It was previously reported that combining GlcN with ibuprofen in a racemic mixture enhanced the potency of ibuprofen, but increasing the dose of GlcN did not further increase the antinociceptive effect of the drug, suggesting a threshold effect. 11 Therefore, the aim of the current study was to investigate the effect of GlcN on modulating the serum level of paracetamol in rats in an attempt to enhance the BA of paracetamol. The involvement of GlcN in the absorption, protein binding, and metabolism of paracetamol was further evaluated to reveal the mechanism by which GlcN affects paracetamol pharmacokinetics.

Glucosamine (GlcN) (2‐amino‐2‐deoxy‐d‐glucose) is an amino monosaccharide that is synthesized naturally inside the cells as GlcN‐6‐phosphate via the hexosamine pathway following the combination of glutamine with fructose‐6‐phosphate using glutamine–fructose‐6‐phosphate amidotransferase. 8 The end‐product of the hexokinase pathway is the formation of uridine‐diphosphate‐N‐acetylglucosamine, which is considered important for the formation of glycolipids, proteoglycans, and glycosaminoglycans. 9 As glycosaminoglycans are major components of joint cartilage and GlcN is not toxic, several clinical trials were directed toward providing further evidence that GlcN supplementation could beneficially influence cartilage structure and alleviate arthritis. 8 , 10 Most of these clinical studies, however, revealed conflicting results on the efficacy of GlcN. Moreover, the concomitant use of painkillers in most clinical studies was unfortunately neglected as an important factor in the efficacy determinations of GlcN.

Paracetamol is the most common analgesic and antipyretic drug in the world. The therapeutic dose undergoes a heavy metabolism (80%–90%) by the liver in a short period of time and therefore lowers its bioavailability (BA). 1 In humans, paracetamol is metabolized in the liver mainly by glucuronide conjugation (40%–50%) and sulfate conjugation (30%–40%), whereas a lesser portion (<20%) is metabolized by cytochrome P450 enzymes (CYP450) to highly reactive intermediate form (N‐acetyl‐p‐benzoquinone imine). The latter form is rapidly detoxified by covalent binding to reduced glutathione (GSH). 2 However, at higher doses of paracetamol, the liver GSH is depleted and the reactive intermediate reacts with other molecules in liver cells leading to hepatotoxicity. 3 In addition, depleting sulfhydryl levels induces lipid peroxidation and leads to decreased liver total antioxidant capability. 4 , 5 Furthermore, paracetamol‐reactive metabolite not only produces hepatic damage, but is also capable of damaging the kidney's medulla and tubules, and reducing the glomerular filtration rate. 6 , 7

Noncompartmental approach was followed for the pharmacokinetic evaluation of the data. The concentrations were calculated and plotted versus time to construct the serum time profiles of paracetamol. The area under the curve (AUC) calculation was based on the trapezoidal rule. The maximum concentration ( C max ), and the time required for the maximum observed concentration ( T max ) were deducted directly from the serum concentration–time curves. The elimination half‐life ( t 1/2 ) was estimated from slopes of the terminal segments of logarithmically transformed serum levels against their corresponding times. The mean residual time (MRT) was calculated by dividing the area under the first moment curve (AUMC) by the AUC. Serum profiles are expressed as mean ± SEM for each group or time points. Data in tables are expressed as mean ± SD. Student's t ‐test was used to analyze the difference between two groups using SPSS 17 software. p < 0.05 was considered significant.

Rats were fed orally with GlcN solution (12 mg/mL) for 2 days, and 2 h prior to paracetamol administration, a 53‐mg/kg of GlcN was also administered. Different oral doses of paracetamol (70, 700, and 2800 mg/kg) were administered and twenty 4 h later, rats were sacrificed, and blood samples were collected, left to clot, centrifuged, and sera were analyzed for liver function tests.

Following the preparation of the protein and ligands structures, the ligands were docked into the previously identified active site using AutoDock (version 4.2). Lamarckian Genetic Algorithm was employed for the conformational sampling of the ligand structures, whereas the protein was treated as rigid (default settings used). Docked conformations were rated by the AutoDock scoring function that includes terms for van der Waals, hydrogen bond, and electrostatic interactions, in addition to internal energy of the ligand.

