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
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.
3.1. Determination of ivermectin solubility and reconstitution time
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.
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 , , . Hence, its use in higher doses may be associated with many systemic adverse events. The present work aimed, on one hand, to prepare an 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 .
Ivermectin-hydroxy propyl-β-cyclodextrin lyophilized formulation was prepared in a 1:200 wt ratio. The lyophilized ivermectin formulation showed a 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 a crystalline pattern for the pure drug to an amorphous pattern for a lyophilized formulation which revealed fast dissolution of the lyophilized powder.
This study also demonstrated the safety of different doses of inhaled ivermectin formulation with the 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 Suzanne. Mansour buy ivermectin | buy ivermectin India | buy ivermectin | buy ivermectin India | ivermectin tablet for humans | ivermectin tablet price ||ivermectin 12 mg tablet price in India | ivermectin buy online | where to buy ivermectin for humans | ivermectin dosage | where to buy ivermectin UK | ivermectin uses | ivermectin | Stromectol |buy ivermectin online | buy ivermectin online UK | buy ivermectin online NZ | buy ivermectin online south Africa | buy ivermectin online Malaysia | Buy Stromectol (ivermectin) Online at Lowest Price | Buy Ivermectin for Covid 19 Over the Counter | Buy Ivermectin for Humans and Ivermectin 3mg |