Week 4, 7 and 8 - Stability of Drugs + Medicines, Quantitative Assessment of API Degradation and Metabolism + Lead Optimisation

Question 1

What is Stability

Answer 1

Chemical stability - each API retains its chemical integrity + potency (stays within specified limit)

Physical stability - inc. aggregation state, structure, dissolution, disintegration
- leads to sedimentation, cracking, caking, change in drug release etc.

Microbiological stability - sterile, resistant to antimicrobial growth (can use antimicrobial agents)
- contamination; environment containing water, machinery, people involved in manufacturing

Question 2

List the 6 factors affecting stability of drugs / medicines

Answer 2

1. Humidity (hydrolysis + oxidation/reduction)
2. Oxygen (oxidation / autoxidation)
3. Light degradation (photo degradation, photons case radical formation, oxidation)
4. Temperature (accelerate hydrolysis, oxidation, reduction)
5. pH (acids + base catalyst can increase chemical degradation)
6. Metal ions (oxidation)

All can effect drugs stability + lead to degradation
Many dugs are chiral, chirality required to maintain potency

Question 3

Explain how hydrolysis affects stability

Answer 3

• Most common drug instability
• Caused by humidity (H2O), temperature
• Nucleophilic attack by water
  - lactam > ester > amide > imide (NH
  = ester more prone to hydrolysis than amide
  = from COOH (carboxylic acid)
  - lactam = ring with NH | amide = O=C-NH2
• Rate of hydrolysis depend on pH

PREVENTION:
- Buffers
- Co-solvents
- Dessicants (absorb H2O)

Question 4

Explain how oxidation affects stability

Answer 4

Autoxidation - oxidation occurring at room temp.
- oxidation forms free radicals (very reactive)
- oxidation is promoted by oxidising agents, light, metal ions
RH = R* + H* (initiation)
R* + O2 = RO2* (propagation)
RO2* + RH = ROOH + R*
- ROOH (hyperoxide) react + produce stable oxidation products
- termination occurs when O2 runs out = free radicals combine + produce unreactive product (process ends)

PREVENTION:
- Air tight container with inert gas (e.g. Nitrogen)
- Antioxidants (suppress oxidation)
- Ascorbic acid (controls oxidation as it undergoes rapid oxidation + forms safe, stable impurity)
- Chelators (EDTA) prevent metal ions from causing oxidation (chelates ions)

• = radical

Question 5

Explain how photodegradation (light) affects stability

Answer 5

UV energy is very high = radiation is high
- light from natural sunlight

• UV can cause free radical formation (causes homiletic split)
  - drug susceptible to photolysis are susceptible to oxidation
• Photolysis can change activity of drug

PREVENTION:
- Amber glass bottles (prevent light from penetrating / filter out UV)
- Airtight container protected with an inert gas

Question 6

Explain how dimerisation and polymerisation affects stability

Answer 6

• 2 molecules of the same drug react to form a polymer or dimer
• causes toxicity when multiple begin to crosslink

Question 7

Explain how isomeric changes affects stability

Answer 7

• some isomers are more active than other forms = loss of activity
• can become more susceptible to degradation (light, humidity, O2)

isomeric changes = e.g. L-isomer vs D-isomer

Question 8

What is the importance of Quality Assurance (QA) (against these risks)

Answer 8

• Ensures have an ideal medicine that is safe and effective for patient
  - i.e. correct product, correct strength, not degraded, packaged + labelled correctly, free from contamination

Product has to be:
- pure (no contaminants, impurities)
- uniformity of dosage form (consistency in colour, shape, size)
- bioavailability
- stability (should retain its properties till expiry date)
- APIs should be on label

• Records are made throughout manufacturing process to ensure product meet standards + followed requirements (need to keep documents)

Question 9

What does Good Manufacturing Practice (GMP) and Quality Control (QC) ensure

Answer 9

• that products are consistently produced
• products are made to the quality standards required for their use + marketing authorisation

QC is a part go GMP
- consists of sampling, testing and checking products at each. level of manufacturing to ensure they meet standards / requirements

