Abstract
Cystic fibrosis is a serious life-threatening hereditary disease that occurs due to a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Ivacaftor (IVA) is a drug that targets the mutated CFTR protein. IVA is highly hydrophobic (log P = 5.6) with poor aqueous solubility (0.05 μg/mL) and is formulated as an amorphous solid dispersion tablet formulation under the brand name Kalydeco®. The recommended daily dose of Kalydeco is twice per day with a high fat meal to aid in IVA’s absorption. In this research, we studied the application of cyclodextrins (CDs) to improve the dissolution of IVA. Phase solubility studies between IVA and four different CDs (α-, β-, γ-, and hydroxypropylβCD [HP-β-CD]) were conducted and it showed a significant improvement in IVA’s aqueous solubility with HP-β-CD. Solid state characterizations confirmed the formation of IVA/HP-β-CD inclusion complexes. In vitro dissolution studies were conducted at pH = 6.8 and showed improvement in IVA’s rate and extent of dissolution with IVA/HP-β-CD (1:2) complexes in comparison to un-complexed IVA. In vivo pharmacokinetics in mice showed 2- fold increase in area under the curve (AUC) after the oral administration of IVA/HP-β-CD complex in comparison to Kalydeco tablets. In addition, HP-β-CD extended the release of IVA from the IVA/HP-β-CD complexes with a longer Tmax of 7.05 h compared to 2.96 h with Kalydeco® tablets. These results demonstrate that CD inclusion complexes of IVA using HP-β-CD can be a successful alternative technology to improve the solubility of IVA while extending its release.
Keywords: cyclodextrin, ivacaftor, cystic fibrosis, inclusion complex, CFTR
Graphical Abstract
1. Introduction
Cystic fibrosis (CF) is a serious hereditary genetic condition affecting approximately 70,000 individuals worldwide, 30,000 of which are in the United States of America. This condition results in deterioration of multiple organs such as the lungs, kidneys, intestines, and liver due to the build-up of mucus at their epithelial cell surface (1,2). As a result, individuals with CF rely on symptomatic treatments such as bronchodilators, antibiotics, and physical therapy to help mitigate the negative effects of the disease (3). In 2012, a new class of drugs for the treatment of CF was introduced which helps treat the underlying genetic cause of the disease. This class of drugs is called CF transmembrane conductance regulator (CFTR) modulators (4). Ivacaftor (IVA) is the first drug to be approved in this class which targets a specific mutation in the CFTR protein (class III gating mutation). To improve the poor aqueous solubility of IVA (<0.05 μg/mL), it is formulated as an amorphous solid dispersion (ASD) with hypromellose acetate succinate (HPMCAS) polymer using a spray drying technique and is available in a tablet form under the brand name Kalydeco®. The recommended Kalydeco® dosage is 150 mg twice daily for adults with a high fat meal to aid the absorption of IVA (5,6). This is due to IVA’s short half-life (12 h) and its high hydrophobicity (logP = 5.6) (5,7). Frequent administration of medications can result in sharp fluctuations of the drug concentrations in the blood which can cause unwanted adverse effects. In addition, patients’ adherence to the treatment decreases with more frequent drug administrations (8,9). Previous reports have shown low adherence levels of CF patients to the Kalydeco treatment regimen with only 61% of the adult patients and less than 50% of the pediatric patients adhering to the medication (10–12).
