WEBINAR: Preclinical Considerations in Cell and Gene Therapy Product Development

Presented by Absorption System’s Chief Business Officer, Vatsala Naageshwaran

About this webinar:

In vitro and in vivo preclinical evaluation of cell and gene therapy (CGT) products contributes significantly to characterization of the product benefit versus risk profile. The field is currently expanding rapidly, with many candidate therapies advancing into clinical testing and many more progressing through preclinical pipelines. As the number of early-stage candidate products continues to increase, so does the need to define effective preclinical development strategies that support clinical translation. Given the diverse and often multi-functional nature of these therapies, it is not possible to define a one-size-fits-all preclinical program for cell and gene therapy. Instead, preclinical studies need to be customized to support the feasibility of the proposed administration route and appropriate application procedure, address the candidate product’s specific therapeutic properties, and fully characterize any potential safety concerns.

Learning Objectives

  • Discover the diversity of cell and gene therapy products
  • Describe the preclinical testing approaches to guide product development
  • List the regulatory requirements to support an Investigational New Drug (IND) submission for a first-in-human (FIH) clinical trial and a Biologics License Application (BLA).

WEBINAR: The ABCs and 123s of the BCS

The Biopharmaceutics Classification System, or BCS, is a framework for classification of drugs according to two fundamental properties of the drug substance, i.e., permeability and solubility.

The BCS class of a drug substance has important implications for formulation development, and a BCS-based biowaiver can eliminate the need for clinical bioequivalence studies, which would otherwise be required for changes in formulation, manufacturing site, or manufacturing method.

High permeability can be demonstrated either in vivo or in vitro; the latter can be faster and cheaper, especially for drugs subject to first-pass metabolism or with high inter-subject variability. This webinar will cover the basics of the BCS as well as nuances that are not included in the FDA’s BCS guidance, learned from interactions with the FDA in support of dozens of biowaiver applications.

In this webinar you’ll learn:

  • Why it’s important to know the BCS class of a drug
  • The significance of the BCS for both generic drugs and NCEs
  • The importance of an accurate high-permeability internal standard for comparison with a test drug for in vitro permeability classification

Who should attend:

  • Formulation scientists
  • Pharmacologists
  • Pharmacokinetics experts
  • Pharmaceutical product managers

Determining the influence of particle size of drug compounds on dissolution and absorption using the IDAS system

The pharmaceutical industry is feeling the acute effects of disruption caused by the COVID-19 pandemic. With most human trials on hold, drug developers and generics companies will be looking for new ways to progress product portfolios. This series of blog posts aims to highlight BCS and BCS-based biowaiver applications as such an opportunity.

In our previous blogs, we highlighted BCS and BCS-based biowaivers. Furthermore, we discussed experimental approaches to guide drug development of complex compounds. In this blog series, we detail on drug-specific characteristics and conditions that can be studied using the IDAS system.

One of the factors influencing the dissolution of a finished drug product, and the subsequent permeation of the API (active pharmaceutical ingredient), is the particle size of the formulation. For drug development, it is essential to investigate various sizes to determine the best possible formulation for a new drug. Particle size is especially crucial for poorly soluble drugs, which have slow and incomplete dissolution in gastrointestinal fluids.

The nanonization of compounds may help increase the bioavailability of a poorly soluble compound. The smaller size results in larger total surface area of the particles, increasing the amount available to permeate. However, testing various particle sizes to identify the optimal formulation can be costly and time-consuming if it is performed in healthy human subjects.

The IDAS system for assessment of the effects of particle size on dissolution and permeation

Our in vitro system, IDAS (In Vitro Dissolution Absorption System), allows for a quick and cost-effective assessment of how particle size affects the dissolution and permeation of a drug. IDAS consists of a dissolution chamber and a permeation chamber, which are separated by a polarized monolayer of human Caco-2 cells. The Caco-2 cell line is a colon carcinoma cell line and serves as a biorelevant barrier, permitting the simultaneous assessment of drug dissolution and permeation through this cell layer. Various buffers can be used in the dissolution chamber to mimic gastrointestinal conditions in the body.

Different formulations can be tested simultaneously, allowing for fast and accurate assessment with a better correlation than standard dissolution tests between the in vitro drug product release characteristics and in vivo performance. The system permits testing of drug products from all BCS classes, including poorly soluble compounds.

