**3. Results and Discussion**

### *3.1. Fourier Transformed Infrared Spectroscopy*

TAC showed absorption bands due to stretching vibrations of O-H at 3438 cm−1, C=O (ester and ketone) at 1738, 1723, and 1692 cm−1, C=O (keto-amide), and C=C at 1637 cm−1, C–O (ester) at 1172 cm−1, and C–O–C (ether) at 1087 cm−1. SAIB showed a strong absorption band at 1744 cm−1, which is related to its ester carbonyl. Major absorption bands of TAC appeared with reduced intensity in the PM due to dilution with the excipients. Furthermore, TAC absorption bands at 1738 and 1723 cm−<sup>1</sup> were masked by SAIB at 1644 cm−1. In the case of CAD (F16), many characteristic absorption bands of TAC disappeared, broadened, or shifted to a new wavenumber. The absorption band of TAC at 783 and 1692 cm−<sup>1</sup> disappeared, which may indicate the phase transformation of the drug. Similarly, F17, an ASD formulation based on HPMC, showed similar vibration bands (Figure 2). Phase transformation of the drug was further supported by XRPD and DSC data.

### *3.2. X-ray Powder Diffraction*

TAC showed characteristics reflection peaks at 8.4, 10.2, 11.1, 11.6, 12.5, 13.6, 14.0, 15.2, 17.0, 18.0, 18.9, and 23.4◦ 2θ values. SAIB showed halo diffractograms, characteristics of amorphous or glassy materials. The degree of crystalline to amorphous phase ratio varied with TAC to SAIB. The crystalline phase decreased with SAIB content in the mixture. The crystalline phase completely disappeared when TAC: SAIB ratio was 1:1 or higher (Figure 3). Adding the excipients did not change the amorphous nature of the drug in the SAIB. XRPD was collected from 6 to 15◦ 2θ value as reflection peaks of the drug before 15◦ were not interfered with by the excipients (lactose, microcrystalline cellulose, etc.). Characteristics reflection peaks of the drug appeared with reduced peak height in the PM due to dilution with the excipients. In the case of F16 (SAIB) and F17 (HPMC) formulations, the drug peaks completely disappeared due to the formation of an amorphous phase (Figure 4).

### *3.3. Differential Scanning Calorimetry*

TAC showed a sharp melting endothermic peak at 131.5 ◦C that indicated the drug's crystalline nature. The melting peak of the drug concurred with the literature-reported value [32]. SAIB showed no thermal event confirming its glassy nature. The addition of TAC to SAIB causes the transformation of the drug from crystalline to amorphous phase. However, the extent of phase transformation depends on the TAC to SAIB ratio. Height of the drug melting endothermic peak decreased with an increase in SAIB proportion in the formulation. This was due to drug solubilization in the glassy SAIB. The drug melting peak completely disappeared when TAC: SAIB ratio was 1:1 or higher (Figure 5).

**Figure 2.** FTIR spectra of tacrolimus, physical mixture (PM), placebo, CAD, and ASD formulations (A: amorphous drug, C: Crystalline drug).

**Figure 3.** X-ray powder diffractogram of tacrolimus, SAIB, and various solutions/dispersions of drug and SAIB.

**Figure 4.** X-ray powder diffractogram, physical mixture, placebo, CAD, and ASD formulations.

**Figure 5.** DSC thermograms of tacrolimus, SAIB, and various solutions/dispersions of drug and SAIB.

DSC thermogram of CCS and MCC exhibited shallow peaks in the region 60–130 ◦C, which could be due to physically adsorbed water. However, SLS showed multiple thermal events that may be due to its complex nature or the presence of numerous polymorphic forms. It is a mixture of sodium alkyl sulfate, mainly lauryl [33]. SLS showed thermal events at 103.9, 111.1, and 200.8 ◦C, which matched with the literature-reported values [34]. The PM showed additive thermograms with some differences from thermograms of individual components. The drug peak intensity was reduced due to dilution with excipients. Additionally, the drug melting peak was shifted to 134.2 ◦C due to excipients acting as impurities, thus causing a shift in melting point to a higher temperature. Additionally, the thermal event peak at 200.8 ◦C became broad and shifted to 180.8 ◦C. This was possibly due to the melting of the drug and partial dissolution that resulted in a broad peak at lower

