logging in or signing up LUNGS targeted drug delivery systems monika29390 Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: Embed: Flash iPad Copy Does not support media & animations WordPress Embed Customize Embed URL: Copy Thumbnail: Copy The presentation is successfully added In Your Favorites. Views: 1058 Category: Education License: All Rights Reserved Like it (0) Dislike it (0) Added: April 20, 2012 This Presentation is Public Favorites: 4 Presentation Description No description available. Comments Posting comment... By: phani.t (10 month(s) ago) my mail id is email@example.com Saving..... Post Reply Close Saving..... Edit Comment Close By: phani.t (10 month(s) ago) if eu dont mind,pls send me eur ppt Saving..... Post Reply Close Saving..... Edit Comment Close By: ali2008 (12 month(s) ago) okkkkkkkkkkkkkk very gooood Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript PowerPoint Presentation: Modern College of Pharmacy (For Ladies) Moshi, Pune. LUNGS TARGETED DRUG DELIVERY SYSTEMS Delivered by, Research supervisor, Miss. Monika Chavanke Dr. Kuldeep Ramteke M.Pharm. (I I Sem.) Assistant Professor Dept of Pharmaceutics Dept of Pharmaceutics Modern College of Pharmacy (For Ladies) Moshi, Pune .PowerPoint Presentation: CONTENT Anatomy of lungs Receptors present in lungs Advantages and Disadvantages Optimal site of deposition for treatment of lung diseases Barriers to macromolecule to absorbed Mechanisms of particle deposition in the airways: Different system used to target lungs Methods used to identify the deposition of inhaled products Ex vivo models of drug absorption across the lungs Applications Marketed preparations Conclusion References 2PowerPoint Presentation: Gross anatomy of lung Lungs are a pair of respiratory organ situated in thoracic cavity. Right and left lungs are separated by mediastinum, texture – spongy color- young brown, adults - mottled black due to deposition of carbon particles. weight- right lung – 600gm, left lung-550 gm 7PowerPoint Presentation: CONT…… Covered by a double layer of membrane that makes movement in the thoracic cavity easier. The outer layer is the parietal pleura and the inner layer is the visceral pleura. The space between the two pleurae is called the pleural cavity Consist of an apex (top section), hilum (middle section) and base (lower section) 8PowerPoint Presentation: A schematic of airway branching in the human lung. 9PowerPoint Presentation: Receptors present in lung ronchopulmonary Dysplasia COPD Copy of Asthma Cryptogenic Organizing Pneumonia (COP) Cystic Fibrosis Rapidly adapting lung stretch receptors: often referred to as cough or irritant receptors. SO ₂ , NH ₂ , cigaratte smoke. Pulmonary and bronchial C- fibre receptors : bradikinin , ether, halothane, histamine Cystenyl leukotrienes receptors: Montelukast , zafirlukast (antagonist) β₂ receptors: β₂ agonist like salbutamol , terbutaline (bronchodilators) Histamine receptors: large airways Muscarinic receptors: Ml receptors: pirenzepine M2 receptors: gallamine M3 receptors: hexahydrosiladifenidol 7. Taste receptors: open the airways, warn against poisons 10advantages in delivering drugs to the lungs: advantages in delivering drugs to the lungs Improve efficacy Reduce unwanted systemic side effects, Large surface area for absorption, Thin alveolar epithelium permitting rapid absorption, Absence of first-pass metabolism, Rapid onset of action High bioavailabity . It is needle free pulmonary delivery. It requires small and fraction of oral dose. Low concentration in the systemic circulation is associated with reduced systemic side effects. Avoidance of gastrointestinal 11DISADVANTAGES OF PULMONARY DRUG DELIVERY: DISADVANTAGES OF PULMONARY DRUG DELIVERY 1) Oropharyngeal deposition gives local side effect. 2) Patient may have difficulty using the pulmonary drug devices correctly 3) Drug absorption may be limited by the physical barrier of the mucus layer. 4) Various factors affect the reproducibility on drug delivery on the lungs, including physiological and pharmaceutical barrier. 5) The lungs are not only accessible surface for drug delivery complex but also delivery devices are required to target drug delivery. 