Posaconazole analytical method development

The analytical method for Posaconazole was developed and validated with regard to the ICH guidelines. The mobile phase and the mobile phase ratio for the analytical method development were chosen based on published studies [36]. The mobile phase consisted of methanol:water at a ratio of 75:25 v/v. Other system parameters for the same are mentioned in “Posaconazole analytical method development.” Supporting figure S2 represents a typical chromatogram of Posaconazole with a retention time of 6.16 ± 0.3 min. The retention time obtained for Posaconazole was comparable to that reported in other studies [60, 61]. Figure 1 gives the chromatographic peaks for the forced degradation study. No significant peaks were observed in any of the forced degradation conditions. As seen in Supporting table S2, minor degradation was observed in acidic, basic, and thermal conditions. Thus, Posaconazole can be well tolerated in the mentioned conditions without any significant amount of degradation. Determining the drug degradation profile for actives is a crucial determinant of the conditions used for formulation development and manufacturing. The developed analytical method should be able to differentiate and detect the presence of chromatographic peaks in the case of drug degradation. Thus, the method developed herein is a simple, efficient, and practical analytical method for the detection of Posaconazole.

Fig. 1

Forced degradation chromatograms of Posaconazole. HPLC chromatograms of untreated Posaconazole a and chromatograms of forced degradation study of Posaconazole under acidic b, basic c, oxidative d, thermal e, and enhanced light f conditions

Linearity

Linearity in response to the developed HPLC method with change in concentration of the analyte (i.e., Posaconazole) was studied around the concentration range of 10 to 60 ppm. The method was observed to be linear in the concentration range of 10–60 ppm (Supporting figure S3). Establishing linearity of the developed analytical method is imperative to its use in determining the drug content during formulation analysis. This aspect makes the analytical method reliable and sensitive. In the graph plotted for concentration (ppm) against AUC (Supporting figure S3), the coefficient of determination (R2) was found to be 0.999 and the regression equation was found to be:

Since the value of the coefficient of determination (R2) was found to be ~ 1; we concluded that the method was linear with a proportionate increase in the absorbance with increasing concentration of the analyte.

Limit of detection (LOD) and limit of quantitation (LOQ)

Detection limit tells us whether the analyte is able to be detected and quantified with an acceptable level of confidence. The least analyte concentration that can be detected in the sample is called LOD, whereas, LOQ is the least concentration of the analyte in the sample that can be determined quantitatively [62]. These detection limits are often associated with obtaining the correct result with a 95% probability [63]. LOD and LOQ of Posaconazole were observed to be 0.18 µg/ml and 0.57 µg/ml, respectively. In any circumstances, if the concentration of Posaconazole were to fall below 0.18 µg/ml or below 0.57 µg/ml, then the proposed HPLC method will not be able to detect or quantify it, respectively.

Precision and accuracy

In analytical terms, precision can be defined as the closeness of values to each other in a set of replicate experiments. Whereas accuracy is the closeness of experimental measurements to a specific or pre-defined value [64]. Intraday and interday precision were carried out three times on the same day and for three consecutive days, respectively. Relative standard deviation (RSD) was calculated and found to be less than 2% (%RSD of 0.003 and 0.006 for intraday and interday precision, respectively). A lower value of the %RSD elucidates the higher precision of the developed HPLC method. For determining the accuracy of the method, a standard of 40 ppm was spiked to 80 and 120% with Posaconazole (32 and 48 ppm, respectively), and the percent recovery was calculated (Supporting table S3). % Recovery was calculated in terms of the injected concentration of Posaconazole which was compared to the output AUC given by the HPLC system. This was then back-calculated to the concentration in terms of ppm. In totality, lower %RSD coupled with good recovery values of Posaconazole ascertain the precision and accuracy of the developed method.

Robustness

Robustness elucidates the reliability of the developed method. It is the capacity of the method to remain unaffected by deliberate, small changes in method parameters [65]. The robustness of the developed HPLC method was studied by varying the analyst, column temperature, and injection volume. The %RSD of the variations implemented on the method was found to be below 2% (Supporting table S4). %RSD values for analyst change and temperature change were 0.18% and 1.7%, respectively. A low value of %RSD indicates that the developed method is robust and would give un-biased results even with small variations in method parameters. The % increase or decrease in the mean AUC was found to be proportionate with respect to iterations in the injection volume. 50% (10 µl) and 150% (30 µl) injection volume change resulted in a ~ 47% and ~ 147% change in the mean AUC post-HPLC analysis.

Thus, the HPLC method for the routine analysis of Posaconazole was successfully validated for various parameters, and %RSD of all the tested parameters was found to be within the specified range of 2%.

