Colon targeted layer-by-layer self-assembled film: pharmacokinetic analysis of model drugs after oral administration

doi:10.21203/rs.3.rs-1750266/v1

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Colon targeted layer-by-layer self-assembled film: pharmacokinetic analysis of model drugs after oral administration

https://doi.org/10.21203/rs.3.rs-1750266/v1

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The aim of the study was to investigate the pharmacokinetic parameters of 5-fluorouracil (5FU) and moxifloxacin (MF) after oral administration using layer-by-layer assembled film in enteric coated capsule. The LbL film was prepared by sequential layering of chitosan and sodium alginate polyelectrolytes containing either 5FU or MF. The films were characterized for physical characteristics, drug loading and release behaviour. Pharmacokinetic studies were performed in the rat model for three different drug concentrations after oral administration and compared with intravenous administration. The results showed that the thickness of 10 bilayerd film was 147 ± 11.66 and 212.3 ± 7.19 µm after 5FU and MF loading, respectively. The amount of 5FU and MF loaded was found to be 1.93 ± 0.48 and 4.64 ± 0.33 mg/cm², respectively. The DSC and PXRD results showed crystalline nature of 5FU and MF after entrapment in LbL film. The LbL film with backing layer provided directional release of 5FU and MF, where 63.81 ± 4.52% and 101.38 ± 5.08%, respectively was released in 24 h. 5FU showed non-linear pharmacokinetics compared with linear pharmacokinetics showed by MF after oral administration. There is a dose dependent increase in C_max after oral administration of 5FU and MF LbL film. The T_max was found to be 720 and 840 mins for 5FU and MF after oral administration. The mean residence time and AUC_0-tat 45 mg/kg were 871.4 ± 6.45 min and 198.6 ± 5.03 x 10³min. ng/mL and 1267 ± 142.4 min and 1590 ± 55.60 10³min. ng/mL for 5FU and MF, respectively. Biodistribution studies showed significantly (p<0.01) greater disposition of 5FU within colon tissue after administration using oral LbL film. Taken together, colon targeted LbL film can be developed for oral administration of drugs for local and systemic applications.

Layer-by-layer assembled film

5-fluorouracil

moxifloxacin

pharmacokinetics

colon

The delivery of therapeutics through oral route is most desirable. However, the conventional dosage forms such as tablets, capsules and solutions may not be suitable for a few classes of molecules with unfavorable physicochemical and biopharmaceutical properties¹. There is a need to develop novel dosage forms suitable for the oral delivery of varied drugs. The innovations in oral dosage form development includes nano-dispersions, and oral patches^2–5. The nano-dispersions are liquid preparations containing nanoparticles to improve rate of dissolution, mucoadhesion, specific cell targeting, lymphoid cell uptake, and to improve stability^6–11. On the other hand, oral patches are solid dosage forms that contain drugs and would adhere to different parts of the gastrointestinal tract by design^12–14. Attributed to the macro-scale design characteristics, patches are amenable to loading of varied drugs, control the drug release, provide mucoadhesion at different gastrointestinal sites, responsive to stimuli such as pH, enzymes, microbiota, and external triggers ^15,16.

In general, oral patches with two dimensional architecture are made of polymers. The patches could be layered with three distinct properties. The layers include core, adhesive and backing layer¹⁷. The drug loaded core is expected to control the rate of drug release. The bioadhevise layer would adhere to the mucosal site within the gastrointestinal tract. The backing layer would limit the amount of drug diffused into the luminal side to achieve directional drug release¹⁸. The pharmacokinetic profile of the drug would depend on its solubility and permeability characteristics and diffusivity through the patch. Among the different types of patches developed for oral administration, layer-by-layer self-assembled films show promise in their ability to carry greater payload and control the drug release characteristics¹³. LbL films are prepared by sequential layering of oppositely charged polyelectrolytes. The characteristics such as polyelectrolyte molecular weight, charge density, concentration, casting solution pH, viscosity, drying time and the number of bilayers can be controlled to achieve a patch with desirable mechanical strength and stability^19–22. Depending on the choice of polyelectrolytes, one of the polymer could act as a mucoadhesive layer²³.

Earlier studies have demonstrated preparation and application of oral patches for a variety of small molecules and large molecules^13,24. However, there is a need to study the pharmacokinetic profiles of drugs administered using oral patches. Here, we report the development of LbL film for oral administration of two model drugs, 5-fluorouracil and moxifloxacin (Fig. 1). 5FU is a chemotherapeutic agent that is widely used in the treatment of oral, gastric, colorectal, liver, pancreatic, ovarian and breast cancer²⁵. 5FU belongs to the Class III of biopharmaceutical classification system (BCS) with good solubility and poor permeability²⁶. Attributed to its poor permeability, 5FU is clinically available as injectable dosage form²⁷. The physicochemical properties of 5FU include molecular weight 130.08 g/mol, aqueous solubility 10 mg/ml, log P (-)0.83, pKa 8.0 and 13²⁸.