melting. The CAD and ASD formulations did not show an endothermic peak of the drug as observed in the PM, indicating conversion of the crystalline drug to the amorphous phase in formulations F16 and F17. However, a broad and shallow doublet appeared at 116–124 ◦C, which could be related to the glass transition temperature of the drug (Figure 6).

**Figure 6.** DSC thermograms of tacrolimus, physical mixture, placebo, CAD, and ASD formulations.

### *3.4. Dissolution*

The dissolution specification of immediate release TAC capsule is 85% in 90 min in 900 mL dissolution media (water containing 1 in 20,000 HPC, pH adjusted to 4.5) as per USP [35]. This criterion was used to develop the CAD formulation of TAC. The drug dissolved in 2 h was 5.4 ± 0.9% from the pure TAC. Various ratios of TAC to SAIB and excipients were explored to increase rate and extent of the dissolution. F1 formulation contained a drug-to-SAIB ratio of 1:1. It showed almost no dissolution due to the hydrophobic nature of the drug and SAIB, and formed a blob in the dissolution medium. To disperse the blob and increase the surface area available for dissolution, LMH or MCC was added to the formulation in various ratios from 1:5 to 1:20 drug to LMH (F2 to F4) or MCC (F5 to F7). Dissolution was 1.9% in 2 h from the F3 formulation with TAC: SAIB: LMH ratio of 1:1:10. Increasing or decreasing the LMH in the formulations did not increase the dissolution. The dissolution was almost negligible when TAC: SAIB: LMH (F2) was 1:1:5. Increasing the LMH proportion in the formulation F4 from 1:10 to 1:20 did not significantly increase dissolution. The dissolution was 3.7 ± 1.4% in 2 h from F4 formulation, respectively.

On the other hand, addition of MCC in the formulations caused a significant increase in dissolution compared to LMH-based formulations. Dissolution was 20.6 ± 0.9 and 22.4 ± 2.2% in 2 h from F5 and F6, respectively (Figure 7A). These formulations contained TAC: SAIB:MCC 1:1:5 and 1:1:10, respectively. The dissolution in MCC and LMH-based formulations can be explained by solubility, porosity/pore formation phenomenon, and adsorbed layer thickness. LMH must dissolve first to make pores for the dissolution medium to penetrate through the formulation matrix in order to dissolve the drug. This is not the case for MCC since it is porous, and a dissolution medium can easily penetrate through the matrix [36]. TAC-SAIB solution was physically adsorbed over the surface of LMH or MCC. The thickness of adsorbed TAC-SAIB layer would be thicker in LMH due to its non-porous nature. Thus, less surface area would be available compared to MCC-based formulations. Dissolution is proportional to the surface area as per the Noyes-Whitney equation [37]. Further, an increase in MCC in the formulation did not increase the extent of dissolution but increased the rate. For example, the drug dissolved was 20.4 ± 1.6% in 2 h in the F7 formulation (Figure 7A). A faster dissolution rate in the F7 formulation with an increase in MCC was due to the thinner adsorb layer of the TAC-SAIB solution. Thus, a higher surface area was available for the drug to dissolve.