Question 10

How do stabilisers minimise degradation concerns

Answer 10

Hydrolysis
- add buffer (prevents pH changes)
- pH affects hydrolysis rate
- co-solvents
- replacing water with other solvent = ↓ hydrolysis
- complexing agent
- slows down hydrolysis

Oxidation:
- antioxidants (e.g. ascorbic acid, citric acid)
- slow down / suppress oxidation reaction
- chelating agents (e.g. EDTA)
- prevents metal ions causing oxidation
- inert gas (e.g. nitrogen)
- prevents oxidation

Preservatives:
- prevents antimicrobial growth

Low temp.
- slows down rate of hydrolysis, oxidation

Question 11

How do storage conditions minimise degradation concerns

Answer 11

• Air tight container and inert gas
  = prevents oxidation and photolysis
• Amber glass bottle
  = prevents photolysis
• Desiccant (e.g. in lid of effervescent tablets)
  = prevents hydrolysis

Question 12

What are instability issues with protein / peptide drugs

Answer 12

Inc. monoclonal antibodies, insulin, vaccines e.g. influenza

• Most are parenteral (non-mouth) as converting them to solid dosage could denature them
  - liquid formulations have more problems with hydrolysis + oxidation
• Need to prevent degradation as drug wont work if its denatured

PROBLEMS:
- hydrolysis (can cause changes from natural to -ive charge)
- oxidation (e.g. if have Sulfur + O2 = SO2, remove S to avoid problem)
- photolysis (if have many double bonds)
- denaturing
- aggregation (antibodies close together will aggregate)
- cross-linking (if have cysteine = disulphide bonds = aggregation + polymerisation)

Question 13

How is the shelf life (t90) of a drug estimated and how does it differ in climate zones

Answer 13

Shelf life - the amount of time a product retains acceptable chemical, physical and microbiological stability

• Most reactions are 1st order (rate = k x [A] ~ A = API)
  - will get a linear line when use natural log
  - most drug decompositions follow 1st orders kinetics

t90 = the time taken for 10% of API to degrade / loss (90% of API is still present)
- drug is only valid if have API >90% (minimum level)
- Equation: (100/90) / k = 1.11/ k = ln(1.11) / k
- ln(1.11) / k tells you how long it will take for your drug to degrade by 10%

Question 14

Why do we use Stability Testing (stress testing)

Answer 14

So product produced can meet / satisfy diff. climate conditions around the world
- temp; 21ºC to 30ºC and humidity; 35 t0 70%
Ensures product quality is good, safe for patient and its stability (for packaging)
- can be used to predict shelf life (t90)
- evaluates how likely it is for product to degragrade
- find out where impurities are from

• testing involves storing product in realistic worst-case conditions to test thermal stability + sensitivity to moisture e.g very humid, very high temp. etc.
  - done to see how stability of a drug will be affected (e.g. ↑ humidity = ↑ hydrolysis rate)

Accelerated Stability Testing
- speeds up testing process during development
- testing is done for min. 3 months (6 month if there’s changes)
- test product at diff. temps (use elevated temp. from 40ºC)
- use Arrhenius equation to work out k = can work out t(90)

Question 15

What is the role of stationary phase in HPLC

Answer 15

Stationary phase binds to diff. components in the sample
- phase is fine silica particles
- silica particles are non polar

• Use ultrasound to break up drug formulation i.e. tablet + to separate and disperse silica particles
• ↑ retention time by making silica less polar / more non-polar
  - do this by adding alkyl groups (CH3)

Question 16

What is the role of mobile phase in HPLC

Answer 16

Mobile phase flows through stationary phase + carry the components in the sample
- phase is polar, use water + organic solvent (e.g. ethanol)
- organic solvent helps dissolve drug / sample (e.g. acetonitrile for GTN)

• ## ↑ retention time if use more polar solvent (water) / ↓ organic solvent

Question 17

What are the functions of components in a High Performance Liquid Chromatography (RP HPLC)

Answer 17

A technique used to separate impurities from API as it is sensitive + selective (purify)
Identify, analyse and QUANTIFY drug / API
Quantify by measuring peak height OR area under peak

