Interviews

Drug Discovery Research in Academia - Olfactory Signals Research using Nematodes

- Please inform us of the origin of your drug discovery research in academia, especially your olfactory signal research using nematodes.

Rather than being interested in medical research, I was interested in basic research on the olfaction of nematodes. My first article was published in Nature and reported on the RAS/MAPK pathway activated in the olfactory nerve as my interest at the time was olfactory signaling. Thereafter, my research shifted to studies on the types of odors preferred or disliked by nematodes, and the circumstances whereby changes in nematode actions occur. In these studies, I performed several analyses on nematodes' behaviors as they approached and escaped from a certain odor.

- In terms of olfaction, dogs are said to be superior to humans but it seems nematodes also have a very good olfactory sensory system.

Regarding the number of receptor genes, there are 350 to 400 in humans, 800 in dogs, and more than 1200 in nematodes. When I investigated whether nematodes could react to a small amount of odor using components that dogs could detect, nematodes responded at very low concentrations. Therefore, I concluded that nematodes have the same odor detection ability as dogs.

- Is the receptor gene that nematodes have more than 1200, always expressed? Or is the receptor protein expressed by any stimulus.

I think that these genes are always expressed, but I have not actually carried out such investigation. Most of them are presented as receptors because they are similar in sequence and almost none have a known function. For example, the correspondence between odorants (ligands) and receptors in nematodes has been confirmed only for two or three types. Moreover, research on mammalian olfactory receptors has progressed considerably more than research on those of nematodes. In fact, such assessments are delayed in nematode research.

- Is the memory function based on the receipt of certain odorant stimuli, which nematodes either like or dislike?

Nematodes have a simple neural circuit. Further, because the odorant stimulus is transmitted to a slightly more upstream nerve, a command is given to approach or escape from the odor.

- Let us discuss your career. After completing the master's course at the University of Tokyo, you joined the Suntory Holdings Limited. Did you work as a regular researcher there or was your research on nematodes?

I joined Suntory because I wanted to see the outside world for once. When I was a member of the Department of Biological Sciences, Graduate School of Science, the University of Tokyo, most of the students advanced to the doctoral course. Genuinely, I did not want to take the same route as everyone else (Lol). At that time, Suntory was developing a project to create blue roses, and I was interested in the project. So, at the job interview, I told them that I would like to make blue roses! and I was hired. However, I was assigned to the product development department, where I was involved in the development of Japanese tea products for the entire year. Although product development in a food manufacturing company is a "star department" and the job itself was interesting, I felt distanced from research. Although I enjoyed my work, I felt like I gave up my research on the way and wanted to resume this path for my doctoral course. As a result, I resigned in one year and returned to the same laboratory.

- I believe your experience of joining Suntory was beneficial to you.

Certainly. Carrying out research in a university laboratory for a long time creates a standard way in which we operate. However, business enterprises are repeatedly taught to work efficiently in a short time. This was a good opportunity for learning.

- This is a painful story for researchers in academia.

R & D of N-NOSE^®

- Could you shed light on how basic research on the olfactory sensory functions of nematodes ultimately progressed to the development of N-NOSE^®. First, could you talk about the background of the development?

Originally, I was part of the Faculty of Science, so I was honestly only interested in basic research. We employed nematodes to study the mechanism of olfaction. If there was a turning point, this would be the time I had my own laboratory. By having a laboratory, I had to acquire research funds. However, there were many researchers who carried out basic research on nematode olfaction, and I thought it was quite difficult to acquire the research funds. Around that time, I got the idea that the superior olfactory sensors of nematodes could be useful to society. I thought research such as laying nematodes on the pale odor of a certain component for a long time to see whether their lifespan would be extended, or whether their learning abilities are extended, might be useful for humans in the future. While completing many applications for research grants on such themes, I learned about the use of dogs for detecting cancers. I thought that the presence of a cancer odor would make nematodes more useful and started to do some research on this topic.