12PowerPoint Presentation: . The therapeutic effect of therapies is dependent upon the dose deposited and its distribution within the lung. Large airways bronchi Bronchioles alveoli β 2 adrenergic receptors present in high density in the airway epithelium from the large bronchi to the terminal bronchioles. Airway smooth muscle has a lower β-receptor density, greater in the bronchioles than bronchi . Optimal site of deposition for treatment of lung diseases 13PowerPoint Presentation: For a macromolecule to be absorbed from the lung into the blood it must pass through a number of dynamic barriers in the following order. Surfactant: The molecular monolayer that spreads at the air/water interface is may cause large molecules to aggregate which could enhance engulfment and digestion by airspace macrophages. Surface lining fluid : Once through the surfactant layer a macromolecule must dissolve in the surface lining fluid that lies over the epithelium. This fluid acts as a reservoir for lung surfactant and appears to contain many of the components of plasma. 15PowerPoint Presentation: i . . Epithelium: The most significant barrier to absorption, this simple layer of cells varies from thick columnar cells in the airways to extremely thin and broad cells in the alveoli Interstitium and basement membrane: The interstitium is the extracellular space inside tissues. Epithelial and endothelial cells are attached to a tough but thin layer of interstitial fibrous material known as the basement membrane Vascular endothelium: The final barrier to systemic absorption is another monolayer of cells that make up the walls of small blood and lymph vessels. The permeability of this second cell barrier varies with the type of blood vessel but even the tightest regions are thought to be more permeable to macromolecules than pulmonary epithelium. 16PowerPoint Presentation: Mechanisms of particle deposition in the airways: Sedimentation:- By the settling under gravity the particles may deposited. It becomes highly important for particles that reach airways where the airstream velocity is relatively low, e.g. the bronchioles and alveolar region. The fraction of particles depositing by this mechanism it may dependent upon the time the particles use in these regions. Brownian diffusion : Is also more common in regions where airflow is very low or absent, e.g. in the alveoli. This mechanism is depend on local respiratory track region geometry, particle diameter, air stream characteristics. 17PowerPoint Presentation: Inertial impaction: This is the main deposition mechanism for particles >10 μm in the upper tracheobronchial regions. particles with higher densities or larger diameter and those travelling in airstreams of higher velocity will show superior impaction Electric charge effect: During mechanical generation of aerosol electric charge may be produced on particles. charge may be of two types Image charge field: is due to response between charges of opposite sign on airway surface and particle that create attraction and deposition. space charge force: is the repulsion between like charge inhaled particles that may direct their motion toward airway walls resulting in deposition . 18PowerPoint Presentation: Particles bigger than 10 μm will have impact in the upper airways and are rapidly removed by Swallowing, coughing and muco ciliary processes. The particles in the size range 0.5–5 μm may break away from impaction in the upper airways and may deposit by sedimentation and impaction in the lower TB and A regions. If the aerosol particle size is between the 3 and 5 μm then deposition it mainly occur in the TB region. If the particles are smaller than the 3 μm then appreciable deposition in the A region is likely to occur Interception: mainly for aggregates and fibers. Interception occurs when a particle contacts an airway surface due to its physical size or shape. Unlike impaction, particles that are deposited by interception do not deviate from their air streamlines. Interception is most likely to occur in small airways or when the air streamline is close to an airway wall. 19Different mechanism by which drug absorbs : Different mechanism by which drug absorbs Passive Diffusion : T he diffusion rate is both compound specific and region selective. Absorption of lipophilic-transcellular diffusion. Hydrophilic compounds- paracellular diffusion. lipophilic drugs-absorbed rapidly and hydrophilic drugs more slowly. Drug Transporters : Transporters of the solute carrier (SLC) family facilitate transport across the cell membrane and most commonly enhance uptake of compounds into cells. counter-transport of another ion or the membrane potential Eg : organic anion transporters (OAT and OATP) and organic cations transporters (OCT) 20PowerPoint Presentation: ABC transporters: Eg : multidrug-resistance proteins (MDR), multidrug-resistance associated proteins (MRP), and breast cancer resistance protein (BCRP). Vesicle-Mediated Transport: Membrane vesicles within the alveolar epithelial type I and type II cells involved in macromolecule transport across the alveolar epithelium, caveolae present in the alveolar type I cells. The