Example: Effect of particle size of oral indomethacin formulations

In this example, we assessed the effect of particle size of oral indomethacin formulations on dissolution and permeation. We dosed indomethacin into the IDAS dissolution chamber as nano-sized and micro-sized formulations at equal API levels.

We measured the dissolution and permeation by liquid chromatography–mass spectrometry (LC-MS/MS) to determine the dissolution constant (kd) and the permeation constant (kp) simultaneously, by modeling the concentration-time profiles using a Nelder-Mead simplex algorithm. The dissolution chamber contained Hanks’ balanced salt solution (HBSS) supplemented with 15 mM glucose (HBSSg) at pH 5.75, and the permeation chamber contained HBSSg supplemented with 4.5% BSA at pH 7.4, mimicking blood plasma.

Figure 1: In vitro measurements of indomethacin dissolution and permeation from micro-sized and nano-sized (submicron) formulations using IDAS.

Figure 2: Simultaneous modeling of indomethacin dissolution and permeation profiles.

We found that nano-sized formulation increased the kd by more than 300%, the kp by 30%, and the maximum dissolution by 17% compared to the micro-sized formulation (Figures 1 and 2).

What’s more, in vivo, orally administered nano-sizing increased the plasma Cmax of indomethacin (an index of the rate of absorption) by 25% without changing the total exposure (area under the curve, AUC). These data show that IDAS enables accurate in vitro-in vivo correlations.

Physiologically relevant modeling

As shown in the example, IDAS allows the assessment of the impact of particle size on the dissolution and permeation of drugs. Given that various formulations can be tested simultaneously while obtaining high-quality data that are translatable to the in vivo situation, the system is an efficient and accurate tool for formulation development.

Here to help

Connect with us to discuss your formulation development needs and to see how we can help design experiments that help save you time and money. Determining the effect of particle size is just one of the many uses of IDAS. We previously discussed some other applications of IDAS in previous blogs, with an emphasis on testing the phenomenon of supersaturation, and the effects of increased viscosity due to food intake, on dissolution and permeation.

Potency Assays for Cell and Gene Therapy

Approved biological products are required to be accompanied by analytical tests to demonstrate safety, purity, and potency. Manufacturing and testing processes for approved products are validated and performed under current Good Manufacturing Processes (cGMP).

Potency is defined as “the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result.”1

In vivo models used during proof-of-concept and efficacy studies provide an early readout of potency by measuring a desired physiological response in animals. For product approval, in vitro assays must be developed providing a quantifiable readout that can be validated. The in vitro approach of introducing a gene into a cell line and then demonstrating its expression and functional activity has been used for over a decade in the evaluation of small molecule drug-drug interaction properties. When a small molecule drug is developed, the interaction potential of the molecule as a substrate, inhibitor, or inducer of specific drug transporters is evaluated. The U.S. Food and Drug Administration (FDA) issued the first drug transporter concept paper in 2004, and it issued a draft guidance in 2006. This draft guidance was most recently updated in 2017.2

PODCAST: Potency Assays for CGT Products

In this podcast, we talk to Vibhor Gupta, Associate Director, Business Operations (Cell & Gene Therapy), about the importance of robust, reliable, and accurate potency assays for cell and gene therapy products. Dr. Gupta discusses the significance of regulatory requirements and some of the major roadblocks encountered along the development route.

Topics covered in this podcast:

  • What is a potency assay in terms of cell and gene therapy products and why is it critical?
  • How early should you start looking into working on potency assay development?
  • What are the major roadblocks with potency assay development, qualifications and validation?
  • What are the regulatory requirements for a potency assay for different phases of a clinical study?
  • What services do Absorption Systems offer that can help with the development of potency assays?

Listen Here!

INFOGRAPHIC: Gene Therapy R&D Roadmap – Tools and Techniques

This infographic explores the tools and techniques used during the AAV gene therapy development pathway, detailing the necessary steps during the process; from rAAV generation and in vitro and in vivo testing through to assessing the host immune response.

Transporters In Drug Development Based On Recent ITC Recommendations


Transporters are membrane proteins highly expressed in various organs of humans. They play a pivotal role in the absorption and disposition of both endogenous and exogenous molecules. In the last decade, a lot of focused research has been conducted to understand potential clinical drug-drug interactions mediated by transporters. Regulatory agencies (including the US FDA and EMA) made mandatory the assessment of new drugs as substrates and inhibitors of drug transporters. Recently, the International Transporter Consortium (ITC) updated their recommendations through a series of white papers based on the latest research. This article discusses the importance of emerging transporters of clinical relevance.

