Pharmacokinetic Exploration of Sublingual Route for the Enhanced Bioavailability of Tacrolimus with Rabbit as Animal Model

A big challenge for clinicians in organ transplantation is the rejection of allograft and the patients are often required to comply with the lifelong use of immunosuppressants. Since the 21^st century, tacrolimus has been proven to be a boon in the area of organ transplantation and has presented superior results when compared to other immunosuppressant drugs in terms of effective tolerance, reduced side effects, decreased risk of organ rejection and improved organ survival in post-transplant patients.¹ It was first approved in USA to prevent organ rejection in patients receiving allogeneic liver in 1994, kidney in 1997, and heart in 2006.²

Tacrolimus binds to a cytosolic protein known as FKBP 12 which then specifically and competitively inhibits calcineurin, resulting in the calcium dependent inhibition of T-helper cell dependent B-cell proliferation, T-cell activation and, thus the formation of lymphokines including interleukins, γ-interferons etc. The suppression of cytotoxic lymphocytes is the main cause of graft rejection.

Tacrolimus is a class II Biopharmaceutics Classification System drug. Tacrolimus exhibits very low aqueous solubility and extensive presystemic metabolism resulting in poor oral bioavailability (approximately 21%). Its pharmacokinetic profile exhibits large inter/intrasubject variability. The half-life of tacrolimus is 8.7 to 11.3 hr and the mean t_max was reported to be as 1.5 to 2.0 hr. The drug is a known substrate for the enzymes- P450 3A4(CYP3A4) and P-gp.³

The low and erratic oral bioavailability of tacrolimus and the incidence of nausea, vomiting, and gastroparesis in posttransplant patients necessitate alternative routes of administration especially in the early postoperative period to improve its therapeutic efficacy. Oral mucosal administration may be used for patients who face difficulty to tolerate oral drug therapy in both inpatient and outpatient settings. Sublingual delivery allows for direct entry of the drug into the systemic circulation, thus bypassing barriers such as the aqueous milieu of the gastrointestinal tract, P-gp and P450 3A4 (CYP3A4), and the first-pass metabolism in the liver.⁴

To our knowledge, studies on the pharmacokinetic parameters after the administration of sublingual dosage forms of tacrolimus have not been reported so far. The present investigation reports the improved pharmacokinetics of tacrolimus from sublingual fast-dissolving film and tablet formulations compared to the marketed formulation with rabbit as the animal model.

Tacrolimus monohydrate was procured from Yarrowchem (Mumbai, India). Lox spray was purchased from Neon laboratories, and Tincture benzoin from Wallis Pharmaceutical. Carboxy methyl cellulose, was procured from Himedia Lab Pvt. Ltd., (Mumbai, India). Acetonitrile and water (HPLC grade) were purchased from Sigma-Aldrich Chemicals (Bangalore, India).

The preparation and evaluation of tacrolimus-loaded fast-dissolving films and tablets had already been discussed in earlier publications by Mohanan J. and co-authors.^5,6 To brief, to enhance the solubility of the drug, firstly, inclusion complexes with β-cyclodextrin were developed and characterized followed by their formulation into fast-dissolving films and tablets for sublingual administration. The optimized formulations were further selected for their in vivo bioavailability study in rabbits. The composition and method of preparation of the optimized formulations are given in Table 1.

The optimized film and tablet formulations in closed vials were placed in a humidified chamber at room conditions and at 40±2°C and 75±2% relative humidity for 6 months.

The film formulations were evaluated for appearance (in terms of transparency, homogeneity, and flexibility), uniformity of drug content, in vitro disintegration time and in vitro drug dissolution studies. The tablet formulations were evaluated for uniformity of drug content, friability, weight variation, in vitro disintegration time, and in vitro drug dissolution studies.

The in vivo pharmacokinetic study was conducted at Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India after obtaining approval from the Institutional Animal Ethics Committee (no. IAEC/68/ SRIHER/802/2022). The experiments were performed following their protocol.

The environmental temperature was maintained in the range of 19°C-23°C and the relative humidity at 50%-70%. 12-15 air changes/hr were maintained in animal confinements. The animals were exposed to artificial light for a period of 12 hr and to darkness for 12 hr. Rabbits were housed in stainless steel cages individually and they were provided with a laboratory feed diet and water ad libitum.

An equivalent drug dose of 1.5mg/kg body weight was administered sublingually in the form of film and tablet. Orally, the same dose of the marketed formulation (Prograf Capsules) was given as a suspension in 0.3% carboxy methyl cellulose (Figure 1).

