a) Macro molecular conjugates, b) Particulate drug carriers
Lock in mechanism of E2-CDS provided by introduction of a targetor moiety that exploits a 1,4-dihydrotrigonelline (green) Trigonelline (red) type conversion. The partition log P and distribution log D coefficients illustrates partition properties that occur during sequential metabolism. The half life in various tissues for T*-E2 formed after i.v administration of E2-CDS in rats. Because of lock in elimination from brain is considerably slower than other organs
Uncoated SLNs, SLN coated with hydrophilic polymers like polysorbates, SLN coated with both hydrophilic polymer (like P.E.G.) and a specific uptake linker monoclonal antibodies/thiamine/glucose. Available SLNs in general circulation immediately after oral administration. SLNs left after 1st metabolism. SLNs left after encounter with another RES organ “spleen”. Final S.L.N's made available to the CNS after passing BBB. This figure shows the fate of different types of SLNs after oral administration. The SLN can bypass the RES removal because of their small particle size, moreover their RES detection could be further decreased by providing a hydrophilic coat e.g. polysorbates, PEG, Poloxamer F 68, Brig 78. This will result in an increased circulation time and thus higher chances to be taken up by the target organ. The nanoparticles statistically keep on circulating until the hydrophilic coating is dissolved; when they are either removed by the liver or are taken up by the target organ. The hydrophilic coating prevents their interaction with the blood plasma proteins (opsonins) and thus with the membranes of macrophages. The binding of SLNs to the target site e.g. the brain can be improved by placing certain ligands e.g. thiamine on to their surface, these thiamine ligands could bind to the thiamine receptors and gain access to the brain by receptor mediated transcytosis
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 liposomes3. The surface of the liposome is covered by thousands of strands of 2000 Da polyethylene glycol (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.
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 accumulatein the target organ or tissue selectively andquantitatively, independent of the site and methods ofits 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 targetCurrently, the concept of magic bullet includes acoordinated 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 areaswith leaky vasculature, or Enhanced Permeability and Retention-EPR-effect),•Physical targeting (based on abnormal pH value and/or temperaturein the pathological zone),•Magnetic targeting (or targeting of a drug immobilized onparamagnetic materials under the action of an external magneticfield), and•Targeting using a specific ‘ vector’ molecules (ligands having anincreased affinity toward the area of interest).
a) Macro molecular conjugates, b) Particulate drug carriers
Targeting Moieties•Antibodies•Lectins and other proteins•Lipoproteins•Hormones•Charged molecules•Polysaccharides•Low-molecular-weight ligands
Brain TargetingDelivery of drugs to the brain is a major challenge because it istightly segregated from the circulating blood by a uniquemembranous barrier, the blood–brain barrier (BBB).The brain and spinal cord are lined with a layer of specialendothelial cells that lack fenestrations and are sealed with tightjunctions that greatly restrict passage of substances from thebloodstream than endothelial cells in capillaries elsewhere in thebody. These endothelial cells, together with perivascular elementssuch as astrocytes and pericytes, constitute the BBB.BBB is often the rate-limiting factor in determining permeation of
Characteristics of the BBB are indicated: (1) tight junctions that seal the pathwaybetween the capillary (endothelial) cells; (2) the lipid nature of the cell membranes ofthe 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 thatremoves molecules from the blood; (7) the efflux pumps which extrude fat-solublemolecules that have crossed into the cells
The factors affecting particular substance to cross BBBDrug 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 generallyaccepted i.e. the value of log Po/w nearing 2 is considered optimal.•However, increasing the lipophilicity with intent to increasepermeability would increase the volume of distribution (Vd) andalso 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 degreeof hydrogen bond formation that occurs with surrounding water.•The presence of certain chemical moieties in drug like terminalamide, primary amines or amides and hydroxyl group favorshydrogen bond formation resulting in a decreased lipophilicity.•Thus for a compound to be transported through the BBB, thecumulative 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 capillarypermeability and drug transfer to the brain and for large drugmolecules with molecular weight above 400 Da or for those withstrong polarity, the capillary permeability will remain lowregardless of the lipophilicity
Several specialized transport mechanisms of solute transfer acrossendothelial cells and into the brain interstitium are also present withinthe BBBCarrier system for monosaccharides, monocarboxylic acid, neutralamino acids, basic amino acid, acidic amino acids, amines, purinebases, nucleosides, vitamins and hormones.The more lipophilic substances that are present in the blood candiffuse passively directly through the lipid of the cell membrane andenter the endothelial cells and brain by this means.
