Evaluation of mucosal immune profile associated with Zileuton nanocrystal-formulated BCS-II drug upon oral administration in Sprague Dawley rats

Abstract

Nanocrystal drug formulation involves several critical manufacturing procedures that result in complex structures to improve drug solubility, dissolution, bioavailability, and consequently the efficacy of poorly soluble Biopharmaceutics Classification System (BCS) II and IV drugs. Nanocrystal formulation of an already approved oral drug may need additional immunotoxic assessment due to changes in the physical properties of the active pharmaceutical ingredient (API). In this study, we selected Zileuton, an FDA-approved drug that belongs to BCS-II for nanocrystal formulation. To evaluate the efficacy and mucosal immune profile of the nanocrystal drug, 10-week-old rats were dosed using capsules containing either API alone or nanocrystal formulated Zileuton (NDZ), or with a physical mixture (PM) using flexible oral gavage syringes. Control groups consisted of untreated, or placebo treated animals. Test formulations were administrated to rats at a dose of 30 mg/kg body weight (bw) once a day for 15 days. The rats treated with NDZ or PM had approximately 4.0 times lower (7.5 mg/kg bw) API when compared to the micron sized API treated rats. At the end of treatment, mucosal (intestinal tissue) and circulating cytokines were measured. The immunological response revealed that NDZ decreased several proinflammatory cytokines in the ileal mucosa (Interleukin-18, Tumor necrosis Factor-α and RANTES [regulated upon activation, normal T cell expressed and secreted]). A similar pattern in the cytokine profile was also observed for the micron sized API and PM treated rats. The cytokine production revealed that there was a significant increase in the production of IL-1β and IL-10 in the females in all experimental groups. Additionally, NDZ showed an immunosuppressive effect on proinflammatory cytokines both locally and systemically, which was similar to the response in micron sized API treated rats. These findings indicate that NDZ significantly decreased several proinflammatory cytokines and it displays less immunotoxicity, probably due to the nanocrystal formulation. Thus, the nanocrystal formulation is more suitable for oral drug delivery, as it exhibited better efficacy, safety, and reduced toxicity.

Keywords:

• Nanocrystal drug formulation
• zileuton
• biopharmaceutics classification system (BCS-II)
• active pharmaceutical ingredients (API)
• physical mixture (PM)
• inflammatory cytokines
• immunotoxicity
• excipients
• mucosal immunity

Introduction

The bioavailability of oral drugs depends on the physiochemical properties (including solubility, surface charge, ionization, lipophilic properties) of the active pharmaceutical ingredient (API), the dosage form, and the physiological factors (luminal pH, intestinal surface area available for absorption, and permeability in the gastrointestinal tract (GIT) (Dahan et al. 2010; Dahlgren and Lennernäs 2019; Fairstein, Swissa, and Dahan 2013). Available drug solubility data show that approximately 40% of approved and 70% of pipeline drugs are poorly soluble in aqueous solution, which limits their solubility to achieve optimal bioavailability and therapeutic efficacy (Censi and Di Martino 2015; Gupta, Kesarla, and Omri 2013). Poorly soluble drugs are erratically absorbed by the GIT resulting in poor bioavailability and delayed onset of action. To improve the drug solubility, several new approaches are currently being explored, such as nanocrystal formulation. This platform produces nano sized crystalline drug. Due to the large surface area offered by size reduction, the solubility and bioavailability of poorly soluble BCS II and IV drugs are significantly improved (Nagarwal et al. 2011). This increased solubility facilitates a high amount of drug entering the circulation, rapidly achieving high bioavailability. Nanocrystals have been suggested as more suitable for oral drug delivery systems as they exhibit better safety, patient compliance, ease of dosing, and larger surface area for drug absorption in the intestinal epithelium (Ensign, Cone, and Hanes 2012; Pridgen, Alexis, and Farokhzad 2015). Global drug delivery market analysis data show that oral administered drug delivery accounts for approximately 38% and it will continue to dominate the pharmaceutical market (www.americanpharmaceuticalreview.com).

In general, nanocrystal-formulation provides several beneficial outcomes including increased solubility, tissue targeting, reduced intake of oral drug load, and increased-binding capacity in the GIT (Gao et al. 2013; Pawar et al. 2014; Su et al. 2018). Since 1995, the US FDA has approved more than 80 nanomedicines in the form of nanocrystals, lipid based nanoparticles (NPs), polymer based NPs, dendrimer based NPs, protein based NPs, and inorganic NPs for clinical use and 563 are currently in clinical trials or other stages of review (Jarvis, Krishnan, and Mitragotri 2019; Shan et al. 2022). Since 1996, 31 nanomedicines have been approved by the European Medicines Agency (EMA) (Jarvis, Krishnan, and Mitragotri 2019). Globally around 33% of nanomedicines are in clinical phase-I, and 24% of them are in phase-II. These clinical trials mostly focus on cancer (around 54%) and infectious diseases (9%) with the remainder focusing on cardiovascular and neuronal diseases (Shan et al. 2022). According to research and market analysis, nanocrystal-based drugs showed the highest market growth rate compared to other nanotechnology-based drug delivery system. Notably, reformulations of drugs into a nanocrystal form requires shorter time and less monetary investment than traditional drug discovery. Research and market analysis predict that nanocrystals will account for around 60% of drugs, with market value of above 300 billion by 2028 (Jarvis, Krishnan, and Mitragotri 2019). The nanocrystal approach has also been utilized in nutritional science. Currently, several nano-nutritional dietary supplements (Coenzyme Q10, rutin, hesperidin, and apigenin) are available for consumer use (Shegokar and Müller 2010).

Although a nanocrystal platform provides several advantages, due to its complexity a knowledge gap exists between this platform and its clinical translational. As the result of nanocrystal complexity, these formulations may display different safety and efficacy profiles from the parent drug, resulting in different pharmacokinetic (PK) and pharmacodynamic (PD) profiles when compared to micron sized parent drugs. For example, studies have shown that positively charged lipid nanovesicles induced cerebral edema, but neutral or negatively charged vesicles did not show a similar effect (Lockman et al. 2004). Consequently, nanocrystal formulated drugs need additional toxicity evaluations to assess the interaction of nanocrystals in biological systems, their distribution in organelles, biocompatibility, etc. Further, nano-sized particles possess higher free energy and are less stable, which may cause an increased concentration in the intestine that can lead to aggregation and potentially result in cytotoxicity, altered intestinal barrier function, and adverse effects. In addition, nanoparticles <150 nm can be internalized by many cell types via opsonization and can then be cytotoxic and/or immunotoxic (Hillaireau and Couvreur 2009; Schäfer et al. 1992). Nanocrystal drug interaction with the GIT can also affect local microenvironmental factors such as the level of bradykinin, vascular endothelial growth factor (VEGF), prostaglandins, and matrix metalloproteinases (MMPs) (Maeda et al. 2000). The risk factors projected to be associated with nanocrystals are due to their smaller size, surface chemistry (charge), biophysical changes on the surface that are a result of nanoformulation of the API, interaction with the larger mucosal surface of the GIT, immunotoxicity, interaction with biological organelles, and the biodegradation of nanocrystal drugs/excipients/ingredients. Nanocrystal drugs can also interact with protein molecules in the plasma due to their modified surface charges; this can promote the formation of a nanomaterials-protein corona complex, which may cause undesirable immune activation (Caracciolo 2015). In this study, Zileuton (an FDA-approved drug) was selected and formulated as a nanocrystal drug to evaluate immunotoxicity.

