Exploring the impact of physicochemical properties of liposomal formulations on their in vivo fate.

Liposomes, vesicles composed of a phospholipid bilayer, are considered a remarkably advanced drug delivery system due to their unique properties, including their biocompatibility and biodegradability, and their capability to reduce toxicity of encapsulated drugs. The in vivo fate of an encapsulated drug in the form of liposome depends on both the drug and the liposome characteristics and the patient pathophysiology. In this review, the impact of the physicochemical properties of liposomes (lipid composition, size and charge) on their pharmacokinetics (systemic absorption, distribution and clearance) was discussed. In the rest, a comprehensive overview of different mechanisms of liposomal uptake by the cells (fusion, lipid transfer, and endocytosis) was provided. The importance of lipid composition and size of liposomes, cell type, and protein corona for each uptake pathway was explained.

Efficacy assessments of tretinoin-loaded nano lipid carriers in acne vulgaris: a double blind, split-face randomized clinical study.

Here, we assessed the efficacy and safety of Nano lipid carrier (NLC) drug delivery system containing tretinoin (NLC-TRE) in comparison with the conventional 0.05% tretinoin cream (TRE cream) in mild to moderate acne vulgaris. A stable and appropriate NLC-TRE formulation was prepared using a high-pressure homogenizer and particle characterization and physicochemical properties were evaluated under accelerated conditions. Efficacy assessment was performed via a split-face clinical study, by comparing the number of acne lesions, porphyrin production and skin biophysical parameters in both sides of the face randomly treated with NLC-TRE and TRE cream. Plasma concentration of tretinoin after topical application of NLC-TRE was measured for primary safety evaluation. We acquired a stable, spherical nanoparticles with particle size of 118.5 nm, PI equal to 0.485 and ZP of - 44.7 mV. The rate of decrease of acne lesions was significantly higher in NLC- TRE side (p value < 0.001). The size and intensity of porphyrin production in pilosebaceous follicles were significantly reduced only on NLC-TRE side (p value < 0.01). The plasma concentration of the tretinoin, after 8 weeks' application remained lower than the toxic levels. The NLC-TRE formula provides better efficiency and good loading capacity of TRE in the drug delivery system.

Investigation of MDMA Inhibitory Effect on CytochromeP450 3A4 in Isolated Perfused Rat Liver Model Using Tramadol.

Purpose: MDMA (methylenedioxymethamphetamine) is a synthetic compound, which is a structurally derivative of amphetamine. Also, it acts like an amphetamine, structurally, and functionally. MDMA uses mechanism-based inhibition, to inhibit isoenzyme CYP2D6. It can also inhibit other isoenzymes contributing to its metabolism, including CYP3A4 which is the most important member of the cytochrome P450 superfamily. Since more than 50% of drugs are metabolized by CYP3A4, its inhibition may cause harmful and even lethal drug interactions. Tramadol, as an opioid-like analgesic, is mainly metabolized into O-desmethyl tramadol (M1), by CYP2D6 and undergoes N-demethylation to M2, by CYP2B6 and CYP3A4. Due to the significant potential of abusing tramadol, either alone or in combination with MDMA, the rate of its toxicity and side effects may increase following possible MDMA relevant enzyme inhibition. Methods: Different doses of MDMA (1-10 mg/kg) were intraperitoneally administered to Wistar male rats of both control and treatment groups. Then, after one hour, their isolated livers were perfused by perfusion buffer containing tramadol (1 µg/mL). Afterward, perfusate samples were collected. They were analyzed by HPLC to determine the concentrations of tramadol and its metabolites. Results: MDMA administration in treatment groups reduced M1 production. On the other hand, by following the treatment with different MDMA doses, the M2 metabolic ratio increased by 46 to 101%. Conclusion: it seems that the regular doses of MDMA cannot inhibit the CYP3A4 activity.

Impact of A Cargo-Less Liposomal Formulation on Dietary Obesity-Related Metabolic Disorders in Mice.

