Chaoyong Ma, Business Development Manager, Phylonix

Could you start off by introducing your company and yourself?



Phylonix Pharmaceuticals is a contract research organization providing drug screening services to pharmaceutical and biotech companies. The Company uses the zebrafish model organism to help customers validate targets and test compounds for efficacy and toxicity.

My scientific background is in molecular biology and genetics. For the past several years I have been doing strategy consulting and business development in the biotech industry.

What is your actual role now within Phylonix?

As the Business Development Manager in the Company, my major role is identifying opportunities for our zebrafish bioassays, and pursuing and managing collaborations with biotech and pharmaceutical companies.

Do you use zebrafish for whole animal screening and can you tell us a bit of background to it?

We focus on using zebrafish as the animal model for our drug screening services because it is very suitable for a lot of drug screening assays. Zebrafish are very small at the early stages, so we can do the screening at a relatively high throughput with very small amount of drugs. This is important because at the early stage of drug discovery the available amount of compounds is often limited, and there are a large number of compounds to test.

The animal is also transparent in its early stages and allows us to observe the drug effects directly using visual assessment, and it is also easy to perform high throughput in vivo screening because of the transparency. Zebrafish also share a lot of similarity in physiology with humans because both are vertebrates, as compared to invertebrate models such as the fruit fly Drosophila or the nematode worm C. elegans. In vivo zebrafish assays have a lot of advantages compared to in vitro assays or assays using invertebrate models. It can bridge the gap between in vitro assays and assays using mammals.

What type of assays have you developed?

We have been doing target validation, toxicity testing, and certain efficacy testing, such as drug effects on angiogenesis, apoptosis, neuroprotection, and cardiac function. Target validation by gene knockdown using Morpholinos is fast and the effect of gene knock down can be thoroughly assessed in the transparent zebrafish. Other techniques, such as whole mount antibody staining and in situ hybridization, can be easily done with this small animal model. Toxicity testing is a major application of this model, partly because it has been used for toxicity testing for a long time in environmental sciences and we have adapted this model to test toxicity for pharmaceutical drugs, which can be used for testing general toxicity or specific toxicity in internal organs and tissues.

As mentioned earlier, zebrafish is transparent at early stages so we can see the effect on internal organs directly, and perform further analysis by histology and staining without complicated process. For the same reason, certain efficacy can be quickly and thoroughly studied. For example, we can screen large numbers of compounds for effects on angiogenesis by a whole animal ELISA using a proprietary monoclonal antibody that is specific for activated endothelial cells.

Would you say that this method of screening gives you a lot more information about the drug candidate?

In vivo assays provide much more information compared to in vitro assays. We can see drug effects on different organs and tissues simultaneously in the whole animal; for example we can see drug effect on the liver, intestines and cardiac in the same animal. We can do a lot of multi-parameter analysis in a physiological context, which is really difficult to do in the in vitro system.

Are there any disadvantages in vivo, compared to in vitro?

In vitro assays can be done at really high throughput. While zebrafish in vivo assays can be done at relatively high throughput compared to mammalian studies, the throughput is lower than in vitro assays.

How fast is the throughput using the Zebrafish assays?

It depends on the assays. For example, some assays rely on dye labeling; you can just put in a dye and that will label the specific tissue and then we can read it out using plate readers. For those assays we can easily screen 100’s or 1000’s of compounds within several weeks.

Are you currently developing any more assays?

We continuously develop more assays that are useful for the pharmaceutical and biotech industries. For example, recently we have developed a family of assays for neuroprotection and neurotoxicity and assays for angiogenesis. We are also creating a number of disease models using a variety of techniques in zebrafish, including gene knockdown, chemical treatment, ectopic gene expression, and mutants

Are any other companies using this method of screening?

Zebrafish has become more mainstream in the past several years as a tool for drug testing. There are now a few pharmaceutical companies that have internal zebrafish labs doing drug discovery research. There are also a few other biotech companies that are using the zebrafish model.

We differentiate ourselves by focusing on providing research services to other companies. We help our customers screen compounds on simple fee-for-service basis.