Solubility and contact angle analysis of Posaconazole in surfactants

Developing a suspension system requires the use of a surfactant as a stabilizer to prevent aggregation of the colloidal dispersion [41]. Optimizing an ideal surfactant which caters the needs of the active and provides it with enhanced stability is of utmost importance. The first step in selecting an appropriate surfactant was to study its solubility and wetting capacity with Posaconazole. Secondly, being an ophthalmic formulation, keeping an eye on the surfactant concentration which goes in the final formulation is a crucial checkpoint to avoid irritation and increase its tolerability. Hence, we decided to use surfactants at their respective strengths based on previously identified values which are considered to be safe for ophthalmic use according to the inactive ingredients guide (IIG) [66]. The next step was to study the solubility of Posaconazole in the shortlisted surfactants. The solubility of Posaconazole was studied in various surfactants based on their safety in ophthalmic preparations per IIG limits. Since we were not aiming to solubilize Posaconazole and rather just suspend it, we aimed for surfactants with minimal solubilization capacity. Supporting table S1 gives the solubility in ppm of Posaconazole in the surfactant solutions prepared as per the IIG limits of the surfactants permitted for ocular instillation. Whenever an interface between a solid and liquid is formed, contact angle plays an imperative role. The contact angle is the angle formed between the liquid surface and the outline of the contact surface which dictates the wetting capacity of a solid by a liquid. The lower the contact angle, the better is the wetting [67]. Contact angle measurements were performed to ascertain the wetting tendency of the surfactant solutions with Posaconazole.

As seen from Supporting tables S1 and S5, Posaconazole is minimally soluble and has the least contact angle with Tween 20 and Pluronic F 127. In order to enhance the stability of the nanosuspension, it is necessary that the suspended particles have good wetting and minimum solubility in the surfactant. To achieve this, the selected surfactant should minimally solubilize the drug and provide maximum wetting. Thus, surfactants in which Posaconazole had the least solubility and minimum contact angle were chosen. Hence, Tween 20 and Pluronic F 127 were chosen as surfactants to stabilize the formulation.

QbD approach for formulating the nanosuspension

Microfluidization technology was utilized for the formulation of a nanosuspension. Microfluidizer (Microfluidics M-110P) is a high shear fluid processor which is unique in its ability to achieve uniform particle size reduction. Advantages of this emerging technology over other existing methods of nanosizing include improved stability of the active, optimized characteristics, and efficient delivery of lipophilic drugs to their target. Numerous studies have developed and optimized nanoparticulate formulations by using microfluidization as a top-down approach [68,69,70,71]. A stable nanosuspension can be prepared by optimizing various formulation and instrument parameters. A suitable product and process optimization design was selected based on the number of factors and their respective levels mentioned below.

The target product profile (TPP) consists of the quality attributes of the final product which play a crucial role in governing its safety and efficacy [72]. The TPP of Posaconazole in situ gelling system was selected based on prior knowledge available of the active and similar products thereof in the market. A critical quality attribute (CQA) of the final pharmaceutical product is a chemical, microbiological, physical, or biological attribute that must fall within a range to ensure the desired quality of the product [73]. The TPP and CQAs for Posaconazole in situ gelling nanosuspension are summarized in Supporting tables S6 and S7, respectively. The next step after identifying the CQAs was the scientific correlation and impact analysis of the formulation components, i.e., critical process parameters (CPPs) on the identified CQAs. CPPs are the parameters used for processing the final product which have an impact on the CQAs. This was established by constructing a risk assessment matrix and ranking the effects of CPPs on CQAs as low, medium, and high risk [74].

The CPPs include process attributes, i.e., number of cycles, the drug to surfactant ratio, and homogenization pressure. The selection of an experimental design is postulated on factors and their respective levels. Factors, in this case, are the CPPs and their respective levels are the ranges of their operation. The factors and their levels are listed in the Supporting table S8.

The response which is checked for is the particle size of the final nanosuspension. As seen from the Supporting table S8, there are 3 factors and 3 levels for each factor. Box-Behnken design (BBD) was used as the optimization design in the Design expert software (DoE). Table 2 summarizes the batches suggested by DoE and the particle size response obtained as measured.

Table 2 Summary of the batches of Posaconazole nanosuspension as suggested by the DoE software

Scrupulous selection of excipients, the drug to excipient ratio, and the CPPs have a major role in affecting the CQAs. This in turn affects the quality and efficacy of the final dosage form. Supporting table S9 gives the relationship between CPPs and the magnitude of the effect they have on the CQAs of the final product. The drug to surfactant ratio, homogenization pressure, and number of cycles play a vital role in determining the particle size and release of the final formulation. Based on these correlations, the effects of CPPs on the product CQAs are ranked as low, medium, and high risk. The goal of QbD was to convert high-risk factors into medium and low risk.

