Science Pool

Patients around the world have limited or restricted access to biopharmaceutical medicines. Reducing production costs while still maintaining high quality standards will help increase the affordability of biologics and ensure more patients benefit from these life-saving medicines.

Traditional biomanufacturing facilities have failed to deliver biopharmaceutical products with sufficiently low Cost of Goods Manufactured (COGM) to allow greater patient access. These facilities have been built with fixed capacity and a focus on large-scale fed-batch manufacturing. Scaling-up processes to large-scale fed-batch manufacturing facilities involves considerable risk, resource, and upfront costs. Such facilities often lack flexibility which limits the products that can be produced within them and can leave valuable production assets idle for periods of time.

Manufacturing costs link directly to capacity utilization and product demand. There is a historical precedent within the biopharmaceutical industry of operating with excess capacity but we must recognise this comes with a financial penalty. We must address this challenge if we are to respond to global healthcare emergencies, changes in the way healthcare systems are managed and greater demand for global access to biotherapeutics.


Continuous Bioprocessing Platforms and Modular Facility Designs

The industry needs new flexible biomanufacturing concepts to quickly react to market fluctuations and achieve a higher predictability of costs. Modern biopharmaceutical productions facilities use building and manufacturing technologies, such as modular construction, to minimize clean room space utilization and reduce footprints. They allow faster speed to market with a lower upfront capital investment and are readily expandable when product demand is better understood. Continuous manufacturing platforms can be integrated into these facilities for low-cost bioprocessing using mammalian cell hosts in perfusion bioreactors linked to continuous downstream trains. Production costs remain low irrespective of facility mass output, the product quality attributes are consistent and manufacturing footprints are minimized.

Just-Evotec Biologics has developed a low-cost manufacturing facility design utilizing modular cleanroom pod technology that we call J.POD®. The J.POD facility design features individual pre-fabricated cleanroom pods arranged in a controlled, non-classified ballroom to minimize the cleanroom footprint of operations that would have previously taken place in a large ballroom. Media and buffer preparations, cell expansion, upstream, downstream and post viral are all housed in separate pods. The design minimizes fixed utility infrastructure and instead relies on single-use continuous upstream and downstream operations.

Biomanufacturing Facility Cost Comparisons

We developed process models for a fed-batch process in a traditional stainless-steel facility, a fed-batch process in a single-use facility and three continuous processes in a J.POD facility. The models were created using the Biosolve software (Biopharm Software Ltd) to show the benefit of the J.POD facility design on the COGM of an antibody biologic. We used Net Present Cost (NPC) to compare scenarios. NPC estimates cash flows by computing operational costs and discounting over time using a capital parameter. It does not include revenues in the accounting of cash flows and assumes capital costs are sunk costs incurred at the beginning of the project.



Figure 1 shows the expected costs from operating the different facility types and assumes their throughput increases at a rate of 250 kg/year up until a peak value. The range selected was representative of market demands for typical biopharmaceutical manufacturing facilities. Jumps in the NPC correspond to points when the capacity of a facility is reaches and new builds are needed.

We can draw the following conclusions from the results.

1. J.POD facilities achieve remarkable production outputs despite their small footprint because of the high productivity of the continuous perfusion process.
2. Fixed CapEx comprises a large proportion of the total costs in a stainless-steel facility. J.POD facilities apply single-use technologies resulting in a shift from fixed to variable costs.
3. The fully continuous J.POD facility gives the lowest costs with the stainless-steel facility returning the highest expenditure over the range.
4. The stainless-steel facility is not being operated at its optimum utilization rate over the production output range modelled that leads to inefficiencies.

Driving Up Access to Biotherapeutic Medicines

We believe that modern biomanufacturing facilities must have smaller processing spaces, higher production throughputs and lower production costs. Modelling shows how our J.POD facilities have the lowest initial build and operating costs as well as the ability to control operating costs. These facilities are outperforming older manufacturing platforms in terms of cost and utilization. They are becoming an essential component of strategies for reducing costs and driving up access to biotherapeutic medicines. 

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Antimicrobial resistance (AMR) is one of the biggest health threats worldwide. Key to countering AMR is the development of novel anti-infective drugs. The limitations of animal models and clinical trial design have emphasised the importance of nonclinical pharmacokinetics/pharmacodynamics (PK/PD) platforms which provide a detailed understanding of the relationship between the fate of the antimicrobial compound in the body (PK) and the impact of exposure to the compound on the target microbes (PD). This allows us to optimise dosing regimens to maximise the efficacy of antimicrobial compounds (microbial killing) while minimising toxicity and the risk of the emergence of AMR.

What is the HFIM?

The HFIM is a system of pumps, tubing and microfibers that mimics the body, allowing in vitro assessment of anti-infective compounds under more relevant conditions. It consists of a central reservoir and tubing used as a circulating system, and a hollow fibre cartridge with thousands of permeable capillaries. The extra capillary space (ECS) outside the fibres within the cartridge contains the target organism. During operation, the drug-infused growth medium in the central reservoir is continuously pumped to the hollow fibre cartridge, rapidly passing through the capillaries into the ECS. This continuous flow ensures that nutrients, oxygen, and test compounds are continuously refreshed while waste products are removed. To simulate drug clearance, fresh medium is added to the central reservoir effectively diluting the drug from the system. Accordingly, this balance of drug supply/clearance can effectively simulate the drug’s PK profile.

Why choose the HFIM as PK/PD model?

