Safety of inhaled ivermectin as a repurposed direct drug for the treatment of COVID-19...
Safety of inhaled ivermectin as a repurposed direct drug for the treatment of COVID-19: A preclinical tolerance study
The COVID-19 pandemic is arguably the world's most serious health epidemic and the biggest threat since the Second World War. Currently, available protocols for managing COVID-19 patients depend mainly on supporting patients, alleviating symptoms, and preventing respiratory and other organ failures. Although remdesivir, received Food and Drug Administration (FDA) authorization for the treatment of hospitalized COVID-19 patients, there are currently no other specific therapies approved by the FDA  for this indication. Thus, the world is in great need of developing novel medications or repurposing (repositioning) existing ones for other therapeutic applications to develop safe and efficient treatments for COVID-19. Numerous previously available medications used as treatments for malaria (chloroquine and hydroxychloroquine) , , SARS-CoV (lopinavir and ritonavir) , , influenza viruses (favipiravir and oseltamivir) , , virus C hepatitis (ribavirin and sofosbuvir) ,  and helminth/parasitic infections (ivermectin) were tested for treatment of COVID-19 , .
Ivermectin an FDA-approved antiparasitic drug that is used to treat several neglected tropical diseases, including onchocerciasis, helminthiases, and scabies ,  has demonstrated an excellent safety profile. Ivermectin is a mysterious multifaceted 'wonder' drug that keeps shocking and exceeding expectations . It was repositioned as a cancer drug ,  and showed potent antiviral activity against Zika , HIV-1, and dengue  viruses. Ivermectin was reported to inhibit the replication of SARS-CoV-2 in cell cultures  possibly through an RNA-dependent RNA polymerase (RdRp)-ivermectin complex, which is recognized as the most possible target for the in-vitro anti-SARS-CoV-2 activity of ivermectin , thus inhibiting coronavirus replication and transcription inside the host cell . Noteworthy, available pharmacokinetic data from clinically relevant and excessive dosing studies indicate that the SARS-CoV-2 inhibitory concentrations for ivermectin are much argued. Some authors reported that effective concentrations are not likely attainable in humans  and suggested that the required plasma concentrations necessary for the antiviral efficacy as detected in-vitro requires the administration of 100-fold the doses approved for use in humans ,  due to its poor solubility  and bioavailability . While others reported that ivermectin achieves lung concentrations over 10-fold higher than its reported EC50 . Even though ivermectin tends to accumulate in lung tissue, expected systemic plasma, and lung tissue concentrations are much lower than the in-vitro calculated half-maximal inhibitory concentration (IC50) against SARS-CoV-2 (~2 µM) . SARS-CoV-2-induced lung inflammation or injury could further greatly affect the ability of ivermectin to accumulate in the lung cells due to changes in the pulmonary microenvironment by inflammation provoked alterations in body temperature, enzymatic activity, and pH . Hence, the advantages of lung accumulation for ivermectin may be hampered during treatment of severe SARS-CoV-2 infection
Furthermore, ivermectin neurotoxicity has been raised by Chaccour et al., especially in patients with COVID-19-induced hyperinflammation. Furthermore, drug interactions with potent CYP3A4 inhibitors (such as ritonavir) necessitate a careful evaluation of co-administered medications. Finally, evidence indicates that achieving significant ivermectin plasma levels with COVID-19 activity would necessitate potentially toxic rises in ivermectin doses in humans .
Local ivermectin administration directly to the lung may represent a potential approach for the difficulties caused by the multiple biological barriers encountered in drug delivery. Over the past two decades, pulmonary drug delivery has gained much interest, offering an interesting route having several advantages over other drug delivery routes including high drug-loading efficiency, and enhanced absorption to the lung epithelium making the inhalation route an ideal drug delivery approach .
Many pharmaceutical researchers are interested in cyclodextrin (CD) complexation as its effectiveness has been demonstrated in improving the solubility, stability, and bioavailability of a variety of lipophilic active compounds , , , , , . CDs can form stable complexes with protein hydrophobic moieties that are vulnerable to aggregation, participate in hydrogen bonding with proteins, and have an intrinsic surfactant-like effect . Researchers investigated the possibility of using CDs to improve the solubility of non-polar medications for inhalation therapy. The superiority of hydroxypropyl-β-cyclodextrin (HP-β-CD) over other CD derivatives has been identified, and it has been documented to demonstrate surface-active properties that are needed for effective protein surface protection through spray freeze-drying , , .
HP-β-CD was used to formulate inhaled dry powder for salbutamol, and results confirmed the successful application of CDs in promoting lung delivery of drugs . Furthermore, Guan et al., reported the successful use of naringenin-HP-β-CD inhalation solution for nebulization to achieve a rapid response with a reduced dose for the treatment of cough . Milani et al. reported the ability to enhance the stability and aerosolization for the freeze-dried IgG formulation using HP-β-CD which acted as a water-replacement agent or a surfactant . The use of HP-β-CD in the treatment of chronic obstructive pulmonary disease has been reported via the inhalation route, owing to its ability to decrease the production of CXCL-1, a potent chemotactic agent for neutrophils in various inflammatory conditions and LPS-induced peribronchial inflammation .