main route is through transcytosis , Involving caveolae and clathrin coated pits. Fig: Schematic of drug transporters in lung cells 21PowerPoint Presentation: Different system used to target lungs Controlled drug delivery to lungs: Microsphere DENDRIMER Liposome nanoparticles Gene delivery using polymeric nano-carriers Peptide-Mediated delivery 22PowerPoint Presentation: Liposome can also be produced with a wide range of size and will incorporate both hydrophilic and lipophilic drugs. The drug carrying capacity, release rate and deposition of liposome in the lungs is dependent upon lipid composition, size, and charge and drug/lipid ratio. Most liposomal carrier–drug systems have been investigated in aqueous systems using nebulisation. DPI liposomal formulations have been produced by lyophilisation followed by milling or spray drying. Encapsulation of drugs in liposome delivered to the lungs results in increased residence time and/or a decrease in toxic side effects. Liposomes 23Microparticles:: Microparticles: Microparticles are hollow spherical particles in which drugs encapsulated. Microparticle are in the 0.1–500μm range. Microparticles are physically and chemically more stable than liposomes , allowing higher loading of drugs. synthetic polymers - polylactic acid (PLA) - polylactic -co-glycolic acid (PLGA). Natural polymers - albumin, gelatin, chitosan and dextran. Coating can be used to alter the properties in vivo. Such as mucoadhesive polymers - chitosan and hydroxypropylcellulose increase the residence time of peptide drug carriers in the lungs. 27Micelles:: Micelles: Drugs can be trapped in the core of a micelle and transported at concentrations even greater than their intrinsic water solubility. A hydrophilic shell can form around the micelle, effectively protecting the contents. The advantages of polymeric micelles include better stability than surfactant micelles, ability to solubilize an adequate amount of hydrophobic drugs, prolonged circulation times in vivo, and capability to accumulate in the target organs. 28Nanoparticles:: Nanoparticles: Nanoparticles use for both dispersed liquid droplet dosage forms such as metered dose inhalers and nebulizers, and dry powder formulations. constituted of polymers or lipids and drugs bound either at the surface of the particles either encapsulated into the vector. Traditional techniques such as spray drying and grinding, and more recent advances in supercritical fluid extraction, precipitation, and solvent extraction have been employed to produce nanoparticle formulations for pulmonary delivery 2 29Solid lipid nanoparticles (SLN):: Solid lipid nanoparticles (SLN): Three possible loadings of drugs can be envisaged: ( i ) Dispersion of drugs into the particle; (ii) core-membrane model containing a membrane improved with the drug; (iii) core-membrane model containing a core improved with the drug. In vitro release studies have showed that an encapsulated drug into solid lipid nanoparticles can diffuse during a period of time ranging from 5 to 7 weeks. Drugs like prednisolone , diazepam and camptotecin have been incorporated into SLN for pulmonary applications. 2 31Dendrimer-based nanoparticles for lung delivery: Dendrimer -based nanoparticles for lung delivery Dendrimers are polymers, which have hyperbranched structures, with layered architectures. Dendrimers are globular repeatedly branched macromolecules that exhibit controlled patterns of branching with multiple arms extending from a central core. They are used in drug delivery and imaging at a size typically ranging from 10 to 100 nm in diameter. Encapsulation of therapeutic agents, particularly peptide therapeutics. 32PowerPoint Presentation: 33PowerPoint Presentation: Methylprednisolone (200 mg) + mixture stirred for 72 h while the precipitation of dicyclohexylurea (DCU) . filtered to obtain a clear solution. The solvent was evaporated to obtain methylprednisolone-glutarate . 2) conjugate from the previous reaction dissolved in anhydrous dimethyl sulfoxide +DCC (110 mg) +N- hydroxy benzotriazole ( HOBt ). stirred for 45 min to activate the unreacted carboxylic acid group of the MP–GA conjugate. the precipitate of DCU was observed. The reaction mixture was filtered to remove DCU. The filtrate was then dialyzed against DMSO for three days by replacing DMSO daily (dialysis membrane cut-off 1000 Da ) to remove unreacted compounds. The dialyzed product was dried under vacuum to obtain the conjugate.Microspheres: Microspheres Both targeted and sustained drug release Hydrophilic and lipophilic drugs can be entrapped or incorporated Albumin microspheres: These are biodegradable colloidal particles that can be prepared by either physical denaturation or chemical cross-linking of albumin droplets. The coating of albumin MS with surfactants could decrease the interaction with mucus layer and possibly increase the deposition in the lower airways Particle size range between 1.94 ± 1.47 and 3.42 ±1.51 μm have been prepared using a high-speed homogenization and heat denaturation process. Poly ( glycolide and/or lactide ) (PGL) microspheres: These are prepared by the polymerization of lactide and/or glycolide monomers via polyester linkages, the hydrolysis of which will result in the breakdown of the polymers to produce non-toxic substances. 