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Ilindra Industrial pharmacySlide 2: Introduction: Endogenous proteins and peptides play an important role in the regulation and integration of life processes and act with high specificity and potency. For example, in the form of enzymes, hormones, antibodies and globulins, they catalyze, regulate and protect the body chemistry, while in the form of haemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other chemical substances within the body. Till recently, injections (i.e. intravenous, intramuscular or subcutaneous route) remain the most common means for administering these protein and peptide drugs. Patient compliance with drug administration regimens by any of these parenteral routes is generally poor and severely restricts the therapeutic value of the drug, particularly for disease such as diabetes .Slide 3: Among the alternate routes that have been tried with varying degrees of success are the oral, buccal , intranasal , pulmonary , transdermal , ocular and rectal . Among these, oral route remains the most convenient way of delivering drugs. Designing oral peptide and protein delivery systems has been a persistent challenge to pharmaceutical scientists because of their several unfavorable physicochemical properties : large molecular size, susceptibility to enzymatic degradation, short plasma half-life, ion permeability, immunogenicity, and the tendency to undergo aggregation, adsorption, and denaturation . Consequently, the absolute oral bioavailability levels of most peptides and proteins are less than 1 %. The challenge here is to improve the oral bioavailability from less than 1% to atleast 30-50% .Slide 4: Designing and formulating a protein and peptide drug for delivery though GI tract requires a multitude of strategies. The dosage form must initially stabilize the drug making it easy to take orally. It must then protect the drug from the extreme acidity and action of pepsin in the stomach. In the intestine, the drug should be protected from the plethora of enzymes that are present in the intestinal lumen. Pharmaceutical Approaches: Chemical modification : A chemical modification of peptide and protein drugs improves their enzymatic stability and/or membrane penetration of peptides and proteins. It can also be used for minimizing immunogenicity.Slide 5: Protein modification can be done either by direct modification of exposed side-chain amino acid groups of proteins or through the carbohydrate part of glycoproteins and glycoenzymes .Slide 6: Modifications of individual amino acids combined with the substitution of one more L-amino acid with D-amino acids can significantly alter physiological properties. This was demonstrated by vasopressin analogs 1-deamino-8-D-arginine vasopressin (DDAVP) and [Val4, D-Arg8], arginine -vasopressin ( dVDAVP ), hereafter called desmopressin and deaminovasopressin , respectively. While the former involves deamination of the first amino acid and replacement of the last L- arginine with D- arginine , the latter also has the fourth amino acid changed to valine . Nobex corporation has developed a proprietary insulin compound modified with small polymers (chemical name of the Nobex insulin is hexyl -insulin- monoconjugate 2 or "HIM2"), in which a single amphiphilic oligomer is covalently linked to the free amino group on the Lys-β 29 residue of recombinant human insulin via an amide bondSlide 7: Enzyme inhibitors : The choice of protease inhibitors will depend on the structure of these therapeutic drugs, and the information on the specificity of proteases is essential to guarantee the stability of the drugs in the GI tract. The quantity of co-administered inhibitor(s) is essential for the intestinal permeability of a peptide or protein drug. For example, enzyme degradation of insulin is known to be mediated by the serine proteases trypsin , a- chymotrypsin and thiol metalloproteinase insulin degrading enzymes. The stability of insulin has been evaluated in the presence of excipients that inhibit these enzymes. Representative inhibitors of trypsin and a- chymotrypsin include pancreatic inhibitor and soybean trypsin inhibitor, FK-448, Camostat mesylate and aprotinin .Slide 8: Inhibitors of insulin degrading enzymes include 1,10-phenanthroline, p- chloromeribenzoate and bacitracin reported the use of a combination of an enhancer, sodium cholate and a protease inhibitor to achieve a 10% increase in rat intestinal insulin absorption . Thiomers are promising candidates within as enzyme inhibitors . Another approach to enzyme inhibition is to manipulate the pH to inactivate local digestive enzymes . A sufficient amount of a pH-lowering buffer that lowers local intestinal pH to values below 4.5 can deactivate trypsin , chymotrypsin and elastase .Slide 9: Absorption enhancers : Absorption enhancers are the formulation components that temporarily disrupt the intestinal barrier to improve the permeation of the drugs. Numerous classes of compounds with diverse chemical properties, including detergents, surfactants, bile salts, Ca 2+ chelating agents, fatty acids, medium chain glycerides , acyl carnitine , alkanoyl cholines , N-acetylated α-amino acids, N-acetylated non-α-amino acids, chitosans , mucoadhesive polymers, and phospholipids have been reported to enhance the intestinal absorption of large polypeptide drugs . A bsorption enhancers act as detergents / surfactants to increase the transcellular transport of drugs by disrupting the structure of the lipid bilayer rendering the