| Osthole (Ost) is an active coumarin compound extracted from Cnidium monnieri (L.) Cusson. Modern pharmacological studies have shown that Ost exhibited various pharmacological effects, including antispasmodic, hypotensive, anti-arrhythmic, enhancing immune function, anti-cancer, anti-osteoporosis, anti-inflammatory, anti-apoptotic, anti-allergy. Recently, Ost was reported to be effective for the treatment of alcoholic fatty liver disease. It has been recognized as the drug belonging to Biopharmaceutics Classification System â…¡. For such hydrophobic compound, poor solubility would resulted in a slow dissolution and hence low and erratic oral bioavailability, which may limit its further clinical application. In the present studies, we raised that not only pharmaceutic technology can improve bioavailability, but reasonable component compatibility can also improve poor solubility and bioavailability. The rationality of component compatibility displays not only in synergy and attenuated efficiency, but also in interaction in pharmacokinetics and physiological disposition.Pharmaceutic technology is usually used to improve the solubility of poorly soluble drugs. Based on the physicochemical property and biological characteristics of Ost, so far, only few formulation strategies for improving dissolution of Ost have been investigated, such as such as cyclodextrin technology, emulsion technology, liposomes technology, traditional solid dispersion technology, nonionic vesicle technology and other semi-synthetic structural transformation method. And only cyclodextrin technology reported about in vivo pharmacokinetic performance of Ost. In the present studies, we attempted to ues new hydrophilic polymers to improve dissolution and bioavailability of Ost by preparation of solid dispersions with hot melt extrusion (HME) technique. In comparison with traditional methods for preparation of solid dispersions, HME, as a promising novel technology for improving the bioavailability of water-insoluble drugs, presents many advantages for pharmaceutical applications. It can be used as a continuous process with the absence of organic solvents and subsequent drying steps, which makes scaling-up easier. In addition, intense blending and agitation during process prevent the aggregation of drug particles suspending in the molten polymer, leading to a more homogeneous dispersion of fine particles. In vitro characterizations, dissolution studies and in vivo pharmacokinetic studies were performed in our study to enhance the solubility and bioavailability of Ost.Reasonable component compatibility can also improve the efficacy and bioavailability of drugs. Its rationality mainly lies in the mutual influence of main active ingredients on Pharmacology and Biopharmaceutics, including interaction in absorption, distribution, metabolism, excretion.Glycyrrhizic acid (GL) is the main active ingredient in licorice. Its molecular consists of a lipophilic triterpenoid aglycon conjugated with a hydrophilic disaccharide glycon containing β(1â†'2) linked D-glucuronic acids. GL has recently been of great interest to researchers due to its ability to form water-soluble complexes with various hydrophobic compounds. It is assumed that GL could form cyclic dimer structures containing hydrophobic cavities, which allows formation of complexes of the "host-guest" type similar to cyclodextrine inclusion complexes. In addition, it exhibited pharmacological effects similar to osthole like anti-inflammation and hepatoprotective effect. It is supposed that application of GL together with Ost may increase the solubility, strengthen therapeutic action, enhance the bioavailability of Ost, and may have interaction in absorption, distribution, metabolism and excretion. Therefore, this experiment clarified the compatibility rationality of the natural solubilizing agent GL using with Ost from the perspective of pharmacology and biopharmaceutics.First, the basic nature of osthol was investigated. The apparent oil-water partition coefficient P of Ost in n-octanol-water was6562.19(logP=3.82), indicating high hydrophobicity. Different pH value had no effect on the apparent partition coefficient of Ost, suggesting that it has good absorption throughout the gastrointestinal tract. A rapid, simple and accurate LC-MS/MS method was eatablished to determine the concentration of Ost in rat plasma after oral and intravenous administration, and the absolute bioavailability of Ost was only15.65%.In vitro characterizations were performed with differential scanning calorimetry (DSC), X-ray powder diffraction (XPRD), Fourier transform infrared (FT-IR) spectroscopy, and in vitro dissolution studies. Based on the above results, in vivo pharmacokinetic studies of Ost solid dispersions prepared with plasdone S-630, HPMC and Eudragit EPO were conducted in rats after a single oral dose. In comparison to the untreated Ost coarse powder and the physical mixture with polymers, the solid dispersions prepared with Plasdone S-630or HPMC-E5(drug/polymer:1:6) showed a significant enhancement of dissolution rate (～3-fold higher D30). In addition, such preparations exhibited a significant decreased Tmax,～5-fold higher Cmax and～1.4-fold higher AUC when comparing with Ost coarse powder. Therefore, the suitable polymers for preparing Ost solid dispersions were Plasdone S-630and HPMC.The rationality of component compatibility can be expressed not only in pharmacodynamic mechanism, but also in biopharmaceutics and pharmacokinetics.Our preliminary pharmacodynamics effect study indicated that osthole combined with glycyrrhizic acid were more effective in treating and prevention from alcoholic fatty liver when comparing with Ost coarse powder. Therapeutic dosage regimen was used to select the optimal ratio (GL/Ost) in treating alcoholic fatty liver. Considering the comprehensive indicators analysis, the most suitable ratio (GL/Ost) was1:2.25. Then, the ratio was verified again in rats, and showed consistence with the ratio (GL/Ost:1:2.25).The rat liver tissue samples was extracted after oral administration, and the expression of three key proteins (PPARa, CPT-1, RXRa) in the samples was determined by western blotting technique to verify the rationality of the selected optimal ratio. The results are consistent with the preliminary results from rats, indicating that the combination of glycyrrhizin can enhance the effect of treating alcoholic fatty liver. Thus, it further validated the pre-selected ratio (GL/Ost:1:2.25) from molecular level.After exploring the underlying mechanism