D. PROTEIN FORMULATIONS 2

Question 1

properties of protein and peptide drugs

Answer 1

• large, hydrophilic molecules 0.5-150 kDa
• not well transported across biological membranes
• typically <2% bioavailability even for small peptides
• injected polypeptides have a small half-life
• major disadvantages compared with small MW drugs
• physical, chemical, biological instabilities

Question 2

physical stability of proteins and peptide drugs

Answer 2

• easily lose their native 3D structure by denaturation
• due to hydrophobic conditions, surfactants, pH, solvent, temperature, dehydration, lyophilisation
• can happen in the bottle or during manufacturing

Question 3

aggregation/precipitation of drugs

Answer 3

• denatured, unfolded proteins may interact
• aggregation becomes precipitation and we can see large particles
• loss of function

Question 4

adsoprtion of drugs

Answer 4

• polar and non-polar residues are adsorbed at interfaces
  air-water: foaming
  air solid: Insulin adsorbs to many surfaces like plastic tubing or containers so we use delivery pumps, glass or plastic syringes
• to decrease the dose lost

Question 5

chemical stability of proteins and peptide drugs

Answer 5

• deamidation: asparagine and glutamine residues
  hydrolysed to form a carboxylic acid
• oxidation: methionine, cysteine by oxygen in air
  mechanism via oxygen radical
  catalysed by transition metals (e.g. Fe and Cu)
  mediated by antioxidants (e.g. Ascorbate)
  PEG can result in peroxide formation
• photo-oxidation

Question 6

biological stability of protein and peptide drugs

Answer 6

• hydrolysed to amino acids and small peptides in the GIT
• Pepsin (preferred aa: phe, tyr, trp)
• Trypsin, chymotrypsin and aminopeptidases [small intestine]
• Carboxypeptidases - produced in pancreas, act in small intestine
• denatured by gastric acid
• Very few peptides are stable to biodegradation: Cyclosporin (11 residues, cyclic - therefore very small) and TRH (very small)
• Colon: Fewer digestive enzymes but substantial microbial enzymes

Question 7

what is the order for pre-formulation

Answer 7

1. selection of candidate drug (protein)
2. determination of properties eg: MW, 3D structure
3. a) chemical/biological alteration
   b) select pH, salts, solvents, surfactants
   c) liquid or dried formulation
4. stable, active formulation

Question 8

what conditions need to be decided

Answer 8

• physical state, pH , ionic strength, temperature: needs to be active and stable at 37 degrees Celsius in body
• stability enhanced when dry and frozen (i.e. freeze-dried) as water is removed and left with pure protein

Question 9

how do additives affect proteins

Answer 9

• salts decrease denaturation via binding to the protein (also metal ions - calcium)
• polyalcohols (glycerol) stabilise by selective solvation of functional groups on the surface
• surfactants prevent adsorption of proteins at surfaces and aggregation

*additives need to be non-toxic

Question 10

what chemical modifications can proteins have

Answer 10

• synthetic polymers (PEG) which makes proteins more stable in aq environments
• lipids covalently bound to proteins to make proteins more stable in non-aq environments

Question 11

how does primary sequence alteration affect proteins

Answer 11

• specific amino acids changed which doesn’t alter final function of protein
• improved physical and chemical stability

Question 12

what problems are there with parenteral route for proteins

Answer 12

• repeated administration
• patient compliance
• stability of dosage form (liquid is not stable - fridge)

Question 13

what is the most preferable route of administration

Answer 13

oral as easy to administer

Question 14

what is the problem with oral administration

Answer 14

low absorption except for a few cases:
- cyclosporin, TRH, captopril

Question 15

advantages of parenteral route

Answer 15

• controlled drug release
• site-specific delivery

Question 16

what advantages does site-specific delivery have

Answer 16

• Pharmacokinetic advantages
• Improvement of therapeutic index
• Protection from unwanted drug disposition
• Extravascular access (cancer-drugs: don’t want it escaping target area due to side effects)

Question 17

what are the methods of site-specific delivery

Answer 17

• particulate systems: non-soluble, protein delivered to site
  disadvantages: problems with RES as particles can’t cross vascular endothelium
• soluble carriers
  disadvantages: problems with stability/transport

Question 18

how are implantables delivered

Answer 18

• IM or SC route

Question 19

advantage of implantables

Answer 19

drug release for periods up to 1 year

Question 20

types of implantable system

Answer 20

• Polymer gel matrix (biodegradable)
• Polymer fibre system
• Osmotic mini-pumps
• Tablet-type implants
• Automatic feedback system (may need surgery)