Moxifloxacin (MF) is a fourth generation fluoroquinolone antibiotic used against bacterial infections²⁹. MF belongs to the Class I of BCS, where it shows good solubility and permeability³⁰. MF is clinically available as a tablet dosage form with normal antibiotic regimen of 400 mg a day for 5 days against Staphylococcus aureus infections^31,32,33. The physicochemical properties of MF include, molecular weight 437.89 g/mol, aqueous solubility 24.8 mg/ml, log P 0.01, pKa 6.25, 7.9^30,34,35.

5FU remains in unionized form at pH values below 8, whereas MF will be in ionized state at pH below 8^35,36. The entrapment and release behavior of drugs loaded within the polyelectrolyte LbL film will be dependent on the ionization and hydrogen bonding. Therefore, the release and absorption of 5FU and MF from the film is expected to be different.

In an earlier report, we have demonstrated development of 5FU loaded LbL film for directional release and colon tissue adhesion³⁷. The objective of this study was to perform pharmacokinetic analysis of 5FU loaded LbL film after oral administration in colon targeted capsule. MF was used as a model BCS Class I molecule to compare the pharmacokinetic parameters of 5FU.

Materials

5-Fluorouracil, moxifloxacin hydrochloride, chitosan (75–85% deacetylated, MW: 100–300 kDa), sodium alginate (MW: 80 kDa) and polycaprolactone (PCL) were purchased from Sigma-Aldrich chemical company (Bengaluru, India). Eudragit S100 was obtained as a gift sample from Evonik industries (Essen, Germany). Size 9 hard gelatin capsules and dosing applicator were procured from Torpac (New Jersey, USA). Milli Q water (Millipore Inc., USA) with 18.2 MΩ.cm resistivity was used in all the experiments. The chemicals were used without further purification.

Preparation of drug loaded LbL film

The LbL film was prepared by sequential adsorption of chitosan and sodium alginate as described in our previous report ³⁸. Briefly, chitosan (MW: 100–300 kDa, pH 5) and sodium alginate (MW: 80 kDa, pH 4) solutions were prepared at 5 mg/ml concentrations, separately in 0.1 M NaCl solution. Propylene glycol (5%) was added to polymer solutions to serve as a plasticizer. Chitosan solution was cast on a cleaned glass slide and dried for 15 min at room temperature, later the unadsorbed chitosan was decanted by washing the slide with deionized water. Then, sodium alginate solution was cast on the chitosan layer and incubated for 15 min and the unadsorbed alginate was washed off. This process of sequential layering of chitosan and alginate was repeated 10 times to prepare 10 bi-layered LbL film. After drying at 40°C for 6 h, the backing layer made of PCL-chitosan dispersion (95:5 ratio) was cast on one side of the LbL film. The dried film was carefully delaminated and stored in an air-tight container at room temperature. The other side of the film contained chitosan layer that would provide mucoadhesion.

The drug-loaded LbL films were prepared by casting the 5FU or MF dissolved polymeric solutions as described above. 5FU and MF were dissolved to achieve a concentration of 10 mg/ml in both chitosan and alginate solutions.

Characterization of LbL film

The LbL film was measured for thickness at different locations of film using a digital micrometer (IP65, Baker, USA). The sequential adsorption of 5FU or MF containing chitosan and alginate was studied by recording increase in the weight and thickness of film after each bilayer formation. Furthermore, films were characterized using Fourier transform infrared spectroscopy (FTIR) (IRAffinity-1S, Shimadzu, Japan) after adsorption of each bilayer. The pellets were prepared by hydraulic compression of LbL film and KBr mixture (ratio 2:98). The % absorbance of the LbL film was recorded within the range of 4000 − 400 cm^− 1 at 4 cm^− 1 resolution.

Powder X-ray diffractometer (PXRD, Ultima IV, Rigaku, Japan) was used to understand the crystallinity of MF before and after entrapment in the LbL film. The X-ray diffraction pattern was recorded within the range of 5 to 50° at a scan speed of 3°/min. In addition, the thermal transition of neat MF and MF-LbL films were recorded using differential scanning calorimeter (DSC 60, Shimadzu, Japan), where 3–5 mg of sample was hermetically sealed in an aluminum pan and exposed to a temperature range of 30–300°C at 10°C/min heating rate.

