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Drug Targeting Dr. Suresh Bandari
The main complications currently associated with systemic drug administration are • Even biodistribution of pharmaceuticals throughout the body • The lack of drug specific affinity toward a pathological site • The necessity of a large total dose of a drug to achieve high local concentration • Non-specific toxicity and other adverse side-effects. Drug targeting may resolve many of these problems
Drug targeting is the ability of the drug to accumulate in the target organ or tissue selectively and quantitatively, independent of the site and methods of its administration. Drug administration protocols may be simplified; Drug quantity may be greatly reduced as well as the cost of therapy; Drug concentration in the required sites can be sharply increased without negative effects on non-target compartments.
MAGIC BULLET CONCEPT OF PAUL EHRLICH • Drugs would be targeted by virtue of groups having affinity • for specific cells • A ligand would confer specificity on a non-specific reagent
‘‘MAGIC BULLET’’ Two components : • The first one is recognizes and binds the target • The second one provides a therapeutic action in this • target Currently, the concept of magic bullet includes a coordinated behavior of three components: (a) drug; (b) targeting moiety; (c) pharmaceutical carrier;
The principal schemes of drug targeting include • Direct application of a drug into the affected zone, • Passive drug targeting(spontaneous drug accumulation in the areas with leaky vasculature, or Enhanced Permeability and Retention-EPR-effect), • Physical targeting (based on abnormal pH value and/or temperature in the pathological zone), • Magnetic targeting (or targeting of a drug immobilized on paramagnetic materials under the action of an external magnetic field), and • Targeting using a specific ‘ vector’ molecules(ligands having an increased affinity toward the area of interest).
Pharmaceutical carriers • polymers • microcapsules • microparticles • Nanoparticles • lipoproteins • liposomes • micelles
Targeting Moieties • Antibodies • Lectins and other proteins • Lipoproteins • Hormones • Charged molecules • Polysaccharides • Low-molecular-weight ligands
Brain Targeting Delivery of drugs to the brain is a major challenge because it is tightly segregated from the circulating blood by a unique membranous barrier, the blood–brain barrier (BBB). The brain and spinal cord are lined with a layer of special endothelial cells that lack fenestrations and are sealed with tight junctions that greatly restrict passage of substances from the bloodstream than endothelial cells in capillaries elsewhere in the body. These endothelial cells, together with perivascular elements such as astrocytes and pericytes, constitute the BBB. BBB is often the rate-limiting factor in determining permeation of therapeutic drugs into the brain.
Characteristics of the BBB are indicated: (1) tight junctions that seal the pathway between the capillary (endothelial) cells; (2) the lipid nature of the cell membranes of the capillary wall which makes it a barrier to water-soluble molecules; (3), (4), and (5) represent some of the carriers and ion channels; (6) the 'enzymatic barrier 'that removes molecules from the blood; (7) the efflux pumps which extrude fat-soluble molecules that have crossed into the cells
The factors affecting particular substance to cross BBB • Drug related factors at the BBB • Concentration at the BBB and the size, • Flexibility, • Conformation, • Ionization (nonionized form penetrates BBB) • Lipophilicity of the drug molecule, • Cellular enzyme stability and cellular sequestration, • Affinity for efflux mechanisms (i.e. P-glycoprotein), • Hydrogen bonding potential (i.e. charge), • Affinity for carrier mechanisms, and • Effect on all of the above by the existing pathological conditions
The physicochemical characteristics • Log Po/w of the therapeutic agent, the rule of 2 is generally accepted i.e. the value of log Po/w nearing 2 is considered optimal. • However, increasing the lipophilicity with intent to increase permeability would increase the volume of distribution (Vd) and also the rate of oxidative metabolism by cytochrome P450 • Peripheral factors including systemic enzymatic stability, • Plasma protein binding affinity, • Uptake of the drug into other tissues, • Clearance rate, and • Effects of existing pathological conditions are also important.
