Protein Formulations Flashcards
(57 cards)
1
Q
What are the problems with protein delivery?
A
- They are large hydrophilic molecules; issues with absorption (lipophilic desirable)
- Extra-vascular access difficult
- Poor stability (degradation)
- Purity/characterisation of recombinant products
- Complex pharmacoglogical action
2
Q
How do low molecular weight drugs reach their site of action?
A
Diffusion and partition
3
Q
What factors determine biodistribution of macromolecules?
A
- Ability of macromolecules to be transported across endothelium
- Differences in relative blood flow to different tissues
- Elimination by kidneys/metabolism
4
Q
What is the cut-off for glomerular filtration for proteins and why is this beneficial?
A
- 60kDa for proteins
- Serum albumin is 65kDa (essential)
5
Q
What is the cut-off for glomerular filtration for dextrans/polysaccharides?
A
40kDa
6
Q
Where are the receptors located for the removal of old serum proteins/unwanted macromolecules released by dying cells?
A
Receptors in liver on parenchymal cells, and macrophages
7
Q
How does the Asialoglycoprotein receptor (ASGPR) recognise proteins for disposal?
A
- Many proteins are glycoproteins (have CHO residues)
- Terminal sugar residue is sialic acid
- Sugars beneath e.g. D-galactose are recognised by specific receptors such as the ASGPR; protein is ‘exposed’ and marked for removal
8
Q
What materials does the ASGP receptor clear?
A
- Hormones
- Carrier molecules
- Protease inhibitors
- Immunological
9
Q
What hormones does the ASGP receptor clear?
A
- Erythropoietin
- Follicle stimulating hormone (FSH)
- Interferon
10
Q
What carrier molecules does the ASGP receptor clear?
A
- Thyroglobulin (iodine)
- Caeruloplasmin (Cu)
- Transferrin (fe)
- Transcobolamin (vitamin B12)
11
Q
What protease inhibitors does the ASGP receptor clear?
A
- α-1 antitrypsin
- α-2 macroglobulin
12
Q
What other clearance receptor besides ASGPR exist?
A
- N-acetylgalactosamine/mannose receptor on macrophages
- Fucose receptor on liver parenchymal cells
13
Q
How proteins such as albumin/LDL with no sugar residues cleared?
A
They age by becoming acetylated and are then cleared by a receptor recognising acetylated proteins
14
Q
What occurs after proteins bind to clearance receptors?
A
- Proteins are endocytosed, sent to lysosomes
- Low pH of lysosome degrades protein and chops it up
15
Q
How are clearance receptors beneficial for drug delivery?
A
Protein drugs can be cleared from circulation by normal homeostatic mechanisms; specific receptors could be used to target drugs to the liver.
16
Q
What sizes are protein/peptide drugs and what is their bioavailibility?
A
- Large, hydrophilic molecules 0.5 - 100 kDa
-
17
Q
How does the half-life of protein drugs compare with small MW drugs?
A
Much less; from 15 seconds for angiotensin, oxytocin 2 mins and vasopressin 4 mins.
18
Q
Why are protein drugs fairly physically unstable/what causes this?
A
- Can easily lose their native 3D structure (secondary, tertiary, quaternary denaturation)
- Unfolding/denaturation as a result of: hydrophobic conditions/surfactants, pH/solvent/temperature, dehydration/lyphilsation
19
Q
What are the issues concerning adsorption to interfaces with protein drugs?
A
- Polar and non-polar residues (surfactant-like)
- Adsorbed at surfaces e.g:
> Air-water: foaming
> Air-solid: insulin binds to many surfaces; delivery pumps/glass/plastic syringes, plastic tubing/containers.
20
Q
How does aggregation of protein drugs come about and what may this lead to?
A
- Denatured/unfolded proteins may interact
- Aggregation becomes precipitation at a macroscopic level
21
Q
Give examples of protein drugs suffering chemical instability
A
- Deamidation; asparagine and glutamine residues hydrolysed to form carboxylic acid
- Oxidation; methionine, cysteine (can cause disulfide bond breakage)
22
Q
How can protein drugs become unstable with oxidation?
