Protein and peptide drug delivery

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21 April 2020 Dattakala College of Pharmacy 2
PROTEIN AND PEPTIDE
DRUG
DELIVERY SYSTEM

PROTEIN AND PEPTIDE
DRUG
DELIVERY SYSTEM
Presented by
Prof. Hemant Bansode
Department of Pharmaceutics

CONTENTS:
 Introduction
 Importance of pre-formulation
 Formulation consideration
 Toxicity
 Immunogenicity
 Stability and regulatory perspective
21 April 2020 4Dattakala College of Pharmacy

Proteins & Peptides
.
 Proteins:
These are large organic compounds made of amino acids arranged in a linear chain
and joined together by peptide bonds.
Protein > 50 amino acids
 Peptides:
 These are short polymers formed from the linking, in a defined order, of α-amino
acids.
peptide < 50 amino acids
 Introduction:
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 These biopolymers which yield two or more amino acids on
hydrolysis.
 Principal components of the protoplasm of cells and are high
molecular weight compounds.
 Consisting of alpha amino acids connected together by peptide
linkages.
 Peptides are short polymers formed from the linking of amino acids.

 Hormones , Serum protein and enzymes have been used as drug .
Such drugs include synthetic vaccines that promise to offer protection against
carcinogens and toxicants.
• Peptide are small and relatively simple molecules compared to protein.
 Each of these amino acids has a fundamental design composed of a central carbon
(also called the alpha carbon) bonded to:
• a hydrogen
• a carboxyl group
• an amino group
• a unique side chain or R group.
Thus, the characteristic that distinguishes one amino acid from another is its unique side
chain, and it is the side chain that dictates an amino acids chemical properties.

 The unique side chains confer unique chemical properties on amino acids, and dictate
how each amino acid interacts with the others in a protein.

FUNCTIONS
 Transport and storage of small molecules.
 Coordinated motion via muscle contraction.
Mechanical support from fibrous protein.
Generation and transmission of nerve impulses.
 Enzymatic catalysis.
 Immune protection through antibodies
 Control of growth and differentiation via hormones.

 Preformulation and Formulation considerations:
 Pharmacokinetic considerations
 Analytical considerations
 Regulatory considerations

Preformulation and Formulation considerations
 Denaturation stabilizers
 Maximising oral protein and peptide absorption.
 Chemical Modifications :
 Amino acid Modification.
 Hydrophobization.
 Conjugation with polymers

DENATURATION
 Specific confirmation is required for proteins to exert pharmacological and
physiological activities.
 Denaturation is a process of altering protein confirmation.
 Heat, organic solvents, high salt concentration, lyophilization can denature
proteins.
 Protein confirmation refers to the specific tertiary structure.
 It held together by three forces :-
• hydrogen bonding,
• salt bridges, and
• hydrophobic interactions.
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COMMON STABILIZERS
 SERUM ALBUMIN :
 It can withstand heating to 60o C for 10 hours.
 At pH 2 albumin molecule expands and elongates but can return to native
confirmation reversibly. Also, it shows good solubility.

 AMINO ACIDS
 Glycine is most commonly used stabilizer.
 Mechanism of action of amino acids as stabilizers may be one of the following :
1. Reduce surface adsorption.
2. Inhibit aggregate formation.
3. Stabilize proteins against heat denaturation.

SURFACTANTS
 They cause denaturation of proteins by hydrophobic disruption.
 Proteins have tendency to concentrate at liquid/liquid or liquid/air interface.
 Due to this proteins may adopt non native confirmation and such confirmation is
having less solubility.
 Optimal concentration of surfactants for stabilization should be greater than cmc.
 Ionic surfactants are more effective stabilizers than non ionic surfactants.
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POLYHYDRIC ALCOHOLS AND CARBOHYDRATES :
 They contain –CHOH-CHOH- groups which are responsible for stabilizing proteins.
 They stabilize proteins against denaturation by elevated temperature or by freeze
drying or by freeze thaw cycles.
 Many important therapeutic proteins and peptides are derived from blood such as
immune globulin, coagulation factors.
 For viral destruction pasteurization at 60o C for 10 hours is needed.
 Hence thermal stability is needed.
 Long chain polyhydric alcohols are more effective as stabilizers. e.g. sorbitol, xylitol.

 Mechanism of action as stabilizers for polyhydric alcohols is that they have effect
on structure of surrounding water molecules which strengthens hydrophobic
interactions in protein molecules.
 Mechanism of action as stabilizers for carbohydrates is that they provide dry
network that provides significant support for protection
 Polyhydric alcohols used are sorbitol, mannitol, glycerol, PEG.
 Carbohydrates used are glucose, mannose, sucrose, ribose.

ANTI-OXIDANTS
Thiol compounds such as thioacetic acid, triethanolamine, reduced glutathione and
metal chelants such as EDTA are used as antioxidants.
MISCELLANEOUS
 Certain enzymes can be stabilized by using compounds having similar structures
of enzymes.
e.g. Glucose stabilizes glucoamylase while aspargine stabilizes asparginase.
 Compounds forming stable complex through ionic interaction with proteins can
stabilize proteins.
 Calcium is essential for thermal stability of certain amylases or proteases.