Figure 1:
Oral and sublingual administration of test items to rabbits.

Three rabbits of New Zealand white species were used for the study in three phases. Following a period of acclimatization for six days, the animals were administered with the marketed oral formulation (Phase 1). After appropriate washout periods, the animals were administered the sublingual film of tacrolimus (Phase 2) and the sublingual tablets (phase 3). Approximately 1.25 mL of blood sample volumes were collected through the marginal ear vein as per standard protocol (anaesthetized using a local anaesthetic agent) before dosing (0 hr) and at 15 min, 30 min, 1, 1.5, 2, 4, 12, and 24 hr after the administration of each treatment in heparinized tubes. The collected blood samples were centrifuged for 10 min, at 3500 rpm and the plasma separated was kept at temperature appropriately of -80°C until further analysis.

The plasma drug concentrations were measured by the official (USP) HPLC-UV method with slight modifications.⁷ The equipment used was Agilent 1220 infinity gradient LC system.

The standard drug was spiked into rabbit plasma and diluted to obtain concentrations of 100, 200, 300, 400, 500, 600 and 700 ng/ μL. All the solutions were vortexed for 15 sec and centrifuged at 5000 rpm for 15 min. Further, the upper layer was filtered through a 0.2 μm millipore filter and samples were injected into the liquid chromatographic column.

A mean plasma drug concentration-time profile was constructed. The peak plasma tacrolimus concentration, C_max, the time to reach this C_max (t_max), elimination half-life (t_1/2), elimination rate constant (Ke) and the area under the plasma drug concentration-time curve from zero time to 24 hr (AUC_0-24) were determined employing non compartmental analysis. Also the relative bioavailability of test products was determined with respect to the reference product.

The various pharmacokinetic parameters calculated were subjected to a one-way analysis of variance and student t-test in Microsoft excel.

Both the optimized formulations stored at room temperature as well as at 40±2°C/75±2% RH showed satisfactory results and were found to be stable (Tables 2 and 3). The in vitro disintegration time of films and tablets was slightly decreased. Their drug content was found to be slightly reduced but still, they were in the conventional acceptable range of 90-110% of the label claim for end-of-shelf-life specifications. Similar results were reported previously, which may probably be attributed to thermal degradation and/or hydrolysis.^8,9

All the animals during the experiment were found to be normal. No test formulation item-related mortality or morbidity was detected in any of the test animals and they did not exhibit any signs of toxicity. There was no decrease in body weight observed throughout the experimental period.

The calibration curve obtained from the HPLC analysis of the plasma spiked standard drug solutions is presented in Figure 2. The analysis of linear regression of the calibration plot in plasma showed a good linear relationship for the concentration ranges of 100-700 ng/μL of tacrolimus. The regression equation was y= 1.949x -28 with a correlation coefficient (R²) of 0.998.

Figure 2:
Calibration curve of tacrolimus in plasma.

Figure 3 shows the plasma concentration time profiles after the oral administration of a single dose (1.5 mg tacrolimus/kg body weight) of marketed formulation and sublingual administration of the developed film and tablet to rabbits. At each sampling time point, the plasma tacrolimus levels after sublingual administration were significantly higher than those after oral administration.

Figure 3:
Plasma concentration-time graph of various formulations (mean±SEM, n=3 for each formulation).

All the pharmacokinetic parameters which were derived from the plasma concentration profile are given in Table 4. An obvious increase in the plasma levels of tacrolimus from sublingual films and tablets was there when compared to those of the marketed formulation. The C_max obtained with sublingual films and tablets were 290.6467±8.23 ng/mL and 266.2667±9.18 ng/mL respectively which were found to be comparatively higher than that obtained with the marketed formulation (148.95±10.09). T_max observed for the marketed formulation was 1 hr whereas it was found to be 0.5 hr for both the sublingual formulations. Comparable t_1/2 values were obtained for all the formulations. There was a notable difference in the AUC_0-24 between test formulations and the reference product; 1445.57±28.68 ng. hr/mL and 1311.615±67.21 ng. hr/mL respectively for sublingual film and tablet and 696.76±32.13 ng. hr/mL for marketed formulation. Moreover, there was approximately a 2 and 1.8 -fold increase in bioavailability observed for film and tablet respectively compared to reference marketed formulation.

