Drug Targeting Organ-Specific Strategies

BDNF, brain derived neurotrophic factor; CBF, cerebral blood flow; GDNF, glial cell line derived neurotrophic factor; NGF, nerve growth factor; TfR, transferrin receptor; VIP, vasoactive intestinal polypeptide.

demonstration of a pharmacological effect with a vector-mediated drug delivery strategy, because VIP-containing nerve fibres are abundant around intracerebral small arteries and arterioles. This peptide acts as a potent vasodilator when applied topically to intracranial vessels and plays an important role in the modulation of cerebral blood flow (CBF). However, as its receptors are expressed on the vascular smooth muscle cells, which are beyond the blood–brain barrier, no effects on CBF are usually seen after systemic administration of VIP.

A metabolically stabilized analogue of VIP was constructed which could be biotinylated at a single site. Brain delivery of the biotinylated VIP analogue by the OX26–avidin vector resulted in the desired pharmacological effect. A significant increase in CBF of 65% could be demonstrated after systemic administration of the chimeric peptide.The effect was seen both in anaesthetized rats under controlled respiration after intracarotid infusion as well as in conscious animals after i.v. bolus injection. When an equal dose of the peptide alone without a vector was injected (12 g kg–1 for the intracarotid infusion or 20 g kg–1 in the i.v. study) there was no measurable effect on CBF compared to control animals. In contrast, the well established peripheral effects of VIP on glandular blood flow in the thyroid gland or the sali-

46 2 Brain-Specific Drug Targeting Strategies

transient forebrain ischaemia was induced in rats by bilateral clamping of the carotid arteries. In order to achieve BDNF delivery with the chimeric peptide approach it was necessary to modify the peptide by ‘pegylation’, i.e. the coupling of multiple PEG residues. Native BDNF is a basic peptide with rapid clearance from plasma. The poor pharmacokinetic properties persisted after coupling to OX26–streptavidin but could be overcome by pegylation. The PEG-BDNF could be delivered through the BBB by vector-mediated transport as efficiently as the OX26 antibody itself [90].Animals treated for 1 week after the ischaemic insult with chimeric peptide (biotinylated PEG-BDNF coupled to OX26-SA) at a daily dose of 250 µg kg–1 were fully protected from neuronal loss in the hippocampal CA1 region.

Oligodeoxynucleotides (ODN) represent another class of hydrophilic macromolecular drug candidates, which require transcellular as well as intracellular delivery where brain cell targeting is concerned. Due to their highly charged, anionic character they also have the potential to impair the pharmacokinetics of the delivery system when used as the drug constituent of chimeric peptides. Coupling of a biotinylated phosphodiester ODN to OX26NLA increases hepatic clearance of the complex and limits brain uptake by lowering the AUC [99]. On the other hand, phosphorothioate-modified ODNs show high plasma protein binding which may contribute to the low BBB transport measured for a PS-ODN/OX26-SA chimeric peptide. In contrast, the neutral peptide backbone of peptide nucleic acids makes these compounds good drug candidates for chimeric peptides and allows for a substantial vector-mediated effect on brain targeting (28-fold increase, [100]). A potential therapeutic application of the ODN approach is the delivery of an antisense oligonucleotide to the rev gene of HIV-1 through the BBB. The feasibility of such an approach was recently demonstrated using the OX26–avidin fusion protein [88].

2.4.2.7 Chimeric Peptide Radiopharmaceuticals

The potential of chimeric peptides for delivery of radiopharmaceuticals across the BBB, either for diagnostic or therapeutic purposes, has been explored in studies with radiolabelled synthetic amyloid peptide and with EGF. Aß peptide in solution deposits specifically on preexisting amyloid plaques and vascular amyloid.A pharmacokinetic study in Rhesus monkeys with the insulin receptor antibody 83-14 as a vector showed brain accumulation of radiolabelled [125I]-Aβ only after vector-mediated delivery. The peptide alone was unable to cross the BBB. In the monkeys, analysis of brain sections by phosphorimager quantitation of radioactivity resulted in images comparable to scans obtained with the non-metabolized glucose analogue 2-deoxyglucose [101]. Labelling with a suitable radioisotope should enable quantitative detection by a neuroimaging method such as SPECT.