The loading efficiency of 5FU or MF in LbL film was measured by suspending a film of 1 cm² area in 2 ml of Milli-Q water. The sample was subjected to continuous vortex mixing overnight followed by centrifugation at 10000 rpm for 10 min to precipitate the undissolved matrix. The supernatant was collected and diluted before HPLC analysis. The efficiency of the extraction procedure was measured by preparing a film containing a known amount of 5FU or MF and subjected to a similar process of sample preparation as mentioned earlier. Extraction efficiency was calculated by the following Eq. (1)

\(Extraction efficiency \left(\%\right)= \frac{extracted amount of 5FU/MF}{total amount of 5FU/MF spiked } \times 100\) - (1)

Preparation of pH-sensitive capsule loaded with LbL film

The 5FU or MF film was cut into a 6 mm circular disc using a biopsy punch and placed in a size 9 hard gelatin capsule (7–10 discs depending on animal weight). Capsules were coated with Eudragit S100 as described in our earlier report ³⁷. Briefly, the pH-sensitive coating solution was prepared freshly by dissolving 10% w/v of Eudragit S100 and 50% w/v of triethyl citrate (plasticizer) in a mixture of isopropyl alcohol and acetone in 1:2 ratio. Glyceryl monostearate (0.5% w/v) was used as anti-tacking agent. The weight buildup (%) was measured by checking the weight of capsule before and during the process. The capsules were coated till 55% weight buildup was attained. The whole process was carried out at room temperature and capsules were stored in a self-seal plastic pouch until utilized for studies.

Preparation of colon simulated fluid

Colon simulated fluid (CSF) was used as the release medium for in vitro drug release studies. The preparation of CSF involves two steps. Initially, 5.5 g of tris(hydroxymethyl) aminomethane (Tris) and 8.8 g of maleic acid was dissolved in 1 liter of Milli-Q water and its pH was adjusted to 7.8 (using 10 N NaOH). Then, bile salts (mixture of equal quantities of sodium cholate and sodium deoxycholate) of 0.113 g were dissolved. Secondly, 25 ml of the above solution was taken into a 100 ml round bottom flask to which 0.222 g of phosphatidylcholine and 0.026 g of palmitic acid, previously dissolved in 3 ml of dichloromethane was added and exposed to vacuum at 40°C to evaporate dichloromethane. The two solutions were mixed under stirring. Finally, 3 g of bovine serum albumin was added resulting in a turbid solution.

In vitro drug release studies

In vitro release of MF from LbL film was determined using Franz diffusion cell apparatus (PermeGear Inc., USA). The dialysis membrane of molecular weight cut-off 2 kDa (Himedia, India) was sandwiched between the donor and receptor compartment. The receptor compartment was filled with 5 ml of CSF and allowed to attain a temperature of 37 ± 0.5°C using a heater-assisted water circulator. The LbL film of 8 mm diameter circular disc (equivalent to an area of 0.5 cm² containing 2.3 mg of MF) was placed in the donor compartment and charged with 20 µl of CSF to wet the film. The film was placed such that chitosan layer is facing the receptor compartment. Samples of 0.3 ml were collected from the sampling port at the pre-determined time intervals, including 0.25, 0.5, 1, 2, 4, 6, 8, 12, 16, 20 and 24 h. In a different experiment, the MF release from the backing layer side was analyzed by placing the film in the donor chamber where backing layer faced the receptor compartment. The amount of MF in the samples was quantified using an RP-HPLC analytical method.

HPLC analytical method for 5FU and MF

The analytical method for detecting 5FU and MF was developed using RP-HPLC system (LC-20AD, Shimadzu, Japan) supported with photodiode array detector (SPD-M40, Shimadzu, Japan). C18 analytical column (Shim-Pack solar 150 mm × 4.6 mm, with a particle size of 5 µm, Shimadzu, Japan) was used to separate sample components chromatographically. For 5FU analysis, sample was eluted with mobile phase constituting acidified water (pH 3.2, adjusted with perchloric acid) and methanol in a ratio 95:5 at a flow rate of 1 ml/min. The analyte, 5FU was detected at 266 nm wavelength after an injection volume of 10 µl. Standard concentrations of 5FU were prepared in the range 20 to 100 ng/ml in Milli-Q water. The regression equation, y = 167615x + 652.7 (R² = 0.9998) was obtained by plotting area under peak against concentration.

For MF analysis, the mobile phase included a mixture of 0.5 mM sodium phosphate buffer and acetonitrile in the ratio of 82:18 pumped at a flow rate of 1 ml/min. The maximum absorbance of MF was recorded at 294 nm with an injection volume of 10 µl. The concentration of MF in test samples was calculated against a calibration curve obtained for a standard drug at the range of 50 to 1600 µg/ml concentration with regression equation y = 126961x-1498 and correlation coefficient of R² = 0.999.

