04_Microbial Spoilage Infection Risk and Contamination Control 2.pdf

11-Oct-23 Microbial Spoilage, Infection Risk & Contamination Control Hugo and Russell’s Pharmaceutical Microbiology, 8th Edition...

11-Oct-23 Microbial Spoilage, Infection Risk & Contamination Control Hugo and Russell’s Pharmaceutical Microbiology, 8th Edition Chapter 17 – Page 273 Mahmoud Alkawareek, PhD 1 Introduction In addition to the API, pharmaceutical products also contain a variety of other ingredients (i.e. excipients) which make the formulation: - Easy to manufacture - Stable - Safe - Effective - Convenient to the patient Products made in the pharmaceutical industry must meet high microbiological specs, i.e. if they are not sterile, they should have no more than a minimal microbial population at the time of product release. Mahmoud Alkawareek, PhD 2 1  11-Oct-23 Introduction Occasionally some product batches with unacceptable contamination (level or type of organism) escape QA net. The consequences will be: - Product spoilage rendering it unsuitable for use - Potential health hazard to patients perhaps resulting in outbreaks of medicament-related infections. - Financial loss: Direct: wastage of individual batches Indirect: damaging publicity through product recall Most commonly, contamination occurs with opportunistic m.o. (e.g. Pseudomonsa spp) which has resulted in the spread of nosocomial infections in compromised patients. Less common is the contamination with pathogenic m.o. (e.g. Salmonella) & products contaminated with toxic microbial metabolites. Mahmoud Alkawareek, PhD 3 Spoilage-- Chemical & Physicochemical Deterioration Of Pharmaceuticals As major contributors to the natural recycling process, m.o. have the ability to degrade a wide range of compounds; such degradation usually occurs at relatively mild physicochemical conditions. Mixed microbial communities are more effective biodeteriogens than individual species alone, why? Because the initial attack of complex substrates by one group of m.o. renders the them susceptible to further deterioration by secondary m.o. Mahmoud Alkawareek, PhD 4 2  11-Oct-23 Spoilage-- Chemical & Physicochemical Deterioration Of Pharmaceuticals Novel pathways to attack synthetic chemicals may also emerge under environmental selection pressures! But the degradation rate of these ‘xenobiotics’ can vary greatly: t0.5 (phenol)≈ few hours, while t0.5 (halogenated pesticides)≈ several years! The overall rate of deterioration of a chemical depends on: - Molecular/chemical structure of the compound - The physicochemical properties of a particular environment - Type & quantity of microbes present - Whether the metabolites produced can serve as source of usable energy & precursors for biosynthesis, and hence the multiplication of m.o. Mahmoud Alkawareek, PhD 5 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖Therapeutic Agents Active drug constituents may be degraded to less potent, inactive or toxic forms. Some drugs may be metabolized by m.o. and serve as substrates supporting the growth of more m.o., examples on these drugs are - Alkaloids (morphine, atropine), analgesics (aspirin & paracetamol), barbiturates and steroid esters Although reports of drug destruction by m.o. are not that frequent, there are some notable exceptions such as: - Inactivation of penicillin injection by b-lactamase producing bacteria, - Steroid metabolism in damp tablets & creams by fungi - Microbial hydrolysis of aspirin suspension Mahmoud Alkawareek, PhD 6 3  11-Oct-23 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖Surface Active Agents Anionic Surfactants - Alkali metals & amine soaps of fatty acids are considered stable due to slight alkaline pH of the formulations, but once diluted into sewage are readily degraded - Alkyl/alkylbenzene sulphonates & sulphate esters are metabolized by multi-step oxidation followed by ‘ring fission’. - Ease of degradation decreases with increasing chain length & complexity of branching of the alkyl chain Mahmoud Alkawareek, PhD 7 Pharmaceutical Ingredients Susceptible to Microbial Attack - Surface Active Agents Non-ionic Surfactants - Examples: alkyl polyoxyethylene alcohol emulsifiers, sorbitan esters (Spans) and polysorbates (Tweens) - Readily metabolized by wide variety of m.o. - Again, increasing chain length & branching decrease ease of attack - Lipolytic cleavage of fatty acids from sorbitan esters, polysorbates & sucrose esters is followed by degradation of the cyclic nuclei, producing small molecules readily utilizable by m.o Mahmoud Alkawareek, PhD 8 4  11-Oct-23 Pharmaceutical Ingredients Susceptible to Microbial Attack - Surface Active Agents Ampholytic Surfactants - Examples: surfactants based on phosphatides, betaines & alkylamino substituted amino acids - Reasonably biodegradable Cationic Surfactants - Examples: cetrimide and benzalkonium chloride (both are QACs) - Usually used as antiseptics and preservatives - Only slowly degraded at high dilutions (i.e. in sewage) Mahmoud Alkawareek, PhD 9 Pharmaceutical Ingredients Susceptible to Microbial Attack - Surface