The fit summary given by the software scans the data and studies the correlation of the factors with the pattern of response obtained. The p-value for the linear model was found to be less than 0.05 which implies that there exists a linear correlation between the factors and response. The p-value for the lack of fit test was found to be greater than 0.05 which suggests the suitability of the model applied as suggested by the software.

The final equation in terms of coded factors is expressed as follows:

$$}= -33.57 A-0.0129B-12.71C$$

where

A:

drug:surfactant (D:S) ratio

B:

homogenization pressure

C:

number of cycles

The contour plot (Supporting figure S4) is a two-dimensional (2D) plot where the response is plotted against a combination of factors and/or mixture components, thus portraying their relationship. The contour plot in Supporting figure S4 demonstrates the combined effect of the drug:surfactant ratio and the number of cycles of the microfluidizer on the particle size of the nanosuspension. As the drug:surfactant ratio and the number of cycles increase, the particle size decreases [color gradient from green (larger particle size) to blue (smaller particle size)]. The 3D surface plot (Supporting figure S5) conveys the same information as the contour plot (it is a projection of the contour plot giving shape in addition to the color and contour). Both the graphs give an effect of the number of cycles and the D:S ratio on the response, when homogenization pressure is kept at a constant value of 15,000 psi. Both the plots demonstrate that as the D:S ratio and number of cycles increase, the graph moves color gradation shifts from green to blue, indicating inverse correlation.

The graphical optimization or an overlay graph (Fig. 2) indicates a “sweet spot” (yellow patch) where the response falls within the set criteria. The bright yellow color indicates the settings that are acceptable, whereas the grey color defines unacceptable parameter settings. The parameter settings include the CPPs which fall within the “acceptable” criteria when the response (particle size) is between 300 and 500 nm. The numerical optimization solution (white flag) represents the factor settings with the predicted response (particle size). Figure 2 gives the design space (in yellow) with a flag which corresponds to the parameters with which the validation batch is taken. The results of the batch along with the confidence intervals are specified in Supporting table S10. Validation batch results shown for a selected batch in the design space as generated by the DoE demonstrate that the response falls well within the range.

Fig. 2

Optimization graph highlighting the design space wherein the response is within the desired range

The model validation was successfully carried out and the design space was thus optimized. Figure 2 demonstrates that when the homogenization pressure is kept at a constant value of 15,000 psi coupled with varying the drug:surfactant ratio and the number of cycles such that they fall within the range highlighted by the yellow patch, the particle size will fall between 300 and 500 nm. By using this approach, we can reduce the highly variable impact of the listed CPPs on the particle size (Supporting table S9). Thus, the factors which were high risk in the risk assessment matrix, i.e., homogenization pressure, number of cycles, and D:S ratio, were optimized to a set range and converted to low risk as summarized in Supporting table S11.

Optimization of GG concentration for in situ gellingViscosity determination

Anton Paar rheometer was used to determine the viscosity of 0.2, 0.4, and 0.6% w/v GG solutions alone and when in contact with artificial tear fluid. Figure 3 gives viscosity measurements of the prepared nanosuspension with viscosity represented on the X axis, shear rate on the Y axis, and shear stress on the Z axis. The rate at which the probe rotates was termed as shear rate and the stress applied by the rotating probe on the GG solution was termed as shear stress. As seen in Fig. 3a and c, 0.4% w/v GG solution shows higher viscosity compared to 0.2% GG solution. Also, GG solution when in contact with ATF (Fig. 3b, d, f) demonstrates higher viscosity when compared to GG solution in the absence of ATF (Fig. 3a, c, e). As a general trend, the viscosity was observed to decrease as the shear rate increased. Also, the shear stress applied on the substance by the probe increases as the shear rate increases. This corroborates the fact that cross-linking of GG occurs when it comes in contact with sodium and calcium ions present in the ATF which leads to increased viscosity in the presence of ATF [75]. Also, viscosity increases as the concentration of GG increases. This phenomenon would lead to cross-linking and gelling of GG when in contact with natural tear film, which would give rise to the enhanced retention time of the formulated eye drops when instilled.

