What's new

What's New

13 | Feb | 2024

Chinese

Preclinical toxicology studies are required for every therapeutic development program as these studies answer fundamental questions on the local and systemic effects of the test drug on the patient. Typically, tox studies are performed in small and large animal models and use defined endpoints. The guidance issued by the FDA has clearly stated the minimum requirements for preclinical toxicology studies include PK/PD profiling, acute toxicity studies in two species (rats and dogs are the most commonly used) and short-term toxicity studies to evaluate continued and potentially delayed onset adverse effects1. Traditional toxicology studies have been more observational and record the ADME characteristics, biodistribution and PK/PD profiling along with optimal dose ranges that have acceptable off target effects. However, there is an increasing shift towards a more active investigational toxicology approach that can be either prospective or retrospective2. Prospective investigative toxicology, as the name suggests, is performed during the discovery stage to quickly identify promising drug assets that have low toxicity and can move forward into efficacy evaluation. This approach supports the concept of “fail early and fail fast” so that assets with unacceptably high levels of toxicity are removed early from the development pipeline, thus saving significant time and downstream costs. These prospective studies are typically performed in translational in vitro models that range from simple 2D cell culture models and 3D organoids to highly complex microphysiological systems (MPS) such as organ-chips3. The retrospective approach is focused on understanding the mechanism of action of adverse effects identified in in vivo animal models or clinical trial patients. These studies can use multiomics-based approaches to review global changes in gene and protein expression profiles in response to drug exposure combined with ADME, histopathology and PK/PD data. The retrospective analysis is very useful to design next-generation therapies that can bypass the signaling triggers that cause off-target effects.

Prospective investigative toxicology studies are recently gaining traction due to the interest in responsible animal use and regulatory willingness to accept data generated in in vitro and ex vivo models. The FDA Modernization Act in the US and the activities by European medical agencies to promote animal-free testing has accelerated the development of complex in vitro model systems to predict toxic effects. It is important to note that in vitro model development has moved at different paces depending on the organ. For example, lung MPS model development was very rapid in response to the COVID-19 pandemic, while liver and kidney MPS development are moving at a slower pace in part due to tissue complexity. The availability of high-quality input materials impacts the pace of development – for example, researchers are dependent on hepatocellular carcinoma (HepG2) cells or primary hepatocytes to test therapies for drug induced liver injury (DILI), which is a critical tox readout. These models are not fully representative of the in vivo state and, in the case of primary hepatocytes, supply and quality continue to be issues. The development of reliable, high-quality iPSC-derived hepatocytes has been a challenge but as reprogramming technologies continue to improve, it is likely that this challenge will be solved. Another example is the development of translational complex kidney models. Simple 2D overexpression models have been used for several years to study drug-drug interactions (DDI) but the recapitulation of kidney glomeruli in vitro is a complex issue. Nephrons, the functional units of the kidney, consists of over 20 cell types that are arranged in a complex structure4 but MPS platforms typically use 2 cell types – epithelial cells and endothelial cells. Micro-physiological systems (MPS) for kidney cell culture were first reported in 2013 with the development of a kidney chip5 that showed expression of uptake and efflux transporters, resulting in accurate and reproducible responses to known transporter inhibitors such as cimetidine. Bioprinting is another technology that is being investigated to develop a 3D model of the kidney for the use in investigative toxicology studies4.

It is clear that the development of complex in vitro models for investigative toxicology is on an accelerated pace. As the development of input materials and culture systems continue to improve and evolve, the combination of biology and engineering will result in human in vitro systems that recapitulate the in vivo state to better predict off target effects.

References:

1https://www.fda.gov/drugs/investigational-new-drug-ind-application/drug-development-and-review-definitions#

2https://www.nature.com/articles/s41573-022-00633-x

3https://www.altex.org/index.php/altex/article/download/1163/1280/6097

4https://portlandpress.com/essaysbiochem/article/65/3/587/228946/Bioprinting-of-kidney-in-vitro-models-cells

5https://pubmed.ncbi.nlm.nih.gov/23644926/

02 | Feb | 2024

Chinese

Imaging methodologies are critical in the diagnosis and prognostic monitoring of solid tumors in humans. Methods such as CT (computed tomography), MRI and PET are widely used in humans and these methods have continued to improve in terms of resolution, sensitivity and data analysis. More recently, the use of AI enablement was reported to improve detection of different solid tumors including skin, breast and head and neck1. Additionally, the combination of different imaging modalities such as MRI and PET have been shown to increase accuracy of tumor detection1. The use of imaging methods to noninvasively detect and monitor tumors in preclinical oncology animal models is becoming more widespread especially due to the translational value of the protocols, tracers and data analysis methods2. Similar to humans, multi-modal preclinical imaging can be used to obtain data on various tumor characteristics including size, morphology, metabolic activity, vasculature and inflammation2.

