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Development and Execution of In Vivo Bioassays

Bioassays are involved in each stage of drug discovery, starting from Target Identification until discovering the Lead compound. Bioassays provide valuable information which displays the therapeutic potency of a drug under investigation. The data generated during bioassay also plays a vital role in drug development and quality control of finished biological products. Properly designed Bioassays help in assessing the biological effect, activity, signal transduction process and receptor binding ability of drug product or biologic on a biological target (proteins) when compared to a reference or standard over a suitable biological system.

The pharmaceutical and biotech companies involved in drug discovery and development are continuously challenged with developing biologically relevant assays for the analysis of multiple potential mechanisms. The process involves use of quality critical reagents, use of specific cell lines and purified test drug and reference drug products which at times may become a constraint. Most of these activities require sufficient time which may become a limiting factor to biopharma manufacturers. It is worth outsourcing activities to reputed CRO service providers to save time in developmental efforts and also to have an unbiased opinion on the functional activities of the drug product.

Veeda Group has qualified and experienced scientists to design, develop, execute, and validate the Bioassays for companies and provides premier bioassay services (in vitro and in vivo) that generate meaningful data to support pharmaceutical and biotech companies in their drug discovery and development journey.

Veeda Group’s experience in Development and Execution of Bioassays include:

  • Plaque Reduction Neutralization Test (PRNT assay)
  • In Vitro Skin Sensitization Human Cell Line Activation test (h-CLAT assay)
  • Nab Assay
  • Assay Development (Pharmacodynamics, Pharmacokinetics, Immunogenicity and Biomarker Assessment)
  • In Vivo Bioassays for drug molecules like Luteinizing Hormone, Epoetin, HCG, Recombinant FSH, β-HCG, and Insulin.
  • ADCC assay for biosimilars and different other assays like Ex Vivo assay, Cell based assay, Receptor Binding Assay, Cytokine Release Assay and ADA assay.

Veeda Group provides Integrated Discovery, Development & Regulatory Services with its multiple technology platforms:

  • Exploratory toxicology studies
  • Regulatory toxicology studies
  • In vitro Bioassays
  • Ex vivo Bioassays

The group also has the experience to handle diverse range of Biotherapeutics like Therapeutic Monoclonal Antibodies, Insulin & Insulin Analogues, Cytokines, Low Molecular Weight Heparins, Biosimilars, Hormones & Biomarkers. Veeda group has demonstrated capabilities to develop recombinant proteins such as non-glycosylated proteins and glycoproteins derived from either bacterial or mammalian host expression systems.

Bioassays in Preclinical Drug Development

Biological assays or bioassays are an essential tool in preclinical drug development. Preclinical bioassays can be in vivo, ex vivo, and in vitro. In vivo bioassays provide a more realistic and predictive measure of functional effects of tests with reference drug products or standard material of defined potency along with the application of statistical tools, study-specific lab techniques, and adherence to the well-designed study protocol. These assays capture the complexity of target engagement, metabolism, and pharmacokinetics of novel drugs better than in vitro bioassays.

The most commonly used experimental mammals in in vivo efficacy assays are mice and rats. Occasionally other species may be used depending on the sensitivity & suitability of the assays.

Development and Validation of Bioassays

Bioassays are used as a screening method to identify the signals that indicate desired biological activity from a set of compounds. In general, two different types of signals can be generated by a bioassay, a linear dose-response, and a sigmoidal (S-shaped) dose-response. Since one solution does not fit all bioassays, it is good to evaluate and analyze the data to develop a precise approach to carry out each bioassay. The life cycle stages of a bioassay are divided into:

Stage 1: Method design, development, and optimization

Stage 2: Procedure performance qualification

Stage 3: Procedure performance verification (fit for purpose)

Developing a bioassay that meets regulatory requirements and gets drug product registered is a very complex process. Developing a bioassay, includes many strategies and tactical designs like selecting the correct in vivo platform, proper method or plate design, data analysis, system/ sample sustainability strategy, method implementation, method performance and monitoring. There are several steps to be followed for development and validation of bioassays such as dose response and curve-fitting selection, development of reference, calculation of potency, bioassay characterization, design of bioassay calculator, standardization and automation of bioassay and finally evaluation.