35Aerosol: Aerosol Advantages: Administration of drug is easy. Dose can be removed without contamination. The medication can be delivered direct to affected area such as spray, Stream quick breaking foam or stable foam. Irritation produce by mechanical application of topical medication is reduced or eliminated. Aerosol administration gives very efficient and quick relief. Component of aerosol formulation: Propellant Product concentrate 36Drug contained in MDI: Drug contained in MDI In this technique, a medication is mixed in a canister with a propellant, and the preformed mixture is expelled in exact measured amounts upon actuation of the device. Correct use of MDIs requires that patients learn how to organize exhalation and inhalation with actuation of the device. 37Nebulizers: Nebulizers deliver atomized aqueous drug solution by air jet or ultrasonic mechanisms, typically delivered continuously over multiple breaths. Used mostly in hospital or ambulatory care settings for elderly, infant, or critically ill patients In jet nebulizers, an aerosol is prepared by a high velocity air stream from a pressurized source directed against a thin layer of liquid solution. Ultrasonic nebulizers include the vibration of a piezoelectric crystal aerosolizing the solution. Air jet nebulizers 38DPI(Dry Powder Inhalers): DPI( Dry Powder Inhalers ) used micronized medication blended with a large lactose-based carrier. With no propellant DPIs have relied on the force of patient inhalation for delivery, which has limited their use in patients with compromised lung capacity or compliance challenges, including children. Lactose blends DPIs also have low lung deposition efficiency, and the delivered dose is dependent upon a patient generated flow rate 39protein and peptide delivery: protein and peptide delivery The vast majority of protein-based pharmaceuticals are given parenterally (including intravenous, intramuscular, subcutaneous, and intraperitoneal injections). Many are delivered as solutions with the exception of recombinant vaccines and insulin. approaches for improving stability is the use of freeze-drying 40Examples of Proteins/Peptides for Inhalation: Examples of Proteins/Peptides for Inhalation Disease State Peptide/Protein Adult Respiratory Distress Syndrome Surfactant Proteins (approved) Cystic fibrosis (CF) DNase (approved) Emphysema/CF Alpha-1-antitrypsin Secretory leukoprotease inhibitor Lung transplant Cyclosporin A Cancer/Pneumocystis carnii Interferon-γ Interleukin-2 Osteoporosis Calcitonin Parathyroid hormone Anemia Erythropoetin Diabetes insipidus dDAVP (1-deaminocysteine-8- D- arginine vasopressin) 42Targeted gene delivery to the lung: Targeted gene delivery to the lung In pulmonary gene therapy, the nucleic acid cargo needs to be delivered to cells in the target region of the lung. . Successful gene therapy for many diseases requires appropriate levels of transgene expression in specific cell types to enhance treatment efficacy and to avoid unwanted side effects caused by expression in inappropriate cell types. For clinical success, gene vectors need to be delivered in a specific manner to the target region of the organ (first order) and to the specific cell types (second order) in the target region. Once inside the specific cells, the gene vectors need to enter the nucleus for expression (third order, intracellular). Inside the nucleus, the episomal vectors need to be addressed to the nuclear matrix for sustained expression and/or episomal replication. For integrating vectors, the target sites for integration must be accessible and support transgene expression 43gene delivery by physical methods: gene delivery by physical methods Generally, the efficiency of gene transfer systems is limited by three major impediments: insufficient enrichment of the applied gene vector at the target tissue; its intracellular trafficking; and Nuclear uptake and intranuclear trafficking to active sites of transcription either with or without genomic integration. Magnetofection : Magnetic targeting