On The Rate and Extent of Drug Delivery to the Brain

To define and differentiate relevant aspects of blood-brain barrier transport and distribution in order to aid research methodology in brain drug delivery. Pharmacokinetic parameters relative to the rate and extent of brain drug delivery are described and illustrated with relevant data, with special emphasis on the unbound, pharmacologically active drug molecule. Drug delivery to the brain can be comprehensively described using three parameters: Kp,uu (concentration ratio of unbound drug in brain to blood), CLin (permeability clearance into the brain), and Vu,brain (intra-brain distribution). The permeability of the blood-brain barrier is less relevant to drug action within the CNS than the extent of drug delivery, as most drugs are administered on a continuous (repeated) basis. Kp,uu can differ between CNS-active drugs by a factor of up to 150-fold. This range is much smaller than that for log BB ratios (Kp), which can differ by up to at least 2,000-fold, or for BBB permeabilities, which span an even larger range (up to at least 20,000-fold difference). Methods that measure the three parameters Kp,uu, CLin, and Vu,brain can give clinically valuable estimates of brain drug delivery in early drug discovery programmes.     

Blood–Brain Barrier Transport of Therapeutics via Receptor-Mediation

Drug delivery to the brain is hindered by the presence of the blood-brain barrier (BBB). Although the BBB restricts the passage of many substances, it is actually selectively permeable to nutrients necessary for healthy brain function. To accomplish the task of nutrient transport, the brain endothelium is endowed with a diverse collection of molecular transport systems. One such class of transport system, known as a receptor-mediated transcytosis (RMT), employs the vesicular trafficking machinery of the endothelium to transport substrates between blood and brain. If appropriately targeted, RMT systems can also be used to shuttle a wide range of therapeutics into the brain in a noninvasive manner. Over the last decade, there have been significant developments in the arena of RMT-based brain drug transport, and this review will focus on those approaches that have been validated in an in vivo setting.     

A Redox-Based System that Enhances Delivery of Estradiol to the Brain: Pharmacokinetic Evaluation in the Dog

The pharmacokinetics of a dihydropyridine–pyridinium salt-type chemical delivery system (CDS) for brain-targeted delivery of estradiol (E₂) were examined in dogs. Parameters evaluated in vitro included stability in buffers and biological fluids and plasma protein binding. In vivo studies examined drug and metabolite concentrations in plasma, urine, and cerebrospinal fluid as well as in selected brain regions. The administered lipophilic E₂-CDS disappeared very quickly from plasma and was not detected in urine. The oxidized drug form, E₂-Q⁺, was excreted unchanged or as a conjugate in the urine for as long as 2 weeks. Plasma levels were below assay detection limits at later times. Pharmacokinetic analysis of urine E₂-Q⁺ levels allowed estimation of a half-life of 2.2 days. Amounts of E₂-Q⁺ excreted into the urine were proportional to the dose but averaged only 13.9% of the dose, indicating that other routes of excretion must be considered. CSF levels were below the limit of detection for both E₂-CDS and E₂-Q⁺, however, brain tissue concentrations of E₂-Q⁺ were similar in several brain regions of individual animals examined 1 or 3 days after drug dosing.     

Drug Targeting to the Brain

The goal of brain drug targeting technology is the delivery of therapeutics across the blood–brain barrier (BBB), including the human BBB. This is accomplished by re-engineering pharmaceuticals to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Certain endogenous peptides, such as insulin or transferrin, undergo receptor-mediated transport (RMT) across the BBB in vivo. In addition, peptidomimetic monoclonal antibodies (MAb) may also cross the BBB via RMT on the endogenous transporters. The MAb may be used as a molecular Trojan horse to ferry across the BBB large molecule pharmaceuticals, including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene medicines. Fusion proteins of the molecular Trojan horse and either neurotrophins or single chain Fv antibodies have been genetically engineered. The fusion proteins retain bi-functional properties, and both bind the BBB receptor, to trigger transport into brain, and bind the cognate receptor inside brain to induce the pharmacologic effect. Trojan horse liposome technology enables the brain targeting of non-viral plasmid DNA. Molecular Trojan horses may be formulated with fusion protein technology, avidin–biotin technology, or Trojan horse liposomes to target to brain virtually any large molecule pharmaceutical.     