Furthermore, Gasteiger–Marsili partial charges were assigned to the ligand structures, which were downloaded from the PubChem database as a minimized 3D structure. 26 However, for chitosan molecules, the 2D structure were built using ChemSketch (Toronto, Canada) that afterward underwent a conformational enumeration process via OMEGA (Openeye, Santa Fe, New Mexico), where the lowest energy conformer was selected for docking. Amino groups in GlcN and chitosan were assigned as protonated.

The protein structure of the human cytochrome P450 2E1 (CYP2E1) was downloaded from the protein data bank (PDB, ID: 3E6I and 3GPH; http://www.pdb.org ). 21 , 22 Next, all water molecules were removed, and partial charges were assigned to all atoms using Kollman united atom model in the AutoDock Tool program. 23 The partial charge of the Heme's iron atom was assigned manually as +1.130. 24 Subsequently, the active site for paracetamol docking was identified by the cocrystallized ligand (PDB: 3E6I) and a grid box of a 50 × 50 × 50 Å 3 size was created with a grid spacing of 0.375 Å using AutoGrid. 25 Different grid size was adopted in the chitosan docking into CYP2E1 (PDB: 3GPH); 50 × 50 × 80 Å 3 box size centered at the heterocyclic ring of the cocrystallized ligand was used in the monomer (i.e., GlcN) and dimer docking, whereas 60 × 74 × 60 Å 3 box size centered at the middle of the hydrophobic chain of the cocrystallized ligand was used in the trimer, tetramer, and pentamer docking.

For the determination of serum protein binding, 150 μL of fresh serum was spiked with a suitable volume of paracetamol solutions to get 10 μg/mL final paracetamol concentration. Standard sample was prepared by spiking of 150 μL phosphate buffer (0.2 M) with paracetamol solution to get 10 μg/mL as a final concentration. Samples were then incubated in a water bath at 37°C for 90 min. After that, samples were ultrafiltrated using 30 μm filter tubes (Millipore, Billerica, MA, USA) following centrifugation at 2500 g for 25 min. Serum proteins and bound drug were detained in the upper compartment of the filter tube, whereas free drug was collected in the lower compartment of the filter tube. 20 The concentration of unbound paracetamol in the lower compartment of the filter was determined by HPLC, applying the same methods described above. This experiment was repeated six times.

Fasting rats were killed by high dose of inhaled ether. The small intestine was removed and washed several times by normal saline (0.9% NaCl) and placed in oxygenated Krebs buffer at 37°C. The intestine was everted on a glass rod; one end of the intestine was clamped and the whole intestine was filled with oxygenated Krebs buffer through the other end, which was then sealed. The everted intestine was divided into sacs of 2.5–3 cm length by using silk braided sutures; few centimeters of ileum and duodenum were discarded. 17 Sacs, then, were divided into four groups ( n = 7 in each group) and soaked in a shaking water bath (60 cycles/min) for 15 min at 37°C. At the end of 15 min, sacs were transferred to 50 mL flask containing 20 mL of oxygenated paracetamol solution and incubated as above. At the appropriate time points (5, 10, 15, 20, and 30 min), sacs were removed, washed several times in saline (0.9% NaCl solution) and blotted dry. Samples were drawn from inside the sacs by a needle and placed in Eppendorf tubes and kept in the refrigerator until analysis. 18 Samples were diluted and then analyzed using a UV method at a wavelength of 257 nm using UV–Vis spectrophotometer (Beckman Coulter, Brea, California, USA). 19

A simulated physiological solution, Krebs buffer, was prepared and oxygenated with carbogen gas (95% O 2 + 5% CO 2 ). After oxygenation, CaCl 2 was added, and the pH was adjusted to 7.4 using 1 M NaOH. Paracetamol solutions were dissolved in Krebs buffer to give a concentration of 0.5 mg/mL, whereas three soaking solutions were prepared by dissolving: GlcN, chitosan 30 kDa, sodium lauryl sulfate (SLS) in 100 mL normal saline to a final concentration of 5 mg/mL. The pH was adjusted to 5 by concentrated HCl.

Paracetamol solutions (5 μM): paracetamol–GlcN mixture solution (5 μM) with (1:4) ratio and paracetamol–propranolol (a known inhibitor to paracetamol metabolism) were prepared. In each run, the above three conditions, in addition to a negative control, were tested. Samples were diluted in 600 μL of 0.1 M phosphate buffer and incubated in a shaking water bath at 37°C and incubated for 5 min. Following incubation, the reaction was started by adding 100 μL of liver homogenate and 250 μL of cofactors solution or phosphate buffer. Then, a volume of 100 μL was withdrawn from each tube and added to Eppendorf‐containing 50 μL perchloric acid at 0, 5, 15, 30, and 60 min, vortexed, and placed on ice. The control group, on the contrary, was examined at 0 and 30 min only to ensure that no metabolic reaction occurred and to compare it with two other groups. 16 Samples, then, were centrifuged at 2500 g for 15 min and each supernatant was injected in the HPLC system as described above. This experiment was repeated three times from three different rat livers.