of gene delivery is achieved by application of a magnetic field to superparamagnetic iron oxide particles associated with the gene vectors. Electroporation : The method allows specific targeting in that the area to which the gene is transferred is delineated by the positioning of the electrodes Sonoporation : 44PowerPoint Presentation: Technique principle Material Advantages Limitations Magnetofection Magnetic field assisted transport into cells Magnetic nanoparticles ± transfection reagent, magnetic field High efficiency Economic Applicable to viral and non-viral vectors Magnetic gradient field strongly decreases with distance Electroporation Electric field induced cell membrane permeabilisation and/or electrophoretic mobility Electrodes and pulse generator High efficiency No gene transfer agent needed Toxicity, invasiveness (electrodes) Sonoporation Ultrasound induced cell membrane permeabilisation Ultrasound probe ± gas microbubbles Imaging during treatment New microbubble formulations Efficacy, toxicity to be established 45Biological targeting of specific lung cells: Biological targeting of specific lung cells Non-viral systems viral vector systems Retroviruses Adeno viruses Adeno-associatd viruses 46PowerPoint Presentation: Non-viral systems : cationic polymers such as PEI, dendrimers , or carbohydrate-based polymers such as chitosan. cell transfection . lactoferrin as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells was investigated. 2. viral vector systems: Retroviruses: Adenoviruses: Adeno-associated viruses: Retroviruses: Moloney murine leukaemia virus (MLV)-based vectors were the first type of recombinant retrovirus used for gene delivery. long-term safety studies in animal models will be necessary before the pulmonary applications of these vectors can be investigated in humans. 47PowerPoint Presentation: 2.Adenoviruses: Adenovirus-based (Ad) vectors have been the most extensively studied recombinant viral systems for gene transfer. adenoviruses replicate as episomal elements in the nucleus of the host cell and consequently there is minimal risk of insertional mutagenesis The first generation Ad vectors ( FGAd ) with the E1 region deleted have been the most extensively used vector for pulmonary gene transfer. Various strategies have been proposed to improve adenoviral entry into airway epithelia, including calcium phosphate co-precipitates, EGTA, EDTA, polycations , polidocanol , sodium caprate , l-α- lysophosphatidylcholine (LPC) and other agents.. 48PowerPoint Presentation: Adeno-associated viruses : Adeno-associated viruses (AAV) are members of the Parvovirus family, dependent on a helper virus, usually adenovirus, to proliferate. They are capable of infecting both dividing and non-dividing cells, and in the absence of a helper virus integrate into a specific point of the host genome at a high frequency. Total length inserted no more than 4.7kb Main interest is due to lack of pathogenisity. ability to transduce non-dividing cell. Prolong the trans gene expression. 49Methods used to identify the deposition of inhaled products: Methods used to identify the deposition of inhaled products Fluorescence/bioluminescence imaging systems for pulmonary gene delivery: The pulmonary delivery and the gene expression can be evaluated using fluorescence of indocyanine green (ICG) as a fluorescent label and thedetection of luciferase activity, respectively, by using a nondestructivereal time in vivo imaging system 50Gamma scintigraphy: Gamma scintigraphy a) Two-dimensional gamma scintigraphy: radiolabelled Teflon particles. usually 99 m Technetium. If two inhalation products deliver the same amount of drug and have similar whole lung and regional deposition patterns then their clinical effect within the airways should be the same. ( b) SPECT (single photon emission computed tomography): It is similar to two-dimensional gamma scintigraphy except that the gamma camera rotates through 360⁰. This allows a full three-dimensional reconstruction of the lungs 51PowerPoint Presentation: Takes much longer so the radiation dose has to be higher. The technique is difficult to apply to multidose inhaled products. The increased imaging time may affect the actual distribution due to mucociliary clearance, coughing and absorption into the systemic circulation. (c) PET (Positron emission tomography): It is now possible to directly incorporate a radiolabel into the drug molecule. The ones recently used are positron emitters such as 11 C or 18 F. Disadvantages: the positron emitters used so far have short half-lives and the method is very expensive. 