Drug distribution and binding

The distribution of drugs in the body depends on their lipophilicity and protein binding. Low plasma binding or high tissue binding or high lipophilicity usually means an extensive tissue distribution. In pharmacokinetics, the distribution is described by the parameter V, the apparent volume of distribution. This parameter measures the relative distribution of drugs between tissue and plasma and depends on the plasma and tissue binding and the lipophilicity of the drug. If the distribution of drug throughout the body is slow, V in the terminal phase will also depend on the clearance of the drug. At equilibrium, V will theoretically not be lower than 7 L in a 70-kg person, but it has no upper limit. The extent to which a drug distributes affects the half-life of the drug and the fluctuation of the concentration at steady state on multiple dosing, but not the average steady-state concentration.     

In vitro adsorption of sodium pentobarbital by SuperChar, USP and Darco G-60 activated charcoals

This study was designed to examine the in vitro adsorption of sodium pentobarbital by three activated charcoals. Solutions of sodium pentobarbital (20 mM) were prepared in distilled water and in 70% sorbitol (w/v). Radiolabeled (14C) sodium pentobarbital was added to each solution to serve as a concentration marker. Two ml of each drug solution was added to test tubes containing 40 mg of either Darco G-60, USP, or SuperChar activated charcoal. The drug-charcoal mixtures were incubated at 37 degrees C for O, 2.5, 5, 7.5 or 10 min. Equilibrium, indicated by a constant percentage of drug bound for two consecutive time periods, was established immediately for the aqueous mixtures and for Darco G-60 in sorbitol. The time to equilibrium was prolonged for USP (2.5 min) and SuperChar (5 min) in the presence of sorbitol. In the second series of experiments, solutions of sodium pentobarbital (1.25 to 160 mM) were prepared in either distilled water or sorbitol. Amount of drug bound by 10 to 320 mg of activated charcoal within a 10 min incubation period was determined. Scatchard analysis determined maximum binding capacity (Bmax) and dissociation constants (Kd) for each activated charcoal. In water, Bmax (mumoles/gm) was greatest for SuperChar (1141), followed by USP (580) and Darco G-60 (381), while the Kd's did not differ. Sorbitol did not change the Bmax or Kd of USP or Darco G-60, but the additive significantly decreased the Bmax (717) and increased the Kd for SuperChar (3.3 to 10.1 mM). The results suggest that relative binding capacity of activated charcoal is directly proportional to surface area, and that sorbitol significantly reduces sodium pentobarbital binding to SuperChar.     

An investigation of the factors controlling the adsorption of protein antigens to anionic PLG microparticles

This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface.     

大孔吸附树脂在EC-SOD纯化中的作用

目的本文主要论述了大孔吸附树脂在EC-SOD的分离与纯化中所起的作用。该研究在大孔吸附树脂的应用方面有很大的开创性意义。方法研究了非极性大孔吸附树脂在蛋白纯化中去除杂蛋白及DNA碎片、色素等成份的效果。结果研究结果表明在33℃,上样量在30%总吸附量,上样速度在20mL.min-1、pH值为7时有很好的吸附解吸附效果,蛋白回收率90%以上。结论非极性大孔吸附树脂对于粗蛋白的纯化去除杂质效果显著,快速方便,成本低,更适于产业化。     