Pharmacokinetic studies

All the animal experiments were performed after protocol approval by the institutional animal ethics committee (IAEC) of BITS Pilani, Hyderabad Campus (BITS-Hyd/IAEC/2021/10). The male Wistar rats aged 7 weeks with an average weight of 200 ± 20 g were procured and housed at the animal facility maintained at room temperature of 25 ± 2°C and relative humidity of 65 ± 5%. The rats were allowed to have free access to food and water and were acclimatized for 7 days.

Rats were randomly divided into four groups and subjected to overnight fasting by placing them in fasting cages supplied with water ad libitum. Later, animals were orally administered with 5FU or MF loaded LbL film encapsulated in the pH-sensitive capsule at a dose of 15, 30 or 45 mg/kg using a dosing applicator (X-9 syringe, Torpac, USA). At pre-determined time points (0.5, 2, 4, 6, 8, 10, 12, 14, 16, 20 and 24 h), rats were anesthetized using isoflurane and oxygen mixture (5 and 21%, respectively) at a flow rate of 1 l/min (Rodent anesthesia system RAS-4, PerkinElmer, USA). The blood samples (250 µl) were collected from retro-orbital plexus into a tube containing 25 µl of dipotassium ethylene diamine tetraacetic acid salt (2% w/v in saline). The blood samples were immediately centrifuged at 5000 rpm for 10 min at 4°C (CM-12 plus, REMI, Mumbai, India) and the supernatant plasma was separated and stored at -80°C until analyzed. Similarly, control group was administered with 5FU or MF solutions of 15, 30 and 45 mg/kg body weight intravenously through the tail vein using 30G needle syringe.

Biodistribution studies

Biodistribution studies were performed for 5FU and MF individually at 30 mg/kg body weight. After overnight fasting, drugs were orally administered through LbL film encapsulated in pH-sensitive capsule. Rats were anesthetized at the time point corresponding to the T_max of drug and blood was collected by heart puncture into an anticoagulant containing tube. The plasma was collected and processed as described in the earlier section. Furthermore, rats were sacrificed by cervical dislocation and the major organs including heart, lungs, liver, spleen, kidney, stomach, small intestine, caecum, and colon were harvested. The tissues were washed with ice-cold PBS and surface dried using a blotting paper. The tissues were stored at -80°C until analyzed for the drug content. The control groups were intravenously administered with 5FU or MF through tail vein at the same dose.

Biological sample preparation and HPLC analytical method

The 5FU from the plasma matrix was extracted using the protein precipitation technique. 5FU (10 µl) was added to the blank plasma (90 µl) at different concentrations including 10000, 5000, 2500, 1250, 625 and 312.5 ng/ml, and mixed well. The effective final concentrations were 1000, 500, 250, 125, 62.5 and 31.25 ng/ml. Later, 900 µl of acetonitrile was added to the 100 µl of plasma at room temperature. Samples were vortexed and sonicated for 15 min, followed by centrifugation at 12000 rpm for 30 min. The supernatant (800 µl) was collected and subjected to solvent evaporation at 45°C under vacuum for 3 h (SCAN VAC, Labogene, Denmark). The residue was reconstituted in 150 µl of RP-HPLC mobile phase and then centrifuged at 12000 rpm for 30 min. The supernatant was collected and analyzed using the HPLC method described earlier. For analyzing plasma samples, mobile phase was adjusted to the ratio 98:2 (acidified water: methanol) and injection volume was increased to 50 µl. The concentrations of test sample were calculated against the calibration curve prepared for concentrations ranging from 31.25 ng/ml – 1000 ng/ml. The regression equation obtained was y = 34167x + 13994 with a correlation coefficient of R² = 0.9999.

While for MF, the blank plasma was spiked with similar concentrations (mentioned above) of standard MF solutions and was protein precipitated using 900 µl of the mixture of methanol and acetonitrile (3:7) to the 100 µl of plasma. After thorough mixing, samples were sonicated for 15 min and centrifuged at 12000 rpm for 30 min. The supernatant of 800 µl was collected and subjected to solvent evaporation at 45°C under vacuum for 3 h. The residue was reconstituted in 150 µl of HPLC mobile phase and then centrifuged at 12000 rpm to remove any undissolved particles. The RP-HPLC method used for MF analysis was modified for analyzing plasma samples. The injection volume was increased to 50 µl and the calibration curve was plotted with dilutions in a range of 31.25 ng/ml – 1000 ng/ml. The regression equation and correlation coefficient were y = 277386x – 20398 and R² = 0.9993, respectively.