52PowerPoint Presentation: One of the most commonly used ex vivo methods is the isolated perfused lung model (IPL). The lungs are isolated from the body of an animal (generally rats or guinea pigs) and housed in an artificial system. Peristaltic pumps carry perfusate ( autologous blood or a buffered artificial medium equilibrated with a mixture of oxygen and carbon dioxide) to and from the organ which is housed at 37 °C and the system can be maintained at a specific pressure. Drugs/delivery systems are administered via the tracheal port. Sampling of the perfusate allows the rate of absorption of the drug to be determined. Limitations :short viable periods of 2–3 h, the likely absence of tracheo –bronchial circulation and the high level of training and expertise required to set-up the IPL. 55Animal models: Animal models Rodents such as mice, rats and guinea pigs Rats and guinea pigs are frequently used because a variety of dosing techniques that require a small amount of the drug can be employed and they are good models for a number of respiratory disease states. Biology of the guinea pig have revealed that the physiology of their pulmonary tract is quite similar to that of humans, particularly the response of the lung to inflammatory stimuli as well as the dermal response to both acute and chronic inflammatory mediators. Therefore guinea pigs are regarded as a good model of bronchoconstriction / bronchodilation in the evaluation of drugs used to treat asthma. 56Methods of aerosol administration IN ANIMALS: Methods of aerosol administration IN ANIMALS 1.Direct administration methods: It include liquid instillation, spray instillation and dry powder insufflations. In general, they involve visualization of the trachea of the animal with the help of a laryngoscope to place a thin stainless steel tube in the trachea, near the carina, to administer drugs. For small rodents, the procedure is straight-forward but for large animals, including rabbits, dogs, pigs and monkeys, some surgical procedures such as tracheotomy may need to be performed to accommodate the device 57Cont….: Cont…. For spray instillation, the MicroSprayer : The atomizer in the tip of a long, thin, stainless steel tube generates a plume of liquid aerosol that can be deposited in the airways and deep lung. The droplet size is influenced by the physicochemical characteristics, such as viscosity of the solutions/ suspension to be delivered. 58Passive inhalation: Passive inhalation Deliver aerosolized drugs to conscious animals by passive inhalation employ exposure chambers that can be for whole-body, head-only and nose-only. A modified whole-body exposure chamber was made using a conventional metabolism chamber with a polypropylene tube inserted to restrain the animal. This chamber has the advantage of a small volume sufficient for the animal to receive a large dose of aerosol based on its inspiratory flow. The main advantage of head-only and nose-only exposure systems is the reduction or elimination of multiple exposure routes. Major disadvantages include adequate exposure seals for the face or neck of the animal and stress related to the restraint necessary for these studies 60PowerPoint Presentation: (B) The nose-only exposure chamber A) The Modified Whole-Body exposure chamber 61CURRENT APPLICATIONS OF PULMONARY DRUG DELIVERY: CURRENT APPLICATIONS OF PULMONARY DRUG DELIVERY 1)Application of pulmonary drug delivery in Asthma and COPD. 2) Recent role pulmonary delivery in Patients on ventilators 3) Pulmonary delivery in cystic fibrosis 4) In migraine 6) Angina pectoris 7) Recent use of pulmonary drug delivery in Transplantation 8) In emphysema 10) In Pulmonary arterial hypertension 11) In acute lung injury 62References: References 1. N.R. Labiris , M.B. Dolovich , Pulmonary drug delivery, Part I: Physiological factors affecting therapeutic effectiveness of aerosolized medications, 2003;65-72. 2. J. S. Patton, C. S. Fishburn and J. G. Weers , The Lungs as a Portal of Entry for Systemic Drug Delivery, Proceedings of the american thoracic society, 2004; 338–344. 3 . J. S. Patton, C. S. Fishburn , and J. G. Weers , The Lungs as a Portal of Entry for Systemic Drug Delivery , Proceedings of the american thoracic society, 2004; 340-352. 4. J. Heyder , Deposition of inhaled particles in the human respiratory tract and consequences for regional targeting in respiratory drug delivery, Proc Am Thorac Soc, 2004; 315–320. 5. B. V. Wichert, R.J. Gonzalez- Rothi , Amikacin liposomes : characterization, aerosolization and in vitro activity against Mycobacterium avium-intracellulare in alveolar macrophages, Int. J. Pharm , 78 ,1992; 227-235.PowerPoint Presentation: THANK YOU You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.