It is the most capable in vitro system for PK/PD determination for anti-infective compounds, against bacteria and fungi. It is a dynamic model capable of simulating almost any given concentration-time profile for one or more compounds, even if they have very different half-lives.. The Hollow Fibre Infection Model is not limited by in vivo model availability, compatibility of PK profiles, dosing or sampling frequency, or study duration, which is extremely important for understanding PK/PD relationships and the risk of AMR over clinically relevant treatment times. Various cartridges with fibres manufactured from different materials are available to optimise the HFIM for microbial growth and compound performance.

In conclusion, the HFIM is a versatile in vitro PK/PD platform which can accelerate the development of antibacterial and antifungal compounds, contributing to the fight against AMR. 

If you’d like to learn more about the uses of the Hollow Fibre Infection Model you can download our white paper here

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Rapid turnaround is essential for ADME-Tox services, especially during drug discovery. However, turnaround time for the assay isn’t the only consideration. The time required to prepare the quotation may delay the scheduling of the assay and, in turn, the study start date.

The new Cyprotex e-Store provides a solution to this. Registered users of the e-Store are able to view instant pricing for a comprehensive range of ADME-Tox services. As well as having access to detailed protocols and the latest editions of our educational guides, users also benefit from special offers only available through the e-Store.

Online ordering is simple. Previous orders can be tracked through the e-Store and it is easy to re-order your favourite assays. All of this information can be accessed outside business hours. A range of payment options are available on the e-Store including credit card, existing purchase order or invoice. These features allow services to be ordered and scheduled the same day so saving you time.

Despite this automated approach, you are still supported by a dedicated scientific study manager and account manager who are on-hand to guide you at all stages of your study.

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The industry gold-standard in vitro cell based test system for studying Breast Cancer Resistance Protein (BCRP) or P-glycoprotein (P-gp) transporters is the polarized cell monolayer system using either Caco-2 cells or MDCK-MDR1 cells (Elsby et al., 2022), grown on semi permeable membrane inserts to form a brush border membrane barrier separating two experimental compartments (apical and basolateral) of equivalent pH (7.4). Apparent permeability (P_app, cm/s x10^-6) of the test article is determined in the apical to basolateral (A-B) and the basolateral to apical (B-A) direction and from this an efflux ratio is calculated (B-A P_app/A-B P_app). An efflux ratio greater than 2 which is reduced by greater than 50% with a corresponding reduction in B-A P_app in the presence of a reference inhibitor (Atkinson et al., 2022) indicates the test article is a substrate of the efflux transporter being investigated. The bidirectional P_app values and subsequent efflux ratios of the known P-gp substrate quinidine, determined in polarized MDCK-MDR1 cell monolayers across a concentration range is summarized in Table 1 (left) and demonstrates that in the MDCK-MDR1 polarized monolayer test system quinidine has been determined to be a substrate of the P-gp transporter.

Table 1: Mean P_app values and corresponding efflux ratios of quinidine (left) and N-methyl quinidine (right) across MDCK-MDR1 monolayer cells in the presence and absence of reference inhibitor cyclosporin A

Despite polarized monolayers being the industry gold standard test system for studying P-gp and BCRP interactions, there are limitations and risk of inconclusive results and false negatives if the test article being studied exhibits low P_app indicating inherently low passive membrane permeability. This is due to the physico-chemical characteristics of the test article limiting cellular entry required to access the efflux transporter on the apical membrane. This is true for the quinidine oxidative metabolite, N-methyl quinidine (NMQ). The presence of the methyl group results in lower lipophilicity and therefore passive permeability without diminishing its ability to bind P-gp. As summarized in Table 1 (right) bidirectional P_app values for the more polar NMQ are very low and although efflux ratios determined are around two, a 50% reduction in efflux ratio with corresponding decrease in B-A P_app is not observed in the presence of inhibitor therefore the result is inconclusive at best, or at worst could be interpreted as not a substrate of the P-gp transporter.

Where assessment of a test article in polarized monolayer cells indicates a compound has inherently low passive permeability, membrane vesicles have the membrane orientated “inside out”, this results in the intracellular binding site of the transporter being positioned on the outside of the vesicle (outwardly facing), therefore compounds do not need to cross a membrane in order to interact with the transporter. As such for efflux transporters such as P-gp or BCRP, substrates will be actively taken up into the vesicle and, due to poor permeability, will remain inside the vesicle for quantification. As demonstrated in Table 2, using P-gp membrane vesicles as an in vitro test system for P-gp substrate identification, NMQ is clearly demonstrated to be, and subsequently correctly identified as, a substrate of the P-gp transporter.

Table 2: Mean uptake rate and corresponding uptake ratios of N-methyl quinidine using P-gp expressing membrane vesicles in the presence of ATP or AMP in the absence and presence of the reference inhibitor cyclosporin A. Figure: Mean uptake rate of N-methyl quinidine into P-gp expressing membrane vesicles in the presence of ATP or AMP in the absence and presence of the reference inhibitor (I) cyclosporin A

Furthermore, if data from P-gp or BCRP substrate identification studies in polarized cell monolayers indicate a compound has inherently low passive permeability then any P-gp or BCRP inhibition data generated in the same in vitro test system should also be scrutinized and a follow up inhibition study in membrane vesicles considered, particularly if no inhibition was observed in the cells.

Whilst membrane vesicles have their advantages over polarized cell monolayers as a test system to identify P-gp or BCRP substrates that are poorly permeable, it is not advisable to utilize membrane vesicles as a first-choice test system. This is because for lipophilic test articles such as quinidine, sufficient passive permeability allows the substrate to move freely across the membrane and would not be trapped within the vesicle lumen, therefore there is a risk of inconclusive results and false negatives.