Preclinical imaging methods can be segmented into the following types: MRI, CT, ultrasound, photoacoustic (PAT) imaging, PET, SPECT and optical imaging (fluorescent and bioluminescent imaging)2,3. MRI is considered the gold standard of imaging modalities and has been shown to have the best tissue resolution that can be enhanced with specific tracers2. Additionally, there are various subtypes of MRI that are tailored to measuring specific characteristics – for example, tissue oxygen levels can be measured via functional tissue oxygen-level dependent MRI that could be used to monitor response to radiotherapies2. The use of contrast agents such as gadolinium chelate allow the visualization of changes in blood vessel architecture in tumors and there are ongoing studies to use gadolinium-based agents to identify cell surface receptors in tumor cells2. Certain imaging methods are more suited for specific tissues types – for example, CT imaging is the optimal method to identify lung lesions due to excellent contrast between air and tissues2. Clinically, ultrasound imaging is the method of choice to detect pancreatic cancers in both human and animal models. Preclinically, ultrasound is also sued used to guide orthotopic model development by helping researchers inject cells in the correct tissue space2 and can be combined with PAT imaging to provide physiological data on the tumor. The basic principle of PAT imaging uses short laser pulses to irradiate tumor tissues leading to heat induced tissue expansion that creates acoustic waves4. The acoustic waves can be measured using ultrasound4.

PET imaging is used to monitor physiological changes in metabolic activity, vasculature etc. and uses radiolabeled tracers such as 18F-fludeoxyglucose (FDG) to monitor glucose uptake in tumors. Since tumors are more metabolically active than surrounding tissues, 18F-FDG PET imaging is a useful method to monitor tumor size and evaluate changes in tumor metabolism after therapeutic intervention5. While 18F-FDG is the most well-known tracer, PET imaging can be performed using multiple radiopharmaceutical tracers and some of the tracers can also be used for SPECT imaging which uses a gamma camera instead of a positron emission scanner. One of the key advantages of using PET and SPECT imaging is that radiolabeled tracers can be used to monitor specific reception expression levels or physiological markers2. For example, 18F-fluorothymidine can be used to monitor DNA synthesis and cell proliferation in tumors. Given the huge focus in immune-oncology, “immuno-PET” has emerged as a specific imaging method where antibodies to select receptor targets or T-cell targeting molecules can be labeled with radiopharmaceutical tracers to monitor the response to specific checkpoint inhibitor therapies2,5. One such reported tracer is a 64Cu-labeled Axl antibody that was used to monitor the efficacy of an hsp90 inhibitor (17-AAG) to downregulate Axl regulation in triple negative breast cancer6.

In vivo optical imaging methods such as fluorescent and bioluminescent require the insertion of a fluorescent tag or a luciferase enzyme into tumor cells or the therapeutic modality2. The tags can be inserted into microbes, viruses, antibodies, peptides etc. so noninvasive luminescent imaging is an easy way to track tumor cells or therapeutic modalities in an animal model. While several fluorescent proteins are used in preclinical studies, one challenge is autofluorescence in specific tissues that can obscure or interfere with the fluorescent signal2. Bioluminescent imaging using luciferase reporters has gained significant traction in preclinical in vivo studies and there is active research to engineer more sensitive luciferase enzymes that have more catalytic activity and improved emission signals7

References:

1https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8945965/

2https://aacrjournals.org/cancerres/article/81/5/1189/649702/Preclinical-Applications-of-Multi-Platform-Imaging

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3687654/

4https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515147/#

5https://www.itnonline.com/article/role-pet-imaging-preclinical-oncology

6https://pubmed.ncbi.nlm.nih.gov/29097911/

7https://pubs.acs.org/doi/10.1021/acschembio.1c00549

16 | Jan | 2024

Chinese

Lipid nanoparticles (LNPs) are vesicle composed of lipids that are used to deliver a wide range of therapeutic modalities including nucleic acids (DNA, mRNA, siRNA), antibiotics and small molecules (such as doxorubicin)1. The most well-known application of LNP drug delivery are the mRNA COVID-19 vaccines developed by Pfizer/BioNTech and Moderna. Fundamentally, LNPs are spherical vesicles composed of ionizable lipids whose charge changes in response to pH2. LNPs have neutral charge at physiological pH, which facilitates entry into cells but have positive charge at acidic pH to promote complex formation with negatively charged nucleic acids. LNPs are internalized into cells via endocytosis and release the payload in the cytoplasm upon exposure to low pH2. LNPs can take various forms including liposomes, nano-emulsions, solid lipid nanoparticles, nanostructured lipid carriers, and lipid polymer hybrid nanoparticles1. Liposomes are best known for delivering chemotherapies such as doxorubicin and paclitaxel for cancer treatment and lipid polymer hybrid particles have also been used to deliver docetaxel for treatment of various cancers1. The nanostructured lipid carriers and solid nanoparticles have been used to deliver nucleic acid therapies. Apart from therapies, LNPs are gaining interest in cosmeceuticals which is an unregulated space that primarily consists of skin and hair care products3. LNPs have desirable properties for topical applications as they adhere well to the skin and easily disperse across the tissues. However, since this space is not overseen by regulatory agencies like the FDA or EMA3, it is important for manufacturers to manufacture and test the LNPs to ensure high quality standards.