- Did you have any concerns that nematodes might have limited exposure to odor components as they live in solution? However, this may be less relevant because cancer cells are in body fluids.

Even if we derive very hard strategies, there are many parts where the results will be unknown until you actually perform the experiment. So, I took it easy and began by examining whether nematodes reacted to the odor of cancer. Regardless of the biological significance of the nematodes' reactions, it is important to determine whether nematodes react to the odor. Therefore, I felt that I could obtain good results if I attempted the assessment. Since I took pride in that, at that time, I conducted the most research analysis on whether nematodes would approach a certain odor or escape from it, I thought I could have the result after one to 1-2 months of trial and error.

- Your story reminds me of that of Dr. Yamanaka when he discovered the Yamanaka factor. Academically speaking, has the substance of cancer that nematodes respond to been discovered?

I also want to know that, as a researcher. Many researchers worldwide are trying to identify the odor of cancer. Despite decades of research and numerous papers, all of them are said to be fake. I was a basic researcher, not an entrepreneur. Therefore, I tried to identify cancer odors using nematodes; however, the cancer odor was too weak to be detected by the machine. Research is still ongoing, but it is quite difficult to carry out and it is also difficult to move forward. Other research teams might be in a more difficult situation because they do not use nematodes. I think they compare substances that can be detected by the machine alone, and they obtain false results. We use nematodes and I think we are coming closer to detecting cancer odor, but honestly, it is rather difficult.

- In the case of cancer, specific exosomes and miRNAs have been applied to drug discovery. Is there a mechanism for catching particles, such as extracellular vesicles, in nematodes?

I really want to know that, too. Currently, it is difficult to identify the substance of odors. As a result, we aim to identify the receptors that receive odor. Because the receptor is a gene, it is possible to determine this receptor. As different cancer types are said to have different odors, we conduct research using the corresponding receptor and it works well so far. It may also allow us to explore candidate odor substances from receptors.

- Thank you very much. It seems the basic part is still under development, and we can expect more in the future.

Certainly. I think it is a very interesting theme.

- I feel the general public thinks biomaterials would not be suitable for high-throughput screening and diagnosis. N-NOSE^® might not be high-throughput, could you tell us how fast and how many samples can be processed by N-NOSE ^®?

In terms of throughput, the behavioral analysis of nematodes was conducted manually in the laboratory. Previously, more analog work, such as individually counting the number of nematodes approaching the odor and the number of nematodes that escaped, using the human eyes to calculate the index. Last summer, we developed a fully automated analysis instrument. Currently, basically all behavioral analyses of nematodes are performed by the machines. Human tasks only involving carrying Petri dishes and specimen samples. The automated analysis machine can achieve a 50- to 100-fold throughput. We now own a few analysis machines, and 500 thousand screening tests are available per year.

- Did you develop the analysis machines in a tie-up manner with a vendor company?

That is correct. We cannot make machines obviously. However, it would have taken a longer time if I remained at a university. In that sense, by establishing a company and collecting funds, we could launch this development.

Establishment of HIROTSU Bio Science Inc.

- We are working on accelerating the practical application of drug discovery and development in academia. From that standpoint, how you established a company with your seed is the most important and interesting point. Could you explain the background of this venture?

In March 2015, we published a paper on the detection of cancer odor using the olfactory sensory function of nematodes. As I was still teaching at a university's School of Science, I left the practical application to another person. Moreover, I had no idea how to create a venture company. Thereafter, I received several offers for practical applications. My concern was if I left it to a company, especially a large company, the screening test might be sold at a high price of 100,000 yen. I wanted to sell the test at a lower price, which meant I had to get involved to some extent.

