 M9657, a novel tumor-targeted conditional anti-CD137 agonist displays MSLN-dependent anti-tumor immunity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.757 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A792-A792

Author(s):

Chunxiao Xu ◽

Brain Rabinovich ◽

Amit Deshpande ◽

Xueyuan Zhou ◽

Frederic Christian Pipp ◽

...

Keyword(s):

Bispecific Antibody ◽

Animal Experiments ◽

Immune Memory ◽

In Vitro Assays ◽

Therapeutic Window ◽

Cytotoxic T Cell ◽

Functional Assay ◽

Dose Dependent

BackgroundThe costimulatory receptor CD137 (also known as 4-1BB and TNFRSF9) plays an important role in sustaining effective cytotoxic T cell immune responses and its agonism has been investigated as a cancer immunotherapy. In clinical trials, the systemic administration of the 1st generation CD137 agonist monotherapies, utomilumab and urelumab, were suspended due to either low anti-tumor efficacy or hepatotoxicity mediated by recognized epitope on CD137 and FcγR ligand-dependent clustering.MethodsM9657, a bispecific antibody was engineered a tetravalent bispecific antibody (mAb2) format with the Fab portion binding to the tumor antigen Mesothelin (MSLN) and a modified CH2-CH3 domain as Fc antigen binding (Fcab) portion binding to CD137. M9657 has a human IgG1 backbone with LALA mutations to abrogate the binding to Fcγ receptor. The biological characteristics and activities of M9657 were investigated in a series of in vitro assays and the in vivo efficacy was investigated in syngeneic tumor models with FS122m, a murine-reactive surrogate with the same protein structure of M9657.ResultsM9657 binds efficiently to both human and Cynomolgus CD137 as well as MSLN. In the cellular functional assay, M9657 displayed MSLN- and TCR/CD3 interaction (signal 1)-dependent cytokine release and tumor cell cytotoxicity associated with Bcl-XL activation and immune memory formation. FS122m demonstrated potent MSLN- and dose- dependent in vivo anti-tumor efficacy (figure 1). Comparing with 3H3, a Urelumab surrogate Ab, FS122m displayed an improved therapeutic window with significantly lower for on-target /off-tumor toxicity.ConclusionsTaken together, M9657 exhibits a promising developability profile as a tumor-targeted immune agonist with potent anti-cancer activity, but without systemic immune activation.Ethics ApprovalAll animal experiments were performed in accordance with EMD Serono Research & Development Institute (protocol 17-008, 20-005) and Wuxi AppTec Animal Care and Use Committee (IACUC) guidelines.Abstract 757 Figure 1FS122m displayed dose-dependent anti-tumor efficacy

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An in vitro model for investigation of vitamin A effects on wound healing

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000692 ◽

2021 ◽

pp. 1-6

Author(s):

Hoda Keshmiri Neghab ◽

Mohammad Hasan Soheilifar ◽

Gholamreza Esmaeeli Djavid

Keyword(s):

Wound Healing ◽

Endothelial Cell ◽

Vitamin A ◽

In Vitro Model ◽

Cellular Proliferation ◽

Endothelial Cell Migration ◽

Normal Fibroblast ◽

Anti Inflammatory ◽

Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inﬂammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inﬂammation responses.

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A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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Teknologi Esktrasi dan Cara Pemisahannya untuk Mendapatkan Kembali Karotenoid dari Minyak Sawit : Suatu Tinjauan

Jurnal Riset Teknologi Industri ◽

10.26578/jrti.v9i1.1707 ◽

2016 ◽

Vol 9 (1) ◽

pp. 80-95

Author(s):

Agus Sudibyo ◽

Sardjono Sardjono

Keyword(s):