Brain Uptake of Nonsteroidal Anti-Inflammatory Drugs: Ibuprofen, Flurbiprofen, and Indomethacin

Purpose To determine the roles of blood–brain barrier (BBB) transport and plasma protein binding in brain uptake of nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, flurbiprofen, and indomethacin. Methods Brain uptake was measured using in situ rat brain perfusion technique. Results [¹⁴C]Ibuprofen, [³H]flurbiprofen, and [¹⁴C]indomethacin were rapidly taken up into the brain in the absence of plasma protein with BBB permeability–surface area products (PS_u) to free drug of (2.63 ± 0.11) × 10⁻², (1.60 ± 0.08) × 10⁻², and (0.64 ± 0.05) × 10⁻² mL s⁻¹ g⁻¹ (n = 9–11), respectively. BBB [¹⁴C]ibuprofen uptake was inhibited by unlabeled ibuprofen (K_m = 0.85 ± 0.02 mM, V_max = 13.5 ± 0.4 nmol s⁻¹ g⁻¹) and indomethacin, but not by pyruvate, probenecid, digoxin, or valproate. No evidence was found for saturable BBB uptake of [³H]flurbiprofen or [¹⁴C]indomethacin. Initial brain uptake for all three NSAIDs was reduced by the addition of albumin to the perfusion buffer. The magnitude of the brain uptake reduction correlated with the NSAID free fraction in the perfusate. Conclusions Free ibuprofen, flurbiprofen, and indomethacin rapidly cross the BBB, with ibuprofen exhibiting a saturable component of transport. Plasma protein binding limits brain NSAID uptake by reducing the free fraction of NSAID in the circulation.     

胰岛素脂质体的制备及在电致孔下的经皮渗透

采用反相蒸发法制备了平均粒径122.7nm的胰岛素脂质体,并考察了电致孔经皮转运情况.结果表明,在电致孔条件下,胰岛素脂质体2h累积渗透量是载药脂质体被动扩散的1倍;是原药及其与空白脂质体混合物的2～3倍.     

脑靶向给药系统研究进展

血-脑屏障阻碍药物进入脑组织,不利于中枢神经系统疾病的治疗.本文介绍了近年来脑靶向给药系统的研究进展,包括通过受体(如载脂蛋白受体、转铁蛋白受体等)介导的主动靶向系统、被动靶向系统(如纳米粒、碳纳米管等)及其他靶向系统(如磁性微粒、阳离子制剂等).     

Blood–brain Barrier Transport of Non-viral Gene and RNAi Therapeutics

The development of gene- and RNA interference (RNAi)-based therapeutics represents a challenge for the drug delivery field. The global brain distribution of DNA genes, as well as the targeting of specific regions of the brain, is even more complicated because conventional delivery systems, i.e. viruses, have poor diffusion in brain when injected in situ and do not cross the blood–brain barrier (BBB), which is only permeable to lipophilic molecules of less than 400 Da. Recent advances in the “Trojan Horse Liposome” (THL) technology applied to the transvascular non-viral gene therapy of brain disorders presents a promising solution to the DNA/RNAi delivery obstacle. The THL is comprised of immunoliposomes carrying either a gene for protein replacement or small hairpin RNA (shRNA) expression plasmids for RNAi effect, respectively. The THL is engineered with known lipids containing polyethyleneglycol (PEG), which stabilizes its structure in vivo in circulation. The tissue target specificity of THL is given by conjugation of ∼1% of the PEG residues to peptidomimetic monoclonal antibodies (MAb) that bind to specific endogenous receptors (i.e. insulin and transferrin receptors) located on both the BBB and the brain cellular membranes, respectively. These MAbs mediate (a) receptor-mediated transcytosis of the THL complex through the BBB, (b) endocytosis into brain cells and (c) transport to the brain cell nuclear compartment. The present review presents an overview of the THL technology and its current application to gene therapy and RNAi, including experimental models of Parkinson’s disease and brain tumors.     