Cyclodextrins (CDs) are cone-shaped cyclic oligosaccharides consisting of 6-, 7- or 8- dextrose subunits (α-, β-, or γ-, respectively) and are characterized by having a hydrophobic interior and a hydrophilic exterior. The hydrophobic interior can form inclusion complexes with hydrophobic guest molecules through no-covalent hydrophobic interactions and the hydrophilic exterior can interact with water molecules through hydrogen bonding. CDs have been widely used in the pharmaceutical industry to improve the solubility of poorly soluble active pharmaceutical ingredients (APIs). In addition, CDs inclusion complexes can improve the stability and extend the release of APIs (13–15)
Previously, we developed long-acting IVA-loaded polymeric microparticles that maintained stable IVA plasma levels in mice for over 4 weeks following a single subcutaneous injection which could eliminate the need for the twice daily administrations of IVA (16). Herein, we further investigated the application of CDs as an alternative way to improve the solubility and bioavailability of IVA while extending its release by forming IVA-CD inclusion complexes that can ultimately reduce the dosing burden on CF patients and improve their quality of life. To select the appropriate CD for forming the inclusion complexes with IVA, we conducted molecular docking and phase solubility studies with α-, β-, γ- and HP-β-CD. We then studied the in vitro dissolution behavior of IVA-cyclodextrin complex in comparison to free IVA and, finally, we conducted in vivo pharmacokinetics of IVA following the oral administration of either IVA-cyclodextrin complex or Kalydeco® tablets to mice.
2. Materials and methods
IVA was purchased from Vertex pharmaceuticals (Boston, MA). α-, β-, γ-, and HP-β-CD, in addition to polysorbate 80 (Tween 80) were purchased from Sigma Aldrich (St. Louis, MO). Water was purified using a Nanopure water purification system (ThermoFisher, Waltham, MA). All solvents used were of analytical grade.
2.1. Molecular docking studies
Molecular docking simulation was conducted following a previously published protocol (15). BIOVIA Discovery Studio Visualizer v20.1.0.19295 (Accelrys) program was used to retrieve 3D structures of α-, β and γ--cyclodextrin from the protein crystal structures of PDB ID: 4FEM, 1Z0N, and 5E70, respectively, deposited in the RCSB Protein Data Bank (PDB) (http://www.rcsb.org). Three-D structure of HP-β-CD was prepared by ChemDraw (version: 22.0.0.22). Three-D confirmation of IVA [PubChem: 16220172] was retrieved from the NCBI-PubChem Compound database. In this study, α-, β-, γ--, and HP-β-CD were considered as targets and IVA was the ligand. In a separate docking study, CYP3A4 (PDB ID: 7UF9) was used as a target and HP-β-CD was the ligand. Energy minimization of IVA and HP-β-CD was conducted by the Merck molecular force field 94 (MMFF94) and Gasteiger was the charge calculation method. Rigid docking was carried out to minimize error generated from active rotatable bonds of the ligands (17). AutoDock Tools (ADT), a free graphic user interface of MGL software packages v1.5.7rc1, was used to supplement polar hydrogens to the target structures (18). Grid box parameters (Supplementary Table 1) were set by ADT [1]. Molecular docking simulation was conducted by the AutoDock Vina (version 1.1.2) following the same parameters mentioned by Naguib et al., 2021 (15,19). The equations used for the binding affinity/binding constant calculation were derived under the assumption of a 1:1 stoichiometry. Therefore, results will serve as an approximation in comparison purposes. BIOVIA Discovery Studio Visualizer and PyMOL molecular graphic system (https://pymol.org/) were used to analyze binding mode and hydrogen bond formation.
2.2. Phase solubility studies
The phase solubility studies were performed according to a previously described method (20,21). Solutions of increasing concentrations of α-, β-, γ-, and HP-β-CD were prepared in Nanopure water in the range of 0 – 50 mM (n=3 per concentration), except for β-CD (up to 16 mM), due to its limited water solubility. An excess amount of IVA was added to all the aqueous CD solutions (n=3). All samples were shaken for 2 days in a rotary incubator (37 °C, 300 rpm). On the 3rd day (after reaching equilibrium), samples were centrifuged at 20,000 xg for 10 minutes and then filtered using PTFE membrane syringe-tip filters (0.22 μm) to remove undissolved IVA. The filtered samples were then analyzed using an Agilent HPLC-UV (Agilent Infinity 1100, Santa Clara, CA) to determine IVA concentration according to Nakhla et al (16). The HPLC-UV method consisted of a reversed phase C18 column (150 mm, 4.6 i.d.) operating in isocratic mode (60: 40, acetonitrile:water v/v) with 0.1% trifluoroacetic acid (TFA). The flow rate was maintained at 1 mL/min and detection was done at 309 nm. Briefly, IVA stock solution was prepared in methanol and dilutions to 6 calibration standards was made using acetonitrile: water (50: 50 v/v) in the range of 0.1 – 50 μg/mL and 50 μL were injected to the HPLC-UV. The calibration standards and their respective peak areas correlated linearly, and the fitted linear-regression equation was as follows: , where Y is the peak area and X is IVA’s concentration. The R-squared value was 0.999 indicating a good correlation. The solubility of IVA was plotted against the increasing concentrations of all four CDs.