Various types of LNPs have been used to deliver different therapeutics, antibiotics and sedatives since the 1990s. A majority of the therapies use liposomes4, and the first LNP based siRNA (Patisiran) therapy was approved in 2018 for the treatment of hereditary transthyretin amyloidosis2. The mRNA based COVID-19 vaccines that were approved in 2021 also used LNPs to deliver mRNA targeting the spike protein of the SARS-CoV2 virus. However, it is important to note that LNPs have pros and cons. One of the key advantages of LNPs is the low toxicity rate since the lipids are biocompatible and do not trigger significant toxicity. Structurally, LNPs are very stable and are amenable to tissue targeting. Depending on the target tissue or organ, LNPs can be directly administered via nebulization to the lung or direct injection into the eye5. LNPs have natural tropism to the liver so they are well-suited to target hepatic diseases and this property is being used to engineer LNPs to deliver payloads to the liver at high efficiency. Additionally, LNPs can be targeted to immune cells such as T-cells via specific antibodies such as anti-CD45. Currently, there is active research to develop next-generation LNPs that have specific tissue targeting properties. LNPs also have certain disadvantages and the major challenge is the low drug loading and delivery efficiency. While this is not a major issue for vaccines, it is of concern to deliver drugs in sufficient quantity to exert a therapeutic effect. LNPs also have short blood circulation time and are susceptible to removal by macrophages causing a low number of LNPs reaching the target tissues. While LNPs are considered to be the most clinically advanced nonviral gene delivery method, the current status of the field restricts LNP use to specific applications but the fields of use are likely to grow with improved next-gen LNPs.

References:

1https://pubs.acs.org/doi/10.1021/acsmaterialsau.3c00032

2https://www.nature.com/articles/s41578-021-00281-4

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8864531/

4https://www.biochempeg.com/article/283.html

5https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9322927/

04 | Dec | 2023

Chinese

Antibody drug conjugates (ADCs) are targeted therapies that consist of a monoclonal antibody (mAb) linked to a chemotherapeutic via a linker. The mAb binds to a target receptor antigen on tumor cells and the ADC complex is internalized into tumor cells resulting in the release of a chemotherapeutic drug that kills tumor cells. The main advantages of developing an ADC are low off target effects and an expanded therapeutic index of the chemotherapy resulting in more effective tumor killing. The concept of using a mAb to deliver a cytotoxic payload to tumor cells is not new but early attempts to develop clinically effective ADCs were unsuccessful due to a few reasons such as poor linkage resulting in separation of the payload, off-target toxicity due to nonspecific antibody binding, immune responses resulting in rapid clearance and low residency time etc1. Additionally, ADCs can be developed against specific membrane bound receptors that have an antigenic extracellular domain that does not get released or shed into the extracellular environment or vasculature1. ADC targets are typically overexpressed in tumor cells compared to normal cells so the continued discovery of differentially expressed biomarkers will help the development of novel ADCs. Currently, most ADCs target receptors or ion channels that are difficult antigen targets, but improvements in antibody discovery methods have helped improve the quality of therapeutics antibodies used in ADCs.

Another critical element of developing an ADC is the linker technology. Using a weak or incorrect linker could result in early release of the payload causing systemic toxicity or cause aggregation of the ADC complexes2. Currently, there are 2 classes of linkers – cleavable and non-cleavable3. Cleavable linkers are primarily cleaved in one of 3 ways – protease, reduced pH or in the presence of reduced glutathione2,3. Enzyme mediated linker cleavage is commonly used and about two-thirds of approved ADCs employ this appriach2. Non-cleavable linkers typically fall in 2 categories – thioether and maleimidocaproyl and are generally considered to be superior to cleavable linkers3. ADCs with non-cleavable linkers are dependent on cellular lysosomal degradation so release of the chemotherapeutic agent only occurs in tumor cells. Therefore, ADCs with non-cleavable linkers have more stability in the vasculature and have a larger therapeutic index and there is active research to develop new and improved linkers.