Both method development and validation of bioassays include three fundamental areas:

  1. Pre-study (Identification and Design Phase) validation
  2. In-study (Development and Production Phase) validation
  3. Cross validation or method-transfer validation

During method development, assay conditions and procedures are selected that minimize the impact of potential sources of invalidity. Coming to the statistical validation for an in vivo assay, it involves four major components:

  1. Adequate study design and data analysis method
  2. Proper randomization of animals
  3. Appropriate statistical power and sample size
  4. Adequate reproducibility across assay runs.

Parallel group design, randomized block design, repeated measures design, and crossover design are the basic types of experimental designs used in in vivo assay. Following are the key-factors that should be kept in mind while designing an in vivo assay

  • All meaningful biological effects (pharmacologically) should be statistically significant.
  • If biologically relevant assays are not present, then a range of plausible effects can be considered.
  • The key endpoints should be well defined before the beginning of the assay.
  • Animals should be allocated randomly in appropriate manner to the treatment groups
  • The dose levels should be selected appropriately. Dose and curve-fitting selection is among the most critical aspects of bioassay development. The dose is determined depending on the type of model used in the signal to fit the data. For Sigmoidal designs, a four- or five-parameter logistics (4PL or 5PL) model fits the data, whereas, for linear design, a parallel line analysis (PLA) model fits the data.

For a 4PL model, nine doses are recommended:

  1. Three doses in the lower asymptote
  2. Three doses in the upper asymptote
  3. Three doses in the linear range

In contrast, for a PLA model, a minimum of four doses is recommended. A minimum of three consecutive doses is required to plot the dose curve.

  • Selection of control groups and time points to collect samples should be optimal.
  • The design strategies should minimize variability and maximize information.

To understand design, developments, and statistical validation of in vivo bioassay in more details, reach out to us at https://www.veedacr.com . One can also read the guidelines mentioned by NIH by visiting the link:

https://www.ncbi.nlm.nih.gov/books/NBK92013/pdf/Bookshelf_NBK92013.pdf

References

  1. A. Little, “Essentials in Bioassay Development,” BioPharm International 32 (11) 2019
  2. Padmalayam, Ph.D., Assay development in drug discovery
  3. Zwierzyna M, Overington JP (2017) Classification and analysis of a large collection of in vivo bioassay descriptions. PLoS Comput Biol13(7): e1005641. https://doi.org/10.1371/journal.pcbi.1005641
  4. White JR, Abodeely M, Ahmed S, Debauve G, Johnson E, Meyer DM, Mozier NM, Naumer M, Pepe A, Qahwash I, Rocnik E, Smith JG, Stokes ES, Talbot JJ, Wong PY. Best practices in bioassay development to support registration of biopharmaceuticals. Biotechniques. 2019 Sep;67(3):126-137. doi: 10.2144/btn-2019-0031. Epub 2019 Aug 5. PMID: 31379198.
  5. F Chana and Hursh D, Bioassays through the Product lifecycle: Perspectives of CDER and CBER reviews.
  6. Haas J, Manro J, Shannon H, et al. In Vivo Assay Guidelines. 2012 May 1 [Updated 2012 Oct 1]. In: Markossian S, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Bookshelf URL: https://www.ncbi.nlm.nih.gov/books/

Introduction

Cancer is a deadly disease leading to the death of many individuals across the globe. Biotech and Pharmaceutical researchers are carrying out extensive studies to develop drugs to treat cancer. However, the current medications used in cancer treatment have many loopholes. They are toxic, lack specificity, and have short half-lives. The difficulty in administering complex oncology molecules, along with the above hurdles, has led to side effects, noncompliance, and patient inconvenience of many current treatments for cancer. Liposomes are Nano-sized drug delivery systems that have shown to be quite effective in improving the selectivity of cancer chemotherapeutic agents.

However, clinical trial experts face many challenges when designing a Bioequivalence (BE) study for generic oncology drugs. It includes selecting the study population, selecting the individual dose for patients, selecting the required study design (cross-over vs. steady-state design), processing samples at investigator sites due to sampling uncertainty, high patient dropout rates, and stringent regulatory guidelines. A bioequivalence study is generally conducted in healthy volunteers if the drug has shown a safety profile in a healthy population and is not a narrow therapeutic index drug. However, the same is not ethically and medically acceptable in most anticancer drugs because of cytotoxicity in a healthy population. Moreover, the regulatory requirements also vary from region to region.

How to design a study for a generic oncology product on liposome injection involving cancer patients?