1. Fine silica particles are packed into column (stainless steel)
2. Have a pump connected to 2 solvents (H2O/ organic solvent)
   - sucks up the solvents pumps it into column
3. The injector injects small amounts of the sample quickly into column
4. Sample flows through column / silica into detector
   - analytes separated when interact with stationary phase / silica
5. UV Detector will detect the absorbance of the analytes passing through
6. Detector display the peaks of each component (amount of analyte)
   - displays one peak at a time
   - peak = retention time (of the component) = time taken for analyte to pass through column
   - area under peak = the AMOUNT that passed the detector

x-axis = retention time
y-axis = detector intensity

Question 18

Why do we use reverse phase (RP) HPLC

Answer 18

Reverse phase is a type of separation (also have normal phase)
- stationary phase (silica - C18) = non-polar | mobile phase = polar

• easier to use + have precise control over organic solvent
• most polar analyte will come out first
• drug bind to silica for specific time until partioned when more organic solvent is added

Isocratic Method: keep composition of 2 solvents on mobile phase same through e.g. 20%/80% or 50%/50%

Gradient Method: composition of each solvent varies over time
- start with H2O and mix in organic solvents
- less steep gradient = better / high resolution
- more organic = analytes elude faster = ↓ retention time = peaks closer

Question 19

Why do we need to use internal and external calibrants in HPLC

Answer 19

External:
Run a series of standards, construct a calibration curve and then determine unknown samples from that curve
- area under peak is calculated automatically
- calculate conc. from the ratio of your sample peak areas and the standard peak areas
1. prepare standard solutions with known conc.
2. perform HPLC
3. plot area (y-axis) vs conc. (x-axis) graph

Internal:
Use / add a standard (compound( that has similar stricture to the API = get 2 peaks which are similar but distinguishable
- more precise, more reliable + less chance of error
1. Add standard to mixture
2. The areas are compared + used to calculate the conc.
- use same sample preparation for sample and standers

Question 20

How do we use chromatographic data to evaluate purity / degradation of sample

Answer 20

• Use internal or external calibrants
• Use placebo samples (without API) to distinguish background peaks
  = peaks caused by excipients, impurities etc.

Question 21

What does BPCRS mean

Answer 21

British Pharmacopoeia Chemical Reference Sample

Question 22

How can you increase / decrease the chemical stability of a lead compound

Answer 22

Increase Stability:
- Remove ESTERS
- they’re less stable due to electron withdrawing nature of O
- they’re more prone to hydrolysis
- C is attacked by H2O (Nu: ) = COOH (c.acid) formed
- they’re very reactive = not wanted in many drugs

• Replace ester with a more stable group (e.g. amide)
• Increase steric hinderance around carbonyl to block Nu: attack
  - molecule conversion is prevented

More stable molecules can have ↑ duration of action

Question 23

How can you increase / decrease the metabolic stability of a lead compound

Answer 23

Increase Stability:
- Remove sites on drug where metabolism is likely to occur
- e.g. H on aromatic rings, H adjacent to aromatic rings and H adjacent to heteroatoms

• Introduce groups at susceptible sites to prevent / block metabolism
  - e.g. replace all H with F (fluorine) as F isn’t susceptible to radical abstraction
  - C-H bonds become C-F bonds
• Reduce lipohilicty of molecules
  lipophilic molecules more likely to bind to CYP)
• Use bioisosteres to shift the position of the susceptible group so its unrecognisable by the metabolic enzyme BUT still recognised by its target
  - used for important susceptible groups that can’t be replaced

Reducing metabolism (clearance) will ↑ drugs lifetime in body + ↑ half life

How Metabolism Occurs
- In liver drug will bin to CYP
- Forms drug-CYP complex
- Oxidative reaction occurs to from metabolite (metabolite-CYP)
- Metabolite dissociates from CYP + allows new drug molecule to bind

Question 24

Why does GTN tablets (sublingual) have shorter half life compared to GTN spray

Answer 24

Once tablets open API begins to evaporate
- as GTN is volatile = loses its potency rapidly
- tablets are stored in container which opens all the time where as liquid in a closed container

Keep in cool conditions
- avoid degradation

Store in glass container
- prevents GTN being absorbed by packaging