ADC payloads have historically been available chemotherapies that inhibit cell proliferation, but recently, novel payloads have been used. One example is Enhertu whose payload is a topoisomerase I inhibitor that can inhibit DNA replication in tumor cells4. Another example is Lumoxiti, an ADC targeting hairy cell leukemia whose payload is a Pseudomonas exotoxin A4. Lumoxiti has been recently discontinued due to low market uptake but is an example of creative payload design.

Currently, there are 11 approved ADCs in the US and over 150 ADCs in clinical trials5. 2 ADCs (Mylotarg™ and Blenrep™ were discontinued due to failure to meet endpoints in post-marketing approval clinical trials but Mylotarg was re-approved at a lower dose5. Due to the clinical success of ADCs, biopharma companies are investing significantly in the space leading to the renaissance of ADCs6. Several large pharma companies such as Pfizer and Astra Zeneca have announced large acquisition or ADC asset deals signaling that pharma companies are interested in developing and commercializing ADCs6. There are a couple of major reasons why pharma is interested in ADC assets. The ADC technology platforms have improved significantly and the current third generation of ADCs demonstrate high target specificity while evading the immune system. Additionally, newer ADCs with superior linker technology can carry more payload. One example of a superior ADC is Enhertu that was approved in December 2019 for HER2-positive metastatic breast cancer6 that has a drug antibody ratio or DAR of 8. The Phase III clinical trial data for Enhertu showed a stunning 72% reduction in disease progression6 and was a major clinical success. From an economic point of view, ADCs are difficult therapies to develop biosimilars due to the multiple components, so ADC developers have a longer window to generate revenue and have more pricing power6.

Given the technological advancements in monoclonal antibody development, linker chemistry and payloads along with a track record of clinical success and high barrier to entry for biosimilars, there is no doubt that ADCs are experiencing a true renaissance and this is positive news for many cancer patients with limited therapeutic options.

References:

1https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359697/

2https://www.biochempeg.com/article/87.html

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8628511/

4https://ascopubs.org/doi/full/10.1200/EDBK_281107

5https://pubmed.ncbi.nlm.nih.gov/37639687/

6https://www.biospace.com/article/biopharma-bets-big-on-antibody-drug-conjugates/

15 | Nov | 2023

Chinese

The drug development and manufacturing industry in China has historically focused on generic drugs and Chinese CROs have heavily depended on drug development business from Western countries. However, these trends have changed significantly in the past several years, and it is becoming clear that the biopharma sector in China is growing rapidly with a focus on technology innovation and first-in-class drugs. This change started in 2015, when China’s regulatory agency, the National Medical Products Administration (NMPA), started a series of reforms and changes to accelerate in-country drug development and expand clinical trials 1. One of the key reforms was the decoupling of drug development and production where the drug developer does not have to be the drug manufacturer2. This decoupling allows companies to focus on innovative drug discovery without the need to divert resources to process development, scale up manufacturing, quality control and lot release of the drug product. Secondly, the Center for Drug Evaluation (CDE) issued guidelines for conducting clinical trials across multiple therapeutic areas including oncology and rare diseases2, thus encouraging more in-country trials of novel therapies. Additionally, the funding environment to support Chinese biopharma companies has grown significantly in the past several years. There has been a rapid increase of available capital through VC firms and more relaxed regulations for companies to go public including the formation of the STAR Board3 in Shanghai.

Notably, in 2023 there have been several licensing deals where Chinese biotech companies have developed and licensed drug assets to big pharma and conversely, have in-licensed several drug assets4. This bidirectional licensing activities suggests that Chinese biopharma companies have gained traction on the world stage as developers of high-quality therapeutic assets. Licensees include top pharma companies such as GSK, Takeda and AstraZeneca and the total value of the top 10 licensing deals range from $2 billion to $700 million4. However, the deals where Chinese biotech are the licensees tend to have a lower total value and range across multiple disease areas including oncology, infectious disease and liver disease4. Unsurprisingly, as the number of biotechs developing licensable assets increases, China based CROs are also growing likely to accommodate the increased outsourcing needs.

The 2022 top global CROs list includes 3 Chinese companies – Wuxi AppTec, Pharmaron, and AsymChem5. The revenue growth projections of China based CROs continues to be strong with an anticipated market growth 13% in 2021 to 19% in 20245. While China based CROs have always been known to have deep expertise in chemistry and small molecule drug development, in the past few years there has been a rapid growth in advanced modalities including monoclonal antibody-based therapies. Importantly, the CROs are no longer dependent on business from North America and Europe since Chinese biopharma companies are outsourcing to in-country CROs. This trend likely started during the COVID-19 pandemic but has continued to hold strong as Chinese CROs have developed impressive end-to-end capabilities across the drug discovery continuum. It is estimated that about 40% of WuXi Biologics, a leading Chinese CRO, client base is in country6. Another key development has been that China joined the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use in 2017, allowing the use of data from clinical trials run in China to be used in filings to multiple regulatory agencies6. Since this development, the clinical CROs based in China can compete with global clinical CROs as they can generate usable data at a more economical price tag compared to several North American and European CROs6.

