Development of drug delivery system for intrathecal administration and its therapeutic effect on cerebral vasospasm and ischemia

To date, the pharmacologic approach to cerebral vasospasm and ischemia has been hampered in part by an inability to attain sufficiently high concentrations of drugs in the cerebrospinal fluid (CSF). To overcome this limitation of current drug therapy, we have developed a sustained-release preparation of the protein kinase inhibitor fasudil. Experimental cerebral vasospasm in rats and dogs was induced by double injection of autologous arterial blood into the cisterna magna. Focal cerebral ischemia in rats was induced by middle cerebral artery occlusion using an intraluminal suture technique. A single intrathecal injection of liposomal fasudil can maintain a therapeutic the drug concentration in the CSF due to the sustained-release property of liposomes, significantly decreasing intact size of acute ischemia and decreasing vasoconstriction of the basilar artery in cerebral vasospasm. This novel approach for the treatment of cerebral vasospasm and ischemia may have significant potential for use in the clinical setting.     

Encapsulation of an antivasospastic drug, fasudil, into liposomes, and in vitro stability of the fasudil-loaded liposomes.

The objectives of this work were to develop a liposomal fasudil, an antivasospastic drug, as a possible means to deliver the encapsulated drug to the brain, and to characterize the stability of the liposomal formulation in vitro. Transmembrane electrochemical gradients of H+ or ammonium sulfate were created, and their effect on the uptake of fasudil into preformed hydrogenated soy phosphatidylcholine/cholesterol (HSPC/CHOL) liposomes were examined. Fasudil was successfully loaded into preformed liposomes in response to sulfate ion (SO4(2-)) and, in part, by H+. Encapsulation levels approaching 100% could be achieved up to a drug to lipid ratio of 0.364 (mol/mol). A stability study of the fasudil-loaded liposomes was performed by storage at 4 degrees C in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffer (pH 7.4) and by incubation in human cerebrospinal fluid (CSF) at 37 degrees C. The formulations were stable with respect to drug retention as well as size alteration, for the period studied. A leakage study clearly showed the sustained release properties of the fasudil-loaded liposomes in human CSF. We recently reported that the intrathecal administration of liposomal fasudil significantly decreased ischemia, with no obvious adverse effect in a rat model [Neurol. Med. Chir. 41 (2001) 109]. Taken together, efficient encapsulation of fasudil into preformed liposomes, their long-term stability at 4 degrees C and the sustained release characteristics in CSF indicate that fasudil-loaded liposomes could be potential candidates for further clinical evaluation.     

Treatment of Focal Cerebral Ischemia with Liposomal Nerve Growth Factor

Liposomal nerve growth factor (NGF) was used for the treatment of focal cerebral ischemia in a rat model. Positive charge inducing agents of sphingosine (SP) and stearylamine (S) were formulated in the liposomal NGF. Dose-response of intraventricular injection of liposomal NGF showed significant reduction in infarct volume at the dose of 5 and 10 μ g/rat of NGF. The liposomal NGF formulated with SP or S demonstrated similar results in the reduction of total infarct volume in rats. When we increased the molar ratio of SP and S from 0.15 to 0.3, the infarct volume from rats showed a similar value as that of the control treated with NGF solution. Liposomal NGF was given prior to the development of ischemia. We found that NGF was effective in prevention of neuronal death. The NGF concentrations in brain for liposomal NGF were maintained in a level significantly higher than those for NGF solution. This was attributed to the positively charged liposomal NGF bound effectively in brain ventricle and caused longer retention time than free NGF for localization in brain. Therefore, the effect of liposomal NGF on reduction of infarct volume was significant. We assumed that the transportation of NGF might go through the cerebrospinal fluid pathway throughout the ventricular system and subarachnoid system to cerebral cortex to produce a therapeutic effect on ischemia.     