A
- Oxygen in air
- Mechanism via oxygen radical
- Catalysed by transition metals (Fe and Cu)
- Mediated by antioxidants e.g. Ascorbate
- PEG can result in peroxide formation
- Photo-oxidation
23
Q
What barrier with biological stability do protein drugs face?
A
- Protein drugs hydrolysed to AAs and small peptide by GIT
- Gastric acid; denaturation, pepsin enzyme (phe, tyr)
- Small intestine; trypsin, chymotrypsin, carboxypeptidases, aminopeptidases (pancreas)
- Colon; substantial microbial enzymes
24
Q
Give examples of peptides stable to biodegredation by the GIT.
A
- Cyclosporin (11 residues, cyclic); immunosuppressant
- TRH (thryotrophin releasing hormone); stimulates release of thyrotropin (thyroid-stimulating hormone/TSH) and prolactin from the anterior pituitary.
25
What are the preformulation aspects for protein drugs?
- Selection of candidate drug (protein)
- Determination of properties
- Chemical/biological alteration, Select pH, salts, solvents, surfactants, Liquid or dried formulation,
- Stable, active formulation
26
What needs to be considered with preformulation in regards to appropriate conditions?
- Physical state
- pH (affects ionisation of groups e.g. COOH/NH2)
- ionic strength
- temperature
27
How does freeze-drying affect stability?
Enhances stability (lyophilisation)
28
How does the addition of salts affect protein formulation?
Decrease denaturation via binding to the protein (also metal ions); stabilises structure
29
How do polyalcohols (e.g. glycerol) affect protein formulation?
They stabilise the protein via selective solvation (dissolution); of hydrophobic/hydrophilic groups
30
How do surfactants affect protein formulation?
They prevent adsorption of proteins at surfaces and aggregation (also phospholipids, fatty acids, other proteins); preventing interacting and keeping protein in solution.
31
What chemical modifications are available for proteins and how they do help?
- Adding synthetic polymers - PEG (polyoxyethylene); increases solubility - more 'membrane friendly'
- Lipids covalently bound to protein
32
What does primary sequence alteration ential for protein formulation?
Specific amino acids can be changes; improving physical and chemical stability
33
What are the issues with parenteral administration with protein drugs?
- Repeated administration required (short term)
- Patient compliance
- Stability of dosage form
e. g. Insuin
34
Why are non-parenteral routes preferred and how are proteins not suited?
- Highly desirable (especially oral)
| - Proteins have v. low absorption/bioavailability apart from a few select peptides
35
Which peptides can be administered non-parenterally and why?
- Cyclosporin
- TRH
- Captopril
>>> Short chain hydrophobic moieties (unusual)
36
What are the advantages of parenteral delivery?
Site-specific delivery:
- Pharmacokinetic advantages
- Improvement of therapeutic index
- Protection from unwanted drug disposition
- Extravascular access
37
What are the parenteral methods of site-specific delivery?
- Particulate systems; attach to polymer etc. (lipid membranes, coating proteins)
>>> Problems with RES (reticuloendothelial); particles can't cross vascular endothelium
- Soluble carriers; but have transport/stability problems (polymer of sorts)
38
What types of implantable system are there for protein drugs?
- Polymer gel matrix (biodegradable)
- Polymer fibre system
- Osmotic mini-pumps (respond to osmotic conditions)
- Tablet-type implants
- Automatic feedback systems
39
How do implantables deliver protein drugs and for how long do they last?
- Via the intramuscular/subcutaneous route
| - Drug release for periods of up to one year
40
What are some examples of implantable protein delivery systems?
- Zoladex (LHRH analogue); injectable biodegradable polymer matrix > 1 month
- Levonorgestrel; polymer fibre system > 6 months
41
How is Zoladex/Goserelin administered and form does it take?
