In contrast, for the SIF study throughout the incubation period the relative deviations (%) of mitragynine was found to be no greater than ±5%.The drug dissolution profiles over time for capsule filled with mitragynine standard in SIF and SGF are shown in Figure 2 . In SGF, mitragynine dissolution was inconsistent at early dissolution time points and only 80% of the drug was released up to 2 h. The RSD of >50% was observed at early dissolution time points (5 to 15 min) but for the rest of the period the RSD was <20%. As for SIF, the drug release was very slow and incomplete; as only 22% of the drug was released up to 2 h. A RSD of >20% was noted throughout the dissolution period.

As for HPLC validation study, the within day and day to day HPLC precisions and accuracies were found to be less than 5%. The calibration curve of mitragynine was linear over the concentration range of 0.16–20 μg/mL (r> 0.999). The lower limits of detection and quantification were 0.1 μg/mL and 0.16 μg/mL, respectively. This method was used for quantification of mitragynine in partition coefficient study. The n-octanol/water partition coefficients (logP and logD) obtained under various conditions are detailed in Table 3 . Mitragynine has logP and logD (pH 4) values of 1.70 and 0.78, respectively.

Analysis was performed on three different occasions to evaluate the repeatability of the techniques. The inter-day pKvalues obtained from different methods and with different concentrations are disclosed in Table 2 . The solubility of mitragynine was found to increase from 18.7 ± 0.4 μg/mL in buffer at pH 9, to 64.6 ± 1.2 μg/mL in water and 88.9 ± 1.6 μg/mL in buffer pH 7, while being the highest at pH 4 buffer of 3.5 ± 0.01 mg/mL. For stability studies, the percentage recovered at 0.5 h, 6 h and 24 h of mitragynine in the buffer systems were 100.6 ± 0.3%, 91.8 ± 1.0% and 88.8 ± 1.4% for pH 4 buffer; 99.2 ± 0.4%, 96.1 ± 1.9% and 101.9 ± 0.9% for pH 7 buffer; 101.3 ± 2.6%, 97.8 ± 0.3% and 95.5 ± 2.5% for pH 9 buffer.

2.2. Discussion

a determination. Mitragynine stock solutions in the range 5–40 μg/mL were tested in various buffers. In agreement with values suggested for poor solubility compounds [ a value for mitragynine which is in agreement with an in silico predicted value of 8.3 [ a values determined by conventional and microplate spectrophotometer methods were comparable and showed good assay repeatability ( Poorly water soluble alkaloids, such as mitragynine, require a suitable strength of stock solution for pKdetermination. Mitragynine stock solutions in the range 5–40 μg/mL were tested in various buffers. In agreement with values suggested for poor solubility compounds [ 17 ] the optimal testing concentration was found to be 50 μM (20 μg/mL) for conventional UV and 12.5 μM (5 μg/mL) for microplate method. The present study is the first to determine the pKvalue for mitragynine which is in agreement with anpredicted value of 8.3 [ 18 ]. The pKvalues determined by conventional and microplate spectrophotometer methods were comparable and showed good assay repeatability ( Table 2 ). There are several advantages for the microplate spectrophotometer method over the conventional assay. The former is rapid and cost effective, and requires only a small volume of sample and a low drug concentration for spectrum analysis, hence making the method suitable for analysis of mitragynine.