The frozen tissue samples were thawed at room temperature. The tissue sample (0.5 g) was minced and homogenized in 2 ml of PBS using a probe homogenizer (IKA T10 basic, Ultra-Turrax, USA). The calibration plot was developed for each tissue homogenate by making different dilutions as discribed above. The homogenate was subjected to protein precipitation, followed by vacuum drying and analyzed using the RP-HPLC method described for plasma sample analysis.

Pharmacokinetic parameters

The pharmacokinetic parameters were analyzed in non-compartment model using Winnolin 64 software. The plasma concentration – time profiles of each group were analyzed and parameters such as t_1/2, clearance, volume of distribution, mean residence time and AUC were calculated. The relative bioavailability of 5FU and MF administered orally using LbL film was calculated using the following Eq. (2)

\(Relative bioavailability \left(\%\right)=\frac{{AUC}_{o-\infty } Oral}{{AUC}_{o-\infty } I.V}\) - (2)

Statistical analysis

The results were reported as mean ± standard deviation (SD). Statistical significance between the groups was analyzed using Student's t-test or analysis of variance (GraphPad Prism V5). A probability of p < 0.05 was considered statistically significant.

Characterization of LbL film

The thickness of blank film, 5FU and MF-LbL film was found to be 69.1 ± 3.4, 147 ± 11.66 and 212.3 ± 7.19 µm, respectively. Figure 2 shows the digital photograph of blank and drug-loaded LbL films.

Figure 3 shows increase in weight and thickness of LbL film with the sequential adsorption of drug loaded chitosan and alginate bilayers. Interestingly, the presence of MF showed greater absolute growth in thickness and weight of LbL film compared to 5FU. Figures 4a and 4b show the DSC thermograms and PXRD diffraction pattern of 5FU and 5FU loaded LbL film. Figure 4d shows DSC thermograms of MF and MF-LbL film. Both MF and MF-LbL film showed an endothermic peak in the range of 50–100°C, corresponding to the loss of unbound water. While the neat MF showed a sharp endothermic peak at 226.3°C followed by exothermic peak representing melting point and thermal degradation, respectively³⁹. Figure 4e shows PXRD pattern of MF with multiple intense peaks at 6.0, 8.7, 10.3, 13.6, 14.6, 17.5, 20.4, 23.7, 26.7, and 29.3°. MF-LbL film showed diffraction peaks at 8.7, 10.0, 17.4, 24.1, and 26.5° corresponding to MF indicating presence of MF in crystalline state⁴⁰. Moreover, the diffraction peaks at 31.6 and 45.4° in MF-LbL film correlate to the NaCl added to polymer solutions⁴¹. Figure 4c and 4f shows characteristic absorbance peaks of 5FU and MF loaded LbL film after each bilayer deposition. The characteristric FTIR peaks of 5FU appears in the range of 3000–3500, 1600–1820 and 1000–1400 cm^-1 corresponding to secondary amide stretching, carbonyl group stretching and C-F bond stretching vibrations, respectively³⁷. The 5FU-LbL film showed all the characteristic peaks, among which the broad peak at 3100–3300 cm¹ was found distinguishable between different bilayers. The MF shows intense absorbance for C-N and aromatic C = C stretching at 1300–1500 and 1400–1600 cm^-1, respectively^40,42, along with C = O and C-F stretching which are also present in 5FU. The MF-LbL film showed broad peaks in the range 1000–1200 cm^-1 wavenumbers attributed to vibrations of C-F bond, and absorbance in the range 1250–1500 and 1500–1700 cm^-1 represents vibration of aliphatic C-N and carbonyl group, respectively. The instensity of absorbance peaks in MF-LbL film was found 10 times greater than 5FU-LbL films which can be correlated to the observations of greater weight and thickness of MF-LbL films.

The loading efficiency of 5FU and MF in LbL film was found to be 1.93 ± 0.48 and 4.64 ± 0.33 mg per cm² area, respectively.

5FU and MF release studies from LbL film

The in vitro release of 5FU and MF was studied using Franz diffusion cell. Figure 5 shows the 5FU and MF release profiles from the chitosan and PCL-side of LbL film. After 6 h of study, 43.7 ± 11.0% and 7.87 ± 2.25% of MF was observed to be released from the chitosan-side and PCL side, respectively. The complete release of MF was observed at 24 h from the chitosan side. The cumulative amount of MF released from the chitosan-side was significantly (p < 0.0001) greater than the PCL-side. The drug release behavior of 5FU from LbL film was reported in our earlier publication³⁷. A similar observation of restricted drug release from the PCL (backing layer) side was observed for 5FU. The 5FU has shown initial burst release followed by controlled release behavior. Whereas, MF did not show burst release from LbL film.