Around 339 million world-wide people have asthma, and its prevalence varies between males and females. Asthma prevalence is higher in males around the age of 13 (about 65%), and in adulthood this tendency lessens. In contrast, the manifestation of asthma in females is higher (approximately 65%) in adulthood compared to childhood (Fuseini and Newcomb 2017; Schatz and Camargo 2003; Wang et al. 2020). Airway inflammation in asthma is primally due to the interferon gamma, Interleukin-17, tumor necrosis factor-alpha and interleukin-1beta (IFN-γ, IL-17, TNF-α, and IL-1β) production by activated immune cells. Zileuton (micron sized) is being used as a prophylactic drug to treat chronic asthma as it prevents the production of leukotriene from arachidonic acid (arachidonic acids are metabolized into leukotrienes by lipoxygenase) by inhibiting the 5-lipoxygenase enzyme (Bailie et al. 1995). 5-Lipoxygenase is also known to modulate colitis through the regulation of intestinal adhesion molecule expression and neutrophil migration. It is prescribed as 2 tablets of 600 mg twice daily, thus making a total of 2400 mg of Zileuton per day. Zileuton has been experimentally used to treat colonic inflammation, chronic obstructive pulmonary disease (COPD), upper airway inflammation, atopic dermatitis, rheumatoid arthritis and inflammatory bowel disease (Bertrán et al., 1996; Mazzon et al. 2006; Sorkness 1997; Zarif et al. 1996). Around 5% of patients develop hepatotoxicity due to Zileuton therapy (Watkins et al. 2007). An animal study had shown that metabolites of Zileuton interact with cellular proteins that in turn affect the cellular function and are the probable reason for liver injury (Joshi et al. 2004). Liver toxicity was found to correlate with higher doses of Zileuton (275 mg/kg bw) (Joshi et al. 2004). Even extended-release tablets show several adverse effects including stomach upset, diarrhea, and muscle pain (Qiu, Hui, and Cheskin 1997). A nanocrystal formulation of this drug would increase the solubility, which in turn may decrease side effects due to lower drug load. To assess the safety of the nanocrystal-formulation of this drug and compare it to micron sized API, excipients must be considered. Excipients refer to the inactive ingredients added to a drug product to act as stabilizers or binders. While they are typically inert, they may have unintended side effects, necessitating the evaluation of their impact on the GIT as well.

The FDA has interest in nanotechnology to address questions related to the safety, effectiveness, and behavior of nanomaterials in biological systems that may change as size decreases (www.nano.gov/sites/default/files/NNI-FY22-Budget-Supplement.pdf). The available literature indicates that biological interaction of larger molecules may not necessarily predict the interaction of nanosized molecules in biological systems. The objective of the current study was to address the knowledge gap with respect to safety of NDZ by comparing it with the micron sized active pharmaceutical ingredient and physical mixture (API and PM) using an animal model. Thus, the PM (Zileuton API, Kollidon VA64, Dowfax 2A1 and trehalose) used in the nanocrystal drug formulation, the API, PM, and Kollidon VA64 were individually examined for biological interactions. The goal of this study is to generate science-based evidence for the evaluation of immunotoxic of this nanocrystal formulation.

Materials and methods

Crystalline zileuton (>99% purity) was purchased from Beijing Mesochem Technology Co. Ltd., China. Trehalose was obtained from Gattefose (Lyon, France). Kollidon VA (vinyl acetate) 64 was obtained from BASF Chemical Company (Ludwigshafen, Germany). Dowfax 2A1 was obtained from Dow Chemical Company (Midland, MI, USA). HPLC grade solvents were purchased from Fisher Scientific (Waltham, MA, USA). Inspire C18 column (4.6 × 100 mm, 5 μm) was gifted by Dikma Technologies Inc (Foothill Ranch, CA, USA). Gelatin capsules was purchased from Torpac, (Fairfield, New Jersey, USA), and polyurethan dosing tubes (#9 capsules, 85 mm long, sterile) were obtained from Instech Laboratories, Inc (Plymouth Meeting, PA, USA).

Preparation of nanocrystalline formulation

Zileuton (BCS class II drug) was selected as a model drug for the preparation of nanocrystal formulation for use in a rat model. Detailed method development and validation of wet media milling, spray drying of nanocrystalline Zileuton formulations, and solid-state analysis were as previously reported (Jog and Burgess 2019).

Wet media milling

The nanocrystalline Zileuton suspension was manufactured using wet media milling. Briefly, Zileuton (1% w/v) was suspended in distilled water containing stabilizers – KollidonVA64 (0.75% w/v) and Dowfax2A1 (0.05% w/v). The prepared suspension was stirred for 30 minutes, and the micro-suspension (100 ml) was milled using a wet media mill (Microcer® Netzsch, bead diameter: 0.05 mm ZetaBeads® Nano (yttrium-stabilized, high purity zirconium oxide powder)) at a milling speed of 1200 rpm, pump speed of 92 rpm, and milling time of 18 min. The temperature of the nanosuspension was maintained at 2–8 °C using two cooling bath recirculators (one attached to the milling and the other to the suspension re-circulation chambers).

Spray drying

Solid dry powder of nanocrystalline zileuton was prepared via spray drying using a B-290 spray dryer (Buchi Labortechnik AG). The spray dryer was equilibrated using distilled water. Once the spray dryer was equilibrated, distilled water was replaced with the nanosuspension formulation. The nanosuspension formulation was spray dried at an inlet temperature of 118 °C, an aspirator rate of 90%, and a feed flow rate of 10%, using trehalose as an excipient [drug: sugar (1:1%w/w)] to prevent nanocrystal aggregation. Spray-dried powder was collected from the collection chamber and immediately evaluated for particle size, drug loading, and solid-state characteristics.