Treatment of focal cerebral ischemia with liposomal nerve growth factor

Liposomal nerve growth factor (NGF) was used for the treatment of focal cerebral ischemia in a rat model. Positive charge inducing agents of sphingosine (SP) and stearylamine (S) were formulated in the liposomal NGF. Dose-response of intraventricular injection of liposomal NGF showed significant reduction in infarct volume at the dose of 5 and 10 μ g/rat of NGF. The liposomal NGF formulated with SP or S demonstrated similar results in the reduction of total infarct volume in rats. When we increased the molar ratio of SP and S from 0.15 to 0.3, the infarct volume from rats showed a similar value as that of the control treated with NGF solution. Liposomal NGF was given prior to the development of ischemia. We found that NGF was effective in prevention of neuronal death. The NGF concentrations in brain for liposomal NGF were maintained in a level significantly higher than those for NGF solution. This was attributed to the positively charged liposomal NGF bound effectively in brain ventricle and caused longer retention time than free NGF for localization in brain. Therefore, the effect of liposomal NGF on reduction of infarct volume was significant. We assumed that the transportation of NGF might go through the cerebrospinal fluid pathway throughout the ventricular system and subarachnoid system to cerebral cortex to produce a therapeutic effect on ischemia.     

法舒地尔对蛛网膜下腔出血后大鼠脑皮质Bcl-2和Bax 蛋白表达的影响

摘要： 目的 探讨法舒地尔对蛛网膜下腔出血 （SAH） 后大鼠脑皮质 B 淋巴细胞瘤-2 （Bcl-2） 和 Bax 蛋白表达的影响。方法 30 只大鼠随机均分为假手术组、 SAH 组及 SAH+法舒地尔组。采用枕大池二次注血法制备大鼠 SAH 模型, 假手术组将自体血替换为生理盐水, SAH+法舒地尔组为第 2 次注血后 30 min 腹腔注射法舒地尔注射液, 剂量为 3 mg/kg。24 h 后比较各组大鼠术后的一般情况和神经行为学评分, TUNEL 染色法观察脑皮质细胞凋亡情况, 免疫组化染色和 Western blotting 检测 Bcl-2 和 Bax 蛋白表达情况。结果 与假手术组相比, SAH 组和 SAH+法舒地尔组大鼠出现明显的神经运动功能障碍体征; SAH 组神经行为学评分 （2.68±0.31） 低于假手术组 （5.00±0.00）, SAH+法舒地尔组 （3.27±0.35） 高于 SAH 组 （P＜0.05）。SAH 组和 SAH+法舒地尔组均可见明显的神经细胞凋亡, 且 SAH+法舒地尔组凋亡程度轻于 SAH 组。 SAH 组的 Bcl-2 表达水平低于假手术组, 而 Bax 表达水平高于假手术组 （P＜ 0.05）; SAH+法舒地尔组 Bcl-2 表达水平较 SAH 组升高, 而 Bax 表达量较 SAH 组降低 （P＜0.05）。结论 法舒地尔可明显改善 SAH 引起的神经功能损伤, 其作用机制可能与调节凋亡相关蛋白 Bcl-2和 Bax 的表达有关。     

Inhibitory effect of hydrocortisone on cerebral salt wasting after subarachnoid hemorrhage in rats

Cerebral salt wasting (CSW) frequently occurs concomitantly with subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, reduces total blood volume, aggravates cerebral vasospasm and causes cerebral ischemia after SAH. This study examined the inhibitory effect of hydrocortisone on CSW in rat SAH models. Hydrocortisone had an inhibitory effect on CSW because hydrocortisone functioned in a dose-dependent manner to inhibit the increase in sodium excretion and sodium/potassium ratio after SAH onset. We conclude that hydrocortisone is a useful drug for the treatment of CSW after SAH.     

