As illustrated in Fig. 6, lungs of (a) normal-control rats (S) revealed the normal histological lung architecture, normal alveoli, bronchioles and thin interalveolar septa. Similar observations were noticed in groups received (b) non-medicated formula (Cd), (c and d) ivermectin with doses of 0.05 and 0.1 mg/kg (I0.05 and I0.1, respectively). However, lungs of rats treated with (e and f) ivermectin in a dose of 0.2 mg/kg (I0.2) revealed mild histopathological alterations with slight focal thickening of the interalveolar septa and perivascular few inflammatory cells infiltration. Furthermore, severe pulmonary damage was seen in examined sections from rats treated with ivermectin dose of 0.4 mg/kg (I0.4) with (g) marked interalveolar septa thickening with inflammatory cells, (h) edema in the interlobular septa associated with inflammatory infiltrate, as well as focal aggregations of inflammatory cells in (h) lymphocytes and (i) macrophages. Similarly, lungs of rats treated with 0.8 mg/kg ivermectin (I0.8) exhibited more severe histopathological alterations demonstrated as (j) congestion of pulmonary blood vessels and focal hemorrhage, (j and k) marked thickening of interalveolar septa with inflammatory cells and (l) multifocal aggregations of inflammatory cells (mainly lymphocytes and macrophages) associated with perivascular inflammatory cells infiltration. These data are summarized as scoring of the collective and individual structural changes in panels m and n, respectively.
Fig. 6. Photomicrographs of lungs in ivermectin-treated rats (H&E; 200X). Sections of (a) normal control (S) group showing normal histological architecture of lung parenchyma with normal alveoli (AV) and bronchioles (B). Sections of (b) non-medicated (Cd), (c) I0.05 and (d) I0.1 demonstrating no histopathological alterations. Sections of (e & f) I0.2, (g, h & i) I0.4 and (j, k & l) I0.8 representing thickening of interalveolar septa with inflammatory cells(black arrow), perivascular inflammatory cells infiltration (blue arrow), interlobular edema (yellow arrow), congestion of pulmonary blood vessel (red arrow), hemorrhage (grey arrow) and focal inflammatory cells aggregation (asterisk) (scale bar = 100 μm). Panels m and n summarize scoring of the collective and individual changes of 5 randomly chosen non-overlapping fields, respectively using Kruskal-Wallis followed by Dunn’s multiple comparisons test (p < 0.05). S: saline, Cd: cyclodextrin, I0.05, I0.1, I0.2, I0.4, I0.8: ivermectin (0.05, 0.1, 0.2, 0.4. 0.8 mg/kg, respectively), C: congestion, E: edema, H: hemorrhage, TAS: thickening of interalveolar septa, IF: inflammatory cells infiltration.
Discussion
The usage of ivermectin in the management of COVID-19 is controversial. Literature existing pharmacokinetic and pharmacodynamic data show that SARS-CoV-2 inhibitory concentrations for ivermectin are not possibly achievable in humans due to its poor solubility and bioavailability [55], [56], [57]. Hence, its use in higher doses may be associated with many systemic adverse events. The present work aimed, on one hand, to prepare a HP-β-CD lyophilized readily soluble ivermectin formulation and, on the other hand, to assess the effect of intratracheal administration of this formulation on biochemical and histopathological changes in the lungs. It is postulated that ivermectin inhaled formulation is effective in SARS-CoV-2 infections. Hence, assessment of the risk–benefit profile of inhaled ivermectin is obliged [58].