In summary, polarized cell monolayers are the gold standard for studying P-gp and BCRP interactions as indicated in regulatory guidances and as such should be the first choice of test system for identifying P-gp or BCRP substrates and inhibitors. However, if results indicate the compound has inherently low passive permeability then P-gp or BCRP expressing membrane vesicles should be considered as an alternative follow up in vitro test system to mitigate against the clinical implications of false negatives.

References:

Atkinson, H., Mahon-Smith, K. and Elsby, R. (2022) ‘Drug permeability and transporter assessment: Polarized Cell Lines’, The ADME Encyclopedia, pp. 401–412. doi:10.1007/978-3-030-84860-6_142.

Elsby, R. et al. (2022) Studying the right transporter at the right time: An in vitro strategy for assessing drug-drug interaction risk during drug discovery and development. Expert Opin Drug Metab Toxicol 18(10):. 619–655. doi:10.1080/17425255.2022.2132932.

Can we age healthier?

Due to better hygiene and medicines, the aging population has been growing steadily over the last 30 years. By the year 2031 1.4 billion people will be aged 60 or over, comprising one in six of the worlds population.. This figure will reach 2.1 billion by 2050, with 426 million being aged 80 or more. Unsurprisingly, the UN declared 2021 -2030 the Decade of Healthy Aging. The aging global population brings considerable societal challenges. In developed nations, old age increases the financial pressure on healthcare systems because healthcare spending rises sharply with age. This is in part due to the increased use of medications with advanced years, but also the associated support and care costs.

Increasing healthspan

However, the underlying problem is not life span but healthspan. Being of advanced age does not necessarily mean being frail, sick and in need of care. Already today, there are many healthy seniors living an active life. To promote this notion, the WHO set the goal to provide every person in every country in the world the opportunity to live a long and healthy life. This is encapsulated perfectly with the definition of healthy aging being ‘the process of developing and maintaining the functional ability that enables well-being in older age’. Functional ability means having the capabilities to be and to do what people have reason to value, i.e. meeting not only basic needs but also allowing them to learn, grow, and make decisions, to be mobile, to build and maintain relationships, and to contribute to society. This is very different from life extension calculated as accumulation of human years.

What is biological aging?

Aging can be defined as a time-dependent decline in body function and is observed in virtually all living organisms. The accumulation of cellular damage due to dysfunction in multiple biochemical systems increases the susceptibility to disease and ultimately results in death. This is most likely a result of evolution, once an organism has reached sexual maturity to enable reproduction and raising offspring, it makes no biological sense to invest more energy in maintaining the organism. However, the case is more complex for long-lived mammals, with offspring that need to be protected for a protracted time to enable them to reach sexual maturity. Hence mammals have evolved sophisticated and very efficient repair and maintenance mechanisms in order to correct any cellular dysfunction. With this perspective in mind, aging can be defined as the gradual deterioration of this biological maintenance. We can then view improving healthspan through the lens of slowing this decline or improving restorative or regenerative capacity within tissues and organs.

Already, science has identified a number of factors that can contribute or accelerate the aging process and these include genetic predisposition, obesity, smoking and the status of the gut microbiome. Downstream of these drivers is frequently low-level chronic inflammation – ‘inflammaging’ that contributes to the gradual deterioration in cellular and tissue function. The immune system itself is also subject to decline over time, meaning that many of the protective and repair mechanisms of the adaptive and innate immune system become less effective over time. A combination of these factors are thought to contribute to the common diseases associated with advanced age, such as cancer, cardiovascular disease, chronic liver and kidney diseases, type-2 diabetes and dementia. We are all familiar with how these diseases lead to a reduced life expectancy, but also significant impairment in the individual’s quality of life.

At the cellular and molecular level, there are key biochemical mechanisms that promote gradual deterioration of function across all cell types, that we believe underpins loss of physiological performance - consistent with the process of aging. These are well recognized as the classical ‘hallmarks of aging’. These include the emergence of cellular senescence and the senescence-associated secretory phenotype- which drives much of the chronic inflammation in tissues. Telomere shortening, genomic instability and the accumulation of genetic damage which can lead to tumour formation, epigenetic changes, exhaustion of stem cells, altered cell-cell communication, loss of control of proteostasis, aberrant nutrient sensing and mitochondrial dysfunction. In addition there are more generalised drivers of the aging process such as those linked to dysbiosis.

There is therefore no single driver of biochemical and physiological aging, but the concerted influence of an array of many contribuing factors at play. These complex systems can seem daunting to address, but within each of these pathways there are potential points for pharmacological intervention that constitute targets for drug discovery programs. The goal of pharmacotherapy can therefore be viewed as intervening in key node points to slow the accumulation of dysfunctional processes and maintain cellular integrity for longer. Targeting these fundamental pathological mechanisms will be key to providing a systems-wide (i.e. holistic) benefit to the individual.

How to prolong health span?

It is known that medical interventions, good health practice, refraining from smoking, eating appropriately, and exercising, can all help protect our health. But as mentioned above, there are opportunities to improve healthspan, through targeting key points within the pathways that are recognized as the classical hallmarks of aging.