Strategies for Intranasal Delivery of Therapeutics for the Prevention and Treatment of NeuroAIDS

Intranasal drug administration is a noninvasive method of bypassing the blood–brain barrier (BBB) to deliver neurotrophins and other therapeutic agents to the brain and spinal cord. This method allows drugs that do not cross the BBB to be delivered to the central nervous system (CNS) and eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways. Intranasal delivery occurs by an extracellular route and does not require that drugs bind to any receptor or undergo axonal transport. Intranasal delivery also targets the nasal associated lymphatic tissues (NALT) and deep cervical lymph nodes. In addition, intranasally administered therapeutics are observed at high levels in the blood vessel walls and perivascular spaces of the cerebrovasculature. Using this intranasal method in animal models, researchers have successfully reduced stroke damage, reversed Alzheimer’s neurodegeneration, reduced anxiety, improved memory, stimulated cerebral neurogenesis, and treated brain tumors. In humans, intranasal insulin has been shown to improve memory in normal adults and patients with Alzheimer’s disease. Intranasal delivery strategies that can be employed to treat and prevent NeuroAIDS include: (1) target antiretrovirals to reach HIV that harbors in the CNS; (2) target therapeutics to protect neurons in the CNS; (3) modulate the neuroimmune function of moncyte/macrophages by targeting the lymphatics, perivascular spaces of the cerebrovasculature, and the CNS; and (4) improve memory and cognitive function by targeting therapeutics to the CNS. Presented at an NIMH workshop “HIV Preclinical–Clinical Therapeutics Research Meeting,” May 5–16, 2006.     

Dose and Time-Course Evaluation of a Redox-Based Estradiol-Chemical Delivery System for the Brain. I. Tissue Distribution

Brain-enhanced delivery and sustained release of estradiol (E₂) may be potentially useful in the treatments of vasomotor hot flushes and prostatic adenocarcinoma and for fertility regulation. Therefore, we have evaluated a redox-based estradiol-chemical delivery system (E₂-CDS) for the brain. The mechanism of this drug delivery is based on an interconvertible dihydropyridinepyridinium salt redox reaction. In this study, we investigated the dose- and time-dependent effects of E₂-CDS on the tissue distribution of E₂-Q⁺ and E₂, the inactive (intermediate) and active metabolites, respectively, of the E₂-CDS. Ovariectomized rats received a single iv injection of E₂-CDS at 0.01, 0.1, or 1.0 mg/kg or an E₂ dose of 0.7 mg/kg or the drug's vehicle, 2-hydroxypropyl--cyclodextrin (HPCD), on day 0. Tissue samples including brain and peripheral tissues were then analyzed for both E₂-Q⁺ and E₂ at 1, 7, 14, 21, or 28 days following the E₂-CDS administration. Initially, both E₂-Q⁺ and E₂ were detected in all tissues analyzed. The dose-distribution and time-course study demonstrates that (1) at 24 hr (1 day) after administration of E₂-CDS, all tissues showed a dose-proportional increase in concentrations of E₂-Q⁺ and E₂; (2) the enzymatic oxidation of E₂-CDS to E₂-Q⁺ was dose dependent over the 100-fold dose range examined; and (3) the disappearance of E₂-Q⁺ as well as E₂ was slow in whole brain and hypothalamus, with an apparent t = 8–9 days, while both of these metabolites were rapidly cleared from plasma, liver, fat, anterior pituitary, kidney, lung, heart, and uterus. Finally, when the kinetic behaviors of E₂-CDS and E₂ were compared on molar basis, the E₂-CDS (1.0-mg/kg dose) produced E₂ concentrations in brain tissue which were 81- and 182-fold greater than those achieved following equimolar E₂ (0.7 mg/kg) injection at 1 and 7 days, respectively. These data demonstrate that the E₂-CDS is much more effective than E₂ itself in delivering the estrogen to the brain. Collectively, these data support the concept of the brain-enhanced delivery and sustained release of E₂ using the redox-based chemical delivery system.     

Production of Soluble and Active Transferrin Receptor-Targeting Single-Chain Antibody using Saccharomyces cerevisiae

Purpose This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. Methods The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. Results Secretion of OX26 scFv was optimal when expression was induced at 20°C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 ± 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 ± 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. Conclusions The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood–brain barrier(BBB).     