Characterization of nanocrystalline formulation

Particle size analysis

Particle size measurements of nanocrystalline formulations were performed using a Zetasizer Nano ZS90 (Malvern Instruments, Dynamic Light Scattering (DLS)). Briefly, the liquid or spray-dried formulations were suspended in a saturated and filtered (0.2 μm PVDF membrane filter) solution of Zileuton in a 30% glycerin solution to avoid any discrepancy resulting from the dissolution of the nanoparticles during measurement. The viscosity of this dispersant solution was measured via a Brookfield viscometer (Model DV-III) and the particle size of the re-dispersed and liquid nanocrystalline suspensions was calculated. All solutions were analyzed in triplicate and reported with standard deviation.

Solubility of Zileuton

Equilibrium solubility was determined in polymer Kollidon VA64 fine under continuous shaking for 48 h at 37 °C. Briefly, 10 mg of the drug was added to each vial containing 10 ml of the Kollidon VA64 fine solution (0.2% w/v solution). After 48 h, 1 ml samples were withdrawn from each vial, filtered through 0.22 µm PVDF (polyvinylidene fluoride) filters, and analyzed using the RP-HPLC method. The solubility of Zileuton in polymer Kollidon VA64 fine is 242.55 µg/ml.

Powder X-ray diffraction (PXRD)

PXRD was utilized to determine the crystallinity of the spray-dried samples. X-ray diffraction patterns were obtained using an X-ray diffractometer (Model D5005, Bruker AXS Inc., Madison, WI) with Cu-kα radiation, a voltage of 40 kV, and a current of 40 mA. All the scans were performed with a scanning rate of 2°/minute with steps of 0.02° from 5° to 40° at 2θ ranges. The above figure displays an overlay of the PXRD diffraction profiles of the neat macrocrystalline zileuton (raw crystalline drug), a physical mixture (Zileuton, KollidonVA64 fine, Dowfax2A1 and trehalose) as well as, the optimized spray-dried nanocrystalline zileuton formulation.

The characteristic diffraction peaks of neat macrocrystalline zileuton drug (most stable – anhydrous polymorphic form I) are 9.8°, 15.7°, 17.7°, 19.7°, 22.48°, 22.5°, 26.5°, 29°, and 31.3°. As shown in the above figure, the optimized spray-dried nanocrystalline zileuton formulation showed a similar diffraction pattern as the neat drug but with reduced intensity. The decrease in the intensity is characteristic of the nanoparticles, due to their small particle size.

Polarized light microscopy

The spray-dried samples were dispersed in the immersion oil and placed on microscope slides. The samples were analyzed using an Olympus BH2 polarized microscope with a Q-imaging camera, accessories (Berek compensator) and software. All the pictures were obtained at 10X resolution. Berek compensator was used for a different background.

HPLC analytical method

The quantification of zileuton in the plasma and ileum was conducted using a Shimadzu-HPLC system with a UV detector. The absorbance wavelength was set at 260 nm. The mobile phase was a mixture of 0.1% trifluoro acetic acid in water and acetonitrile at a 40:60% v/v ratio. A C18 Inspire 5 μm analytical column (4.6 × 100 mm) was used with a flow rate of 1 ml/min and the column temperature was maintained at 40 °C using a column heater. All samples were analyzed in triplicate and reported as the drug loading ± standard deviation.

In vivo study design

The animal study was approved by the NCTR Institute Animal Care Use Committee (IACUC). The strategy for the life stage animal experimental groups and test materials-exposure is provided in experimental schematic of Figure 1(A,B).

Figure 1. Schematic diagrams showing study design and experimental design. (A) Two control groups were used in this study, one group that did not receive any treatment and a second group received an empty capsule (placebo control). Three experimental groups received respective test compounds (30 mg/kg bw). The NDZ and PM treatment groups received 30 mg/kg bw. API, however, had 4.0 times less drug load when compared to the micron size treated rats. The Kollidon group received an amount of Kollidon that was equivalent to that present in the nanocrystal formulated drug or physical mixture. Placebo control group is n = 4 and remaining groups are n = 3. (B) This diagram shows animal quarantine, experiment timeline, drug treatment duration and sample collection schedules.

In brief, this study consisted of examining the effects of several test materials (experimental groups) and two control groups as shown in Figure 1(A). Sprague Dawley (SD) rats were selected for this study since they share many anatomical and physiological features with humans. Ten-week-old males and females (approximately 150-to-200-gram weight) SD rats were obtained from Charles River Co. (Wilmington, MA, USA) and were grouped (2–3 per cage) separately for one week for acclimatization. The rats were fed with NTP 2000 food pellets, and the test materials were orally gavaged. The reason to choose the 30 mg/kg bw dose is based on the following facts. The in vitro dissolution profiles (Figure 2(B)) for nanosuspension (∼98% dissolved in 1.5h) and spray dried crystalline nanodrug (∼95% dissolved in 4h) showed superior dissolution rate (16 times) compared to micron sized API (∼6% dissolved in 24h). The other reason we choose 30 mg/kg bw dose is because of the efficacy of nanocrystal formulated Zileuton showed 4-times higher efficacy than parent micron sized API. To mimic human consumption of medicine, all test materials were filled in 9h clear gelatin capsules (Torpac, Fairfield, New Jersey, USA) under sterilized conditions according to the weight of an individual rat (30 mg/kg bw). These filled capsules were orally administered after the morning feed using flexible oral gavage syringes (Polyurethane dosing tubes #9 capsules, 85 mm long, sterile, Instech Laboratories, Inc. Plymouth Meeting PA, USA). After administration of the first dose, animals were housed in metabolic cages for 24 h to collect blood samples (at various time points) as well as fecal and urine samples for PK/PD studies (Figure 1(B)).

Figure 2. Characterization of nanocrystalline zileuton. (A) The stability of the spray dried nanocrystalline zileuton was evaluated via particle size analysis (Zetasizer, DLS) to ensure absence of aggregation prior to use either in vitro or in vivo experimental procedure. The spray dried nanocrystalline zileuton particle size: 571 nm ± 47 nm. (B) The in vitro dissolution profiles for nanosuspension (∼98% dissolved in 1.5h) spray dried crystalline nanodrug (∼95% dissolved in 4h) and macro-crystalline drug (∼6% dissolved in 24h); (C) PXRD diffraction profiles of the neat macrocrystalline zileuton (raw crystalline drug), physical mixture of the drug (zileuton), polymer (KollidonVA64 fine), surfactant (Dowfax2A1) and sugar (trehalose) and the optimized spray-dried nanocrystalline zileuton formulation. (D) PLM images of the neat macrocrystalline zileuton (raw crystalline drug), physical mixture (zileuton, KollidonVA64 fine, Dowfax2A1 and trehalose) and the optimized spray-dried nanocrystalline zileuton formulations. Note: All images are of 10× magnification. Berek compensator was used for a different background.

