Sinteza in karakterizacija karbamatnih analogov polisorbatov 20 in 80 - Raščan, Primož (2021)
Biološka zdravila sodijo v skupino najbolj prodajanih zdravil in število novih zdravil iz te skupine je v strmem porastu. Biološka zdravila so zelo heterogena skupina, ki zaobjema tudi rekombinantne biološke učinkovine, izmed teh pa so najpogostejša protitelesa. Pri formulacijah z zadnjimi je v zadnjih letih opazen trend povišanja koncentracij, ki pogosto presegajo 100 mg/ml. Pri visokih koncentracijah imajo proteini še večjo tendenco k denaturaciji in agregaciji. Da bi te procese preprečili, se k raztopinam proteinov dodaja PAS, predvsem PS20 ali PS80, ki sta prisotna pri več kot 80 % formulacij. Slednja sta neionski PAS, ki med drugim učinkovito preprečujeta denaturacijo proteinov oz. njihovo agregacijo. Uporabljata se v širokem razponu koncentracij, ki je v večini primerov v območju od 0,01 mg/ml do 1,00 mg/ml. K temu pripomore njun visok HLB in nizka CMC. Po drugi strani pa imata PS20 in PS80 tudi slabosti, saj sta oba dovzetna na oksidacijo in hidrolizo. Velik problem predstavlja predvsem hidroliza esterske vezi, ki je lahko encimsko ali neencimsko katalizirana. Produkti hidrolize izgubijo svoje amfifilne lastnosti, kar vodi v koloidno nestabilnost oz. so sami slabo topni in agregirajo (npr. maščobne kisline). Z namenom povečanja stabilnosti PS smo v magistrski nalogi pripravili štiri analoge PS20 in PS80, pri čemer smo estersko skupino zamenjali za kemijsko stabilnejši karbamat. V prvi fazi eksperimentalnega dela smo iskali ustrezne reakcijske pogoje in pogoje izolacije, ki bi omogočili iz PS20 oz. PS80 pridobiti nezaestren POE sorbitan, na katerega smo v drugi fazi vezali ustrezen izocianat. Uporabili smo dodecil in oktadecil izocianat. Samostojno smo pripravili tudi izocianat iz oleilamina in mešanico izocianatov, ki smo ju prav tako uporabili pri sintezi analogov. Karbamatnim produktom smo določili CMC in izračunali HLB vrednosti ter tako dokazali površinsko aktivne lastnosti, ki so ključne za stabilizacijo sistema. Na koncu smo želeli primerjati njihovo kemijsko stabilnost v primerjavi s PS20 in PS80. Le-te neposredno nismo dokazali, čeprav se ta nakazuje. S tem smo odprli pot do novih potencialnih polisorbat karbamatov za stabilizacijo terapevtskih proteinov. Biopharmaceuticals are one of the best-selling groups of drugs and the number of new drug applications is in incline. Biologics are a very heterogeneous group that also encompasses therapeutic proteins, of which the most common are antibodies. For formulations with the latter, a trend of increasing concentrations, often exceeding 100 mg/ml, has been observed in recent years. At high concentrations, proteins have an even greater tendency to denature and aggregate. To prevent these processes, surfactants, especially polysorbates PS20 or PS80, are added to protein solutions in more than 80% of formulations. The latter are non-ionic surfactants that, among other things, effectively prevent the denaturation of proteins or. their aggregation. They are used in a wide range of concentrations, in most cases ranging from 0.01 mg/ml to 1.00 mg/ml. Their high HLB and low CMC contribute to this. On the other hand, PS20 and PS80 also have disadvantages, as they are both susceptible to oxidation and hydrolysis. A major problem is the hydrolysis of the ester bond, which can be enzymatically or non-enzymatically catalyzed. The products of hydrolysis lose their amphiphilic properties, which leads to colloidal instability or are the products poorly soluble and aggregate (e.g. fatty acids). In order to increase the stability of PS, we prepared four analogs of PS20 and PS80 in the master's thesis, replacing the ester group with a more chemically stable carbamate. In the first phase of the experimental work, we developed appropriate reaction conditions and isolation procedure, that allowed us to obtain un-esterified POE sorbitan in high yield and purity from commercially available PS20. In the second phase corresponding isocyanate was reacted with un-esterified POE sorbitan to produce carbamate. Commercially available dodecyl and octadecyl isocyanate were used, however we also prepared an oleylamine isocyanate and a mixture of isocyanates from corresponding amines. CMC was determined for carbamate products and HLB values were calculated, thus proving the surface-active properties that are crucial for system stabilization. In the end, we tried to compare their chemical stability to PS20 and PS80. We could not directly prove this, although our experiments indicated this. This research paves the way for new potential polysorbate carbamates to stabilize therapeutic proteins.