EGF receptors are abundantly expressed by gliomas and present a target both for diagnostic imaging and radio-immunotherapy. A cerebral implant model in rats bearing human U87 gliomas was utilized to test the brain delivery of [111In]-labelled EGF by vector mediated transport with OX26 following i.v. injection. Brains were sampled after 2 h and cryosectioned for subsequent autoradiography. The tumours were clearly visualized on these autoradiographs, but only when the labelled EGF was given as a chimeric peptide, not when injected without the vector [102].

48 2 Brain-Specific Drug Targeting Strategies

An antibody used for brain targeting of immunoliposomes has to meet several requirements. First, the antibody should recognize a structure which is present exclusively at the blood–brain barrier. Second, the antibody should be able to cross the blood–brain barrier by an active transport mechanism such as receptor-mediated transcytosis. Third, the epitope against which the antibody is targeted should preferably not be species specific. Fourth, high quantities of the antibody should be available. The OX26 mAb [38] meets several (but not all) of the above requirements. In vitro experiments have demonstrated that OX26-immuno- liposomes can be taken up specifically by living RG2 rat glioma cells overexpressing the rat transferrin receptor despite their particulate size of approximately 90 nm [113]. The fluores- cent-labelled OX26-immunoliposomes accumulated within an intracellular (endosomal) compartment [114]. Similar results were obtained by incubation of fluorescent OX26-im- munoliposomes with freshly isolated rat brain capillaries [115] which revealed binding to the luminal and basolateral membranes of the brain endothelium.

2.4.3.3 Drugs of Interest for Targeting to the Brain

Brain delivery of the anticancer drug daunomycin provides an example of the in vivo application of OX26-immunoliposomes [111]. Different formulations of [3H]-daunomycin were i.v. administered to rats either as the free drug or encapsulated in conventional liposomes, sterically-stabilized liposomes, or PEG-conjugated immunoliposomes (Table 2.3). Plasma samples were taken at defined time points and after 1 h the animal was killed and drug concentrations in brain tissue were determined.

Free daunomycin and not PEG-conjugated liposomes containing daunomycin, disappear rapidly from the circulation. Plasma clearance of the liposome was reduced 66-fold by PEGconjugation. Coupling 29 OX26 monoclonal antibodies per PEG-liposome partially reversed the effect of PEG-conjugation on plasma clearance.

Analysis of the blood–brain barrier permeability surface area (PS) product indicated that daunomycin, and to a lesser degree conventional liposomes, have the potential to penetrate the blood–brain barrier. However, brain tissue accumulation of free daunomycin or conventional liposomes was poor, being the result of their high systemic plasma clearance. The use of PEG-conjugated liposomes reduced the blood–brain barrier PS product value to zero. No brain uptake of the PEG-liposomes was observed, despite their marked increase in plasma circulation time. Conversely, the use of PEG-conjugated OX26 immunoliposomes increased the blood–brain barrier PS product, relative to PEG-liposomes, resulting in increased brain uptake. Thus, optimal brain delivery of daunomycin was achieved using OX26 immunoliposomes (see Table 2.3). Titration of the amount of OX26 conjugated per liposome (n between 3 and 197) revealed an increase in plasma clearance and a decrease in the systemic volume of distribution of immunoliposomes at higher OX26 concentrations. Highest PS product values and brain tissue accumulation was observed for immunoliposomes with 29 OX26 mAb. At higher OX26 densities on the liposome, a saturation effect was observed resulting in a reduction in volume of distribution, PS product and brain tissue accumulation of OX26 immunoliposomes.

Recently the OX26 immunoliposomes were used in a gene delivery approach to transport expression vectors for luciferase or β-galactosidase through the BBB [116]. The plasmids

Table 2.3. Pharmacokinetics of different formulations of [3H]-daunomycin after i.v. administration to rats.