To understand the effect of SAIB on the dissolution, TAC: SAIB proportion was decreased from 1:1 to 1:0.1 (F8) and 1:0.75 (F9) while keeping the proportion of TAC: MCC constant (1:10). Compared to F6, rate and extent of the dissolution in F8 and F9 decreased significantly. The dissolution was 4.1 ± 0.2 and 15.3 ± 1.0% in 2 h from F8 and F9, respectively (Figure 7A). A decrease in dissolution can be explained by crystallinity of the drug, which was supported by diffractograms and thermograms (Figures 3 and 5). These formulations exhibited characteristic reflection peaks of the drug without MCC. Thus, a lower proportion of SAIB was insufficient to keep the drug in its molecular form. Dissolution was further increased by adding CCS outside the CAD formulation, which means CCS was added to the formulation after CAD manufacturing. Addition of CCS resulted in a significant increase in dissolution from 22.4 ± 2.2 (F6) to 50.2 ± 2.2% (F10) in 2 h (Figure 7B). Proportional ratio of TAC:SAIB:MCC:CCS was 1:1:10:2 in F10. An increase in dissolution in F10 can be explained by the fast dispersion of the capsule compact that resulted in a significant increase in rate and extent of the dissolution. The addition of CCS during the manufacturing step significantly impacted the dissolution. CCS was added during the CAD manufacturing step and, at the same time, increased the proportion of SAIB in the formulation. F11 formulation proportional composition was TAC:SAIB:MCC:CCS 1:1.5:10:2. The dissolution of TAC was increased from 50.2 ± 2.2 (F10) to 72.8 ± 5.6% (F11) in 2 h. Adding CCS during manufacturing step resulted in a thin coating of the formulation over the insoluble excipients, which increased the surface area available for dissolution. Further, an increase in SAIB proportion relative to the drug from 1:1.5 (F11) to 1:2 (F12) while keeping MCC and CCS constant did not increase dissolution. On the contrary, it decreased

the dissolution, which can be explained by the hydrophobic nature of SAIB and the thick coating of the TAC-SAIB solution over the excipients and, thus, decreased available surface area for dissolution (Figure 7B). Surfactants were explored to increase rate and extent of the dissolution in formulation F11. SLS was added during the CAD manufacturing step in the ratio 1:1 ratio with respect to TAC in the formulation F13. The dissolution rate and extent were increased from 72.8 ± 5.6% (F11) to 81.3% in 2 h in F13 (Figure 7B). Surfactants are known to improve the dissolution of hydrophobic compounds by forming micelles structures [38]. Dissolution of 85% in 1.5 h can be achieved by increasing CCS proportion from 1 to 1.5 (F14) and 2 (F15) with respect to the drug. Increasing CCS resulted in 86.2 ± 1.4% dissolution in 1.5 h from the F14 formulation. However, increasing CCS to two proportions with respect to the drug in F15 did not result in a significant change in dissolution and did not achieve ≥85% dissolution in 1.5 h (Figure 7C). Formulation F14 was considered the optimized formulation that met the USP dissolution criterion [35]. Finally, F14 was converted from capsule to tablet dosage form to understand its impact on dissolution. The dissolution was increased from 86.2 ± 1.4 to 91.1 ± 6.6% in 1.5 h by changing the dosage forms (Figure 7C). This behavior can be explained by the tablet's disintegration and the capsule's bursting. The bursting time was about 5-10 min compared to less than 1 min disintegration time of the tablet. To compare with the HPMC formulation, the HPMC-based formulation (F17) was prepared by replacing the SAIB with HPMC and was compositionally identical to the F16 formulation. The dissolution was 89.2 ± 2.1% in 1.5 h from the HPMC-based formulation. The dissolution of tablet formulation with crystalline drug (F18) without SAIB or HPMC was 4.8 ± 1.1% in 2 h (Figure 7C).

**Figure 7.** Dissolution profiles of (**A**) F5-F9, (**B**) F10-F13, and (**C**) F14-F18 formulations.

### *3.5. Stability*

The stability conditions exposed samples showed no significant changes in the physical and chemical attributes of the CAD and ASD formulations. FTIR spectrum indicated insignificant changes in the F16 formulation on exposure to high humidity and temperature conditions, which suggested no chemical interactions and maintenance of the amorphous phase of the drug during storage. This was supported by the nonappearance of the absorption bands of TAC at 783 and 1692 cm<sup>−</sup>1. Similar findings were observed in the F17 formulation, and the spectrum was identical to before exposed sample (Figure 8).

