神经毒素-I纳米粒鼻腔给药的脑内递送研究

许多分子质量较大的药物如肽类、蛋白质、基因和抗生素等很难通过血脑屏障(blood brain barrier,BBB).进入脑内发挥作用[1,2].常用于提高脑内药物浓度的方法有局部脑室植入、利用高渗溶液或血管活性物质增加BBB开放等[3],但这些方法具有一定损伤性,仅适用于短期治疗药物.     

Brain targeted nanoparticulate drug delivery system of rasagiline via intranasal route

The aim of the present study was to prepare and evaluate a rasagiline-loaded chitosan glutamate nanoparticles (RAS-CG-NPs) by ionic gelation of CG with tripolyphosphate anions (TPP). RAS-loaded CG-NPs were characterized for particle size, size distribution, encapsulation efficiency and in vitro drug release. The mean particles size, polydispersity index (PDI) and encapsulation efficiency was found to be 151.1?±?10.31, 0.380?±?0.01 and 96.43?±?4.23, respectively. Biodistribution of RAS formulations in the brain and blood of mice following intranasal (i.n.) and intravenous (i.v.) administration was performed using HPLC analytical method. The drug concentrations in brain following the i.n. of CG-NPs were found to be significantly higher at all the time points compared to both drug (i.n.) and drug CG-NPs (i.v.). The Cmax (999.25?ng/ml) and AUC (2086.60?ng?h/ml) of formulation CG-NPs (i.n) were found to be significantly higher than CG-NPs (i.v.) and RAS solution (i.n.). The direct transport percentage (DTP%) values of RAS-loaded CG-NPs (i.n.) as compared to drug solution (i.n.) increased from 66.27?±?1.8 to 69.27?±?2.1%. The results showed significant enhancement of bioavailability in brain, after administration of the RAS-loaded CG-NPs which could be a substantial achievement of direct nose to brain targeting in Parkinson's disease therapy.     

Cationic liposomes for gene delivery: novel cationic lipids and enhancement by proteins and peptides

Cationic liposome-DNA complexes, also called "lipoplexes", constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations to emulate in vivo conditions.     

Bioavailability of intranasal neostigmine: comparison with intravenous route

The pharmacokinetics of intranasal (i.n.) neostigmine was compared with the intravenous (i.v.) route in 6 healthy volunteers with a mean age of 25.5 years and a mean weight of 65.8 kg in a crossover design. The doses used were 0.5 mg i.v. and 21.6 mg i.n. Neostigmine was determined by HPLC. The plasmatic profiles of the two routes were similar. An early peak of plasmatic concentration after i.n. administration was observed. The absolute bioequivalence of the i.n. route was ten-fold greater than the oral one.     

Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex

Abstract

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.     

Anchoring cationic amphiphiles for nucleotide delivery: significance of DNA release from cationic liposomes for transfection

We have designed and synthesized lithocholic acid-based cationic amphiphile molecules as components of cationic liposomes for gene transfection (lipofection). To study the relationship between the molecular structures of those amphiphilic molecules, particularly the extended hydrophobic appendant (anchor) at the 3-hydroxyl group, and transfection efficiency, we synthesized several lithocholic and isolithocholic acid derivatives, and examined their transfection efficiency. We also compared the physico-chemical properties of cationic liposomes prepared from these derivatives. We found that isolithocholic acid derivatives exhibit higher transfection efficiency than the corresponding lithocholic acid derivatives. This result indicates that the orientation and extension of hydrophobic regions influence the gene transfection process. Isolithocholic acid derivatives showed a high ability to encapsulate DNA in a compact liposome-DNA complex and to protect it from enzymatic degradation. Isolithocholic acid derivatives also facilitated the release of DNA from the liposome-DNA complex, which is a crucial step for DNA entry into the nucleus. Our results show that the transfection efficiency is directly influenced by the ability of the liposome complex to release DNA, rather than by the DNA-encapsulating ability. Molecular modeling revealed that isolithocholic acid derivatives take relatively extended conformations, while the lithocholic acid derivatives take folded structures. Thus, the efficiency of release of DNA from cationic liposomes in the cytoplasm, which contributes to high transfection efficiency, appears to be dependent upon the molecular shape of the cationic amphiphiles.     

Comparative cellular pharmacokinetics and pharmacodynamics of siRNA delivery by SPANosomes and by cationic liposomes

Mechanistic understanding of intracellular trafficking is important for the development of small interfering RNA (siRNA) delivery vehicles. Here, we describe a novel methodology to quantitatively analyze nanocarrier-mediated disposition of siRNA. Cellular uptake and cytoplasmic release of siRNA over time were quantified by measuring the fluorescence intensities of fluorescently-labeled siRNAs and molecular beacons using flow cytometry. This method was used to investigate the cellular pharmacokinetics (PK) of siRNA delivery by SPANosomes (SP) and by cationic liposomes (CL). The results showed that the superior pharmacodynamic (PD) response of SP was because it enhanced transport of siRNA into the cytoplasm compared to the CL. The divergent cellular pharmacokinetic profiles of the two formulations were associated with different cellular entry pathways. These findings can facilitate the rational design of more efficient siRNA delivery vehicles in the future.From the Clinical EditorIn this paper the authors describe a novel methodology to quantitatively analyze nanocarrier-mediated disposition of small interfering RNA, comparing SPANosomes with cationic liposomes as delivery systems with different entry pathways.     

