Peptides/ proteins Flashcards
Why are peptides and proteins injected IV or SC?
due to enzymatic and pH-dependent degradation in GIT,
low epithelial permeability and instability during formation
Common routes of admin of peptide/proteins
nasal cavity (rich blood supply)
buccal cavity
rectal cavity (bypass first metabolism)
transdermal
What is good and bad in biological pharmaceuticals (like peptides)?
It has the molecular characteristics to promote interaction with target
BUT
it lacks the ability to reach the target
What are the issues in peptide/proteins?
stability and barriers
They are highly polar, soluble and rapidly excreted (short half life)
why are peptides/proteins substrates for peptidases?
because they cannot cross biological barriers very well (due to N-terminal amino, C-terminal carboxy, and side chains COOH,NH2,OH)
Challenges in delivery of biological pharmaceuticals
Physical (BBB, GI)
Chemical (pH change)
Biochemical (digestive enzymes/ flora in GI)
What happens in physical instability (4 types)
proteins lose 3D structure
Aggregation, adsorption, denaturation, preciptation
Aggregation
This is irreversible (self-association is reversible - insulin is selfassociated then dissociated after SC dosing)
When it is too acidic/ too much salt (negative charge), the interaction between negative and positive charges in the folded polypeptides become weak, so it falls out (conformation change) and bind to each other with similar shape
They drop out fluid and cause precipitation
Denaturation
Reversible or irreversible
Disruption of secondary and tertiary structure of protein
By heating/cooling/freezing/pH extremes/ contact with denaturants
two CYS-SH bond together and these can unfold leading to adsorption, aggregation, precipitation, chemical reactivity
What are some examples of irreversible denaturation in physical instability?
increase in ionic strength of protein solution (neutralisation of surface charge for repulsion so aggregate formation)
Increase in temperature increases flexibility/ unfolding
(collision increases and aggregate formation)
Adsorption
Insulin frosting on the glass surface
leads to further denaturation and precipitation (physical blocking of delivery ports)
How to prevent protein adsorption?
Avoid glass containers
Coat glass container with another protein
Add surfactants (sticks to glass)
Minimise air exposure (decrease protein denaturation)
Agitation or application of other shear forces can cause denaturation
What happens in chemical instability of peptides/proteins?
covalent modification in protein or AA residue to make new molecules via bond breaking/forming/rearrangement/ substitution
What are the chemical instabilities?
deamination, oxidation, racemisation
What happens in deamination
hydrolysis of side chain in glutamine and ASPARAGINE
to make COOH
(acid catalysed hydrolyssi –> base structure comes off and the acidic group is attached by H-bonds)
Oxidation
major cause of protein degradation
Occurs on side chain of His, Met, Cys, Trp, Tyr
Racemisation
Base- catalysed racemisation occurs in all AA residues EXCEPT GLYCINE (chiral at carbon atom with the side chain - fixed in space)
What are the biochemical barriers in delivery of peptides?
Celluar (epi/endothelial), metabolic, immunological, lymphatic drainage
Difference between epi/endothelia
epi= covering cutaneous, mucous, serous SURFACES
endo - lining membrane of cavities (heart/lung)
GI barriers for oral delivery
intestinal lumen, mucosa, liver, chemical/physical instability by acids, epithelial barrier, enzymatic barrier (proteases cause hydrolysis of peptide bonds)
Biochemical barrier - enzymes
most degradation by peptides need contact with brushborder membrane or uptake into intestinal mucosal cells
Brush-border peptidases
(active mainly against tri/tetra and higher peptides)
Intracellular peptidases (predominantly against DI)
Endopeptidases (cleave internal peptide bonds, for lage peptides or with blocked ends)
Exopeptidases (cleave one or more residues from terimini of peptide)
Biochemical barier - efflux system - P-gp
MAJOR BARRIER by limiting absorption
P-glycoprotein (multi-drug resistance)
Acts on atp-dependent efflux pump, reducing intracellular accumulation/ transceullar flux of drugs
Restrict transcellular reflux of some molecules to GIT
Physical barrier - intestinal mucosa structure
Paracellular (between)
transcellular (through) -
What is the main physical barrier?