Intrathecal chemotherapy with antineoplastic agents in children

Intrathecal chemotherapy with antineoplastic agents is mainly utilised in children with leukaemia and lymphoma, and in selected brain tumours. In these diseases, intrathecal use is restricted to methotrexate (MTX), cytosine arabinoside (Ara-C) and corticosteroids. A number of other agents are, at the present time, under evaluation. Intrathecal MTX administered sequentially with systemic high dose MTX infusion prolongs therapeutic cerebral spinal fluid (CSF) levels of the drug. Prolonged therapeutic CSF levels can also be achieved by giving repeated small intrathecal doses of MTX over an extended period in selected patients, with an implanted Ommaya reservoir. In the CSF, the metabolic inactivation of Ara-C is significantly lower than in plasma with a CSF clearance similar to the rate of CSF bulk flow. A slow-release formulation of Ara-C may be given intrathecally, resulting in a prolonged cytotoxic concentration in the CSF. CNS relapse and neurotoxicity in patients with acute lymphoblastic leukaemia, especially younger children, may be reduced by using age-related dosing of intrathecal MTX and Ara-C. Hydrocortisone is used in combination with MTX and Ara-C for so-called 'triple intrathecal chemotherapy' in the treatment of meningeal leukaemia. Intrathecal thiotepa does not appear to be advantageous over systemic administration in patients with brain and meningeal leukaemia. Monoclonal antibodies, reactive with tumour-associated antigens, can be used as delivery systems for chemotherapeutic agents and radionuclides. However, the development of this new approach is currently under evaluation in larger clinical studies. Neurological adverse effects may be expected with intrathecal chemotherapy and are increased by high dose systemic therapy, concomitant cranial radiotherapy or meningeal infiltration by neoplastic cells. Inadvertant intrathecal administration of antineoplastic agents that are indicated for systemic administration only, is dangerous and may result in a fatal outcome.     

Tumour-selective drug delivery via folate receptor-targeted liposomes

Tumour cell-targeted liposomal delivery has the potential to enhance the therapeutic efficacy and reduce the toxicity of anticancer agents. Folate receptor (FR) expression is frequently amplified among human malignancies. FR is, therefore, potentially useful as a tumour marker for targeted drug delivery. FR-mediated liposomal delivery has been shown to enhance the antitumour efficacy of doxorubicin both in vitro and in vivo, and to overcome P-glycoprotein-mediated multi-drug resistance. In addition, FR-targeted liposomes have shown utility as effective delivery vehicles of genes and antisense oligodeoxyribonucleotides to FR(+) tumour cells. Both solid tumours and leukaemias can potentially benefit from FR-targeted drug delivery. Multiple mechanisms might contribute to greater therapeutic efficacy for FR-targeted liposomes, such as FR-dependent cytotoxicity and antiangiogenic activity. Further investigation of this promising drug delivery strategy is clearly warranted.     

Targeted thermosensitive liposomes: an attractive novel approach for increased drug delivery to solid tumors

Introduction: Currently available chemotherapy is hampered by a lack in tumor specificity and resulting toxicity. Small and long-circulating liposomes can preferentially deliver chemotherapeutic drugs to tumors upon extravasation from tumor vasculature. Although clinically used liposomal formulations demonstrated significant reduction in toxicity, enhancement of therapeutic activity has not fully met expectations.

Areas covered: Low drug bioavailability from liposomal formulations and limited tumor accumulation remain major challenges to further improve therapeutic activity of liposomal chemotherapy. The aim of this review is to highlight strategies addressing these challenges. A first strategy uses hyperthermia and thermosensitive liposomes to improve tumor accumulation and trigger liposomal drug bioavailability. Image-guidance can aid online monitoring of heat and drug delivery and further personalize the treatment. A second strategy involves tumor-specific targeting to enhance drug delivery specificity and drug internalization. In addition, we review the potential of combinations of the two in one targeted thermosensitive-triggered drug delivery system.

Expert opinion: Heat-triggered drug delivery using thermosensitive liposomes as well as the use of tumor vasculature or tumor cell-targeted liposomes are both promising strategies to improve liposomal chemotherapy. Preclinical evidence has been encouraging and both strategies are currently undergoing clinical evaluation. A combination of both strategies rendering targeted thermosensitive liposomes (TTSL) may appear as a new and attractive approach promoting tumor drug delivery.     