The lipophilicity of a given drug is inversely related to the degree of hydrogen bond formation that occurs with surrounding water. • The presence of certain chemical moieties in drug like terminal amide, primary amines or amides and hydroxyl group favors hydrogen bond formation resulting in a decreased lipophilicity. • Thus for a compound to be transported through the BBB, the cumulative number of hydrogen bonds should not go beyond 8–10. • Therefore for small drugs increasing lipophilicity i.e. decreasing hydrogen bonds has a positive impact on capillary permeability and drug transfer to the brain and for large drug molecules with molecular weight above 400 Da or for those with strong polarity, the capillary permeability will remain low regardless of the lipophilicity
Several specialized transport mechanisms of solute transfer across endothelial cells and into the brain interstitium are also present within the BBB Carrier system for monosaccharides, monocarboxylic acid, neutral amino acids, basic amino acid, acidic amino acids, amines, purine bases, nucleosides, vitamins and hormones. The more lipophilic substances that are present in the blood can diffuse passively directly through the lipid of the cell membrane and enter the endothelial cells and brain by this means.
These solutes, and in many cases their metabolites, are actively removed from the CNS by efflux transporters. Various efflux transport pathways like P-glycoprotein and active organic acid present in choroids plexus may also be active in brain endothelial cells efflux systems are present in the BBB to remove unwanted substances, On the other hand the presence of the tight junctions and the lack of aqueous pathways between cells greatly restrict the movement of polar solutes across the cerebral endothelium
The molecules that can freely diffuse through this capillary endothelial membrane can passively cross the BBB, and this ability is closely related to their lipid solubility (lipophilicity/ hydrophobicity). • Practically all drugs currently used to treat brain disorders are lipid-soluble and can readily cross the BBB following oral administration. • The BBB also has an additional, enzymatic aspect: solutes crossing the endothelial cell membrane are subsequently exposed to numerous degrading enzymes within these cells.
These cells also contain many mitochondria – metabolically active organelles – and active transport can significantly alter both inward and outward transport for compounds. • The BBB is highly efficient and makes the brain practically inaccessible to lipid-insoluble compounds. • Brain-delivery of such compounds, therefore, requires a strategy to overcome the BBB. • Delivery of compounds such as neuropeptides or oligonucleotides is further complicated by their metabolic lability.
Functions of the BBB • Firstly, maintaining internal environment of the brain, i.e. maintaining brain interstitial fluid (ISF) and the cerebrospinal fluid (CSF) composition within extremely fine limits, far more so than the somatic extracellular fluid, so that the neurones can perform their complex integrative functions. • BBB protects the brain from fluctuations in ionic composition that can occur after a meal or exercise, which could disturb synaptic and axonal signaling. • The barrier helps to keep the centrally and peripherally acting neurotransmitters separate.
A major function of the BBB is neuroprotection. Over a lifetime CNS will be exposed to a wide range of neurotoxic metabolites and acquired xenobiotics, which may cause cell damage and death. As neuronal replacement is virtually absent in the CNS of mammals, any enhancement of neuronal death will result in accelerating degenerative pathologies and advance natural debilitation with age. • Finally the continual turnover and drainage of CSF and ISF by bulk flow helps to clear larger molecules and brain metabolites, thus maintaining brain microenvironment
Strategies for Brain Targeting Mechanisms for drug targeting in the brain involve going either "through" or "behind" the BBB. Neurosurgical or Invasive Strategies BBB disruption Disruption of BBB by osmotic means (Hyperosmolar solutions), Intraventricular drug infusion Intracerebral Implants Biodegradable implants, Physiologic based strategies Psuedo nutrientseg: L-dopa Cationic antibodies: These undergo Absorption mediated trancytosis through BBB owing to positive charge. Chimeric peptides
Pharmacologic Strategies Chemical Delivery system Nanocarriers for active targeting of the brain Liposomes Polymeric micelles. Polymeric nanoparticles Lipid nanoparticles . Biochemically by the use of vasoactive substances such as bradykinin, Localized exposure to high intensity focused ultrasound (HIFU). Cell-penetrating peptides and Brain transport vectors
