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structure of proteins and barriers affecting protein and peptide drug delivery system


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2 CONTENT Introduction Types of Proteins Structure of protein -Primary structure -Secondary structure -Tertiary structure -Quaternary structure Barriers affecting protein and peptide drug delivery system - Enzymatic Barrier -Intestinal epithelial barrier -Capillary endothelial barrier -Blood brain barrier References

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3 Introduction Proteins are the most abundant organic molecule of the living system. The term protein was derived from a greek word ‘ proteios ’ meaning holding the first place. Proteins on complete hydrolysis yield L- α -amino acids. Therefore proteins are the polymers of L- α -amino acids. Peptides are short chains of amino acid monomers linked by peptide (amide) bonds, the covalent chemical bonds formed when the carboxyl group of one amino acid reacts with the amino group of another.

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4 CLASSIFICATION OF PROTEINS Functional Classification of Proteins Structural protein: Keratin Catalytic protein: Hexokinase Transport protein: Hemoglobin Hormonal protein: Insulin Contractile protein: Actin , Myosin Genetic protein: Nucleoprotein Defense protein: Immunoglobulins

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5 Chemical Classification of Proteins Simple Proteins : Composed of only amino acid residue. e.g: Albumins, Globulins Conjugated Proteins: Besides amino acid they also contain a non-protein moiety. E.g: Nucleoproteins, Glycoprotein c . Derived Proteins : They are denaturated or degraded products of simple or conjugated protein. e.g Proteoses , Peptones, Polypeptides and Peptides.

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6 3. Nutritional Classification of Proteins Complete Protein: These are the proteins having all ten essential amino acids in the required proportion by the human body to promote good growth. Eg. Egg, Milk etc Partially incomplete Protein: These proteins are partially lacking one or more essential amino acid and hence promote moderate growth. Eg. Wheat and Rice protein Incomplete Protein: These proteins completely lack one or more essential amino acid and do not promote growth at all. Eg. Gelatin and Zein .

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7 STRUCTURE OF PROTEIN Primary Structure: This the linear arrangment of amino acids in a protein and the re is presence of covalent linkages such as peptide bond between amino acids. Secondary Structure: Areas of folding or coiling within a protein; examples include alpha helices and pleated sheets, which are stabilized by hydrogen bonding. Tertiary Structure: The final three-dimensional structure of a protein, which results from a large number of non-covalent interactions between aminoacids. Quaternary Structure: Non-covalent interactions that bind multiple polypeptides into a single, larger protein. Hemoglobin has quaternary structure due to association of two alpha globin and two beta globin polyproteins

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9 PRIMARY STRUCTURE Primary Structure - This describes the unique order in which amino acids are linked together to form a protein. Proteins are constructed from a set of 20 amino acids. Generally, amino acids have the following structural properties: A carbon (the alpha carbon) bonded to the four groups below: A hydrogen atom (H) A Carboxyl group (-COOH) An Amino group (-NH 2 ) and A "variable" group or "R" group All amino acids have the alpha carbon bonded to a hydrogen atom, carboxyl group, and amino group. The "R" group varies among amino acids and determines the differences between these protein monomers.

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10 SECONDARY STRUCTURE The conformation of polypeptide chain by twisting or folding is referred to as secondary structure. There are two types of secondary structure: α helix β sheet. The α Helix : α helix is the most common spiral structure of protein. The structure was proposed by Pauling and Corey. The α helix is stabilized by extensive hydrogen bondings. Hydrogen bond is formed between H atom attached to peptide N and O atom attached to peptide C.

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12 All the peptide bonds, except the first and last in a polypeptide chain, participate in hydrogen bonding. Each turn of α -helix contains 3.6 amino acids and travels 0.54 nm. The spacing of each amino acid is 0.15nm. Certain amino acids disrupt the α -helix. E.g.- Proline Proline OH O H H 2 N + C C H 2 C CH 2 H 2 C

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α - helix has two helical structure Right handed Left handed 13

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14 A regular element of secondary structure in proteins, in which two or more extended strands of the polypeptide chains lie side by side (running either parallel or antiparallel). These strands are held together by a regular array of hydrogen bonds between backbone NH and C=O groups, to form a ridged planar surface. The amino-acid side chains alternately face to the opposite sides of the sheet. Adjacent polypeptide chains in a β sheet can be either parallel or antiparallel . β S heet:

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The β conformation of polypeptide chains. 15 C-terminal C-terminal N-terminal

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16 C-terminal C-terminal C-terminal

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17 TERTIARY STRUCTURE The tertiary structure of a protein is a description of the complex and irregular folding of the peptide chain in three dimensions. These complex structures are held together by a combination of several molecular interactions that involves the R-groups of each amino acids in the chain. These interactions include : Hydrogen bonds between polar R- groups Ionic bonds between charged R-groups Hydrophobic interactions between nonpolar R-groups Covalent bonds: The R-group of the amino acid cysteine contains a sulfur atom and this sulfur atom is capable of forming a covalent bond with another sulfur atom on a different cysteine molecule somewhere else on the chain. This bond is known as a disulfide bond and it acts as to stabilize the tertiary structure of those proteins that have such bonds.