Plasma clearance (Cl), blood–brain barrier permeability surface area product (PS) and accumulation as % injected dose detected in brain tissue (%ID g-1 tissue) at 1 h after administration. Results show free [3H]-daunomycin (Daunomycin), [3H]-daunomycin encapsulated in conventional liposomes (Liposomes), sterically stabilized liposomes (PEG–liposomes), immunoliposomes (29 OX26, where 29 designates the number of OX26 mAb conjugated per liposome) and control immunoliposomes where the OX26 mAb was replaced by a non-specific isotype control antibody (IgG2a). Values are means ± SEM of n = 3 experiments.

were physically entrapped inside the neutral liposomes rather than being complexed on the surface of cationic liposomes. Gene expression was demonstrated in brain cells beyond the BBB, indicating both penetration of the liposomes through the BBB in vivo and escape from the endosomal compartment by an as yet unidentified mechanism.

In conclusion, the use of an immunoliposome-based drug delivery system allows for targeted delivery of a small molecule such as daunomycin or plasmids to the rat brain in vivo. Further experiments will be needed to clarify the subcellular routes and compartments involved in the transcytosis mechanism, as well as the eventual release mechanism in the target cell.

2.5 Conclusions

Various strategies to circumvent or to overcome the BBB for brain-directed drug therapies are under evaluation. It can be predicted that for broad clinical application noninvasive methods will be required, in particular for chronic diseases where long-term treatment is necessary. The utilization of physiological transport mechanisms at the BBB in experimental models generated evidence that pharmacological effects can be achieved with this approach. In order to be useful as drug delivery systems in humans, several steps are necessary. The transport capacity must be increased, which is possible through improved vectors and optimized coupling strategies. In order to avoid potential immunogenicity of antibody-based vectors from murine sources, humanization techniques are now being applied [117]. Further developments may include specific targeting to neuronal or non-neuronal cells, and efficient intracellular release mechanisms.

50 2 Brain-Specific Drug Targeting Strategies

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Drug Targeting Organ-Specific Strategies. Edited by G. Molema, D. K. F. Meijer Copyright © 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29989-0 (Hardcover); 3-527-60006-X (Electronic)

3Pulmonary Drug Delivery: Delivery To and Through the Lung

Anne H. de Boer, Grietje Molema, Henderik W. Frijlink

3.1 Introduction

The respiratory tract is one of the oldest routes used for the administration of drugs. Anaesthetics, aerosolized drugs, smoke or steam have been inhaled for medical purposes for centuries. Over the past decades inhalation therapy has established itself as a valuable tool in the local therapy of pulmonary diseases such as asthma or COPD (Chronic Obstructive Pulmonary Disease) [1]. This type of drug application in the therapy of these diseases is a clear form of targeted drug delivery: the major advantages are a rapid onset of the therapeutic effect, a lowering of the required dose (as compared to systemic administration) and a reduction in unwanted side-effects (increased therapeutic index). Currently, over 25 drug substances are marketed as inhalation aerosol products for local pulmonary effects and about the same number of drugs are in different stages of clinical development. Furthermore, there are some drugs that are not marketed as inhalation aerosols per se but are formulated as such by pharmacists.

The majority of the marketed products are used for asthma and COPD.Typical agents that are used for these indications are β2-agonists such as salbutamol (albuterol), Terbutalin or formoterol, corticosteroids such as budesonide, Flixotide or beclomethasone and mast-cell stabilizers such as sodium cromoglycate or nedocromil.

Patients suffering from cystic fibrosis often use various aerosolized drugs. To reduce the viscosity of the mucus in the airways, recombinant human deoxyribonuclease is used. This enzyme is the first recombinant protein that has been developed for specific delivery to the lungs via the airways. It has a local action on the mucus in the airways and its absorption is minimal. Another drug that decreases the viscosity of the mucus is acetylcysteine. Aerosolized antibiotics are a further group of therapeutics that is widely used by cystic fibrosis patients. Solutions of antibiotics like tobramycin or colistin are used in nebulizers to prevent exacerbation of the disease. Pentamidine has been used for the prophylaxis of Pneumocystis pneumonia in patients infected with HIV virus, while chronic rejection of lung transplants provided a reason to develop an aerosol formulation of cyclosporine A.

The latest and probably one of the most promising applications of pulmonary drug administration is its use to achieve systemic absorption of the administered drug substances. Particularly for those drug substances that exhibit a poor bioavailability when administered by the oral route, as for example peptides or proteins, the respiratory tract might be a convenient port of entry [2]. For this application a more or less contradictory situation occurs: ‘delivery (into the lung) is required to obtain systemic absorption followed by a non-targeted