Chemical Delivery Systems Brain-targeted chemical delivery systems (CDSs) represent a rational drug design approach that exploits sequential metabolism not only to deliver but also to target drugs to their site of action. By localizing drugs at their desired site of action, one can reduce toxicity and increase treatment efficiency. The CDS concept evolved from the prodrug concept in the early 1980s, but was differentiated by the introduction of target or moieties and the use of multistep activation. The cunning aspect of these brain-targeted systems is that, in addition to providing access by increasing the lipophilicity, they exploit the specific bidirectional properties of the BBB to ‘lock’ inactive drug precursors in the brain on arrival, preventing exit back across the BBB
CDSs are inactive chemical derivatives of a drug, being obtained by one or more chemical modifications. The introduced bioremovable moieties can be categorised into two types. A targetor (T) moiety is responsible for targeting, site-specificity and lock-in; whereas modifier functions (F1...Fn) serve as lipophilizers, protect certain functions, i.e., necessary molecular properties to preventpremature, unwanted, metabolic conversions. The CDS is designed to undergo sequential metabolic conversions, disengaging the modifier function(s) and finally the targetor, after the moiety has fulfilled its site- or organ-targeting role
Lock in mechanism of E2-CDS provided by introduction of a targetor moiety that exploits a 1,4-dihydrotrigonelline (green) Trigonelline (red) type conversion. On hydrolysis trigonelline converts to active drug.
During the past decade, the system has been explored with a wide variety of drug classes, and considerably increased brain exposure as well as brain targeting (i.e. brain vs systemic exposure) have been obtained in several cases; for example, 3’-azido-3’-deoxythymidine (AZT)-CDS, ganciclovir-CDS and benzylpenicillin-CDS. AZT-CDS administration in rats simultaneously increases brain exposure 32-fold and decreases blood exposure threefold as compared with AZT administration. Among all CDSs, the estradiol chemical delivery system (E2-CDS) is in the most advanced investigation stage. Following earlier clinical trials (Phase I and II),
Molecular packaging: brain delivery of Neuropeptides • Delivery of peptides through the BBB is even more challenging than delivery of other drugs, because peptides tend to be rapidly inactivated by the ubiquitous peptidases. • For a successful delivery, three issues have to be solved simultaneously: • enhance passive transport by increasing the lipophilicity, • ensure enzymatic stability to prevent premature degradation, and • exploit the lock-in mechanism to provide targeting. • Successful brain deliveries have already been achieved using this strategy for a Leu-enkephalin analog, thyrotropin-releasing hormone (TRH) analogs and kyotorphin analogs
It is of particular significance for TRH delivery because the corresponding process might require up to five or six consecutive metabolic steps. Therefore, selection of a suitable spacer moiety, which is inserted between the targetor and peptide units to ensure correct timing for targetor release, proved important for the efficacy of TRH-CDSs.
Dopamine is also classed as a monoamine neurotransmitter and is concentrated in very specific groups of neurons collectively called the basal ganglia. Dopaminergic neurons are widely distributed throughout the brain in three important dopamine systems (pathways): the nigrostriatal, mesocorticolimbic, and tuberohypophyseal pathways. A decreased brain dopamine concentration is a contributing factor in Parkinsonﾕs disease, while an increase in dopamine concentration has a role in the development of schizophrenia.
The first group regulates movements: a deficit of dopamine in this (nigrostriatal) system causes Parkinson's disease which is characterized by trembling, stiffness and other motor disorders, while in the later phases dementia can also set in. The second group, the mesolimbic, has a function in regulating emotional behavior. The third group, the mesocortical, is involved with various cognitive functions, memory, behavioral planning and abstract thinking, as well as in emotional aspects, especially in relation to stress. The earlier mentioned reward system is part of this last system. Disorders in the latter two systems are associated with schizophrenia.