450 metabolism and associated with increase in non-specific binding to plasma proteins that limit blood brain barrier (BBB) penetration [ in vivo brain penetration; this partly could explain the psychotropic properties of mitragynine reported in literature [ We found that acetic acid, Tris base and sodium carbonate buffer systems provided an improved buffering capacity over phosphate buffers which are commonly used in the range of pH 3 to 12 [ 19 20 ]. In addition, these buffers did not absorb UV at the selected analytical wavelength, thus no interference was encountered in the spectrophotometer assay. In both assay procedures, similar buffer solutions were used for UV spectrum analysis. The UV absorbance spectra of various absorbing species at different pH showed a gradual decrease in absorbance beyond pH 7.0 for both methods. The mitragynine absorption spectrum was higher in the acidic pH when compared to the basic media. Mitragynine is a weak basic compound and it exhibits a basic character that is attributed to an amine group. This is supported by the fact that it is highly soluble at acidic pH, but the solubility was extremely reduced in basic media. Mitragynine exists predominantly in an ionized form at lower pH; hence a higher solubility was seen at lower pH. In the present study, mitragynine lipophilicity was determined by the octanol/water) partition method. Lipophilicity is expressed in several different ways including terms such as logP and logD. LogP is calculated as a ratio of concentrations of unionised compound between the octanol and water. LogD reflects the distribution of both neutral and ionized species of a drug at a particular pH between the aqueous and organic phase, and therefore is pH dependent. Mitragynine has an intermediate lipophilicity, with a logP value of 1.70. Highly lipophilic compounds are more exposed to Pmetabolism and associated with increase in non-specific binding to plasma proteins that limit blood brain barrier (BBB) penetration [ 21 ]. Those drugs with intermediate lipophilicity often exhibit highestbrain penetration; this partly could explain the psychotropic properties of mitragynine reported in literature [ 22 23 ]. We previously reported a low content of mitragynine (31–444 µg/mL) in ketum decoctions often used by drug users to wean themselves off opiate withdrawal effects [ 24 ]. This may further explain the psychotropic properties of mitragynine owing to its intermediate lipophilicity which favours high BBB penetration. However, this warrants further detailed investigations using appropriate BBB studies.

It is also important to note that mitragynine is a basic drug and likely to be charged in the intestinal lumen; as such to understand its lipophilic properties under various pH influence, logD values at pH 4, 7 and 9 were further determined. Despite being highly ionized in acidic solution, the drug has a logD value of 0.78 at buffer pH 4 thus indicating its preference to be distributed into lipid layer. The poor water solubility of mitragynine could be partly attributed to its lipophilicity properties. With regards to stability, mitragynine is stable at pH 7 and 9 for a period of 24 h. However, at pH 4 it appears to degrade over time and there is a high possibility the drug is degradable in gastric juice. In our previous study, we demonstrated the low absolute oral bioavailability (≈3%) of mitragynine in rats [ 11 ]. In the highly acidic environment of the stomach, drugs that have weak base mainly exist in their ionic form and are not readily absorbed through the cell membranes of the GI tract [ 25 26 ]. Mitragynine being a basic drug is highly solubilised and ionized in the stomach, thus making it less bioavailable. In addition, poor oral bioavailability was also likely to be caused by its degradation in the highly acidic gastric juice.

max and half-life varied from 0.4–2.3 µg/mL and 3.8–9.4 h respectively after an oral dose ranging from 20–50 mg/kg [12,13, et al employed mitragynine salt solution for toxicity testing and reported no adverse reactions up to 920 mg/kg (mice) and 806 mg/kg (rats); a dose far higher than the doses reported by other investigators [10,13,9,10,12, in vitro solubility for the poorly soluble drug but its solubility in vivo might be totally different. With mitragynine, being a poor water soluble alkaloid, there is a possibility of this compound getting precipitated in the gut when given at large doses. Taking all possibilities into account, this may give an explanation for the wide-ranged disparity in the reported MG preclinical studies. Other investigators have also reported the pharmacokinetic properties of mitragynine in rats after oral administration but the pharmacokinetic parameters varied largely among these studies. The reported value of Cand half-life varied from 0.4–2.3 µg/mL and 3.8–9.4 h respectively after an oral dose ranging from 20–50 mg/kg [ 11 14 ]. These investigators used 1% acetic acid (pH 4.7), propylene glycol or 1% cremophor in saline respectively as a vehicle to facilitate the solubility of mitragynine for either oral or intravenous (i.v.) drug administration. In a separate study, Mackoemployed mitragynine salt solution for toxicity testing and reported no adverse reactions up to 920 mg/kg (mice) and 806 mg/kg (rats); a dose far higher than the doses reported by other investigators [ 9 15 ]. This is probably attributed to the fact that in salt solution mitragynine is highly in an ionized state and not readily absorbed from stomach. Further to this, the drug is not protected from degradation by the highly acidic gastric juice. This probably explains the non- manifestation of toxicity even at higher doses tested. Similar variability in analgesic and toxicity studies were also encountered when investigators employed various vehicles such as 4% acacia gum, tween 20% and 1% acetic acid adjusted to pH 4.7 for either oral or intraperitoneal (i.p.) drug administration [ 8 15 ]; this was done without proper understanding of its physicochemical properties and rationally this would have an effect on the outcome of their pharmacological studies [ 8 14 ]. The use of co-solvents or surfactants might enhance thesolubility for the poorly soluble drug but its solubilitymight be totally different. With mitragynine, being a poor water soluble alkaloid, there is a possibility of this compound getting precipitated in the gut when given at large doses. Taking all possibilities into account, this may give an explanation for the wide-ranged disparity in the reported MG preclinical studies.