Fig. 3

Viscosity measurements of 0.2% w/v GG solution a with and b without the presence of ATF; 0.4% w/v GG solution c with and d without the presence of ATF; 0.6% w/v GG solution e with and f without the presence of ATF determined with a rheometer

Mucoadhesion testing

Texture analyzer was used to measure the mucoadhesion of the prepared formulations (with 0.2, 0.4, and 0.6% w/v solutions of GG) with freshly excised goat lens mucosa as the membrane. Numerous studies have utilized excised goat cornea to test the mucoadhesion of the prepared nanoformulation [46, 54, 76, 77]. Mucoadhesion is measured in terms of the peak force (N) required by the probe to detach the formulation from the mucous membrane and the work of adhesion, i.e., the inter-particulate force of adhesion between the particles of the formulation.

As seen from Supporting table S12 and Supporting figure S6, peak force (N) and the work of adhesion (N.s) were 0.00866 N and 0.14 N.s for the 0.4% w/v GG ATF which therefore showed the maximum mucoadhesion. As per the viscosity results, 0.6% w/v GG ATF had maximum viscosity, i.e., it forms a more viscous system when in contact with the tear fluid. Although, when we studied the ease with which the solutions of various concentrations of GG were dispensed by an eye dropper bottle, we found that plain 0.6% w/v GG alone formed a very viscous system which restricted its smooth flow from the eye dropper bottle. Hence, 0.4% w/v was chosen as the optimized concentration of GG for in situ gelling. Higher mucoadhesion gives rise to enhanced retention of the formulation and more contact time with the cornea. Similarly, an optimum value of the mucoadhesion is a balance between its ease to come out of the dropper bottle without any resistance to flow and enhancement in the contact time.

Ocular irritation studies (hen’s egg test–chorioallantoic membrane—HET-CAM)

HET-CAM is a widely used test to determine the irritancy potential of the developed formulations or test substances. Many studies have reported its use in studying the eye irritancy potential of pharmaceutical formulations [78,79,80]. HET-CAM was performed in order to study the ocular irritation of the prepared formulations which is dependent on the active as well as the excipients used at their respective concentrations. The potential irritancy score, which gives the intensity of irritation caused by the test substance, was calculated using the time required for the endpoints (hemorrhage, vascular lysis, and protein coagulation) to develop on the CAM surface based on the equation stated earlier. A PI score of 0–0.9 is deemed to be non-irritant; a score of 1–4.9 is termed to be slightly irritant; a score of 5–8.9 causes moderate irritation; and a score of 9–12 causes severe irritation [81]. Table 4 gives the scores for the respective groups and the inferences based on them.

As seen in Table 3, it is evident from the PI score that the positive control shows severe irritation, and the negative control shows no irritation indicating that the CAM surface is responsive to the tested substances and that the assay is reliable. Also, Posaconazole nanosuspension is found to be non-irritant with a PI score of 0.85. This study gives us a proof of concept that the developed formulation would be well tolerated by the eye at the respective quantities of the active and excipients used.

Table 3 HET-CAM potential irritancy score and inference deduced based on the time required for the endpoints to developIn vitro drug release

In vitro drug release was studied for the developed nanosuspension with and without the surfactant Tween 20 in the external media (simulated artificial tear fluid). Tween 20 was added in order to facilitate the release and dissolution of Posaconazole which is a BCS class II drug [51]. Various other studies have reported the use of Tweens as surfactants to facilitate the in vitro release and dissolution of BCS class II dugs [82,83,84]. Figure 5a, b denote the % cumulative release v/s. time for both the suspension and the in situ gelling nanosuspension with and without Tween 20. Herein, we wanted to compare the in vitro drug release from the prepared Posaconazole in situ gelling nanosuspension against the Posaconazole coarse suspension as the control. The aim was to obtain a discriminating release pattern to differentiate the release profiles of both the formulations being tested. Our second aim was to determine whether Posaconazole formulated as an in situ gelling nanosuspension (nanosized Posaconazole) increases its release and dissolution as compared to its coarse (non-size reduced Posaconazole) counterpart. Ideally, particle size reduction is known to increase the dissolution and thus the bioavailability of the drug by increasing its surface area available for solvation [85].

As seen in Fig. 4a, it is evident that in the absence of the surfactant, the drug release is comparatively less (~ 2% for the coarse suspension and ~ 10% for the in situ gelling nanosuspension); however, a discriminating release pattern is observed between the coarse suspension and the in situ gelling nanosuspension. From Fig. 4b, it can be concluded that with the addition of a surfactant, the release is enhanced but the release pattern is not discriminating (~ 10% for the coarse suspension and ~ 25% for the in situ gelling nanosuspension). The reason being that the surfactant increases the dissolution of the drug from both the coarse suspension and the in situ gelling nanosuspension at a similar rate. It should also be noted that we observed a tenfold and a threefold increase in the dissolution of Posaconazole in both the scenarios, i.e., with and without the addition of Tween 20 in the external media respectively, by the in situ gelling nanosuspension as compared to the coarse suspension. This observation further corroborates the finding that nanosizing aids in increasing the dissolution of a poorly soluble BCS class II drug.