Current therapeutic options for obesity often require pharmacological intervention with dietary restrictions. Obesity is associated with underlying inflammation due to increased tissue macrophage infiltration, and recent evidence shows that inflammation can drive obesity, creating a feed forward mechanism. Therefore, targeting obesity-induced macrophage infiltration may be an effective way of treating obesity. Here, we developed cargo-less liposomes (UTS-001) using 1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC (synthetic phosphatidylcholine) as a single-agent to manage weight gain and related glucose disorders due to high fat diet (HFD) consumption in mice. UTS-001 displayed potent immunomodulatory properties, including reducing resident macrophage number in both fat and liver, downregulating liver markers involved in gluconeogenesis, and increasing marker involved in thermogenesis. As a result, UTS-001 significantly enhanced systemic glucose tolerance in vivo and insulin-stimulated cellular glucose uptake in vitro, as well as reducing fat accumulation upon ad libitum HFD consumption in mice. UTS-001 targets tissue residence macrophages to suppress tissue inflammation during HFD-induced obesity, resulting in improved weight control and glucose metabolism. Thus, UTS-001 represents a promising therapeutic strategy for body weight management and glycaemic control.

A phospholipid-based formulation for the treatment of airway inflammation in chronic respiratory diseases.

Inflammation, the major hallmark of all chronic respiratory diseases is generally managed by inhaled corticosteroids. However, long term high dose treatment can result in significant side effects. Hence, there is a medical need for non-steroidal anti-inflammatory therapies to address airway inflammation. Phospholipids have been shown to reduce inflammation in several inflammatory conditions; however, their clinical translation has been limited to liposomal formulations traditionally used as drug carriers and their biological activity has not been investigated. Here we report the first application of empty liposomes as an anti-inflammatory treatment in airway inflammation. In the current study, liposomes (UTS-001) were prepared from cholesterol and a synthetic phospholipid (DOPC). The formulation was characterised in terms of size, charge, polydispersity index, morphology and stability as colloidal suspension and freeze-dried nanoparticles. Time-dependant uptake of UTS-001 in airway epithelial cells was observed which was inhibited by nystatin demonstrating that the uptake is via the caveolae pathway. In-vitro, in primary nasal epithelial cells, UTS-001 treatment successfully attenuated IL-6 levels following TNF-α stimulation. Consistent with the in-vitro findings, in-vivo, in the ovalbumin model of allergic airway inflammation, UTS-001 significantly reduced total immune cell counts in bronchoalveolar lavage fluid and reduced airway hyperresponsiveness in response to increasing doses of methacholine challenge. Therefore, our results establish UTS-001 as a potential anti-inflammatory treatment that may be useful as a therapeutic for lung inflammatory diseases.

The effect of surface treatment on the brain delivery of curcumin nanosuspension: in vitro and in vivo studies.

Background: Curcumin, a bioactive component with multiple characteristics, has been shown to have many therapeutic effects. However, there are several limitations regarding the use of curcumin such as instability, low solubility, poor bioavailability, and rapid elimination. Different approaches have been used to solve these problems. Materials and methods: In this study, surface-modified nanosuspension (NS) is investigated as a novel brain delivery system. Two different methods were used for the preparation of nanosuspensions with two different stabilizers. The surface of the nanosuspensions was coated with D-α-tocopheryl polyethylene glycol 1,000 succinate (TPGS) and Tween 80 using physical adsorption. Curcumin NSs were prepared using two different top-down techniques by high-pressure homogenizer and probe sonicator. A validated sensitive and selective high-performance liquid chromatography method using fluorescence detection was used for the determination and quantification of curcumin. Pharmacokinetics and biodistribution of curcumin NSs and solutions after intravenous administration in rats were studied. Results: Higher levels of curcumin in the brain were detected when Tween 80-coated NS was used compared with the curcumin solution and TPGS coated NS (TPGS-NS) (P-value<0.05). Absorption of ApoE and/or B by Tween 80-coated nanoparticles (NPs) from the blood were caused transferring of these NPs into the brain using receptor-mediated endocytosis. Distribution of TPGS-NS in the brain compared with the curcumin solution was higher (P-value<0.05). Higher levels of curcumin concentration in the liver, spleen, and lung were also observed with TPGS-NS. Conclusion: The results of this study indicate that the surface-coating of NSs by Tween 80 may be used to improve the biodistribution of curcumin in the brain.

Correlation between plasma concentrations of tramadol and its metabolites and the incidence of seizure in tramadol-intoxicated patients.