It is clear that China continues to be a competitive player in the preclinical and clinical drug development sector and is expected to grow at a significant pace. This growth is fueled by available capital, supportive government regulations, lucrative licensing deals and an aggressive strategy from Chinese CROs to support domestic biopharma companies while penetrating Western markets.

References:

1https://globalforum.diaglobal.org/issue/june-2021/chinas-new-era-of-reform-transforming-regulatory-professionals/

2https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10326289/

3https://www.pharmexec.com/view/china-invests-in-building-biotech

4https://baipharm.chemlinked.com/news/2022s-top-10-cross-border-licensing-deals-involving-chinese-biopharma-companies

5https://baipharm.chemlinked.com/news/china-contract-research-organization-cro-2022-review-and-outlook

6https://www.spglobal.com/marketintelligence/en/news-insights/latest-news-headlines/chinese-drug-contract-research-companies-see-surge-in-domestic-demand-63832298

06 | Nov | 2023

Chinese

The drug development pipeline is increasingly moving towards complex therapies such as monoclonal antibodies and their derivatives, cell and gene therapies and nucleic acid (DNA and RNA) therapies. The critical requirement to support clinical trials and successful commercialization of advanced therapies is high quality consistent manufacturing that is primarily outsourced by pharma to CDMOs (contract development and manufacturing organization). The rapid growth in the complex therapies pipeline has fueled the need for manufacturing capacity at CDMOs that is supported by experienced talent and establishing quality systems. Not surprisingly, the pharmaceutical CDMO market is experiencing high growth with a current marker size of $95B in 2022 that is expected to grow to over $170B in a decade with an estimated 6.2% CAGR1. While estimated market sizes do vary across reports, most reports suggest that the CDMO market is expected to double in the next decade or so.

Historically, drug developers have been primarily based in North America and Europe (NA and EU) who have partnered with CDMOs in the same geographies2. This has largely been due to the interest in building and maintaining a close bond between drug developers and CDMOs. CDMOs in North America and Europe have had access to premier talent pools and manufacturing expertise across multiple advanced modalities. It is estimated that about 37% of CDMOs have comprehensive end-to-end portfolios2, which, helps foster strategic partnerships with drug developers. However, a survey in 2014 revealed an interesting trend that drug developers did not consider geographic location to be a key consideration for selecting a CDMO3 suggesting that the CDMO globalization trend was only a matter of time.

The dominant position of North America and European CDMOs is being challenged by an increasing trend towards globalization especially in Asia. There are a few key reasons for this globalization trend – capacity, supply chain and expertise. Drug developers are increasingly concerned about a “capacity crunch” at NA and EU CDMOs that can delay manufacturing and negatively impact the race to be first to market4. This has led to drug developers towards CDMOs in China and India that have available capacity. Additionally, the COVID-19 pandemic had a significant impact on supply chain availabilities and costs of raw materials and consumables resulting in large price increases and manufacturing delays5. The rush towards manufacturing COVID-19 vaccines and antiviral therapies has helped equip several Asia based CDMOs with the infrastructure and knowhow to manufacture advanced biologics and complex therapies at a high quality.

Specifically, China is experiencing extremely high growth in the CDMO sector at an estimated 32% CAGR6. One of the major reasons that triggered this growth is a legislative change in 2015 where Chinese drug developers did not have to build in-house manufacturing facilities and could use CDMOs to manufacture drugs for approval6. This allowed the CDMO sector to grow rapidly and develop advanced capabilities to manufacture biologics therapies. India is another growing CDMO geography and this is largely being fueled by low costs and the availability of skilled labor6. Other emerging geographies for CDMO outsourcing include Latin America and Africa but these seem to be initially focused on vaccine production.

An interesting trend that is being observed is the shift of Asian CDMOs to North America and Europe. Several savvy CDMOs that were originally based in Asia (primarily China) are recognizing the need to have a presence in drug development hubs in the US and other countries. This shift is helping increase the profiles of CDMOs that originated in Asia but are now global organizations with end-to-end capabilities to support drug development and manufacturing globally. As this trend increases, it is clear that the CDMO world will move towards a virtual global space where physical distances are irrelevant and drug developers will have a broad range of global CDMO partners to accelerate advanced therapies to market.

