Question 1:1. Methods for determining toxicity in anti-cancer cells
- a) Chemical and preparation- in this method, reference materials are utilized for evaluation research of the 3T3 NRU (neutral red uptake) essay. During this process, the test materials that are used should be water-soluble. A culture medium is also used to directly prepare suitable dilations. After that, the solutions obtained are tested in at least five concentrations in a range that is individually selected for each use.
- b) Cell culture – during this process, Balb/c3T3 cells are carefully prepared and maintained in Dulbecco's modified Eagle’s medium (DMEM) that is supplemented with 2 mM Glutamx, 100 mg/mL streptomycin, 100 UI/mL penicillin, and 10% calf serum. RPMI-160 is also used to prepare H460 cells that are supplemented with fetal bovine serum, 2 mM Glutamx, and 100 mg/mL streptomycin. Each of the cells is then cultured in a humid atmosphere containing 5 % carbon IV oxide (CO2) (Joann et al., 2005).
- c) NRU (neutral red uptake) essay- during this process, the test is conducted in a modified form. Afterward, Balb/c3T3 cells are then seeded using 96 plates of a density of 5!103 cell/walls. After a duration of 24 hours, the growing cells that to the bottom of the plates are treated using different concentrations that are freshly prepared test compounds. For the substances that are dissolved in DMSO, it is found out such a solution did not have a huge impact on the growth of the cells. The reason for that is because the normal complete medium has a huge influence as compared to the use of 0.2% DMSO. After a duration of 48 hours, the solutions are then removed from the plates before washing them using 200mL PBS or well.
- d) MTT essay – in using this method, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] study is conducted on the cell that are cultured in well or PBS plates. The cells are then seeded using the plates maintained at a 5!103. After a duration of 24 hours, preincubation is performed for all the cells that have attached to the bottom of the plates. In order to influence exponential cell growth, different concentrations are used to treat them. 2% DMSO is also used as the control experiment because it does not influence cell growth.
The plates are then incubated for at least 4 hours before dissolving the purple formazan crystals that could have been formed in 10 % sodiumdodecylsulfate (SDS) (Joann et al., 2005). Once dissolved, it takes 24 hours incubation at room temperature. After that, the absorbance rate is measured using spectrophotometric microplate reader PowerWaveXS. All the cells tested contain the reagents used except for that in the control wells
1:1. Drugs used and how much to dose to implement
Toxicity- in researching innovative anticancer drugs, there is the use of cytotoxicity tests as the main screening method. These are HTS (high throughput screening) tests that reveal compounds that have high cytotoxic activities. Despite that anticancer medicines are largely manufactured for the purpose of killing cells, clinically such activity is selective to tumor cells. That indicates that it will be logical to carry it out at the initial stage screening. Such a procedure is important because it assists in selecting compounds that are least toxic amongst those that are more active.
On the other hand, by first determining the dosage for test compounds, there are various chemical and physical factors that will have to be taken into consideration. They include partition coefficient, molecular weight as well as the toxicity of all the related chemicals. Failure to do that, the oral activity cannot make any biological significance to chemical effects. Acute toxicity examinations have the propensity of providing preliminary information regarding the toxic characteristic of materials for which there are no other toxicology data that is available. The reason that information is paramount is that it aid in the handling of cases such as accidental ingestion of relatively large materials. Possible targeted tissues can also be evaluated by enhancing repetitive dose toxicity analysis.
Efficacy – The attrition rate of the anticancer medicines subjected to medical testing is unacceptably higher. For the case of MM (multiple myeloma), it postulated that that is as a result of preclinical models that have been realized to overemphasize the general antiproliferative activities of medicines. The same is also as a result of the medical tests that could have been conducted in refractory end-stage patients. Because of that, it is validated that the use of VK*MYC transgenic mouse is one of the fruitful models for predicting single-agent drug activities in MM (multiple myeloma) (Marta et al., 2012). It has a 57% positive predictive value for medical activity as well as a 86% negative predictive value for medical inactivity.
There are four successful agents that are prioritized for assessment in medical trials. Ideally, the transplantation of VK*MYC transgenic mouse tumor cells into congenic mice chosen was effective because it responded only to a combination of medicines consisting of end-stage drug-resistant MM (Sheng et al., 2019). Therefore, it is predicated that standardized agents, hypoxia-activated medicines, bromodomain inhibitors, and histone deacetylase will have the potential of demonstrating drug efficacy in treatment.
