Preservation and PET in pharmaceutical chemistry by University of Portsmouth

Preservation and PET Overview

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Preservation and PET
By the end of this lecture you should be able to:

• Understand the meaning of preservation and the need for it
• Understand the difference between physical and chemical preservation
• Describe the common methods of physical preservation and their key features
• Give an account of different types of chemical preservatives, discuss their features and applications
• Discuss the main activity limiting factors for preservatives
• Define PET, explain how it is performed and how its outcomes are interpreted and applied.Preservation

Preservation systems are employed to prevent or reduce microbial contamination in pharmaceutical
products AFTER production.
Not an alternative to cover for inadequacies of manufacturing.
Efficacy of preservation is tested using a "challenge test" on the final product in its container.

Physical and Chemical Preservation Methods

Physical Preservation Factors

Physical
Water
content
1
Redox
potential
PH
Temperature

Chemical Preservation Factors

Chemical
Preservatives
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Physical Preservation: Water Content

Water is they key requirement for microbial growth in pharmaceutical products.
Water activity (Aw) is the measure of available water in the product.
Water activity ranges from 1.00 for pure water to 0.00 for a bone-dry material. E.g., a tablet in a sealed container
has Aw of 0.3-0.4 whereas syrup BP (66.7% sucrose in water) has Aw of 0.86.
Most organisms need an environment with water content >75% (Aw > 0.75) to grow.

Minimum Water Activity for Microorganisms

Microorganism
Minimum Aw required
Pseudomonas sp.
0.96
G-ve bacteria
0.95
G+ve bacteria
0.8-0.9
Moulds and yeasts
0.65
Reducing the Aw below optimal level (< 0.75)
" increase in lag phase
" decrease in growth rate
decrease in number of microorganisms at the stationary phase
At very low Aw no growth occurs
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Aw =
Vapour pressure of product
Vapour pressure of water

Microbial Growth Phases

Stationary phase
Number of cells (log)
Death
(decline)
phase
Log
(exponential)
phase
Lag phase
Time
Different phases of microbial growth

Physical Preservation: pH, Temperature, Redox Potential

pH Effects on Microorganisms

pH
· Microorganisms can survive at pH between 3-11.
· Optimal growth is at near neutral pH (6-8).

Temperature Effects on Microorganisms

Temperature
. Most microorganisms grow best at ambient
temperatures (15-45 ℃).
· Storing the products in fridge (2-8 ℃) reduces
microbial growth.

Redox Potential Effects on Microorganisms

Redox potential
· Determined by oxygen content and ingredients.
•
Many aerobic and facultative anaerobic bacteria
multiply at relatively low redox potentials.
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Ideal Chemical Preservative Characteristics

Broad spectrum
activity
Rapid antimicrobial
activity
Chemically stable at
wide pH range (5-8)
Microbiologically
effective at wide pH
range
Ideal chemical
preservative
Cost-effective
Compatible with
excipients and
packaging
safe (not toxic)
Not alter physical
properties of the
product (color, taste,
smell)
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No preservative will meet all these criteria!

Types of Chemical Preservatives

Essential oils
Acids
Metals
Alcohols
Chemical
preservatives
Quaternary
ammonium
compounds
Phenols
Hydroxybenzoates
Sulphites
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Chemical Preservatives: Combinations and Chelating Agents

Combination of Preservatives

Combination of preservatives:
" To increase the spectrum of activity
To enhance the activity of an agent (synergism)
Antagonism may also occur between antimicrobial agents.

Chelating Agents

Addition of chelating agents such as EDTA to the formulation (no intrinsic microbiological activity):
Bind with divalent and trivalent cations in cell wall (mostly in gram negative bacteria)
Cell wall becomes more permeable to preservative agents
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Preservatives: Activity Limiting Factors

pH =pKa + log10
[salt]
[acid]
Benzoic acid
(pKa = 4.2 at 25 ℃)
at pH 4.2: 50% ionised
at pH 6.2: 99% ionised
at pH 2.2: 1% ionised
Solution pH /
preservative pKa
effects
Partition into oil
phase
(lipophilic
preservatives)
Micellization
(lipophilic
preservatives)
YE
Concentration
Effective
Concentration
adsorption to
suspended particles
Entry into container
and/closure
(leaching)
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Natural Preservatives: Essential Oils

