FEQ group continues working to make the processing of tacrolimus cheaper, which prevents antibody attacks on grafts
At least since the end of 2016, the press has been reporting problems in the distribution of an essential immunosuppressant for patients who have received kidney, liver, pancreas and heart transplants: tacrolimus, which combined with other drugs prevents antibodies from attacking the so-called graft. ; If it is not taken regularly, the person may lose the transplanted organ and, in some cases, die. In Brazil, which has the largest public organ transplant system in the world, tacrolimus – also recommended for autoimmune diseases such as rheumatoid arthritis, bronchial asthma and dermatological disorders such as vitiligo – is included in the SUS list of high-cost medications (Sistema Unified Health System).
A group of researchers from Unicamp has been working in recent years to reverse this scenario, producing tacrolimus in the laboratory, using alternative sources that increase the production of the drug and lower the cost for the health system. “Dependence on medication also brings psychological suffering to the transplant recipient, who faces a queue at the SUS hoping to get the medication or not. The person only keeps graft survival under control thanks to the continuous supply of the medicine”, emphasizes Professor Marco Aurélio Cremasco, who coordinates research at the Faculty of Chemical Engineering (FEQ).
The Pharmaceutical Assistance Coordination, a São Paulo body with the mission of guaranteeing and expanding access to medicines for the population, released a note stating that it had requested, for the first quarter of 2019, 592,2 thousand 5mg tacrolimus tablets and 10,3 million of 1mg, but received from the federal government, respectively, 436,1 thousand and 6,4 million units. Research carried out in Campinas, where Unicamp's Hospital de Clínicas (HC) is located, one of the main transplant centers in the country, raised prices for tacrolimus on the market ranging from R$680 to R$1.234 for 100 1 mg capsules - the high The cost is attributed, among other issues, to production not yet consolidated in Brazil, to a low production of tacrolimus in fermentation and to the complex purification process.
Marco Aurélio Cremasco has already supervised two doctoral studies and one master's degree involving tacrolimus, defended in 2018 and 2019, with another master's thesis in progress. “Tacrolimus is a secondary metabolite, produced via fermentation of several bacteria of the genus Streptomyces, in particular the S. tsucubaensis. This drug was discovered in the 1980s by researchers from Fujisawa Pharmaceuticals, led by Professor [Tohru] Kino, who verified its immunosuppressive activity for the kidney and then the liver. In 1994, the FDA [Food and Drug Administration] adhered to medication. Previously, transplant recipients used cyclosporine to prevent graft rejection, but it was discovered that tacrolimus is around ten times more effective, which brings a huge advantage, such as reducing the dose.”
Fermentation
It was up to student Jean Vinícius Moreira to produce tacrolimus in a liquid medium using bacteria S. tsukubaensis, imported from Germany, from the fermentation of maltose and glucose as primary carbon sources, as well as soy peptone (substrate) and steep corn liquor as nitrogen sources – and with success. In this doctoral thesis, the author provides a history of tacrolimus, since it was identified in a sample of broth fermented by that bacteria, isolated from soil in the Mount Tsukuba region, in Japan. “In 1987, scientists from Fujisawa Pharmaceutical Company had already elucidated the chemical structure of tacrolimus (FK506), as well as its potent immunosuppressive effect, the fermentation conditions, the drug purification techniques and the physicochemical and biological characteristics.”
Jean Moreira remembers that the Fujisawa group was a pioneer, but once the patent expired, other companies entered the market, which has high economic potential. “In 2016, the sale of medicines derived from tacrolimus reached 1,8 billion dollars, a value that represents 15,4% of the global market for immunosuppressants. At the In Brazil, where 95% of transplants are carried out with public resources, tacrolimus was initially approved for sale with specific indications for rescue in kidney and liver transplants. Between 2010 and 2014, InCor (USP Heart Institute) studied the use of tacrolimus in heart transplants and, in 2015, the drug began to incorporate immunosuppression protocols for the heart as well.”
In the researcher's opinion, his thesis contributed to making the Unicamp group's original idea viable, which was to first produce tacrolimus, which no one had done in academia in Brazil. “We achieved a maximum productivity of between 112 and 116 milligrams per liter. As therapeutic protocols generally indicate 1mg to 2mg per day for the transplant recipient, 112mg seems to be a lot. However, this amount is in the fermented broth, and the entire purification process is still missing until the drug reaches the patient – considering the losses, the result is a low value. In any case, we can say that I reached a high productivity throughout the research, starting from 10 mg in the first fermentation to a scale twelve times larger.”