Phase Ι cofactors were used to show GlcN effect on paracetamol phase Ι metabolic reaction. 15 Phase Ι and ΙΙ cofactors were used together to study the combination of the two phases metabolic reactions. Cofactor solutions were prepared by dissolving the corresponding cofactors in 2% NaHCO 3 : phase Ι cofactors (NADPH regeneration system) consisted of: NADP (1.7 mg/mL), glucose‐6‐phosphate (7.8 mg/mL), glucose‐6‐phosphate dehydrogenase (6 units/mL), whereas phase ΙΙ cofactors consisted of: uridine‐5‐α‐d‐diphosphate glucuronic acid (1.9 mg/mL), and 3‐phosphoadenosine 5‐phosphosulfate (100 μg/mL). 15

Rat livers were excised, washed with cold saline, and 5 g of liver tissue weighed and homogenized in 10 mL ice‐cold homogenizing solution (33 mM KCl, 8 mM MgCl 2 , 100 mM phosphate buffer pH 7.4) for 1 min. 14 The mixture was centrifuged for 20 min at 6500 g at 4°C, and the resulting supernatant was transferred into another tube and centrifuged for a further 20 min at 6500 g at 4°C. The protein concentration in the liver homogenate was determined by the Lowry method. Aliquots were diluted twice in 0.1 M phosphate buffer to reach the working protein concentration of 30 mg/mL and were stored at −80°C until use.

Each sample (150 μL) was transferred to an Eppendorf tube and 20 μL of 30% perchloric acid was added to precipitate the proteins. Samples were vortexed for 30 s and centrifuged (ALC 4239R, high‐speed refrigerated centrifuge; ALC International SrI, Cologno Monzese, Italy) at 2500 g for 10 min. The supernatant was transferred to HPLC by injecting a 25‐μL volume injected into the column autosampler. The accuracy and precision were within 10% of the tested concentration range and all measured paracetamol concentrations were above the lower limit of quantification (0.2 μM). 13

Animals were divided into groups and oral administrations of freshly prepared solutions was performed using stainless steel oral gavage needles (Harvard Apparatus, Kent, UK). Following oral administrations, blood samples were collected from rat's tail at different time intervals (0.5, 2, 3, 4, 6, and 8 h). Blood was left to clot, centrifuged for 10 min at 2000 g, and then serum was separated and transferred directly into Eppendorf tubes, and kept in a freezer at −20°C until analysis.

Paracetamol or paracetamol–GlcN mixture solutions were prepared by preparing paracetamol reference and GlcN solutions. Paracetamol solution was prepared by dissolving 380 mg paracetamol and 100 mg NaOH in 50 mL distilled water. The GlcN‐containing solutions were prepared by dissolving accurate weights of 540, 1619, 2159, 4317, and 6480 mg of GlcN, or none, in 30 mL distilled water to get molar ratios (paracetamol–GlcN) of (1:1, 1:3, 1:4, 1:8, and 1:12). Later, paracetamol solution was added drop wise into GlcN solution under efficient stirring. During the additional process, the pH of the mixture was monitored and adjusted to 3 by concentrated HCl.

Adult nonpregnant female Sprague–Dawley rats with an average weight of 220 ± 20 g were purchased from Yarmouk University (Irbid, Jordan). Animals were accommodated at the University of Petra's Animal House (Amman, Jordan) under controlled temperature (22°C–24°C), humidity (55%–65%), and a 12‐h light/dark photoperiod cycle. Rats were offered standard pellet diet (Jordan Feed Company Ltd., Amman, Jordan) and served tap water ad libitum . All rats were acclimatized for 14 days before experimentation. All experiments were carried out in accordance with University of Petra's Institutional Guidelines on Animal Use that adopts the guidelines of the Federation of European Laboratory Animal Sciences Association (FELASA). The animal study protocols were revised and approved by the Ethical Committee of the Higher Research Council at the Faculty of Pharmacy and Medical Sciences, University of Petra (Amman, Jordan).