- Into the upper abdominal wall via S.C.
- A sterile white/cream in a coloured cylinder 1mm in diameter & 5mm long, preloaded into a special single-use syringe
- Continuous release for 28 days for palliative treatment of advanced prostate carcinoma
42
How do peptidase inhibitors affect protein drug formulation?
- Improve stability/oral bioavailibility
| E.g. amistatin promotes absorption of enkephalins
43
How is saturation/bypassing of intestinal peptidases achieved?
- Increase dosage or load with another peptide
- Enteric coating e.g. acid-resistant acrylic resin (stomach)
- Use of azo polymers
44
How do penetration enhancers work? What are their disadvantages?
- Improvement of passive absorption either transcellular or paracellular
- Problems with irritation/tissue damage
45
List 3 penetration enhancers for insulin.
- Ionic/non-ionic detergents
- Bile salt surfactants
- EDTA and other chelating agents (coordinate bonds to metal ions)
46
How can reduction of hepatic first pass clearance/biliary excretion be achieved?
- Saturation of specific enzymes
| - Promotion of lymphatic uptake
47
How does cyclosporin bioactivity change when given in an olive-oil based formulation? Why is it so?
- From 1% to 20-50%
| - Reduction of hepatic first pass clearance through promotion of lymphatic uptake
48
What factors influence non-passive/paracellular transport for protein drugs and what does it affect?
- Carrier-mediated for di- and tri- peptides
- Endocytic/transcytotic for proteins
- M-cell (Peyer's patch, small intestine; can't take up enough for effective dose though)
- Role of P-glycoprotein efflux pump
- Transport across tight junctions (paracellular; widen junctions?)
>>> Affects oral bioavailability of peptides/proteins.
49
How can oral bioavailibility of peptides/proteins be improved?
- Peptidase inhibitors
- Saturation/bypassing of intestinal peptidases
- Penetration enhancers
- Reduction of hepatic first pass clearance
- Non-passive and paracellular transport
50
What factors must be considered with the pharmacological action of peptides?
- Dose-response relationships non-standard
- Multi-faceted cascade processes
- Hormone/paracrine/autocrine actions (short and long distance, local vs. systemic)
- Differences in biological action in male/female:
> How much?
> How often?
> Where to?
51
How do the actions of LHRH change with zero-order kinetics and pulsatile delivery? What does this suggest?
- Zero-order; antagonist
- Pulsatile; agonist
Zero order controlled release not always optimal; pulsatile/complex delivery kinetics beneficial sometimes.
52
What factors are considered when designing non-zero order delivery systems to achieve optimal pattern?
- Trigger (marker of disease state)
| - Recognition of trigger by system
53
What are the issues with delivery insulin subcutaneously?
- Fails to mimic endogenous insulin secretion
| - Non-compliance
54
How is insulin delivered to the lungs?
- Spray-dried insulin powder with stabilisers in blister
- Blister loaded at teh base of the inhaler and punctured by actuation
- Fluidization/deaggregation in aerosolisation chamber by compressed air
- Patient inhales particle cloud through slow deep breath
55
How do the pharmacokinetics/dynamics of inhaled insulin compare with insulin SC?
- Serum concentrations peak earlier and decay more rapidly
- Onset of action quicker and duration of action prolonged compared to rapid-acting insulin analogues
- Patients can inhlae insulin just 10 minutes before meal but still require bedtime SC injection
56
What are the potential non-respiratory adverse effects of inhaled insulin and how do they come about?
- Hypoglycaemia (frequency + severity similar to SC injections)
- Increase in insulin antibodies (no clinical effects so far but long-term?)
Bioavailibility of inhaled insulin; 15-30% (unabsorbed dose = side effects)
57
What are the potential respiratory adverse effects of inhaled insulin?
- Cough, increased sputum
- Decrease in lung function in some patients (recommend patients undergo lung function tests and periodically thereafter)