Vitamin A ◽

Palm Oil ◽

Beta Carotene ◽

Cost Effective ◽

Raw Material ◽

Extraction And Separation ◽

Molecular Product ◽

Industrial Use ◽

Palm Oil Mill ◽

Selection Of

Crude palm oil (CPO)is the richest natural plant source of carotenoids in terms of retinol (pro-vitamin A) equivalent, whereas palm oil mill effluent (POME) is generated from palm oil industry that contains oil and carotenes that used to be treated before discharge. Carotenoids are importance in animals and humans for the purpose of the enhancement of immune response, conversion of vitamin A and scavenging of oxygen radicals. This component has different nutritional functions and benefits to humaan health. The growing interest in the other natural sources of beta-carotene and growing awareness to prevent pollution has stimulated the industrial use of CPO and POME as a raw material for carotenoids extraction. Various technologies of extraction and separation have been developed in order to recover of carotenoids.This article reports on various technologies that have been developed in order to recover of carotenoids from being destroyed in commercial refining of palm oil and effects of some various treatments on the extraction end separation for carotenoid from palm oil and carotenoids concentration. Principally, there are different technologies, and there is one some future which is the use of solvent. Solvent plays important role in the most technologiest, however the problem of solvents which are used is that they posses potentiaal fire health and environmental hazards. Hence selection of the most safe, environmentally friendly and cost effective solvent is important to design of alternative extraction methods.Chemical molecular product design is one of the methods that are becoming more popular nowadays for finding solvent with the desired properties prior to experimental testing.ABSTRAKMinyak sawit kasar merupakan sumber karotenoid terkaya yang berasal dari tanaman sawit sebagai senyawa yang sama dengan retinol atau pro-vitamin A; sedangkan limbah pengolahan minyak sawit dihasilkan dari industri pengolahan minyak sawit yang berisi minyak dan karotene yang perlu diberi perlakuan terlebih dahulu sebelum dibuang. Karotenoid merupakan bahan penting yang diperlukan pada hewan dan manusia guna memperkuat tanggapan terhadap kekebalan, konversi ke vitamin A dan penangkapan gugus oksigen radikal. Dengan berkembangnya ketertarikan dalam mencari beta-karotene yang bersumber dari alam lain dan meningkatnya kesadaran untuk mencegah adanya pencemaran lingkungan, maka mendorong suatu industri untuk menggunakan CPO dan POME sebagai bahan baku untuk diekstrak karotenoidnya. Berbagai macam teknologi guna mengekstrak dan memisahkan karotenoid telah dikembangkan untuk mendapatkan kembali karotenoidnya. Makalah ini melaporkan dan membahas berbagai jenis teknologi yang telah dikembangkan guna mendapatkan kembali senyawa karotenoid dari kerusakan di dalam proses pemurnian minyak sawit secara komersial dan pengaruh beberapa perlakuan terhadap ekstrasi dan pemisahan karotenoid dari minyak sawit dan konsentrasi karotenoidnya. Pada prinsipnya, berbagai teknologi yang digunakan untuk mengekstrak dan memisahkan karotenoid terdapat perbedaan, dan terdapat salah satu teknologi yang digunakan untuk esktrasi dan pemisahan karotenoid adalah menggunakan bahan pelarut. Pelarut yang digunakan mempunyai peranan yang penting dalam teknologi ekstrasi; namun pelarut yang digunakan untuk mengekstrak tersebut mempunyai persoalan karena berpotensi mengganggu kesehatan dan membahayakan cemaran lingkungan. Oleh karena itu, pemilihan jenis teknologi yang aman, ramah terhadap lingkungan dan biaya yang efektif untuk penggunaan pelarut merupakan hal penting sebelum dilakukan desain metode/teknologi alternatif untuk esktrasi karotenoid. Pola produk molekuler kimia merupakan salah satu metode yang saat ini menjadi lebih populer untuk mencari pelarut dengan sifat-sifat yang dikehendaki sebelum diujicobakan. Kata kunci : karotenoid, ekstrasi, pemisahan, teknologi, minyak sawit kasar, limbah industri pengolahan sawit.

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In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207321666180220125406 ◽

2018 ◽

Vol 21 (3) ◽

pp. 215-221

Author(s):

Haroon Khan ◽

Muhammad Zafar ◽

Helena Den-Haan ◽

Horacio Perez-Sanchez ◽

Mohammad Amjad Kamal

Keyword(s):

In Silico ◽

Docking Studies ◽

In Vivo Studies ◽

In Vitro Assays ◽

Chronic Obstructive ◽

Lipoxygenase Inhibitors ◽

Lipoxygenase Inhibition ◽

In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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Repurposing of auranofin against bacterial infections: An In silico and In vitro study

Current Computer - Aided Drug Design ◽

10.2174/1386207323666200717155640 ◽

2020 ◽

Vol 16 ◽

Author(s):

Nidhi Sharma ◽

Arti Singh ◽

Ruchika Sharma ◽

Anoop Kumar

Keyword(s):

Broad Spectrum ◽

In Silico ◽

Antibacterial Agent ◽

Bacterial Infections ◽

In Vitro Assays ◽

Infection Model ◽

Synergistic Activity ◽

Bacterial Strains ◽

Molecular Docking Study

Aim: The aim of the study was to find out the role of auranofin as a promising broad spectrum antibacterial agent. Methods: In-vitro assays (Percentage growth retardation, Bacterial growth kinetics, Biofilm formation assay) and In-silico study (Molegro virtual docker (MVD) version 6.0 and Molecular operating environment (MOE) version 2008.10 software). Results: The in vitro assays have shown that auranofin has good antibacterial activity against Gram positive and Gram negative bacterial strains. Further, auranofin has shown synergistic activity in combination with ampicillin against S. aureus and B. subtilis whereas in combination with neomycin has just shown additive effect against E. coli, P. aeruginosa and B. pumilus. In vivo results have revealed that auranofin alone and in combination with standard drugs significantly decreased the bioburden in zebrafish infection model as compared to control. The molecular docking study have shown good interaction of auranofin with penicillin binding protein (2Y2M), topoisomerase (3TTZ), UDP-3-O-[3- hydroxymyristoyl] N-acetylglucosaminedeacetylase (3UHM), cell adhesion protein (4QRK), β-lactamase (5CTN) and arylsulphatase (1HDH) enzyme as that of reference ligand which indicate multimodal mechanism of action of auranofin. Finally, MTT assay has shown non-cytotoxic effect of auranofin. Conclusion: In conclusion, auranofin in combination with existing antibiotics could be developed as a broad spectrum antibacterial agent; however, further studies are required to confirm its safety and efficacy. This study provides possibility of use of auranofin apart from its established therapeutic indication in combination with existing antibiotics to tackle the problem of resistance.

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