Surface modified methotrexate loaded erythrocytes for enhanced macrophage uptake

The unique property of macrophages to distinguish between 'mature self' and 'senescent self' was applied for the enhanced macrophage uptake upon loading drugs into erythrocytes and provoking environment within which it gets translated into senescent cells. Two different approaches were applied one by depleting the sialic acid from the surface of cells and another by inducing hemichrome formation in the cells. Erythrocytes encapsulation of methotrexate was carried out by preswell dilution technique. Surface modification of erythrocytes was performed using desialation and hemichrome induction by treating with trypsin (Tt) and phenylhydrazine (PhT) respectively. Desialation technique was optimized in terms of concentration of trypsin, incubation temperature and time period. Both of these surface modified erythrocytes were characterized for in vitro macrophage uptake. In vivo organ localization was assessed by recording amount of drug present in different organs. The macrophage uptake of both surface modified erythrocytes was enhanced by almost 3-5 and 5-6 times with Tt and PhT cells, respectively. These new surface modified carriers are found to be excellent candidate for enhanced macrophage uptake and anticipated to be useful for any RES tumors.     

Reduced hepatic toxicity, enhanced cellular uptake and altered pharmacokinetics of stavudine loaded galactosylated liposomes.

The aim of the present investigation was to reduce the hepatic toxicity, enhance the cellular uptake and alter the pharmacokinetics of stavudine using galactosylated liposomes. beta-D-1-Thiogalactopyranoside residues were covalently coupled with dimyristoyl phosphatidylethanolamine, which was then used to form liposomes. The galactosylated liposomal system was assessed for in vitro ligand-specific activity. The drug release from liposomes was studied by dialysis method. Ex vivo cellular uptake study was performed using liver parenchymal cells harvested from male albino rats. Changes in hematological parameters, hepatic enzymes, hepatomegaly, plasma and tissue distribution of the formulations (free stavudine solution, uncoated liposomal and galactosylated liposomes) were determined using albino rats. Percent cumulative drug release in 24h was low (34.8+/-2.6%). Enhanced hepatic cellular d4T uptake (27.96+/-2.41pg d4T/million cells) was seen in case of galactosylated liposomal d4T. Galactosylated liposomes maintained a significant level of d4T in tissues rich in galactose specific receptors and had a prolonged residence (11.44+/-1.25h) in the body resulting in enhanced half-life of d4T (23.07+/-1.25h). This formulation did not show either hematological or hepatic toxicity. Galactosylation of liposomes alter the biodistribution of encapsulated drug thereby delivering the drug to cells bearing galactose specific receptors.     

Changes in liver histology during methotrexate therapy of psoriasis correlated to the concentration of methotrexate and folate in erythrocytes

To investigate, whether an assessment of the folate and methotrexate concentrations in erythrocytes could be used to indicate incipient histological liver changes, these 2 parameters were related to liver histology in series of liver biopsies (means 4.3 biopsies) from 31 long-term methotrexate-treated psoriasis patients. Logistic regression analysis showed that the probability of changes in liver histology during methotrexate therapy increased significantly with increasing erythrocyte methotrexate concentrations and with decreasing erythrocyte folate concentration. The application of these variables as a decisive criterion when to institute liver biopsy surveillance needs further studies, but can probably help reduce the number of liver biopsies to be taken during long-term methotrexate therapy considerably.     

Enhanced hepatocyte uptake and liver targeting of methotrexate using galactosylated albumin as a carrier.