These solutes, and in many cases their metabolites, are activelyremoved from the CNS by efflux transporters.Various efflux transport pathways like P-glycoprotein andactive organic acid present in choroids plexus may also beactive in brain endothelial cells efflux systems are present in theBBB to remove unwanted substances,On the other hand the presence of the tight junctions and thelack of aqueous pathways between cells greatly restrict themovement of polar solutes across the cerebral endothelium
The molecules that can freely diffuse through this capillaryendothelial membrane can passively cross the BBB, and thisability is closely related to their lipid solubility (lipophilicity/hydrophobicity).Practically all drugs currently used to treat brain disordersare lipid-soluble and can readily cross the BBB following oraladministration.The BBB also has an additional, enzymatic aspect: solutescrossing the endothelial cell membrane are subsequentlyexposed to numerous degrading enzymes within these cells.
These cells also contain many mitochondria – metabolicallyactive organelles – and active transport can significantly alterboth inward and outward transport for compounds.The BBB is highly efficient and makes the brain practicallyinaccessible to lipid-insoluble compounds.Brain-delivery of such compounds, therefore, requires astrategy to overcome the BBB.Delivery of compounds such as neuropeptides oroligonucleotides is further complicated by their metaboliclability.
Functions of the BBB•Firstly, maintaining internal environment of the brain, i.e.maintaining brain interstitial fluid (ISF) and the cerebrospinalfluid (CSF) composition within extremely fine limits, far moreso than the somatic extracellular fluid, so that the neurones canperform their complex integrative functions.•BBB protects the brain from fluctuations in ionic compositionthat can occur after a meal or exercise, which could disturbsynaptic and axonal signaling.•The barrier helps to keep the centrally and peripherally actingneurotransmitters separate.
•A major function of the BBB is neuroprotection. Over a lifetimeCNS will be exposed to a wide range of neurotoxic metabolites andacquired xenobiotics, which may cause cell damage and death. Asneuronal replacement is virtually absent in the CNS of mammals,any enhancement of neuronal death will result in acceleratingdegenerative pathologies and advance natural debilitation with age.•Finally the continual turnover and drainage of CSF and ISF bybulk flow helps to clear larger molecules and brain metabolites,thus maintaining brain microenvironment
Strategies for Brain TargetingMechanisms for drug targeting in the brain involve going either"through" or "behind" the BBB.Neurosurgical or Invasive StrategiesBBB disruptionDisruption of BBB by osmotic means (Hyperosmolar solutions),Intraventricular drug infusionIntracerebral ImplantsBiodegradable implants,Physiologic based strategiesPsuedo nutrients eg: L-dopaCationic antibodies: These undergo Absorption mediatedtrancytosis through BBB owing to positive charge.Chimeric peptides
Pharmacologic StrategiesChemical Delivery systemNanocarriers for active targeting of the brainLiposomesPolymeric micelles.Polymeric nanoparticlesLipid nanoparticles .Biochemically by the use of vasoactive substances such asbradykinin,Localized exposure to high intensity focused ultrasound (HIFU).Cell-penetrating peptides and Brain transport vectors
Chemical Delivery SystemsBrain-targeted chemical delivery systems (CDSs) represent a rationaldrug design approach that exploits sequential metabolism not only todeliver but also to target drugs to their site of action.By localizing drugs at their desired site of action, one can reducetoxicity and increase treatment efficiency.The CDS concept evolved from the prodrug concept in the early1980s, but was differentiated by the introduction of target or moietiesand the use of multistep activation.The cunning aspect of these brain-targeted systems is that, in additionto providing access by increasing the lipophilicity, they exploit thespecific bidirectional properties of the BBB to ‘lock’ inactive drugprecursors in the brain on arrival, preventing exit back across theBBB
CDSs are inactive chemical derivatives of a drug, being obtained byone or more chemical modifications.The introduced bioremovable moieties can be categorised into twotypes.A targetor (T) moiety is responsible for targeting, site-specificity andlock-in; whereas modifier functions (F1...Fn) serve as lipophilizers,protect certain functions, i.e., necessary molecular properties toprevent premature, unwanted, metabolic conversions.The CDS is designed to undergo sequential metabolic conversions,disengaging the modifier function(s) and finally the targetor, after themoiety 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 toactive drug.