Blood collection

Blood was collected in a heparinized tube from all rats before oral gavage and these samples served as base line samples. Blood samples were collected at 0, 1, 2, 4, 6, and 24 h after oral gavage of the test compounds (Figure 1(B)). Plasma was separated for various biochemical assays and stored at −80 °C until use.

Collection of biological samples

At the end of the study, terminal blood was collected by cardiac puncture and was immediately processed into plasma and stored at −80 °C. Intestinal tissue was also collected and immediately snap frozen on dry ice and other biochemical end point analysis and stored at −80 °C until further use.

Protein extraction from the rat intestinal tissue

Ileal tissue was minced using a sterile surgical knife and transferred to Miltenyi Biotech M-tube (Miltenyi Biotec, Inc., Auburn, CA, USA) containing ice-cold sample lysis buffer supplemented with Factor-I and Factor-II (Bio-Rad, Hercules, CA). The tissue was then homogenized in a gentle MACS dissociator. After lysis, the samples were centrifuged at 4 °C for 10 minutes at 60 g and the clear homogenate was transferred to 2 ml Eppendorf tubes and centrifuged at 4 °C for 15 minutes at 15,000 g. The clear supernatant was transferred into a new vial and the protein concentration was measured using Bradford reagent (Bio-Rad, Hercules, CA, USA). The final concentration of protein was adjusted to a 900 µg/ml using lysis buffer and stored at −80 °C until cytokine profiling using the Bio-Rad 23 plex panel (Bio-Rad, Hercules, CA, USA).

Measurement of cytokine levels in ileal tissue extract and plasma samples

Cytokine levels in plasma and ileal tissue samples were measured using a Bio-Rad 23 plex panel Multiplex Immunoassay System (Bio-Rad, Hercules, CA, USA), which uses a bead-based, multiplex system to quantify specific target proteins. Plasma or protein lysates were thawed on ice and plates were prepared for cytokine analysis following the manufacturer’s instructions with slight modification (Gokulan et al. 2018). Plates were read on a Bio-plex 200 instrument using Bio-Plex Manager software (Bio-Rad, Hercules, CA, USA). Standards, Blank, serum and tissue lysate samples were diluted using sample dilution buffer as suggested by the manufacture.

Histopathological assessment of intestinal ileal tissue

For histopathological assessment, intestinal tissue was fixed in neutral buffered formalin. Next, 5 µm sections were prepared and stained with hematoxylin-eosin stain. To evaluate possible physical changes in the villi of intestinal epithelial cells, the ratio of the height of the villi to the depth of the crypts (V:C) was estimated. The quantitative measurement of the intestinal villus was also performed from the lamina propria. To evaluate possible polymorphonuclear cell migration (PMN) into the villi, the granulocytic cellular infiltration of the villi in ileum samples was determined by the approximate average numbers of granulocytes per villus in the lamina propria. For comparison purposes, the numbers of granulocytes in each of ten villi was counted for each sample to obtain an average number of PMN cells per villus.

Data analysis

Multiplex cytokine profiling data was exported and analyzed using the Bio-Plex Data Pro Software (Bio-Rad, Hercules, CA, USA) containing Mann–Whitney/Kruskal–Wallis statistical test. Rats gavaged with an empty capsule (placebo) control group were compared with other experimental groups and untreated control groups. All data are expressed as the mean ± standard deviation of the mean (SD).

Results

Physical characterization of Zileuton nanocrystal drug

Zileuton nanocrystal was formulated and characterized in Dr. Diane J. Burgess’s lab (University of Connecticut). The stability of the spray dried nanocrystalline zileuton was evaluated via particle size analysis (Zetasizer, DLS) to ensure absence of aggregation prior to in vivo use. The spray dried nanocrystalline zileuton particle size was 571 ± 47 nm and the drug loading 23.8 ± 0.08% w/w (Figure 2(A)). The in vitro dissolution profiles for the nanosuspension (∼98% dissolved in 1.5h) and spray dried crystalline nanodrug (∼95% dissolved in 4h) showed a superior dissolution rate (16 times) when compared to the micron sized drug (∼6% dissolved in 24h) (Figure 2(B)). The characteristic diffraction peaks of neat macrocrystalline zileuton drug (most stable – anhydrous polymorphic form I) are 9.8°, 15.7°, 17.7°, 19.7°, 22.48°, 22.5°, 26.5°, 29°, and 31.3°. As shown in the Figure 2(C), the optimized spray-dried nanocrystalline zileuton formulation showed a similar diffraction pattern as the neat drug but with reduced intensity. The decrease in the intensity is characteristic of the nanoparticles, due to their small particle size. The spray dried samples were dispersed in the immersion oil and placed in a polarized light microscope to analyze the particle size. Figure 2(D)). The large rod and plate-shaped crystals (sharp birefringence) was observed for the neat drug (macrocrystalline zileuton − 20 μ particle size). This sharp birefringence was also observed in the physical mixture. The birefringence was evident in the spray-dried nanocrystalline Zileuton formulations. However, the large rod and plate-shaped macrocrystals were converted to uniform sized nanocrystals during the milling process which was evident in the spray-dried nanocrystalline Zileuton formulations.

Ileal cytokine levels

The objective of this study was to evaluate the intestinal immune response upon exposure of NDZ in comparison with the micron sized API, and the physical mixture. First, we investigated whether treatment of empty capsule (gelatin) alone had any effect on the intestinal cytokine production by comparing with untreated control group. Hereafter in this manuscript, empty gelatin capsule treated rats will be mentioned as placebo control. Likewise, the effect of Kollidon VA64 on the intestinal cytokine profiles was compared with the untreated or placebo control. In addition, whether there was any sex-dependent difference in the cytokine production among the controls and the experimental groups was assessed. The results of ileal cytokine profiles were classified into four categories: (i) cytokines involved in intestinal inflammation; (ii) cytokines involved in intestinal anti-inflammation; (iii) cytokines involved in regulation of cell growth, proliferation, and differentiation; and (iv) cytokines involved in Type-2 inflammation in asthma.

Effect of test compounds on the production of proinflammatory cytokines

Intestinal epithelial cells are constitutively secreting Interlekin-1α (IL-1α), which exhibits various biological functions. An elevated level of IL-1α has been corelated with several intestinal inflammatory diseases. To evaluate the effect of the NDZ on the intestinal immune response, IL-1α in the ileal tissue extract was measured. The results show that ileal tissue extracts of control and NDZ groups had similar levels of IL-1α in the female rats (Figure 3(A)). A very similar pattern was also observed in API and PM treated female rats. Interleukin 1β (IL-1β) is a proinflammatory cytokine and high-levels of IL-1β are reported in patients with inflammatory diseases. In the present study, IL-1β cytokine profiles revealed that female rats administered with NDZ had significantly (p < 0.05) higher level of IL-1β in the ileal tissue (Figure 3(B)). Surprisingly the empty capsule or Kollidon VA64 treated group had significantly (p < 0.05) decreased levels of IL-1β in female rats (2-fold reduction) when compared to the control groups (Figure 3(B)). Similarly, API or PM treated female rats also had significantly (p < 0.05) higher level of IL-1β than control groups (Figure 3(B)).