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18 Spiral ribbons → α -helices Flat arrows → β -sheet Red color → Key functional group Yellow color → Disulfide bond

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19 QUATERNARY STRUCTURE Some proteins contain two or more separate polypeptide chains, or subunits, which may be identical or different. The arrangement of these protein subunits in three-dimensional complexes constitutes quaternary structure. The monomeric subunit are held together by non-covalent bonds namely hydrogen bonds, hydrophobic interactions, and ionic bonds. These proteins play significant role in the regulation of metabolism and cellular function. E.g.- Deoxyhemoglobin, aspartate transcarbomylase, lactate dehydrogenase.

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21 BARRIERS TO PROTEIN AND PEPTIDE DRUG DELIVERY INTRODUCTION: A barrier is a physical structure which blocks or impedes something. The successful delivery of proteins and peptides based pharmaceuticals is primarily determined by its ability to cross the various barriers. Barriers to proteins and peptide drug delivery are: Enzymatic Barriers Intestinal epithelial barriers Capillary endothelial barriers Blood brain barriers

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22 ENZYMATIC BARRIERS: Enzymatic barrier is the most important barrier that limits the absorption of protein and peptide drug from GIT. The enzymatic degradation is brought about by two ways: Hydrolytic cleavage of peptide bonds by proteases, such as insulin degrading enzyme, angiotensin converting enzyme and renin. Chemical modification of protein such as- phosphorylation by kinases, carbamylation of proteins, degradation by chymotrpsin.

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23 DENATURATION: The phenomenon of disorganization of proteins is called denaturation. Denaturation results in the loss of secondary, tertiary and quaternary structure of the proteins. But the primary structure of the protein remains undisturbed. Various solvents, salts of heavy metals, urea and salicylates are the agents responsible for denaturation. UBIQUITINATION: Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Ubiquitination is a process in which the ubiquitin protein gets attached to the subtrate protein and marks it for degradation.

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24 INTESTINAL EPITHELIAL BARRIER There are several mechanism that are involved in the transport of protein/peptide drug across the intestinal epithelium. 1. Passive and carrier mediated transport Stereoisomerism, side-chain length and N- and C-terminal substitution are reported to affect dipeptide absorption. L-Ala-L-phe and L-Leu-L-Leu were found to absorb more rapidly than their D-isomers. Acidic or basic dipeptides have lower affinity compared to neutral dipeptides for peptide transport.

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25 2.Endocytosis It is an energy using process by which cells absorb molecules by engulfing them. With the help of this process cellular internalization of protein and peptides takes place. There are different pathways for endocytosis. Phagocytosis: Phagocytosis is a specific form of endocytosis which involves the vesicular internalization of solids. Pinocytosis: Pinocytosis is a mode of endocytosis in which small particles are brought into the cell, forming an invagination, and then suspended within small vesicles. Receptor mediated endocytosis: Receptor-mediated endocytosis (RME), is a process by which cells internalize molecules by the inward budding of plasma membrane vesicles containing proteins with receptor sites specific to the molecules being internalized

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27 3.Transcytosis A mechanism for transcellular transport in which a cell encloses extracellular material in an invagination of the cell membrane to form a vesicle (endocytosis), then moves the vesicle across the cell to eject the material through the opposite cell membrane by the reverse process (exocytosis). The transport mechanism by which most proteins reach the Golgi apparatus or the plasma membrane.

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28 4.Paracellular movement Paracellular transport refers to the transfer of substances across epithelium by passing through the intercellular space between the cells. It is in contrast to transcellular transport, where the substances travel through the cell, passing through both the apical membrane and basolateral membrane. The paracellular pathway allows the permeation of hydrophilic molecules that are not able to permeate through the lipid membrane for the transcellular pathway of absorption. However, this is only applicable for small molecules, as larger molecules will not be able to fit through the pores in the tight junctions. Thus ,the passage of water across the tight junction is capable of carrying the dissolved drug or facilitate the transport of macromolecules.