Drug bioactivation, covalent binding to target proteins and toxicity relevance

A number of therapeutic drugs with different structures and mechanisms of action have been reported to undergo metabolic activation by Phase I or Phase II drug-metabolizing enzymes. The bioactivation gives rise to reactive metabolites/intermediates, which readily confer covalent binding to various target proteins by nucleophilic substitution and/or Schiff's base mechanism. These drugs include analgesics (e.g., acetaminophen), antibacterial agents (e.g., sulfonamides and macrolide antibiotics), anticancer drugs (e.g., irinotecan), antiepileptic drugs (e.g., carbamazepine), anti-HIV agents (e.g., ritonavir), antipsychotics (e.g., clozapine), cardiovascular drugs (e.g., procainamide and hydralazine), immunosupressants (e.g., cyclosporine A), inhalational anesthetics (e.g., halothane), nonsteroidal anti-inflammatory drugs (NSAIDSs) (e.g., diclofenac), and steroids and their receptor modulators (e.g., estrogens and tamoxifen). Some herbal and dietary constituents are also bioactivated to reactive metabolites capable of binding covalently and inactivating cytochrome P450s (CYPs). A number of important target proteins of drugs have been identified by mass spectrometric techniques and proteomic approaches. The covalent binding and formation of drug-protein adducts are generally considered to be related to drug toxicity, and selective protein covalent binding by drug metabolites may lead to selective organ toxicity. However, the mechanisms involved in the protein adduct-induced toxicity are largely undefined, although it has been suggested that drug-protein adducts may cause toxicity either through impairing physiological functions of the modified proteins or through immune-mediated mechanisms. In addition, mechanism-based inhibition of CYPs may result in toxic drug-drug interactions. The clinical consequences of drug bioactivation and covalent binding to proteins are unpredictable, depending on many factors that are associated with the administered drugs and patients. Further studies using proteomic and genomic approaches with high throughput capacity are needed to identify the protein targets of reactive drug metabolites, and to elucidate the structure-activity relationships of drug's covalent binding to proteins and their clinical outcomes.     

多孔羟基磷灰石微球对药物的吸附性质

目的考察多孔羟基磷灰石微球对药物的吸附性质。方法采用溶液吸附法研究市售羟基磷灰石微球对药物的吸附性质。以氯化锶为标记物,用扫描电镜-能谱分析仪定点扫描微球内锶的分布情况,氮吸附法测定微球的比表面积和孔径。结果可与羟基磷灰石上的钙离子螯合的药物吸附量相对较高,而依靠氢键或电荷间相互作用、发生物理吸附的药物吸附量偏低,难溶性药物吸附药量普遍低于水溶性药物。市售微球孔径约为30 nm,属于介孔材料,药物多分布在微球的表层。结论阐明了羟基磷灰石微球对不同类型药物的吸附特征,为其作为药物载体提供依据。     

大孔吸附树脂对淫羊藿总黄酮的吸附性能研究

目的研究不同大孔树脂对淫羊藿总黄酮及淫羊藿苷的吸附及解吸附性能,为分离淫羊藿总黄酮提供树脂选择的依据.方法选择11种常用大孔吸附树脂,以淫羊藿总黄酮和淫羊藿苷为指标,考察不同大孔树脂对两成分的比吸附量和解吸率.同时考察了11种大孔树脂的静态吸附动力学行为.结果对淫羊藿总黄酮比吸附量超过100 mg·g-1的树脂有HPD-300、HPD-400、HPD-500、HPD-600、S-8、AB-8;解吸率超过95%的有D101、HPD-600、AB-8、HPD-400、HPD-500、D201;HPD-600、HPD-500和AB-8为淫羊藿总黄酮快速吸附树脂.结论不同树脂对淫羊藿总黄酮的吸附量、解吸率和速度有很大差异,综合各项试验结果,HPD-600、HPD-500、AB-8为分离淫羊藿总黄酮的最佳吸附树脂.     

A computational study of the binding of propidium to the peripheral anionic site of human acetylcholinesterase

Combined docking and molecular dynamics (MD) simulations were carried out in order to investigate the binding mode of propidium at the human acetylcholinesterase (HuAChE) peripheral site. Two different docking protocols followed by cluster analyses were performed, allowing the identification of five high-populated and low-energy configuration families. To dynamically explore the behavior of the ligand at the peripheral HuAChE binding site, six complexes (five low-energy and one high-energy) were submitted to 2.5 ns of MD simulations. The representative propidium/HuAChE binding modes were chosen on the basis of both the docking energy score and the dynamic stability of the complexes throughout the MD simulations. The most stable poses of propidium at HuAChE PAS were similar to those experimentally determined with the murine enzyme. We therefore suggest that the present modeling protocol might be used in the dynamic investigation of the interactions of a small-molecule inhibitor with a surface-like binding site of a protein. Finally, because of the biological relevance of the target studied here, the present results can be of interest for the rational design of molecules potentially useful in the treatment of the Alzheimer's disease.     