Figure 3. Effect of Zileuton nanocrystal drug, active pharmaceutical ingredient, and physical mixture on the production of the proinflammatory cytokines. (A and C) IL-1α in female and male, respectively, (B and D) IL-1β in female and male, respectively, (e and h) TNF-α in female and male, respectively, (F and I) IL-18 in female and male, respectively and (G and J) RANTES in female and male, respectively in the rat ileal tissue upon oral gauge. The experimental groups were compared with both control groups (untread and placebo) and the Kollidon treated group for statistical analysis. *Indicates the statistical significance. p < 0.05 or below considered as statistical significance.

On the other hand, NDZ treatment significantly (p < 0.05; 2-fold) decreased the level of IL-1α in the intestinal tissue extracts in the male experimental groups when compared to the control groups (Figure 3(C)). A similar pattern of IL-1α level was observed in the male rats treated with API and PM. In male rats, the intestinal tissue extracts had an elevated of IL-1β, however, this was not statistically significant (Figure 3(D)). The same observation was also found in the male rats treated with API and PM. Notably, the intestinal tissue extracts of female rats had much higher levels of IL-1β than male rats (Figure 3(B)). Kollidon treated male rats had significantly p < 0.05) decreased level of IL-1β compared to control groups. A similar pattern was also observed in the female rats (Figure 3(B,D)).

The level of other proinflammatory cytokines including tumor necrosis factor- α (TNF-α), interleukin-18 and RANTES (regulated on activation, normal T-cell expressed and secreted) was analyzed. The results show that intestinal tissue extracts of female rats treated with NDZ had significantly (p < 0.05) decreased levels of TNF-α, IL-18 and RANTES when compared to controls (Figure 3(E,F,G)). A similar pattern was also observed in the female rats treated with API and PM. TNF-α levels for all three experimental female rats had 4 to 5-fold lower concentrations compared to controls (Figure 3(E)). The intestinal IL-18 concentration showed that NDZ, API, and PM treatment decreased the level of IL-18 by more than 10-fold when compared to controls. Kollidon treated female rats also had significantly (p < 0.05) decreased levels of IL-18 compared to controls (Figure 3(F)). Intestinal tissue extracts of all three experimental groups had significantly (p < 0.05) decreased (6 to 8-folds) level of RANTES in females compared to controls (Figure 4(G)).

Figure 4. Effect of Zileuton nanocrystal drug, active pharmaceutical ingredient, and physical mixture on production of the anti-inflammatory cytokine IL-10. (A) females and (B) males. The experimental groups were compared with control groups and Kollidon treated group for the statistical analysis. *Indicates the statistical significance. p < 0.05 or below considered as statistical significance.

Similar to female rats, male rats treated with NDZ, API and PM had significantly (P < 0.05) decreased levels of TNF-α, IL-18 and RANTES in the intestinal tissue extracts (Figure 3(H–J). The treatment of NDZ or API decreased the level of IL-18 approximately 10 to 15-fold when compared to the control rats (Figure 4(H)). In other words, NDZ or API had significant impact on the production of IL-18 in the males (10 to 15-fold decrease) when compared to the females (approximately 10-fold decrease). These results together suggests that low dose of NDZ significantly impacted the production of proinflammatory cytokines in the intestinal tissue.

Effect of test compounds on the production of intestinal anti-inflammatory cytokines

The production of anti-inflammatory cytokines upon treatment of NDZ, or API was analyzed in ileal tissue extract (Figure 4(A)). The results show that intestinal tissue extracts had 2-fold higher level of IL-10 in the females when compared to the control and Kollidon treated group (Figure 4(A)). Intestinal tissue extracts of the male rats treated with NDZ, or API had an elevated level of IL-10 when compared to the control groups, however, this was not statistically significant (Figure 4(B)).

Effect of test compounds on the production of cytokines involved in regulation of cell growth, proliferation, and differentiation

The macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF), macrophage inflammatory proteins (MIP-1α) and MIP-3α are involved in the regulation of cell proliferation and differentiation. Ileal extracts from female rats treated with NDZ or API had significantly (p < 0.05) higher levels (2-fold higher) of M-CSF when compared to the control groups (Figure 5(A)). Levels of VEGF (p < 0.5) were also significantly elevated in female rats treated with NDZ, 5 to 6-fold higher compared to controls (Figure 5(B)). Significantly lower levels of VEGF in the Kollidon treated females was also observed when compared to the control groups. Male rats displayed similar findings (Figure 5(C,D)). The production of MIP-1α and MIP-3α was decreased approximately 1.5-fold in all experimental groups in both males and females, however, no statistical significance was observed when compared to the controls (data not shown).

Figure 5. The effect of Zileuton nanocrystal drug, active pharmaceutical ingredient, and physical mixture on the cytokines involved in cell growth, proliferation, and differentiation (M-CSF and VGEF) upon oral gauge to rats. (A and B) immune response in the females (C and D) males. The experimental groups were compared with control groups and the Kollidon treated group for the statistical analysis. *Indicates the statistical significance. p < 0.05 or below considered as statistical significance.

Effect of test compounds on the production of type-2 inflammation markers in asthma

In most asthmatic patients Th2 cells predominantly produce lymphokines and chemokines (IL-4, IL-5, and IL-13) in response to allergens; these cytokines regulate airway inflammation. Due to the increasing evidence of a lung-gut axis, the presence of Type-2 cytokines in the ileal tissue extract was evaluated. In the female rats no treatment groups impacted the production of IL-4 (Figure 6(A)). In contrast, the level of IL-5 was significantly (p < 0.05) decreased in the NDZ, API, and PM treated rats when compared to untreated control (Figure 6(B)). In male rats treated with NDZ, API, or PM there was a significant decrease in the production of IL-4 when compared to controls (Figure 6(C)). The effect on the level of IL-5 in the ileal tissue extract of male rats treated with NDZ, API, or PM was also significantly lower when compared to controls (Figure 6(D)). Surprisingly, in the placebo control group or Kollidon treated male rats IL-5 could not be detected in the ileal tissue.

Figure 6. The effect of Zileuton nanocrystal drug, active pharmaceutical ingredient, and physical mixture on Th2 type cytokines (IL-1-α, IL-4 and IL-5). (A and B) immune response in females and (C and D) males. The experimental groups were compared with the control groups and the Kollidon treated group for statistical analysis. *Indicates the statistical significance. p < 0.05 or below considered as statistical significance.

