Ivermectin is already deployed to treat a variety of infections and diseases, most of which primarily afflict the world&#;s poor. But it is the new opportunities with respect to ivermectin usage, or re-purposing it to control a completely new range of diseases, that is generating interest and excitement in the scientific and global health research communities.
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Ivermectin is registered for human use primarily to treat Onchocerciasis and strongyloidiasis, and, in combination with albendazole, to combat Lymphatic filariasis, as well as being increasingly used &#;off-label&#; to combat a variety of other diseases. Oral treatments are commonplace, but ivermectin doses have also been given successfully per rectum, subcutaneously and topically (Figure 5). Ivermectin has now been used for over three decades to treat parasitic infections in mammals, and has an extremely good safety profile, with numerous studies reporting low rates of adverse events when given as an oral treatment for parasitic infections.50 Several problematic reactions have been recorded, but they are generally mild and usually do not necessitate discontinuation of the drug.
Figure 5Ivermectin has been formulated in a variety of ways, for example, as an injectable solution for livestock (a); donated as tablets for human use to treat Onchocerciasis (b); and as a commercial tablet preparation for scabies and strongyloidiasis (c). (Photo credits: Andy Crump). A full color version of this figure is available at The Journal of Antibiotics journal online.
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In addition to the gradual appreciation of the diverse and invaluable health and socioeconomic benefits that ivermectin use can provide, research is currently shedding light on the promise that the drug still harbors and the prospects of it combatting a new range of diseases or killing vectors of various disease-causing parasites.
The following are an indication of the divergent disease-fighting potential that has been identified for ivermectin thus far:
Myiasis is an infestation of fly larvae that grow inside the host. Surgical removal of parasites is often the only remedy but unavailable to many of the needful people who live in poor, rural tropical communities where myiatic flies thrive. Oral myiasis has been successfully treated with ivermectin,51 which has also been used effectively as a non-invasive treatment for orbital myiasis, a rare and preventable ocular morbidity.52
Globally, approximately 11 million individuals are infected with Trichinella roundworms. Ivermectin kills Trichinella spiralis, the species responsible for most of these infections.53
Ivermectin is highly effective in killing a broad range of insects. Comprehensive testing against 84 species of insects showed that avermectins were toxic to almost all the insects tested, including the vectors of malaria and critical neglected tropical diseases such as leishmaniasis and trypanosomiasis (see below). At sub-lethal doses, ivermectin inhibits feeding and disrupts mating behavior, oviposition, egg hatching and development.54, 55
Mosquitoes (Anopheles gambiae) that transmit Plasmodium falciparum, the most dangerous malaria-causing parasite, can be killed by the ivermectin present in the human bloodstream after a standard oral dose.56, 57, 58, 59 Meanwhile, it has been demonstrated that even at sub-micromolar levels, ivermectin inhibits the nuclear import of polypeptides of the signal recognition particle of P. falciparum (PfSRP), thereby killing the parasites. Consequently, in combination with other anti-malarial agents, ivermectin could become a useful, novel malaria transmission control tool.60, 61 The use of ivermectin as an additional malaria control weapon is now receiving increased attention, driven by the growing importance of outdoor/residual malaria transmission and the threat of insecticide resistance. One outcome has been the creation of the &#;Ivermectin Research for Malaria Elimination Network&#;.62
Ivermectin has been proposed as a possible rodent-bait feed-through insecticide to help control the Phlebotomine sandfly vectors that transmit Leishmania parasites.63, 64 Experiments to test the impact of ivermectin on one blood-feeding sandfly vector, Phlebotomus papatasi, demonstrated that they die if the blood feed is 1&#;2 days post treatment. Although Leishmania major promastigotes have been shown to die or lose their infectivity after exposure to ivermectin, it does not have a major impact against L. major. Nevertheless, ivermectin is more effective in killing promastigotes than rifampicin, nystatin and erythromycin.65, 66 For cutaneous leishmaniasis, ivermectin is more effective than other drugs (including pentostam, rifampicin, amphotericin B, berenil, metronidazole and nystatin) in killing Leishmania tropica parasites in vitro and by subcutaneous inoculation, with accelerated skin ulcer healing.60 When combined with proper surgical wound dressing, ivermectin shows significant promise for curing cutaneous leishmaniasis.67
Tsetse flies (Glossina palpalis) fed on ivermectin-treated animals die within 5 days, demonstrating that ivermectin has promise to help control these African trypanosomiasis vectors.68, 69 Effective in killing tsetse flies, experiments in mice infected with Trypanosoma brucei brucei parasites have also shown that ivermectin treatment doubled their survival time, suggesting that there is scope for investigating the use of ivermectin in the treatment of African trypanosomiasis from several aspects.70
When dogs infected with Trypanosoma cruzi parasites suffered a tick infestation, ivermectin treatment eliminated the ticks but had no impact on either the dogs or their infection. Triatomine bug vectors of T. cruzi feeding on the dogs relatively soon after treatment displayed high mortality, which declined rapidly as the interval between ivermectin treatment and blood feed increased.71
Schistosoma species are the causative agent of schistosomiasis, a disease afflicting more than 200 million people worldwide. Praziquantel is the sole drug available for controlling schistosomiasis, with schistosome-resistant parasites now becoming an increasingly worrying problem.72, 73 Ivermectin is a potent agonist of glutamate-gated chloride channels and as glutamate signaling has been recorded in schistosomes,74, 75 there may be an ivermectin target in the tegument. Workers in Egypt evaluating the effect of ivermectin on mice infected with Schistosoma mansoni, concluded that ivermectin has promising anti-schistosomal effects. It has potential due to its schistosomicidal activity on adult worms, especially females, and its ovicidal effect, in addition to its impact in improving hepatic lesions.76, 77 It has also been reported that ivermectin can kill Biomphalaria glabrata, intermediate host snails involved in the schistosomiasis re-infection cycle, reinforcing the prospect of using ivermectin to help control one of the world&#;s major neglected tropical diseases.78, 79
Bedbugs are parasitic insects of the Cimicidae family that feed exclusively on blood. Cimex lectularius, the common bedbug, feeds on human blood, with infestations increasing significantly in poor households across North America and Europe. Ivermectin is highly effective against bedbugs, capable of eradicating or preventing bedbug infestations.80
Although the broad-spectrum anti-parasitic effects of ivermectin are well documented, its anti-inflammatory capacity has only relatively recently been identified. Ivermectin is used &#;off-label&#; to treat diseases associated with Demodex mites, such as blepharitis and demodicosis, oral ivermectin, in combination with topical permethrin, being a safe and effective treatment for severe demodicosis.81 Demodex mites have also been linked to rosacea, a chronic skin condition that manifests as recurrent inflammatory lesions. Long-term treatment is required to control symptoms and disease progression, with topical medicaments being the first-line choice. Ivermectin 1% cream is a new once-daily topical treatment for rosacea lesions, more effective and safer than all current options,82 which has recently received approval from American and European authorities for the treatment of adults with rosacea lesions.