Bioavailability – the bioavailability is regarded as being the overall result of absorbing, distributing, metabolism, as well as the ADME excretion. Following oral administration, absorption is used to describe the capability of compounds to move and pass into the organized circulation. Distribution is used to describe how efficient compounds pass into the targeted tissues. Metabolism is the rate at which compounds or nutrients are eliminated from the circulation system after absorption (Hana, 2012). Excretion refers to the rate at which compounds are removed from systematic circulation and finally from the body.
Methods
Mouse models, studies and transcriptomic profiling
Chronologically analyzed tumor growth information obtained from syngeneic mouse, CDX, and PDX tumor models are used to assess several efficacy and points as well as to come up with better statistical methods to model MCTs. Ideal, the general development of mouse models entails the selection of freshly dissected patient tumors into systematically measured chunks. These chunks are then engrafted subcutaneously on flanks of immunocompromised mice such as NOG, NOD/SCID, BALC/c, and so on. A caliper is used twice a week to monitor their growth to be in the position of establishing the initial stage of the PDX model. The growth of tumors is then harvested when they have attained a length of about 500-700 mm3. Several engrafted aid in the production of successive passages for the model. For the case of the syngeneic model and CDX model, the cell suspension is usually injected into the immunocompetent mice and immunocompromised mice to stimulate tumor (Sheng et al., 2019). During this time, pharmacological dosing commences when such tumors have attained the required volume.
Category efficacy endpoints in mouse studies
In this research, for main endpoint models are examined including the Response Evaluation Criteria In Solid Tumors (RECIST) method, a 3-category, the 4 response mRECIST method, a 5-cat method. Afterward, the RECIST based method classifies drug or medicine responses into a complete response CR, progressive disease (PD), stable disease (SD), and partial response (PR) based on the tumor’s RVT (relative tumor volume). Metastasis is not taken into consideration because it seldom occurs in several subcutaneous implantations. The 3-cat criteria or method is used for the purpose of classifying responses to objective response (OR), progressive disease (PD), and stable disease (SD) based on relative tumor volume (RTV). The mRECIST criteria take into account the general growth kinetics of the tumors 10 days following initiation of treatment. It also assists in classifying responses to PD, PR, SD, and CR using two main RVT parameters mainly the best average response and best response. The 5-cat criteria aid in the classification of responses of the maintained PD, PR, SD, and CR (MCR) based on the RVT ratio (Sheng et al., 2019). In defining CR and MCR, RTV is equal to 0 (zero) to aid in designating the disappearance of the measurable mass of the tumors to replace the principle (TV<0.10 cm3) utilized in Houghton.
Continuous efficacy endpoints in mouse studies
In this study, 4 continuous endpoints are described. The first one is the PFS (progressive-free survival) which is considered to be the volume of the tumor doubling time that is obtained through linear interpolation of the tumor growth statistics. The next one is the RTV ratio which is an RTV ratio that is obtained between the vehicle group and drug group at a particular time of the day and is equal to RTVt /RTVC. In this case, RTVt refers to the relative tumor volume between a certain day and initiation of treatment day and RTVc is the vehicle group. The third category is the tumor growth inhibition (TGI) which is computed as 1- RTVt /RTVC. The fourth category is the tumor’s growth rate ratio that is the comparison between drug group and vehicle group (Kt/Kc) in which Kt/Kc is the tumor growth rate obtained as a result of modeling their growth data. An AUC endpoint ratio can also be introduced can be introduced to understand how growth rate ratios can be reduced when the tumors are made to develop under exponential kinetics (Sheng et al., 2019). With the AUC endpoints, unique treatment methods are used to compute continuous endpoints using the syngeneic model, CDXs model, and PDXs model for the selected mice.
Pharmacodynamic and pharmacokinetic models – in order to understand the genetic basis of cancer and the molecular pharmacology of newly developed anti-cancer drugs require the use of various approaches that can address some of the primary key issues of medicines or drugs. That will incorporate understanding the development process of drugs, including pharmacodynamic (PD) and pharmacokinetic (PK) relationships.