At that time, I had the most expertise and knowledge of the test, so I thought the practical application would be delayed if I were not involved. Without thinking of its own suitability as a manager, I changed my mind and decided to take the lead to make this technology ready for practical use. When I was interviewed in 2015, I answered at ease, "It would be in practical application in 10 years." However, in a practical sense, I thought it might not be possible to put it into practical application forever, not in 10 years as it was. I decided to start a business half-year after our paper was published. The first venture company was set up in Fukuoka; however, it failed without the occurrence of activities within a year. Thereafter, in August 2016, I decided to launch the next venture company in Tokyo and assume the position of the president. From that point, the current business was on track.

- When you first started a business in Fukuoka, were you still teaching at the university?

Yes, at that time, I was teaching at Kyushu University, and I had dual work. I ended my time at the School of Science but honestly, I knew nothing about business. Moreover, all advisors said, "Do not be the top of the company." They thought that a university faculty could not manage the business, and I accepted their opinion, but this opinion led to failure. Just around the time I closed the first venture company, I met the starting members of the current company. We agreed to start a business in Tokyo to aim for a world. I was strongly encouraged to be president as I embodied the technology, and I decided to do so despite being skeptical. I think the decision to be president was a good one, in terms of raising funds, as it was not convincing if executives, without knowledge of the technology, explained the business. It is not common for a forefront researcher to become president. However, as far as our company, it was the main factor that enabled its success. As I carried out my own research based on my own motivation without input from advisors around me, I thought it would be better to move the plan forward in a way that suites my own technology, while understanding what advisors say was a textbook. I would like to advise other researchers to take action with confidence and without fear.

- Between 2015 and 2016, the University of Tokyo started to provide support, and various systems became available for venture companies. Did you receive any support at that time?

Unfortunately, at the university where I worked, almost no support was provided. The situation was quite different from that of the University of Tokyo or Kyoto University. The University of Tokyo and Kyoto University were very enthusiastic about supporting venture companies originating from universities. At the other universities, I think there was almost no real support even if they ostensibly clarified support systems. Even with the support of a university, management of a company cannot be achieved through their support alone. Thus, once you start a venture company, you must be determined to live on your own rather than rely on a university.

- I have the impression that universities in the U.S. provide solid support. The University of Tokyo and Kyoto University provide a small amount of funding support, but I believe this is still insufficient.

That is correct. In addition, in terms of intellectual property, universities become obstacles. The patent for my research on nematodes was held by Kyushu University. I negotiated to buy the patent, but it did not work; this is because universities think about intellectual properties slightly different from the general public. It is not always the case that IP departments in universities have staff who are familiar with patents; therefore, universities should respond more flexibly to intellectual properties if they want to support ventures.

- Are you saying there are cases where the organization and operation in universities can be obstacles with regard to intellectual property?

Yes. I think these obstacles are still present at universities.

- What was the most difficult point in establishing a company?

The failure of the first venture company was a good lesson. First, I learned that the most straightforward path is for the person who embodies the technology to be the leader of the venture. In venture companies around me, there are cases where university faculty members are chief technology officers (CTOs), but there are only few cases where they are the president of the company. I feel a little disappointed that no matter the extent taken by the people in management to explain the business, it seems their goal is to make money. It would be good if more researchers took a plunge and became presidents to lead the business. I cannot say I have not had a hard time as the leader, but I think many people have come to understand our business. The second is how to raise funds. The textbook says to collect money from venture capital, but I tried to raise funds via other means and struggled in that aspect. However, I was not bothered by pressure from the venture capital. I think it is better to raise funds in a way that suits yourself.

- If you do not mind, please tell us specifically how you raised the funds.

Compared with the United States and other countries, venture capital in Japan invests a markedly lower investment amount; I could say a two-digit difference. Nevertheless, their demands are at the same level as those of their US counterparts. In addition, I have the impression that they interfere with the research. Because they did not understand our research content, we were told to not conduct unnecessary research when taking the company public. However, as a researcher, I am familiar with the fact that "useless research is worthwhile, and if we do not conduct that kind of research, we will not be trusted." Unfortunately, the venture capital did not understand that and asked us to be single-minded and to proceed to practical application. If I abided by their request, I thought that we could not take the company public. I then decided to raise funds outside of venture capital. However, only few companies, besides venture capitals, offered risk money. Accordingly, we set up a strategy, that is, while borrowing money from banks or receiving investment, we hired many researchers and conducted proof-of-concept experiments and a company invested to us at the time that practical application was nearly close.