A study investigated the impact of ivermectin on allergic asthma symptoms in mice and found that ivermectin (at 2&#;mg&#;kg&#;1) significantly curtailed recruitment of immune cells, production of cytokines in the bronchoalveolar lavage fluids and secretion of ovalbumin-specific IgE and IgG1 in the serum. Ivermectin also suppressed mucus hypersecretion by goblet cells, establishing that ivermectin can effectively curb inflammation, such that it may be useful in treating allergic asthma and other inflammatory airway diseases.83
Nodding syndrome (NS) is a mysterious and problematic form of epilepsy that occurs in parts of South Sudan and northern Uganda. It is also endemic in a locus in Tanzania but, there, the prevalence is low and stable.84, 85 The condition has serious socioeconomic implications and, like other forms of epilepsy, generates profound social stigma.86 The obvious outward feature of NS, which afflicts children and adolescents, is a paroxysmal bout of forward and downward head movement, the nodding episodes representing epilepsy seizures.87 Children with NS display varying levels of mental retardation, often alongside notable stunted growth and failure to develop secondary sexual characteristics (hyposexual dwarfism). Affected children are outwardly healthy until the nodding episodes begin, with several dying due to uncontrolled seizures.84 The cause of NS remains unknown but there appears to be an unexplained link with Onchocerciasis infection.88, 89, 90 The African Programme for Onchocerciasis Control, which operated in the three afflicted countries, adopted mass drug administration of ivermectin in . However, it was not always possible to operate in conflict-affected regions. After the civil war in northern Uganda ceased, biannual ivermectin distribution in districts affected by both Onchocerciasis and NS since has coincided with a substantial drop in the number of new NS cases. No new cases were reported in , although there is no conclusive evidence to prove any connection.91
Many neurological disorders, such as motor neurone disease, arise due to cell death initiated by excessive levels of excitation in central nervous system neurons. A proposed novel therapy for these disorders involves silencing excessive neuronal activity using ivermectin. Because of its action on P2X4 receptors, ivermectin has potential with respect to preventing alcohol use disorders92 as well as for motor neurone disease.93 Indeed, in , Belgian scientists applied for a patent, &#;Use of ivermectin and derivates thereof for the treatment of amyotrophic lateral sclerosis&#; (Publication No.: WO//A3), to cover &#;the use of ivermectin and analogs, to prevent, retard and ameliorate a motor neuron disease such as amyotrophic lateral sclerosis and the associated motor neuron degeneration&#;.
Recent work has elucidated how ivermectin binds to target receptors and helped explain its selectivity for invertebrate Cys-loop receptors. Combined with emerging genomic information, species sensitivity to ivermectin can now be predicted and the molecular basis of ivermectin resistance has become clearer. In humans, Cys-loop neurotransmitter receptors, particularly those activated by GABA, mediate rapid synaptic transmission throughout the nervous system and are crucial for intercellular communication. They are key factors in fundamental physiological processes, such as learning and memory, and in several neurological disorders, making them attractive drug targets.94 Improved understanding of the stereochemistry of ivermectin binding will facilitate the development of new lead compounds, as anthelmintics as well as treatments for a wide variety of human neurological disorders.95, 96
Recent research has confounded the belief, held for most of the past 40 years, that ivermectin was devoid of any antiviral characteristics. Ivermectin has been found to potently inhibit replication of the yellow fever virus, with EC50 values in the sub-nanomolar range. It also inhibits replication in several other flaviviruses, including dengue, Japanese encephalitis and tick-borne encephalitis, probably by targeting non-structural 3 helicase activity.97 Ivermectin inhibits dengue viruses and interrupts virus replication, bestowing protection against infection with all distinct virus serotypes, and has unexplored potential as a dengue antiviral.98
Ivermectin has also been demonstrated to be a potent broad-spectrum specific inhibitor of importin α/β-mediated nuclear transport and demonstrates antiviral activity against several RNA viruses by blocking the nuclear trafficking of viral proteins. It has been shown to have potent antiviral action against HIV-1 and dengue viruses, both of which are dependent on the importin protein superfamily for several key cellular processes. Ivermectin may be of import in disrupting HIV-1 integrase in HIV-1 as well as NS-5 (non-structural protein 5) polymerase in dengue viruses.99, 100
