| Pyridostigmine bromide (PB) acts as a competitive reversible inhibitor of cholinesterase and prolongs the hydrolysis of endogenous acetylcholine. PB is used for the symptomatic treatment of myasthenia gravis and antagonism of nondepolarizing neuromuscular blockers. PB may also be used after abdominal surgery flatulence and urinary retention. However, the clinical usage of PB has been limited by poor oral bioavailability. The low bioavailability may be due to the strong hydrophilicity but slight liposolubility of PB. Therefore, it is significant important for clinical to improve the bioavailability. In recent decades, phospholipid complex was used as a new drug delivery system. Phospholipid is one of the component of cell membranes. It has strong liposolubility. The liposolubility of hydrophilic drug can be increased by complex with phospholipid, thus enhancing its bioavailability. This article clearly based on the biological pharmacy characteristics of pyridostigmine bromide, designed and optimized the preparation technology of a novel pyridostigmine bromide-phospholipid complex (PBPLC), and in-depth study of its physical, chemical and biological characteristics. These properties will be compared with the free pyridostigmine bromide to confirm that the oral bioavailability of pyridostigmine bromide was increased by complexed with phospholipid. Specific research includes the following sections:In the first section, the preparation method of PBPLC was studied, and central composite design- response surface methodology was used to optimize the preparation technology. The PBPLC was prepared by solvent evaporation methord, and evaluated by complex rate of drug and phospholipid. We first use single factor method to investigate preparation technology, and optimize the significant factors by central composite design. The data was fitted to a linear regression model and a quadratic model. Then, using response surface method selected optimal conditions and forecast. The optimal values for phospholipid-to-drug ratio, reaction temperature and drug concentration should be 8, 40°C and 4 mg·ml-1, respectively. Under the optimal conditions, the complex rate of PBPLC was (84.02±1.68)%, and compared with the predict result of quadratic model was less than 2%. So, central composite design- response surface methodology can be used to as a quick and easy method to optimize the preparation technology of PBPLC, and has a good predictability.In the second section, the physicochemical properties of PBPLC were studied. The physicochemical properties of PBPLC were investigated by differential scanning calorimetry (DSC), ultraviolet (UV) spectroscopy, Fourier transformed infrared (FT-IR) spectroscopy. The particle size and Zeta potential of PBPLC was determined by photon correlation spectroscopy. And N-octanol/water partition coefficient of PBPLC was also investigated. The DSC curve of PBPLC showed that the characteristic endothermal peak of PB disappears, and the phase transition temperature was lower than that of phospholipids. These results indicated that PB and phospholipids may have some hydrogen bonding and intermolecular interactions. The spectrum profile of PBPLC was quite similar to that of PB, it suggest that the physical interactions between PB and phospholipids may be too weak to be dectected by the imaging probes, and the chromophore structure has not changed. The infrared spectra showed that there was a significant difference between the physical mixture and the complex. The spectrum of the physical mixture is nearly an additive effect of the spectra of PB and phospholipids. Compared with that of the physical mixture, some characteristic absorption peaks of PB are disappeared or masked by phospholipids in PBPLC. The result of particle size and zeta-potential of PBPLC showed that the particle size of the optimized formulation is about 204.6 nm with a polydispersity index (PDI) of 0.258, and the zeta-potential is -25.12 mV. The N-octanol/water partition coefficient of PBPLC was about 17 times more than that of PB material, which was more than that of physical mixture. These data indicate that lipophilicify of PBPLC increases remarkably. These physicochemical properties of PBPLC showed that there has some interaction between PB and phospholipid, rather than just a simple physical mixture. In the third section, the pharmacokinetics and bioavailability of PBPLC in SD rat were investigated. Accurate, reliable and simple method for determination of the PB content in SD rat plasma was established, and the method in line with in vivo drug analysis requirements by the study methodology. Pharmacokinetic parameters were calculated using the DAS 2.1.1 statistical software. The result clearly show that the plasma drug concentration-time curves of PBPLC and free PB after oral administration were both in accordance with the two-compartment open model. Pharmacokinetic parameters of PBPLC compared to free PB were Tmax 2 h vs 2 h; Cmax 22.79 vs 6.00μg·ml-1; and AUC0–∞7128.21 vs 1772.36μg·min·ml-1, respectively. In conclusion, the oral bioavailability of PB increased remarkably when administered as a PBPLC compared to that of free PB. This result is likely due to the higher lipophilicity and permeability of PBPLC.In the forth section, the intestinal permeability of PBPLC was studied using in situ intestinal absorption characteristics. In situ experimental model - in rat intestinal perfusion method was used to study the rat intestinal absorption. Dual-wavelength absorption elimination method was used to eliminate the influence of phenol red to the determination of PB. Compared to free PB, the better intestinal permeability was obtained by the PBPLC. The result showed that the absorption rate constant (Ka) and effective permeability (Peff) of PBPLC are 2.32 times and 2.52 times that of free PB, respectively. Indicated that phospholipid can improve the absorption of PB.
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