During the past decade, the system has been explored with a widevariety of drug classes, and considerably increased brain exposureas well as brain targeting (i.e. brain vs systemic exposure) havebeen obtained in several cases; for example, 3’-azido-3’-deoxythymidine (AZT)-CDS, ganciclovir-CDS andbenzylpenicillin-CDS.AZT-CDS administration in rats simultaneously increases brainexposure 32-fold and decreases blood exposure threefold ascompared with AZT administration.Among all CDSs, the estradiol chemical delivery system (E2-CDS)is in the most advanced investigation stage. Following earlierclinical trials (Phase I and II),
Molecular packaging: brain delivery of NeuropeptidesDelivery of peptides through the BBB is even more challenging thandelivery of other drugs, because peptides tend to be rapidly inactivatedby 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, andexploit the lock-in mechanism to provide targeting.Successful brain deliveries have already been achieved using thisstrategy for a Leu-enkephalin analog, thyrotropin-releasing hormone(TRH) analogs and kyotorphin analogs
It is of particular significance for TRH delivery becausethe corresponding process might require up to five or sixconsecutive metabolic steps.Therefore, selection of a suitable spacer moiety, which isinserted between the targetor and peptide units to ensurecorrect timing for targetor release, proved important forthe efficacy of TRH-CDSs.
HO NH2 HO Dopamine• 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 Parkinsons disease which is characterizedby trembling, stiffness and other motor disorders, while in the laterphases dementia can also set in. The second group, the mesolimbic, hasa function in regulating emotional behavior. The third group, themesocortical, is involved with various cognitive functions, memory,behavioral planning and abstract thinking, as well as in emotionalaspects, especially in relation to stress. The earlier mentioned rewardsystem is part of this last system. Disorders in the latter two systemsare 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 beappropriate to administer dopamineProblem: Dopamine does not cross BBB, since it is too polar HO NH3+ Polar groups Mostly protonated HO to the corresponding Dopamine ammonium salt
If dopamine is too polar to cross the BBB, how can L-DOPA cross it? HO NH3+ HO NH3 + Polar groups Mostly protonatedPolar groups Mostly protonated to the corresponding HO O O HO to the corresponding ammonium salt ammonium salt H Dopamine L-DOPA Polar group 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 DopaDecarboxylase.This is an example of a “prodrug”, that is, a molecule thatis a precursor to the drug and is converted to the actual drug at anappropriate place in the body.In actual practice, L-DOPA is almost always coadminstered togetherwith an inhibitor of aromatic L-amino acid decarboxylase, so it doesn’tget converted to dopamine before it crosses the BBB.The inhibitor commonly used is carbidopa, which does not cross theBBB itself. The inhibitor also prevents undesirable side effects ofdopamine release into the PNS, including nausea. H HO H+ N 3 HO N N 2 H HO O HC 3 C 2 OH O HO H L O A -D P C r id p ab o a
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 theBBB.• Opening of tight junctions due to the presence of nanoparticles.• Transcytosis of nanoparticles through the endothelium.Furthermore, coating of these polymeric nanoparticles with polysorbate hasbeen reported to improve the brain bioavailability. Some of the proposedmechanisms 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
But, there are various problems associated with the use of these polymericnanoparticles• 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 BBBand their limitation(s) especially toxicity and stability, another suitableoption for drug delivery into the brain would be SLNs.