As the previous experiments showed that GlcN increases the BA of paracetamol by reducing its liver metabolism and it is known that paracetamol metabolites induces hepatocyte damage, the following experiments were performed to show that preadministrating GlcN reduces paracetamol‐induced hepatocyte injury. Hepatocyte injury was observed by elevation of serum ALT, AST, ALP, and GGT levels following increasing paracetamol administration (0, 70, 700, and 2800 mg/kg) ( p < 0.01) (Figs. 6 a–6d). Serum albumin levels, however, did not change (data not shown). Furthermore, the percent increase of ALT and AST was much higher than percent increase of ALP and GGT levels. Preadministering GLcN significantly reduced the increase of ALT and AST induced by paracetamol dosage ( p < 0.01; Figs. 6 a and 6 b). On the contrary, GLcN increased GGT levels with paracetamol dose of 700 mg/kg only (Figs. 6 c and 6 d).

Chitosan docking was performed with a different chain length and into a more open active site adopted by the human CYP2E1 protein as compared with the structure used in the paracetamol docking. The human CYP2E1 active pocket could accommodate up to two GlcN molecules (the dimer even has a better score than the monomer), and then the binding energy started to increase proportionally with the molecular weight and thus binding becomes less favorable (Table 5 ).

The GlcN molecule also possesses a pretty good binding mode in the human CYP2E1 active site, especially that it formed bidentate hydrogen bonding with the Thr303 side chain (by its hydroxyl and tertiary amine groups). Moreover, it appeared to be easily coordinated with the iron atom by one of its hydroxyl groups (both in a 3.2‐Å distance). GlcN (AutoDock score: −3.6 Kcal/mol) seemed to be less favorable than paracetamol (AutoDock score: −5.3 Kcal/mol), and this is probably because of the hydrophobic nature of the human CYP2E1 active site that better suits the latter molecule (Fig. 5 ; Table 5 ).

Cytochrome P450 2E1 is the main isoenzyme that is responsible of the paracetamol metabolism. 27 Similar to the cocrystallized ligand (indazole), paracetamol forms a hydrogen‐bonding interaction with the side chain of Thr303, the only polar residue lining the human CYP2E1 active site. Paracetamol is further stabilized by forming several hydrophobic interactions with the side chains of Phe106, Ile115, Phe116, Phe298, and Phe478 (Fig. 5 ; Table 5 ).

The absorption curve of paracetamol versus paracetamol–GlcN (1:4) concentration in everted rat intestinal sac after incubation in solution containing paracetamol concentration of 0.5 mg/mL (a). The data are presented as mean ± SEM. (b) Shows the absorption curve of paracetamol in the everted rat intestinal sac versus time after incubation for 15 min in normal saline; GlcN concentration (5 mg/mL); chitosan 30 kDa (5 mg/mL), and SLS (1 mg/mL), followed by paracetamol (0.5 mg/mL) incubation. The data are presented as mean ± SEM ( n = 7) (* p < 0.05).

To assess paracetamol metabolism in the presence of GlcN at 1:4 ratio, in vitro liver homogenate assay was used. GlcN reduced paracetamol phase I metabolism by 46% ( p < 0.05) within 60 min (Fig. 3 a). In phase Ι and ΙΙ metabolisms, GlcN also significantly reduced paracetamol metabolism by 53% within 60 min ( p < 0.005) (Fig. 3 b). In comparison to propranolol (an inhibitor to paracetamol metabolism), GlcN influence was significantly more than propranolol on reducing paracetamol metabolism by liver homogenate (Fig. 3 b; p < 0.05).

Serum concentration versus time curves of paracetamol in rats after a single oral dose of 10 mg/kg as paracetamol in normal‐fed rats and in rats fed with GlcN for 2 days prior to administration of paracetamol. Rats were fed with GlcN solution (12 mg/mL) for 2 days prior the experiment and a single dose 2 h prior to receiving paracetamol. The data are presented as mean ± SEM ( n = 5).

Finally, GlcN solution (12 mg/mL) was fed to rats for 2 days and 53 mg/kg of GlcN was also given orally 2 h prior to paracetamol administration with a 10‐mg/kg dose. Paracetamol AUC and C max values in GlcN‐fed rats significantly increased by 165% and 88% ( p < 0.0001) (Fig. 2 ; Table 4 ). This increase in AUC and C max values were not associated with any change in t 1/2 or MRT (Table 4 ).