Up until recently, avermectins were also believed to lack antibacterial activity. However, in , reports emerged that ivermectin was capable of preventing infection of epithelial cells by the bacterial pathogen Chlamydia trachomatis, and to do so at doses that could be used to counter sexually transmitted or ocular infections.101 In , researchers confirmed that ivermectin was bactericidal against a range of mycobacterial organisms, including multidrug resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the authors suggesting that ivermectin could be re-purposed for tuberculosis treatment. Although other researchers found that ivermectin does not possess anti-tuberculosis activity, the results were later shown to be non-comparable due to differences in testing methods, with the original findings being confirmed by further work in Japan.102, 103, 104 Unfortunately, the potential use of ivermectin for tuberculosis treatment is doubtful due to possible neurotoxicity at high dosage levels. Ivermectin was also reported to be bactericidal against M. ulcerans,105 although other researchers found no significant activity against this bacterium.106
There is a continuously accumulating body of evidence that ivermectin may have substantial value in the treatment of a variety of cancers. The avermectins are known to possess pronounced antitumor activity,107 as well as the ability to potentiate the antitumor action of vincristine on Ehrlich carcinoma, melanoma B16 and P388 lymphoid leukemia, including the vincristine-resistant strain P388.108
Over the past few years, there have been steadily increasing reports that ivermectin may have varying uses as an anti-cancer agent, as it has been shown to exhibit both anti-cancer and anti-cancer stem cell properties. An in silico chemical genomics approach designed to predict whether any existing drugs might be useful in tackling glioblastoma, lung and breast cancer, indicated that ivermectin may be a useful compound in this respect.109
In human ovarian cancer and NF2 tumor cell lines, high-dose ivermectin inactivates protein kinase PAK1 and blocks PAK1-dependent growth. PAK proteins are essential for cytoskeletal reorganization and nuclear signaling, PAK1 being implicated in tumor genesis while inhibiting PAK1 signals induces tumor cell apoptosis (cell death).
PAK1 is essential for the growth of more than 70% of all human cancers, including breast, prostate, pancreatic, colon, gastric, lung, cervical and thyroid cancers, as well as hepatoma, glioma, melanoma, multiple myeloma and for neurofibromatosis tumors.110
Globally, breast cancer is the most common cancer among women but treatment options are few. Ivermectin suppresses breast cancer by activating cytostatic autophagy, disrupting cellular signaling in the process, probably by reducing PAK1 expression. Ivermectin-induced cytostatic autophagy also leads to suppression of tumor growth in breast cancer xenografts, causing researchers to believe there is scope for using ivermectin to inhibit breast cancer cell proliferation and that the drug is a potential treatment for breast cancer.111 Triple-negative breast cancers, which lack estrogen, progesterone and HER2 receptors, account for 10&#;20% of breast cancers and are associated with poor prognosis. Tests using a peptide corresponding to the SIN3 interaction domain (SID) of MAD, found that the SID peptide selectively blocks binding of SID-containing proteins to the paired α-helix domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial&#;mesenchymal transition. An in silico screen identified ivermectin as a promising candidate as a paired α-helix domain-binding small molecular weight compound to inhibit SID peptide, ivermectin phenocopying the effects of SID peptide to block SIN3-paired α-helix interaction with MAD, inducing expression of CDH1 and ESR1, and restoring tamoxifen sensitivity in mass drug administration-MB-231 human and MMTV-Myc mouse triple-negative breast cancers cells in vitro. Ivermectin addition led to transcriptional modulation of genes associated with epithelial&#;mesenchymal transition and maintenance of a cancer stem cell phenotype in triple-negative breast cancers cells, resulting in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo.112
It has been reported that ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells and it has also been suggested that ivermectin synergizes with the chemotherapy agents cytarabine and daunorubicin to induce cell death in leukemia cells, with researchers claiming that ivermectin could be rapidly advanced into clinical trials.113 This potential has been supported by reports that ivermectin displays bioactivity against chronic lymphocytic leukemia cells and against ME-180 cervical cancer cells.114 Additionally, ivermectin has been shown to potentiate doxorubicin-induced apoptosis of drug-resistant leukemia cells in mice.115 Cancer stem cells are a key factor in cancer cells developing resistance to chemotherapies and these results indicate that a combination of chemotherapy agents plus ivermectin could potentially target and kill cancer stem cells, a paramount goal in overcoming cancer.