2.3. Preparation and characterization of IVA: HP-β-CD complexes
IVA: HP-β-CD inclusion complexes (1:2 molar ratio) were prepared using a simple kneading method (22). Briefly, 2 moles (3.083 g) of HP-β-CD were transferred to a mortar and a mixture of water and ethanol (1:1, v/v) was added while kneading the mixture using the pestle until a viscous paste was formed. An amount of IVA equivalent to 1 mole (0.3925 g) was gradually added to the paste while continuously kneading the mixture for 50 minutes to evaporate the ethanol. The paste was then left under a fume hood for 4 h to allow for further evaporation of ethanol. The paste was freeze-dried using a Freezone® lyophilizer and the dried complex was ground into fine powder using a pestle. To calculate the drug loading (DL), the IVA complex was dissolved in water at 1 mg/mL IVA and the solution was diluted (10-fold) with acetonitrile: water (50:50, v/v) then injected in the HPLC to determine IVA concentration.
Drug loading was calculated using Eq. 1:
| (1) |
Theoretical drug loading was calculated using Eq. 2:
| (2) |
Scanning electron microscopy (SEM)
The surface morphology of the IVA complex, HP-β-CD, IVA and their physical mixture were examined using a Hitachi S-4800 scanning electron microscope (SEM) (Hitachi High Technologies, Ontario, Canada) (23). Samples were spread onto a carbon double-adhesive tape that was mounted on an aluminum stub, and the surface was coated with gold and palladium using an argon beam K550 sputter coater (Emitech Ltd., Kent, U.K.). SEM photomicrographs were captured at 1.5 kV accelerating voltages.
Differential scanning calorimetry (DSC)
DSC thermograms were obtained using a DSC instrument (TA Instruments model Q20, New Castle, DE) coupled with a refrigerated cooling system (RCS90) (24). A quantity of approximately 3–5 mg of IVA complex, HP-β-CD, IVA, and their physical mixture was transferred to standard aluminum pans covered with Tzero lids. An empty aluminum pan covered with Tzero lids was used as a reference. Pure dry nitrogen was used as purge gas (set at 20 psi pressure and 40 mL/min flow rate). Samples were scanned in the temperature range of 25 – 350 °C using a 10 °C/min heating rate.
Powder X-ray diffraction (PXRD)
The X-ray powder diffraction patterns of the IVA complex, HP-β-CD, IVA, and their physical mixture were obtained using a Siemens D5000 diffractometer (25). A Cu Kα X-ray with λ =1.51418Å was used as an X-ray source. Diffractograms were recorded in the range 5° to 50° at 2ϴ values using a step size of 0.02° and a dwell time of 0.5 s.
Fourier transform infrared spectroscopy (FT-IR)
The FT-IR spectra of IVA, the IVA complex, and HP-β-CD were recorded in the scanning range of 4000 cm−1 to 400 cm−1 using a Perkin Elmer FT-IR spectrometer (Waltham, MA) at room temperature (26). Samples were ground with KBr (3:300) into a fine powder and transferred to an oven at 300 Fahrenheit to remove moisture before being compressed into fine discs and scanned by the spectrometer.
2.4. In vitro dissolution studies
In vitro dissolution experiments were conducted using a USP dissolution Apparatus-II (VK700, Vankel Technology group, Inc., North Carolina, USA) (27). An amount equivalent to 2 mg IVA of either pure IVA or IVA complex was transferred to the dissolution medium consisting of 500 mL deionized water with 0.002% Tween 80. The paddle speed was set at 50 rpm and the temperature was set at 37 °C. Samples were collected at a specific height in the dissolution vessel over 4 hours, filtered using PTFE filters (0.45 μm) and then injected into the HPLC to determine IVA concentration.