Study overview

Veeda Clinical research completed an open-label, randomized, two-treatment, two-period, two-sequence, single-dose, multicentric, fasting, cross-over bioequivalence study of Doxorubicin Hydrochloride Liposome Injection 2 mg/mL in ovarian cancer patients for an Indian based Sponsor Company towards submission to USFDA. The study was completed within the stipulated timeframe with meticulous project management. In both periods, the subjects received a 50mg/m2 Single dose (intravenous infusion) of  Doxorubicin Hydrochloride Liposome Injection 2 mg/mL (either test or reference product) , according to the randomization schedule created before the trial, on the first day of the chemotherapy cycle. The wash out period was at least 28 days between each consecutive dosing period. Each cycle began with the collection of serial blood samples, a total of 25 blood samples were collected with the last blood sample collected at 360.00 hours in each period. Blood samples starting from 72.00 hours till 360.00 hours were collected on an ambulatory basis in each period to determine free and liposomal encapsulated doxorubicin plasma concentrations for PK analysis.

Subjects inclusion and exclusion criteria

The study involved female patients between the age of 18-65 years who had ovarian cancer (confirmed through cytological and histopathological tests) and who were already receiving or scheduled to start therapy with the reference listed drug (RLD) or the reference standard product. The four major inclusion criteria in this study were:

  • Subjects with Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2
  • Subject with Left Ventricular Ejection Fraction ≥ 50%
  • Subjects with a life expectancy of at least three months determined by checking liver, kidney, and bone marrow function.
  • Subjects who had recovered from minor (at least one week) and major (at least four weeks) surgery.

Women who were pregnant, or lactating, or planning for a family were excluded from the study. A total of 18 parameters were judged under exclusion criteria. Some of the major exclusion criteria were:

  • Impaired cardiac function with the occurrence of unstable angina/arrhythmia/ myocardial infarction/ Qtc prolongation/ coronary artery bypass graft surgery/ heart failure/ symptomatic peripheral vascular disease within the last six months
  • Known history of brain metastasis.
  • Pre-existing motor or sensory neurotoxicity of a severity ≥ grade 2 according to NCI criteria
  • Positive test result for hepatitis and HIV

Reporting and handling of adverse events

The investigators reported six serious adverse events (SAE) during the entire study. Fever with diarrhea was reported in two subjects. Another two subjects were observed with Nausea, Vomiting, and weakness. Fever due to hospitalization and Non-neutropenic fever with acute gastroenteritis were also found in one subject each. At the time of writing this article, all SAEs have been resolved after constant follow-up with the patient.

During the study, hypersensitivity reaction due to Doxorubicin Hydrochloride Liposome Injection was avoided by administering Prophylactic Antiemetic and Dexamethasone Injection 8mg.

Conclusion

The study was successful as the test product showed bioequivalence with the reference product. The pharmacokinetic parameters like Cmax, AUC0-t, and AUC0-∞ were within the range of 80.00 to 125.00%. Veeda Clinical research provided end to end services in identifying and selecting the clinical trial sites, prepare and submit regulatory documents like protocol, ICF, CRF, and Clinical Study Report to drug regulatory authority on behalf of the sponsor company. Trained and experienced nurses and investigators regularly monitored the oncology patients who participated in the study. The study was completed successfully with less patient dropouts, abiding by the principles of Good Clinical Practice. Finally, the product was approved by USFDA. Experienced personnel including Principal and Clinical investigators team at different sites, the project management team, CRAs, phlebotomists, nurses, medical writing team, and the bioanalytical team of Veeda Clinical Research is responsible for successfully completing this clinical trial.

Hi, I am Mansi Shah, a clinical research nurse with over 9 years of experience. I started my practice in 2013 at Sterling Hospital after completing my GNM nursing course. I have been working with Veeda since 2015. As a clinical research associate, my day mainly constitutes of assisting in research activities and ensuring volunteer safety, protection, and that volunteers are well supported throughout the research study

Even though every research is unique and every day is varied, I’m a seasoned Senior Research Associate and my duty is to be right alongside the research participants in the journey, from the day of admission to their dosing and till the time they get discharged. However, the most important task of a clinical research nurse involves determining consent of suitable volunteers, who consent to proceed with the study. I have to ensure that the volunteer understands what the research seeks to accomplish and the protocols associated with it. After educating the volunteers, I have to check her/his eligibility through OVIS and double-check it through routine screenings like blood and urine tests.