- Is this an unusual way of receiving funding?

I do not recommend the way we acquired funding to everyone as it was risky up to the point of collaboration with the first business company. However, after that point, our stockholders are the business companies and banks only, and a venture capital was not involved.

- At that point, we could say that was a fair and square way, similar to in an ordinary company.

Yes. There were no stockholders who interfered with our research. It is thought that venture companies do not conduct basic research because they only carry out research for practical application; however, for our company, preparing the environment in which no one could interfere with researchers who wanted to performed basic research, was one of the keys to our success. The decision to not use a venture capital would have been impossible if I followed the textbook. In any case, it is better to carefully consider how to raise funds.

- Thank you for discussing the difficulties in drug discovery in academia and fundraising. What do you think is the reason for the bottleneck in drug discovery in Japanese academia?

In my case, I worked on a testing method, and not drug discovery. As a result, the cost for clinical trials was lower. For the clinical trial, I developed a strategy to give my right to write a paper to a physician. Accordingly, the clinical study changed in its form to a physician-initiated clinical trial, and I did not need to pay for that. With regard to drug discovery, billions are needed and how to earn that cost, which many venture companies suffer. It is probably impossible to proceed with clinical research using grants alone. I think that this is the reason for this bottleneck. In the case of drug discovery, venture companies may be good at finding seeds, but they do not have enough funding for further clinical trials. I think that practical applications will be delayed unless research and development is carried out in collaboration with a major pharmaceutical company with financial power.

- Currently, pharmaceutical companies are also looking for seeds. Therefore, is the point to create a flow in which venture companies discover seeds and carry out clinical research in tie-ups with pharmaceutical companies?

Many venture companies are listed at the seed stage to raise funds, but it is difficult to conduct clinical trials while making deficits without any remarkable outcomes. It is better to clarify the roles of each organization, such as universities, venture companies, and mega-pharma.

Achievements and prospects of N-NOSE^®

- Let me change the topic here and have you to talk about the achievements and prospects of N-NOSE^®.

We refer to N-NOSE^® as a primary screening method, which is different from other cancer tests. As symbolized by the conventional five major cancer tests, it is thought that people must take a test based on each cancer type. We explain N-NOSE^® as the first screening, which extensively examines the whole body and determines the possibility of cancer before proceeding to the next test. The number of people that understand the screening test is increasing, as we started to provide explanations. Moreover, our business has become full-fledged since the introduction of fully automated machines in November of last year (2020), and approximately 70,000 people have undergone screening tests to date. This is a very large number of new tests, but it is available in limited regions. Previously, a mechanism to freeze and transport urine was unavailable. As a result, it was difficult to build a pickup and delivery network. Freight services were only available from some metropolitan cities, such as Tokyo and Fukuoka, and there were many people who wanted the test but could not receive it. However, the freight service has expanded nationwide and has been available since May 2021. People from 47 prefectures are now able to take the test, and we expect that the number of tests will increase.

- This is similar to the problem of vaccine transportation for COVID-19. The problem of distribution was solved, and an increase in the demand was expected.

In the current situation, the rate of cancer screening at medical institutions is extremely low. However, as the perception that cancer is a rare disease has not changed, everyone should think cancer screening is important. N-NOSE^® only requires a small amount of urine and can be tested at home. We plan to expand the service to collect urine from homes nationwide. N-NOSE^® can be used for cancer screening at home. N-NOSE^® is a well-suited test for the situation of the coronavirus pandemic. I believe it is important to establish a platform for those who want to receive the test.

- Thank you very much. Specifically, how many types of cancers can be detected by N-NOSE^®?