**Figure 8.** FTIR spectra of CAD and ASD formulations before and after storage at 25 ◦C/60% RH and 40 ◦C/75% RH conditions (A: amorphous drug).

This was further supported by DSC and XRPD data. The diffractogram of F16 and F17 formulations did not show appearance of characteristics reflection peak of the drug at 8.4, 10.2, 11.1, 11.6, 12.5, and 13.6◦ that could have indicated amorphous to crystalline reversion (Figure 9). Similarly, the melting peak of the drug in the region 130–135 ◦C did not appear in thermograms of the exposed F16 and F17 formulations (Figure 10). The data of orthogonal techniques indicated no transformation of the amorphous drug to its crystalline form on exposure to high humidity and temperature condition.

**Figure 9.** X-ray powder diffractogram of CAD and ASD formulations before and after storage at 25 ◦C/60% RH and 40 ◦C/75% RH conditions.

No significant change in dissolution was expected as no reversion of the drug phase was observed. Both formulations (F16 and F17) met the USP limit of 85% in 90 min after exposure to stability conditions (Figure 11). The dissolution changed from 91.1 ± 6.6% to 90.1 ± 1.8 and 98.5 ± 1.9% after exposure of F16 to 25 ◦C/60% and 40 ◦C/75% RH, respectively. Similarly, the dissolution changed from 89.2 ± 2.2% to 96.6 ± 0.2 and 95.4 ± 2.7%

after exposure of F17 to stability conditions. An increase in the dissolution rate was observed at 30 min, possibly due to decreased disintegration time after exposure.

**Figure 10.** DSC thermograms of CAD and ASD formulations before and after storage at 25 ◦C/60% RH and 40 ◦C/75% RH conditions.

**Figure 11.** Dissolution profiles of (**A**) F16 and (**B**) F17 formulations before and after storage at 25 ◦C/60% RH and 40 ◦C/75% RH conditions.

### *3.6. Pharmacokinetics*

Oral absorption of TAC is incomplete and variable. The absolute bioavailability of TAC is 17 ± 10% in adult kidney transplant patients, 22 ± 6% in adult liver transplant patients, 23 ± 9% in adult heart transplant patients, and 18 ± 5% in healthy volunteers [39]. Comparative bioequivalence was performed among F16 (SAIB-based CAD), F17 (HPMCbased ASD), and Control formulation (F18 formulation contained crystalline drug without SAIB or HPMC). The pharmacokinetic profiles of F16 and F17 formulations were similar and almost superimposable but different from F18 (Figure 12). The drug was detectable up to 72 h post-dosing but below the quantification limit of 4 ng/mL after 12, 8, and 6 h from F16, F17, and F18, respectively.

The pharmacokinetic parameters of F16 and F17 were also very similar. Maximum plasma concentration (Cmax) and AUC0–<sup>∞</sup> (area under the plasma concentration curve) of F16 and F17 were 38.0 ± 11.4 and 35.9 ± 5.9 ng/mL, and 270.3 ± 222.2 and 224.7 ± 55.3 ng/mL.h, respectively. The time to achieve (Tmax) Cmax was one hour for both formulations. No statistical differences were observed in the pharmacokinetic parameters of both formulations (*p* < 0.05). On the other hand, the pharmacokinetic profile of F18 was completely different from CAD and ASD formulations. Time to achieve Cmax was longer in PM compared to CAD and ASD formulations, and was reached in 2 h. Furthermore, the AUC of F18 was lower than F16 and F17 formulations which also concurred with dissolution data. Longer Tmax and lower Cmax and AUC0–<sup>∞</sup> value in F18 were due to crystalline drug that accounted for lower in vitro and in vivo dissolution compared to CAD and ASD formulations. CAD and ASD formulation can achieve supersaturation due to the amorphous nature of the drug [5,40,41]. Tmax, Cmax, and AUC0–<sup>∞</sup> of F18 were two h, 20.6 ± 0.6 ng/mL, and 142.9 ± 10.8 ng/mL.h, respectively. The formulations (F16 and F17) exhibited Cmax and AUC 1.7–1.8 and 1.5–1.8 folds of the F18 formulation, respectively. Higher values of pharmacokinetic parameters in CAD and ASD formulations were due to faster and higher dissolution. Dissolution is the rate-determining step in the absorption and bioavailability of BCS class II drugs such as TAC [9,42]. Furthermore, statistically significant differences (*p* < 0.05) were observed between the pharmacokinetic parameters of CAD and ASD (F16 and F17) and F18 formulations. Therefore, more drug is expected to be bioavailable from CAD and ASD formulations compared to F18.