Targeted Delivery of Complexes of Biotin–PEG–Polyethylenimine and NF-κB Decoys to Brain-derived Endothelial Cells <Emphasis Type="BoldItalic">in Vitro</Emphasis>

Purpose To evaluate the effect of re-directing the uptake mechanism of polyplexes containing oligodeoxynucleotide (ODN) decoys to nuclear factor kappa B (NF-κB) from absorptive-mediated to receptor-mediated endocytosis. Materials and Methods Complexes of ODNs and a co-polymer of biotin–polyethylenglycol and polyethylenimine (BPP) were targeted to brain-derived endothelial cells with a conjugate of antibody 8D3 and streptavidin (8D3SA). Size and stability of ODN/BPP complexes was measured by dynamic light scattering. Cellular uptake was studied by confocal microscopy. Cell viability and pharmacological effects were investigated on murine bEnd5 cells stimulated with tumor necrosis factor. Results ODN/BPP complexes showed sizes of 116 ± 2.3 nm, which increased by 40 nm when coupled to 8D3SA, and were stable in physiological fluids. Targeted complexes were internalized intact into endosomal compartments. Treatment conditions, which yielded significant inhibitory effects on mRNA expression of VCAM-1, ICAM-1, IκBα and iNOS by bEnd5 cells, did not affect viability. At 0.5 μM, decoy ODN significantly inhibited monocyte adhesion to bEnd5 monolayers when delivered as 8D3SA-targeted complex, while higher concentrations of untargeted complex were ineffective. Conclusions The complex of NF-κB decoys and BPP, which can be targeted to transferrin receptors, is a promising drug candidate for neuroinflammatory diseases affecting the blood–brain barrier.     

Assessment of Blood–Brain Barrier Permeability Using the <Emphasis Type="BoldItalic">In Situ</Emphasis> Mouse Brain Perfusion Technique

Purpose  To assess the blood–brain barrier (BBB) permeability of 12 clinically-used drugs in mdr1a(+/+) and mdr1a(−/−) mice, and investigate the influence of lipophilicity, nonspecific brain tissue binding, and P-gp-mediated efflux on the rate of brain uptake. Methods  The BBB partition coefficient (PS) was determined using the in situ mouse brain perfusion technique. The net brain uptake for 12 compounds, and the time course of brain uptake for selected compounds ranging in BBB equilibration kinetics from rapidly-equilibrating (e.g., alfentanil, sufentanil) to slowly-equilibrating (fexofenadine), was determined and compared. Results  There was a sigmoidal relationship in mdr1a(−/−) mice between the log-PS and clogD_7.4 in the range of 0–5. The brain uptake clearance was a function of both permeability and blood flow rate. The brain unbound fraction was inversely proportional to lipophilicity. Alfentanil achieved brain equilibrium approximately 4,000-fold faster than fexofenadine, based on the magnitude of PS×fu,brain. Conclusions   In situ brain perfusion is a useful technique to determine BBB permeability. Lipophilicity, ionization state, molecular weight and polar surface area are all important determinants for brain penetration. The time to blood-to-brain equilibrium varies widely for different compounds, and is determined by a multiplicity of pharmacokinetic factors.     

脂质体肺部给药研究进展

就脂质体雾化肺部给药剂型的稳定性、靶向性、安全性作一综述     

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Dose and Time-Course Evaluation of a Redox-Based Estradiol-Chemical Delivery System for the Brain. II. Pharmacodynamic Responses

Clinically, brain-enhanced delivery and sustained release of estradiol (E₂) are desirable for effective treatments of menopausal hot flushes and prostatic adenocarcinoma and for fertility regulation. Thus, we conducted studies to determine the dose- and time-dependent effects of a brain-enhanced estradiol-chemical delivery system (E₂-CDS) on anterior pituitary hormones secretion in ovariectomized (OVX) rats. The E₂-CDS has consistently demonstrated preferential retention of its intermediate metabolite (E₂-Q⁺ ), with slow release of E₂ in the brain but rapid clearance from peripheral tissues. Animals received a single iv injection of E₂-CDS at doses of 0.01, 0.1, or 1.0 mg/kg or an E₂ dose of 0.7 mg/kg on day 0. The responses of plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL) were then evaluated at 1, 7, 14, 21, or 28 days after drug administration. The E₂-CDS caused a dose- and time-dependent suppression of LH and FSH throughout the time course studied. The maximum LH and FSH reduction occurred at 7 days postinjection. Plasma LH and FSH were significantly suppressed by 86 and 58% on day 7, respectively, and were suppressed by 35% (LH) or were at preinjection levels (FSH) at 28 days following the single injection of a 1.0-mg E₂-CDS dose. An equimolar E₂ dose suppressed LH and FSH by only 29 and 20% on day 7, respectively which were not significantly different from time 0 values. Plasma PRL increased significantly on day 14 with the 1.0-mg E₂-CDS dose but levels returned to preinjection values by 28 days after drug administration. Lower doses of the E₂-CDS did not affect PRL concentrations. Plasma GH concentrations were not altered in response to the E₂-CDS at any dose or time. Also, anterior pituitary and uterine weights increased in a dose- and time-dependent manner in response to E₂-CDS administration. Collectively, these data demonstrate that the E₂-CDS effects on gonadotropins suppression are dose and time dependent and this duration of suppression is consistent with the long half-lives of the E₂-CDS metabolites in the brain.     