The optimum outcome for an immunosuppressant like tacrolimus is when its physicochemical and pharmacokinetic limitations are successfully overcome. The extensive presystemic metabolism in the intestine and liver has greatly influenced the in vivo potential of tacrolimus which has led to its inadequate therapeutic efficacy. Therefore, the issue of the drug’s poor and unpredictable oral bioavailability would ideally be resolved by obtaining optimal concentration in blood with a minimal dose.

Currently reported target therapeutic concentrations of tacrolimus in different transplant types usually range from 5-20 ng/mL, though it may vary for specific organs transplanted¹⁰ and for the first 2-3 months following organ transplantation, the target trough concentrations in the blood are 8 to 10 ng/ mL.¹¹ A decreased trough level of tacrolimus during the initial post-transplant periods has been associated with a greater rate of acute rejection. Moreover, lower allograft survival has been linked to increased intra-individual variability in tacrolimus trough concentration.¹²

Presently tacrolimus is available as solutions and solid dispersion-based capsules for oral administration and as IV infusions. Commercial formulations for oral use are reported to have only 25% bioavailability.¹³ Intravenous tacrolimus administration requires a continuous infusion and is associated with nephro as well as some neurotoxicities.¹⁴

There are many studies reported for the enhancement of oral bioavailability of tacrolimus. A pharmacokinetic study performed in rats by Lee et al. (2016) revealed that a supersaturable formulation was effective in boosting its dissolution and oral absorption.¹⁵ Nekkanti et al. (2016) conducted an in vivo study in rats with a proliposome formulation of tacrolimus and showed a considerable improvement in the rate and absorption.¹³

Research efforts have also been made by exploiting alternate routes for its administration. Lee et al. (2019) determined various pharmacokinetic parameters of tacrolimus following intramuscular injection in cynomolgus monkeys. The study suggested intramuscular injection as an alternative dosing route.¹² Sakai et al. (2004) successfully explored the rectal route and met with improved pharmacokinetics of tacrolimus.¹⁶

Several human clinical studies have been reported on the utility of sublingual tacrolimus administration in the prevention of allograft rejection.^17–19 To our knowledge, no previous studies on the pharmacokinetics of any tacrolimus formulation in animal models have been reported.

The present study has explored the sublingual pharmacokinetics of tacrolimus employing rabbits as the animal model. Considerably high drug concentration in plasma was attained (Figure 3) with sublingual administration compared with oral, as can be seen from the significantly higher AUC_0-24, 1445.57±28.68 ng.hr/mL for the film (p<0.05) and 1311.615±67.21 ng.hr/mL for the tablet (p<0.05) in comparison with for marketed formulation (696.76±32.13 ng.hr/mL). C_max was also significantly increased (290.65±8.23 ng/mL, p< 0.05 for film and 266.2667±9.18 ng/ mL, p<0.05 for tablet) compared to the marketed formulation (148.95±10.09 ng/mL) which may be due to quick and better drug absorption through sublingual mucosa. There was a shift for t_max from 1 hr to 0.5 hr with oral and sublingual routes respectively, but these differences were not statistically significant (p>0.05). The notable hike observed for relative bioavailability (~2 and 1.8 fold increase for film and tablet than that of reference marketed formulation) indicates that more amount of the drug got entry into the blood circulation and the reason may be attributed to the inherent advantages of the sublingual route including bypassing of presystemic metabolism, high permeability, etc.

Moreover, the pharmacokinetic parameters obtained from sublingual film were also compared with the paramerters of tablets. Even though AUC_0-24 and t_max of the film were higher than that of the tablet, the difference was concluded to be statistically insignificant (p>0.05). But the differences observed in the case of C_max between them were found to be statistically significant (p<0.05).

Successful development of dosage forms of drugs possessing poor physicochemical and pharmacokinetic properties has been always a big challenge for researchers. The current research work demonstrated the utility of the sublingual route for the improved therapeutic outcome of a poorly bioavailable drug candidate like tacrolimus. The in vivo performance of previously developed fast-dissolving films and tablets was compared to marketed oral formulation in rabbits. The sublingual administration resulted in an improved pharmacokinetic profile than the oral route with higher relative bioavailability. So, sublingual tacrolimus can be considered as an alternative to oral administration which may lead to better patient compliance, reduced medication cost, and improved bioavailability.

The authors acknowledge the State Board of Medical Research, Government Medical College, Kozhikode, Kerala, India, for providing financial support for the present study (No. 2574/ sbmr/mcc/kkd/2018).