2.5. In vivo pharmacokinetic studies
The in vivo pharmacokinetics of IVA was studied in mice following the oral administration of the IVA complex or Kalydeco® tablets or the intravenous administration of free IVA. A total of N = 63 female BALB/c mice were split into three groups. The first group was administered IVA dissolved in 10% Tween 80 in PBS (pH = 7.4) intravenously via tail vein injection at a dose of 5 mg/kg. The remaining 2 groups were administered an equivalent IVA dose of 25 mg/kg of either crushed Kalydeco® tablets or IVA complex (both suspended in PBS) by oral gavage. At predetermined time points (5, 15, 30, 60, 180 min, 6 h, and 24 h), mice were sacrificed from each group, and blood samples were collected via cardiac puncture into pre-heparinized tubes. Samples were centrifuged at 5000 xg for 10 minutes to separate the plasma. IVA was determined in the plasma samples following protein precipitation using acetonitrile. Briefly, 10 μL lumacaftor (internal standard – 0.01 μg/mL) was spiked into the plasma samples and 1 mL acetonitrile was added followed by high-speed shaking for 10 minutes. Samples were then centrifuged at 5000 xg and the supernatant was separated, evaporated using a stream of nitrogen and then reconstituted in 100 μl acetonitrile:water (50:50, v/v). The reconstituted samples were sonicated for 5 minutes and centrifuged again; then the supernatant was injected into the HPLC to determine IVA concentration. Supplementary Table 2 and Supplementary Figure 1 contain the standard curve results of IVA in plasma samples. Non-compartmental and compartmental pharmacokinetic analyses were conducted using PKsolver (an Excel Ad-in) (28). All animal experiments were approved by the University of Iowa Institutional Animal Care and Use Committee (IACUC).
2.6. Statistical analysis
All experiments were performed in triplicate. Statistical analysis and figures were done using GraphPad Prism software (GraphPad, La Jolla, CA). Ordinary one-way ANOVA followed by Dunnet’s multiple comparison test was used to compare the maximum solubility of IVA using different CDs in the phase solubility studies where a p-value of less than 0.05 was considered a statistically significant difference.
3. Results and discussion
3.1. Molecular docking studies
To evaluate the role of cavity size in the formation of IVA/CD complexes with higher binding affinities, we used molecular docking simulations to precisely calculate the likely order of complex formation with different CDs, namely α-, β-, HP- β- and γ-CD. In general, all predicted binding energy values are negative which indicates stable and spontaneous complex formation. There was a relatively higher value in the case of the complex formed with γ-CD in comparison to other types of CD (Figure 1), which may be explained by the larger cavity size of the former which can accommodate large portions of IVA leading to more hydrophobic interactions. However, since hydrogen bond formation is crucial for the stabilization of the complex (15), and contributes to overall hydrophilicity and improved solubilization, we decided to evaluate other cyclodextrin derivatives with hydrophilic surface functional groups. Thus, we chose HP-β-CD which contains hydroxypropyl (HP) group substitutions on the surface. The HP group substitutions substantially improved the aqueous solubility of the CD compared to the unmodified CDs.
Figure 1 –
Molecular docking simulation studies between IVA and alpha-, beta-, and HP beta- and gamma-CD. A. Predicted binding free energies of IVA to different types of CD. B. 3D structures of docked IVA molecule with each CD. C. 2D diagram of docked IVA with each CD representing the hydrophobic interactions between the drug and CD. The blue lines represent the carbon-hydrogen bonds, the purple lines represent the hydrophobic interactions, and the green lines represent the conventional H-bonds. H-bond and hydrophobic interaction distances were generated using BIOVIA discovery studio visualizer, and PyMOL, respectively.