References:

1https://www.globenewswire.com/en/news-release/2023/06/07/2683991/0/en/Pharmaceutical-CDMO-Market-Size-Will-Expand-to-USD-172-02-BN-by-2032.html

2https://www.strategyand.pwc.com/de/en/industries/health/2022-global-cdmo-study.html

3https://www.pharmtech.com/view/biomanufacturing-outsourcing-globalization-continues

4https://www.bioprocessonline.com/doc/top-trends-in-biomanufacturing-for-0001

5https://www.bioprocessonline.com/doc/s-bioprocessing-year-in-review-key-takeaways-0001

6https://www.cphi-online.com/emerging-regional-markets-show-promise-as-cdmo-news114072.html

13 | Oct | 2023

Chinese

The drug development process has several critical milestones. One of the milestones is pharmacokinetics (PK) studies, which is the study of how a given drug interacts with the body. PK studies typically evaluate the ADME or absorption, distribution, metabolism and excretion of a new therapy. Crossing the BBB poses a significant challenge for several therapies that target brain diseases including neurodegenerative diseases and brain tumors. PK studies performed in the CNS are especially important to ascertain how much of a given drug crosses the blood brain barrier (BBB)1 to have a therapeutic effect on brain tissues. If the drug cannot cross the BBB efficiently, then it is likely to have limited therapeutic efficacy at an acceptable dosage. It is important to understand the barriers between blood, CSF and the extracellular fluid (ECF) to appreciate the complexities of drug transport into the brain. The BBB separates blood flow from brain tissue and is a tight barrier with no gap junctions or pores, while the BCSFB (blood CSF barrier) is more porous and supports vesicular pinocytosis for the transport of biomolecules including drugs2. Since CSF freely interacts with blood and is in fact produced from blood plasma, drugs that are administered systemically are detected in CSF2. CSF can deliver drugs to specific areas of the brain3. The most common administration methods are lumbar puncture and ICV or intracerebroventricular injection3. Measurement of the available drug concentration and metabolites in CSF after administration are typical readouts to assess ADME characteristics.

Typically, PK studies are performed in primate models that have similar brain anatomy and physiology as humans. PK studies are complex and require analysis at multiple time points to map out the effect of the drug on the body, so it is essential to use minimally invasive methods for repeated sampling of biofluids. There are a couple of different methods to access biofluids in the CNS. One approach is CSF sampling through lumbar puncture or through the cisterna magna, and another approach is through microdialysis where a probe is placed in the tissue of interest to facilitate sampling4. Both approaches have their uses and limitations. CSF sampling typically uses lumbar puncture for repeated sampling of the spinal CSF, while microdialysis samples ECF around the tissue of interest4. Secondly, microdialysis is more widely performed in rodent models with limited use in nonhuman primates, while CSF sampling is well established in nonhuman primates. Microdialysis methods are useful to analyze the immediate environment surrounding a brain tumor or brain region of interest while CSF sampling provides a more global picture of free or unbound drug concentrations. Typically, samples from a microdialysis probe are used to evaluate changes in secreted proteins and neurotransmitters and locally expressed biomarkers. On the other hand, CSF sampling can be used to evaluate global biomarker changes as the sampling is typically done at a site that is distal to the tissue of interest. Interestingly, some reports have shown no significant differences in drug PK characteristics between CSF samples and ECF samples acquired through microdialysis5. Therefore, it is important to select the appropriate sampling method depending on the experiment objective and animal model of choice. In summary, sampling and analyzing the CSF are essential to evaluate both direct drug delivery and drug pharmacokinetics in the CNS.

References:

1https://pubmed.ncbi.nlm.nih.gov/15381336/

2https://link.springer.com/article/10.1007/s10928-013-9301-9

3https://www.sciencedirect.com/science/article/pii/S0169409X21000685

4https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388052/

5https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151035/

02 | Oct | 2023

Chinese

CNS (central nervous system) tumors are primarily located in the brain with some tumors in the spinal cord. CNS tumors can be of various types and are typically named for the cells that are involved – for example, astrocytomas are tumors growing in astrocytes1. Globally, over 308,000 people are estimated to be diagnosed with a CNS tumor and about 25,000 adults in the US are expected to be diagnosed per year2. Additionally, over 4,000 children are diagnosed with a brain tumor and most cases have a poor prognosis2. Given the limited therapeutic options for CNS tumors, early and accurate diagnosis of tumors is critical to improve prognosis and survival rates.

Accessing the brain tissue directly is complicated and invasive but the cerebrospinal fluid (CSF) is a viable alternative to assess cancer biomarkers. CSF is a body fluid that circulates over the brain and down the spinal cord. Since it comes into contact with brain tissue and tumors, secreted biomolecules or cancer cells diffuse into the CSF and can be detected using established analytical or cell based assays3. CSF sampling is typically done using a lumbar puncture where a needle is inserted into the spinal cord between the vertebrae. However, cancer cells and secreted biomarkers are not typically found in abundance in the CSF and the analysis may not always be reliable. Therefore, there is a need for more sensitive assays to identify low abundance biomarkers or cancer cells3.