Ivermectin inhibits proliferation and increases apoptosis of various human cancers. Over-expression of P2X7 receptors correlates with tumor growth and metastasis. However, ATP release is linked to immunogenic cancer cell death, in addition to inflammatory responses caused by necrotic cell death. Exploiting ivermectin as a prototype agent to allosterically modulate P2X4 receptors, it should be possible to disrupt the balance between the pro-survival and cytotoxic functions of purinergic signaling in cancer cells. Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to ATP-rich tumor environments, providing an explanation of the tumor selectivity of purinergic receptor modulation, confirming ivermectin&#;s potential to be used for cancer immunotherapy.116 Activation of WNT-TCF signaling is implicated in multiple diseases, including cancers of the lungs and intestine, but no WNT-TCF antagonists are in clinical use. A new screening system has found that ivermectin inhibits the expression of WNT-TCF targets. It represses the levels of C-terminal β-catenin phosphoforms and of cyclin D1 in an okadaic acid-sensitive manner, indicating its action involves protein phosphatases. In vivo, ivermectin selectively inhibits TCF-dependent, but not TCF-independent, xenograft growth without side effects. Because ivermectin has an exemplary safety record, it could swiftly become a useful tool as a WNT-TCF pathway response blocker to treat WNT-TCF-dependent diseases, encompassing multiple cancers.117
Researchers have recently reported a direct interaction between ivermectin and nematode and human tubulin, even at micromolar concentrations. When added to human HeLa cells, ivermectin stabilizes tubulin against depolymerizing effects and prevents replication of the cells in vitro, although the inhibition is reversible. This suggests that ivermectin binds to and stabilizes mammalian microtubules. Ivermectin thus affects tubulin polymerization and depolymerization dynamics, which can cause cell death. Again, given that ivermectin is already approved for use in humans, its rapid development as an anti-mitotic agent offers significant promise.118
The increasing resistance to antiparasitic drugs and limited availability of new agents highlight the need to improve the efficacy of existing treatments. Ivermectin (IVM) is commonly used for parasite treatment in humans and animals, however its efficacy is not optimal and the emergence of IVM-resistant parasites presents a challenge. In this context, the physico-chemical characteristics of IVM were modified by nanocrystallization to improve its equilibrium water-solubility and skin penetration, potentially improving its therapeutic effectiveness when applied topically. IVM-nanocrystals (IVM-NC) were prepared using microfluidization technique. The impact of several process/formulation variables on IVM-NC characteristics were studied using D-optimal statistical design. The optimized formulation was further lyophilized and evaluated using several in vitro and ex vivo tests. The optimal IVM-NC produced monodisperse particles with average diameter of 186 nm and polydispersity index of 0.4. In vitro results showed an impressive 730-fold increase in the equilibrium solubility and substantial 24-fold increase in dissolution rate. Ex vivo permeation study using pig's ear skin demonstrated 3-fold increase in dermal deposition of IVM-NC. Additionally, lyophilized IVM-NC was integrated into topical cream, and the resulting drug release profile was superior compared to that of the marketed product. Overall, IVM-NC presents a promising approach to improving the effectiveness of topically applied IVM in treating local parasitic infections.
Previous studies have explored various dermal delivery routes for IVM, including solid lipid nanoparticles ( Guo et al., ), lipid nanocapsules ( Ullio-Gamboa et al., ), microemulsions ( Das et al., ), and nanoemulsions ( Da, ). However, to the best of our knowledge, this research represents the first attempt to investigate the use of nanocrystals for reformulating IVM into a topical product to enhance its dermal delivery and antiparasitic efficacy. The objective of this work was to design, optimize, and characterize IVM-NC as a potential therapeutic alternative for the treatment of parasitic skin infections. The optimized formulation selected through statistical design was subjected to lyophilization to improve its physical stability and facilitate its incorporation into topical products, such as cream. This formulation was evaluated through a series of in vitro and ex vivo studies to determine its effectiveness compared to IVM raw material (IVM-RM) and physical mixtures (PM) of IVM with selected stabilizers. Furthermore, the optimized IVM-NC topical formulation was compared to the commercially available Soolantra Cream (10 mg/g, Galderma International), and its stability was assessed.
Ivermectin (IVM) is a mixture of avermectin A 1a , 5-O-demethyl-22,23-dihydro-(component H 2 B 1a ), and avermectin A 1a , 5-O-demethyl-25-de(1-methylpropyl)-22,23-dihydro-25-(1-methylethyl)-(component H 2 B 1b ) belonging to the macrocyclic lactone family ( Sharun et al., ; USP, ). It is a broad-spectrum antiparasitic drug against a variety of parasites. It has been a popular drug of choice for the treatment of different types of parasite infestations, such as scabies, rosacea, head lice, trichuriasis, river blindness (onchocerciasis), lymphatic filariasis, and strongyloidiasis ( Das et al., ; Atmakuri et al., ). It is available in several commercial forms including creams, lotions, and tablets, however, their effectiveness is limited due to IVM low water solubility and poor ability to penetrate the skin. IVM is classified as a Biopharmaceutics Classification System (BCS) Class II drug, and it is also a substrate for P-glycoprotein, which may assign it as BCS Class IV as well ( Das et al., ; Starkloff et al., ). In treating parasitic infections such as scabies, where the parasites are located in the deeper layers of the skin, topical IVM has been found to be more effective compared to oral administration ( Das et al., ). However, prolonged use of antiparasitic agents can lead to the emergence of resistant organisms ( Bruschi, ). Therefore, it is crucial to develop new formulations that can improve IVM dermal delivery and enhance its antiparasitic efficacy.
All measurements were carried out in triplicates and values were presented as mean ± SD. Statistical analysis was conducted using Prism 9 (Version 9.5.1, GraphPad Software, San Diego, CA, USA). A two-tailed unpaired Student's t-test was used to compare two groups, while one-way ANOVA followed by Tukey post hoc test was employed for multiple comparisons. A p-value <0.05 was considered statistically significant.
An isocratic reversed-phase validated HPLC method ( USP, ) was used for the detection and quantification of IVM using photodiode-array (PDA) detector (Waters Nova Framingham, MA, USA) equipped with a quaternary pump, auto-sampler unit, and UV detector set at 254 nm. The stationary phase was a reversed-phase C18 column (4 μm particles size) (Waters Nova-Pak C18, Framingham, MA, USA). The mobile phase was a mixture of acetonitrile, methanol and water (53:27.5:19.5) and the flow rate was 1 mL/min. The mobile phase was filtered through a 0.45 μm pore-size membrane filter. The run time was 5 min, and the injection volume was 10 μL. The regression equation for the calibration curve was as shown in eq. (2) with a regression coefficient of R 2 = 0.. The assay was linear in the concentration range of 0.&#;1.0 mg/mL.