Pharmacokinetics of 5FU

The plasma concentration of 5FU administered through IV route and oral route was determined using the RP-HPLC method. The lower limit of quantitation (LLOQ) of 5FU spiked in plasma was found to be 31.25 ng/ml. Supplementargy Figure S1a shows the overlay of representative chromatograms of blank plasma and 5FU spiked plasma. Figures 6a and 6b show the plasma concentration – time profile after IV and oral administration of 5FU at a dose of 15, 30 and 45 mg/kg body weight.

Table 1 shows the pharmacokinetic parameters of 5FU at 15, 30 and 45 mg/kg doses administered through IV route and LbL film after oral delivery. The C_max of 5FU after IV administration was found to be 7835 ± 978.8, 21355 ± 723.52 and 45271 ± 2863.1 ng/ml for 15, 30 and 45 mg/kg, respectively. The C_max reduced when 5FU was delivered orally through LbL film at 46.18 ± 14.60, 369.2 ± 25.90 and 437.4 ± 15.22 ng/ml for 15, 30 and 45 mg/kg, respectively. The AUC and C_max values increaseed with the increase in dose for both IV and orally administered 5FU. However, the increase in C_max between oral 30 and 45 mg/kg was not proportional. This resulted in the reduced relative bioavailability of 5FU to 18.56 ± 1.03% at the higher dose of 45 mg/kg while 39.63 ± 6.34% was obtained at 30 mg/kg dose. From the pharmacokinetic studies of different doses of 5FU, it was understood that 5FU undergoes non-linear pharmacokinetics after oral route of administration (Fig. 7). The lag time of 6 h was observed for LbL film. In IV route, 5FU was found to be eliminated in biphasic nature where there was a rapid declining initial phase. The t_1/2 obtained for 5FU when administered through IV route is 10.38 ± 0.80, 26.43 ± 3.33 and 27.82 ± 3.96 min for 15, 30 and 45 mg/kg, respectively. On the other hand, the t_1/2 and mean residence time (MRT) of 5FU administered through LbL film was longer than when administered through IV route.

Table 1

Pharmacokinetic parameters of 5FU administered through intravenous and oral route.
	IV route
Parameters	15 mg/kg	30 mg/kg	45 mg/kg	15 mg/kg	30 mg/kg	45 mg/kg
Ke (1/min)	0.07 ± 0.01	0.03 ± 0.003	0.03 ± 0.002	-	0.01 ± 0.001	0.01 ± 0.001
t_1/2 (min)	10.38 ± 0.80	26.43 ± 3.33	27.82 ± 3.96	-	219.7 ± 58.36	208.7 ± 10.65
T_max (min)	5	5	5	720	720	720*
C_max x 10³ (ng/mL)	7.83 ± 0.98	21.36 ± 0.72	45.27 ± 2.86	0.05 ± 0.02*	0.37 ± 0.03*	0.44 ± 0.02*
AUC_0 − t x 10³ (min x ng/mL)	181.3 ± 8.89	460.2 ± 39.20	1152.9 ± 56.24	7.26 ± 1.12*	168.0 ± 19.47*	198.6 ± 5.03*
AUC_{0 − inf} x 10³ (min x ng/mL)	181.6 ± 8.57	462.0 ± 38.36	1155 ± 57.41	-	182.5 ± 27.81	214.1 ± 2.09
MRT (min)	15.54 ± 3.05	15.73 ± 2.28	23.74 ± 1.06	750.2 ± 21.43*	824.6 ± 9.24*	871.4 ± 6.45*
V_d (ng)/(ng/mL)	386.7 ± 45.06	281.2 ± 9.70	348.8 ± 21.86	86378 ± 24754*	20382 ± 1489*	36038 ± 1269*
Relative BA (%)				-	39.63 ± 6.34	18.56 ± 1.03*
K_e – elimination rate constant; MRT – mean residence time; V_d – volume of distribution. Asterisk (*) represents that the values are significantly different compared with corresponding values after intravenous administration at p < 0.001.

Pharmacokinetics of MF

The plasma concentration of MF was determined using RP-HPLC method with the lower limit of quantitation (LLOQ) of 62.5 ng/ml. Supplementary Figure S1b shows representative chromatograms of blank and MF spiked plasma. Figures 6c and 6d show the plasma concentration-time profile of MF administered through IV and oral route. Table 2 shows pharmacokinetic parameters summarized from the non-compartmental analysis. The T_max of MF administered using LbL film in oral route was found to be 14 h (840 min) which was longer than that observed for 5FU oral route administration. There was no significant difference between AUC_0−∞ of IV and LbL film, resulting in the oral bioavailability of 107.55 ± 16.34, 97.32 ± 9.40 and 93.75 ± 20.85% for 15, 30 and 45 mg/kg doses, respectively. The AUC in both IV and oral route increased linearly with the increase in dose indicating linear pharmacokinetics of MF (Fig. 7b). Similar to 5FU, the t_1/2 and mean residence time of MF administered through LbL film was significantly longer compared to IV route.