tight junctions in CNS
What is paracellular pathway
between cells
- aqueous channel, extracellular route across EPIthelia,
- Passive, driven by graidents and hydrostatic pressure
- increase HYDROPHILICITY
- Generally by water and small solutes
What is transcellular pathway
Through the cells for intestinal absorption
- Passive (rare), carrier mediated (substrate specific), vesicular
- increase LIPOPHILICITY,
- improve reabsorption
What are the vesicular mechanisms in intestinal absorption in transcellular pathway?
- endocytosis when peptide is too large to be absorbed by di/tri transporters
- pino (fluid filled endocytosis, no interaction between polypeptide, apical membrane)
- phago
- receptor mediated endocytosis (binding of peptides, proteins to plasma membrane before getting into vesicles)
- Passive diffusion
Physical barrier: mucous function
coat the muosa
lubricate and protect
effect peptide absorption (washes away)
Delivery to CNS involves 3 factors, they are
BBB, CNS entry, associated factors, transporters
What is the distance of mean inter-capillary distance in brain?
almost 40um (very small) instantaneous solute equilibration throughout the brain for small molecules (eg. glucose)
Does peptide/protein go through BBB?
No,
tight junctions in brain capillary endothelium
brain capillary endothelial cells contain few pinocytotic vesicles
Cerebral capillary transport
- Tight junctions (seal pathway between the capillary cells) - arachnoid cells line the meninges
- lipid membranes (barrier to water-soluble)
- ions channels/carriers
- enzymatic barrier (remove molecule from blood)
- efflux pumps (extrude fat-soluble molecules)
Transport mechanisms in the brain
paracellular :diffusion of small water soluble ones
transcellular lipophilic: diffusion of lipid soluble ones
saturable transport: transfer of essential POLAR solutes like glucose, AA, nucleosides
Specific receptor-mediated endocytosis: insulin, transferrin, cytokines
Adsorptive endocytosis: cationisation of albumin
Effux transport: pumping out faster than accumulating
Carrier mediated transport across BBB
Monoclonal antibodies to transferrin receptor (act as dimer, it has high affinity binding sites for Fe2+ ions, most of the receptor in the extracellular space)
Glucose - GLUT1 (smaller)
- Na+ independent
Solution to delivery problems - what are the 3 broad approaches of chemical modifications?
- Non-peptide natural product that mimics/antagonises biological activity (Ang ii antagonist)
- use a novel route/ co administer with enzyme inhibitor
- Develop a peptidomimetic that resembles target protein but reduce metabolism and optimise activity
How to limit peptide bond hydrolysis in vivo
- Add steric bulk form (Alkyl group or 4o amino acid - to increase incidence of CIS configuration of amide bond
- -> conformation/steric changes - Replace the scissile (RNH-COR) bond altogether - in ACE, renin
- -> subtle conformational changes - Modify peptide conformation so that it s no longer recognised by proteases (constraining peptide backbone)
4. Cyclize peptide (use covalent linkages) - amide/ side chains(NH2, COOH) --> conformational constraint - simple and effective
Enhancement of passive diffusion cross intestinal mucosa
sufficient hydrophilicity
highly potent and hydrophilic peptides
(more lipophilic –> more interaction with cell membrane)
Enhancement for transcellular pathway
need an optimal lipophilicity
- energy necessary for a solute to enter from aqueous phase to membrane phase
- decrease in h-boding potential for peptide permeation improves transcellular passage
Minimisation of substrate activity towards intestinal efflux systems
difficult to identify characteristic structural element needed for substrate activity
LIPHILICITY - substrate feature
WATER SOLUBILITY
high hydrophobic drugs that are sparingly soluble in water are not substrates
Inhibition of efflux pumps
co-administer 2nd generation inhibitor, lacking pharmacological activity (verapamil (R-isomer)