BACKGROUND: Seizure is one of the important symptoms of tramadol poisoning, but its causes are still unknown. The aim of this study is to find a relationship between tramadol and the concentrations of its metabolites versus the incidence of seizures following the consumption of high doses of tramadol. METHODS: For this purpose, the blood samples of 120 tramadol-intoxicated patients were collected. The patients were divided in two groups (seizure and non-seizure). The concentrations of tramadol and its metabolites (M1, M2 and M5) were measured by using a high-performance liquid chromatography method. The relationship between tramadol and the levels of its metabolites and seizure incidences was also investigated. RESULTS: In 72% of the patients, seizures occurred in the first 3 h after the ingestion of tramadol. The seizure incidences were significantly correlated with the patients' gender, concentrations of tramadol, M1 and M2 and the history of previous seizures (p<0.001). The average concentration of M2 was significantly higher in males (p=0.003). A previous history of the use of sedative-hypnotics and the co-ingestion of benzodiazepines and other opioids were shown to significantly decrease the rate of seizure. The rate of seizure was directly related to the concentrations of tramadol and its metabolites. Higher M2 concentration in males can be considered a reason for increased incidences of seizures in males. The plasma concentration of M1 affected the onset of seizure. CONCLUSIONS: Therefore, it can be concluded that differences in the levels of the metabolites can affect the threshold of seizure in tramadol-intoxicated patients.

Simultaneous measurement of formic acid, methanol and ethanol in vitreous and blood samples of postmortem by headspace GC-FID.

BACKGROUND: Formic acid (formate) is the main reason for toxicity and death through methanol poisoning. The simultaneous determination of methanol, ethanol, and formate in the body can help to discover the cause of death and is useful in the diagnosis of acute methanol poisoning. The measurement of formate is not yet available in Iran. With regard to the increasing rate of methanol poisoning and its related mortality in Iran, as well as the main role of formate in methanol poisoning, this study was designed to set up an analytical method for the concurrent determination of ethanol, methanol, and formate. METHODS: Following the modification of a previously developed gas chromatography method, vitreous and blood samples of 43 postmortem cases with a history of methanol intoxication were collected over a period of 2 years at the Legal Medicine Organization of Mashhad. Thereafter, ethanol, methanol, and formate concentrations were measured by headspace GC/FID. Formate esterification was performed by the methylation of formate with sulfuric acid and methanol. In order to confirm the esterification method for the production of methyl formate, we used gas chromatography with a mass detector (GC/MS) because of its higher sensitivity and accuracy. Furthermore, the correlations between formate and methanol concentrations in blood and vitreous samples, and between formate and methanol were investigated. RESULTS: A significant relationship was found only between methanol concentrations in blood and vitreous samples (P < 0.03). CONCLUSIONS: In postmortems, with the passage of time since alcohol ingestion, the measurement of only methanol concentration cannot determine the degree of toxicity or the cause of death. Therefore, using the present analytical method and measurement of formic acid, we can estimate the degree of toxicity and cause of death.

Changes in CYP2D enzyme activity following induction of type 2 diabetes, and administration of cinnamon and metformin: an experimental animal study.

1. Alterations in the activity of hepatic cytochrome P-450 isoenzymes result in changes in the pharmacokinetic behavior of drugs. This study was designed to explore the impact of type II diabetes, metformin and cinnamon on the activity of CYP2D isoenzyme. 2. Streptozotocin-nicotinamide-induced diabetic and normal rats were gavaged by cinnamon and/or metformin for 14 days. Using isolated perfusion of rat livers, the metabolic activity of CYP2D in the study groups was evaluated based on the oxidative biotransformation of tramadol hydrochloride. 3. The metabolic ratios of O-desmethyltramadol, the product of CYP2D-mediated metabolism of tramadol, in normal and diabetic control rats were found to be 0.33 ± 0.12 and 0.29 ± 0.07, respectively. Cinnamon significantly reduced the mentioned ratio in both normal and diabetic rats (0.13 ± 0.05 and 0.15 ± 0.04) and metformin increased the reduced activity in diabetic rats (0.37 ± 0.09 versus 0.29 ± 0.07). 4. In conclusion, it is evident that this study has shown the significant inhibitory effect of cinnamon on CYP2D. This finding suggests that it should be taken into consideration the possible metabolism-related pharmacokinetic drug-cinnamon interactions. 5. Additionally, type 2 diabetes condition reduced the enzyme activity and metformin consumption reversed this reduction; however, the significance of the latest is not clear.

Evaluation of the Ecstasy influence on tramadol and its main metabolite plasma concentration in rats.