In Parkinson’s disease, there is degeneration of the substantia nigra which produces the chemical dopamine deep inside the brain
Since PD is related to a deficiency of dopamine, it would be appropriate to administer dopamine Problem: Dopamine does not cross BBB, since it is too polar
If dopamine is too polar to cross the BBB, how can L-DOPA cross it? L-DOPA is transported across the BBB by an amino acid transport system (same one used for tyrosine and phenylalanine)
Once across, L-DOPA is decarboxylated to dopamine by Dopa Decarboxylase.This is an example of a “prodrug”, that is, a molecule that is a precursor to the drug and is converted to the actual drug at an appropriate place in the body. In actual practice, L-DOPA is almost always coadminstered together with an inhibitor of aromatic L-amino acid decarboxylase, so it doesn’t get converted to dopamine before it crosses the BBB. The inhibitor commonly used is carbidopa, which does not cross the BBB itself. The inhibitor also prevents undesirable side effects of dopamine release into the PNS, including nausea.
Polymeric nanoparticles suitable delivery systems for brain. • The mechanisms for nanoparticle mediated drug uptake by the brain include: • Enhanced retention in the brain–blood capillaries, with an adsorption on to • the capillary walls, resulting in a high concentration gradient across the BBB. • Opening of tight junctions due to the presence of nanoparticles. • Transcytosis of nanoparticles through the endothelium. • Furthermore, coating of these polymeric nanoparticles with polysorbate has been reported to improve the brain bioavailability. Some of the proposed mechanisms by which the polysorbate coating is effective, include: • Solubilization of endothelial cell membrane lipids and membrane • fluidization, due to surfactant effects of polysorbates. • Endocytosis of polymeric nanoparticles due to facilitated interaction with • BBB endothelial cells. • Inhibition of efflux system, especially P-gp.
But, there are various problems associated with the use of these polymeric nanoparticles • Residual contamination from the production process, for example by • organic solvents, • Polymerization initiation, • Large polymer aggregates, • Toxic monomers and toxic degradation products, • Expensive production methods, • Lack of large scale production method and • A suitable sterilization method e.g. autoclaving. • Considering the success of nanoparticles to pass through the BBB and their limitation(s) especially toxicity and stability, another suitable option for drug delivery into the brain would be SLNs.
SOLID LIPID NANOPARTICLES SLNs constitute an attractive colloidal drug carrier system. SLNs consist of spherical solid lipid particles in the nanometer range, which are dispersed in water or in aqueous surfactant solution. They are generally made up of solid hydrophobic core having a monolayer of phospholipid coating. Advantages of SLNs over polymeric nanoparticles (and other delivery systems like liposomes) The nanoparticles and the SLNs particularly those in the range of 120–200 nm are not taken up readily by the cells of the RES (Reticulo Endothelial System) and thus bypass liver and spleen filtration. 2. Controlled release of the incorporated drug can be achieved for upto several weeks. Further, by coating with or attaching ligands to SLNs, there is an increased scope of drug targeting.
3. SLN formulations stable for even three years have been developed. 4. High drug payload. 5.Excellent reproducibility with a cost effective high pressure homogenization method as the preparation procedure. 6.The feasibility of incorporating both hydrophilic and hydrophobic drugs. 7. The carrier lipids are biodegradable and hence safe. 8. Avoidance of organic solvents. 9. Feasible large scale production and sterilization.
Use of ligands. Ligands or homing devices that specifically bind to surface epitopes or receptors on the target sites, can be coupled to the surface of the long-circulating carriers. Certain cancer cells over express certain receptors, like folic acid (over-expressed in cells of cancers with epithelial origin), LDL (B16 melanoma cell line shows higher expression of LDL receptors) and peptide receptors (such as somatostatin analogs, vasoactive intestinal peptide, gastrin related peptides, cholecystokinin, leutanising hormone releasing hormone). Attaching suitable ligands for these particular receptors on to the nanoparticles would result in their increased selectivity Allen et al. postulated that the presence of specific ligands on the surface of nanoparticles could lead to their increased retention at the BBB and a consequent increase in nanoparticle concentration at the surface of BBB. While attempting to prove their assumption, they prepared coated nanoparticles from Brij 78, and emulsifying wax, with thiamine ligand (linked to DSPE via a PEG spacer).
