Peptides/ proteins

Question 1

Why are peptides and proteins injected IV or SC?

Answer 1

due to enzymatic and pH-dependent degradation in GIT,

low epithelial permeability and instability during formation

Question 2

Common routes of admin of peptide/proteins

Answer 2

nasal cavity (rich blood supply)
buccal cavity
rectal cavity (bypass first metabolism)
transdermal

Question 3

What is good and bad in biological pharmaceuticals (like peptides)?

Answer 3

It has the molecular characteristics to promote interaction with target

BUT
it lacks the ability to reach the target

Question 4

What are the issues in peptide/proteins?

Answer 4

stability and barriers

They are highly polar, soluble and rapidly excreted (short half life)

Question 5

why are peptides/proteins substrates for peptidases?

Answer 5

because they cannot cross biological barriers very well (due to N-terminal amino, C-terminal carboxy, and side chains COOH,NH2,OH)

Question 6

Challenges in delivery of biological pharmaceuticals

Answer 6

Physical (BBB, GI)
Chemical (pH change)
Biochemical (digestive enzymes/ flora in GI)

Question 7

What happens in physical instability (4 types)

Answer 7

proteins lose 3D structure

Aggregation, adsorption, denaturation, preciptation

Question 8

Aggregation

Answer 8

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

Question 9

Denaturation

Answer 9

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

Question 10

What are some examples of irreversible denaturation in physical instability?

Answer 10

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)

Question 11

Adsorption

Answer 11

Insulin frosting on the glass surface

leads to further denaturation and precipitation (physical blocking of delivery ports)

Question 12

How to prevent protein adsorption?

Answer 12

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

Question 13

What happens in chemical instability of peptides/proteins?

Answer 13

covalent modification in protein or AA residue to make new molecules via bond breaking/forming/rearrangement/ substitution

Question 14

What are the chemical instabilities?

Answer 14

deamination, oxidation, racemisation

Question 15

What happens in deamination

Answer 15

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)

Question 16

Oxidation

Answer 16

major cause of protein degradation

Occurs on side chain of His, Met, Cys, Trp, Tyr

Question 17

Racemisation

Answer 17

Base- catalysed racemisation occurs in all AA residues EXCEPT GLYCINE (chiral at carbon atom with the side chain - fixed in space)

Question 18

What are the biochemical barriers in delivery of peptides?

Answer 18

Celluar (epi/endothelial), metabolic, immunological, lymphatic drainage

Question 19

Difference between epi/endothelia

Answer 19

epi= covering cutaneous, mucous, serous SURFACES

endo - lining membrane of cavities (heart/lung)

Question 20

GI barriers for oral delivery

Answer 20

intestinal lumen, mucosa, liver, chemical/physical instability by acids, epithelial barrier, enzymatic barrier (proteases cause hydrolysis of peptide bonds)

Question 21

Biochemical barrier - enzymes

Answer 21

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)

Question 22

Biochemical barier - efflux system - P-gp

Answer 22

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

Question 23

Physical barrier - intestinal mucosa structure

Answer 23

Paracellular (between)

transcellular (through) -

Question 24

What is the main physical barrier?

Answer 24

tight junctions in CNS

Question 25

What is paracellular pathway

Answer 25

between cells - aqueous channel, extracellular route across EPIthelia, - Passive, driven by graidents and hydrostatic pressure - increase HYDROPHILICITY - Generally by water and small solutes

Question 26

What is transcellular pathway

Answer 26

Through the cells for intestinal absorption - Passive (rare), carrier mediated (substrate specific), vesicular - increase LIPOPHILICITY, - improve reabsorption

Question 27

What are the vesicular mechanisms in intestinal absorption in transcellular pathway?

Answer 27

- 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

Question 28

Physical barrier: mucous function

Answer 28

coat the muosa lubricate and protect effect peptide absorption (washes away)

Question 29

Delivery to CNS involves 3 factors, they are

Answer 29

BBB, CNS entry, associated factors, transporters

Question 30

What is the distance of mean inter-capillary distance in brain?

Answer 30

``` almost 40um (very small) instantaneous solute equilibration throughout the brain for small molecules (eg. glucose) ```

Question 31

Does peptide/protein go through BBB?

Answer 31

No, tight junctions in brain capillary endothelium brain capillary endothelial cells contain few pinocytotic vesicles

Question 32

Cerebral capillary transport

Answer 32

- 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)

Question 33

Transport mechanisms in the brain

Answer 33

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

Question 34

Carrier mediated transport across BBB

Answer 34

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

Question 35

Solution to delivery problems - what are the 3 broad approaches of chemical modifications?

Answer 35

1. Non-peptide natural product that mimics/antagonises biological activity (Ang ii antagonist) 2. use a novel route/ co administer with enzyme inhibitor 3. Develop a peptidomimetic that resembles target protein but reduce metabolism and optimise activity

Question 36

How to limit peptide bond hydrolysis in vivo

Answer 36

1. Add steric bulk form (Alkyl group or 4o amino acid - to increase incidence of CIS configuration of amide bond - -> conformation/steric changes 2. Replace the scissile (RNH-COR) bond altogether - in ACE, renin - -> subtle conformational changes 3. 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 ```

Question 37

Enhancement of passive diffusion cross intestinal mucosa

Answer 37

sufficient hydrophilicity highly potent and hydrophilic peptides (more lipophilic --> more interaction with cell membrane)

Question 38

Enhancement for transcellular pathway

Answer 38

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

Question 39

Minimisation of substrate activity towards intestinal efflux systems

Answer 39

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

Question 40

Inhibition of efflux pumps

Answer 40

co-administer 2nd generation inhibitor, lacking pharmacological activity (verapamil (R-isomer)