A usual day starts with a doctor assigning me my duty as per the study slots. I go to my designated location and mind the volunteers and check their vitals. I hope and work to minimize the risk of adverse events during the research, but the risk is always there. Identifying adverse events at the earliest possible time requires disciplined training and an in-depth understanding, thereby minimizing risk to research participants. With the nature of conducting novel research, the risk of adverse events is always there and the way we counteract it by having ICU wards with doctors and nurses on standby so that we can treat any complication with haste. Volunteer Safety is of the utmost importance to me and to Veeda and we take all the necessary measures, in terms of personnel skills and our infrastructure, to ensure the same. 

Besides taking care of research participants, documenting and recording information during clinical trials is the most important responsibility that a research nurse has. And we at Veeda ensure the Quality and reproducibility of data by taking meticulous approach and following highest level of integrity.

I am extremely passionate about my job as, I feel, I am a part of something that is larger than us & larger than my role. I wanted to be a part of it, as I get to be a part of the research that aims to test an experimental practice onto willing volunteers and see it becoming a part of standard practice, therefore, saving many lives to come in the future. 

A day in the life of a CRA

Hi, I am Gangichatti Laxman Kumar & I work as Clinical Research Associate with Veeda and this is how a day in my life looks like

Although I’m based out of Hyderabad, I might be visiting a site that’s in a completely different part of the country by the time you’ll read it. This blog is supposed to walk you through a typical day in the life of a CRA.

A Clinical Research Associate plays a crucial role within the pharmaceutical businesses. A CRA is responsible for pre study qualification visits, reviewing the study progress, checking the quality & accuracy of data collection, compliance of patients to trial visits, and will ensure good clinical practices are maintained throughout the trial

After successfully completing Pharma-D, I started working as a Safety Associate to the regulatory bodies, after that I switched to clinical research operations and started working as a CRA in Oncology, NeurologyEndocrinology, Cardiology and General Medicine. I have also worked in the department of BA/BE trials where I experienced a multi-functioning team and finally moved to Veeda Clinical Research where I got the opportunity to work in BA/BE studies as well as Late Phase Trials in the field of Oncology.

Being a CRA, I have to spend a significant amount of time traveling to and fro to all the research sites that I have been assigned with which are spread throughout the country, I visit 4 to 5 sites in a day.The very fact that I have to be constantly on the move, which happens to be a part of my job, adds a travel aspect into the mix that always remains fresh.

I believe Social interaction plays an important role in learning and with this role I get to interact with a lot of people,from site coordinators to doctors to project managers, which has proven to be quite effective in my cognitive learning.

My standard operating day comprises of monitoring and supervising data files as a part of source data verification process to ensure that the site is entering data accurately and in a timely manner.The safety of a patient is of the utmost importance at Veeda and I, along with my staff, regularly assess patient notes to ensure safe undertaking of procedures as per the protocol.

Every role comes with its own set of challenges and the role of a CRA is no different. Veeda offers workplace flexibility, which helps me deal with challenges calmly & efficiently. Being a CRA, I practice a fast-paced lifestyle but for me, the sense of accomplishment I get from tackling all those challenges is what makes me choose this line of profession every time.

INTRODUCTION

In our last blog on Master protocols, we discussed the definition of master protocol, the types and advantages of using Master Protocol in clinical trials. In today’s article, we will like to present before you the parameters that are kept in consideration while designing a master protocol for oncology drugs and biologics. During the preparation of master protocols, different parameters are kept in consideration like:

  • Specific Design Considerations
  • Biomarker Development Considerations
  • Statistical Considerations
  • Safety Considerations
  • Regulatory Considerations

SPECIFIC DESIGN CONSIDERATIONS IN MASTER PROTOCOLS

  1. Use of a single common control arm

FDA recommends the use of a single control arm with the current System Organ Class (SOC) while developing a master protocol where multiple drugs are assessed in a single disease.SOC for the target population can be changed during the conduct of the trial if there is a new drug approval or scientific evidence that makes it unethical to randomize patients based on the previous SOC. During such a situation, FDA recommends the sponsor to suspend patient enrollment until the protocol, the SAP, and the protocol informed consent document are modified to include the new SOC as control.