Maximising oral protein and peptide absorption
 Amino acid modifications:
 Metkephamid, an analog of methionine enkephalin with substitution of glycine₂
by l-alanine and modified methionine.
 Readily penetrated across the nasal mucosa with 54% bioavailability relative to
subcutaneous administration but was orally inactive.

Hydrophobization
 Hydrophobization of peptides may be attempted by two approaches.
 The first is peptide backbone modification to include more of hydrophobic amino
acids.
 The second would be covalent conjugation of a hydrophobic moiety—for example,
a lipid or polymeric tail.
 Increasing the hydrophobicity by surface modification using lipophilic moieties may
be of particular benefit to transcellular passive or active absorption.
 Membrane penetration or attachment, respectively; or it may simply aid in the
increased stability of the protein.

 EXAMPLE
• Lipophilic modification of TRH by covalent conjugation of lauric acid to this
tripeptide(Lau-TRH).
• The derivative was more stable in rat plasma and was rapidly converted to TRH in
the intestinal mucosal homogenate.
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DEVELOPMENT OF DELIVERY
SYSTEMS FOR PEPTIDE AND
PROTEIN BASED
PHARMACEUTICALS
 Pharmacokinetic considerations
 Analytical considerations
 Regulatory considerations

 Analytical considerations:
 Many tests are required for stability of protein products to assure identity, purity,
potency and stability of formulation.
 Due to complexity of proteins bioassay are required to assess potency of the
formulation.
 Bioassay are of two types : in vitro and in vivo.
 In case of in vitro bioassays response of cells to hormones and growth factors is
monitored.
 In case of in vivo bioassay pharmacological response of animals to proteins is
monitored : e.g., post injection blood sugar in rabbits is monitored for bioassay
of insulin.

 Preformulation consideration:
 Initial objective in the preformulation development of drug is its physical and
chemical properties.
 Quantitative characterization of the physical properties of these compounds.
A) Purity,
B) Solid state properties,
C) Solution phase properties,
D) Solubility,
E) Chemical stability,

 Purity :
 Nafarelin acetate drug substance contain three predominant type of impurities…
1. Peptide,
2. Acetic acid and its salt,
3. Water.
 The peptide impurities result from the inevitable carrgover of small amount of
intermediates and side products in to the final product.
 Acetic acid is present because nafarelin is isolated by precipitation .
 Water is usually not considered as an impurity.
 Batch to batch variations in the peptide impurities present in nafarelin acetate were
carefully monitored by HPLC.

 U.V. SPECTROSCOPY :
 Proteins containing aromatic amino acid residues such as phenyl alanine, tyrosine,
tryptophan can be detected by U.V. spectroscopy.
 Ultraviolet spectroscopy can be used for in process quality control.
 Protein aggregates scatter U.V. light and absorbance increases.
 Hence U.V. spectroscopy can be used to monitor protein aggregation.

 ELECTROPHORESIS:
 Most often used technique for protein products is sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE).
 Proteins are denatured by boiling in the SDS solution.
 All charges of protein are masked by negative charge of dodecyl sulphate.
 Thus protein moves on polyacrylamide gel strictly on basis of size of protein
molecule.
 This technique is useful for determining molecular weight of proteins.
 For visualization of proteins on the gel reagents used are silver nitrate, coomassie
brilliant blue dye.
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 LIQUID CHROMATOGRAPHY:
 To study stability of proteins and peptides HPLC is useful technique.
 Various modes used are….
1. Normal Phase HPLC
2. Reverse Phase HPLC
3. Ion Exchange
4. Chromatofocusing

 Solid -state properties:
 Crystalinity:
 Do not exhibit a strong tendancy to crystalize and are isolated as amorphous
powder.
 Isolation method are …
1. Lyophillization.
2. Precipitation.

 Solution phase properties:
 Ionization constant:
 It contains 3 ionisable amino acid residue….
Histidine,
Tyrosine,
Arginine ,
 Pka of Tyrosine residue by UV- spectrophotometry is 9.92,
 Pka of Histidine residue by Potentiometric titration is 5.9,
 Pka of Arginine residue is not determined but is expected to be similar to that of
free arginine.

 The state of ionization in solution can affect its ability to ion pair ,aggregate and bind
to surface , which in turn can affect the solution properties described below..
 Absorption :
 Loss of peptides and proteins from solution to various surface is common
phenomenon and should be addressed early in the preformulation activities.
Losses are practically significant at low concentration.
 In case adsorption is due to ionic interaction of the peptide with the Silanol groups
on the glass surface , it can be prevented by Silylating organosilanes such as Prosil -28.

 Stability problem:
 Protein stability is another common problem in protein expression. .
 It is also an important topic in purification, formulation, and storage.
 Properly folded proteins are usually stable during expression and purification.
 Sufficient amount of intact protein should be obtained
 Some of them are so unstable that sufficient amount of protein cannot be obtained.
 Many factors such as amino acid sequence of the protein, protein construction, host
cell strain, expression and purification conditions may affect protein stability.