BACKGROUND: Tramadol is prone to be abused alone, or in combination with 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy). It was reported that 95% of people with a history of substance abuse in the United States used tramadol in 2004. According to the WHO report in 2016, there was a growing number of tramadol abusers alone or in combination with psychoactive substances such as MDMA in particular in some Middle East countries. Higher concentrations of tramadol in plasma may lead to adverse drug reactions or lethal intoxication. In this study, the effect of MDMA on the pharmacokinetics of tramadol was examined in male rats. METHODS: The effect of MDMA on Tmax, Cmax, area under the curve, elimination rate, and half-life of tramadol and its metabolites was examined. Two control and two treatment groups were designed. The treatment groups received MDMA 18 h before the administration of tramadol. Jugular vein blood samples were analyzed by high-performance liquid chromatography with fluorescent detector to determine the concentrations of tramadol and its metabolites. Independent-sample t-test was used to define the differences between pharmacokinetic parameters of control and treatment groups. RESULTS: When tramadol administered intraperitoneally, the absorption rate of this drug was reduced, and a lower Cmax (40%) with longer Tmax (eight-fold) was achieved. MDMA exerted greater inhibitory effects on cytochrome P450 3A4 (CYP3A4) than on cytochrome P450 2D6 (CYP2D6). The M2 metabolite ratio was reduced by half, and because of the inhibition of M2 production, the M1 plasma concentration slightly increased. CONCLUSIONS: According to the obtained data, MDMA treatment affected the absorption, distribution and metabolism phases of tramadol. This treatment increased the concentration of tramadol if administered intravenously and can latent the absorption of tramadol in oral route. However, MDMA was introduced as CYP2D6 inhibitor; in this study, MDMA inhibited CYP3A4 isoenzymes as well. This finding is important for the compounds that are metabolized through CYP3A4. It can be proposed that in abusers of MDMA who only receive tramadol for medical or nonmedical purposes in short intervals, the dangers of the intravenous administration of tramadol should be considered, and if tramadol is administered orally, the desired effect may not be achieved at the routine dose.

Evaluation of the route dependency of the pharmacokinetics and neuro-pharmacokinetics of tramadol and its main metabolites in rats.

Tramadol hydrochloride is a centrally acting analgesic used for the treatment of moderate-to-severe pain. It has three main metabolites: O-desmethyltramadol (M1), N-desmethyltramadol (M2), and N,O-didesmethyltramadol (M5). Because of the frequent use of tramadol by patients and drug abusers, the ability to determine the parent drug and its metabolites in plasma and cerebrospinal fluid is of great importance. In the present study, a pharmacokinetic approach was applied using two groups of five male Wistar rats administered a 20mg/kg dose of tramadol via intravenous (i.v.) or intraperitoneal (i.p.) routes. Plasma and CSF samples were collected at 5-360min following tramadol administration. Our results demonstrate that the plasma values of Cmax (C0 in i.v. group) and area under the curve (AUC)0-t for tramadol were 23,314.40±6944.85 vs. 3187.39±760.25ng/mL (Cmax) and 871.15±165.98 vs. 414.04±149.25µg·min/mL in the i.v. and i.p. groups, respectively (p<0.05). However, there were no significant differences between i.v. and i.p. plasma values for tramadol metabolites (p>0.05). Tramadol rapidly penetrated the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) (5.00±0.00 vs. 10.00±5.77min in i.v. and i.p. groups, respectively). Tramadol and its metabolites (M1 and M2) were present to a lesser extent in the cerebrospinal fluid (CSF) than in the plasma. M5 hardly penetrated the CSF, owing to its high polarity. There was no significant difference between the AUC0-t of tramadol in plasma (414.04±149.25µg·min/mL) and CSF (221.81±83.02µg·min/mL) in the i.p. group. In addition, the amounts of metabolites (M1 and M2) in the CSF showed no significant differences following both routes of administration. There were also no significant differences among the Kp,uu,CSF(0-360) (0.51±0.12 vs. 0.63±0.04) and Kp,uu,CSF(0-∞) (0.61±0.10 vs. 0.62±0.02) for i.v. and i.p. pathways, respectively (p>0.05). Drug targeting efficiency (DTE) values of tramadol after i.p. injection were more than unity for all scheduled time points. Considering the main analgesic effect of M1, it is hypothesized that both routes of administration may produce the same amount of analgesia.