Nasal administration of albuterol: an alternative route of delivery

The use of metered-dose inhalers for the delivery of albuterol, a beta2-selective adrenergic agonist, is associated with drawbacks, especially in children and the elderly. This investigation was designed to assess the effectiveness of albuterol delivered intranasally and to compare this delivery route with intratracheal and intravenous delivery. Three parameters of pulmonary function (peak maximal expiratory flow, maximal expiratory flow at 50% vital capacity, and total lung capacity) in anaesthetized, artificially ventilated guinea pigs were used to determine the degree of protection produced by albuterol against bronchoconstrictor responses provoked by acetylcholine. The heart rate was also measured. Although intranasal albuterol induced a slower protective action during the very initial phase of absorption, the drug was shown to be equally effective when administered either intranasally or intratracheally. In contrast, despite a significant effect initially in the case of intravenous albuterol, its ability to influence pulmonary function faded rather rapidly. No statistically significant differences in heart rate could be detected among the different treatment groups. In conclusion, intranasal albuterol may offer an alternative to metered-dose inhalers for the treatment of acute bronchospasm and for prevention of exercise-induced asthma, especially for children and the elderly.     

鼻腔给药促进盐酸美普他酚在大鼠体内的吸收及脑转运

目的考察大鼠鼻腔给药后血和脑脊液中盐酸美普他酚 (MEP)的浓度，并与口服比较。方法采用连续采集法收集脑脊液样品，用HPLC-荧光检测器测定各生物样品中MEP的浓度。结果鼻腔给药后药物迅速吸收入血，并在血和脑脊液中达到高浓度，而MEP口服后体内药浓很低。鼻腔给药后血和CSF的AUC值分别为口服的7.375和15.6倍。结论MEP鼻腔给药具有起效快、生物利用度高的特点，有望成为口服的替代途径。     

Brain targeted intranasal delivery of tramadol: comparative study of microemulsion and nanoemulsion

Abstract

Objective: The present investigation was aimed to develop and compare microemulsion and nanoemulsion for brain targeted intranasal delivery of tramadol to achieve maximum therapeutic efficacy in treatment of episodic and emergency pain.

Methods: Tramadol microemulsion (TME) and tramadol nanoemulsion (TNE) were developed and evaluated for physical properties. Ex vivo diffusion and nasal toxicity of TME and TNE were assessed by using sheep nasal mucosa. Biodistribution, pharmacokinetic and pharmacodynamic studies in mice were also performed.

Results: Globule sizes of TME and TNE were 16.69?±?3.21 and 136.3?±?4.3?nm, respectively. TNE was found be safe with respect to multiple dosing via nasal route. Both TME and TNE were stable during accelerated stability studies. AUC_(0→24) in mice brain for TME and TNE was significantly higher as compared with tramadol solution. TME and TNE displayed significantly higher antinociceptive effect for a period of 16?h as compared with tramadol solution.

Discussion: TME and TNE were delivered to brain, circumventing BBB in brisk manner, establishing immediately the minimum effective concentration required for therapeutic response. Significant enhancement in antinociceptive effect was observed after intranasal delivery of TME and TNE.

Conclusion: Intranasal administration of TME and TNE would be effective in management of episodic and emergency pain treatment.     

A new vaginal delivery system of amphotericin B: a dispersion of cationic liposomes in a thermosensitive gel

Amphotericin B (AmB) is used in the treatment of fungal infections; however, its clinical use is limited by its toxic side effects. In this study, AmB-loaded cationic liposome gels were formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE:DOTAP:CH?=?4:5:1 in thermosensitive gel composed of poloxamer 407 (P407) and poloxamer 188 (P188). To enhance the solubility of AmB, 6 mol% of distearoyl phosphatidyl ethanolamine–polyethylene glycol was added prior to encapsulation of the drug into liposomes. Scanning electron microscopy was used to observe the AmB encapsulated cationic liposome gels. In vitro release, stability and cytotoxicity of AmB in cationic liposome gels were evaluated. The particle size and zeta potential of AmB-loaded liposomes were in the range of 400–500?nm and 40–60 mV, respectively. The thermosensitive gel at the ratio of P407:P188?=?15:15 (w/w) gelled at 37°C, approximating body temperature. Encapsulation efficiency of AmB was ～50–60%, which was influenced by the ratio of AmB to lipid. Moreover, AmB-loaded cationic liposome gels were more stable and less toxic than free AmB. From these results, cationic liposome gel formulations may be useful for vaginal delivery of AmB.     

