| Memantine HC1, the first drug approved for the treatment of patients with moderate to severe Alzheimer’s disease, is postulated to exert its therapeutic effect through its action as a low to moderate affinity uncompetitive (open-channel) NMDA receptor antagonist which binds preferentially to the NMDA receptor-operated cation channels. In this paper, methods for concentration assay of memantine in biological matrix were developed. The pharmacokinetic processes of memantine in vivo were systematically studied. The absorption, distribution and excretion of memantine in rats after oral administration, the linear pharmacokinetic characteristics and the single-dose and the multiple-dose pharmacokinetics of memantine in Chinese healthy young men and women were investigated. Plasma and urine metabolic perturbations between AD and healthy old person were studied using ultra performance liquid chromatography/mass spectrometry (UPLC/MS) and metabonomics approach.14 biomarkers were found and the identification of Alzheimer’s disease (AD) biomarkers may allow for a less invasive and more accurate diagnosis as well as serving as a predictor of future disease progression and treatment response in clinical study.1 Pharmacokinetic study of memantine HCI in ratsThe pretreatment for the determination of memantine in rat plasma was investigated. The feasibility of using cloud-point extraction (CPE) as a simple and effective means of recovery of memantine from rat plasma before LC-MS analysis has been established. Meanwhile, a traditional liquid-liquid extraction method coupled with HPLC-MS for the determination of memantine was also evaluated. The LLOQs of both methods were 1 ng/mL, and the mean recoveries of CPE and liquid-liquid extraction were 95.7% and 85.4 %, respectively. The established method for the determination of memantine in matrix laid the root for following study.The pharmacokinetic processes of memantine in rats after oral administration (2.5 mg/kg) and intravenous injection with same doses were investigated. the maximum concentration were 183.3±36.6 and 243.1±79.9 ng/mL; AUC0-t were 815±79 and 881±193 ng·h/mL, AUC0-∞were 837±82 and 924±1964 ng·h/mL, t1/2 were 4.56±0.73 and 4.78±1.08 h, respectively; The AUCo-t parameters obtained were then compared and analyzed. Bioavailability of this drug, determined as 92.6%, was very high.The tissue distribution of memantine in rats after an oral administration of 2.5 mg/kg was investigated. The results presented a quick and dispersive affinity to rat tissues of this drug. Only 10 min after administration, concentrations of memantine in most of the tissues already reached a high level. The high drug concentration with 197.4±85.5 ng/g in target brain organ was detected, which indicated a sound penetration through the BBB (blood-brain barrier). Because of oral administration, besides stomach, small intestine, and liver, with the concentration of 86.7±18.8,15.01±8.78 and 9.64±2.18μg/g, respectively. Higher drug concentrations were also found in lung and spleen.The excretion of memantine from rats after an oral administration of 2.5 mg/kg was investigated. In urine, the cumulative amount of prototype drug was 87.5±35.4μg, which amounted to 15.9% of the given dosage indicating a major urinary excretion of the parent compound. The cumulative amount in feces for memantine was only 4.132±2.407μg which amounted to only 0.8% of the given dosage within 72 h after oral administration. The excretion of memantine from bile was slight and constant, with the cumulative amount of 885±495 ng at 16 h after dosing. All the parent compound content determined was about 17% of the given dosage. The data above suggested that most of the drug administrated has been metabolized, and enteropatic recycling of memantine is weak in rats.2 Pharmacokinetic study of memantine in health volunteersThe pharmacokinetics of memantine in health volunteers was investigated. After the determination of memantine in plasma, the single-dose and the multiple-dose pharmacokinetics of memantine in Chinese healthy young men and women were estimated.Thirty healthy volunteers were enrolled, and divided into three groups at random with 5 men and 5 women in each group. For each group, oral administration of 5 mg,10 mg and 20 mg were adopted, respectively. After oral administration of single dose of 5 mg, the pharmacokinetic process of drug in men and women were characterized:Tmax (h) were 5.40±1.95 and 6.00±1.41, Cmax (ng/mL) were 5.932±0.730 and 6.477±0.734, t1/2(h) were 70.75±12.78 and 66.98±11.77, AUC0-t (ng·h/mL) were 484.5±71.9 and 487.9±96.0, AUC0-∞(ng·h/mL) were 535.5±95.9 and 544.6±94.1 for men and women, respectively. After oral administration of single doses of 10 mg, the pharmacokinetic parameters were as follows:Tmax (h):5.60±1.67 and 6.40±0.89, Cmax (ng/mL):11.47±2.31 and 11.73±1.78, t1/2(h):63.11±8.53 and 64.02±16.82h, AUC0-t (ng·h/mL):875±315 and 904±171, AUC0-∞(ng·h/mL):922±303 and 942±167 for men and women, respectively. After oral administration of single doses of 20 mg, the pharmacokinetic parameters