The third sets of experiments were performed to compare paracetamol–GlcN complexes with lower ratios (1:4, 1:3, and 1:1) on paracetamol BA using a single dose of 30 mg/kg paracetamol. Results showed that paracetamol–GlcN (1:4, 1:3, and 1:1) preparations did not induce a significant change in paracetamol AUC or C max ( p > 0.05) (Table 3 ). The latter changes were 21%, 8%, and 2%, respectively, for AUC and 19%, 8%, and 0% for C max , respectively. In addition, none of the tested preparations affected the T max value of paracetamol, t 1/2 , or MRT values of paracetamol (data not shown).

To gauge the paracetamol–GlcN mixture (1:4) on different doses of paracetamol, a set of experiments were performed to evaluate such an effect on paracetamol BA. Results showed that at higher doses of paracetamol (30 and 90 mg/kg) with GlcN at 1:4 did not induce a significant increase ( p > 0.05) in paracetamol AUC and C max as compared with the increase induced by 10 mg/kg dose (Table 2 ). This insignificant increase was 21% and 23% for AUC, and 9% and 14% for C max , respectively.

Different sets of experiments were carried out in different doses of paracetamol with and without GlcN to show the effect of GlcN on paracetamol BA. The first sets of experiments were performed to compare paracetamol–GlcN at ratios of 1:12, 1:8, and 1:4 on paracetamol BA using a single dose of paracetamol (10 mg/kg). Results showed that paracetamol–GlcN at a ratio of 1:4 significantly increased paracetamol AUC and C max by 100% and 66%, respectively ( p < 0.05). This significant increase in AUC and C max , however, was not seen at 1:8 and 1:12 of paracetamol–GlcN ratios (Fig. 1 ; Table 1 ). Conversely, none of the tested preparations affected the T max value of paracetamol, t 1/2 , or MRT values of paracetamol (data not shown).

DISCUSSION

The present study was performed to investigate the effect of GlcN on paracetamol pharmacokinetics and metabolism. In rats and following oral administration of different doses of paracetamol–GlcN racemic mixtures, GlcN at 4:1 to paracetamol 10 mg/kg significantly increased the BA of paracetamol. This increase, however, was either less or not observed at higher doses of paracetamol or different ratios to GlcN. Persiani et al.28 have shown that GlcN absorption in humans is reduced if given in high doses. Pharmacokinetics and BA parameters showed that C max of GlcN was linear only within dose 750–1500 mg but not at higher doses (e.g., 3000 mg). Furthermore, GlcN undergoes extensive hepatic first‐pass metabolism resulting in a relatively low bioavailability (20%).8, 28 If GlcN doses used in this study is converted to human adult, the 1:8 and 1:12 paracetamol–GlcN ratios corresponds to over 7000 mg of GlcN. This may be the reason that no effects on AUC or C max values were seen at these ratios.

The different paracetamol–GlcN ratios’ experiments pointed out that GlcN effect on paracetamol BA is dose‐dependent. Furthermore, feeding GlcN to rats for 2 days prior to paracetamol administration increased paracetamol AUC and C max even further than administration of paracetamol–GlcN mixture. This could be explained by that GlcN needs more time (2–3 h) to reach peak serum concentration29 than that of paracetamol (0.5–1 h),2 and GlcN oral feeding may saturates the enzymatic liver sites for paracetamol metabolism before paracetamol reaches liver and becomes metabolized. This agrees with other studies that proved that sulfotransferases are rapidly saturated, whereas glucuronosyl transferases have the higher capacity for paracetamol‐conjugate formation.30 As 10 mg/kg in rats corresponds to 700 mg in adult (within the therapeutic dose), this may suggest that higher doses used in the study 30 and 90 mg/kg (corresponds to 2100 and 6300 mg/dose in humans) may saturate the glucuronidation and sulfation process of paracetamol. This shows that paracetamol–GlcN effect was not dependent on complexation or structure changing but depends on a drug‐reducing metabolism interaction.

Earlier studies have pointed that the combination of GlcN with ibuprofen as a racemic mixture reduced the effective dose of ibuprofen by 58%,11 but increasing the dose of GlcN did not further increase the antinociceptive effect when combined with ibuprofen suggesting a threshold effect. In the same study, the authors studied the racemic mixture of GlcN with paracetamol on a paracetamol dose of 146 mg/kg (14 times higher than in the present study). The antinociceptive effect of the latter high‐paracetamol dose was actually antagonized by GlcN.31 Thus, it can be concluded that the effective dose of the drug and its racemic ratio mixture with GlcN is crucial to have either positive or no effect on the drug.