Plasma cytokine levels

To determine drug effect on cytokines circulating in the bloodstream, the cytokine levels from plasma samples collected 24 h post-drug treatment (Day-1) were evaluated. When comparing the cytokine levels of the placebo control and the Kollidon treated female rats, the control groups had a significantly (p < 0.05) lower levels of several proinflammatory cytokines (GM-CSF, IL-1β, IL-17, and TNF-α) (Figure 7(A)). Kollidon treated female rats also had a significantly (p < 0.05) higher level of IL-18 compared to controls (Figure 7(B)). Female rats that received NDZ had significantly (p < 0.05) reduced level of IL-18 and IL-17 and an elevated level of VEGF when compared to the controls (Figure 7(C)). On the other hand, the placebo control and the Kollidon treated male rats had significantly p < 0.05) reduced plasma cytokines (GM-CSF, IL-1 β, IL-17, MCP-1, TNF- α and IL-18) when compared to the untreated control (Figure 7(D,E)). Similar to female rats, male rats treated with NDZ displayed significantly reduced (p < 0.05) IL-18 and RNATES levels and an increased level of VEGF when compared to controls (Figure 7(F)). Notably, although NDZ treatment significantly decreased the level of VEGF in ileal tissue, the plasma VEGF levels were approximately 20-fold higher when compared to controls in both sexes. Similarly, both ileal tissue and plasma samples had reduced levels of IL-18 after NDZ treatment in both sexes.

Figure 7. The effect of Zileuton nanocrystal drug, active pharmaceutical ingredient, and physical mixture on secretion of plasma cytokines. (A, B, and C) show the immune response in females and (D, E, and F) male. The left panel shows the effect of empty capsule and Kollidon on serum cytokine production after 24 h of treatment. The mid panel shows the IL-18 production. The right panel shows the effect of NDZ on production of IL-18, VEGF, IL-17 and RANTES. The experimental groups were compared with the control groups or the Kollidon treated group for statistical analysis. *Indicates the statistical significance. p < 0.05 Or below considered as statistical significance.

Impact of test compounds treatment on the ileal mucosa characteristics including transepithelial migration of immune cells across intestinal epithelium

To evaluate possible physical changes in the villous of intestinal epithelial cells after drug treatment, the ratio of the villous to crypt (V:C) was estimated in the histopathological sections. In rodents, normally the ratio of V:C is approximately 3–5:1. The V:C for all samples labeled as ileum was judged to be within the range of 3–5:1 and were normal, with no physiological changes observed due to treatment (data not shown). To evaluate possible polymorphonuclear cell migration into the villi resulting from treatments, the granulocytic cellular presence (infiltration) of the villi in samples labeled as ileum were categorized according to the approximate average numbers of granulocytes per villus observed in the lamina propria. The number of granulocytes in each of ten villi per sample were averaged. The infiltration of granulocytes within the villi was graded as follows: 1–9 as minimal; 10–19 as mild; 20–29 as moderate; and 30 or more as marked. The PMN scores fell within the minimal and mild category. The results revealed that only male rats administrated with NDZ, API and PM had mild PMN infiltration (Figure 8). Control and Kollidon VA64 treated groups also had significantly (p < 0.5) low levels of PMN filtration when compared to the experimental groups.

Figure 8. The numbers of granulocytes in each of ten villi per sample were averaged to determine the number of granulocytes per villus, which were graded as follows: 1–9 as minimal; 10–19 as mild; 20–29 as moderate; and 30 or more as high. *Indicates the statistical significance. p < 0.05 or below considered as statistical significance.

Discussion

Nanocrystal formulations are emerging as potential therapeutics due to several advantages over traditional BCS II and IV micron sized drugs. The nanocrystal formulated drugs may display distinct and complex PK and PD profiles that may have dissimilar safety and efficacy when compared to the micron sized parent drug. In biological systems, nanocrystal formulated drugs <150 nm can be internalized by immune cells that can subsequently trigger a deleterious immune response (Giannakou et al. 2016; Zolnik et al. 2010). The epithelial layer of the intestine operates as a physical barrier, and it is also responsible for nutrients and water absorption (Odenwald and Turner 2013; 2017). The intestinal mucosa also serves as the largest immune organ in the body, and it maintains the cellular function and intestinal hemostasis by regulating proinflammatory and anti-inflammatory cytokines (Odenwald and Turner 2013, 2017). Many factors are capable of altering the cellular functions and intestinal homeostasis, including intestinal mucus layer damage, intestinal permeability, and imbalance in cytokine production (Scaldaferri et al. 2012). Analysis of proinflammatory/anti-inflammatory cytokines can be utilized to predict mucosal immunotoxicity of nanocrystal formulated drugs. The present study addresses the safety and risk assessment of gastrointestinal immunotoxicity of nanocrystal formulated Zileuton.

Zileuton has been used to treat several inflammatory conditions in the respiratory tract (COPD and upper air way inflammation), intestines (colonic), skin (atopic dermatitis), and joints (rheumatoid arthritis). The orally ingested drug interacts with the ileal mucosa to produce mediators that could impact extra-intestinal effects. Therefore, it is essential to investigate the interaction of nanocrystal drugs with intestinal mucosa to assess any impact on cytokine production that may impact guidelines for nanocrystal formulations. The purpose of the present study was the evaluation of ileal cytokines and the impact of nanocrystal drug on any morphological aberration in the ileal mucosa, including changes in villi length and mononuclear migration.

Results of the present study indicate a sex-dependent differences in the secretion of some selective cytokines/chemokines during zileuton treatment; specifically, treatment of NDZ and API in rats significantly increased the level of IL-1β, but only in females. IL-1 is a lymphocyte activating factor that can induce acute phase immune response (Rosenstreich et al. 1978). In ulcerative colitis (UC) high levels of IL-1 have been reported clinically in the colorectal mucosa (Nakamura et al. 1992). In a transgenic mouse model, it was previously reported that knocking out either or both IL-1β and IL-18 protected the mice from trinitrobenzene sulfonic acid (TNBS) induced colitis (Impellizzeri et al. 2018). IL-1β and IL-18 cytokines, which both use a common signal transduction pathway MyD88 (Myeloid differentiation primary response 88), promote the proinflammatory response in various cell types (Sivakumar et al. 2002) (Adachi et al. 1998; Nakanishi et al. 2001). IL-18 is an important molecule in regulating the host-microorganism homeostasis in the intestine and is also a determining factor for inflammatory bowel disease (IBD) (Kim et al. 2013). IL-18 has been classified as a member of the IL-1 family, which modulates the production of INF-γ from Th1 cells and other immune cells by activating the MyD88 signaling pathway (Adachi et al. 1998; Nakanishi et al. 2001). Elevated levels of IL-18 have also been reported in asthma patients, with the clinical severity positively correlating with increased free IL-18 concentration (Tanaka et al. 2001).