Transporters of Clinical Relevance: The updated recommendations of the ITC for transporter screening by organ, considering drug-drug interaction potential, liver toxicity, drug-induced vitamin deficiency, and disposition of biomarkers, are presented below.

OCT1 is responsible for the hepatic uptake of metformin and its subsequent pharmacological effect.  Therefore, inhibition of OCT1 can greatly impact metformin pharmacodynamics (but not its pharmacokinetics, since it is eliminated almost exclusively by the kidney). Based on clinical evidence, evaluation of substrate and inhibition potential for OCT1 is suggested during drug development as per the decision trees shown below.


OATP2B1 has low- and high-affinity binding sites and is expressed in the intestine and liver. Notable clinical substrates of OATP2B1 are atorvastatin and rosuvastatin. OATP2B1 shows pH-dependent transport. Hence, in addition to screening at pH 7.4, it is also recommended to screen substrates at pH 5.5.

OAT2 is expressed in the liver and kidney. Recent studies have shown that OAT2 is responsible for uptake of creatinine (a renal function biomarker) into the renal proximal tubule. As there is no selective inhibitor of OAT2 in vivo, it is difficult to clearly establish the role of OAT2 in creatinine disposition.

Thiamine Transporters (THTR) 1 and 2 are responsible for uptake and distribution of thiamine (vitamin B1) and are expressed in the intestine, blood-brain barrier, and kidney proximal tubule. THTRs interact with drugs like metformin, trimethoprim, and fedratinib. Inhibition of THTR2 has a significant impact on thiamine intestinal absorption and renal reabsorption, resulting in thiamine deficiency. Many reported cases of drug-induced thiamine deficiency, which ultimately caused Wernicke’s encephalopathy, were due to inhibition of the thiamine disposition pathway.

Hepatic bile acid transporters (BSEP, NTCP, MRP2, MRP3, MRP4, & OST α/β) contribute to the disposition of bile acids, and drug interactions involving these transporters may cause cholestasis and liver injury. Hepatic bile acid levels are regulated through FXR-SHP and FXR-FGF19 pathways, which control multiple adaptive mechanisms for bile acid detoxification.  Another transporter, ASBT, is responsible for reabsorption of bile acids from the intestine and its inhibition is not involved in cholestasis.


In addition to the transporters (P-gp, BCRP, OAT1, OAT3, OCT2, MATE1, MATE2-K, OATP1B1, and OATP1B3) recommended by regulatory agencies for screening, the following transporters may also be considered: OCT1 & OATP2B1.

  • Clinical evidence has emerged for screening OCT1 for substrates and inhibitors
  • Uptake by OATP2B1 may be evaluated for unexplained mechanisms of possible drug-drug interactions
  • Assessing drug interactions with THTR2 in vitro to enable monitoring of thiamine deficiency in susceptible populations
  • OAT2 as a mechanism of creatinine renal secretion
  • Mechanistic understanding of drug effects on multiple transporters involved in bile acid homeostasis, in addition to BSEP inhibition


  1. Zamek-Gliszczynski MJTaub MEChothe PPChu XGiacomini KMKim RBRay ASStocker SLUnadkat JDWittwer MBXia CYee SWZhang LZhang YInternational Transporter Consortium. Transporters in drug development: 2018 ITC recommendations for transporters of emerging clinical importance. Clin Pharmacol Ther. 2018. 104(5): 890-899
  2. Webinar presented by Dr. Zamek-Gliszczynski, Senior Fellow and Director, DMPK, GlaxoSmithKline
  3. Absorption Systems’ Transporter Reference Guide, 2018 4th Edition, Absorption Systems,
  4. In Vitro Metabolism- and Transporter- Mediated Drug-Drug Interaction Studies Guidance for Industry, USFDA, October 2017, https://www.fda.gov/regulatory-information/search-fda-guidance-documents/vitro-metabolism-and-transporter-mediated-drug-drug-interaction-studies-guidance-industry
  5. Guideline on the investigation of drug interactions, EMA, January 2013, https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-investigation-drug-interactions_en.pdf

Clinical Probes and Endogenous Biomarkers as Substrates for Transporter Drug-Drug Interaction Evaluation

Perspectives From the International Transporter Consortium


Transporter studies are performed at various stages of drug development ‒ discovery to first-in-human (FIH) phase (in-vitro substrate and inhibition studies), FIH to proof of concept (POC) phase (clinical DDIs), and POC to new drug application (NDA)/post-marketing phase (product labeling and post-marketing surveillance). Currently, in-vitro transporter inhibition assays are conducted to determine the risk for potential clinical DDIs.