Gene targeting technology & gene therapy of the brain Many serious disorders of the CNS that are resistant to conventional small-molecule therapy could be treated, even cured, with gene therapy of the brain. Current approach include delivery of the therapeutic gene to the brain by drilling a hole in the head followed by insertion of the gene incorporated in a viral vector. The advantage of craniotomy-based gene delivery is that the gene can be expressed in a highly circumscribed area of the brain with an effective treatment volume of 1–10 μl. This craniotomy based delivery does not enable the expression of the therapeutic gene widely throughout the brain or even to a relatively localized area such as a brain tumor, which could have a volume greater than several milliliters. Viruses have been the vector of choice because the virus-coat proteins trigger endocytosis of the virus into the target brain cell. The two most commonly used viral vectors are adenovirus or herpes simplex virus (HSV). The problem with both these viruses is that, because they are common, humans have a pre-existing immunity. This immunity generates an inflammatory response
Gene targeting technology Craniotomy and viruses are first-generation brain gene delivery systems. Gene therapy of the brain use delivery systems that are both noninvasive and non-viral. A brain gene delivery system should enable widespread expression of a therapeutic gene throughout the brain following a simple intravenous injection. First, the exogenous gene packaged in a non-viral plasmid vector is interiorized within a nanocarrier, much like exogenous genes are packaged in the interior of viruses. This protects the therapeutic gene from the endonucleases in the body. Second, the nanocarrier is non-immunogenic and formed by either natural lipids or other non-immunogenic polymeric substances. Third, the nanocarrier carrying the exogenous gene is stable in the bloodstream with optimal plasma pharmacokinetics following an intravenous injection. (The rapid RES uptake can be blocked by pegylation. The pegylated liposomes are stable in the bloodstream and have long blood circulation times).
Fourth, the surface of the nanocarrier is modified that triggers transcytosis across microvascular endothelial barriers such as the BBB and then endocytosis into target neurons or glial cells in brain. (Targeting through the BBB and neuronal plasma membrane is accomplished by tethering the tips of 1–2% of the PEG strands with a targeting monoclonal antibody (MAb) to form an immunoliposome). Owing to expression of the transferrin receptor (TfR) on both the BBB and the neuronal plasma membrane, the use of an anti-TfR MAb causes the pegylated immunoliposome to undergo transport through both the BBB and the neuronal plasma membrane in vivo. The liposomal lipids fuse with the endosomal membrane inside neurons, which releases the plasmid into the cytosolic space of target neurons, where it can then diffuse to the nuclear compartment. The only immunogenic component of the formulation is the MAb and the immunogenecity of murine MAbs in humans can be eliminated with genetic engineering and ‘humanization’ of the MAb.
β-Galactosidase histochemistry of a rat brain removed 48 h after a single intravenous injection of a β-galactosidase gene carried by a plasmid that is packaged in the interior of 85 mm liposomes. The surface of the liposome is covered by thousands of strands of 2000 Da (PEG), and this stabilizes the liposome in the blood and prolongs the circulation time in the plasma. Approximately 2% of the PEG strands that project from the liposome surface are tethered to a monoclonal antibody that targets the transferrin receptor. This receptor is expressed both on the brain capillary endothelium, which forms the blood–brain barrier in vivo, and on the neuronal plasma membrane. Targeting the immunoliposomes to the transferrin receptor enables transport across both the blood–brain barrier and the neuronal plasma membrane in vivo. The use of gene targeting technology enables widespread expression in the brain of an exogenous gene following a single intravenous administration of a non-viral gene formulation.
Nice presentation All the credit goes to Dr. Suresh Bandari Thank you.