Ivermectin is classified according to biopharmaceutical classification system (BCS) as class 4/3 drug, which is practically insoluble in water and has low permeability [59]. The prepared lyophilized ivermectin formulation showed 127-fold increase in drug solubility than the drug alone (p < 0.05). The enhancement of solubility is attributed to the complexation of the drug with the hydrophobic cavity of HP-β-CD, which increases the drug aqueous solubility [60], [61]. HP-β-CD molecule has primary, and secondary hydroxyl groups located on the narrow rim and wider rim of the molecule [62], respectively, responsible for its hydrophilicity [63]. Due to its hydrophilic outer surface and large number of hydrogen bond donors and acceptors, HP-β-CD can form hydrogen bonds with several drugs. Furthermore, ivermectin molecule has several exposed hydroxyl and ester groups [55), which can act as a hydrogen bond donor with the hydroxyl groups in HP-β-CD to form complex. HP-β-CD was chosen for this study because of its high water solubility (50 times greater than β-CD), low parenteral toxicity, and high biocompatibility and pharmacological inactivity, allowing it to be administered parenterally, orally, ophthalmically, and by inhalation [64]. Wang et al., presented a novel approach for complexing hydrophobic drugs as Ketoprofen and nitrendipine with HP-β-CD using lyophilization [60].
On the other hand, the drug in lyophilized form revealed 4-fold increase in drug solubility than the drug in a physical mixture (p < 0.05). This can be explained by the fact that, HP-β-CD in physical mixture acts only to decrease the interfacial tension between the drug and the aqueous medium, while the lyophilization process results in a complete inclusion complex with the drug and increases the aqueous solubility [65]. This improved solubility of the lyophilized powder is necessary for rapid reconstitution in aqueous media before use.
The rapid reconstitution and solubility of the lyophilized formulation are attributed to the amorphous complexation of the drug with HP-β-CD, which confirmed the results obtained by the X-ray diffraction study. Vass and his team formulated a reconstitution dosage form of voriconazole with HP-β-CD using an electrospinning process. The prepared complex showed complete reconstitution and a clear solution after 30 s of vigorous shaking [66].
The mutual crosstalk between aggravated pulmonary inflammation and fibrosis has been also pinned down here. Intratracheal instillation of ivermectin in doses of 0.2–0.8 mg/kg markedly enhanced the protein expression of the fibroproliferation marker, N-PCP-III, which goes in line with a previous report [77]. N-PCP-III is considered as an early response to lung injury [78] that liberated during the conversion of type-III procollagen to type-III collagen and correlates positively with the grade of pulmonary fibrosis [79]. Bejermer et al. showed that patients with idiopathic pulmonary fibrosis exhibited higher N-PCP-III levels with deteriorated lung function than those with stable disease [80]. These results were further supported as mentioned above by the elevated SP-D serum levels. Changes in SP-D structure and function have been involved in a wide assortment of pulmonary diseases. These include pneumonia [81], acute respiratory distress syndrome [82], cystic fibrosis [83], and interstitial fibrosis [84].
Another important consequence for the exacerbated pulmonary inflammation is the TNF-α mediated stimulation of the cell surface glycoprotein, ICAM-1 [85], [86] as observed herein following the administration of ivermectin in doses of 0.2 to 0.8 mg/kg. ICAM-1 performs a crucial role in the influx of neutrophils into the lung [87]. Increased levels of ICAM-1 have been noticed in cases of airway inflammation disorders and in many patients, ICAM-1 levels reflect the severity of the disease [88]. Furthermore, it was reported that ICAM-1 levels are positively correlated with increased pulmonary fibrosis [89]. To the authors knowledge, this is the first report examining the effect of ivermectin administration on ICAM-1 level.
The current investigation further demonstrated that intratracheal administration of ivermectin formulation in doses of 0.2 to 0.8 mg/kg was accompanied by an upshot in the gene expression of MCP-1, an important member of the chemokine family. MCP-1 is crucial in the development of inflammation [90]. Chronic and acute inflammation was reported to increase MCP-1 expression [91], [92]. MCP-1 stimulates mononuclear cells, macrophages, and induces cytokine expression by binding to its major receptor—CCR2 [93], that may perpetuate the inflammatory process as aforementioned. In contradistinction, topical treatment with ivermectin in several skin infections was shown to reduce the levels of MCP-1 [94], [95].
Conclusion
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.
Credited to reham N Shamma