OContain alcohols, aldehydes, esters, ketones, or terpenes
· e.g. peppermint oil
x High concentration:
· affecting organoleptic properties
" might not be miscible with the rest of the excipients in the formulation
x Expensive
v Natural, better accepted by the patients.
? Are likely to exert more than one activity (can be advantage or disadvantage)
" e.g. peppermint oil can have positive GI effects
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Synthetic Preservatives: Acids

· Weak carboxylic acids
· e.g. Benzoic acid, Sorbic acid
· Optimally active in unionised form i.e. pH <2-3 units below pKa
· Active against bacteria but less active against yeasts and fungi
. Used primarily in foods and oral drug products (pH < 5) ; sufficient low toxicity

Acid Preservative Properties

Agent
In-use
concentration
(%w/v)
Formulation
usage
Optimal pH
range
Water
solubility
Partition
coefficient
Antimicrobial
Activity
(1:highly active - 3:weakly active)
G +ve
G -ve
Moulds
Yeasts
Acids and salts
Benzoic acid
Sorbic acid
0.1
Oral, topical
2-5
1 in 350
3 - 6
1
2
3
3
0.2
Oral, topical
<6.5
1 in 700
3.5
2
2
2
1
O
OH
OH
pH =pKa + log10
[salt]
[acid]
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Benzoic acid, pKa:4.2
Sorbic acid, pKa:4.8

Synthetic Preservatives: Alcohols

Usually arylalkyl or substituted aliphatic alcohols
" e.g. ethanol, benzyl alcohol, phenoxyethanol, phenylalcohol, chlorbutol and Bronopol
Can be used for topical, oral, ophthalmic and parenteral formulations.
Disrupt the cell membrane of microorganisms
More active against bacteria and less active against yeasts and fungi
Have broad antimicrobial activity
Ethanol and isopropyl alcohol: skin antiseptics & disinfectants
Highly water miscible (can be also used as co-solvents)
Their activity is reduced upon dilution (optimal concentration range) and by organic material.
Low toxicity and Low cost
! Flammable

Alcohol Preservative Properties

Agent
In-use
concentration
(%w/v)
Formulation
usage
Optimal pH
range
Water
solubility
Partition
coefficient
Antimicrobial
Activity
(1:highly active - 3:weakly active)
G +ve
G -ve
Moulds
Yeasts
Alcohols
Benzyl alcohol
1.0
<5
1 in 25
1.3
1
3
3
3
Bronopol
0.01- 0.1
Parenteral, topical
Oral, topical
Parenteral, topical
5-7
1 in 4
0.11
2
1
3
3
Chlorbutol
0.3-0.5
<4
1 in 130
1
1
3
2
Ethanol
20 - 70
Oral
Miscible
1
1
2
2
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1.0
Broad
1 in 43
2
1
3
3
Phenylethanol
0.25-0.5
Topical
Parenteral, ophthalmic
<7
1 in 50
2
1
3
3

Synthetic Preservatives: Phenols

e.g. Phenol, Cresol, Chlorocresol
Kill the microorganisms by damaging the cell membrane, protein denaturation and cell lysis.
Introduction of chlorine and methyl groups results in chlorocresol and chloroxylenol respectively that lack tthe toxic
and corrosive properties of phenol while enhancing and prolonging its antimicrobial activity.
I Chlorocresol: Used as bactericide in injections and O/W emulsions (creams)
Chloroxylenol: Antiseptic
Phenol:
Diluted to <1% w/v to be less caustic
Used in gargles and lotions.
· Dissolved in glycerol for ear drops (Phenol Glycerin)
Phenols have their greatest activity in the unionised form (acidic conditions).
O Inactivated by dilution and organic material.