et al. reported in their study on the addictive potential of mitragynine in rats where such effects were observed at high (30 mg/kg) and moderate (10 mg/kg) doses, but were absent at mild (5 mg/kg) and low (1 mg/kg) doses [ et al. in his psychosocial study reported that ketum users developed addiction only after prolonged, frequent and chronic consumption of high doses of ketum drinks prepared as leaves concoction, thus not mitragynine per se [ i.e. , mediated via opiate receptor) of mitragynine being less potent than the M. speciosa crude extract [ Besides the above reported pharmacological studies, the addictive potential of mitragynine has been recently reported in humans and rodents [ 5 6 ]. Yusofreported in their study on the addictive potential of mitragynine in rats where such effects were observed at high (30 mg/kg) and moderate (10 mg/kg) doses, but were absent at mild (5 mg/kg) and low (1 mg/kg) doses [ 6 ]. Their study involved intraperitoneal injection of mitragynine dissolved in the surfactant Tween 80. To the best of our knowledge, the first human study on the effects of mitragynine was performed by Grewal, but nothing on addictive properties was reported [ 27 ]. In Grewal’s study, the volunteers received mitragynine acetate (50 mg in 4 occasions) or ketum powdered leaves (0.65–1.3 g) with 20 mL of distilled water, in which case the solubility of the drug could be a limiting factor to oral absorption. Of late, Singhin his psychosocial study reported that ketum users developed addiction only after prolonged, frequent and chronic consumption of high doses of ketum drinks prepared as leaves concoction, thus not mitragynine 5 ]. Interestingly, in a receptor interaction study using HEK 293 cells, ketum powder demonstrated a 350-fold lower affinity for the µ opiate receptor when compared to morphine [ 28 ]. This partly could explain the fact that regular consumption of high dose ketum drinks is required to observe abuse liability in humans. On the other hand, there is also a report on the antinociceptive effect (, mediated via opiate receptor) of mitragynine being less potent than thecrude extract [ 29 ]. Presently, we cannot exclude that existing discrepancies between and within studies may be due, but not limited to, the route or the mode of administration. This further highlights the necessity to establish the physicochemical characteristics of mitragynine in order to prepare a suitable pharmaceutical formulation which would provide adequate absorption for investigations in future.

With reference to mitragynine stability, the drug was found to be stable in SIF compared to SGF over time of incubation (37 °C). A deviation above 5% of the original drug content, such as measured in the manner presented in this study is suggestive of drug instability in the GI tract. Mitragynine was not stable in SGF (pH ≈ 1.2) since drug loss was observed (>20%) at 20 min onwards after the onset of the incubation period. A similar drug loss was reported for mitragynine in SGF (26%) [ 30 ]. In the present study mitragynine degradation was further evident as large variation in drug release (>50%) was apparent in SGF dissolution profile with a total drug release up to 2 h which was only 80%. This is most likely due to the fact that the drug started to breakdown immediately upon release from its capsule after the onset of dissolution.

However, mitragynine was stable up to 3 h throughout the incubation period in SIF (pH 6.8) with <5% deviation. Based upon these results, mitragynine is considered stable in SIF (3 h). Since the exposure of drug substances at 37 degrees Celsius to SIF (3 h) mimics the in vivo drug contact with these fluids, it was concluded that mitragynine could resist the pH and enzymatic conditions of the intestine fluid. However in SIF, mitragynine dissolution over time was prolonged and incomplete. After onset of the dissolution, only 22% of drug was released in 2 h. The drug in its pure basic form though stable in SIF, demonstrated a very poor aqueous solubility. SIF with a pH of 6.8, along with its chemical constituents and presence of enzyme certainly had an effect on the drug solubility. This was reflected in the low solubility of the drug in SIF when compared to its high solubility in acidic media. Though the amount of drug substance that goes into solution in SGF is very much higher than SIF, mitragynine in its ionized form is unlikely to be absorbed from the stomach.