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29 CAPILLARY ENDOTHELIAL BARRIER To cross the capillary endothelium ,the protein/peptides must pass between the cells or alternatively traverse across the endothelial cell themselves. There are two types of cappilaries Fenestrated capillaries have pores in the endothelial cells (60-80 nm in diameter) that are spanned by a diaphragm of radially oriented fibrils and allow small molecules and limited amount of proteins to diffuse. Sinusoidal capillaries are a special type of fenestrated capillaries that have larger openings (30-40 μm in diameter) in the endothelium. These types of blood vessels allow red and white blood cells (7.5μm - 25μm diameter) and various serum proteins to pass through using a process that is aided by a discontinuous basal lamina.

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30 Solutes that traverse the endothelial cell membrane may get modified or metabolized by cytoplasmic enzymes. Thus, the endothelial passage poses metabolic or enzymatic barrier to the solute passage. Due to this, the failure of circulating dopamine, ammonia and fatty acids occur to enter brain. While endothelial tight junction also serves as a major extracellular barrier to solute exchange, these junctions in general hamper the transcapillary movement of macromolecular tracers injected into plasma or interstitial fluid.

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31 Mechanism for solute transit Carrier mediated transport In the brain cappilary endothelium eight independent nutrient transport system have been indentified: - hexose carrier - moncarboxylic carrier - neutral amino acid carrier - carrier for lysine, arginine, ornithine - choline carrier - adenine/guanine carrier - port for purine and uracil nucleosides - carrier for aspartic and glutamic acid Of all the carriers mentioned above, only neutral amino acid carrier holds greater interest with regard to drug delivery.

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32 2. Receptor mediated transport Endothelial receptor mediated transport often exists for various polypeptides including insulin and transferin The endothelial barrier is modulated by several physiological parameters. Angiotensin, bradykinin, histamine and serotonin increases vascular permeability by opening large gaps in the endothelial junctions or post cappilary venules. Inflammatory agents, vasopressive agents, certain hormones and hypertensive agents relax the endothelial barrier.

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33 BLOOD BRAIN BARRIER The blood brain barrier (BBB) is a dynamic interface that separates the brain from the circulatory system and protects the central nervous system (CNS) from potentially harmful chemicals while regulating transport of essential molecules and maintaining a stable environment. Endothelial cells restrict the diffusion of microscopic objects (e.g., bacteria) and large or hydrophilic molecules into the cerebrospinal fluid (CSF), while allowing the diffusion of small hydrophobic molecules (O 2 , CO 2 , hormones).

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34 Circulatory molecules with different transport mechanism across BBB. Lipophilic molecule – Lipid mediated transport Acidic drugs, peptides and highly Lipophilic drugs – Plasma protein mediated transport. Bulk flow transcytosis could be pinocytocis or tubulocanalecular molecular transport. This transport system is minimal with regards to activity profile under physiological conditions but may turn out to be significant under certain pathological circumstances. Carrier-mediated transport enables molecules with low lipid solubility to traverse the blood-brain barrier. Glucose from blood enters the brain by a transport protein. Glucose is the primary energy substrate of the brain.  Glucose transport protein (GLUT-1) is highly enriched in brain capillary endothelial cells.  These transporters carry glucose molecules through the blood brain barrier.

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35 Specific nutrient transport system could be utilized for brain capillary wall permeation in order to deliver the therapeutic agents selectively to the CNS. Most classical example is the systemic treatment of parkinson’s disease using L-DOPA, a metabolic precursor of dopamine. This utilizes neutral amino acid transport system. The permeability can also be increased by intra arterial injection of hyper osmolar solution which disrupts inter- endothelial tight junctions. This approach was used with success to deliver antibodies to brain. Cationization using hexamethyldiamine or anionization by succinylation could enhance the uptake of protein in brain.

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36 REFERENCES Vyas S.P. And Khar Roop K., “Controlled Drug Delivery Concepts and Advances”; Vallabh Prakashan , Delhi. Page no. 503-570. Satyanarayana U., Chakrapani U., “Biochemistry”, 3 rd edition, 2011, Books and Allied (P) Ltd, Kolkata, page no 43-59. Ratnaparkhi M.P.,* C haudhari S.P., P andya V.A, P eptides and Proteins in Pharmaceuticals, International J ournal of Current P harmaceutical Research,vol 3, issue 2, 2011 . Richard A. Hawkins ,Robyn L. O'Kane,Ian A. Simpson and Juan R. Viña , Structure of the Blood–Brain Barrier and Its Role in the Transport of Amino Acids, J. Nutr . January 2006 vol. 136 .

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