Current assays to identify cancer cells include cytology analysis where CSF samples are analyzed under a microscope, and flow cytometry analysis to identify cancer cell surface markers. A few recent studies have demonstrated that circulating tumor cells (CTCs) in the CSF can be detected used the FDA approved CellSearch® system4. The CellSearch system was originally approved to detect CTCs in breast, colorectal and prostate cancer5, but it has also been successfully used to identify CTCs in breast cancer related brain metastases6. It is likely that as more studies with larger patient cohorts are performed, CTC detection in the CSF may become a standard diagnostic tool to identify brain metastases as well as CNS tumors.

CSF samples are rich in different biomarkers that are typically proteins or microRNAs. Changes in protein composition in CSF from normal vs cancer patients can be measured using ELISA or IHC based assays as well as proteomic analysis or mass spectrometry. A study in 2006 used a mass spectrometry-based method to identify elevated levels of carbonic anhydrase as a marker for gliomas6. Several studies have compared normal and malignant patient samples and have identified levels of specific markers3. While the results of these studies show promise, it will be important to thoroughly validate tumor type specific biomarkers to meet regulatory requirements for diagnostic testing. MicroRNAs (miRs) are short noncoding RNA fragments that bind to the 3’ end of mRNA and inhibit protein translation. There has been an explosion of interest in developing miR based therapies and several miRs have been identified as high potential drugs for specific tumor types. However, as of now, no miR based therapies have been approved by the FDA but the interest in the biopharma industry continues to grow. Identifying miRs in CSF samples is of high interest as diagnostic biomarkers especially since panels of miRs can be used to diagnose specific CNS tumor types. An example of this panel type approach was reported in 2012 where 7 miRs were used to accurately identify glioblastoma and metastatic brain cancer7.

References:

1https://www.mayoclinic.org/diseases-conditions/brain-tumor/symptoms-causes/syc-20350084

2https://www.cancer.net/cancer-types/brain-tumor/statistics#

3https://jcmtjournal.com/article/view/1321

4https://academic.oup.com/clinchem/article/68/10/1311/6661459

5https://www.cellsearchctc.com/

6https://pubmed.ncbi.nlm.nih.gov/17078017/

6https://pubmed.ncbi.nlm.nih.gov/22492962/

22 | Sep | 2023

Chinese

Genotoxins are chemicals or drugs or any entities that cause damage to chromosomes, DNA or RNA. The damage can result in mutations, single or double stranded DNA breaks and impaired transcription and translation. If the damage occurs in somatic cells, the consequences can include the development of tumors, cell death and inflammation but if the damage occurs in germ cells, it can cause heritable diseases, reproductive issues and birth defects. Drugs with genotoxic potential cause damage that may or may not be repaired by cellular mechanisms so if the repair mechanisms are not able to adequately repair the damage, mutations are generated that may have disease causing potential.

Due to the significant potential impact of genotoxic damage, it is critical to test new therapies for genotoxic stress potential. Since the endpoints of genotoxic testing are defined, several relatively simple bacterial and mammalian cell models are available1 One of the earliest genotoxic tests was the bacterial Ames assay which assesses genotoxic potential by measuring mutations in specific strains of Salmonella bacteria that carry a mutation in the gene required to synthesize the amino acid histidine. The bacteria are cultured in media containing histidine and then exposed to the candidate drugs. The mutagenic potential of drugs is evaluated by determining if they cause reverse mutations and allow the bacteria to metabolize histidine in the culture and the number of bacterial colonies is a gauge of high, medium or low mutagenic potential2.

Currently, two assays are popularly used to assess genotoxic stress – the Comet assay and the Micronucleus assay. The Comet assay uses single-cell gel electrophoresis assay to assess genotoxicity. The assay principle measures single- or double-stranded DNA breaks caused by drugs as cleaved DNA fragments migrate out of the cell when current is applied (ie. electrophoresis) while the undamaged DNA remains in the cell and forms the head of the comet. The denatured undamaged and cleaved DNA are stained with a DNA intercalating dye and visualized using fluorescence. While the Comet assay is simple and rapid and can be run on almost any eukaryotic cell, it does not shed any light on the mechanism of genotoxicity. The micronucleus test is also widely used to assess genotoxicity as micronuclei are essentially extra-nuclear bodies that include damaged chromosome fragments that result from chromosomal aberrations or genotoxic stress of specific drugs3. The chromosomal fragments from the micronuclei are not included in the nucleus after mitosis or meiosis so the genotoxic potential of drugs can be determined by counting the number of micronuclei. In many cases, the Comet assay and Micronucleus assay are both performed to assess the potential of drugs to cause DNA damage as well as chromosomal aberrations4. An interesting study from 2013 compared the Comet assay and Micronucleus for sensitivity and found that the Comet assay required higher doses of the test drugs and is less sensitive4. Nevertheless, both assay types provide valuable data on genotoxic stress. Research into the underlying mechanisms of genotoxicity is limited but some work has been done on drugs such as dacarbazine that is a chemotherapeutic approved to treat melanoma and Hodgkin’s lymphoma5. Dacarbazine is known to cause DNA methylation that impact transcription and translation.