Effects of cyclosporine A on the hepatobiliary disposition and hepatic uptake of etoposide in an isolated perfused rat liver model.

PURPOSE: A recirculating isolated perfused rat liver model was used to investigate the hepatobiliary disposition of etoposide and the effects of cyclosporine A (CyA) on the pattern of drug disposition in the bile and uptake in the liver. METHODS: The portal vein, bile duct, and superior vena cava were cannulated in four groups of rats. The perfusions were conducted in the control group, which only received 10 µg/ml etoposide, and the tested groups which received etoposide and CyA in 0.4, 2, and 10 mg/kg doses. Perfusate and bile samples were collected up to 180 min. RESULTS: The determination of etoposide in the samples and homogenized liver by the high-performance liquid chromatography method showed that the administration of CyA led to significant changes in the hepatic excretion (E h), hepatic clearance (CL h), and half-life (T 1/2) of etoposide in the CyA 2 and 10 mg/kg treatment groups but not in 0.4 mg/kg group. The volume of the bile decreased to 64 and 45 % and biliary clearance (CL b) of etoposide reduced by 73 and 82 % in 0.4 and 2 mg/kg CyA group, respectively, when compared with the control group. CONCLUSIONS: These results demonstrated the dose-dependant non-specific inhibitory effects of CyA on p-glycoproteins, multidrug resistance protein 2, bile salt export pump, and organic anion-transporting polypeptide, the drug transporters responsible for etoposide hepatobiliary disposition, hepatic uptake, and bile formation in rat.

A Rapid and Sensitive HPLC-Fluorescence Method for Determination of Mirtazapine and Its two Major Metabolites in Human Plasma.

A rapid and sensitive HPLC method has been developed for the quantification of mirtazapine (MRZ), a noradrenergic and specific serotonergic inhibitor antidepressant (NaSSA) and its two major metabolites N-desmethyl mirtazapine (NDM) and 8-hydroxymirtazapine (8-OHM) in human plasma. The separation was achieved using Chromolith C18 column and a mobile phase of acetonitrile: phosphate buffer (pH = 3, 20:80, v/v) in isocratic mode at a flow rate of 2 mL/min. A fluorescence detector was set at 290 and 350 nm for excitation and emission, respectively. Zolpidem was used as the internal standard. Liquid-liquid extraction was applied for sample clean up. All analytes were eluted in less than 5 minutes with LOQ of 1 ng/mL for MRZ and 2 ng/mL for both NDM and 8-OHM. The developed method was successfully applied to quantify MRZ and its metabolites in plasma of a healthy volunteer.

Pharmacokinetics of mirtazapine and its main metabolites after single intravenous and oral administrations in rats at two dose rates.

BACKGROUND: Mirtazapine (MRZ) is a human antidepressant drug metabolized to 8-OH mirtazapine (8-OH) and dimethylmirtazapine (DMR) metabolites. Recently, this drug has been proposed as a potential analgesic for use in a multidrug analgesic regime in the context of veterinary medicine. The aim of this study was to assess the pharmacokinetics of MRZ and its metabolites DMR and 8-OH in rats. FINDINGS: Eighteen fasted, healthy male rats were randomly divided into 3 groups (n = 6). Animals in these groups were respectively administered MRZ at 2 and 10 mg/kg orally and 2 mg/kg intravenously. Plasma MRZ and metabolite concentrations were evaluated by HPLC-FL detection method. After intravenous administration, MRZ was detected in all subjects, while DMR was only detected in three. 8-OH was not detected. After oral administration, MRZ was detected in 3 out of 6 rats treated with 2 mg/kg, it was detected in 6 out of 6 animals in the 10 mg/kg group. DMR was only detectable in the latter group, while 8-OH was not detected in either group. The oral bioavailability was about 7% in both groups. CONCLUSIONS: The plasma concentration of the MRZ metabolite 8-OH was undetectable, and the oral bioavailability of the parental drug was very low.

Pharmacokinetics Alterations of Midazolam Infusion versus Bolus Administration in Mechanically Ventilated Critically Ill Patients.