3.2. Phase solubility studies
Phase solubility studies of IVA in solutions of α-, β-, γ-, and HP-β-CD were conducted to determine which CD could form the most stable and hydrophilic complexes with IVA resulting in its improved solubility. Phase solubility diagrams of IVA with each of the 4 CDs are shown in Figure 2. Phase solubility diagrams are classified into “A” and “B” type diagrams (47). B- type diagrams are seen when the drug-CD complex has limited solubility in the medium. B-type curves are further classified into “Bi” and “Bs” type diagrams, where “i” stands for “insoluble” and “s” stands for “some solubility”. “Bi” type diagrams occur when the drug-CD complex is insoluble in water, where at any drug concentration the complex starts precipitating resulting in low drug solubility. “Bs” type diagrams occur when the complex has some limited solubility in water. Both α and γ-CD did not enhance IVA solubility and were characterized by a B-type curve. IVA α-CD solubility curve shows a (Bi) type curve (Figure 2) where IVA did not demonstrate a significant increase in solubility at increasing α-CD concentrations. IVA γ-CD solubility curve shows a (Bs) type curve where IVA demonstrated an initial linear increase in drug solubility followed by a saturation (plateau) phase then a precipitation or decrease in drug solubility. In contrast, both IVA/β-CD and IVA/HP-β-CD solubility curves showed A-type phase solubility diagrams. IVA β-CD solubility curve was characterized by linear increase in IVA solubility at increasing CD concentrations suggesting an AL-type phase solubility diagram, where each CD molecule encapsulates one IVA molecule causing a linear increase in the solubility of IVA at higher CD concentrations (1:1 ratio). Due to the medium cavity size of β-CD, it is generally used to form inclusion complexes with small molecules (average M. Wt. 300 g/mol). β-CD formed stable interactions with IVA, However, it has limited water solubility (16 mM) which hinders its capacity to further improve the solubility of IVA. IVA/HP-β-CD solubility curve has shown an “Ap” type phase solubility diagram where at higher concentrations each CD molecule can take up two IVA molecules causing an exponential increase in solubility and a positive deviation from linearity. HP-β-CD, which is similar to β-CD except that it possesses HP group substitutions, has a higher aqueous solubility than β-CD due to the hydrogen bonding that form between the hydroxypropyl groups and water molecules. IVA solubility was approximately 40 – 100 times higher at 50 mM HP-β-CD compared to the other CDs (48 μg/mL for HP-β-CD vs 1.2, 0.4, and 0.5 μg/mL for α-, β-, and γ-CD, respectively, p<0.0001). These results indicate that HP-β-CD is the most suitable CD to improve the solubility of IVA.
Figure 2:
A. Phase solubility diagrams of IVA with all 4 CDs (α-, β-, γ-, and HP-β-CD). Inset: the difference between solubility diagrams between α-, β-, and γ-CD using different Y-axis scale. B: the maximum solubility of IVA at the highest concentration of each CD. (**** p< 0.0001)
3.3. Characterization of IVA: HP-β-CD (1:2) complexes
Drug loading (DL)
The average calculated DL (±SD) of IVA in the complex formulation was 115 μg/mg (± 1.4). This value represents both the complex and un-complexed IVA in the formulations.
Scanning electron microscopy (SEM)
The goal of SEM is to visualize the surface morphology of prepared formulations and their raw materials (29). SEM photomicrographs of IVA/HP-β-CD inclusion complexes, IVA, HP-β-CD, and their physical mixture are shown in (Figure 3). IVA appeared as irregularly shaped crystalline structures with different sizes and rough surfaces, while HP-β-CD existed in its typical spherical particles with some cavity structures (30). The physical mixture showed both the crystalline structures of IVA and the spherical particles of HP-β-CD, indicating no interaction between them. In the case of the IVA/HP-β-CD inclusion complexes, the original morphology of both IVA and HP-β-CD could not be found, instead, the powder appeared as homogenous, organized blocky structures with smooth surfaces, and it was impossible to visually identify the rough crystals of IVA, nor the spherical structures of HP-β-CD. The observed change in particle morphology in the IVA/HP-β-CD inclusion complexes suggests that there was an interaction between IVA and HP-β-CD which indicates the formation of the inclusion complex. The appearance of crystalline blocky structures has been reported previously in literature to confirm the formation of inclusion complexes (29,31–34).