Antivasospastic effects of hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage

We investigated the anti-vasospastic potential of fasudil's active metabolite, hydroxyfasudil, a Rho-kinase inhibitor, after subarachnoid hemorrhage (SAH) and also its effect on hemorheological abnormalities following cerebral ischemia. Chronic cerebral vasospasm was produced using a two-hemorrhage canine model. On day 7, angiographic vasospasm was observed in all animals, and intravenous administration of hydroxyfasudil (3 mg·kg(-1)·30 min(-1)) significantly reversed the vasospasm (predose diameter of the basilar artery, 57.9% ± 2.0% of the baseline before the injection of blood; postdose diameter, 64.5% ± 1.9%). The viscosity of whole blood was significantly increased 24 h after 1 h middle cerebral artery occlusion in rats. Hydroxyfasudil (3 and 10 mg/kg, i.p.) significantly decreased blood viscosity. The specificity of hydroxyfasudil was examined against a panel of 17 protein kinases using ELISA analysis. Hydroxyfasudil inhibited Rho-kinase α and β at a concentration of 10 μM by 97.6% and 97.7%, respectively. No other protein kinase was inhibited with 10 μM hydroxyfasudil by over 40%. The present results indicate hydroxyfasudil is a selective inhibitor of Rho-kinase. The results also suggest that hydroxyfasudil contributes to the potency of fasudil to prevent cerebral vasospasm and hyperviscosity and suggest the potential utility of hydroxyfasudil as a therapeutic agent for patients with SAH.     

Liposomal ophthalmic drug delivery system I. Triamcinolone acetonide

The potential of liposomes as an ophthalmic drug delivery system was investigated in the rabbit. Triamcinolone acetonide as a model compound for lipophilic drugs was encapsulated into large multilamellar vesicles and instilled into the eye. A suspension form served as a control preparation. Drug distribution was determined at various time intervals. Compared to the suspension, the liposomal form produced significantly higher drug levels in the ocular tissues up to 5 h after the drug administration. The results suggest that liposomal encapsulation of the drug may be a superior drug delivery system for ocular therapy.     

A novel method to calculate the extent and amount of drug transported into CSF after intranasal administration

The aim of this paper is to establish a novel method to calculate the extent and amount of drug transported to brain after administration. The cerebrospinal fluid (CSF) was chosen as the target region. The intranasal administration of meptazinol hydrochloride (MEP) was chosen as the model administration and intravenous administration was selected as reference. According to formula transform, the extent was measured by the equation of X(A)CSF, infinity/X0 = Cl(CSF) AUC(0-->infinity)CSF/X0 and the drug amount was calculated by multiplying the dose with the extent. The drug clearance in CSF (Cl(CSF)) was calculated by a method, in which a certain volume of MEP solution was injected directly into rat cistern magna and then clearance was assessed as the reciprocal of the zeroth moment of a CSF level-time curve normalized for dose. In order to testify the accurateness of the method, 14C-sucrose was chosen as reference because of its impermeable characteristic across blood-brain barrier (BBB). It was found out that the MEP concentrations in plasma and CSF after intranasal administration did not show significant difference with those after intravenous administration. However, the extent and amount of MEP transported to CSF was significantly lower compared with those to plasma after these two administrations. In conclusion, the method can be applied to measure the extent and amount of drug transported to CSF, which would be useful to evaluate brain-targeting drug delivery.     

注射用缓控释给药系统的研究进展

注射用缓控释给药系统因可延长药物的作用时间、减少注射次数、改善患者的顺应性而成为当前药剂研究的一大热点。现根据给药途径的不同，介绍了近年来注射用缓控释给药系统在静脉、肌内、皮下、鞘内及靶部位给药方面的研究状况，涉及微球、脂质体、纳米粒及原位凝胶等剂型，评价了该类制剂开发中存在的问题和潜在的医疗风险。     

Recent advances in tumor vasculature targeting using liposomal drug delivery systems

Tumor vessels possess unique physiological features that might be exploited for improved drug delivery. The targeting of liposomal anticancer drugs to tumor vasculature is increasingly recognized as an effective strategy to obtain superior therapeutic efficacy with limited host toxicity compared with conventional treatments. This review introduces recent advances in the field of liposomal targeting of tumor vasculature, along with new approaches that can be used in the design and optimization of liposomal delivery systems. In addition, cationic liposome is focused on as a promising carrier for achieving efficient vascular targeting. The clinical implications are discussed of several approaches using a single liposomal anticancer drug formulation: dual targeting, vascular targeting (targeting tumor endothelial cells) and tumor targeting (targeting tumor cells).     