Fig. 4

% Cumulative drug release vs time graph for the coarse and in situ gelling nanosuspension without surfactant a and with 1% w/v Tween 20 b

Ex vivo corneal retention study

Franz diffusion cell assembly was used to determine the localization of Posaconazole in various chambers of the diffusion cell. This was done to simulate in vivo conditions to study whether Posaconazole would be retained in the corneal tissue, above the tissue, or in the receptor chamber (penetrates from the tissue into the receptor chamber). Our major aim was to determine the corneal retention of Posaconazole to serve as a drug depot and hence provide sustained effect in conditions like fungal keratitis. To our advantage, albumin is one of the major components of the corneal tissue and Posaconazole predominantly binds to albumin [86, 87]. This essentially implies that Posaconazole would preferentially bind to the cornea. Firstly, retention of Posaconazole above the cornea would imply that under in vivo conditions, it would be cleared off via tear drainage. This would ultimately result in the loss of the drug. Secondly, if Posaconazole was found to traverse the cornea (isolated goat cornea in this case) and reach the receptor chamber, it would possibly mimic the same route in in vivo conditions and be localized in the posterior chamber of the eye. In the third scenario, Posaconazole could be localized in the cornea. We aimed at achieving this condition, wherein, maximum localization of Posaconazole in the corneal tissue would give efficacious corneal clearing in fungal keratitis in both prophylactic and treatment conditions. The percentage amount of drug permeated in the receptor chamber, in the tissue, and above the tissue was calculated and a graph was plotted denoting the % distribution of the drug. Figure 5 denotes the retention of the drug in the coarse suspension and the in situ gelling nanosuspension. The majority of the drug from the nanosuspension (~ 70%) is retained in the tissue followed by retention of drug above the tissue (~ 20%) and a minimum amount of the drug permeated in the receptor chamber (< 10%). The imperative function of the prepared formulation is prophylaxis and treatment of fungal keratitis. In order to serve this purpose, the prepared formulation should deposit the majority of the drug in the cornea so as to inhibit the fungus from penetrating it. Deposition of the drug above the cornea would also aid the intended function; however, due to the high clearance rate of the cornea, establishing an enhanced drug concentration would be a problem. Also, permeation of the drug in the receptor chamber would imply permeation inside the cornea into the posterior chamber in vivo. This would also not serve the desired property of the prepared formulation acting as a prophylactic aid.

Fig. 5

Region of drug retention in Franz diffusion cell for the coarse suspension and in situ gelling nanosuspension

Antifungal assay

Antifungal assay was carried out using the optimized formulation and the activity was compared with the available marketed formulation. The agar cup diffusion method was used to find the antifungal efficacy of the prepared formulation. This was done by measuring the zone of inhibition (ZOI) of the Posaconazole nanosuspension against Candida albicans. Studies have reported the use of the agar cup diffusion method to determine the antifungal efficacy by the zone of inhibition method [88,89,90]. The antifungal efficacy of the formulation was calculated in terms of the ZOI (mm).

0.9% NaCl and placebo were used as controls along with a widely used marketed ophthalmic nanosuspension of itraconazole (0.5% w/v). We compared our Posaconazole nanosuspension at the same concentration as the marketed itraconazole preparation, i.e., 0.5% w/v. The ZOI is a circular area surrounding the cup, in which when the antifungal is instilled, the fungi do not grow. Hence, the higher the ZOI higher is the susceptibility of the fungi to the antifungal formulation [91]. As seen in Table 4, the ZOI of the Posaconazole nanosuspension was greater as compared to that of the marketed itraconazole nanosuspension. It can be deduced that the prepared Posaconazole nanosuspension has a greater efficacy against C. albicans as compared to that of the marketed antifungal nanosuspension available for ocular use.

Table 4 Zone of inhibition of different groups in the antifungal assayStability of the nanosuspension

We further performed stability studies on the developed Posaconazole in situ gelling nanosuspension. We checked for parameters like appearance, drug content, particle size, and pH of the nanosuspension for a period of 3 months on storage at the mentioned conditions. Table 5 represents the stability data for 1 month, 2 months, and 3 months of the Posaconazole nanosuspension which was kept at room conditions (30 °C/65% RH) and at accelerated conditions (40 ºC/75% RH) of temperature and humidity. The nanosuspension was observed to be stable for a period of 3 months at both room and accelerated stability conditions.

Table 5 Characterization and stability results of the Posaconazole nanosuspension