Figure 3:
Scanning electron microscopy (SEM) photomicrographs of IVA/HP-β-CD (1:2) complexes, IVA, HP-β-CD, and their physical mixture.
Differential scanning calorimetry (DSC)
It is well established that when guest molecules are entrapped in the cavity of CD, changes occur in their DSC curves, mainly because their melting point endothermic peak either shifts to a different temperature or disappears, indicating the loss of the crystalline structure of the molecule and the formation of inclusion complexes (35). The thermograms of IVA complexes, IVA, HP-β-CD and their physical mixture are shown in Figure 4A. IVA showed a sharp endothermic peak at 318 °C corresponding to the melting point of its crystalline form. It also exhibits a small endothermic peak at 192 °C and another exothermic peak at 224 °C, which likely correspond to the polymorphic nature of IVA, as shown previously (36). HP-β-CD showed a broad endothermic band ranging from 60 to 110 °C corresponding to its amorphous nature and can be related to the release/loss of water molecules from the cavity (34,37,38). The physical mixture showed a small exothermic peak of IVA at about 234 °C (Figure 4A), and a broad endothermic band corresponding to HP-β-CD indicating minimal if any interaction between them. In contrast, the endothermic peak of IVA completely disappeared in the IVA/HP-β-CD complex, which was due to the formation of non-covalent interactions between IVA and HP-β-CD resulting in the conversion of IVA from a crystalline into an amorphous state. In addition, the broad endothermic peak corresponding to loss of water in HP-β-CD was reduced, which is also an indication of inclusion complex formation (26).
Figure 4:
A- Differential scanning calorimetry (DSC) thermograms; arrow denotes exothermic peak of IVA. B- Powder X-ray diffractograms (PXRD); note the broad peaks at 2θ° values of about 19 and 25. C- Fourier-transform infrared spectroscopy (FTIR) spectra.
Powder X-ray diffraction (PXRD)
Upon the formation of an inclusion complex between a crystalline drug and CD, the drug molecule loses its crystalline state and converts into an amorphous state and, as a result, the diffractogram of the inclusion complex would show the disappearance of the crystalline peaks of the guest molecule (35). The diffractograms of IVA complexes, IVA, HP-β-CD, and their physical mixture are shown in (Figure 4B). IVA showed sharp, intense peaks corresponding to its crystalline nature. In contrast, due to the amorphous nature of HP-β-CD, it showed two broad peaks at 2θ° values of about 19 and 25 (31). The physical mixture showed an overlap between the sharp crystalline peaks of IVA and the 2 broad peaks of HP- β-CD, indicating no interaction between the two. In contrast, IVA complexes showed that most of the characteristic crystalline peaks of IVA disappeared, and the rest of them are attenuated, indicating the conversion of most IVA into an amorphous form due to its entrapment in the HP- β-CD cavity upon complex formation.
Fourier transform infrared spectroscopy (FT-IR)
Figure 4C shows the FTIR spectra of IVA complex, IVA and HP-β-CD. The HP-β-CD spectrum showed absorption bands at 3404 cm−1 (for -OH stretching vibration), 2930.1 cm−1 (for C-H stretching vibration), 1035.5 cm−1 (for C-O stretching vibration), and 1646.1 cm−1 (for H – O – H bending vibration). The IVA spectrum showed absorption bands in the region 1800 – 1200 cm−1 characteristic to C=O ketone, C=C aromatic, and C-N amine, as well as that characteristic to N-H amine at 3300 cm−1. The IVA/HP-β-CD complex spectrum showed only the absorption bands characteristic to HP-β-CD functional groups while the absorption bands corresponding to the functional groups of IVA completely disappeared, suggesting the entrapment of IVA inside HP-β-CD cavity and the formation of the inclusion complex. The disappearance of the absorption bands corresponding to IVA’s structure in the inclusion complex could be due to the limited vibration capacity of IVA when entrapped into the host molecule (HP-β-CD) due to the non-covalent hydrophobic interactions between the two (21, 33, 39). It has been reported (29,40) that the change or disappearance of absorption bands corresponding to the functional groups of the guest molecule can confirm the formation of the inclusion complex. Moreover, the absence of new absorption bands indicates no conformational changes in IVA’s structure due to the lack of covalent interactions with HP-β-CD (21,27,40).