To compare the release of IVM from the compounded IVM-NC cream (1% w/w IVM) to marketed Soolantra Cream (1% w/w IVM), in vitro release experiments were conducted. The study utilized a water bath shaker (Grant OL5 Aqua Pro, UK) and involved placing equal amounts of both creams (0.5 g) in dialysis bags (cellulose membrane, Mw cut off 14,000 Da, Sigma-Aldrich, USA) that were properly tightened. The bags were then placed in 250 mL conical flasks filled with 250 mL PBS (pH = 7.4) ( Ahmed et al., ; Najm et al., ) to mimic the conditions of infected skin, at a temperature of 32 °C. The samples were shaken at 100 rpm to minimize the unstirred water layer effect. At 10-, 30-, 60- and 120-min intervals, samples of 3 mL were withdrawn from each flask and replaced with equal volumes of PBS (pH = 7.4). The samples were then filtered through 0.2 μm filter and analyzed for IVM content by a microplate reader (Synergy H1, BioTek, Germany) at 240 nm ( D'Souza, ). The concentration of the released drug was determined and plotted against time. The measurements were conducted in triplicates (n = 3).
The ex vivo permeation studies were carried out using six Franz cells and a V-series stirrer (V6-CB, PermeGear, USA), as previously reported ( Rawas-Qalaji et al., ; Aodah et al., ; Bafail et al., ; Najm et al., ). To maintain the integrity of the stratum corneum (SC) lipids, temperature control at 32 ± 0.5 °C was crucial and achieved using a water bath (JULABO GmbH, BC4, Germany). Adult pig ear skin was obtained from a local butcher (Ajman, UAE) and used for the study due to its comparable structural and permeation kinetics with human skin. The dorsal skin was separated from the underlying cartilage with forceps and scalpel (blades No. 20 and 11), and cleaned to remove the subcutaneous fat, hair, and blood vessels. The selected intact skin patches were washed with PBS (pH = 7.4), wrapped in aluminum foil, and stored at &#;20 °C. prior to the study, the frozen skin was moistened with PBS (pH = 7.4) until completely defrosted at ambient temperature and placed on Franz cells with a diffusional area of 3.14 cm 2 . The skin patches were divided into two sets, where only one of them was soaked in a solution of acetonitrile and water in the ratio of 3.5:6.5 for 24 h before the experiment (treated skin), while the other set was not exposed to the organic solvent (untreated skin). In order to assess the impact of the organic solvent utilized in IVM solution preparation on the skin integrity, the experiment was conducted on IVM-NC-S using both treated and untreated skin simultaneously. The SC faced the donor compartments, which were filled with 3 mL of IVM-NC-S or IVM solution (prepared in acetonitrile and water in the ratio of 3.5:6.5), while the receptor compartments were filled with 15 mL of PBS (pH = 7.4). Samples of 100 μL were taken from the receptor medium and replaced with fresh medium at specific intervals (1, 2, 4, 8, 12, and 24 h), followed by HPLC analysis. The cumulative drug concentration that permeated into the receptor medium was plotted against time. All experiments were done in triplicates (n = 3), and the mean values (±SD) were recorded.
The physical stability of the optimal IVM-NC-S and IVM-NC-L was evaluated for a period of three months at two different storage conditions: 25 °C/60% relative humidity (RH) using a Climacell stability chamber (MMM Group, Germany) and 5 ± 3 °C using a refrigerator. Prior to storage and after 1, 2 and 3 months, the PS, PDI and ZP of the samples were analyzed. The stability samples were freshly prepared and stored in glass vials wrapped in aluminum foil. All measurements were conducted in triplicates (n = 3) ( Starkloff et al., ; Verma et al., ).
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The morphology of the optimal IVM-NC-S as well as IVM-NC-L were determined by transmission electron microscopy (TEM) operated at a voltage of 200 kV. The sample was loaded on a copper&#;gold carbon grid and left to be air-dried at room temperature. Then, the grid was placed in the vacuum chamber of the electron microscope (JEOL-, Jeol Ltd., Tokyo, Japan) and images were captured using different magnification powers ( Shen et al., ). The IVM-RM was captured through scanning electron microscope (SEM). To prepare the SEM sample, a small amount of dispersed IVM-RM was placed on a clean slide cover and allowed to dry in a vacuum. Afterwards, the sample was attached to carbon tape and coated with gold using a high vacuum sputter module. The coated sample was then scanned, and images were captured using a 3 kV acceleration voltage (Thermo Scientific Apreo SEM, FEI Company, Hillsboro, OR, USA).
Powder XRD patterns of IVM-RM, SDS, IVM-SDS-PM in 1:4 ratio, and the optimal IVM-NC-L were determined using an X-ray diffractometer (D8 Advance, Bruker, Germany). Diffractograms were created using a Cu radiation source (λ = 1. Å) with a maximum voltage of 40 kV and a maximum current of 40 mA over the 2θ range of 5° to 60°.
The United States Pharmacopoeia (USP) monograph method was employed to establish the dissolution profiles of the previously prepared samples outlined in section 2.6.1 (IVM-RM, IVM-SDS-PM, IVM-NC-L), which were equivalent to 20 mg of IVM. A USP paddle apparatus (ERWEKA DT820 Dissolution Apparatus, ERWEKA GmbH, Germany) was used for the dissolution tests, which were conducted at 32 °C in 900 mL of acetate buffer solution with a pH of 5.5, which is the pH of normal skin ( Sheshala et al., ). Samples of 3 mL were taken at different time intervals (5, 10, 15, 20, 25, 30, 35, 40, 45, and 60 min) and replaced with an equal volume of acetate buffer (pH = 5.5). The samples were then filtered through a 0.2 μm syringe filter and analyzed for drug content at 240 nm using a microplate reader (Synergy H1, BioTek, USA). The dissolved drug percentage was determined using the calibration curve equation as before and then plotted against time. All tests were conducted in triplicates (n = 3).