In a transgenic asthma mouse model, high concentration of IL-18 resulted in higher levels of IFN- γ, IL-13 and eotoxin accompanied with inflammatory CD4+ and CD8 cells in the lung airway pathways (Sawada et al. 2013). In the GIT, IL-18 is directly involved in altering the normal function of goblet cells during colitis (Nowarski et al. 2015). A transgenic animal model showed over expression of IL-18 induced the TH1/TH2 cytokines in the airway and increased hypersensitivity. Although an asthmatic animal model was not used in this study, administration of a low dose of NDZ decreased the production of IL-18 in intestinal tissue as well as in systemic circulation. IL-18 plays a significant role in differentiating naïve T-cells into proinflammatory TH1-type, which in turn impairs the production of anti-inflammatory cytokines (Veenbergen et al. 2010). It is also responsible for a shift in the TH1/TH2 immune response, has been correlated with altering the structural integrity of the intestinal barrier, aggravating inflammation, and destroying the intestinal epithelial layer during disease development and susceptibility to CD (Pan, Leng, and Ye 2011) (Zaki, Lamkanfi, and Kanneganti 2011). In contrast, a limited number of studies have shown that IL-18 in fact protects the intestinal epithelial cells from inflammation and colitis development (Salcedo et al. 2010). Our study shows that NDZ treatment significantly decreased the level of IL-18 in both male and female rats. The efficacy of NDZ was very similar to a 4.0 times higher dose of the micron sized API. Although fold difference in the decrease in IL-18 was the same for both sexes, the range of the IL-18 level was much higher in females (124–174 pg/ml) compared to males (99–138 pg/ml) (Figure 3(G,H)). A very similar pattern was also observed in the circulating IL-18, where 5-fold decreased level was observed in experimental groups when compared to the controls. This observation shows the effect of NDZ on decreased production of proinflammatory cytokines in the intestinal as well as in the plasma.

The present study also shows that NDZ significantly decreased the production of another proinflammatory cytokine, TNF- α. TNF-α plays a central role in activating the proinflammatory immune response and is also involved in signaling other immune cells to produce proinflammatory cytokines. Studies have shown that elevated levels of TNF-α and the presence of high numbers of TNF-α secreting cells in patients with intestinal inflammatory diseases including CD, UC, and celiac disease. Elevated levels of TNF-α are known to be involved in compromising the intestinal barrier function by altering the permeability and allowing the luminal antigen to interact with lymphocytes (Al-Sadi et al. 2013; Arrieta et al. 2009; Dionne et al. 1997; Hollander 1999; Reinecker et al. 1993). An earlier study indicated that treatment with anti-TNF-α reverses the intestinal abnormalities by tightening the epithelial tight junctions caused by the high level of the intestinal TNF-α (Suenaert et al. 2002).

In the GIT, proinflammatory immune cells highly express cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and phospholipase A2. The end products of these pathways are responsible for inflammatory reactions (Melstrom et al. 2008; Youn and Gabrilovich 2010). An animal study has shown that 5-LOX expression is correlated with colorectal cancer (Soumaoro et al. 2006). A separate study indicated the administration of Zileuton (1200 mg/kg bw) decreased the proliferation rate of polyp growth both in the intestinal tissue and the colon (Gounaris et al. 2015). This same study showed that Zileuton treatment decreased the systemic level of TNF-α. Here we show that Zileuton nanocrystal treatment (7.14 mg/kg bw) decreased TNF-α level by 6–7-fold in the intestine when compared to controls. Our findings are consistent with this; however, it should be noted that the dose of Zileuton nanocrystal formulated drug is several folds lower than marketed Zileuton yet has a similar effect (Gounaris et al. 2015). Our data indicate that the level of TNF-α is 5-fold lower in rats treated with NDZ when compared to controls for both the male and female rats. This low level of TNF-α is insufficient to activate deleterious immune response in the GIT. Thus, the nanocrystal formulation appears to be superior compared to the micron sized API alone in regulating the TNF-α in intestinal tissue.

It is well documented that specific cytokines (including IL-12, IL-10, and TGF-β) suppress airway inflammation (Hogan et al. 1998; Oh et al. 2002; van Scott et al. 2000). Earlier, a murine asthma model demonstrated that engineered CD4 cells secreting IL-10 directly inhibited airway hyperactivity and inflammation, protecting against allergic disease (Oh et al. 2002). In the present study, SD rats demonstrated that administration of the nanocrystal formulated Zileuton increased the production of IL-10 in all groups. This indicates the nanodrug formulation of NDZ acts in two ways; first, it decreases the production of several proinflammatory cytokines and second, it increases the production of anti-inflammatory cytokines.

Eosinophils and leukocytes are normal residents in the lamina propria of the GI-tract (Mir et al. 2002). In the present study, it was deemed necessary to carefully evaluate the morphological changes in the intestine considering the role of chemoattractant molecules such as eotoxins-1,2,3 and RANTES in the migration of eosinophils from the systemic circulation to the GI-tract (Lamousé-Smith and Furuta 2006). Immunohistochemical analysis (data not shown) of the GIT indicated that NDZ did not cause any intestinal inflammation or immune cells migration. In vivo, secreted RANTES acts as a potent chemoattractant for various immune cells including monocytes, NK cells, memory T-cells, eosinophils, dendritic cells, and basophils. Type-2 asthma is mainly driven by eosinophilic inflammation and leads to the secretion of IL-4, IL-5, and IL-13. These cytokines contribute to changes in the airway by inducing the secretion of various chemokines and cytokines. Additionally, they contribute to recurring eosinophils in the airway and are involved in differentiation, maturation, and maintenance of eosinophils. This results in the release of granular content consisting of proinflammatory cytokines, chemokines, and lipid mediators. These molecules in turn activate the expression of several surface receptors and enhances the inflammatory response (Powell, Chung, and Gravel 1995; Soman et al. 2017). In patients with inflammatory bowel disease (i.e. UC and CD) an elevated level of RANTES (both mRNA and protein) has been reported in the intestine (Ansari et al. 2006). Additionally, RANTES can enhance the antigen specific humoral response on the mucosal surface as well as in serum. This adaptive response is mainly triggered through TH1 and TH2 cells, by activating costimulatory molecules and expressing cytokine receptors. Studies have shown that RANTES can activate lymphocyte migration on the mucosal surface in allergic patients. An animal study showed that nasal immunization induced the production of antigen specific humoral and cellular response (Subrakova et al. 2020). The current study shows that administration of the NDZ significantly decreased the level of RANTES in the intestinal tissue and blood as well. This study demonstrated the inhibitory effect of Zileuton on the production of RANTES on the mucosal surface, which was also consistent with API and PM treatment. Taken together, the data suggest that the low level of RANTES is insufficient to recruit eosinophiles and other immune cells in the GIT, resulting in no ileal inflammation.