Active Agents Relative Ease of Degradation?! (generally speaking) Non-ionic > Ampholytic > Anionic > Cationic Mahmoud Alkawareek, PhD 10 5  11-Oct-23 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖ Organic Polymers Thickening & Suspending Agents - Many of these agents are subject to microbial depolymerization by specific extracellular enzymes yielding nutritive monomers & fragments ✓ Examples: amylases (starch), pectinases (pectin), cellulases (carboxymethylcellulose), proteases (proteins) ✓ An exception is agar (a complex polysaccharide) which is relatively inert and hence is used as a support for solidifying culture media. Synthetic Packaging Polymers - Examples: nylon, polystyrene & polyester - Extremely resistant to microbial attack. Mahmoud Alkawareek, PhD 11 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖Humectants Low molecular weight hygroscopic materials like glycerol & sorbitol Included in some formulations to reduce water loss Readily metabolized unless present in high concentrations Mahmoud Alkawareek, PhD 12 6  11-Oct-23 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖Fats and Oils Extensively attacked when dispersed in aqueous formulations (e.g. oil-in-water emulsions) aided by high solubility of oxygen in oils. Fungal attack can occur in condensed moisture films on the surface of oils in bulk or in water droplets contaminating the oil phase. Lipolytic rupture of triglycerides liberates glycerol & FAs, where FAs then undergo β-oxidation of the alkyl chain producing odiferous ketones Mahmoud Alkawareek, PhD 13 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖ Sweetening, Flavouring & Colouring Agents Sugars & sweetening agents can be readily used as substrates for microbes, but if used at high conc., they inhibit microbial attack by reducing water activity In the past a variety of colouring agents (tartrazine & amaranth) & flavouring agents (peppermint water) were kept as stock solutions to be dispensed & diluted when required. - But many times they got contaminated by, and even supported the growth of, Pseudomonas spp. - Nowadays such stock solutions should be preserved. Mahmoud Alkawareek, PhD 14 7  11-Oct-23 Pharmaceutical Ingredients Susceptible to Microbial Attack ❖Preservatives & Disinfectants Many preservatives & disinfectants can be metabolized by G-ve bacteria, mostly when used below their effective ‘use’ level - Pseudomonas spp can grow in solutions of QAC antiseptics & chlorhexidine which has resulted in infection of patients. - Pseudomonas spp have also metabolized parabens in eye drops & caused serious eye infections. Mahmoud Alkawareek, PhD 15 Observable Effects of Microbial Attack on Pharmaceutical Products Microbial contaminants attack ingredients of a medicine and create substrates necessary for biosynthesis and energy production before they can replicate to levels where obvious spoilage becomes apparent. Early indications of spoilage are organoleptic (e.g. smell & taste) - “Sour” F.A. taste - Earthy taste - Bitter taste - “Fishy” amine smell - “Bad eggs” smell - Discoloration by microbial pigments Mahmoud Alkawareek, PhD 16 8  11-Oct-23 Observable Effects of Microbial Attack on Pharmaceutical Products Loss of viscosity due to depolymerization of thickening & suspending agents like acacia, CMC - This usually results in sedimentation of suspended ingredients Microbial polymerization of sugars and surfactant molecules - Production of slimy, viscous masses in syrups, shampoos and creams, - ‘Gritty’ texture in creams by fungal growth. Mahmoud Alkawareek, PhD 17 Observable Effects of Microbial Attack on Pharmaceutical Products Change of product pH by acidic or basic microbial metabolites, which may result in - Product instability and deterioration - Enhancement of microbial growth which was inhibited by the initial product pH (i.e. secondary attack) Production of gaseous metabolites - Seen as trapped bubbles within viscous formulations. Mahmoud Alkawareek, PhD 18 9  11-Oct-23 Observable Effects of Microbial Attack on Pharmaceutical Products - Example Inadequately preserved o/w emulsion: - Pigments may discolour the product (A) – aqueous phase - Metabolism of surfactants reduces stability and accelerates 'creaming' of oil globules (B) - Lipolytic release of fatty acids from oils lowers pH and encourages coalescence of oil globules (C) → cracked emulsion! - Also observed is a fungal mycelial growth on surface (D) Mahmoud Alkawareek, PhD 19 Factors Affecting Microbial Spoilage of Pharmaceuticals Types and Size of Contaminant Inoculum - Before doing a formula, the formulator should consider the environment and usage to which the product is likely to be subjected during its life and the history of similar medicines, why? to build as much protection as possible against non-standard encounters, such as additional preservation for a syrup if osmotolerant yeast contamination is particularly likely - If microbial failure occurs, identification of the contaminant(s) & knowledge of microbial ecology are very useful in tracking down the defective steps in the