## 4 Discussion  
These studies were designed and performed as a follow-up to the existing reports that suggested a possible carcinogenic effect of ACE-Is on humans, caused by the unknown mechanism. In this context, we intended to verify whether these actions could have been associated with the formation of toxic degradation impurities in the dosage forms of RAM, routinely administered to patients. We considered so, because the correlation between ACE-Is stability and toxicity, despite the existing theoretical rationale, still remains unexplored. We selected RAM as a model molecule due to its widespread clinical use, poor stability profile and incomplete stability information. Hence, to fulfill our research plan, we had to supplement the lack of data on RAM degradation kinetics under dry air and to then perform a well-structured toxicological assessment. Figure 6: The results of bacterial reverse mutation test with TA100 strain with metabolic activation for the nitrosation mixture of DKP (N-DKP) and pure DKP. The first step of our project aimed at providing a detailed description of RAM degradation behavior under the conditions of dry air, combined with the identification of the emerging degradation products. For that purpose, we developed and validated a HPLC stability-indicating protocol, which can now be also successfully applied for similar assays by manufacturers. Using this method, we managed to establish the kinetic parameters of RAM degradation under dry air and then to compare them with the corresponding ones, previously reported for RAM at humid conditions (RH 76%). Here, we found that, at RH0%, the degradation of RAM follows first-order kinetics, meaning that its rate solely depends on the concentration of the parent compound. First-order reactions are preferred for drugs, due to their predictable and easy-to-control progress. On heating, the degradation accelerates, as evidenced by increasing k values with temperature, yet its kinetic order remains unaltered (Table 1). For RAM under humid conditions, similar observations have been previously made. However, with the lack of environmental moisture, the rate of RAM degradation and its thermodynamics changed, as well as its degradation products' yield when compared to RH 76%. In detail, at RH 0%, the reaction progressed more rapidly when compared to RH 76%, as shown a by higher degradation rate constant and shorter half-life. Simultaneously, the E\({}_{\text{a}}\) of RAM degradation under dry air was increased relative to that reported for humid conditions. This means that, under dry air, the initiation of RAM degradation requires a higher energy input. Finally, the RAM degradation pathway was changed, as in the present study, it was found to follow a single intramolecular cyclization with the formation of DKP. On the contrary, the degradation of RAM in the presence of moisture involved two parallel reactions, i.e., hydrolysis and cyclization, with the former one being dominant. Hence, unlike dry air conditions, under humid conditions, the major degradation impurity was RAM diacid. The main reason for these variations between dry and humid environments is the obvious lack of water molecules to participate in hydrolytic bond cleavage in the environment of dry air. Thus, at RH0%, intramolecular cyclization is the only possible degradation pathway. Under humid conditions, in turn, although cyclization actually occurs, it is very limited in scope. This is probably caused by the fact that it requires a higher E\({}_{\text{a}}\) than hydrolysis. Hence, it is not favored. As a consequence, in the presence of moisture, DKP is only minimally formed, while under dry air its yield equals 100%, as shown in our experiments (Figure 1C). Furthermore, despite a high energy barrier between substrates and the activated complex under dry air (high E\({}_{\text{a}}\)), once initiated RAM cyclization proceeds very fast. Probably, this is caused by the two-stage nature of this process, as shown in Figure 5. Firstly, the adoption of an appropriate molecular conformation by RAM occurs.