静态吸附杜仲叶中绿原酸较合适树脂的研究

目的以静态吸附法从杜仲叶提取液中筛选吸附、纯化绿原酸的最佳树脂。方法以绿原酸静态吸附量、解析率、纯度为评价指标分别对NKA-Ⅱ、NKA-9、D-101-1、HPD-100、HPD-600、HPD826、330、AB-8 8种树脂进行考察。结果 NKA-Ⅱ吸附量为18.63 mg.g-1,60%与95%乙醇加合解析率达到70.2%,绿原酸在样品中纯度大于20.30%。结论 NKA-Ⅱ是绿原酸较合适的吸附树脂,可供参考。     

Determination of protein binding byin vitro charcoal adsorption

Certain compounds such as SC-52151 have extensive nonspecific adsorption to the ultrafiltration devices or to dialysis membranes and therefore can not be measured by the conventional ultrafiltration or equilibrium dialysis methods. A new method based on charcoal adsorption was developed to overcome this difficulty. Unlike many conventional methods, which are based on the separation of free drug from bound drug under equilibrium conditions, the new method is operated under nonequilibrium conditions and involves measuring the time course of decline of the percentage of bound drug remaining in plasma while the free drug is being removed by charcoal adsorption. Theoretical aspects of the method and the data processing procedure are presented. SC-98A, a compound with minimal nonspecific adsorption to the ultrafiltration membrane, was used to demonstrate the applicability of this method against the ultrafiltration method. Using this method, the protein binding of SC-52151 in human plasma at 1.0 μg/ml was determined to be in the range of 91.4–97.7% at room temperature.     

Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions.

Loss of drug content during cell culture transport experiment can lead to misinterpretations in permeability analysis. This study analyses drug adsorption to various plastic containers and drug retention in cultured cells under in vitro conditions. The loss of various drugs to polystyrene tubes and well plates was compared to polypropylene and glass tubes both in deionised water and buffer solution. In cellular uptake experiments, administered drugs were obtained from cultured cells by liquid extraction. Samples were collected at various time points and drug concentrations were measured by a new HPLC-MS/MS method. Acidic drugs (hydrochlorothiazide, naproxen, probenicid, and indomethacin) showed little if any sorption to all tested materials in either water or buffer. In the case of basic drugs, substantial loss to polystyrene tubes and well plates was observed. After 4.5 h, the relative amount remaining in aqueous test solution stored in polystyrene tubes was 64.7 +/- 6.8%, 38.4 +/- 9.1%, 31.9 +/- 6.7%, and 23.5 +/- 6.1% for metoprolol, medetomidine, propranolol, and midazolam, respectively. Interestingly, there was no significant loss of drugs dissolved in buffer to any of the tested materials indicating that buffer reduced surficial interaction. The effect of drug concentration to sorption was also tested. Results indicated that the higher the concentration in the test solution the lower the proportional drug loss, suggesting that the polystyrene contained a limited amount of binding sites. Cellular uptake studies showed considerable retention of drugs in cultured cells. The amounts of absorbed drugs in cellular structures were 0.45%, 4.88%, 13.15%, 43.80%, 23.57% and 11.22% for atenolol, metoprolol, medetomidine, propranolol, midazolam, and diazepam, respectively. Overall, these findings will benefit development and validation of further in vitro drug permeation experiments.     

Drug adsorption in human skin: a streaming potential study

The objective of this study was to investigate the drug adsorption process in human skin using in vitro streaming potential measurements. Streaming potential is an electrokinetic phenomenon, which reflects both the charge density and the pore size of a membrane. Thus, the adsorption of charged solutes on the pore walls can be detected as a change of streaming potential, viz., as a change in the slope deltaE/deltaP. In these streaming potential measurements, hydrophilic nadolol and luteinizing hormone-releasing hormone, and lipophilic propranolol and Nafarelin were used as model drugs. As could be expected, the hydrophilic drugs did not change the slope. The more lipophilic propranolol and Nafarelin, instead, changed the slope. Propranolol changed the slope gradually from negative to positive when the concentration was increased from 1 to 10 mM. With Nafarelin, a straight line with a slope of about 0 was obtained at pH 7.3 and an ascending curve at pH 4.2. These results indicate that the negative charges on the pore walls of human skin are blocked by adsorption of the lipophilic cations. The adsorption of lipophilic cations in the skin alters the permselectivity of the skin, which, in turn, may lead to the inhibition of electroosmotic flow across the skin during iontophoresis and to the shut down of transdermal drug permeation of higher molecular weight drugs.