Ivermectin's solubility changed when incorporated in the physical mixture or the lyophilized form (0.0047 ± 0.0004, 0.1431 ± 0.0070, 0.6005 ± 0.0120 mg/mL, respectively). The reconstitution property of the lyophilized powder was evaluated by the addition of 3 mL of normal saline solution in vials containing 200 mg powder. Fig. 1 demonstrats the rapid dissolution of the lyophilized formulation after 5 s of adding the normal saline solution. The lyophilized solution's clarity was compared with the deionized water, which did not show any turbidity or drug crystallization, owing to the high solubility of the lyophilized powder in water, as previously mentioned.
Powder X-ray diffraction
The physical form was evaluated using powder X-ray diffraction for the pure drug, HP-β-CD, formulation physical mixture, non-medicated lyophilized formulation, and the prepared medicated lyophilized formulation (Fig. 2) in the range from 2 to 80° 2θ. Two prominent diffraction peaks revealed in the X-ray diffractogram indicating the drug’s crystalline nature at 2θ of 9.31°, and 13.09°.
X-ray diffraction pattern for: (1) ivermectin, (2) HP-β-CD, (3) physical mixture for ivermectin with HP-β-CD, (4) non-medicated formulation and (5) medicated formulation.
Due to the amorphous nature for HP-β-CD, X-ray diffraction pattern showed low intensities broad and diffused peaks. When the ivermectin and HP-β-CD were physically blended, their diffractogram revealed their combined diffraction patterns. Meanwhile, the physical mixture's diffraction peak intensity decreased, possibly due to powder dilution. The intense characteristic peaks for the crystalline structure for ivermectin were not detected in the diffraction pattern of ivermectin-HP-β-CD lyophilized formulation, but instead, a typical diffuse pattern as that for HP-β-CD was detected. The absence of characteristic ivermectin diffraction peaks, indicates effective transformation into an amorphous state.
To evaluate the relative degree of crystallinity (RDC), pure drug peak at 2θ-value of 9.31 (I°) was used for calculating the RDC. The calculated RDC-values were 0.647, and 0.003, for the formulation physical mixture, and lyophilized formulation, respectively.
3.3. Biochemical studies
Since all findings showed no statistical significance among the normal-control group in which the animals received saline (S) and those receiving the non-medicated cyclodextrin formulation (Cd), all comparisons were conducted against the Cd group.
3.3.1. Effect of ivermectin inhalation on the inflammatory and anti-inflammatory pulmonary cytokines levels
As depicted in Fig. 3, intratracheal administration of ivermectin at doses of 0.05 and 0.1 mg/kg did not result in any significant alteration in the pulmonary contents of the inflammatory cytokines (a) TNF-α, (b) IL-6 and (c) IL-13 as compared to Cd group. However, inhaled ivermectin at dose of 0.1 mg/kg has markedly declined levels of the anti-inflammatory molecule (d) IL-10 as compared to Cd group. In a comparable manner, starting from the ivermectin dose of 0.2 mg/kg, a profound dose-dependent increase in the levels of the inflammatory cytokines namely, TNF-α, IL-6 and IL-13 paralleled by a profound reduction in the anti-inflammatory IL-10 was distinguished.
Ivermectin-hydroxy propyl-β-cyclodextrin lyophilized formulation was prepared in 1:200 wt ratio. The lyophilized ivermectin formulation showed 127 and 30-fold increase in drug solubility compared to drug alone and drug in the physical mixture, respectively. Ivermectin X-ray diffraction patterns changed from crystalline pattern for pure drug to amorphous pattern for lyophilized formulation which revealed fast dissolution of the lyophilized powder.
This study also demonstrated the safety of different doses of inhaled ivermectin formulation with recommendation that lower doses namely, 0.05 and 0.1 mg/kg can be used as a potential treatment for COVID-19. Moreover, the current work was the first to show the probable deleterious impacts of higher doses of inhaled ivermectin (0.2, 0.4 and 0.8 mg/kg) on the lungs. This could be partially attributed to increased inflammatory and profibrotic states, as well as distorted lung architecture. The value of ivermectin in COVID-19 cases, however, requires further investigations to prove its risk/benefit profile.
Crredited to suzan M