Currently, 15 types of cancers are indicated. We would like to identify the type of cancer in the future, but we have not yet reached this point. Studies of hematologic and childhood cancers have been increasing presented at recent academic conferences. We would also like to increase the number of indications for cancer in the future.

- To improve the accuracy of identifying cancer types in the future, do you start from receptors, such as cloning receptors, and proceed to the use of more accurate methods, such as genetically-modified nematodes?

Yes. By genetic modification and knocking down a certain receptor, nematodes react in a different manner based on the cancer type. Research is progressing considerably, and we are working quickly to put it into practical application.

Future Innovations of HIROTSU Bio Science besides N-NOSE^®

- I am interested in the future innovations of your company, besides N-NOSE^®. Have you thought about any new business project that does not use nematodes?

Diseases besides cancer are also reported to have an odor; thus, we are trying to develop tests for other diseases. In addition, nematodes are not good worms, and there are many nematodes that parasitize crops and reduce yield. Therefore, we are currently developing a technology to control them. Several remarkable creatures exist in the world. In the future, it would be beneficial to detect diseases using various organisms, and not just nematodes. I may leave the company and return to the university or build a university, but I would like to expand my possibilities in various directions.

- Are you going to prepare and give lectures as endowed courses and social partnership courses?

Yes, we are already collaborating with Osaka University by creating such courses. I think the potential of universities is higher in nature. Accordingly, I would like to collaborate with other universities and do things companies cannot. We are also working on detecting cancers in pets rather than humans.

- Is that so? This is a very interesting story for us as veterinarians. Could you provide some details?

So far, we have been examining dogs and cats, and the results are quite good. As we might be able to conduct the screening tests in the same manner as that used for humans, we anticipate the practical application of N-NOSE^® for pets in a few years.

- We would be pleased if we can assist you with such practical applications.

Expectations to and Problems in Japanese Academia of Drug Discovery, and Expectations to AMED

- The end of this interview is approaching. Please feel free to talk to us if you have any expectations for drug discovery from Japanese academia, problems with social structures, expectations, or opinions for AMED, etc. at the end. We will post it as is. (Lol)

I have been involved in research at universities in Japan for a long time and I believe that the technology and inventive power used are comparable to those used in other parts of the world. As you all know, the characteristics of the Japanese society (i.e., research in academia will not lead to practical application) is very unfortunate. For practical applications, funding is important; however, organizations are more important. Even if AMED funding is promising, if the operation of the organization does not go well, the research only uses the funds to make a report. In other words, even if AMED grants money to famous researchers at famous universities, if they are not interested in practical applications, they will submit reports to basic research journals, which would be a disappointing result. Thus, to accelerate practical application, it is important to develop human resources and grant funds to researchers who aim to transform the technology into practical applications.

- I feel that witnessing the successes of familiar researchers will inspire me. Other academic researchers will also be interested in drug discovery synergistically. Moreover, the number of researchers who will be motivated to work in drug discovery will increase. I believe there are many success stories, and increasing the opportunities to hear these stories is very important.

I agree. I would like researchers younger than me to do their best to achieve the practical application of their technology. I want academic researchers to know that there are several different strategies to collect research funds besides traditional research funds and I would like to be a successful example of this. There are various strategies besides the strategy I employed; however, explaining without a successful example would not help to paint a clear image. If AMED and other government agencies promote successes and disseminate success stories to younger generations, their technologies will become easier to link to future practical applications.

- This is one of the missions of the catalyst unit. We hope that posting the content of this interview on our website will help Japan's excellent technology to be transformed into practical applications. Thank you very much for taking the time out of your busy schedule.

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※1: N-NOSE^® is a new cancer screening test that uses a superior olfactory sensor in nematodes to detect cancer odor in human urine. A nematode has approximately 1,200 olfactory receptors, which is 1.5-fold higher than that of dogs, with a body length of 1 mm. Nematodes are easy to breed at an inexpensive cost as they are hermaphroditic and feed on Bacillus coli.

July 2021