Table 2

Pharmacokinetic parameters of MF administered through intravenous and oral route.
	IV route
Parameters	15 mg/kg	30 mg/kg	45 mg/kg	15 mg/kg	30 mg/kg	45 mg/kg
Ke (1/min)	0.004 ± 0.0006	0.003 ± 0.0003	0.003 ± 0.001	0.002 ± 0.0003	0.002 ± 0.0003	0.001 ± 0.0003
t_1/2 (min)	195.9 ± 37.75	258.4 ± 25.99	224.0 ± 25.47	356.3 ± 55.58	312.2 ± 38.52	508.6 ± 120.6
T_max (min)	5	5	5	840	840	840*
C_max x 10³ (ng/mL)	5.08 ± 0.10	13.39 ± 0.32	26.65 ± 1.18	0.97 ± 0.19*	1.93 ± 0.15*	2.80 ± 0.20*
AUC_0 − t x 10³ (min x ng/mL)	573.2 ± 48.57	1177 ± 13.70	2241 ± 436.6	546.6 ± 147.3	1017 ± 85.99	1590 ± 55.60*
AUC_{0 − inf} x 10³ (min x ng/mL)	642.3 ± 52.03	1261 ± 9.79	2478 ± 633.9	693.6 ± 145.4	1227 ± 113.8	2246 ± 244.9
MRT (min)	190.2 ± 17.93	140.6 ± 3.99	151.4 ± 30.37	910.5 ± 4.25*	895.7 ± 12.12*	1267 ± 142.4*
V_d (ng)/(ng/mL)	591.2 ± 11.72	448.3 ± 10.64	591.7 ± 25.57	3945 ± 742.3*	3887 ± 287.0*	5640 ± 417.7*
Relative BA (%)				107.5 ± 16.34	97.32 ± 9.40	93.75 ± 20.85
K_e – elimination rate constant; MRT – mean residence time; V_d – volume of distribution. Asterisk (*) represents that the values are significantly different compared with corresponding values after intravenous administration at p < 0.001.

Biodistribution studies

The biodistribution studies were performed for 5FU and MF administered through IV and oral route. Figure 8 shows the disposition of 5FU and MF in different organs and tissues after administering through IV and oral LbL film. The amount of 5FU or MF distributed in different tissues was analyzed using RP-HPLC method. Supplementary Table S1 provides extraction efficiency of 5FU and MF from different organs and tissues. In general, extractional efficiency was at least 80% or more in any sample. When 5FU was administered through IV route, it was found to be disposessed more in liver (69.02 ± 11.64 µg/g) heart (58.38 ± 5.34 µg/g) and kidney (42.58 ± 19.34 µg/g). The caecum and colon tissues had 2.08 ± 1.11 and 4.02 ± 0.42 µg/g of 5FU, respectively. In contrast, when the 5FU bL film was orally administered, 196.8 ± 33.42 and 67.46 ± 29.42 µg/g of 5FU was found in the luminal contents of caecum and colon tissues. Moreover, the amount of 5FU found within the colon tissue was 9.52 ± 2.13 µg/g after administration using LbL film.

The IV administration resulted in deposition of MF in gastroinstestinal tissues stomach, small intestine, caecum and colon at 31.97 ± 7.35, 46.36 ± 4.43, 22.09 ± 6.63, 13.02 ± 3.78 µg/g, respectively more than highly perfused organs. The oral administration of LbL film resulted in MF deposition of 166.78 ± 34.65, 92.46 ± 25.53 µg/g in lumen of caecum and colon regions, respectively.

There is a growing interest in the development of oral patches for drug delivery⁴⁴. The patch-based delivery system has been especially developed to adhere to a site within gastro-intestinal tract and deliver the entrapped drugs for local and systemic applications ^13,14,45. However, there have been no systematic studies reporting the pharmacokinetics and biodistribution of drugs belonging to different BCS classification delivered using colon targeted oral patches. Here, we report the pharmacokinetic analysis of BCS Class III and Class I compounds after oral administration using colon targeted LbL film.