As a result of that, the incorporation of the modern predictive premedical (PD/PK) models into a logically designed early-stage medical tests provide an effective means of relieving bottlenecks medical issues. Therefore, such an understanding will also be based on the need of discussing various considerations on how PD and PK evaluation of anticancer medicines will have to be performed and incorporated into global translational efforts (Oingyu & James, 2011). Research will also have to be conducted using PD/PK models to examine drug disposition as well as the dynamics they have to any targeted tissue or tissues.
Nevertheless, the significance of scrutinizing drug disposition as well as the dynamics they have to the targeted tissues or cells entail supporting the general or continued development of the premedical PD/PK models that can be consequently extrapolated for predicting patient’s pharmacological characteristics. The main physiological based PK/PB models be recognized include hybrid PBPK model, whole-body PBPK model, a two-pore models for examining macromolecules and their clinical applications. More medical data is also required to authenticate the premedical PK-PB/PD tumor based models and therefore encourage a better framework for preclinical translation to medical translation (Oingyu & James, 2011).
Nonetheless, tests such as the use of GMM (genetically modified mouse) models have also be regarded as being the best approach for mimicking the molecular and pathophysiological characteristics of HCC (Femke et al., 2008). Because of that, it is possible to come up with ground-breaking techniques that can be used for the purpose of measuring the concentration of drugs in cell and related biomarkers for drug responses. These are some of the unique models that assist in the assessment of the effects of chemicals agents such as oncogenes in return; GMMs enhance detailed examination of the carcinogenic pathway (Joann et al., 2005). Such a strategy is important in assessing the dependence and cooperativity in Vivo.
From the information collected above, it means that the advantages of conducting essays such as 3T3 NRU (neutral red uptake) essay are what conclude the development of anticancer drugs. Such tests are the ones that have enabled researchers to understand the need for rejecting non-selective genistein derivation and then chose the most suitable structures of additional optimization. Conversely, considering the use of the VK*MYC GEMM model, PD/PK model, and other methods proves them to be successful premedical models that assist in predicting the medical activity of medicines. For instance, in unprocessed and relapsed MM (multiple myeloma), such a strategy is perceived to have the likelihood of providing useful filters (William & Norman, 2012). Such filters are important because they assist in the prioritization of a large percentage of agents for assessment in medical trials.
Conclusion
The research for new drugs takes into account the use of several sophisticated tests that are perceived to foster the production of more and more innovative drugs. In the process of researching for active substances, multiple chemicals will have to be tested before selecting the most appropriate one. Despite that, it should be understood that there often exist high risk as well as expensive projects that can be analyzed. In the long-run, the selection of the most appropriate preliminary test is perceived to have the potential of enabling researchers to diminish expenses that are linked with preliminary clinical tests. Currently, there are various in vitro examinations that are perceived to be suitable in conducting drug toxicity analysis.
References
Femke, H, Isabelle, C, & Hans, V.V. (2008). Experimental mouse models for hepatocellular carcinoma Research. Department of Gastroenterology and Hepatology, Ghent University Hospital, Ghent, Belgium
Hana, S.S. 2012). Accessing The Bioavailability Of Phytochemicals In Caco-2 Cell Model And Developing A Sensitive Method For The Detection And Quantification Of These Compounds. University of Massachusetts Amherst
Joann, P, Krzystof, P, Janusz, S.S, & Aleksander, P.M. (2005). In vitro toxicity evaluation in the development of new anticancer drugs—genistein glycosides. ELSEVIER Press
Marta C,Geoffrey M. M,Victoria M. G,Stephen E. P, Jake S,Marcus L,Keith S, Ricky, W. J & LeiF, P.B (2012). Drug response in a genetically engineered mouse model of multiple myeloma ispredictive of clinical efficacy. The American Society of Hematology
Oingyu, Z & James, M.G. (2011). The Pharmacokinetic/Pharmacodynamic Pipeline: Translating Anticancer Drug Pharmacology to the Clinic. New York
Sheng, Xiaoqian J, Binchen M & Qi-Xiang L. (2019).The design, analysis and application of mouse clinical trials in oncology drug development. BMC Press
William Y. K & Norman E. S. (2012). Drug Efficacy Testing in Mice. Springer-Verlag Berlin Heidelberg