Liver targeting of drugs has wide therapeutic implications due to numerous liver-related diseases. Using conjugates of methotrexate (MTX) to variously galactosylated bovine serum albumin (BSA), we studied whether we could enhance the liver targeting of MTX, a model drug, via galactose receptors selectively abundant on the hepatocytes. Here, we report that the galactosylation of the carrier protein BSA significantly enhanced the hepatocyte uptake and liver targetability of MTX. In vitro, the amount of MTX taken up by rat hepatocytes was positively correlated with the galactose content in BSA. MTX conjugates were relatively stable in plasma, but released MTX with time in liver homogenates. These results imply that the conjugates would exert low toxicity in the blood, but have therapeutic activity in the liver by liberating MTX. In vivo, MTX-galactosylated BSA conjugates (MTX-L(24)BSA) showed significantly different pharmacokinetics from free MTX or MTX-BSA conjugates. The plasma level of free MTX rapidly declined in a biexponential fashion with an apparent terminal half-life of 0.35 h. MTX-BSA conjugates showed the slowest decline with an apparent terminal half-life of 6 h, whereas MTX-L(24)BSA showed a biphasic pattern; a rapid distributive phase with a half-life of 0.567 h and a slow terminal phase. MTX-L(24)BSA showed the highest liver targetability, when evaluated in terms of two indices based on the area under the total amount of radioactivity-time curve (AUQ); Te*(liver), % AUQ(liver) to total AUQ, and te*, the ratio of AUQ(liver) to AUQ(kidney). Compared with free MTX and MTX-BSA, MTX-L(24)BSA showed about twofold higher Te*(liver) of 87.5%. The te* of MTX-L(24)BSA was 25- and fourfold higher than those of free MTX and MTX-BSA, respectively. Moreover, MTX-L(24)BSA showed a gradual increase in the therapeutically active intact form of MTX in the liver while showing the lowest level of intact MTX in the kidney. These results suggest that galactosylated BSA has a great potential as an hepatocyte-directed and more effective liver targeting carrier of drugs for liver diseases.     

喷雾干燥法制备鼻用甲氨蝶呤壳聚糖微球及其特性的考察

目的以壳聚糖为载体材料,甲氨蝶呤为模型药物,制备鼻腔给药甲氨蝶呤壳聚糖微球。方法采用喷雾干燥法制备甲氨蝶呤壳聚糖微球;采用正交设计优化制备工艺,并对药物的包封率、收率、粒径以及释放行为进行考察。结果优化条件下所得微球粒径分布较为均匀,平均粒径为4.0~5.0μm,制成微球后药物可缓慢释放,符合鼻腔给药的要求。结论该方法适用于甲氨蝶呤壳聚糖微球的制备,壳聚糖是良好的鼻用制剂的载体材料,可用于制备脑部靶向鼻黏膜给药制剂。     

Study on formulation variables of methotrexate loaded mesoporous MCM-41 nanoparticles for dissolution enhancement

The aim of this study was to develop methotrexate loaded mesoporous MCM-41 nanoparticles for improved dissolution of methotrexate. The mesoporous MCM-41 nanoparticles act as carrier for drug and increase the solubility of the drug. In order to achieve this objective small pore size MCM-41 nanoparticles have been synthesized followed by drug loading process. The process of drug loading was optimized using full 33 factorial design. With a view to obtain maximum drug loading three variables, concentration of drug solution, stirring rate, and drug:carrier ratio were optimized using a full 33 factorial design. Using statistically designed experiments, the inclusion of methotrexate in MCM-41 nanoparticles was successfully carried out to obtain a drug loading of about 48%. X-ray powder diffraction and differential scanning calorimetry revealed the presence of methotrexate in amorphous form and FT-IR spectroscopy showed the presence of light interactions between the silicate silanols and the drug. The decrease of Brunauer, Emmett and Teller specific surface area and pore volume between free MCM-41 and the inclusion compound was the proof of the presence of methotrexate inside the mesopores. The inclusion compound was submitted to in vitro dissolution tests and a remarkable dissolution rate improvement was observed in comparison to the crystalline drug in all tested conditions.     

A study of the uptake of chloroquine in malaria-infected erythrocytes. High and low affinity uptake and the influence of glucose and its analogues

A study of concentration- and substrate-dependence of chloroquine uptake has been carried out on mouse erythrocytes infected with the chloroquine-sensitive NK65 and the chloroquine-resistant RC strains of Plasmodium berghei. The presence of drug binding sites of high and low affinity in such strains of P. berghei was confirmed. High affinity uptake sites in cells parasitized with chloroquine-sensitive and chloroquine-resistant parasites have similar characteristics, but in the sensitive strain the major component of chloroquine-uptake is at high affinity and dependent on the availability of ATP whilst in the resistant strain the major component of uptake is at low affinity and independent of energy. An absolute increase in the quantity of the low affinity site in erythrocytes parasitized with chloroquine-resistant P. berghei was noted, which may be related to an increase in quantity of parasite membrane.     

Nicotinamide and methionine reduce the liver toxic effect of methotrexate.

Methotrexate is widely used as a therapeutic agent in different diseases. This therapy is connected with various side effects, including liver toxicity. We have developed a mouse model to demonstrate the toxic effects of methotrexate: mice were given 50 mg/kg acetaminophen, which itself has no effect on the liver. If, additionally, methotrexate is applied, there is an increase in the death rate, as well as in glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities. If methotrexate is administered in conjunction with either nicotinamide or methionine, the rise in the death rate and in GOT and GPT activities associated with methotrexate application is markedly reduced. On the basis of these results, it can be concluded that methotrexate therapy should be combined with either nicotinamide or methionine, respectively.     

Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint

A controlled release delivery system that localizes methotrexate (MTX) in the synovial joint is needed to treat inflammation in rheumatoid arthritis (RA). The purpose of this work was to develop and characterize MTX loaded poly(l-lactic acid) (PLLA) microspheres and evaluate in vivo tolerability and MTX plasma concentrations following intra-articular injection into healthy rabbits. MTX loaded PLLA (2 kg/mole) microspheres were prepared using the solvent evaporation method and characterized in terms of size, molecular weight, thermal properties, and release rates into phosphate buffered saline (PBS) (pH 7.4) at 37 degrees C. Biocompatibility was evaluated by observing the swelling of the joints of the rabbits and histological analysis following the injection of the microspheres. MTX concentrations in the plasma and urine samples of rabbits were evaluated by high-performance liquid chromatography (HPLC). MTX loaded microspheres showed a rapid burst phase followed by a slow release phase. MTX loaded and control microspheres were biocompatible and plasma concentrations of MTX were tenfold higher in rabbits injected intra-articularly with free MTX than MTX microspheres. MTX microspheres may retain the drug in the joint by reducing clearance from the joint into the blood.     

Octreotide-targeted liposomes loaded with CPT-11 enhanced cytotoxicity for the treatment of medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, but treatment with irinotecan (CPT-11) is limited because of side effects. MTC is known to overexpress the somatostatin receptor subtype 2 (SSTR2). Octreotide (Oct) is a somatostatin analogue that has a high binding affinity for SSTR and can be used as a tumor-targeting ligand. We prepared Oct-targeted liposomes loaded with CPT-11 using Oct-poly (ethylene glycol) (PEG)-lipid and evaluated Oct-mediated association and cytotoxicity of the liposomes with an MTC cell line TT. The association of higher concentrations of modified Oct-targeted liposomes with TT cells was significantly higher than PEGylated liposomes and was significantly inhibited by empty Oct-targeted liposomes but not by free Oct. With exposure for 96 h, the cytotoxicity of Oct-targeted liposomal CPT-11 (IC50: 1.05 ± 0.47 μM) was higher than free CPT-11 (IC50: 3.76 ± 0.61 μM) or PEGylated liposomal CPT-11 (IC50: 3.05 ± 0.28 μM). In addition, empty Oct-targeted liposomes showed significantly higher cytotoxicity than empty nontargeted liposomes at a concentration where free Oct did not show cytotoxicity, suggesting that Oct as a ligand showed cytotoxicity. Moreover, Oct-targeted liposomal CPT-11 led to significantly higher antitumor activity and prolonged the survival time compared with nontargeted liposomal and free CPT-11 at a one-third dose and lower administration times with free CPT-11. These findings indicated that Oct-targeted liposomes loaded with CPT-11 may offer considerable potential for MTC chemotherapy because cytotoxicity of both CPT-11 and Oct was enhanced by effective cellular uptake via SSTR2.     

Facilitated uptake of zinc into human erythrocytes. Relevance to the treatment of sickle-cell anaemia

The ability of a number of heterocyclic metal chelators to deliver zinc into red cells, to release the liganded zinc to haemoglobin and thereby cause a left shift in the oxygen dissociation curve of intact red cells has been investigated. Incubation of neutrally charged zinc-pyrone and zinc-pyridin-2-one complexes with red cells led to the rapid accumulation of zinc within cells, whereas unliganded zinc in the form of zinc acetate, zinc chloride or zinc sulphate accumulated only slowly. The rate at which zinc was delivered to red cells by pyrone and pyridin-2-one ligands increased with increasing lipid solubility of the ligands. The uptake of zinc into both normal adult and sickle red cells was associated with a dose-dependent increase in the oxygen affinity of haemoglobin. The degree of left shift in the oxygen dissociation curve following the incubation of red cells with zinc-pyrone and -pyridin-2-one complexes suggests that these complexes may find application as agents to increase the oxygen affinity of haemoglobin in sickle cell disease and thereby decrease the probability of intravascular sickling at low tissue oxygen tensions. Ethylmaltol appears to be a particularly useful agent due to its known low toxicity.     