Jean Vinícius Moreira obtained data from 2015 on the cost of transplants in Brazil, which exceeds R$ 1 billion per year, with R$ 300 million of this total being allocated to patient maintenance. “I don't know if the production of tacrolimus meets demand in the country, but we had recurrences of non-supply in 2016 and 2017, and the second one last year, I don't know if it was due to costs or low productivity. The Brazilian pharmaceutical company Libbs produces tacrolimus at its Embu das Artes [SP] unit, while Johnson & Johnson is the world leader in the production of not only the anti-rejection drug but also the ointment used to treat vitiligo and other disorders. dermatological.” Libbs, which entered into a partnership with Fiocruz (Farmanguinhos) to transfer national technology, estimates that 25 thousand kidney and liver transplant recipients consume tacrolimus in the country.
Purification
Wilson Murilo Ferrari, in his doctoral research, completed the next stage for the production of tacrolimus by S. tsukubaensis, with an innovative process for purifying fermented broth. “The aim of the work was to develop a robust and unprecedented process for purifying tacrolimus from any fermentative medium. The processing consists, in general terms, of pre-purifying the broth, with the purification sequence using chromatographic techniques and crystallization of the final product”, explains the author of the thesis. “Initially, I would only work on the purification of tacrolimus, but we thought it necessary to include the production part, adopting different carbon sources, especially Brazilian oils, with Brazil nut oil showing the best results.”
Ferrari explains that the production of tacrolimus is complex precisely because of the purification process, and because it is a molecule with naturally low productivity. “From a technical point of view, in many processes we have problems isolating it: tacrolimus is a large molecule, having more than 40 carbons in its structure, in addition to being produced together with other analogous molecules, sometimes with a single different bond. , which makes it difficult to establish a process that can differentiate small connections. That’s why we’re looking for a refined purification system.”
According to the researcher, a very high purity was reached, greater than 99%. “This is very important, firstly, because the therapeutic window for applying the medicine is very small, that is, the small amount supplied to the patient requires high purity. But it is a very potent immunosuppressant within the body. Due to the low quantity, the priority is quality and optimization of the process to reduce the cost of producing the drug. We can still improve productivity with some adaptations, but the fundamental thing has already been done: developing the entire process, from tacrolimus production to its purification. Obtaining an organ is the biggest difficulty for a transplant, so losing it causes enormous suffering.”
Patent at INPI
Professor Cremasco informs that Ferrari's thesis on the processing of tacrolimus resulted in the filing of a patent at the INPI (National Institute of Industrial Property), and that the bacteria Streptomyces tsukubaensis It is stored at CPQBA (Multidisciplinary Center for Chemical, Biological and Agricultural Research) at Unicamp for continued research. “With the patent filing we open the possibility of expanding the production scale of tacrolimus. We already know the path of the stones and the quest to transfer technology to industry is the responsibility of Inova – Unicamp's Innovation Agency, which has the expertise for this. Currently, we seek to anticipate the scale-up [increased scale] of several stages in the processing of the drug”, highlights the FEQ professor.
Séforah Carolina Marques Silva's master's thesis, recalls Cremasco, brought good results in the addition of national elements to the fermentation process – such as Brazil nut and coconut oil – for the production of tacrolimus. Alessandra Suzin Bertan's master's degree is now underway, which aims to separate the biomass produced in the fermentation of the broth using membrane microfiltration. “During fermentation, tacrolimus is surrounded by, in addition to bacterial biomass, several other components, such as vitamins and sugars”, says Alessandra Bertan, who developed a specific filtration strategy for this purpose. “We are testing the efficiency of the filter in retaining micelles to obtain, in future stages, the purest tacrolimus possible.”
Marco Aurélio Cremasco observes that in research prior to Alessandra's, biomass filtration was vacuum, on a bench, which makes scaling up difficult. “In the future, this filter that we developed will allow us to increase scale, not to mention that it is national technology, which also impacts the final price of the drug. Furthermore, there is an effective technological contribution: the various stages of engineering processing always have something innovative in relation to what is already known, such as the elimination and replacement of process steps. And the end result is social contribution, which I understand is also one of Unicamp's objectives. In other words, we can do science and develop innovative technology with strong social commitment”, concludes the professor.