Development of Nanoparticles for Drug Delivery to Brain Tumor: The Effect of Surface Materials on Penetration Into Brain Tissue

Surface-modified poly(d,l-lactic-co-glycolic acid) PLGA nanoparticles (NPs) were fabricated via nanoprecipitation for obtaining therapeutic concentration of paclitaxel (PTX) in brain tumor. The cellular uptake and cytotoxicity of NPs were evaluated on C6 glioma cells in vitro, and BALB/c mice were used to study the brain penetration and biodistribution upon intravenous administration. Results showed that by finely tuning nanoprecipitation parameters, PLGA NPs coated with surfactants with a size around 150 nm could provide a sustained release of PTX for >2 weeks. Surface coatings could increase cellular uptake efficiency when compared with noncoated NPs, and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) showed the most significant enhancement. The in vivo evaluation of TPGS-PLGA NPs showed amplified accumulation (>800% after 96 h) of PTX in the brain tissue when compared with bare NPs and Taxol^®. Therefore, PLGA-NPs with PLGA-TPGS coating demonstrate a promising approach to efficiently transport PTX across blood-brain barrier in a safer manner, with the advantages of easy formulation, lower production cost, and higher encapsulation efficiency.     

跨血脑屏障纳米递药系统的研究进展

血脑屏障是治疗多种中枢神经系统疾病的主要障碍.随着纳米技术的发展,新型脑靶向递药系统的研究取得了较大进展.该类递药系统可通过受体、转运体、吸附等介导的转胞吞作用,以及暂时破坏血脑屏障结构完整性等多种机制跨越血脑屏障并完成脑内药物递释,达到治疗中枢神经系统疾病的目的.本文综述了近年来跨血脑屏障纳米递药系统的研究进展.     

Rapid Colorimetric Screening of Drug Interaction and Penetration Through Lipid Barriers

Purpose The aims of this study are to develop a rapid colorimetric assay for evaluating membrane interactions and penetration through lipid barriers and to create a platform, amenable to high-throughput screening formats, for predicting the extent of penetration of pharmaceutical compounds through lipid layers. Methods The colorimetric platform comprises vesicles of phospholipids and the chromatic lipid–mimetic polymer polydiacetylene. The polymer undergoes visible, concentration-dependent blue–red transformations induced through interactions of the vesicles with the molecules examined. Results We observe rapid colorimetric transitions induced by addition of pharmaceutical compounds to the chromatic vesicle solutions. We find that the concentration ranges for which the color transitions are induced in the lipid/polymer vesicles are correlated with the degree of lipid interactions and bilayer penetration of the tested compounds. The colorimetric platform could distinguish between three primary types of membrane-permeation profiles: bilayer-surface attachment, membrane penetration, and absence of lipid interactions. Application of complementary bioanalytical techniques corroborated the interpretation of the colorimetric data. Different pharmaceutical compounds were tested by the new assay. The results indicated clearly distinct membrane interaction profiles for molecules expected by conventional methods to have similar membrane-insertion properties (i.e., close log D/log P values). In addition, the new colorimetric assay pointed to similar membrane activities for molecules having highly divergent log Ds. Conclusions The colorimetric assay facilitates “color coding” that could distinguish among different membrane permeation profiles. The data point to the usefulness of the platform for characterization of drug compound interactions with lipid assemblies. The new colorimetric technology constitutes a generic, extremely fast, and easily applicable approach for predicting and screening interactions of pharmaceutical compounds with lipid barriers. Electronic Supplementary Material Supplementary material is available for this article at .