红细胞包蔽吗啡的制备及包蔽效果的研究

目的：了解高渗法制备RBC吗啡载体的可行性。方法：①全血与50％GS混合后以血球压积的变化评价RBC的脱水效果；②利用扫描电镜观察RBC包蔽吗啡前后其形态的变化；③采用放免分析(RIA)方法对RBC一吗啡溶液中RBC内外吗啡的浓度进行测定；④HPLE法分析人RBC内Hb与吗啡的结合率。结果：全血与50％GS按1：0．5混合时的脱水效果最显著；RBC脱水后以及包蔽吗啡后其形态由正常圆盘状变为扁平和皱缩，最后又恢复为正常圆盘状；RBC包蔽吗啡剂量为5mg和10mg时RBC内外吗啡浓度之间均无显著差异。Hb与吗啡在不同浓度混合下均具有一定的结合率。结论：高渗法制备RBC吗啡载体具有较好的包蔽效果，Hb与吗啡结合率的存在可能影响吗啡的作用时效。     

Methotrexate loaded polyether-copolyester dendrimers for the treatment of gliomas: enhanced efficacy and intratumoral transport capability

Therapeutic benefit in glial tumors is often limited due to low permeability of delivery systems across the blood-brain barrier (BBB), drug resistance, and poor penetration into the tumor tissue. In an attempt to overcome these hurdles, polyether-copolyester (PEPE) dendrimers were evaluated as drug carriers for the treatment of gliomas. Dendrimers were conjugated to d-glucosamine as the ligand for enhancing BBB permeability and tumor targeting. The efficacy of methotrexate (MTX)-loaded dendrimers was established against U87 MG and U 343 MGa cells. Permeability of rhodamine-labeled dendrimers and MTX-loaded dendrimers across the in vitro BBB model and their distribution into avascular human glioma tumor spheroids was also studied. Glucosylated dendrimers were found to be endocytosed in significantly higher amounts than nonglucosylated dendrimers by both the cell lines. IC 50 of MTX after loading in dendrimers was lower than that of the free MTX, suggesting that loading MTX in PEPE dendrimers increased its potency. Similar higher activity of MTX-loaded glucosylated and nonglucosylated dendrimers was found in the reduction of tumor spheroid size. These MTX-loaded dendrimers were able to kill even MTX-resistant cells highlighting their ability to overcome MTX resistance. In addition, the amount of MTX-transported across BBB was three to five times more after loading in the dendrimers. Glucosylation further increased the cumulative permeation of dendrimers across BBB and hence increased the amount of MTX available across it. Glucosylated dendrimers distributed through out the avascular tumor spheroids within 6 h, while nonglucosylated dendrimers could do so in 12 h. The results show that glucosamine can be used as an effective ligand not only for targeting glial tumors but also for enhanced permeability across BBB. Thus, glucosylated PEPE dendrimers can serve as potential delivery system for the treatment of gliomas.     

超声辐照增强载羟基喜树碱聚乳酸微泡对肝癌细胞生物学效应

目的:探讨载羟基喜树碱聚乳酸微泡超声辐照下对肝癌细胞的生物学效应。方法:选择人肝癌细胞株Bel-7402为体外模型,采用MTT比色分析法,观察羟基喜树碱聚乳酸微泡对肝癌细胞的生长和增殖抑制情况,评价载药微泡对肝癌细胞的毒性作用;采用激光共聚焦荧光显微镜技术和流式细胞仪技术观察超声辐照下载药微泡对肝癌细胞摄取药物的影响。结果:羟基喜树碱聚乳酸微泡抑制肝癌细胞的生长增殖,且随羟基喜树碱浓度的升高而增强;激光共聚焦荧光照片显示:与未超声的肝癌细胞相比,超声辐照下,肝癌细胞内羟基喜树碱的绿色荧光增强,说明细胞摄取羟基喜树碱的量增加;流式细胞仪数值:细胞内羟基喜树碱荧光值对照组仅37.5,未超声实验组为71.9;超声实验组为91.3。结论:羟基喜树碱聚乳酸微泡对肝癌细胞Bel-7402有毒性作用,且超声辐照对细胞摄取药物有促进作用。其超声辐照下对肿瘤细胞生物学效应的评价可将为其今后的临床超声治疗提供必要依据。     