Developing novel senolytics - challenges and opportunities

One of the hallmarks of aging that has been very well characterized is cellular senescence. This occurs when cells reach the end of their ability to divide resulting in stasis, a state associated with the ‘senescence associated secretory phenotype’ (SASP). Under these conditions, the senescent cells release a cocktail of proinflammatory mediators which in the young targets them for removal by the immune system. However, in the aged, where the immune system itself is subject to gradual decline, senescent cells continue to release the cytokines, chemokines, growth factors and other bioactive components which contribute to inflammaging. As such, the use of senolytic agents which are aimed at killing off senescent cells by suppressing the pathways that keep them alive, is receiving significant attention in the aging field. According to a recent report in Nature Medicine, around 20 clinical trials of senolytic compounds are ongoing. Clearly, blocking the ‘keep me alive’ signals in the cell and triggering cell death offers a compelling approach in the treatment of cancer, but such a powerful pharmacological mechanism carries risk. In keeping with this, at present the majority of the ongoing clinical studies are for very severe indications and not aging. However, it seems likely that if the risk-benefit of such a pharmacological approach in man is understood and favourable, senolytics could find utility in aging.

An additional or maybe complimentary approach would also involve the immune system. A healthy lifestyle suppresses pro-inflammatory mediators and at a very simplistic level, agents which inhibit inflammation may prove beneficial. However, inflammation also plays a very important protective function in the body and so such an approach may not prove advantageous for chronic treatment. Conversely, boosting the performance of the immune cells seems like a more viable approach since it would enable the body to fight off infectious agents and correct and repair cellular and tissue dysfunction in a more effective way. The stem cells giving rise to immune cells reside in the bone marrow and their accessibility means their biology is very well understood, especially with respect to stem cell maturation and differentiation. Manipulation of these precursor cells in a positive way could offer the potential to regenerate the immune system and hence slow many of the downstream effects of inflammaging and the damaging consequences of infectious disease, which we know is more prevalent in the aged population.

Before we go in search of completely novel agents to address aging, there may already be therapeutic agents available which may be beneficial. Metformin and rapamycin are generic and widely used in the treatment of Type 2 diabetes and as an immunosuppressant for organ rejection, respectively. However, several clinical studies have suggested that there are health benefits to these agents which seem to be driven by pharmacology that lies outside of that recognized in their primary indications. In the preclinical setting, both compounds have been reported as extending lifespan in mice, but these findings have proven controversial. Metformin improves insulin sensitivity, so it seems reasonable to assume that metformin reduces the risk of cellular damage and oxidative stress by improving cellular homeostasis. On a more global level, improved glycaemic control will reduce the emergence of some cardiovascular disease and peripheral nerve damage. The immunosuppressive effects of rapamycin can be theoretically linked to a dampening of inflammaging, however it seems that the agent may have a more cryptic pharmacological effect associated with improving energy homeostasis in cells.

The bisphosphonates are a group of compounds used clinically in the treatment of osteoporosis, but there are observational studies emerging from the clinic suggesting that they could have beneficial effects on human health beyond that associated with bone homeostasis.

Collectively, these widely used agents may have uncovered key pathways in which to focus efforts to identify more potent or selective agents to address cellular aging. What is required is a deeper mechanistic understanding of the cellular pharmacology of these drugs to determine where best to intervene. A key approach to address this is the possibility of using phenotypic screens in cellular models which capture one or more of the hallmarks of aging, to determine modes of action of known agents. In addition, such models can also be used in a blind fashion to screen libraries of compounds to uncover completely novel pathways and identify agents that may be beneficial.

One of the big challenges in the search for treatments to improve healthspan is having robust endpoints by which to measure efficacy. Clearly extension in chronological time to death provides a very clear endpoint, but it seems likely that clinical trials aimed purely at increasing longevity are a long way off. As such, there is a growing need to accurately measure biological age, as chronological age fails to capture the heterogeneity of signs and symptoms with which people age. For example, we probably all have family members who we think look and behave much younger than we know their chronological age to be. How do we measure healthspan in the context of a clinical setting? How do we define quality of life? These are big challenges but we do have the ability to measure directly improvements in, for example, heart or liver function, muscle strength, mobility, cognition and the performance of the immune system. It seems probable that the identification of pharmacological agents that improve healthspan will be found via exploration in multiple surrogate indications where hard endpoints can be measured and beneficial or detrimental effects become clear.

We need to continue to develop biomarkers and translational strategies which are able to inform us of whole-body cellular ‘health’ and with the gathering interest in this area, we will likely have an increasing array of tools to more accurately assess biological age over time.

However, we should always remember that patients don’t care about biomarkers. They are interested in whether they ‘feel’ better, i.e. can meet their basic needs, whether they can learn and grow and make decisions, can be mobile, build and maintain relationships and contribute to society. That is the patient’s perspective which is encapsulated perfectly in the goals as stated by the WHO.

So, while there is a growing understanding of potential ways we can measure and improve healthspan, there are some challenges in clinical development of novel anti-aging compounds. Study subjects may be aged but otherwise healthy, leading to ethical considerations associated with treating healthy patients in a preventative manner. There is no regulatory path at present, and there is the fundamental question of who is going to pay for agents that improve healthspan. Currently there is an argument over whether old age can be regarded as a disease or not. This is irrelevant as approval of any novel agents or use of an existing therapeutic in an age-related condition will require properly controlled, randomized clinical trials. Given the likely heterogeneity in such a trial population and the differing rates at which individuals age (i.e. manifest the hallmarks of aging), the trials will need to be large and long in duration. There would be parallels to the many trials in Alzheimer's disease, where the cost is enormous and efficacy hard to find. Moreover, it seems probable that like the thinking around AD, treatments should start early before symptoms appear. An additional confounder is the likely variations in ADME in individuals. We know elderly subjects often have reduced hepatic and renal function which could introduce significant variability in the exposure to novel agents.