The lyophilization process is used to remove the frozen solvent from a sample by sublimation, which involves freezing and then drying the sample at low temperature and pressure . Lyophilization is a pivotal drying process for pharmaceutical and biopharmaceutical products because such a process is energy-efficient, scalable, and the lyophilized finished product has low residual water content . Over the last five years, the use of lyophilization for both pharmaceutical and biopharmaceutical development has increased by approximately 13.5 percent per year . The freeze-drying method is adaptable, cost-effective, and simple to scale up. It's good for heat- and water-labile drugs, and it is supposed to be useful for changing the physicochemical properties of hydrophobic drugs. Furthermore, Doyle et al. evaluated different methods in the preparation of inclusion complexes with β-CD; namely, kneading, co-evaporation, and freeze-drying. Their results confirmed the superiority of the freeze-drying technique in improving the dissolution rate of the poorly water-soluble drug, dexamethasone acetate .
The suggested ivermectin doses in the treatment of COVID-19 are very high and this can increase its incidence of side effects. This problem can be solved by delivering ivermectin to the lung tissue by inhalation. The solubility of ivermectin should be enhanced in such a delivery system to increase its bioavailability. In March 2021, and based on the WHO, the decision to use the ivermectin in COVID-19 patients was inconclusive, and the WHO recommended that the drug can be used within clinical trials . Therefore, in this proposed work, ivermectin lyophilized formulation was developed using hydroxypropyl-β-cyclodextrin as a carrier. The safety of the proposed formulation on lung tissue was tested in male Wistar rats using histopathological and biological evaluations.
2. Materials and methods
2.1. Drugs and chemicals
Ivermectin was kindly provided by EgyEuro Animal Health Company, Egypt and it was originally purchased from North China Pharma Group Aino, China with technical purity of 97%. Hydroxypropyl-β-cyclodextrin (HP-β-CD) was kindly donated by Roquette, France. Tween 80 was purchased from El-Nasr Pharmaceutical Chemicals (Egypt).
Adult male Wistar rats weighing 200 to 220 g were obtained from the National Research Centre's breeding colony (NRC, Giza, Egypt). Before beginning any experimental procedure, animals were required to acclimate for one week in the animal facility of the Faculty of Pharmacy (Cairo University, Egypt). Under a 12:12 light-dark cycle, the rats were given unlimited water and a normal laboratory diet. This work was undertaken in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, Cairo University, Cairo, Egypt (PT-2968; 26/04/2021). All procedures were performed under thiopental sodium (50 mg/kg, i.p.) anesthesia.
2.3. Preparation of ivermectin formulation
Briefly, ivermectin was dissolved in distilled water in the presence of HP-β-CD as a carrier (1:200 wt ratio) to enhance ivermectin solubility. Furthermore, 0.02 w/v% Tween 80 was added to the solution. The prepared solution was frozen overnight at -80 °C, then the frozen solution was lyophilized in a Christ freeze dryer (ALPHA 2–4 LD plus, Germany) under a temperature of -80 °C and vacuum of 7 × 10−2 bar for 24 h. After the freeze-drying process, the dried powder was collected and stored in a tightly closed container.
2.4. Determination of ivermectin solubility
The solubility of the lyophilized ivermectin formulation was compared with the solubility for the drug alone and the formulation physical mixture. The samples were added in excess amounts in well-closed vials containing 3 mL of normal saline solution (reconstitution media for lung delivery). The samples were agitated at room temperature for 72 h using an incubator shaker (IKA KS 4000, Germany). After reaching an equilibrium where the solubility became constant, the samples were filtered using a cellulose membrane syringe filter with a pore size of 0.2 µm (Chmlab Group, Spain) to remove the insoluble ivermectin. After filtration, the samples were measured for drug concentration using an ultraviolet spectrophotometer (Shimadzu Spectrophotometer UV-1800, Japan) at 245 nm.
2.5. Reconstitution test
The reconstitution study of the lyophilized ivermectin formulation was performed in normal saline (reconstitution media for lung delivery). A proper amount of powder (200 mg) equivalent to 1 mg of ivermectin was added into vials containing 3 mL normal saline solution and shaken well for the reconstitution. Images were taken at different times to observe the reconstitution process using a digital camera (Nikon D5200, Japan).