To study the effect of GlcN on metabolism of paracetamol, several in vitro and in silico experiments were performed, namely, on microsomal, cytosolic, and docking into CYP2E1. In these studies, GlcN reduced paracetamol metabolism by 48% through phase Ι CYP 450 and phase I and II by 54%. The latter reduction was even more than propranolol, a known inhibitor to paracetamol metabolism. It has been shown in a human study that propranolol increased the BA of 1500 mg dose of paracetamol by reducing its oxidation and glucuronidation pathways.32 Furthermore, our docking results indicate that GlcN, to some extent, is capable of competing with the paracetamol molecules on the catalytic pocket of the human CYP2E1 protein. Docking was also performed for chitosan where additional GlcN molecules were incrementally added and then docked into the human CYP2E1 active site. In addition to the favorable binding shown by GlcN, the chitosan dimer molecule has also had favorable binding with CYP2E1. However, binding of trimer and larger polymers was unfavorable by CYP2E1 because of (1) the serious clashes formed between these ligands and the protein residues and (2) the high distortion present in the generated ligand poses.

As the current study is performed on rats, it has to be mentioned that CYP2E1 expression shows a quite strong conservation among species with an identity to human CYP2E1 of 80% for rat, mouse, and dog and of 96% for monkey.33 Moreover, CYP2E1 accounts for approximately 6% of total P450 in the liver in human and is involved in the metabolism of 2% of the drugs on the market including paracetamol. On the contrary, the intestinal involvement of the reported interaction between paracetamol and GlcN could be minimal because of the low expression of CYP3A1 in the intestine of female rats.34

All of the above results favor the fact that GlcN might have a protective activity on paracetamol‐induced hepatic injury by reducing the extent of paracetamol metabolism. Thus, we performed a preclinical study and found that GlcN reduced hepatic injury as measured by ALT and AST serum enzymes but it had no effect on the increase of GGT and ALP enzymes. These data suggest that GlcN induces a hepatocyte protection but may not protect against biliary tract damage. Similar studies have shown the antioxidant activity of GlcN by its reducing power and superoxide/hydroxyl‐radical scavenging abilities and increasing the radical scavenging capacity.35, 36 In recent years, numerous studies have shown that chitosan and its derivatives have antioxidative activities both in vivo and in vitro. These studies also showed that the scavenging effect of chitosan on hydroxy radicals inhibited lipid peroxidation and prevented liver injury.37 Furthermore, a very recent study has shown that GlcN reduces genotoxic effect induced by paracetamol in cultured lymphocytes.38

Intervening drugs absorption could take place by changing the speed of gastric emptying and altering the rate of delivery of other drugs to the site of absorption, which influence drug uptake. The combined administration of paracetamol and metoclopramide has also been used to enhance the rate of drug absorption in the treatment of migraine.39 In the present study, the model of the ERIS was adapted to enable the investigation of the absorption effect of GlcN on paracetamol. Furthermore, this model has been well elucidated for its correlation with human absorption and it is well suitable for evaluating the intestinal absorption without the influence of hepatic first‐pass metabolism.40 Meanwhile, the intestinal absorption behaviors of paracetamol in the presence of absorption enhancers were also investigated. Recently, chitosan has been studied as a potential enhancer of mucosal drug absorption. The mechanism of action was proposed to be a combination of muco adhesion and an effect on tight junctions in the epithelium. The absorption‐enhancing capacity was appeared at high‐molecular‐weight chitosan.41 The results herein appeared that paracetamol absorption was not modulated by GlcN at all time intervals. Paracetamol absorption was increased by 27% in the presence of Chitosan 30 kDa. SLS, on the contrary, increased paracetamol intestinal absorption by 38%.

The present study showed that GlcN can increase the relative BA of paracetamol mostly by metabolic enzyme inhibition but not through paracetamol absorption or protein binding. Furthermore, the present study suggests that GlcN may reduce paracetamol liver toxicity, but further testing is warranted. These studies are to determine the GlcN effect on liver GSH level and lipid peroxidation following escalating doses of paracetamol. In addition, further studies of GlcN metabolism and its effect on CYP450 systems are warranted.