We can meet these challenges as we believe there is significant will within society to succeed. We all share the common goal of living long and healthy lives.

If you’d like to hear how Evotec has developed capabilities to measure the hallmarks of aging which can support efforts to identify novel agents to treat age-related disease then reach out to us. You can also learn more from our webinar "Therapeutic approaches for aging and age-related diseases" by Steve England, SVP, Head of in vitro Biology and Disease Area Lead for Aging and Senescence at Evotec.

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This week we celebrate Mitochondrial Awareness Week, a dedicated time to turn our attention to these tiny yet incredibly powerful cellular structures that play a pivotal role in our health and well-being. While the mention of mitochondria might not immediately conjure up images of groundbreaking scientific discoveries, these dynamic organelles deserve a moment in the spotlight. They are the unsung heroes of our cells, and their importance extends far beyond just being the "powerhouses" that produce cellular energy.

In addition to being cellular “powerhouses” and generating the majority of a cells energy in the form of adenosine triphosphate (ATP), mitochondria are also involvement in apoptosis, calcium signalling, regulation of cellular metabolism and proliferation of haem and steroids.

Unfortunately, mitochondria are also a common target for drug-induced toxicity which can have a significant adverse effect on cellular function and overall health. Here are some key reasons why understanding the potential for mitochondrial toxicity is crucial when screening for a drug candidate:

1. Energy Production: Any disruption in mitochondrial function can lead to a decrease in ATP production, which can have detrimental effects on cellular processes and overall cellular health.
2. Cellular Health: Healthy mitochondria are essential for the proper functioning of cells and tissues. Mitochondrial dysfunction can lead to a range of health issues, including neurodegenerative diseases, cardiovascular diseases, and metabolic disorders.
3. Potential Adverse Effects: Drugs that cause mitochondrial toxicity can lead to serious adverse effects in patients. For example, some drugs have been associated with muscle weakness, neuropathy, or liver damage due to their impact on mitochondria. Identifying mitochondrial toxicity during drug screening can help prevent these adverse effects in clinical trials and post-market use.
4. Mitochondrial DNA Damage: Mitochondria have their own DNA (mtDNA), and they replicate independently of the cell nucleus. Drug-induced mitochondrial toxicity can result in mtDNA damage, mutations, or deletions. This can further impair mitochondrial function and contribute to a variety of diseases.
5. Drug Efficacy: In addition to avoiding harm to mitochondria, it's important to consider the potential benefits of a drug on mitochondrial function. Some drug candidates may enhance mitochondrial function, which can be beneficial for diseases characterized by mitochondrial dysfunction, such as certain neurodegenerative disorders.
6. Predictive Toxicology: Early identification of mitochondrial toxicity can help pharmaceutical companies make informed decisions about whether to advance a drug candidate in the development pipeline. Identifying mitochondrial toxicity in the preclinical stages allows for the modification of compounds or the discontinuation of those with high toxicity, potentially saving time and resources.
7. Regulatory Requirements: Regulatory agencies, such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), require thorough safety assessments, including an evaluation of mitochondrial toxicity, during the drug development process. Failing to address mitochondrial toxicity issues can result in regulatory delays or rejection of a drug candidate.

In summary, studying mitochondrial toxicity is essential to ensure the safety and efficacy of drug candidates. It helps prevent potential harm to patients, reduces the risk of adverse effects, and supports the development of drugs that are not only effective but also safe for long-term use.

Cyprotex offer a number of approaches to study mitochondrial toxicity, including:

• Functional Mitochondrial Toxicity Assay (Seahorse)
• Mitochondrial Respiratory Complex Assay using Permeabilised Cells (Seahorse)
• Mitochondrial Biogenesis Assay
• Mitochondrial Glu/Gal Assay
• HCS Mitochondrial Toxicity Assay

In addition, we frequently publish our work in the field of understanding mitochondrial toxicity. You may be interested in:

Poster: A Comprehensive Approach using In Vitro Assays to Detect and Identify Mechanism of Mitochondrial Toxicity

Publication: A Combined In Vitro Approach to Improve the Prediction of Mitochondrial Toxicants

Contact us to find out more about our mitochondrial toxicity services today!

Blood microsampling is a less invasive and simplified alternative to traditional venipuncture for PK/TK sampling, used mainly in small-animal studies. The purpose of this work was to evaluate the possibility of using microsampling technique also to support PK/TK studies in non-human primates.
A comparison of plasma PK parameters was conducted by traditional blood collection from the femoral vein and microsampling from the tail vein of six non-naïve cynomolgus monkeys. Four drugs were selected for this comparison, based on acid-base properties and volume of distribution. 
The results obtained in this work, supported by robust statistics, demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. 
The plasma exposures of the tested drugs are comparable for both sampling techniques and are not influenced by acid-base characteristics and volume of distribution. 
Microsampling used in non-human primates avoids the occurrence of hematomas at the animal sampling site and can also refine practices to limit pain and distress to which animals are exposed (refinement of 3Rs) and, as a result, may reduce the impact of animal stress on PK/TK readouts; moreover, it also provides significant advantages for animal technicians during in life handling.