Currently, several researchers are focusing on IL-18 antibodies as a therapeutic agent for several inflammatory diseases due to IL-18 strong proinflammatory activity. Specifically, anti-IL-18 antibodies are under clinical trial to suppress the proinflammatory activity of IL-18 or IL-18 binding protein. Type-2 asthma is mainly driven by eosinophilic inflammation that leads to the secretion of IL-4, IL-5, and IL-13. In asthma, these cytokines contribute in recruiting eosinophils in the airway and are involved in the differentiation, maturation, and maintenance of eosinophils, resulting airway inflammation (Umetsu and DeKruyff 1997). Earlier studies have shown an increased level of IL-5 in asthma, allergy, and inflammatory diseases (Brusselle, Maes, and Bracke 2013; Tanaka et al. 2004). In the present study, rats administrated with NDZ showed a significant decrease in IL-4 and IL-5 cytokines. Decreased IL-5 production impacts eosinophil growth, differentiation, and maturation. These findings show that the impact is greatest in males. Type-2 asthma can be effectively treated with several therapeutic agents; however, those therapeutic agents are only effective in around 40–50% of asthmatic patients (Hinks, Levine, and Brusselle 2021). In the non-type-2 cases, neutrophils play a significant role by inducing inflammation via IL-17. A murine airway allergic disease model previously indicated that IL-17 induced hypersensitivity in the airway (Chesné et al. 2015). Additionally, in asthmatic patients an elevated level of IL-17 has been reported, which activates airway epithelial cells to secrete chemokines and cytokines to attract neutrophils to the site of inflammation (Blauvelt and Chiricozzi 2018; Chung 2016). In the present study we found that the level of IL-17 in the plasma was significantly decreased in comparison with the placebo control.

High dosage of Zileuton (275 mg/kg BM) is known to cause liver toxicity (Joshi et al. 2004). In the present study, we administrated a dose several fold lower to rats (30 mg/kg bw of NDZ (7.5 mg of API)), which may not be sufficient to cause liver toxicity as reported elsewhere (Joshi et al. 2004; Li et al. 2007; Rodrigues and Machinist 1996; You et al. 2020). Specifically, nanocrystal treatment group rats received 30 mg/kg bw of nanocrystal formulated drug, which contains 7.5 mg of API due to the nanoformulation. Whereas in API treatment group rats received 30 mg/kg bw of API, which is 4 times higher dose of Zileuton in comparison with nanocrystal treated rats. The neat API Zileutons are micron sized molecules; thus, the rats also received higher dose. Hence, it has more chance of interacting with cellular protein and possibly to form a corona formation or it can aggregate and precipitate in the absence of excipients. The less immunotoxicity in the nanocrystal formulation can be explained in two ways (1) in nanocrystal treatment group rats received lower dose of API (7.5 mg/kg), thus the chance of aggregation and precipitation may be less, (2) in the nanocrystal formulation, KollidonVA64 and Dowfax2A1 were used as stabilizers and trehalose was used to prevent nanoparticle aggregation during formulation process. Moreover, the nanocrystal formulated Zileuton may be escaping from the immune cells in the presence of excipients. Hence, overall, the low dose of drug and nanocrystal formulation could collectively attribute to less immunotoxic.

The present study demonstrated that the PMN cell migration score was near normal, suggesting that NDZ is incapable of inducing inflammation. The NDZ drug load was much less in comparison with micron sized API, but the production of the inflammatory mediator was more effective than with API. The decreased production of proinflammatory cytokines by NDZ may be explained in four ways: (1) decreased production of IL-18 and high binding affinity toward IL-18 binding protein together could lead to a low concentrations of free IL-18 (unbound form) in the intestinal tissue that may not be sufficient to induce inflammation and proinflammatory cytokines such as IFN- γ, IL-4, IL-5 and IL-13 and eotoxin; (2) the nanocrystal formulated Zileuton may inhibit eosinophil migration or lipid mediated IL-18 secretion in the intestinal epithelial cells; (3) fewer numbers of PMN migration in GIT indicates less immune activation; and (4) the low level of systemic and intestinal IL-18 concentrations may be insufficient to activate a shift in Th1/Th2 type cells to induce inflammation and proinflammatory cytokine production (Figure 9(A,B)). Further studies to assess the xenobiotic metabolic enzymes in the intestine during these treatments are warranted.

Figure 9. Schematic diagram to show the mechanism of action. (A) Shows how allergens induce inflammasome activation and production of IL-18. Free IL-18 binds to its receptor which in turn activates the Myd88 signaling pathway. MyD88 activation leads to shift in Th1/Th2 ratio and that causes imbalance in production of proinflammatory cytokines from Th2 cells. (B) NDZ directly or indirectly decreases the production of IL-18, resulting in a lower concentration of free IL-18. The decreased level of IL-18 may be insufficient to activate MyD88 signaling pathway; alternatively, less activation can lead to decreased production of several proinflammatory cytokines, both of which prevent immune cell activation.

Conclusion

The results of this study show that the Zileuton nanocrystal formulation has superior efficacy in suppressing several proinflammatory cytokines in the intestinal tissue in comparison with the parent microsize API. Zileuton’s immunosuppressive effect on IL-18 was also observed in the systemic circulation. The Zileuton nanocrystal drug formulation had 4.0 times less API compared to API treated rats, indicating the efficacy of the nanocrystal formulation is likely due to smaller particles size. Additional studies using the murine asthma model to further validate the safety and efficacy of Zileuton nanocrystal formulated drug are warranted.

Author contributions

KG, SK, DJB designed the study and KG, SK, RJ, AB performed the experiments, analyzed the data, and KG, SK, RJ interpreted the results of the experiments. The original manuscript was written by KG, SK and RJ and revised by KG, SK, DJB, SC. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Dr. Joshua Kanungo (NCTR) and Dr. Nathan Koonce (NCTR) for reviewing the manuscript and providing valuable comments and suggestions. We would also like to thank Toxicologic Pathology Associates (TPA) for proving immunohistochemistry work and PMN migration analysis. Additionally, we would like to thank animal care facilities for their help in completing the in vivo studies successfully. Dr. Banu Zolnik’s guidance during the selection of candidate molecule and constructive comments throughout the tenure of the study is highly appreciated.

Disclosure statement

No potential conflict of interest was reported by the author(s). This manuscript reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration.

Data availability statement

The datasets of the study will be available as per the guidelines of US-Food and Drug Administration data sharing policy.

Additional information

Funding

This research was funded in part by a Collaborative Opportunities for Research Excellence in Science (CORES) grant.