 Physical instability:
1)Denaturation
2) Adsorption
3) Aggregation
4) Precipitation
 Chemical instability:
1) Hydrolysis
2) Oxidation
3) β-elimination
4) Racemization
5) Isomerization
6) Disulphide exchange

 Physical instability:
1. Denaturation:
 Denaturation is unique to protein
occurs when their native tertiary and secondary structures are disrupted.
 Preventive measures: It can be prevented by the use of the additives such as salts.
2. Adsorption:
 Protein exhibit a certain degree of surface activity due to that protein adsorb to
surface of containers.
 Consequently biological activity may be lost if such absorption occurs during
manufacturing and storage.
 Preventive measures:
• Modify the surface of glass container.
• Add surfactant.
• Add macromolecules.
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3. Aggregation:
 Certain protein tends to undergo self association resulting in the formation of
multimers
 Protein aggregation is the mainly caused by hydrophobic interaction resulting
from denaturation.
Preventive measures:
• It can be prevented by the use of surfactant, polyols or sugars.

 Chemical instability:
1. Hydrolysis:
undergo deamidation to form free carboxylic acid.
 Neutral and alkaline conditions enhanced the rate of deamidation.
 Preventive measures: -
Hydrolysis can be reduced by reducing the pH.
2. Oxidation:
 Proteins containing methionine, cystein, histidine may be sensitive to oxidation.
Methionine and cysteine may lead to disulfide bond formation and loss of biological
activity.
• Antioxidant: e.g. sodium sulfide, chelating agent,thiols etc.
• Elimination of peroxides.
• Prevention by using thiols carveng.

 β –elimination:
 At temperature above ambient temperature, proteins undergo disulfide cleavage.
 Such reactions occurs at low temperature and high pH.
By using thiol scarvengers such as copper ion, diethylmaleimide.
 Racemization:
• Naturally occurring amino acids are in the L form isomerise into D form leads to the
change in structure and therefore the activity of the proteins.

 Immunogenicity of therapeutic peptides/proteins:
 Consequences of drug-induced immune responses:
Therapeutic proteins (including human proteins) and peptides can induce antibodies in
patients.
 Anti-drug antibody production can :
1. Affect efficacy
2. Affect drug pharmacokinetics
3. Induce autoimmune responses
 Also the potential for hypersensitivity or allergic reactions

Clinical Consequences:
For safety.
For efficacy.
Patient Specific factor that affect Immunogenicity:
Immunologic Status and Competence of the Patient.
Prior Sensitization/History of Allergy .
Route of Administration, Dose, and Frequency of Administration.
Genetic Status .
Status of Immune Tolerance to Endogenous Protein.

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Product-Specific Factors That Affect Immunogenicity:
Product Origin (foreign or endogenous).
Primary Molecular Structure/Post Translational Modifications.
Quaternary Structure: Product Aggregates and Measurement of Aggregates.
Glycosylation / Pegylation.
Impurities with Adjuvant Activity.
Immunomodulatory Properties of the Therapeutic Protein Product.
Formulation.
Container Closure Considerations.
Product Custody.

 Regulatory Perspective of Protein and Peptide Drug
Development:-
 Interactive nature of drug developmental efforts.
 Fundamental issues.
 Networking.
 The demand of novelty.
 obtaining a consensus.
Reassessment of quality assurance.

 Chemistry and manufacturing controls:
 Generalities.
 Source of Therapeutic protein.
1. Peptide extracted from natural source.
2. Semisynthetic peptide.
3. Biosynthetic peptide.
4. Full chemical synthesis.
 Structure of the final product:
1. Natural sequence peptide.
2. Agonistic analog.
3. natural source agonistic analog.
4. Biosynthetic agonistic analog.
5. Antagonistic analog.

Purity:
• Extraneous protein.
• Viral and cellular DNA
• Infectious contamination.
 Stability:
• Stable for long period.

 Animal studies:
• Pharmacological and toxicological studies.
 Preclinical studies.
 Additional Tests.
 Clinical Studies:
 Medical involvement during the preclinical period.
• Extramural Interaction.
• Intramural Interaction.

 Novel Conceptual Approaches to Drug Development:
 Finding the Initial Indication.
 Defining a Developmental Algorithm.
 The FDA gradually adopted the following general algorithm of the drug development.
 List all conceivable indication.
 Eliminate those that are not based on correct rationale.

Product Formulation Route Indication
Lupron Sterilized lyophilized
microspheres Contains Leuprolide
acetate.
i.m. Prostatic
cancer
Pitressin tannate Vasopressin tannate in peanut oil. i.m. Antidiuretic
H.P. Acther gel Adrenocorticotropic harmone in
gelatin
i.m. , s.c. Endocrine
cancer
Neurotropin Growth harmone i.m . Drowsiness
Marketed formulations

 References:
Protein and peptide drug delivery by Vincent H. L. Lee.
Formulation and Delivery of Proteins and Peptides by Jeffrey L. Cleland.

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Protein and peptide drug delivery

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