Are your assays standard, or do you work with your customers to produce an assay specific to them?

When we do our assay development, we identify the need of the industry and try to develop standard assays that can be marketed to many different companies. This is obviously an attractive model as we can multiply the return on investment. At the same time, customer needs are often different even if they are interested in the same category of assays, so while providing services to our customers we also learn from them and work with them to design specific studies that better serve their needs, Some of the studies we have done for our customers were custom designed. Zebrafish is a relatively new system, so I think we are still at in early stage of using the system to really change the drug discovery landscape.

Would you say using zebrafish for screening produces safer drug candidates going into clinical trials?

I think zebrafish in vivo assays can contribute a lot to the drug safety area. Right now a lot of toxicity testing is done in a cell based or even in vitro system prior to the lead optimization step, because studies using mammals are very expensive and very slow. By doing in vivo assays earlier in the drug discovery and development process we can help optimize compounds and select the best compounds to go forward, thus contributing to the reduction of late-stage attrition rate.

So you would say that screening using this method actually reduces the costs as well?

Yes, by doing in vivo assays earlier you can get more predictive data earlier, and that can then help make decisions on selecting which compounds to proceed with and reduce late-stage attrition, thereby reducing the overall costs for drug development. One of the problems facing the pharmaceutical industry is that there are a lot hits from the high-throughput in vitro drug screening programs, and with a large numbers of potential leads it is very difficult to do thorough mammalian studies on all these leads to identify the most promising ones. With in vitro assays it is often difficult to get data that reliably predict in vivo effects, so if you can combine the in vitro assays with in vivo analysis using a relatively high throughput system such as the zebrafish, you can quickly narrow down to the most promising compounds to bring forward to the next stage of the drug discovery process.

How similar are zebrafish to humans genetically?

Genetically there is a very high resemblance. A lot of proteins have almost 100% identity within the functional domain, such as the ATP binding domains and kinase domains. As you know the function of the protein mostly resides on the functional domains, so this explains the very high functional similarity between fish and human homologous proteins. Obviously both species are vertebrates and vertebrates have a lot of common features such as the organ systems.

In our company we have tested a lot of drugs and we have found many of them caused very similar response in zebrafish compared to the reported response in mammals. Similar observations have also been reported in the literature. For example, a research group in the Massachusetts General Hospital tested 100 small molecule drugs that have effects on cardiac rhythm in humans, and found that they also affect heart beat in zebrafish, presumably due to the highly conserved cardiac ion channels in zebrafish as compared to those in humans. We have tested a number of ion channel blockers in zebrafish and have obtained similar results.

Are you going to stick with zebrafish or are there any other animal models that you might be working with?

We are very focused on the zebrafish model and will stick to this model for drug screening. I think it combines the advantages of the in vitro assay and mammalian assays, without being too distant from humans, so I think they are more useful for drug screening compared to other alternative models such as the fruit fly or nematode worm. Those invertebrate animals also have the advantage of being very small and very easily handled and very cost-effective. Also, for some pathways there is high conservation with humans. For example there are studies showing that it is possible to study the insulin and glucose pathway in C. elegans.

However, compared to zebrafish, they obviously have the drawback of being further away from humans. As for other animal models such as rodent, they are much more expensive and assays are much slower. They are good for certain assays but are not suitable for the kind of studies we are doing, which require high speed and low cost.

Do you think generally within pharmaceutical companies we are going to see more of a movement towards whole animal screening?

There are a lot of discussions about this trend, and I think there are also a lot of actual applications being developed, using the whole animal assays to complement high throughput in vitro assays. It has been increasingly recognized that there is a lot of problem with reliance on high throughput in vitro assays; you have got a lot of data, you have got a lot of potential hits, but then what is the next step? It is very expensive to use mammalian models for secondary screening for all these hits, it will cost a lot of money and take a long time and you may not find anything because of a quality problem with the in vitro assays and the lack of physiological context.

By combining high throughput screening with in vivo assays, using animal models such as zebrafish, one can get more reliable data and have more information to make better decisions.

Further Information: http://www.phylonix.com/