To determine the equilibrium solubility of IVM raw material (IVM-RM), IVM physical mixture with SDS (IVM-SDS-PM), and the optimal IVM-NC-L formulation, the shaking flask method was employed (USP general chapter). In this method, an excess amount of powders equivalent to 10 mg IVM was taken from each sample and added to conical flasks containing 10 mL of acetate buffer (pH = 5.5) to ensure supersaturation conditions. The flasks were placed in a benchtop shaking incubator (Labnet International Inc., USA) and shaken at 50 rpm at room temperature for 24 h to reach equilibrium. The resulting samples were filtered through a 0.2 μm filter, diluted with acetate buffer (pH = 5.5) as required, and analyzed for IVM concentration at 240 nm using a microplate reader (Synergy H1, BioTek, USA). The concentration of the dissolved IVM was calculated using the calibration curve equation and then plotted against time. The regression equation for the calibration curve was as shown in eq. (1) with a regression coefficient of R 2 = 0. and the assay was linear in the concentration range of 2&#;20 μg/mL. All tests were conducted in triplicates (n = 3).
The Malvern Instruments' Zetasizer Nano ZS-90 utilizing photon correlation spectroscopy dynamic light scattering (DLS) was used to determine the average PS, PDI, and ZP. Prior to analysis, the IVM-NC-S samples were suitably diluted with purified water, while IVM-NC-L samples were redispersed then diluted in purified water. Additionally, the surface charge of the optimal IVM-NC was quantified by measuring the ZP at a scattering angle of 173° and a temperature of 25 °C. The mean value ± standard deviation (SD) of three replicates was calculated (n = 3).
To compare the drug release of the lyophilized IVM-NC (IVM-NC-L) with the commercial product Soolantra Cream (1% w/w IVM, Galderma, France), the IVM-NC-L was incorporated into a cream formulation. The IVM-NC cream was composed of 1% IVM (w/w), 4.1% (w/w) liquid paraffin, 48% (w/w) white soft paraffin ointment, 10% (w/w) propylene glycol, and 36.9% (w/w) water containing 0.21% citric acid ( Ahmed et al., ).
After selecting the optimal IVM-NC suspension (IVM-NC-S) using the experimental design, it was combined with 1% mannitol as a cryoprotectant and frozen at &#;80 °C using the Innova U725-G freezer (New Brunswick Scientific, Canada). The frozen nanosuspension was then subjected to lyophilization using a freeze drier (VirTis BenchTop Pro with Omnitronics&#;, USA) at &#;102 °C and 200 mT for 36 h to obtain dry powder lyophilizate (IVM-NC-L).
In accordance with the experimental design, IVM-NC were prepared using a top-down approach via the microfluidization technique ( Rawas-Qalaji et al., ; Verma et al., ). Increasing amounts of IVM (ranging from 0. mg to 0.05 mg) and Food and Drug Administration (FDA) approved, biocompatible stabilizers such as Tween® 80, PVA, and SDS were added to 50 mL of purified water, to result in a drug concentration range of 0.025% to 0.1% (w/v) and a stabilizer-to-drug ratio of 1 to 4. The resulting mixture was then processed in a microfluidizer (M-110P V3, Microfluidics Corporation, USA) under a pressure of 30,000 psi for 1 to 5 cycles. In instances where homogenization was necessary as per the experimental design, the mixture was homogenized prior to microfluidization at 10,000 rpm for 10 min using a high-speed homogenizer (T-25 digital Ultra Turrax, IKA-Werke GmbH & Co., Germany).
The chosen model for response prediction was assessed for its significance, and 3D-response surface plots were generated for each response to assess the degree of factor interaction. The outcomes were visually examined. The optimal values for the dependent variables were determined based on the criteria of the smallest PS, lowest PDI, and highest absolute ZP. For optimization, both numerical and graphical analyses were carried out utilizing the desirability function. Desirability values range from 0 to 1, and values closer to 1 were reported to indicate the most desired response ( Oscar et al., ). After selecting the optimal IVM-NC preparation, further investigations were conducted.
presents 34 experimental runs (formulations) generated by the Design-Expert® software, which includes five replications. All responses were fitted concurrently to linear, two-factor interaction (2FI) and quadratic response surface models. The resulting polynomial equations were subjected to automatic statistical validation using analysis of variance (ANOVA), and interactions of three or higher order were disregarded for simplicity. Statistical parameters such as p-value, lack-of-fit p-value, adjusted multiple correlation coefficient (Adjusted-R 2 ), predicted multiple correlation coefficient (Predicted-R 2 ), and multiple correlation coefficient (R 2 ) were assessed to ensure the significance of the selected model. The model with the maximum Adjusted-R 2 and Predicted-R, 2 with a minimal difference between the two parameters and an insignificant lack of fit, was selected. To enhance the model's predictability and eliminate any potential bias, the experimental runs were conducted in random order.