To request a copy of the article, contact the authors. For Evotec: massimo.breda@evotec.com

Katie Haughan, Drug Transporter Sciences Team, Cyprotex 

Drug transporters play a pivotal role in drug-drug interactions (DDI’s), as such regulatory bodies such as the FDA and EMA recommend the study of specific transporters that are known to cause clinical DDI’s. One of these recommendations for orally administrated drugs, unless waivered based on the BCS classification, is the victim (substrate) potential at Breast Cancer Resistance Protein (BCRP) due to evidence that inhibition of intestinal BCRP in DDI can increase the absorption and therefore exposure of sensitive substrate drugs such as rosuvastatin and topotecan. Pharmacogenetics also need to be considered when developing drugs that are BCRP substrates as this can impact the BCRP expression levels between individuals and within the global population and can therefore result in differing pharmacokinetic profiles. Polymorphisms associated with BCRP may or may not impact on the expression or functionality of the transporter, however the prevalence of these can vary dependent on the ethnicity. One example that is of importance for understanding the pharmacogenetic impact is BCRP SNP C421A, which is associated with lower BCRP protein expression, and leads to the ABCG2-Q141K polymorphism which has a higher frequency in Japanese and Chinese populations compared to Caucasian populations [Birmingham et al. 2015, Hua et al. 2012]. As a result, the plasma levels of BCRP substrates such as rosuvastatin in these populations is increased due to greater absorption and therefore this would need to be considered for Cmax estimates and dosage strategies.  Differences in BCRP expression amongst the global population can be a result of many factors such as ethnicity, genetic polymorphisms and disease states.  

The industry gold standard, and regulatory recommendation, for BCRP substrate identification is the use of a polarised cell monolayer system to determine the bidirectional flux of the investigational drug in the absence and presence of a selective inhibitor. To do so there are two cell lines which are favoured across the industry; BCRP over expressing transfected Madin-Derby Canine Kidney cell line (MDCK-BCRP), or the immortalised colorectal adenocarcinoma cell line Caco-2, both of which differentiate and polarise allowing for the expression on a range of proteins and display the in vivo like characteristics such as tight junctions. 

In order to select which cell line to utilise in these regulatory studies there are many aspects to consider, each cell line comes with its own advantages and disadvantages and can favour the outcome of certain classes of compounds. Whilst MDCK-BCRP has advantages over Caco-2 such as short culturing times and higher expression of the transporter of interest, there is a risk that data interpretation may be clouded due to an intrinsic limitation of the MDCK cell line which, for efflux transporter substrate determination, may result in false negatives. This potential error in the reported classification can have implications downstream as being a substrate may reduce the oral absorption and bioavailability of the drug, which may result in therapeutic dose not being achieved.  Whilst transfected MDCK cells have been used for many years in order to assess an investigational drug’s BCRP substrate status, it’s applicability may be limited due to the cells lacking expression of the relevant basolateral uptake transporters that allow polar substrates to enter the cell and in turn interact with BCRP transporter on the apical membrane. Without this uptake mechanism, and in combination with the compounds poor permeability, the basolateral to apical flux would be negligible resulting in an efflux ratio (ER) less than 2 (B-A/A-B).  The initial classification by the FDA is dependent on this ratio with a BCRP substrate having an efflux ratio greater than 2. 

Whilst Caco-2 cells have a considerably longer culture time compared to that of MDCK cells, they have the benefit of being able to correctly identify the efflux compounds that rely upon the interplay between apical and basolateral transporters. Organic solute transporter alpha (OST-α) is expressed on the basolateral membrane of epithelial cells in the small intestine, kidney, liver, and colon amongst other organs aswell as on Caco-2 cells, and this trans-membrane protein allows passive facilitative transport of endogenous and exogenous molecules across the membrane, including polar structures. A primary function of OST-α is the transport of bile acids, and therefore lends itself to the transport of other polar substrates such as the prototypical BCRP substrates rosuvastatin and estrone 3-sulfate across the membrane and into the cell. Once inside the cell the compound then has access to the binding site of the BCRP transporter and the opportunity to be effluxed if it is indeed a substrate. For cells that lack this uptake mechanism, such as MDCK-BCRP, polar compounds have restricted entry to the cell and do not have the opportunity to be effluxed regardless of if they are a substrate of BCRP or not. 

Rosuvastatin is one of the most widely prescribed statins and implicated in BCRP DDI’s due to its victim classification. Rosuvastatin is a polar compound with a logP of 0.13 and therefore has low permeability and is dependent upon uptake mechanisms to enter cells such as MDCK. The flux of rosuvastatin, apical to basolateral, basolateral to apical and the subsequent efflux ratios can be seen in Table 1 [Li et al. 2012] for three cell lines. The B-A secretory apparent permeability (and derived efflux ratio) is significantly higher (and therefore more sensitive) in Caco-2 cells compared to that in the two MDCK cell lines even though the protein expression of the BCRP transporter in Caco-2 cells would be considerably lower than in the transfected MDCK-BCRP cells, demonstrating the crucial requirement of the basolateral uptake mechanism that is present in Caco2, but absent in MDCK-BCRP, to facilitate the interaction of rosuvastatin with intracellular BCRP on the apical membrane.  This factor needs to be taken into consideration when it comes to assessing the efflux potential of an investigational drug using MDCK-BCRP cells. 