There is no randomized study carried out in order to compare their pharmacokinetic parameters although midazolam, as a sedative, has been widely administered via continuous infusion as well as intermittent bolus doses in mechanically ventilated critically ill patients. We prospectively investigated the effect of these two principal methods on pharmacokinetic parameters in 23 of mentioned patients (16 males, 7 females) with the mean (± SD) age of 41.22 ± 17.5. All patients received total dose of 72 mg throughout the test days, 9 of whom received 1 mg/h (continuous infusion) and the rest obtained 4 mg / 4 h (intermittent bolus doses). Blood samples were collected at 8 and 4 h prior to the end time of drug administration (zero time), zero time and 4, 8, 12, 20 and 30 h after it. APACHE (Acute Physiology and Chronic Health Evaluation) II required data was recorded daily and the patients' mean score was 16.26 ± 4.38. The mean (± SD) value of pharmacokinetic parameters of Midazolam in continuous infusion and intermittent bolus doses methods were as follows: (t½ = 17.88 ± 14.65 h, Cl = 21.80 ± 14.95 L/h) vs. (t½ = 19.74 ± 12.45 h, Cl = 29.43 ± 19.45 L/h). Volume of distribution (Vd) was measured in continuous infusion group which was 612.58 ± 582.93 L. The calculated clearance and half-life were found not to be significantly different (p < 0.05). The patients might be exposed to similar undesired effects due to the large volumes of distribution following the administration methods studied.

Study of the pharmacokinetic changes of Tramadol in diabetic rats.

BACKGROUND: Besides the pathological states, diabetes mellitus may also alter the hepatic biotransformation of pharmaceutical agents. It is advantageous to understand the effect of diabetes on the pharmacokinetic of drugs. The objective of this study was to define the pharmacokinetic changes of tramadol and its main metabolites after in vivo intraperitoneal administration and ex vivo perfused liver study in diabetic rat model.Tramadol (10 mg/kg) was administered to rats (diabetic and control groups of six) intraperitoneally and blood samples were collected at different time points up to 300 min. In a parallel study, isolated liver perfusion was done (in diabetic and control rats) by Krebs-Henseleit buffer (containing 500 ng/ml tramadol). Perfusate samples were collected at 10 min intervals up to 180 min. Concentration of tramadol and its metabolites were determined by HPLC. RESULTS: Tramadol reached higher concentrations after i.p. injection in diabetics (Cmax of 1607.5 ± 335.9 ng/ml) compared with control group (Cmax of 561.6 ± 111.4). M1 plasma concentrations were also higher in diabetic rats compared with control group. M2 showed also higher concentrations in diabetic rats. Comparing the concentration levels of M1 in diabetic and control perfused livers, showed that in contrast to intact animals, the metabolic ratios of M1 and M5 (M/T) were significantly higher in diabetic perfused liver compared to those of control group. CONCLUSIONS: The pharmacokinetic of tramadol and its three metabolites are influenced by diabetes. As far as M1 is produced by Cyp2D6, its higher concentration in diabetic rats could be a result of induction in Cyp2D6 activity, while higher concentrations of tramadol can be explained by lower volume of distribution.

Lack of evidence for involvement of P-glycoprotein in brain uptake of the centrally acting analgesic, tramadol in the rat.

PURPOSE: Tramadol Hydrochloride is a widely-used centrally acting analgesic drug, which has some features of being a P-gp substrate. The present study evaluates the functional involvement of P-gp in CNS distribution of tramadol. METHODS: The possible involvement of P-glycoprotein in brain distribution of tramadol was evaluated using a pharmacokinetic approach in two groups of Pgp-inhibited and control rats. Six male Sprague-Dawley rats were used in each group to collect plasma and brain at 1, 5, 10, and 30 min following two tramadol doses of 1 and 10 mg/kg. RESULTS: The brain uptake clearances of tramadol in Pgp-inhibited and control rats were 2.47±0.56 and 2.34±0.56 ml min(-1)g(-1), respectively, for 1 mg/kg and 3.50±0.60 and 3.14±1.02 mlmin(-1)g(-1), respectively, for 10 mg/kg dose. The brain-to-plasma concentration ratio (Kp,app) of more than 1 in all the time points following both the high and low dose cases (sometimes more than 3) indicated the brain accumulation of the drug. Linear correlation was found between tramadol dose and both corresponding plasma and brain concentrations, but the presence of a dose-dependency was not confirmed by the data obtained for brain-to-plasma concentration ratio. CONCLUSION: Considering the results of the previous studies and the present research, it seems that the brain accumulation of tramadol is not affected by P-gp inhibition which implies that there may be some other transport mechanisms involved in BBB transport of tramadol.