During clinical DDI studies, an appropriate probe substrate is selected to tease out the effect of NME inhibition of a transporter, to understand the mechanism of the DDI, and enable extrapolation to other drugs, leading to appropriate product labeling. Due to multiple factors, including the limitations of IVIVE, an overlap of substrate/inhibition for enzymes and transporters, multiple drug binding sites on transporters, and the fact that organ-specific changes in drug exposure may not be reflected in systemic PK, this strategy may result in false-positive and false-negative outcomes.

Instead of using a drug as a clinical probe substrate, a validated endogenous biomarker offers the potential for evaluating NMEs as transporter inhibitors in early clinical studies (by reanalyzing already-collected PK samples), without the need for dedicated clinical DDI studies. A list of desirable characteristics for biomarkers for this purpose are summarized in Table 1.

Based on the above characteristics and data generated so far, ITC has compiled a list of suitable probe substrates and biomarkers to study transporter-related clinical DDIs (Table 2).

Biomarkers listed in the above table are additionally transported by MRP2 and MRP3 (CPI, CPIII, CB), OATP2B1 (CPIII), OAT1 (HDA, TDA), OAT3 (GCDCA-S, HAD, TDA) or NTCP (GCDCA-S), thus reducing their selectivity.

As a perpetrator drug may inhibit multiple transporters, a probe drug cocktail approach has been tested in vitro and in clinics. Examples of cocktails successfully tested are shown in Table 3.

Key Takeaways:

  • ITC proposed the following workflow for the identification, characterization, and validation of an endogenous biomarker

  • ITC proposed the following decision tree for incorporating endogenous biomarkers and probe drugs to assess transporter-related inhibition during drug development


  1. Chu XLiao MShen HYoshida KZur AAArya VGaletin AGiacomini KMHanna IKusuhara HLai YRodrigues DSugiyama YZamek-Gliszczynski MJZhang LInternational Transporter Consortium. Clinical Probes and Endogenous Biomarkers as Substrates for Transporter Drug-Drug Interaction Evaluation: Perspectives From the International Transporter Consortium. Clin Pharmacol Ther.2018, 104(5):836-864
  2. Müller FSharma AKönig JFromm MF. Biomarkers for in vivo assessment of transporter function. Pharmacol Rev. 2018, 70(2): 246-277
  3. Webinar presented by Dr. Zamek-Gliszczynski, Senior Fellow and Director, DMPK, GlaxoSmithKline https://www.absorption.com/kc/webinar-state-of-the-art-in-clinical-transporter-ddi-evaluation/
  4. Absorption Systems’ Transporter Reference Guide, 2018, 4th Edition, Absorption Systems https://www.absorption.com/kc/transporter-reference-guide-4th-edition-download/

E-BOOK: Preclinical Toxicology Reference Guide, 1st Edition

Toxicity is categorized as acute or chronic depending on the exposure of a molecule to biological tissues.

The stepwise preclinical toxicity evaluation in drug development has the primary goal to characterize the potential dose-related adverse effects at the level of tissue, organs, and organ system. Preclinical toxicity and safety evaluation can be helpful to determine a safe starting dose of a pharmaceutical
for human testing.

This guide provides an overview of preclinical and non-clinical safety and toxicology studies necessary to be conducted for a successful IND submission and advance a drug candidate through the development phase and covers the following concepts:


Request your copy today!

E-BOOK: Transporter Reference Guide, 5th Edition

Included in the Guide:

  • Things to know in 2020 based on the new FDA guidance
  • Metabolism in Safety Testing
  • Drug Transporters in Safety Testing
  • Evaluation of DDI Potential of Metabolites
  • Concentration Selection Cheat Sheet
  • Regulatory Requirements
  • Decision Trees
  • Waiving Clinical Endpoints

A closer look at Absorption Systems’  Innovation Corner:

  • IDAS™: Elucidate the role of transporters and other factors affecting absorption
  • CellPort Technologies®: Validated tracking test system with proven reliability
  • CellPortReady Plates™: Assay-Ready Plates, robust test systems for transporter evaluations in your own lab!