颈淋巴阻断加重SAH脑脊液对PC12细胞的损伤

目的探讨脑淋巴引流途径在蛛网膜下腔出血(SAH)后神经元损伤中的作用。方法建立兔SAH及脑淋巴引流阻滞(CLB)模型,于SAH模型建立后5d抽取脑脊液加入培养的PC12细胞中,随机将PC12细胞分为空白对照组、正常对照组(正常脑脊液)、SAH脑脊液组,SAH+CLB脑脊液组,分别于培养0.5、1、2、4h后,用比色法测定乳酸脱氢酶(LDH)释放率,采用免疫荧光染色法激光共聚焦显微镜观察PC12细胞细胞骨架。结果与正常对照组比较,SAH脑脊液组PC12细胞LDH释放率增加;SAH+CLB脑脊液组LDH释放率明显高于SAH脑脊液组。正常脑脊液不引起PC12细胞细胞骨架改变,SAH脑脊液组及SAH+CLB脑脊液组PC12细胞胞质着色较弱,突起变短甚至回缩,胞核呈现凋亡特征,上述表现以SAH+CLB组更为明显。结论脑淋巴引流阻滞加重SAH脑脊液对PC12细胞的损伤,提示通过改善脑淋巴引流途径可减轻SAH继发性脑损伤。     

An insight to osmotic drug delivery

In a typical therapeutic regimen the drug dose and the dosing interval are optimized to maintain drug concentration within the therapeutic window, thus ensuring efficacy while minimizing toxic effects. For many decades treatment of acute disease or a chronic illness has been mostly accomplished by delivery of drugs to patients using various pharmaceutical dosage forms. The immediate release conventional dosage form does not provide the proper plasma concentration of drug for prolonged period. This results in the development of various controlled drug delivery system. Among which the osmotic drug delivery systems (ODDS) are gaining importance as these systems deliver the drug at specific time as per the path physiological need of the disease, resulting in improved patient therapeutic efficacy and compliance. They work on the principle of osmotic pressure for controlling the delivery of the drug. Osmotic drug delivery systems with their versatility and their highly predictable drug release rates offer various biomedical advantages when given parenterally like reduced dose, targeting of site, avoiding gastrointestinal stability, hepatic bypass of drug molecule and follows zero order kinetics. Osmosis is an aristocratic phenomenon that seizes the attention for its exploitation in zero-order drug delivery systems. The release of the drug is independent of pH and physiological factors of the GIT to a large extent. Optimizing semi-permeable membrane characteristics and osmotic agent can modulate delivery of drug from the system. This review highlights the theoretical concept of drug delivery, history, types of oral osmotic drug delivery systems, factors affecting the drug delivery system, advantages and disadvantages of this delivery system, theoretical aspects, applications, and the marketed status.     

Enhanced Therapeutic Effect of Cytidine-5′ -Diphosphate Choline when Associated with GM1 Containing Small Liposomes as Demonstrated in a Rat Ischemia Model