of the synergistic effect from the perspective of the pharmacology, we further investigated the scientific connotation of GL in combination with Ost from the view of biopharmaceutics. In comparison to the untreated Ost coarse powder, Ost combined with GL exhibited a significant～2.3-fold higher Cmax and～1.7-fold higher AUC.In present studies, various models were employed to investigate the effect of GL and its hydrolyzate glycyrrhetinic acid (GA) on the solubilization, absorption, distribution, metabolism and excretion of Ost, thus illuminating the reason for enhanced bioavailability of Ost when combined with GL. Finally, in vivo pharmacokinetic study was conducted to investigate the influence of GA on Ost’s bioavailability, which further complemented the above results and confirmed the main reason for the enhancing bioavailability.In vitro solubility experiment was used to study the effects of GL and its hydrolyzate GA on solubility of Ost. The results showed that the solubility of Ost was higher with the increasing concentration of GL, however, the concentration of GA had little effect on the solubility of Ost. The enhanced solubility by GL was attributed to its structure similar to cyclodextrins. It is assumed that GL at low concentrations could form cyclic dimer structures containing hydrophobic cavities. The existence of cavities allows formation of complexes of the "host-guest" type, which is similar to cyclodextrine inclusion complexes. Besides, the presence of both hydrophobic (triterpene fragment) and hydrophilic (two glucoronic residue) moieties in GL structure suggest that GL might form micelles in aqueous solutions. The increased solubility of osthole in the gastrointestinal tract by GL may significantly contribute to its increased bioavailability in rats.XRD and FTIR study of the binary systems of Ost with GL was to reveal how GL enhanced the solubility of Ost, and results showed that the crystallinity of osthole was decreased to a greater extent in the prepared Ost-GL lyophilized sample and there was the formation of hydrogen bonding between Ost and GL, which thus marked increased the solubility of osthole in GL solution.Caco-2cell monolayer model was performed to evaluate the potential absorption interactions. The results shown that Ost was well absorbed in caco-2cell and no significant changes were observed in the value of the absorptive permeability parameter (Papp(A-B)) and secretary permeability parameter (Papp(B-A)) in the presence of glycyrrhizic acid or glycyrrhetinic acid. Thus, we may conclude that the enhanced bioavailability may not derive from the absorption aspect.Tissue distribution in rats after Ost and Ost combined with GL was employed to evaluate the potential distribution interactions. The results shown that in both groups Ost had the highest distribution in the gastrointestinal tissues. After absorption, Ost had rapid distribution to the various tissues and organs, and then quickly eliminated from these tissues and organs. The overall trend of the two group indicated that Ost was mainly distributed in tissues with rich blood such as the liver, spleen and kidney. Of the three tissues, it had the highest concentration in liver, suggesting that this phenomenon were closely related to hepatoprotective effect of Ost. The high concentration in spleen and kidney may relate to its effects like kidney protection, eliminating dampness, strengthening immune function. By comparing the distribution of Ost at each time point in the two groups, the results indicated that GL affected the distribution of Ost. It is assumed that the improved bioavailability and efficacy when Ost combined with GL was due to the enhanced blood concentration by GL. GL had no significant effect on the distribution of Ost in speen, kidney and stomach. In addition, the enhanced distribution of Ost in liver by GL may lead to the improved efficacy in treating liver disease, since GL significantly enriched Ost’s distribution in liver.The following step was focused on whether there was metabolic interaction, and the rat enterocyte S9and liver S9fractions were utilized. For the metabolic interaction study, when glycyrrhizic acid or glycyrrhetinic acid was added with Ost in the rat enterocyte S9and liver S9fractions, neither of them affected the reduced amount of osthole, suggesting that there was no interaction in the metabolism of Ost.Excretion study in rats was employed to evaluate the potential excretion interactions after oral administration of Ost and Ost combined with GL. The results showed that the cumulative excretion of Ost in urine within24h was0.01%, and in feces was0.25%, suggesting Ost may be primarily excreted as its metabolites. Since the prototype Ost excretion was less than1%, Ost was xtensively metabolized in vivo. When Ost combined with GL, the cumulative excretion of Ost in urine within24h was0.08%, and in feces was0.96%. Although GL increased the excretion of Ost, because Ost excretion in prototype was less than1%, the impact of GL on the excretion of Ost was almost negligible.Finally, in vivo pharmacokinetic study was conducted to investigate the influence of GA on Ost’s bioavailability. The results shown that GA had little effect on Ost’s bioayailability, suggesting the enhanced bioavailability was attrbuted to the effect of GL. The result further complemented the above results and confirmed the main reason for the enhancing bioavailability was due to the improved solubility of Ost by GL.In the present study, HME technology was used to prepare Ost solid dispersions with optimal polymers. The Ost solid dispersions exhibited enhanced solubility and bioavailibility. In additon, GL was used to improve solubility and bioavailibility of Ost. We investigated the scientific connotation of GL in combination with Ost from the view of pharmacodynamics, pharmacology and biopharmaceutics. The results show that GL enhanced efficacy for treatment of alcoholic fatty liver due to different therapeutic targets. When osthole combined with GL, the main reason for the improved bioavailability was the solubilization by GL. The present study indicated that not only pharmaceutic technology can be applied to improve the solubility of poorly soluble drugs, but also component compatibility can be used as well. It is of great importantance for formulation design and bioavailability evaluation to concern about the impact of component compatibility on physiological disposition of drug and the rationality of component compatibility. |
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