PEPTIDE AND PROTEIN DRUG DELIVERY

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PEPTIDE & PROTEIN DRUG DELIVERY K.JAYA RAJ KUMAR AMIST UNIVERSITY MALAYSIA

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DEFINITIONS Peptide: A short chain of amino acid residues with a defined sequence ( e.g.leuprolide ). Protein : polypeptides which occur naturally and have a defined sequence of amino acids and a three-dimensional structure (e.g. insulin). Polypeptide: A longer amino acid chain, usually of defined sequence and length (e.g. vasopressin).

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STRUCTURE OF PEPTIDES AND PROTEINS Proteins have in increasing order of complexity (Figure ) : Primary structure – the order in which the individual amino acids are arranged. Secondary structures – including coiled α-helix and pleated sheets. Tertiary structure – the three-dimensional arrangement of helices and coils. Quaternary forms – the association of ternary forms (e.g. the hexameric form of insulin). Loss of the unique tertiary or quaternary structure, through denaturation , can occur from a variety of insults that would not affect smaller organic molecules. Formulations must preserve the protein structure. Keyword

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HYDROPHOBICITY OF PEPTIDES AND PROTEINS Amino acids have a range of physical properties, each having a greater or lesser degree of hydrophilic or hydrophobic nature. If amino acids are spatially arranged in a molecule so that distinct hydrophobic and hydrophilic regions appear, then the polypeptide or protein will have an amphiphilic nature.

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SOLUBILITY OF PEPTIDES AND PROTEINS The aqueous solubilities of proteins vary enormously, from the very soluble to the virtually insoluble. The solubility of globular proteins increases as the pH of the solution moves away from the isoelectric point (IP), which is the pH at which the molecule has a net zero charge (Figure ).

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At its IP a protein has a tendency to self-associate. As the net charge increases, the affinity of the protein for the aqueous environment increases and the protein molecules also exert a greater electrostatic repulsion. Proteins are surrounded by a hydration layer, equivalent to about 0.3 g H2O per gram of protein (about 2 water molecules per amino acid residue).

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Aqueous solutions of proteins sometimes exhibit phase transitions (Figure ). The phase behaviour of protein solutions is affected by pH and ionic strength. Addition of electrolytes such as NaCl , KCl and (NH4)2SO4 decreases solubility. At high ionic strengths proteins precipitate – a salting-out effect. Organic solvents tend to decrease the solubility of proteins by lowering solvent dielectric constant

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THE STABILITY OF PROTEINS AND PEPTIDES Protein pharmaceuticals can suffer both physical and chemical instability (Figure ): Physical instability results from changes in the higher-order structure (secondary and above). Chemical instability is modifi - cation of the protein via bond formation or cleavage Keyword

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PHYSICAL INSTABILITY Denaturation is the disruption of the tertiary and secondary structure of the protein molecule. can be reversible or irreversible: It is reversible if the native structure is regained, for example on decreasing the temperature when temperature has caused the initial changes. It is irreversible when the unfolding process is such that the native structure cannot be regained. Denaturation

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Aggregation Some proteins self-associate in aqueous solution to form oligomers . Insulin, for example, exists in several states: The zinc hexamer of insulin is a complex of insulin and zinc which slowly dissolves into dimers and eventually monomers following subcutaneous administration, conferring on it long acting properties.

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Surface adsorption and precipitation Adsorption of proteins such as insulin on surfaces such as glass or plastic in giving sets: – can reduce the amount of agent reaching the patient – can lead to further denaturation , which can then cause precipitation and the physical blocking of delivery ports in protein pumps. Denaturation is facilitated by the presence of a large head space allowing a greater interaction of proteins with the air–water interface.