Purpose. Cytidine-5-diphosphate choline (CDPc) was encapsulated in long-circulating unilamellar vesicles (SUVs) to improve the drug's biological effectiveness. Methods. SUVs made up of diaplmitoylphosphatidylcholine/diaplmitoylphosphatidylserine/ cholesterol (7:4:7 molar ratio) and 8 mol % of ganglioside G_Ml were prepared by extrusion through polycarbonate filters (mean diameter 50 nm). The formulation effectiveness was evaluated by an in vivo model of cerebral ischemia on Wistar rats. Results. The enhanced delivery of CDPc into the brain improved the therapeutic effectiveness of the drug. CDPc-loaded SUVs improved the survival rate of ischemized and reperfused Wistar rats (320-350 g) by -66% compared with the free drug. Liposome formulation was also able to effectively protect the brain against peroxidative damage caused by post-ischemic reperfusion. SUVs lowered the conjugated diene levels of the cerebral cortex. The liposomal delivery system did not alter the distribution patterns in the various cerebral lipid fractions of the drug, radiolabeled with ¹⁴C-CDPc. Conclusions. CDPc-loaded SUVs were able to protect the brain against damage induced by ischemia. A possible clinical application is envisaged.     

2种盐酸法舒地尔注射液对大鼠实验性脑缺血保护作用的研究

目的:比较2种盐酸法舒地尔注射液(商品名分别为依立卢、川威)对大鼠实验性脑缺血的保护作用。方法:SD大鼠60只随机分为伪手术组、模型组、依立卢和川威高、低剂量组,分别给予相应药物后建立局部脑缺血模型,观察盐酸法舒地尔注射液对脑缺血大鼠神经功能、脑梗死面积和脑组织病理形态学的影响。结果:与模型组比较,在相同剂量下,2种制剂可相同程度地降低实验性脑缺血大鼠的神经功能评分、脑梗死面积及减轻脑组织病理形态改变(P<0.05或P<0.01);2种制剂之间比较差异无显著性(P>0.05)。结论:2种盐酸法舒地尔注射液对大鼠实验性脑缺血的保护作用相当。     

Bispecific MAb aided liposomal drug delivery

We have developed a new method for specifically delivering liposomal model drugs to tumor cells. Bispecific monoclonal antibodies (bsMAb) (174H.64 x anti-biotin) which can bind tumor-specific antigen and biotin were developed and characterized. Biotinylated stealth liposome loaded with model drug 99mTc-DTPA can bind to the biotin-binding arm of bsMAb. This targeted liposomal delivery strategy was tested in mouse KLN-205 squamous carcinoma model. bsMAbs were administered 24h in advance into tumor allograft bearing mice, which allow them to bind to tumor cells through the anti-tumor binding arm. After clearance of circulating bsMAb, biotinylated stealth liposomes were introduced to specifically bind to the tumor sites where bsMAb localized earlier. The results show that pretargeted bsMAb can enhance liposomal drug targeting by four times, 3.61% dose/g vs. 0.89% dose/g. This bsMAb/liposome strategy show the broad possibilities of selective delivery of cytotoxic drugs or genes to the specific targets.     

国内盐酸法舒地尔治疗蛛网膜下腔出血后脑血管痉挛的有效性和安全性系统评价

目的:评价法舒地尔治疗蛛网膜下腔出血后脑血管痉挛的有效性和安全性。方法:计算机检索中国期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、万方数字化期刊全文数据库、中文科技期刊全文数据库(VIP),纳入法舒地尔治疗蛛网膜下腔出血后脑血管痉挛的随机对照试验(RCT)文献;手工检索其他相关文献。对纳入研究进行方法学质量评价,并采用RevMan5.0软件进行Meta分析。结果:共纳入19个RCT。Meta分析结果显示,对于总有效率和脑血管痉挛发生率,法舒地尔30mg,q8h单用与尼莫地平24mg.d-1单用比较差异无统计学意义;总有效率的RR及其95%CI分别为1.06(0.98,1.16),脑血管痉挛发生率的RR及其95%CI分别为1.17(0.53,2.59);法舒地尔30mg,q8h单用与尼莫地平10mg.d-1单用比较差异有统计学意义;总有效率的RR及其95%CI分别为1.30(1.11,1.52)。结论:目前国内证据表明,应用法舒地尔治疗蛛网膜下腔出血后脑血管痉挛能增加总有效率和减少脑血管痉挛发生率。法舒地尔和尼莫地平的有效性差异受使用剂量和给药途径影响。