Phenol Preservative Properties

Agent
In-use
concentration
(%w/v)
Formulation
usage
Optimal pH
range
Water
solubility
Partition
coefficient
Antimicrobial
Activity
(1:highly active - 3:weakly active)
G +ve
G-ve
Moulds
Yeasts
Phenols
Chlorocresol
0.1
Parenteral, topical
3-5
1 in 260
117- 190
1
2
3
3
Cresol
0.3
Parenteral, topical
3-5
1 in 50
2
3
3
3
Phenol
0.25-0.5
Parenteral, topical
5
1 in 15
2
3
3
3
OH
CI
-OH
H3C
Chlorocresol
H3C
CH3
CI
Chloroxylenol
4
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Synthetic Preservatives: Bisphenols

De.g. Hexachlorophane and triclosan (Irgasan)
-Have Low solubility but enhance the activity of other phenols
OUsed as skin antiseptics
OUsed in creams, cleansing lotions or soaps. Valuable in post-operative infections and cross-infections
! Hexachlorophane is restricted in UK (concentration and product type); used in prophylaxis against
Staphylococcal infections.
x Are absorbed by rubber and plastics (packaging considerations)
x Have little or no sporicidal activity at room temperature.
CI
CI
CI CI
CI
CI
OH
OH
Hexachlorophane
CI
OH
0
CI
CI
Triclosan
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Synthetic Preservatives: Sulfites

Sulfites and metabisulfites are very water soluble
· e.g. Sulfur dioxide, Sodium sulphite anhydrous, Sodium hydrogen sulfite, Sodium metabisulfite, Potassium
metabisulfite
Active against Bacteria, fungi and yeasts.
Their activity increases with decreasing pH and is mostly derived from undissociated sulfurous acid,
which predominates at a pH < 3.
OUsed as preservative and antioxidant in foods, drinks, and drug products. Food/drinks preservatives and
drug products.
ISulfiting agents are commonly used in emergency parenteral drugs such as epinephrine,
norepinephrine, dexamethasone, dobutamine, dopamine, phenylephrine, procainamide, and
physostigmine.
Possibility of allergic reactions or anaphylaxis.
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Sulfite Mechanism of Action and Properties

H2N
H2N
H
H
+
N
N
O
O
N
O3S
N
H
H
Pyrimidines in nucleic acids react with HSO3- to form addition compounds
RSSR +
SO3-2
RSSO3" +
RS"
Cleavage of S-S bonds in amino acids by SO3-2 forms addition compounds

Sulfite Preservative Activity

Agent
In-use
concentration
(%w/v)
Formulation
usage
Optimal pH
range
Water
solubility
Partition
coefficient
Antimicrobial
Activity
(1:highly active - 3:weakly active)
G +ve
G-ve
Moulds
Yeasts
Sulphites
0.1
Parenteral
<4
1 in 2
3
3
2
2
1
3
3
3
2
1
3
3
3
2
2
2
1
1
1
1
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HSO3

Synthetic Preservatives: Hydroxybenzoates (Parabens)

Parabens: esters of p-hydroxybenzoic acid and their salts (methyl, ethyl, propyl, butyl and benzyl esters)
OHigher esters exhibit greatest activity.
OHave low water solubility; use co-solvents such as glycerol to solubilise.
OHigher members have water solubilities only slightly greater than concentrations needed for
preservative effect. Sodium salts are sometimes used (consider pH of formulations).
Parabens have activity in a wide range of pH (3-9). However, hydrolysis (ester bond) occurs in alkaline
solutions.
OMaximum activity achieved by using two (or more) esters to provide higher total concentration in
solution.
· Methyl paraben 0.2%
· Propyl paraben 0.02%

Hydroxybenzoate Preservative Properties

Agent
In-use
concentration
(%w/v)
Formulation
usage
Optimal pH
range
Water
solubility
Partition
coefficient
Antimicrobial
Activity
(1:highly active - 3:weakly active)
G +ve
G-ve
Moulds
Yeasts
Hydroxybenzoates
All members of
the series
and salts
0.4-0.8 (acid)
Oral, topical
Contact lens
solutions
3.0-9.5
Methyl 1 in 500
7.5
1
3
2
2
Ethyl 1 in 1300
1
3
2
2
Propyl 1 in 2500
80
1
3
2
2
Butyl 1 in 6500
280
1
3
2
2
Benzyl 1 in 10000
1
3
2
2
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