design or production process Mahmoud Alkawareek, PhD 20 10  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Types and Size of Contaminant Inoculum - Very low levels of contaminants which are unable to replicate in a product might not cause appreciable spoilage but, if a surge in contaminant bio-burden occur, the built- in protection could be insufficient and spoilage occurs - This surge might arise if: 1. Raw materials were unusually contaminated; 2. A problem of the plant cleaning protocol occurred; 3. Biofilm detached itself from within supplying pipework; 4. There was demolition or maintenance work in the vicinity of the manufacturing site (i.e. dust) 5. Gross misuse of the product during administration. Mahmoud Alkawareek, PhD 21 Factors Affecting Microbial Spoilage of Pharmaceuticals Types and Size of Contaminant Inoculum - Inoculum size alone is not always a reliable indicator of spoilage potential. e.g. a very low level of, aggressive Pseudomonads in a weakly preserved solution may suggest a greater risk than tablets containing fairly high numbers of fungal and bacterial spores. When an aggressive contaminant enters a medicine, there may be an appreciable lag period before significant spoilage begins, the duration of which decreases disproportionately with increasing contaminant loading. However, since there is usually a long delay between manufacture and administration of factory-made medicines, growth and attack could start during this period unless additional steps are taken to prevent it. Mahmoud Alkawareek, PhD 22 11  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Types and Size of Contaminant Inoculum - The isolation of a particular m.o. from spoiled product does not necessarily mean that it was the initiator of the attack. It could be a secondary opportunistic contaminant which had overgrown the primary spoilage organism once the physicochemical properties had been favourably modified by primary spoiler. Mahmoud Alkawareek, PhD 23 Factors Affecting Microbial Spoilage of Pharmaceuticals Nutritional Factors - Many common spoilage microorganisms (i.e. opportunistic m.o.) have simple nutritional requirements and metabolic adaptability This enables them to utilize many of the components of medicines as substrates for biosynthesis and growth, even including trace materials contained in them. Even demineralized water prepared by good ion-exchange methods normally contains sufficient nutrients to allow significant growth of many water-borne Gram-negative bacteria such as Pseudomonas spp. That’s why when such contaminants fail to grow in a medicine it is unlikely to be as a result of nutrient limitation but due to other, non-supportive, physicochemical or toxic properties. Mahmoud Alkawareek, PhD 24 12  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Nutritional Factors - Most acute pathogens require specific growth factors normally associated with the tissues they infect but which are often absent in pharmaceutical formulations. Therefore, they will unlikely multiply in them, but they may remain viable and infective for an appreciable time in some dry products where the conditions are protective. - The use of crude vegetable or animal products in a formulation provides an additionally nutritious environment. Mahmoud Alkawareek, PhD 25 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw) - Microorganisms require readily accessible water in appreciable quantities for growth. - Although some solute-rich medicines such as syrups appear to be 'wet', microbial growth in them may be difficult, why? Since the microbes have to compete for water molecules with the large numbers of sugar and other molecules of the formulation which also interact with water via hydrogen bonding. - An estimate of the proportion of the unbound water in a formulation available to equilibrate with any microbial contaminants and facilitate growth can be obtained by measuring its water activity (Aw). vapour pressure of formulation 𝑨𝒘 = …(under similar conditions ) vapour pressure of water Mahmoud Alkawareek, PhD 26 13  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw), examples: 1. Sucrose Solutions: Mahmoud Alkawareek, PhD 27 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw), examples: 2. NaCl Solutions: - The scale is different for the %’s of different solutes - The greater the (same) solute concentration, the lower is the water activity. Mahmoud Alkawareek, PhD 28 14  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw) - With the exception of halophilic bacteria, most m.o. grow best in dilute solutions (high Aw) and, as solute conc. rises (lower Aw), growth rates decline until a minimal, growth-inhibitory Aw is reached. - Limiting Aw values are of the order of: Gram-negative rods: 0.95 Staphylococci, Micrococci and Lactobacilli: 0.9 Most yeasts: 0.88 Mahmoud Alkawareek, PhD 29 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw) - The Aw of aqueous formulations can be lowered to increase resistance to microbial attack by the addition of high concentrations of sugars or polyethylene glycols. - Since