To create a novel formulation for a pre-existing drug, it is essential to recognize the potential Critical Material Attributes (CMAs) and Critical Process Parameters (CPPs) and have a clear understanding of how they influence the desired Critical Quality Attributes (CQAs) of the final product. In this study, D-optimal response surface design was utilized to optimize IVM-NC using Design-Expert® software (Version 13.0, Stat-Ease Inc., Minneapolis, MN, USA). Three numerical discrete factors and two categorical factors were chosen as CMAs/CPPs to investigate their impact on the selected CQAs. These factors were: (1) number of microfluidization cycles (X 1 ), (2) drug concentration (X 2 ), (3) stabilizer to drug ratio (X 3 ), (4) type of stabilizer (X 4 ) and (5) homogenization prior to microfluidization (X 5 ). The levels of independent variables were chosen in a way that ensures an appropriate design space while also allowing for feasible processing of the NC. ( ) . Three responses were selected to monitor closely for the optimization of the studied factors: (1) particle size (PS, Y 1 ), (2) polydispersity index (PDI, Y 2 ) and (3) Zeta Potential (ZP, Y 3 ). IVM-NC formulations were optimized for the three responses with a target of achieving the smallest PS, lowest PDI and highest ZP to obtain the highest overall desirability value. The optimal independent variables were then used to prepare the optimal IVM-NC for further testing and incorporation into the topical cream.
IVM was received as a gift from Hovione PharmaScience Limited (Taipa, Macau). High Performance Liquid Chromatography (HPLC) grade Acetonitrile (Honeywell, Germany), Ethanol (Honeywell, Germany), Methanol (Sigma-Aldrich, France) and Orthophosphoric Acid 85% (VWR, France) were used. Also, the following chemicals were used: Acetic Acid (Sigma-Aldrich, Germany), Cetostearyl Alcohol (Spectrum Chemical MFG. CORP, USA), d-Mannitol (Sigma-Aldrich, China), Liquid Paraffin (Merck KGaA, Germany), Paraffin Wax (Sigma-Aldrich, Switzerland), Phosphate Buffered Saline (PBS) Tablet (Sigma-Aldrich, USA), Poly(vinyl alcohol) (PVA) with average molecular weight of 30,000&#;70,000 Da (Sigma-Aldrich, Netherlands), Propylene Glycol (Sigma-Aldrich, USA), Sodium Acetate Trihydrate (Sigma-Aldrich, Germany), Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, Japan), Tween® 60 (Sigma Life Science, USA) and Tween® 80 (Sigma-Aldrich, Germany). Soolantra 1% w/w Cream (Lot No. , Expiry Date 09/, Galderma, France) was used in this study as a reference comparable product.
The response surface analysis of the D-optimal design was utilized to optimize the preparation of IVM-NC formulation with desired CQAs. The goal was to minimize the PS and PDI while maximizing the absolute ZP of IVM-NC. The PDI represents the width of particle size distribution of IVM-NC, where values closer to 0 indicate monodisperse NC distributions (Nidhi et al., ). A narrow PDI minimizes the variability in equilibrium solubility between particles of different sizes, and consequently minimizes Ostwald ripening (Starkloff et al., ). Also, particles with ZP values in the range of &#;10 to +10 mV are considered neutral while particles with ZP of more than +30 or less than &#;30 are preferred to ensure particles repulsion, thus preventing their aggregation (Clogston and Patri, ). To achieve this, a simultaneous optimization approach was employed, where the highest priority value was assigned for PS, followed by PDI then ZP.
Based on the optimization results, the IVM-NC formulation prepared with SDS as a stabilizer in a 4:1 SDS to IVM ratio and a concentration of 0.1% (w/v) IVM was found to be the most desirable. The formulation was homogenized and microfluidized for three cycles. The optimized formulation exhibited a desirability value of 0.725, which indicates a high level of optimization for all the CQAs ().
Open in a separate windowThe results suggest that utilizing a QbD approach led to the development of a straightforward method for preparing IVM-NC formulation with the desired PS (<200 nm), appropriate PDI, and ZP. After optimization, the most suitable formulation was selected for further examination. The predicted and observed responses of the optimized IVM-NC formulation are listed in . The observed values fell within the predicted values ± SD range for all responses.
After preparation, the resulting formulation was subjected to lyophilization using a matrix forming agent with cryoprotective action (1% w/v mannitol) to enhance the stability of the IVM-NC and reduce agglomeration and PS growth upon standing (Van Eerdenbrugh et al., ). The lyophilized powder was then subjected to comprehensive physico-chemical characterization tests. Subsequently, the powder was incorporated into a cream preparation to conduct comparable in vitro release studies with the marketed Soolantra cream.
The TEM was employed to visualize the morphology of the optimal IVM-NC formulation in both nanosuspension and lyophilized powder forms. The TEM micrographs of the IVM-NC-S revealed that the particles were spherical with a narrow size distribution. The particles were uniformly shaped and did not display any signs of aggregation. The TEM micrograph and the DLS spectra of IVM-NC-S are shown in -A and B, respectively. Similarly, the TEM micrograph of the IVM-NC-L exhibited comparable characteristics, indicating that the nanosuspension remained stable during the freeze-drying process. The TEM micrograph and the DLS spectra (following redispersion) of IVM-NC-L are shown in -C and D, respectively.
Open in a separate windowIn contrast to the IVM-NC, the IVM-RM exhibited an irregular crystalline particle shape with a size range of 6.68 μm to 15.45 μm, as observed by SEM (-E). The significant reduction in particle size achieved through the nanocrystallization technique used in this study is crucial for improving the pharmacodynamic profile of the drug upon topical application.
A short-term physical stability study was conducted on both IVM-NC-S and IVM-NC-L for three months under two different storage conditions: 25 °C/ 60% RH (accelerated conditions) and 5 ± 3 °C (long-term conditions). The results of the study are illustrated in . The study found that both formulations showed good stability profiles throughout the study period under both storage conditions with statistically insignificant changes related to the PS, PDI and ZP. The findings for IVM-NC-S coincide with the results of other studies that reported remarkable stability for 1% IVM-NC-S at similar accelerated conditions for six months (Starkloff et al., ) and indomethacin nanosuspensions stored at the same accelerated and long-term conditions for 28 days (Verma et al., ).