2.6. Powder X-ray diffraction (XRD)
The crystalline structure of ivermectin pure powder, HP-β-CD, physical mixture, and lyophilized ivermectin formulation, in addition to its corresponding non-medicated formulation, were examined in a Scintag X-ray diffractometer (USA) using Cu-radiation with a nickel filter at a voltage of 45 kV, a current of 40 mA and scanning speed of 0.02°/sec. The reflection peaks between 2θ = 2° and 80°, the corresponding spacing (d, A°) were determined using HighScore Plus, Malvern Panalytical Ltd, UK and the relative intensities (I/I°) were determined by calculating the ratio between the height of a selected peak in the X-ray diffractogram in the lyophilized formulation (I) and its height in ivermectin diffractogram (I°) 
2.7. Lung toxicity study protocol
Forty-two animals were randomly and equally allocated into seven groups as follows; saline (S), non-medicated cyclodextrin formulation (Cd), and ivermectin formulations (I0.05, I0.1, I0.2, I0.4, and I0.8) administered the lyophilized ivermectin-cyclodextrin formula reconstituted in saline in doses of 0.05, 0.1, 0.2, 0.4 and 0.8 mg/kg, respectively for 3 successive days. These doses were selected based on the approved oral doses in humans. Ivermectin was given to rats after conversion of its human equivalent doses according to the formula of Phillips [Human dose normalized to body mass (μg/kg) = Animal drug dose per unit body mass (μg/kg)*(Animal body mass (kg)/ Human body mass (kg))^(1− constant) were 0.67 as the constant] . Rats were anesthetized with thiopental (50 mg/kg; ip) and the concentrations were adjusted so that each animal received 0.1 mL of the solution by intratracheal instillation. All rats were weighed daily, and by the end of the experiment (day 4), rats were deeply anesthetized by an overdose of thiopental. Blood samples were obtained from the heart after chest opening. Sera were separated for the estimation of surfactant protein-D (SP-D) using the corresponding rat ELISA kit and both lungs were quickly harvested. The left lung tissue was preserved in 10% formalin in saline for histological investigation, while the right lung tissue was sectioned into parts and stored at - 80 °C until assessed next for the chosen biochemical parameters using the respective western blot, PCR, or ELISA methods.
2.7.1. Quantification of serum level of SP-D and lung contents of TNF-α, IL-6, IL-13, IL-10, and ICAM-1
Serum levels of pulmonary surfactant protein-D (SP-D, MBS703468), as well as lung contents of tumor necrosis factor-α (TNF-α, MBS2507393), interleukin-6 (IL-6, MBS175908), interleukin-13 (IL-13, MBS355408), interleukin-10 (IL-10, MBS034393), and intracellular adhesion molecule-1 (ICAM, MBS267983), were determined using their respective ELISA kits (MyBioSource, CA, USA) according to the manufacturers’ guidelines.
2.7.2. Assessment of the protein expression of procollagen III N-terminal propeptide (PIII-NP)
For assessment of the protein expression of procollagen III N-terminal propeptide (PIII-NP), the western blot method was used . Briefly, lung tissues were homogenized in phosphate-buffered saline. Then, 10 μg protein from each lung sample was separated using the SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The nitrocellulose membrane was incubated with the anti-PIII-NP antibody (MBS2120628, (MyBioSource, CA, USA) overnight at 4˚C, and the formed blot was detected using enhanced chemiluminescence detection reagent (Amersham Biosciences, IL, USA). Results were expressed as arbitrary units against β-actin using image analysis software (Image J, version 1.46a, NIH, Bethesda, MD, USA).
2.7.3. Estimation of the gene expression monocyte chemoattractant protein-1 (MCP-1)
Gene expression of monocyte chemoattractant protein-1 (MCP-1) was estimated using the qRT-PCR technique. Following total RNA extraction (Invitrogen Life Technologies, Inc, CA, USA), Mx3000P real-time PCR system was used for the qRT-PCR (Stratagene, La Jolla, CA, USA) with a two-phase program including 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Each sample was examined in duplicate. Relative gene expression was calculated using the 2-ΔΔCt method. The primer sequences used for MCP-1 and the reference gene, β-actin, are listed in Table 1.
2.7.4. Histopathological examination of lungs
Lungs were removed and fixed in 10% formalin in saline for 72 h. All specimens were then washed, dehydrated, cleared, and embedded in paraffin. The paraffin-embedded blocks were sectioned at 5 µm thickness and stained with hematoxylin and eosin (H&E) for light microscopic examination (Olympus BX50, Tokyo, Japan) . A blinded pathologist scored the pulmonary histopathological changes in the experimental groups using a scoring scale from 0 to 4 for each lung damage parameter (congestion, edema, hemorrhage, thickening of interalveolar septa, and inflammatory cell infiltration) in five microscopic fields per section/rat (100x total magnification) .
2.8. Statistical analysis
The parametric data were expressed as means ± standard deviations (SD) and analyzed using the one-way analysis of variance (ANOVA) test followed by Tukey's Multiple Comparison Test. The non-parametric data (scores) were interpreted as medians and analyzed using the non-parametric ANOVA Kruskal Wallis test, followed by the posthoc Dunn's test. The GraphPad Prism1 software package for Windows, version 7 (GraphPad Software Inc., CA, USA) was used to carry out all statistical tests and drawings. For all statistical procedures, the degree of significance was held at p < 0.05.
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 demonstrates 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.
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