Furthermore, FDA standards were used to determine bioequivalence among F16, F17, and F18 formulations. The two formulations were considered bioequivalent when the confidence interval (CI) at the alpha level of 90% is 80–125% of the geometric mean of the log-transformed ratio of test/reference or reference/test of Cmax and AUC for regular drug [43]. However, the CI criterion is tightened for narrow therapeutic index (NTI)

drugs to ensure clinical similarity when formulations are switched. FDA-recommended CI criterion is 90–11.1% for NTI [44,45]. The ratio of Cmax of F16/F17 and F17/F18 ranged from 99.7 to 100.3%. Similarly, the AUC of F16/F17 and F17/F18 ranged from 98.0 to 102.0%. Therefore, the F16 and F17 formulations can be considered bioequivalent as they met the CI criterion for both pharmacokinetic parameters. Thus, the clinical performance of F16 would be similar to F17, and these formulations may be interchangeable.

**Figure 12.** Comparative pharmacokinetic profiles of F16 (SAIB), F17 (HPMC), and F18 formulations.

Bioequivalence of the F16 or F17 formulation was also compared with the F18 formulation. The ratio of Cmax and AUC of F16 /F18 and F18/F16 ranged from 84.3 to 118.7 and 90.2 to 110.9%, respectively. Both Cmax and AUC criteria should be met to be considered bioequivalent. Thus, F16 and F18 were not bioequivalent. F17 and F18 were not bioequivalent and interchangeable as they did not meet the CI criteria of pharmacokinetic parameters. The ratio of Cmax and AUC of F17/F18 and F18/F17 ranged from 84.6 to 118.3 and 90.0 to 108.7%, respectively.

### **4. Conclusions**

CAD and ASD formulations offer a tremendous opportunity to convert the poorly insoluble drug into a soluble, dissolvable, and absorbable molecule, provided it maintains its amorphous phase at various stability conditions. The prepared SAIB-based CAD formulation showed that the drug was in the amorphous phase as indicated by XRPD and DSC data. The dissolution can be modulated by SAIB proportion and other excipients to meet regulatory or pharmacopeial requirements. Short-term stability at room temperature and accelerated conditions indicated that the drug's amorphous phase was maintained and met dissolution specifications. The dissolution and stability of the SAIB-based CAD formulation were similar to the HPMC-based ASD formulation. Therefore, the pharmacokinetic and clinical performance of SAIB-based CAD would be similar to HPMC-based ASD formulations. However, long-term stability and human study data must correlate with the current findings.

**Author Contributions:** Investigation, conceptualization, writing—review, and editing, visualization—E.M.M.; investigation, conceptualization—S.D.; conceptualization, review and editing—P.C. and R.A.; writing—review, and editing—M.T.H.N.; conceptualization, writing—review and editing, M.A.K.; conceptualization, formal analysis, writing—review and editing, supervision, project administration—Z.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Eastman Chemicals Inc. research agreement #M1901663.

**Informed Consent Statement:** Not Applicable.

**Acknowledgments:** Authors would like to thank Phillip Cook, Technical Associate, Eastman Chemical Company, for his helpful advice on various technical issues and comments.

**Conflicts of Interest:** The authors declare no conflict of interest.