Open in a separate windowThe water solubility of IVM is extremely poor. The goal of IVM nanocrystallization was to enhance its ability to penetrate the skin to treat parasitic infections. Nanocrystals have been found to enhance skin permeability and drug deposition compared to conventional topical formulations and applied drug solutions (Najm et al., ; Pelikh et al., ). They achieve this by augmenting the drug supersaturation solubility and increasing drug adhesion and retention time at the site of application, thus leading to a higher concentration gradient for passive diffusion of the drug (Ghasemiyeh and Mohammadi-Samani, ). The efficiency of IVM-NC in penetrating the skin layers was evaluated using pig ear skin. Porcine skin from the ear is commonly employed to assess transdermal drug permeation since it shares structural similarities with human skin, including hair growth density, the existence of Langerhans cells, the thickness of the SC and viable epidermis, glycosphingolipids and ceramides contents, and the arrangement of collagen fibers in the dermis (Neupane et al., ).
The permeation profiles of IVM-NC-S and IVM solution in acetonitrile and water (3.5:6.5) through the entire pig ear skin were compared. illustrates the results, indicating that the rate and extent of IVM-NC-S permeation to the receptor chamber were lower than those of IVM solution at 2 and up to 24 h of the experiment, however, the difference was insignificant. The total percentage of IVM that permeated the skin into the receptor chamber was calculated as 34.3% for IVM-NC-S (untreated skin), 25.9% for IVM-NC-S (treated skin) and 44.8% for IVM solution.
Open in a separate windowTo estimate the quantity of IVM retained in the skin, the amount of IVM remaining in the donor chamber at the end of the experiment was determined for both formulations. The results showed that IVM-NC-S using untreated or treated skin had significantly lower amount of drug left in the donor chamber, with 4.3% and 4.6 respectively, and 32.8% for IVM solution (p < 0.05). This might be attributed to the enhanced passive drug diffusion. Given the comparable amount of drug permeated through the skin and the significant difference in the percentage of drug left in the donor chamber, it can be estimated that the IVM retained in the skin is significantly higher for IVM-NC-S (61.3% for untreated skin and 69.5% for treated skin) compared to IVM solution (22.3%), as shown in . Additionally, there was no significant difference between the two IVM-NC-S sets using untreated or treated skin, which indicates that the applied organic solvent (acetonitrile in water, 3.5:6.5) was suitable and did not impact the integrity of the skin.
Open in a separate windowWhile a significant portion of the initial IVM solution remained in the donor chamber, possibly indicating a low concentration gradient, the majority of the dissolved drug passed through the skin into the receptor chamber and was not retained. In contrast, the IVM-NC-S showed high skin retention, indicating its potential for effectively treating skin diseases while minimizing systemic absorption. These findings suggest that a combination of optimized PS and surfactant concentration can further enhance the efficacy of NC for dermatological applications.
This study aimed to evaluate the release of IVM from IVM-NC cream in comparison to the commercially available Soolantra Cream (1% w/w IVM, Galderma, France) by incorporating IVM-NC-L into a cream preparation (1% w/w IVM). It has been reported that incorporation of NC into cream formulations enhances drug penetration into the skin particularly when compared to hydrogels or oleogels thanks to the inherent lipophilic properties of creams (Pelikh et al., ).
The results shown in demonstrate that the IVM-NC cream exhibited a notably superior IVM release profile when compared to the Soolantra cream. Throughout a two-hour observation period, the IVM-NC cream consistently exhibited significantly higher percentages of IVM released at all time points (p < 0.001). For instance, after 60 min, the IVM-NC cream showed an average drug release of approximately 99%, whereas the Soolantra cream only achieved around 25% drug release. These results support the suitability of cream as a vehicle for incorporating IVM-NC. These results are in agreement with the equilibrium solubility and dissolution results, suggesting that nanocrystallization leads to improved solubility and faster dissolution of IVM. Additionally, the use of lyophilized IVM-NC in the cream likely aided in achieving even and uniform distribution of IVM-NC in the cream, leading to quick dissolution of the drug in the cream's aqueous phase and subsequent fast release. Lyophilizates are known to enhance wettability, reduce aggregation, improve particle dispersibility, and speed up dissolution when used in a dosage form or exposed to water (Najm et al., ). Therefore, the incorporation of IVM-NC in a cream as a vehicle offers a potential approach to improve the efficacy of IVM in treating parasitic skin diseases.
Open in a separate windowIn summary, NC formulations hold immense potential for effectively treating topical skin diseases due to their ability to improve skin penetration. This can be attributed to several factors including the small PS, high supersaturation solubility, rapid dissolution, and large surface to volume ratio of nanocrystals which allow for better interaction and adherence at the application site. The major route for drug penetration through the skin is the follicular pathway (Patel et al., ). In particular, particles with sizes ranging from 300 to 700 nm can penetrate into the hair follicle and localize deeper to form a reservoir, enhancing drug retention in the skin (Li et al., ). Furthermore, the presence of surfactants has been shown to act as a skin permeation enhancer by decreasing the intracellular lipid bilayers crystallinity (Parmar and Bansal, ). Therefore, changing the physico-chemical characteristics of IVM by nanocrystallization and lyophilization followed by its incorporation into a cream product is a promising therapeutic approach to improve the pharmacodynamic profile of IVM in topical treatment of parasitic infections.
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