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Additives can coat the surface of glass or bind to the proteins. Serum albumin can be included in the formulation to compete with the therapeutic protein for the binding sites on glass and reduce adsorption. A similar effect can be achieved by the addition of surfactants such as poloxamers and polysorbates to the protein solution. Prevention of adsorption

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Significant denaturation of proteins can occur when the protein solutions are exposed at the air–solution interface. Agitation of protein solutions in the presence of air or application of other shear forces (e.g. in filters or pumps) may lead to denaturation . The inclusion of surfactants can reduce denaturation arising from these processes. Minimisation of exposure to air

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Addition of cosolvents Some excipients and buffer components added to the protein solution are able to minimise denaturation through their effects on solvation . These include polyethylene glycols and glycerol, referred to as cosolvents . These act either by causing the preferential hydration of the protein or alternatively by preferential binding to the protein surface (Figure ):

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Optimimisation of pH To avoid stability problems arising from charge neutralisation and to ensure adequate solubility, a pH must be selected which is at least 0.5 pH units above or below the IP. Since a pH range of 5–7 is usually required to minimise chemical breakdown, this frequently coincides with the IP

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CHEMICAL INSTABILITY Deamidation in deamidation the side-chain linkage in a glutamine ( Gln ) or asparagine ( Asn ) residue is hydrolysed to form a free carboxylic acid. Prevention of deamidation If the deamidation occurs by a general acid–base mechanism then the optimum pH for a peptide formulation will usually be about 6, where both rates are at their minimum. If the deamidation occurs through the cyclic imide intermediate it is preferable to formulate at a low pH since this type of deamidation is base- catalysed . Deamidation

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Oxidation Oxidation is one of the major causes of protein degradation. The side chains of histidine (His), methionine (Met), cysteine ( Cys ), tryptophan ( Trp ) and tyrosine (Tyr) residues in proteins are potential oxidation sites. Methionine is very susceptible to oxidation and reacts with a variety of oxidants to give methionine sulfoxide (RS(OO)CH3) or, in highly oxidative conditions, methionine sulfone (RS(O)CH3). The thiol group of cysteine readily reacts with oxygen to yield, successively, sulfenic acid (RSOH), a disulfi de (RSSH), a sulfi nic acid (RSO2H) and, finally, a sulfonic (cystic) acid (RSO3H) depending on reaction conditions

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An important factor determining the extent of oxidation is the spatial positioning of the thiol groups in the proteins. Histidine is susceptible to oxidation in the presence of metals, primarily by reaction with singlet oxygen, and this constitutesa major cause of enzyme degradation. Both histidine and tryptophan are highly susceptible to photooxidation . Prevention of oxidation In most cases oxidation results in a complete or partial loss of activity. Minimising protein oxidation is essential for maintaining the biological activity of most proteins and avoiding the immunogenic response caused by degraded proteins. A variety of measures may be employed in order to prevent protein oxidation: – temperature reduction, either by refrigeration or by freezing – control of pH if the rate of oxidation is pH-dependent.

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Racemisation All amino acid residues except glycine ( Gly ) are chiral at the carbon atom bearing the side chain and are subject to base catalysed racemisation . Proteolysis Proteolysis involves the cleavage of peptide (–CO–NH–) bonds: Asp is the residue most susceptible in proteolysis. The cleavage of the peptide bonds in dilute acid proceeds at a rate at least 100 times that of other peptide bonds

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Beta-elimination High-temperature treatment of proteins leads to destruction of disulfide bonds as a result of β-elimination from the cystine residue: The inactivation of proteins at high temperatures is often due to β-elimination of disulfi des from the cystine residue. Other amino acids, including Cys , Ser, Thr , Phe and Lys, can also be degraded via β-elimination. The inactivation is particularly rapid under alkaline conditions and is also influenced by metal ions.

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Disulfide formation The interchange of disulfide bonds can result in incorrect pairings with consequent changes of three-dimensional structure and loss of catalytic activity.

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THERAPEUTIC PROTEINS AND PEPTIDES

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There are three main types of insulin preparation: Those with a short duration of action which have a relatively rapid onset (soluble insulin, insulin lispro and insulin aspart ). 2. Those with an intermediate action ( isophane insulin and insulin zinc suspension). 3. Those with a slower/slow action, in onset and lasting for long periods (crystalline insulin zinc suspension) Insulin

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Calcitonin , a peptide hormone of 32 amino acids having a regulatory function in calcium and phosphorus metabolism, is used in various bone disorders such as osteoporosis. Salmon, human, pig and eel calcitonin are used therapeutically. Species differences may be significant – salmon calcitonin is 10 times more potent than human calcitonin . Human calcitonin has a tendency to associate rapidly in solution and, like insulin, form fibrils, resulting in a viscous solution. The fibrils are 8 nm in diameter and often associate with one another. Calcitonin

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