Table 1: Bidirectional (apical-to-basolateral (A-B) & basolateral-to-apical (B-A)) apparent permeability (Papp) and efflux ratios for rosuvastatin in different cell test systems [1]  

*P<0.001, significance level of the difference from the B-to-A transport in Caco-2 

As shown the use of Caco-2 cells for BCRP substrate identification removes a complication seen in MDCK-BCRP cells that is dependent on the physicochemical properties of the compound, however Caco-2 cells come with their own complexities. As Caco-2 are human derived cells they express a range of endogenous human transporters including human P-glycoprotein (P-gp). BCRP and P-gp have a similar substrate profile and therefore the efflux seen in Caco-2 cells may be due to BCRP, Pgp efflux or a combination of both. In order to decipher between the two main efflux transporters a selective P-gp inhibitor such as verapamil can be added to the test system buffer to chemically knockdown P-gp activity. The residual efflux is then considered to be due to BCRP transport however this can be confirmed using a selective BCRP inhibitor, fumitremorgin C (FTC) as shown in Table 2. 

Table 2: Bidirectional (apical-to-basolateral (A-B) & basolateral-to-apical (B-A)) apparent permeability (Papp) and efflux ratios for test compounds and the substrate classifications given per assay condition 

Choosing the right in vitro cell test system for BCRP substrate identification is critical for achieving the correct classification. The two gold-standard approaches have their own pros and cons, each of which can be mitigated if these test system limitations are understood. If a chemical series has a reasonable degree of lipophilicity then MDCK-BCRP should correctly identify a substrate of BCRP. However, as the industry moves towards more metabolically stable molecules and chemical series that have similar low intrinsic permeability and polarity to the clinically relevant BCRP substrate rosuvastatin, the use of Caco-2 cells would be required for the correct identification of BCRP substrates in order to avoid false negatives.

References

1. Birmingham BK, Bujac SR, Elsby R, et al. Impact of ABCG2 and SLCO1B1 polymorphisms on pharmacokinetics of rosuvastatin, atorvastatin and simvastatin acid in Caucasian and Asian subjects: a class effect? Eur J Clin Pharmacol. 2015 Mar;71(3):341-55.  
2. Li J, Wang Y, Zhang W, Huang Y, Hein K, Hidalgo IJ. The role of a basolateral transporter in rosuvastatin transport and its interplay with apical breast cancer resistance protein in polarized cell monolayer systems. Drug Metab Dispos. 2012 Nov;40(11):2102-8.  
3. Hua, W.J., Hua, W.X. and Fang, H.J, The Role of OATP1B1 and BCRP in Pharmacokinetics and DDI of Novel Statins. Cardiovascular Therapeutics,. 2012, 30: e234-e241. 

Rapidly growing tumor cells need a lot of oxygen and nutrients to proliferate, which requires access to the bloodstream. Therefore, tumors induce the formation of new blood vessels through a variety of molecular mechanisms. In normal tissue, there is a delicate balance of pro- and anti-angiogenic factors. In cancers, a process known as the "angiogenic switch" is initiated. As a result, pro-angiogenic signaling becomes dominant, allowing tumors to induce anarchic blood vessel formation. This switch is a critical step in the rapid growth of malignant cells, accompanied by the formation of new blood vessels.

One of the most important initiators of angiogenesis is the family of pro-angiogenic vascular endothelial growth factors (VEGF) and their receptors (VEGFR), which play an important role in both physiological and cancer angiogenesis. All members of the VEGF family stimulate cellular responses by binding to specific tyrosine kinase receptors, the VEGFRs, on the cell surface, causing them to dimerize and become activated. While VEGF-A regulates angiogenesis and vascular permeability by activating VEGFR-1 and VEGFR-2, VEGF-B seems to play a role in the maintenance of newly formed blood vessels under pathological conditions, while VEGF-C and VEGF-D and their corresponding receptor VEGFR-3 regulate lymphangiogenesis, i.e., the formation of lymphatic vessels.

This critical involvement of VEGFRs and their associated signaling pathways in the orchestration of (lymph)angiogenesis makes VEGFRs attractive targets for the treatment of tumors and the prevention of metastasis. However, existing therapies targeting VEGFRs are not very specific and inhibit a broad spectrum of receptor tyrosine kinases including the entire VEGFR family, causing many side effects such as hypertension, proteinuria, hand-foot syndrome, anorexia, and fatigue. While they show good response rates and short-term efficacy, their impact on overall survival is limited, in part because side effects limit the effective dose. This also results in only partial or transient inhibition of VEGFR-3, allowing lymphangiogenesis to serve as a tumor escape mechanism.

Evotec and Kazia therapeutics has therefore started to develop more selective VEGFR-3 receptor tyrosine kinase inhibitors. Its lead candidate, EVT801, an orally available VEGFR-3 inhibitor, is not only highly selective for VEGFR-3, but also the only inhibitor known to inhibit both VEGFR-3 homodimers and VEGFR-3:VEGFR-2 heterodimers. The compound shows low nanomolar inhibitory activity and high selectivity over kinases, various receptors, and ion channels. In November 2022, Evotec scientists reported in Cancer Research Communications that EVT801 showed potent antitumor activity in various in vitro and in vivo models. Moreover, the compound's in vivo efficacy was at least as good as that of the marketed pankinase inhibitors sorafenib and pazopanib. However, unlike sorafenib, EVT801 did not increase blood pressure in monkeys during regulatory toxicology studies or in a rat model of hypertension at doses up to 500 mg/kg, an order of magnitude higher than the pharmacological doses.

The investigators also observed that EVT801 reduced tumor (lymph)angiogenesis, apparently affecting small tumor vessels more significant than larger ones. 