Question 21

examples of implantable - Zoladex

Answer 21

• injectable biodegradable polymer matrix
• SC injection into upper abdominal wall – continuous release over 28 days
• sterile white/cream – coloured cylinder 1 mm in diameter & 5 mm long, preloaded into a special single-use syringe
• indicated for palliative treatment of advanced prostate carcinoma

Question 22

example of implantable: Levonorgestrel

Answer 22

• polymer fibre system
• > 6 months
• for birth control

Question 23

what are the 5 ways oral bioavailability can be improved

Answer 23

1. peptidase inhibitors
2. saturation/bypassing of intestinal peptidases
3. penetration enhancers
4. reduction of hepatic first pass clearance
5. non-passive and paracellular transport

Question 24

peptidase inhibitors

Answer 24

-improve stability 1
- eg: Amistatin promotes absorption of enkephalins

Question 25

saturation/bypassing of intestinal peptidases

Answer 25

• increase dosage or load with another peptide
• enteric coating eg: acid-resistant acrylic resin
• use of azo polymers so drug released further down GIT

Question 26

penetration enhancers

Answer 26

• improvement of passive absorption
• transcellular or paracellular - eg: for insulin
• ionic/non-ionic detergents
• bile salt surfactants
• EDTA and other chelating agents
• problems with irritation/tissue damage
• recently (2020), oral semaglutide (GLP synthetic hormone) for Type 2 diabetes
• sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC)
• increases local pH, promotes absorption in GI epithelium

Question 27

reduction of first pass clearance

Answer 27

• saturation of specific enzymes
• promotion of lymphatic uptake
  e.g. cyclosporin bioavailability increased from 1% →20-50% when drug in olive-oil based formulation so bypassed hepatic clearance

Question 28

non–passive and paracellular transport

Answer 28

• carrier mediated for di- and tri- peptides
• endocytic/transcytotic for proteins
• M-cell (Peyer’s patch, small intestine) but only 1000 molecules could be transported which isn’t enough
• role of P-glycoprotein efflux pump
• transport across tight junctions (paracellular)

Question 29

PKs of protein drugs compared to small drugs

Answer 29

• dose-response relationships non-standard ie: in hormonal systems there is no improved reaction when you get a dose of hormone due to:
• multi-faceted cascade processes
• differences in biological action in males/females:
• How much?
• How often?
• Where to?
• both zero-order or complex delivery systems (non-zero order) may be used depending on situation ie: time of day/season when protein delivered changes actions of protein

Question 30

limitations of SC insulin injection

Answer 30

• insulin SC fails to mimic endogenous insulin secretion
• lack of acceptance of multiple daily injections by patients
• complications: retinopathy, nephropathy, neuropathy

Question 31

properties which promote insulin as a candidate for inhalation delivery

Answer 31

• relatively large hydrophilic molecule (5.6 kDa)
• transported by passive paracellular diffusion through tight junction

Question 32

inhalation delivery for insulin

Answer 32

• 2nd gen dry powder inhalation technology for insulin delivery
• spray-dried insulin powder with stabilisers in blister
• blister loaded at the base of the inhaler and punctured by actuation
• fluidization/deaggregation in aerosolisation chamber by compressed air
• patient inhales the particle cloud through a slow deep breath

Question 33

PKs of inhaled insulin

Answer 33

• serum concentrations peak earlier and decay more rapidly than after SC injection of regular insulin
• onset of action quicker and duration of action prolonged as compared to rapid-acting insulin analogues
• patients can inhale insulin just 10min before food but they still need a bedtime SC injection

Question 34

limitations of inhaled insulin

Answer 34

• relative bioavailability of insulin is 15-30% vs SC injection
• fate of unabsorbed dose

Question 35

non-respiratory effects of inhaled insulin

Answer 35

• hypoglycaemia (frequency and severity similar to SC injections) ie: it wasn’t improved
• increase in insulin antibodies (no clinical effects so far: long-term?)

Question 36

respiratory effects of inhaled insulin

Answer 36

• cough, increased sputum
• decrease in lung function in some patients (recommended patients undergo lung tests before and periodically thereafter)

Question 37

why was Exubera and Afrezza inhaled insulin a failure

Answer 37

they were FDA approved then removed from the market
- not good sales due to sizing of device (Exubera)
- cancer-causing warning
- lung-function concerns

Question 38

what is the only medicinal form of insulin now

Answer 38

solution for injection (can order infusion)