  1. Novel combination of two or more investigational drug

While writing a master protocol, where two or more investigational drugs are involved as a combinational product, the sponsor should summarize the following.

  • Safety of the combinational product
  • Pharmacology of the combinational product
  • Preliminary efficacy data for each investigational drug
  • Rationale for the use of the drugs as a combination product
  • Evidence of any synergistic effect (if any) of the two or more investigational drugs when given in combination.

The FDA strongly recommends that the sponsor ensure that the Recommended Phase II Dose (RP2D) for each drug having antitumor activity should be identified in all cases.

  1. Studies with drugs targeting multiple Biomarkers

Early discussion of biomarker research strategies is highly encouraged by the FDA when the sponsor plans to use one or more biomarkers to guide patient preference for trials. A defined plan for the allocation of eligible patients should be present. Patient selection studies must be analytically checked with well-defined parameters for master protocols involving drugs that target multiple biomarkers.

  1. Adding and stopping treatment arms

Before beginning the trial, the sponsor should make sure that the master protocol and its corresponding SAP identify conditions that would contribute to adaptations, such as introducing a new experimental arm or arms to the study, re-estimating the sample size based on the interim analysis results, or discontinuing the experimental arm on the rules of futility.

  1. Independent Data Monitoring Committee (IDMC)

The master protocol should provide the details of the IDMC that is involved in monitoring the efficacy results and the details of Independent Safety Assessment Committee (ISAC) that is involved in monitoring the safety results. However, the IDMC can perform both the functions of safety and efficacy. For marketing an oncology drug, if the basis of marketing application involves one or more sub studies, FDA recommends the inclusion of independent radiologic review committee to perform blinded tumor-based assessments.

BIOMARKER DEVELOPMENT CONSIDERATIONS

            Master protocols assessing biomarker-defined populations should explain the rationale behind the use of that particular biomarker.The sponsor should employ in vitro diagnostic (IVD) tests that are analytically validated, establish procedures for sample acquisition, handling, and the testing and analysis plans as early as possible. The sponsor may need to submit the IVD’s analytical validation data for FDA(CDRH or CBER) to determine whether the clinical results will be interpretable.

STATISTICAL CONSIDERATIONS

If a sponsor introduces randomization into the design of an umbrella trial, the FDA advises that a standard control arm to be used where possible.Bayesian statistical method or other methods for dropping an arm, modifying sample size, or implementing other adaptive strategies can be used in preparation of master protocols. The SAP should include details on implementation of Bayesian or other methods as described in the FDA guidance for industry Adaptive Design Clinical Trials for Drugs and Biologics and the guidance on Enrichment Strategies for Clinical Trials to Support Approval of Human Drugs and Biological Products.Statistical considerations for master protocols can be strategized in four different ways:

  1. Nonrandomized, Activity-Estimating Design
  2. Randomized Designs
  3. Master Protocols Employing Adaptive/Bayesian Design
  4. Master Protocols With Biomarker-Defined Subgroups

SAFETY CONSIDERATIONS

The sponsor should implement a structured team of ISAC or an IDMC to assess the safety as well as the efficacy of all master protocols.The constitution of this committee and the definition of its responsibilities should be well defined in the IND. A sponsor should not begin a clinical trial until the master protocol has been reviewed and approved by an IRB or IEC. The FDA encourages the use of a central IRB to promote the IRB analysis of master protocols. The sponsor is required to perform a safety review of master protocols more regularly than on an annual basis and supply the investigator with the details.If the master protocol contains proposals to include pediatric patients in the study, the FDA advises that the IRB include a pediatric oncology expert in its team who has expertise with the regulatory criteria for the enrollment of pediatric patients in clinical investigations, including parental approval and consent. The informed consent document should be submitted to the IRB for review.

ADDITIONAL REGULATORY CONSIDERATIONS

Each master protocol should be submitted as a new IND to the FDA. This is done to avoid miscommunication owing to the sophistication of master protocols that may hamper patient safety.If the sponsor is conducting a clinical trial on more than one indication for oncology drugs or biologics, the IND should then be forwarded to the Office of Hematology and Oncology Products at CDER or CBER for approval.

REFERENCE

Master Protocols: Efficient Clinical Trial Design Strategies to Expedite Development of Oncology Drugs and Biologics, Guidance for Industry, Draft Guidance.U.S. Department of Health and Human Services, Food and Drug Administration, September 2018.