were investigated, Tmax (h) were 6.80±1.79 and 7.00±1.16, Cmax (ng/mL) were 26.07±7.82 and 24.42±10.65, t1/2 (h) were 60.86±12.78 and 63.57±4.43, AUC0-t (ng·h/mL) were 1693±658 and 1873±1068, AUC0-∞(ng·h/mL) were 1753±633 and 1978±1067 for men and women, respectively. The ANOVA and linear regression were applied for the main pharmacokinetic parameters among three different groups. AUC0-t, AUC0-∞and Cmax increased in proportional to the dosages and the differences of Tmax or t1/2 are not significant Memantine undergoes linear pharmacokinetic characteristics in the dose range of 5～20 mg. In all single-dose groups, women tended to have higher areas under the plasma concentration-time curve from time zero to the last sampling time, longer time to reach maximum plasma concentration, but the difference was not significant.For the pharmacokinetic study after multiple-dose oral administration,5 men and 5 women were enrolled and underwent oral administration of multiple doses of 5 mg once daily for 14 days. The plateau concentration was achieved after 11 days. On the 14th day, the pharmacokinetic parameters were investigated, Tmax (h) were 6.00±2.00 and 7.60±2.97, Css max (ng/mL) were 18.37±1.31 and 21.01±1.85, t1/2 (h) were 57.67±8.17 and 71.47±20.12, AUCss (ng·h/mL) were 363.9±43.8 and 408.8±71.3, DF (%) were 42.22±12.10 and 46.73±19.87, Accumulation factor (R) were 3.46±0.49 and 4.29±1.21, for men and women, respectively. When the dosage regimen was adopted, memantine underwent medium accumulation in human body. After a long-time administration of memantine, the adverse reaction might be paid more attention to.As the main pharmacokinetic parameters of single-dose and multiple-dose oral administration, Tmax and t1/2 didn’t show significant difference, which indicated that the absorption and elimination of memantine didn’t change with dosage regimen in this study. Except Css max was lower in men than that in women, the other pharmacokinetic parameters didn’t change significantly between different genders.Protein binding rate of memantine in human plasma was determined at three concentration levels (5,20 and 80 ng/mL). Equilibrium dialysis coupled with LC-MS was adopted for the investigation. When the concentration of memantine was lower than 20 ng/mL, the mean protein binding rate was 45.9%.while the concentration was higher than 80 ng/mL, the mean protein binding rate decreased to 33.5%, which indicated that saturation phenomenon may be exist in the protein binding rate of memantine. Because of plus dosage in clinic, the ratio of concentration of free drug to that of plasma drug would increase, then the non-linear pharmacokinetics characters of memantine would appear, which would lead to serious adverse reaction.3 Metabonomic analysis of Alzheimer’s disease by UPLC/MS analysisPlasma and urine metabolic perturbations between AD and healthy old person were investigated using ultra performance liquid chromatography/mass spectrometry (UPLC/MS) and metabonomics approach. The plasma samples were got from 20 AD patients and 20 healthy old persons and the urine samples were got from 9 AD patients and 10 healthy old persons. After protein precipitation, UPLC/MS analysis was applied. The principal component analysis (PCA) of UPLC/MS spectra showed that metabolic changes between two groups. The PCA of UPLC/MS spectra showed that metabolic changes observed between AD and control were clear. Nine potential biomarkers in plasma in correlation with the extent of AD were found including five kinds of lysophosphatidyl choline(LPC C16:0, LPC C18:2, LPC C18:1, LPC C18:0, LPC C20:4), three kinds of sphingosine(Phytosphingosine, Dihydrosphingosine, C16 dihydrosphingosine) and tryptophan. Eight of them belong to lipoid. Nine potential biomarkers in urine in correlation with the extent of AD were found including six kinds of sphingosine(C14DHS,C16 DHS,C18DHS,C20DHS, Phytosphingosine, C20 phytosphingosine), tryptophan, creatinine,and a unkown substance (m/z 402.2). There are four biomarkers existed in both plasma and urine metabolic perturbations. The different mechanisms on the AD correlated with these biomarkers were illuminated:the changes of LPCs, sphingosine, tryptophan were related to the phospholipid metabolism, cermaide metabolism and kynurenine pathway in brain, respectively. The result we got was compared with the United States Patent:US 2009/0029473A1, Lipid profile as a biomarker for early detection of neurological disorders, Washington University in ST.LOUIS,MO(US), Significance of this study was showed. The result of this study supported that lipid profile including almost 1000 lipoid substance could be a biomarker for early detection of neurological disorders. More importantly, the changes of 14 biomarkers in this study were showed, which was not described in the patent, and the metabolic perturbations in urine were not investigated in that patent. The found biomarkers for AD were very important to the study on the pathogenesy, diagnosis and clinical research of AD. |
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