At this time, the field is focused on using these assays to determine if specific chemicals, drugs or environmental toxins can cause DNA damage using simple endpoints but it is likely that more complex assays using next-generation sequencing will be broadly adopted to assess genome-wide genotoxic stress and understand mechanisms and hotspots for DNA damage6.

References:

1https://www.sciencedirect.com/topics/medicine-and-dentistry/genotoxicity-assay#

2https://www.news-medical.net/life-sciences/What-is-Genotoxicity-Testing.aspx

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3708156/

4https://pubmed.ncbi.nlm.nih.gov/23863314/

5https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/genotoxicity

6https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003768/

30 | Aug | 2023

Chinese

Animal models have been the cornerstone of cancer drug development for decades and different types of tumor mouse models have been used extensively to study cancer biology and evaluate single and combination therapies. However, mouse models of cancer have also been widely acknowledged to have limited translational value and in many cases, do not accurately recapitulate tumor biology. This is especially true in the space of immuno-oncology where there are fundamental differences between the mouse and human immune systems. It is important to note that both simple and complex mouse models have a role in oncology drug development and the selection of the model is dependent on the scientific question that is being answered. For example, mice bearing subcutaneous tumors are useful for screening multiple drug assets for efficacy using simple endpoints such as tumor killing1. Once promising assets are identified, more complex models are needed to understand the drug mechanism of action and off target effects.

There are several types of more complex mouse models that can be broadly segmented as transplanted models, carcinogen induced models and genetically modified models. In the past several years, there has been an increased focus on transplanting patient tumors into mouse models. Patient derived Xenografts or PDX models have become the mainstay of oncology drug development primarily due to the availability of patient tumors via biopsy and surgical excisions. The patient tumors can be implanted into animals that have compromised immune systems so that the mouse model does not reject the human tumor – while this model is useful to study tumor growth and development in an in vivo setting, it is not useful to evaluate therapies that target immune cells such as checkpoint inhibitors. Several research model providers have developed humanized mice where components of the human immune system are introduced into immune-compromised mice such as the NSG or NCG models. Human PBMCs (peripheral blood mononuclear cells) isolated from human donors can be injected into the mice to mimic the human in vivo immune response to a xenografted tumor. One such model was reported where colorectal cancer xenografts were implanted into NSG mice that had been injected with human PBMCs2 and the effect of a combination of nivolumab (anti-PD1 therapy) and regorafenib (a multi-kinase inhibitor) was evaluated2. Interestingly, the model was most predictive in an autologous setting where the tumor tissues and PBMCs were from the same patient as the allogeneic model showed nonspecific graft-vs-host issues2. These results suggest that humanized models have a limited role in evaluating response to anticancer therapies and there is an unmet need for robust allogeneic humanized mouse models. Another type of transplant-based mouse model are syngeneic, where the mice with an intact immune system are injected with mouse tumor cells derived from mice with the same genetic background. Essentially, syngeneic models are mouse focused where a mouse tumor is evaluated in the context of a mouse immune system. While this model can be a useful proxy for the human state in some situations. Syngeneic models are reliable and cost-effective and can be used for short-lived efficacy studies. However, there are limited number of syngeneic cell lines and models and in many cases, limited translation to human disease.

Genetically modified mouse models (GEMMs) have been developed for decades and the first reported GEMM was in the 1980s3. The development of GEMMs has expanded rapidly as more advanced gene editing methods have been developed such as Cre-loxP, CRISPR-Cas9, RNA interference etc3. As gene editing methods have become more precise with less off-target effects, GEMMs have become more advanced and recapitulate several hallmarks of the disease state. However, developing GEMMs is an expensive and time-consuming exercise and in many cases, requires detailed knowledge of disease drivers. The genetic engineering required to build a relevant GEMM can be complicated with no guarantee of success. However, once a GEMM is successfully developed, it can be used to study disease development and progression, identify biomarkers for diagnostic use and prognostic monitoring and can be used to evaluate anticancer therapies. SEMMs or somatically engineered mouse models are another type of engineered model where somatic cells in the organ of interest are genetically engineered to express oncogenes or tumor suppressors4.

While there are several types of mouse models of cancer available, selecting the best model is not easy and requires a deep understanding of disease biology4. Multiple types of models may be used in a specific anticancer therapy development program that is dependent on the stage of drug development and the scientific questions that are being asked.

References:

1https://www.nature.com/articles/s41416-019-0495-5

2https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9532947/

3https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286388/

4https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8288236/