Protein delivery PharmDr. Veronika Mikušová, PhD.

Protein structure

• primary structure: amino acid chaines in well- defined 3D structure

• secondary structure: α helices, β sheets

• tertiary structure: proper positioning of the different subunits relativer to each other

• quarternary structure: individual protein molecules interact to build a larger well-defined structure ex: haemoglobin

• Formation and stability of sec., tert. and quarternary are based on relatively weak physical interactions:

▫ Electrostatic interactions

▫ Hydrogen bounding

▫ Van der Waals forces

▫ Hydrophobic interactions

▫ Not on covalent binding

which are relatively weak = protein structure can be rather easily changed (loss of pharmacological properties)

• Amino acid chaines can be modified by covalently attaching non-amino acid sections:

▫ sugars = glycoproteins

▫ phosphate groups

▫ sulphate groups

• These groups may be essential for the pharmacological effect of the protein

Proteins and GIT

• Pharmaceutical protein molecules are large • The GIT muconasal surface is a large interface that is

protected by a monolayer of epithelial cells, connected by tight junctions - it acts as a physical and chemical barrier towards the absorption of proteins and peptides

• Diffusional transport through epithelial barriers (GIT) is slow unless specific transporter molecules are available

• Conditions in GIT are extremely hostile to this proteins = enzymatic degradation is fast

• => large majority of pharmaceutical protein is therefore delivered via parenteral route

Possible mechanisms of proteins

absorption in GIT:

• Passive transport: simple or facilitated diffusion,

along concentration gradient, independent of chemical energy (but low lipophilicity and large Mr limit this process, beneficial for di/tripeptides)

• Active transport: against concentration gradient

• Endocytosis: energy consuming process – engulfment of the molecules for absorption

• Transcellular pathway: across the cells 1. by passive diffusion: depends on physico-chemical

properties of the molecule: size, charge and lipophilicity

2. by a specific carrier: peptide and amino acid transporters transport molecule from lumen into the cell – substrate specificity

• But all compounds absorbed through transcellular pathway are substrate for p-glycoprotein mediated efflux which transports the molecules from inside the cell back into the intestinal lumen

• The lipophilicity of a molecule is one of the main factors that governs transport via the transcellular route as molecules need to pass through the lipid bilayer of the intestinal membrane - a significant challenge

3. Transcytosis, Receptor-mediated endocytosis

• The transepithelial transport of macromolecules by intestinal epithelial cells occurs to a limited extent through absorption into lymphatic circulation via M-cells of Peyer's patches – minimal degradation of proteins - promising mechanism

• Paracellular pathway: transport of peptides across the aqueous channels in the cell junctions

• Not ideal for macromolecules (restricted to relatively small hydrophilic molecules)

• The addition of a novel functionality to the protein or the use of novel delivery systems – may enable optimal absorption

• Proteolytic enzymes: present at various regions along the GIT (trypsin, chymotrypsin, exopeptidases, endopeptidases etc.)

• Acidic nature of gastric medium – denaturation and degradation of protein

• Hydrolytic, irreversible cleavage of proteins into AA and small absorbable oligopeptides

• conserving the integrity of these large molecules is essential to:

▫ ensure an optimal therapeutic effect

▫ and to minimize effects such as the induction of unwanted immune responses (immune response may neutralize therapeutic activity in chronic dosing schedules and cause serious side-effects)

• Many functional groups are available for chemical degradation (and preferred 3D structure is irrevesibly disturbed) by for example:

▫ Heat

▫ pH changes

▫ Changes in ionic strenght

• Preferred shelf life for pharmaceutical produc is at minimum 2 years

• But most protein degrade too fast when formulated as aqueous solutions even when kept in the refrigerator

• Therefore they have to be stored in in a dry form and be reconstituted before administration

• They are usually dried by freeze-drying and the choice of proper excipients during this step (lyoprotectants) is extremely important

Sources of pharmaceutical proteins

• Most proteins used in therapy are produced by: ▫ recombinant DNA technology ▫ or hybridoma technology

• They are known as biotechnology products or bioitech products

• Ex: ▫ Human isulin ▫ Erythropoietin ▫ Monoclonal antibodies ▫ Cytokines ▫ Interferons

Production

• They are all produced in cell cultures by procaryotic or eucaryotic cells (E.coli, mamalian cells as Chinese hamster ovary cells or transgenic animals and genetically modified plants)

• Not all are exactly identical with endogenous product • Clinical trials has proved their efficacy and safety • Isolation of the expressend protein from the culture

medium is a multistep process consisting of several different steps (chromatography and filtration)

• For every protein a tailor made purification protocol has to be developed to remove impurities while ensuring integrity

• Majority of protein drug are biotech-derived molecules

• But there are still proteins of major therapeutic importance isolated from blood from humans or animals ▫ Albumin ▫ Blood clotting factors (factor VIII from the blood of

human volunteers) ▫ Antisera from patients or animals (horses, sheeps)

• Special purification protocols have to be developed, with particular emphasis on reduction of viral contamination

Formulation of pharmaceutical

proteins for parenteral administration

Physical instability • Depends on environmental conditions • Examples:

▫ Elevated temperature – denaturation of proteins in aqueous solutions

▫ Low temperature – may induce destabilization ▫ Adsorption of the protein monomer on the walls of the

container – protein aggregation ▫ Shaking, exposure to shear forces – protein

aggregation (hydrophobic parts of the molecule are exposed to hydrophobic interface air/water –> unfolding of the protein -> aggregation)

Chemical instability

• Full prevention of all chemical degradation reactions is difficult (because of many AA involved)

• Formulator should consider which chemical degradation pathways are relevant

• At neutral pH peptide bonds between AA are stable, only Asn-Gly and Asn-Pro are relatively labile

• Pathways of degradation of protein: ▫ Fragmentation ▫ Isomerisation ▫ Deamidation ▫ Oxidation ▫ Disulphide scrambling ▫ Oligomerization ▫ Aggregation ▫ Cross-linking ▫ Denaturation

Deamidation

• Rather common degradation in water

• Asn and Gln can be deamidated

• Kinetics depends on pH and neighbouring AA

→

Glu Gln

→

Oxidation • Met, Cys, His, Try, and Tyr are sensible • Oxidative milieu may also cause free Cys units to form disulphide

bridges or disulphide bind scrambling ▫ oxidation of Met to sulfoxide can be associated with the loss of

biological activity for many peptide hormones (i.e. corticotropine)

▫ oxidation can be iniciated by metal ions, visible light – photoxidation

▫ pH independent, may increase at low temperature (better solubility of O2 in water)

▫ the most important controling parameter: the degree of solvent accessibility to the side chains of proteins (burried side chains oxidize very slowly)

Isomerization, racemization

• Naturally occuring L-forms turns to D-forms, it changes activity of the protein

• Slow process occuring in vivo

Proteolysis • acidic hydrolysis of

peptide bonds of Asp

• hydrolysis can take place at either N- terminal and/or C- terminal peptide bond

Incorrect disulfide formation

• sulfhydryl groups and disulfide bonds are an important factor affecting the properties of proteins

• the interchange of disulfide bonds can result in incorrect pairings, leading to an altered 3D structure

Denaturation (the loss of the globular or 3D structure)

• thermal denaturation (often depends on pH; for lysozyme 42°C/pH=2, 65°C/pH=6.5)

• cold denaturation (proceeds often below -20°C) • chemical denaturation (urea, guanidinium

hydrochlorid) • pres