SOLID LIPID NANOPARTICLESSLNs constitute an attractive colloidal drug carrier system.SLNs consist of spherical solid lipid particles in the nanometer range, which aredispersed in water or in aqueous surfactant solution. They are generally madeup of solid hydrophobic core having a monolayer of phospholipid coating.Advantages of SLNs over polymeric nanoparticles (and other delivery systemslike liposomes)The nanoparticles and the SLNs particularly those in the range of 120–200 nmare 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 severalweeks. Further, by coating with or attaching ligands to SLNs, there is anincreased 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 pressurehomogenization method as the preparation procedure.6.The feasibility of incorporating both hydrophilic and hydrophobicdrugs.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 thetarget sites, can be coupled to the surface of the long-circulating carriers.Certain cancer cells over express certain receptors, like folic acid (over-expressed incells of cancers with epithelial origin),LDL (B16 melanoma cell line shows higher expression of LDL receptors) and peptidereceptors (such as somatostatin analogs, vasoactive intestinal peptide, gastrin relatedpeptides, cholecystokinin, leutanising hormone releasing hormone). Attaching suitableligands for these particular receptors on to the nanoparticleswould result in their increased selectivityAllen et al. postulated that the presence of specific ligands on the surface ofnanoparticles could lead to their increased retention at the BBB and a consequentincrease in nanoparticle concentration at the surface of BBB. While attempting to provetheir assumption, they prepared coated nanoparticles from Brij 78, and emulsifyingwax, 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 conventionalsmall-molecule therapy could be treated, even cured, with gene therapy of thebrain. Current approach include delivery of the therapeutic gene to the brainby drilling a hole in the head followed by insertion of the gene incorporated in aviral vector. The advantage of craniotomy-based gene delivery is that the gene canbe expressed in a highly circumscribed area of the brain with an effectivetreatment volume of 1–10 μl. This craniotomy based delivery does not enablethe expression of the therapeutic gene widely throughout the brain or even to arelatively localized area such as a brain tumor, which could have a volumegreater than several milliliters. Viruses have been the vector of choice because the virus-coat proteinstrigger endocytosis of the virus into the target brain cell. The two mostcommonly used viral vectors are adenovirus or herpes simplex virus (HSV).The problem with both these viruses is that, because they are common, humanshave a pre-existing immunity. This immunity generates an inflammatoryresponse
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 atherapeutic gene throughout the brain following a simple intravenous injection. First, the exogenous gene packaged in a non-viral plasmid vector isinteriorized within a nanocarrier, much like exogenous genes are packaged in theinterior of viruses. This protects the therapeutic gene from the endonucleases inthe body. Second, the nanocarrier is non-immunogenic and formed by eithernatural lipids or other non-immunogenic polymeric substances. Third, the nanocarrier carrying the exogenous gene is stable in thebloodstream with optimal plasma pharmacokinetics following an intravenousinjection. (The rapid RES uptake can be blocked by pegylation. The pegylatedliposomes are stable in the bloodstream and have long blood circulation times).
Fourth, the surface of the nanocarrier is modified that triggerstranscytosis across microvascular endothelial barriers such as the BBB and thenendocytosis into target neurons or glial cells in brain. (Targeting through the BBBand neuronal plasma membrane is accomplished by tethering the tips of 1–2% ofthe PEG strands with a targeting monoclonal antibody (MAb) to form animmunoliposome). Owing to expression of the transferrin receptor (TfR) on both the BBBand the neuronal plasma membrane, the use of an anti-TfR MAb causes thepegylated immunoliposome to undergo transport through both the BBB and theneuronal 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 canthen diffuse to the nuclear compartment. The only immunogenic component of theformulation is the MAb and the immunogenecity of murine MAbs in humans canbe eliminated with genetic engineering and ‘humanization’ of the MAb.
β-Galactosidase histochemistry of a rat brain removed 48 h after a single intravenousinjection of a β-galactosidase gene carried by a plasmid that is packaged in the interior of85 mm liposomes.The surface of the liposome is covered by thousands of strands of 2000 Da (PEG), and thisstabilizes 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 tetheredto a monoclonal antibody that targets the transferrin receptor. This receptor is expressedboth on the brain capillary endothelium, which forms the blood–brain barrier in vivo, andon the neuronal plasma membrane. Targeting the immunoliposomes to the transferrinreceptor enables transport across both the blood–brain barrier and the neuronal plasmamembrane in vivo. The use of gene targeting technology enables widespread expression inthe brain of an exogenous gene following a single intravenous administration of a non-viralgene formulation.
Nice presentationAll the credit goes to Dr. Suresh BandariThank you.