cell membrane more permeable and/or by increasing the solubility of insoluble drugs .Slide 10: The chelators are believe to exert their action by complex formation with calcium ions, thus rupturing the tight junctions (TJs) and facilitate paracellular transport of hydrophilic drugs.Slide 11: Among the recent absorption enhancers displaying this principle and exhibiting the safest and most effective promising results in enhancing drug delivery is Zonula Occludens toxin or Zot . Zot is a single polypeptide chain of 44.8 kDa , 399 amino acids in length, with a predicted pI of 8.5, of bacteriophage origin, present in toxigenic stains of V. cholerae with the ability to reversibly alter intestinal epithelial TJs, allowing the passage of macromolecules through mucosal barriers . Formulation vehicles : Emulsions protect drug from chemical and enzymatic breakdown in the intestinal lumen. Drug absorption enhancement is dependent on the type of emulsifying agent, particle size of the dispersed phase, pH, solubility of drug, type of lipid phase used etc. the lipid phase of microemulsions is composed of medium chain fatty acids triglycerides increasing the bioavailability of muramyl dipeptides analog .Slide 12: Torisaka et al. have recently prepared a new type of oral dosage form of insulin, S/O/W emulsions, in which a surfactant-insulin complex is dispersed into the oil phase . A critical drawback of this formulation was physical-chemical instability in long-term storage and the requirement for storage at low temperatures . Dry emulsion formulations are typically prepared from O/W emulsions containing a soluble or an insoluble solid carrier in the aqueous phase by spray drying , lyophilization or evaporation . Dry emulsions are regarded as lipid-based powder formations from which an O/W emulsion can be reconstituted . Eiichi Torisaka et al. have developed a unique dry emulsion formulation in which the surfactant-insulin complex was entrapped in the oil phase of the solid formulation. Using a pH-responsive polymer, HPMCP, the dry emulsion was enteric-coated .Slide 13: The influence of pH variability through the stomach to the intestine on the oral bioavailability of peptide and protein drugs may be overcome by protecting them from proteolytic degradation in the stomach and upper portion of the small intestine using pH-responsive microspheres as oral delivery vehicles. Lowman et al ., loaded insulin into polymeric microspheres of poly ( methacrylic -g-ethylene glycol) and observed oral bioavailability in healthy and diabetic rats. In the acidic environment of the stomach, the microspheres were unswollen as a result of the formation of intermolecular polymer complexes. The insulin remained in the microspheres and was protected from proteolytic degradation. While in the basic and neutral environments of the intestine, the complexes dissociated which resulted in rapid microspheres swelling and insulin release.Slide 14: N anoparticles as particulate carriers are used to deliver protein and peptide drugs orally. It is stated that particles in the nanosize range are absorbed intact by the intestinal epithelium, especially, through peyer's patches and travel to sites such as the liver, the spleen and other tissues . The proteins and peptides encapsulated in the nanoparticles are less sensitive to enzyme degradation through their association with polymers . I nsulin was encapsulated in nanospheres using phase inversion nanoencapsulation . The insulin released over a period of appoximately 6 h, was shown to be orally active, and had 11.4% of the efficacy of intraperitoneally delivered insulin . One problem using nanoparticles is the erratic nature of nanoparticles absorption.Slide 15: Liposomes are prone to the combined degrading effects of the acidic pH of the stomach, bile salts and pancreatic lipase upon oral administration . Attempts have been made to improve the stability of liposomes either by incorporating polymers at the liposome surface, or by using GI-resistant lipids . In vitro release of insulin, a model peptide, from liposomes in the bile salts solution was markedly reduced by coating the surface with the sugar chain portion of mucin or polyethylene glycol. Encapsulation of insulin with the sugar chain portion of mucin and that of polyethylene glycol completely suppressed the degradation of insulin in the intestinal fluid, whereas uncoated liposomes suppressed it only partially.Slide 16: Mucoadhesive polymeric systems : Mucoadhesive polymeric systems are the most promising approach among several approaches. Mucoadhesive properties can provide an intimate contact with the mucosa at the site of drug uptake preventing a presystemic metabolism of peptides on the way to the absorption membrane in the gastrointestinal tract. Mucoadhesive polymers are able to adhere to the mucin layer on the mucosal epithelium and thus results in the increase of oral drug bioavailability of protein and peptide drugs . Most of the current synthetic bioadhesive polymers are either polyacrylic acid or cellulose derivatives . Examples of polyacrylic acid-based polymers are carbopol, polycarbophil , polyacrylic acid ( PAAc ), polyacrylate , poly ( methylvinylether -co- methacrylic acid), poly (2-hydroxyethyl methacrylate ), poly( methacrylate ), poly( alkylcyanoacrylate ), poly( isohexylcyanoacrylate ) and poly( isobutylcyanoacrylate ).Slide 17: Cellulose derivatives include carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, methylcellulose, and methylhydroxyethyl cellulose. In addition, seminatural