*3.3. Comparison of Binary and Ternary System Interactions*

In the BSA-ERL binary system, the quenching constants (Table 2) decreased at higher temperatures, suggesting the formation of a complex between BSA and ERL and a static quenching mechanism. Furthermore, the BSA-QUR system quenching mechanism was found to be in accordance with earlier studies [31,46] which reported a static quenching mechanism between them. For the ternary system (BSA-QUR)-ERL, the quenching constant in the presence of QUR for the BSA-ERL system was higher than in its absence. The rise of the quenching constant of the BSA-ERL system can be attributed to the fact that in the presence of QUR, the accessibility of ERL to BSA increases, improving the quenching efficacy of ERL. An earlier study reported a similar phenomenon whereby the quenching efficiency of gliclazide increased in the presence of QUR [43]. The fluorescence quenching plot for the binary system and the ternary system is provided in Figure 2a. In the BSA-ERL system, ERL reduced the fluorescence intensity of BSA by 19%. Whereas in the (BSA-QUR)- ERL system, the fluorescence intensity of BSA reduction was by almost 45%. Therefore, the presence of QUR in the BSA-ERL system further reduced the fluorescence intensity of the BSA-ERL system by 26%. The decrease in the fluorescence intensity of the BSA-ERL system suggests a strong influence of QUR in the BSA-ERL interaction.

**Figure 2.** Quenching curve for BSA (1.5 μM)-ERL(0.00–27.5) (**a**); and BSA (1.5 μM)-ERL(0.00–27.5 μM) in presence of QUR (5.5 μM) (**b**); Quenching curve (**c**) and Binding constant for ERL in the **Figure 2.** Quenching curve for BSA (1.5 µM)-ERL(0.00–27.5) (**a**); and BSA (1.5 µM)-ERL(0.00–27.5 µM) in presence of QUR (5.5 µM) (**b**); Quenching curve (**c**) and Binding constant for ERL in the presence of site markers phenylbutazone and ibuprofen.

Furthermore, QUR in the BSA-ERL system reduced the binding constants of the system, implying that the free drug fraction of ERL may rise in the systemic circulation in the presence of QUR. A study conducted in non-small cell lung cancer patients taking ERL reported that adherence to the treatment regimen of ERL depends on the severity of side effects in these patients [48]. It also reported that side effects were more severe in patients The BSA-ERL binary system's binding constant of <sup>≈</sup> <sup>10</sup><sup>4</sup> suggests moderate binding between the ERL and BSA. Our results corroborated earlier studies for the interaction between BSA and ERL [15]. The binding constants for the BSA-ERL system lowered with a rise in temperature (Table 2). In the other binding system that consisted of BSA-QUR, it was observed that QUR had a strong binding interaction with BSA. Some studies have reported a strong binding interaction between QUR and serum albumin [43,47]. As a result of this interaction between QUR and BSA, ERL, which is moderately bound to BSA, may not displace the bound QUR from the binding sites present on BSA. Therefore, a ternary

presence of site markers phenylbutazone and ibuprofen.

system was developed whereby the interaction between BSA and ERL was studied in the presence of QUR. Finally, it was concluded that QUR considerably affected the BSA-ERL system, and the presence of QUR caused a decrease in the binding constant of the BSA-ERL system from 2.0 <sup>×</sup> <sup>10</sup><sup>4</sup> to 0.2 <sup>×</sup> <sup>10</sup><sup>2</sup> . (Table 2).

The binding Site for ERL was identified using phenylbutazone and ibuprofen as site-specific markers for Site I and Site II of BSA, respectively. Quenching curve and binding constants of the BSA-ERL system in the presence of site markers were compared to the binding constant of the BSA-ERL system in the absence of site markers. Ibuprofen presence did not influence the quenching behavior of the BSA-ERL system (Figure 2b), and binding constants in the presence and absence of ibuprofen were similar (Table 2). Thus, these results rule out the binding of ERL to BSA Site II. However, the presence of phenylbutazone in the BSA-ERL system strongly influenced the quenching behavior and the binding constant of the BSA-ERL system (Figure 2c). Hence, it was concluded that ERL binds to Site I of BSA. Furthermore, since QUR also binds to Site I of BSA [43,47], the presence of QUR in the BSA-ERL system can markedly influence the interaction between the BSA-ERL system.

Furthermore, QUR in the BSA-ERL system reduced the binding constants of the system, implying that the free drug fraction of ERL may rise in the systemic circulation in the presence of QUR. A study conducted in non-small cell lung cancer patients taking ERL reported that adherence to the treatment regimen of ERL depends on the severity of side effects in these patients [48]. It also reported that side effects were more severe in patients with higher plasma area under the curve (AUC) for ERL. One of the most common adverse events related to the ERL treatment regimen is skin disorders, including acneiform rash, xeroderma (dry skin), pruritus, and paronychia. Sometimes, these side effects are so severe and necessitate treatment interruption or cessation [49]. One of the reasons attributed to the severity of these side effects for ERL is its plasma concentrations. Therefore, the severity of side effects influences patients' adherence to the treatment regimen. Since QUR may affect the AUC of ERL in plasma, increasing the plasma concentrations of ERL which might lead to unwanted adverse events affecting patients' adherence to the treatment regimen.