LbL films were constructed with polyelectrolytes originating from natural or synthetic sources. The chitosan and sodium alginate used in preparing LbL film are biodegradable and show mucoadhesive property^46–48. The nature of interactions between the entrapped drug and polyelectrolytes influence the loading efficiency and release behavior from LbL films^49,50. The thickness of LbL film increased by 2.1-fold after 5FU entrapment compared with blank film. Whereas, the increase in thickness of LbL film after MF loading was 3-fold greater compared with blank film. 5FU and MF were found to be in crystalline state after entrapment in chitosan-alginate LbL film. 5FU remains unionized at pH values less than 8⁵¹. The number of hydrogen bond donors and acceptors are only 2 and 2 in 5FU⁴⁹. On the other hand, MF is negatively charged at pH values below 8⁵². This results in electrostatic attractive interactions with positively charged chitosan in LbL film. The electrostatic interactions also caused slow release of MF from LbL film. The highly water soluble 5FU release profile from LbL film showed a two-phase release characteristic, where burst release of 35.51 ± 6.05% was found within 0.5 h. There was a slow release of the remaining 5FU with 63.81 ± 4.52% released from LbL film in 24 h. On the other hand, MF release profile showed a single-phase release characteristic without burst release, where complete release was achieved in 24 h.

The mucoadhesive nature of chitosan surface allows adhesion of LbL film to the colon tissue after release from the Eudragit S100 coated capsule upon oral administration^37,53. Earlier reports showed directional release of 5FU, oxaliplatin and STAT3 siRNA from LbL film after adhesion in the colon tissue^13,37. The plasma concentration of 5FU and MF were detected only after 6 h of oral administration, attributed to the gastro-intestinal transit time to reach large intestine and dissolve the Eudragit S100 to release LbL film ⁴³. The orally administered 5FU LbL film acts as a reservoir within the colon and caecum region. The poor epithelial permeability of 5FU also contributes to the accumulation of 5FU within the colon region. The folic acid conjugated LbL film containing oxaliplatin and STAT3 siRNA was found to bind to the colon tumor lesions and provide tumor remission¹³. Therefore, the prolonged exposure of 5FU within colon tissue can be effective in its localized delivery in colo-rectal cancer conditions^54,55. The 5FU administered orally (5FU-LbL film) showed longer plasma half-life of 12 h. In contrast, 5FU administered through IV route undergoes hepatic metabolism and showed a shorter plasma half-life of 0.083 h⁵⁶. The orally administered 5FU LbL film showed nonlinear pharmacokinetics attributed to the poor relative bioavailability of 5FU.

In the case of BCS Class I compound, MF showed linear pharmacokinetics after oral administration entrapped in colon targeted LbL film. However, similar to 5FU, the T_max of MF after oral administration was achieved at 14 h after dosing. This could be attributed to the slow release of MF from LbL film as demonstrated in vitro release studies. Nonetheless, MF achieved relative bioavailability of > 94% at all the dosing concentrations.

The first line of treatment for colon cancer includes administration of 5FU by intravenous infusion. The current study showed only 4.25 ± 0.20 and 3.05 ± 0.63% of intravenously administered 5FU dose distributed in colon and caecum regions, respectively. While, 21.83 ± 3.44 and 60.19 ± 5.92% of orally administered 5FU dose was retained in the colon and caecum tissues. The deposition of 5FU in the heart decreased after administration through oral LbL film that could decrease cardiotoxicity of 5FU⁵⁷. Taken together, drug delivery through LbL film offers longer residence time and localization of drug within gastrointestinal tract, thereby minimizing unwanted distribution to healthy tissues.

The 5FU and MF loaded LbL film was prepared for colon targeted delivery using Eudragit S100 coated capsule. The drug loading and release behaviour is influenced by the physicochemical properties of 5FU and MF and their interactions with chitosan and alginate in LbL film. Oral administration of 5FU and MF loaded LbL films follow non-linear pharmacokinetics and linear pharmacokinetics, respectively. When compared to IV administration, both 5FU and MF showed greater mean residence time when delivered using LbL film. Overall, LbL film has similar pharmacokinetic parameters for 5FU and MF. In addition, colon targeted LbL film can provided enhanced localized drug delivery in colon region for both poorly and highly permeable drugs.

Ethics approval

All the animal experiments were performed after approval of protocol by the institutional animal ethics committee (IAEC) of BITS Pilani, Hyderabad Campus with approval number BITS-Hyd/IAEC/2021/10.

Consent for publication

Not applicable

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors have no relevant financial or non-financial interests to disclose

Funding

This work was financially supported by the Science and Engineering Research Board, Department of Science and Technology, India under Core Research Grant scheme (EMR/2016/005421).

Authors' contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Leela Sai Lokesh Janardhanam, Ashwini Suresh Deokar, Srivarsha Reddy Bollareddy and Venkata Vamsi Krishna Venuganti. The first draft of the manuscript was written by Leela Sai Lokesh Janardhanam and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable

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Colon targeted layer-by-layer self-assembled film: pharmacokinetic analysis of model drugs after oral administration

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Materials And Methods

Results

Characterization of LbL film

5FU and MF release studies from LbL film

Pharmacokinetics of 5FU

Pharmacokinetics of MF

Biodistribution studies

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