3.4. In vitro dissolution studies
Figure 5 shows the in vitro dissolution profiles of free IVA and IVA/HP-β-CD complexes. Free IVA showed a slower dissolution behavior in comparison to IVA complexes. By 30 minutes, only 20% of IVA was dissolved, and by the end of the experiment only 50% of the drug was dissolved. In contrast, IVA complexes showed a faster dissolution behavior of IVA where by 15 min, 80% of the IVA was dissolved and by 30 minutes 100% of the drug was solubilized. Forming IVA/HP-β-CD inclusion complexes improved the wettability of IVA in the dissolution medium as visually observed during the experiment. In contrast, free IVA (un-complexed) formulation showed poor wettability and could not fully disperse in the dissolution medium which resulted in its poor dissolution. In addition, as confirmed using DSC and PXRD, the presence of IVA in an amorphous form (partially or fully) in the complex formulation resulted in improved dissolution due to the lack of the energy barrier needed to break its crystalline lattice as opposed to the situation with free IVA (38,41). Further investigation is needed to quantify amorphous IVA, and if possible to discriminate between the dissolution behavior of free IVA and IVA/HP-β-CD within the system, using analytical and/or mathematical approaches, as this can shed more light on the mechanisms of drug diffusion and further predict valuable insight of bioavailability, as previously highlighted by Tzanova and co-workers (42,43).
Figure 5:
In vitro dissolution profiles of free IVA vs IVA/HP-β-CD (1:2) inclusion complexes.
This study aims to improve the poor aqueous solubility and dissolution rate of IVA, as the flux through the membrane is solely dependent on the free drug fraction (42), and complexes made with hydrophilic CD are not able to cross membranes (44). While the dissolution rate of IVA is a major determinant of its oral bioavailability, its Caco2 permeation is still moderate to high (11.9 × 10−6 cm/s) (45). Several reports have studied the correlation between the mechanism of membrane permeation and the dissolution enhancement using inclusion complexes (46,47). Further investigation is still needed to highlight the involvement of HP-β-CD in the improvement of IVA membrane permeability.
3.5. In vivo pharmacokinetics studies
Non-compartmental and compartmental PK analysis of IVA following the IV administration of free IVA or the oral administration of IVA complexes or Kalydeco tablets are shown in Supplementary Table 3. Fitting IVA concentrations to 1-, 2- and 3- compartment (CMT) PK models showed that a 2-CMT PK model provided the best-fit for IVA concentrations (R2 = 0.99, AIC = −3.81, Table 1). Figure 6 shows the observed and model-predicted (2-CMT) plasma concentration vs time profile of IVA following the IV administration of free IVA and the oral administration of either IVA complexes or Kalydeco tablets. IVA complexes resulted in higher area under the curve (AUC0 - ∞) (39.73 vs 19.6 μg.h/mL) and a longer Tmax (7.05 vs 2.96 h) compared to Kalydeco tablets (Table 2).
Table 1:
Goodness of fit criteria (model diagnostics) for selecting the best model that fits IVA plasma concentrations following its IV administration to mice.
| Diagnostic criteria | 1 Compartment model | 2 Compartment model | 3 Compartment model |
|---|---|---|---|
| Objective function SS | 1.44 | 0.18 | 0.18 |
| Akaike information criterion (AIC) | 6.59 | −3.81 | 0.18 |
| Schwarz criterion (SC) | 6.48 | −4.03 | −0.14 |
| R2 | 0.95 | 0.99 | 0.99 |
The lower the value of the diagnostic test (SS, AIC, SC), and the closer the R2 value to 1, the better the fit of the model.