The efficacy of EVT801 is expected to depend on the level of VEGFR-3 expression. Interestingly, expression level of VEGFR-3 did not appear to be affected by sorafenib treatment, suggesting that EVT801 could be used in patients previously treated with any VEGFR tyrosine kinase inhibitor. However, these findings need to be confirmed in clinical trials to determine the minimum threshold of VEGFR-3 expression for effective clinical application of EVT801 and for future patient stratification. 

The proposed mechanism of action of EVT801 involves three sequential anti-cancer mechanisms, all of which contribute to the inhibition of tumor growth and metastasis:

• It prevents tumor growth by impairing both tumor angiogenesis and (lymph)angiogenesis, thereby stabilizing the tumor vasculature, reducing metastasis, and reducing hypoxia in the tumor microenvironment.
• It enhances anti-cancer immunity as reflected by a decrease in immunosuppressive cytokines and cells in the circulation and tumor environment.
• It promotes T-cell infiltration into the tumor, ultimately supporting an enhanced and long-lasting anti-tumor immune response.

Taken together, these studies demonstrate that EVT801 is a novel anti(lymph)angiogenic agent that selectively targets VEGFR-3, modulates the tumor microenvironment to induce tumor vasculogenesis (i.e., fewer and overall larger vessels), and enhances immunotherapy.

Based on these promising results, EVT801 was selected to enter clinical trials. It is currently being evaluated as a single agent in a Phase I trial (NCT05114668) sponsored by Kazia and managed by Evotec Clinical Operations. The first stage of this Phase I study is designed as an open‑label, dose escalation trial to assess the safety, tolerability, and pharmacokinetics of EVT801 in up to 48 patients with advanced solid tumors. Details of the study were presented at the AACR Annual Meeting 2023 (Orlando, FL) (Abstract #1015). This dose escalation part will be followed by a biomarker and pharmacodynamics expansion cohort (second stage), including patients with high VEGFR-3 expressing cancers. These study sections may then be followed by a second dose escalation study, in combination with cancer immunotherapies.

The plan is to establish a patient stratification based on VEGFR-3 expression assessed by immunohistochemical imaging and immunofluorescence on tumor tissues before and after treatment. To enable this patient stratification analysis in a clinical setting, Evotec has established and validated a highly specific protocol for VEGFR-3 immunohistochemistry labeling and scoring strategy that is readily transferable to clinical sites.

To refine the VEGFR3 immunohistochemistry signature and to improve patient characterization, Evotec has developed a VEGFR3 mRNA gene signature consisting of 23 genes highly correlated with VEGFR-3 expression. The gene signature will be analyzed using the Fluidigm platform on matched FFPE patient samples. The relationship of key markers at protein and mRNA level will be investigated to potentially establish biomarkers for patient stratification and selection. 

We expect that the ambitious biomarker strategy will help to better understand the effects of EVT801 in humans and may also help to select the most responsive patients and provide early indications of clinical efficacy as a monotherapy (e.g. clear renal cell carcinoma, soft tissue sarcoma and ovarian cancer) or in combination with standard of care (e.g.immune checkpoint therapies). Evotec and Kazia recently presented a scientific poster on this topic.

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Our Drug Transporter Sciences team at Cyprotex are delighted to announce publication of a review article in Expert Opinion on Drug Metabolism and Toxicology entitled ‘Studying the Right Transporter at the Right Time: An In Vitro Strategy for Assessing Drug-Drug Interaction Risk during Drug Discovery and Development’.

As our understanding of the role of drug transporters in clinical drug-drug interactions (DDIs) has developed, the list of transporters requiring in vitro study by regulators has grown to accommodate assessment of risk for new drugs.  Currently, ten transporters require routine study prior to regulatory NDA submission.  Getting the timing wrong for these investigations could result in in vitro data being generated either 1) too early in the drug discovery/development timeline and potentially becoming surplus to requirements if the investigational drug fails for reasons of poor pharmacokinetics (and efficacy) or toxicity, or 2) too late to influence finalisation of the clinical development plan resulting in perhaps unnecessary comedication exclusions that impact patient recruitment and thus delay clinical trials.  In either case, there will be a cost and resource penalty, with the overall impact being considerably cheaper for the former compared with the latter.  To minimize these development risks, project teams should study the right transporters at the right time for their investigational drug and the authors (Dr’s Robert Elsby, Hayley Atkinson, Philip Butler and Rob Riley) have tried to address this in their review article by proposing in vitro strategies that could be employed to either mitigate/remove transporter DDI risk during development through frontloading certain studies, or to manage (contextualize) DDI risk to patients in the clinical setting.

In the article, an overview of clinically relevant drug transporters and observed DDIs is provided, alongside presentation of key considerations/recommendations for in vitro study design when evaluating drugs as inhibitors or substrates of transporters.  Guidance on identifying critical victim comedications and their clinically relevant disposition pathways, and using mechanistic static equations for quantitative prediction of DDI (demonstrating a 97% predictive accuracy for 28 statin DDIs) is also compiled.  To truly alleviate or manage clinical risk, the industry would benefit from moving away from current regulatory qualitative basic static equation approaches to quantitative mechanistic DDI prediction, thereby contextualising risk to ascertain whether a transporter DDI is simply pharmacokinetic or clinically significant requiring intervention.  Furthermore, such a mechanistic approach can be used towards either mitigating perpetrator DDI risk early during candidate selection, or managing clinical risk and aiding patient recruitment by informing labels and potentially providing an alternative to conducting costly clinical interaction studies with co-medications in the future. 

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