齐墩果酸聚氰基丙烯酸正丁酯纳米囊在小鼠体内的肝靶向研究

目的:研究齐墩果酸聚氰基丙烯酸正丁酯纳米囊(OA-PBCA-NC)在小鼠体内的肝靶向性。方法:小鼠尾静脉注射OA-PBCA-NC试验组及齐墩果酸(OA)对照组,HPLC法测定小鼠心、肝、脾、肺、肾各脏器及血浆中OA的含量,比较两组体内分布特点,并进行靶向性评价。结果:秩和检验结果表明静脉注射OA-PBCA-NC试验组与OA对照组比较,其在肝脏的分布差异有显著性,OA-PBCA-NC静脉注射给药后能显著增加OA在肝脏的分布。试验组各肝靶向指标结果均优于对照组。结论:OA-PBCA-NC能增加OA的肝靶向性。     

伊伐布雷定肝脏转运机制的初步研究

目的：探讨伊伐布雷定肝脏转运与OCT1的相关性。方法：将雄性SD大鼠分为对照组与实验组,对照组伊伐布雷定（1 mg·kg^-1）灌胃给药,实验组OCT1抑制剂雷尼替丁（0.8 mg·kg^-1）和伊伐布雷定（1.0 mg·kg^-1）相继灌胃给药,比较2组间伊伐布雷定的药动学参数;采用原代肝细胞,分为对照组：伊伐布雷定（0.2,0.5,1 μmol·L^-1）;实验组：雷尼替丁（0.5,1.0,2.0 μmol·L^-1）+伊伐布雷定（0.2,0.5,1 μmol·L^-1）,探讨雷尼替丁对伊伐布雷定肝脏摄取的影响。结果：合用雷尼替丁后,伊伐布雷定药动学参数C_max、AUC_0-t、AUC_0-∞分别增加了115.41%,128.73%,128.05%,而CLz/F值降低了56.35%,差异有显著性（P<0.05或P<0.01）。雷尼替丁明显抑制肝细胞对伊伐布雷定的摄取,抑制作用随雷尼替丁的浓度增加而增强。结论：伊伐布雷定肝脏转运很可能与OCT1有关。     

Studies on the uptake of low molecular weight monomeric tris-galactosyl conjugates by the rat liver.

We have attempted to direct low molecular weight compounds to the liver via the internalizing asialoglycoprotein receptor on parenchymal cells by conjugation to a monomeric triantennary galactosyl cluster. Acetate and a hypolipidaemic ansamycin were derivatized and the biodistribution of the conjugates was determined 250 sec and 30 min after administration to Wistar rats. The ansamycin conjugate (CGH46) was rapidly cleared from the circulation by the liver; after 250 sec, 64% of the radiolabelled dose was found in the liver compared to 18% in the blood. However, the distribution of the conjugate did not differ significantly from that of unconjugated ansamycin (CGH45). Tris-galactosyl acetate showed no capacity to localize in the liver, with only 2% recovered from that organ 250 sec after administration compared to 38% in the blood and 13-18% in the kidneys, skin and muscle. Extraction efficiency of CGH46 by isolated perfused rat livers was almost 20% of the administered dose and this value was not significantly changed by co-administration of specific inhibitors of the uptake process. It is concluded that derivatization of low molecular weight molecules with monomeric triantennary galactosyl residues is unlikely to increase their affinity for the liver.     

冰片对甲氨蝶呤透过血脑屏障影响的实验研究

目的 研究冰片对甲氨蝶呤(抗肿瘤药)透过血脑屏障的影响.方法 12只日本大耳白兔随机分为试验组和对照组,试验组给予冰片0.7 g·kg-1(75%乙醇溶解)灌胃;对照组以75%乙醇灌胃1 h后,2组均耳缘静脉注射甲氨蝶呤100 mg·kg-1;用高效液相色谱法测定不同时间点血液和脑脊液的甲氨蝶呤浓度,用3P97软件计算药代动力学参数.结果 试验组、对照组在脑脊液药代动力学参数:Cmax分别为15.65、10.06 mg·L-1,AUC0-t分别为45.36、25.91 mg·h·L-1,AUC0-∞分别为46.76、26.23 mg·h·L-1;试验组、对照组在血浆药代动力学参数:AUC0-t分别为512.63、532.71 mg·h·L-1,AUC0-∞分别为515.35、533.90 mg·h·L-1.脑脊液中,试验组甲氨蝶呤的Cmax和AUC0-t、AUC0-∞均明显高于对照组(P<0.05);血浆药代动力学参数与对照组比较无显著性差异;血脑屏障透过率由4.86%提高到8.85%.结论 冰片可促进甲氨蝶呤透过血脑屏障,增加其在脑脊液中的药物浓度;而不改变其血药浓度.