Topical application versus intranasal instillation: a qualitative comparison of the effect of the route of sensitization on trimellitic anhydride-induced allergic rhinitis in A/J mice.

Allergic airway diseases caused by low-molecular weight chemicals including trimellitic anhydride (TMA) have been linked to Th2 cytokines and are characterized by mucus hypersecretion and infiltration of lymphocytes and eosinophils into the airways. The most common route of human exposure to chemical respiratory allergens is inhalation. Most murine models, however, use topical exposure to sensitize mice. The present study tests the hypothesis that topical sensitization on the ears of mice with TMA will induce a qualitatively similar immunologic and pathologic response in the nasal airways after intranasal challenge to that induced after intranasal sensitization and challenge. A/J mice were sensitized topically or by intranasal instillation followed by intranasal challenge with TMA in an ethyl acetate/olive oil vehicle. Intranasal challenge with TMA in mice that were either topically or intranasally sensitized with TMA caused a marked allergic rhinitis, of similar severity, characterized by an influx of eosinophils and lymphocytes. Both the topical and intranasal routes of sensitization also caused significant increases in total serum IgE after intranasal challenge with TMA. In addition, both the topical and intranasal routes of sensitization caused significant increases in the mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13. Collectively, these findings suggest that topical application is effective in sensitizing mice to TMA and induces a nasal airway lesion and associated immune response after intranasal challenge, which is qualitatively similar to that induced by intranasal sensitization and challenge. Skin exposure may be a potential route of sensitization of the respiratory tract to chemical allergens.     

DC-Chol/DOPE cationic liposomes: A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.     

Systemic delivery systems of angiogenic gene by novel bubble liposomes containing cationic lipid and ultrasound exposure

Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice, and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased in the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.     

Brain targeting by intranasal drug delivery (INDD): a combined effect of trans-neural and para-neuronal pathway

The effectiveness of intranasal drug delivery for brain targeting has emerged as a hope of remedy for various CNS disorders. The nose to brain absorption of therapeutic molecules claims two effective pathways, which include trans-neuronal for immediate action and para-neuronal for delayed action. To evaluate the contribution of both the pathways in absorption of therapeutic molecules and nanocarriers, lidocaine, a nerve-blocking agent, was used to impair the action potential of olfactory nerve. An anti-Parkinson drug ropinirole was covalently complexes with ^99mTc in presence of SnCl₂ using in-house developed reduction technology. The radiolabeled formulations were administered intranasally in lidocaine challenged rabbit and rat. The qualitative and quantitative outcomes of neural and non-neural pathways were estimated using gamma scintigraphy and UHPLC-MS/MS, respectively. The results showed a significant (p?≤?0.005) increase in radioactivity counts and drug concentration in the brain of rabbit and rat compared to the animal groups challenged with lidocaine. This concludes the significant contribution (p?≤?0.005) of trans-neuronal and para-neuronal pathway in nose to brain drug delivery. Therefore, results proved that it is an art of a formulator scientist to make the drug carriers to exploit the choice of absorption pathway for their instant and extent of action.     

Pharmacodynamics of acute intranasal administration of verapamil: comparison with i.v. and oral administration

The present study was conducted in order to examine the intranasal administration of verapamil and compare this route to oral and intravenous administration in a 3 way crossover protocol in five dogs. Unanesthetized, adult mongrel dogs were given verapamil intravenously (0.5 mg/kg), orally (2.5 mg/kg) and intranasally (0.75 mg/kg) with at least a 3-4 day washout period between each administration. Blood samples were collected over a 10 hour period and analyzed for verapamil using HPLC with fluorescence detection. A lead II ECG was monitored to determine the effects of verapamil on heart rate and P-R interval. Following intravenous administration, verapamil was distributed according to a two compartment model. Bioavailability (corrected for dose and elimination rate constant) following intranasal administration (36% +/- 7%) was approximately 3 fold that after oral administration (13% +/- 3%). Absorption from the nasal cavity appeared instantaneous compared to an absorption half-life of 50 +/- 6 min after oral administration. All three routes of administration resulted in significant increases in heart rate and increases in the P-R interval. Maximal P-R interval prolongation occurred after peak plasma concentrations of verapamil. The results of this study suggest that the intranasal route is a viable alternative route of administration for verapamil.     

Chlorimipramine: A double-blind comparison of intravenous versus oral administration in depressed patients

A double-blind study was carried out in 30 patients to compare the results of chlorimipramine given orally and intravenously.The speed of onset of action and the overall improvement were excellent for both treatment modalities. No significant differences were found favoring either way of administration. As a consequence, our results do not support previous reports that suggest advantages and rationale for the intravenous usage of chlorimipramine.Also called Anafranil^® abroad and Clomipramine in the U.S.A.This investigation was supported in part by research grant MYP-5106 from the National Institute of Mental Health, U.S. Public Health Service.We thank Ms. Carol Umland, R. N. and Ms. Connie Ball, R. N. for their cooperation and dedication which made possible the successful completion of this study.