bioadhesive polymers include chitosan and various gums such as guar, xanthan , poly( vinylpyrrolidone ), and poly(vinyl alcohol). The system consists of four layered films contained in an enteric capsule. The backing layer is made of a water-insoluble polymer, ethyl cellulose (EC). The surface layer is made of an enteric pH-sensitive polymer such as hydroxypropylmethylcellulose phthalate, Eudragit L100 or S100 and was coated with an adhesive layer. The middle layer, drug-containing layer, made of cellulose membrane is attached to the EC backing layer by a heating press method.Slide 18: Both drug and pharmaceutical additives including an organic acid, citric acid, and a non-ionic surfactant, polyoxyethylated castor oil derivative were formulated in the middle layer. The surface layer was attached to the middle layer by an adhesive layer made of carboxyvinyl polymer. Structure of proteins : Primary Structure : The primary structure of peptides and proteins refers to the linear number and order of the amino acids present. The designation of the order of amino acids is that the N-terminal end (i.e. the end bearing the residue with the free α-amino group) is to the left (and the number 1 amino acid) and the C-terminal end (i.e. the end with the residue containing a free α-carboxyl group) is to the right.Slide 19: Secondary Structure : Proteins fold into two broad classes of structure termed, globular proteins or fibrous proteins. Globular proteins are compactly folded and coiled, whereas, fibrous proteins are more filamentous or elongated. It is the partial double-bond character of the peptide bond that defines the conformations a polypeptide chain may assume. The α-Helix The α-helix is a common secondary structure encountered in proteins of the globular class. The formation of the α-helix is spontaneous and is stabilized by H-bonding between amide nitrogens and carbonyl carbons of peptide bonds spaced four residues apart. This orientation of H-bonding produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis.Slide 20: Typical α-Helix Not all amino acids favor the formation of the (α-helix due to steric constraints of the R-groups . Amino acids such as A, D, E, I, L and M favor the formation of α-helices, whereas, G and P favor disruption of the helix. β-Sheets β-sheets are composed of 2 or more different regions of stretches of at least 5-10 amino acids. The folding and alignment of stretches of the polypeptide backbone aside one another to form β-sheets, is stabilized by H-bonding between amide nitrogens and carbonyl carbons.Slide 21: β-sheets are said to be pleated. This is due to positioning of the α-carbons of the peptide bond which alternates above and below the plane of the sheet . β-sheets are either parallel or antiparallel . In parallel sheets adjacent peptide chains proceed in the same direction (i.e. the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions. Ball and Stick Representation Ribbon Depiction of β-Sheet of a β-SheetSlide 22: Super-Secondary Structure Some proteins contain an ordered organization of secondary structures that form distinct functional domains or structural motifs. Examples include the helix-turn-helix domain of bacterial proteins that regulate transcription and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic transcription regulators. These domains are termed super-secondary structures. Tertiary Structure : Tertiary structure refers to the complete three-dimensional structure of the polypeptide units of a given protein. secondary structures themselves fold into the three-dimensional form of the protein.Slide 23: Secondary structures of proteins often constitute distinct domains. Therefore , tertiary structure also describes the relationship of different domains to one another within a protein. The interactions of different domains is governed by several forces: These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der Waals forces . Quaternary Structure : Many proteins contain 2 or more different polypeptide chains that are held in association by the same non-covalent forces that stabilize the tertiary structures of proteins . Proteins with multiple polypetide chains are oligomeric proteins. The structure formed by monomer-monomer interaction in an oligomeric protein is known as quaternary structure.Slide 24: Oligomeric proteins can be composed of multiple identical polypeptide chains or multiple distinct polypeptide chains. Proteins with identical subunits are termed homo- oligomers . Proteins containing several distinct polypeptide chains are termed hetero- oligomers . Hemoglobin , the oxygen carrying protein of the blood, contains two α and two β subunits arranged with a quaternary structure in the form, α 2 β 2 . Hemoglobin is, therefore, a hetero- oligomeric protein . Structure of HemoglobinSlide 25: Complex Protein Structures : Proteins also are found to be covalently conjugated with carbohydrates( Glycoproteins ) . These modifications occur following the synthesis (translation) of proteins and are, therefore, termed post-translational modifications . Glycoproteins are of two classes, N -linked and O -linked, referring to the site of covalent attachment of the sugar moieties. N -linked sugars are attached to the amide nitrogen of the R-group of asparagine ; O -linked sugars are attached to the hydroxyl groups of either serine or threonine and occasionally to the hydroxyl group of the modified amino acid, hydroxylysine .Slide 26: Thank you……. You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.