Figure 6:
A: IVA plasma concentration vs time profile following the IV administration of free IVA to mice at a dose of 5 mg/kg (n=3, data points are means ± SD). B: Predicted and observed IVA plasma concentration vs time profile following the oral administration of either IVA/HP-β-CD (1:2) complexes or Kalydeco tablets at a dose of 25 mg/kg (n=1–3, data points are means ± SD).
Table 2:
IVA pharmacokinetic parameter estimates following the oral administration of either IVA/HP-β-CD (1:2) complexes or Kalydeco tablets. 2-CMT = 2-compartment PK model parameter estimate; AUC = area under the curve; Cmax = maximum observed concentration; Tmax = time to maximum observed concentration
| Kalydeco | Complex | ||
|---|---|---|---|
| PK Parameter | Unit | 2-CMT | 2-CMT |
| Cmax | (μg/mL) | 2.44 | 2.05 |
| Tmax | (h) | 2.96 | 7.05 |
| AUC (0 – t) | (μg/mL*h) | 19.6 | 33.6 |
| AUC (0 – inf) | (μg/mL*h) | 19.6 | 39.73 |
The prolonged Tmax following administration of the inclusion complex may be correlated with the reported cytochrome inhibitory activity of HP-β-CD (48,49,50), or alteration of membrane permeation kinetics. Further investigation to explain this prolonged exposure of IVA in inclusion complex, which can be beneficial in reducing its dosing frequency, is still needed.
While CD complexation may have contributed to improvement in bioavailability of IVA following a single oral dose of the complex via dissolution enhancement, the effect of inclusion complex formation on the drug permeation (51) still needs further investigation, as mentioned above. As we plan to further investigate drug permeation in future work, some in vitro cell-free simulation techniques like the parallel artificial membrane permeability assay, and the PermeaPad® assay (52), should be considered.
4. Conclusion
IVA/HP-β-CD inclusion complexes (1:2) were successfully developed. DSC and PXRD analysis showed the disappearance of the melting point of IVA, as well as its crystalline peaks, respectively, thus confirming its presence in an amorphous form due to the formation of the inclusion complexes. In addition, SEM photomicrographs showed the formation of blocky structures and FTIR analysis showed the disappearance of the bands corresponding to the functional groups of IVA, both further confirming the successful formation of inclusion complexes. In vitro dissolution experiments revealed a significant improvement in the rate and extent of dissolution of IVA with HP-β-CD complex formulation in comparison to free IVA. In vivo PK studies in mice following the oral administration of either IVA/HP-β-CD complexes or Kalydeco tablets showed a 2-fold increase in its AUC and a 2.3-fold increase in its Tmax. This indicates that IVA/HP-β-CD inclusion complexes successfully improved the oral bioavailability of IVA and prolonged its half-life which could reduce the frequency of its administration, thus improving patient compliance and ensuring successful treatment outcomes.
Supplementary Material
Acknowledgements
This work was supported in part by the National Institute of Health grant (P30CA086862) and the Lyle and Sharon Bighley Chair in Pharmaceutical Sciences grant.
Conflict of Interest:
This work has resulted in a U.S. patent application for Compositions comprising molecules for cystic fibrosis treatment. Patent number: WO2022125963A1 for Aliasger K. Salem, David A. Stoltz, Michael J. Welsh, Sarah Ernst, Youssef W. Naguib, David Nakhla, Aml I. Mekkawy, Nikesh Gupta.
PCT/US2021/062913
Footnotes
Credit author statement:
Conceptualization: DSN, YWN, AKS
Methodology/Investigation: DSN, YWN, SS, AKS
Data Collection: DSN, YWN, DG, NG, WM, TM
Funding acquisition: AKS
Project administration: DSN, YWN, AKS
Supervision: AKS
Writing – original draft: DSN, YWN, SS, AKS
Writing – review & editing: DSN, YWN, SS, AKS
Data and materials availability:
Available upon request
References
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