there are trends to eliminate sucrose from medicines, alternative solutes are used, such as sorbitol and fructose, which are not thought to encourage dental caries. - But, syrup-fermenting osmo-tolerant yeasts were found to spoil products with Aw levels as low as 0.73, while some filamentous fungi can grow at even lower values, such as Aspergillus glaucus (0.61) That’s why even Syrup BP (67% sucrose; Aw= 0.86) has been reported to occasionally fail to inhibit osmo-tolerant yeasts and hence additional preservation may be necessary Mahmoud Alkawareek, PhD 30 15  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw) - Aw can also be reduced by drying, although the dry, often hygroscopic, medicines (tablets, capsules, powders) will require suitable packaging to prevent reabsorption of water and consequent microbial growth. - Some tablet film coatings are now available which greatly reduce water vapour uptake during storage. These might contribute to increased microbial stability during storage in particularly humid climates, although suitable foil strip packing may be more effective, but also more expensive. Mahmoud Alkawareek, PhD 31 Factors Affecting Microbial Spoilage of Pharmaceuticals Moisture Content: Water Activity (Aw) - Without proper packaging, condensed water films can accumulate on the surface of 'dry’ products such as tablets or bulk oils following storage in damp atmospheres with fluctuating temperatures, resulting in high localized Aw which can initiate fungal growth. - Dilute aqueous films similarly formed on the surface of viscous products such as syrups and creams, or exuded by syneresis from hydrogels, reach sufficiently high Aw to permit surface yeast and fungal spoilage. Mahmoud Alkawareek, PhD 32 16  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Redox Potential - The ability of microbes to grow in an environment is influenced by its oxidation-reduction balance (redox potential) since they require compatible terminal electron acceptors to permit function of their respiratory pathways. - The redox potential even in fairly viscous emulsion may be quite high due to the high solubility of oxygen in most fats & oils. Mahmoud Alkawareek, PhD 33 Factors Affecting Microbial Spoilage of Pharmaceuticals Storage Temperature - Spoilage of pharmaceuticals could occur over the range of about -20° to 60°C, although much less likely at the extremes. - The actual storage temperature may determine the spoilage by particular types of microorganisms. - Storage in a deep freeze at -20°C or lower is used for long- term storage of foodstuffs and some pharmaceutical raw materials. - Dispensed total parenteral nutrition (TPN) feeds have also been stored in hospitals for short periods at -20°C to even further minimize the risk of growth of any contaminants which might have been introduced during their aseptic compounding. Mahmoud Alkawareek, PhD 34 17  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Storage Temperature - Reconstituted suspensions and multi-dose eye drop packs are sometimes dispensed with the instruction to store them in domestic fridge (2°- 8°C), partly to reduce the risk of in-use contamination. - On the other hand, ‘water for injection’ is usually held at 80°C or above after distillation (prior to packing and sterilization) to prevent potential regrowth of Gram-negative bacteria, and the release of endotoxins Mahmoud Alkawareek, PhD 35 Factors Affecting Microbial Spoilage of Pharmaceuticals pH - Extremes of pH prevent microbial attack. - Around neutrality bacterial spoilage is more likely, with reports of Pseudomonads and related Gram-negative bacteria growing in antacid mixtures, flavoured mouth washes and in distilled or demineralized water. - Above pH 8, e.g. with soap-based emulsions, spoilage is rare. - For products with low pH levels such as the fruit juice-flavoured syrups (ca. pH 3-4) mould or yeast attack is more likely. Yeasts can metabolize organic acids and raise the pH to levels where secondary bacterial growth can occur. - In food industry low pH adjustment can be made to preserve foodstuffs (pickling, yoghurt), but this is not practical for medicines ( why??? ). Mahmoud Alkawareek, PhD 36 18  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Packaging Design - Packaging should be made in a way to control the entry of contaminants during both storage and use. - The most important dosage form to be protected are the parenteral drugs because of the high risks of infection by this route. - Self-sealing rubber closures must be used to prevent microbial entry into multi-dose injection containers following drug withdrawals. Mahmoud Alkawareek, PhD 37 Factors Affecting Microbial Spoilage of Pharmaceuticals Packaging Design - Wide-mouthed cream jars will allow the entry of fingers with their high bioburden of contamination. Thus, it is better to replace them with narrow nozzle and flexible screw capped tubes. - For medicines which rely on their low Aw to prevent spoilage, packaging such as strip foils must be of water vapour-proof materials with fully efficient seals. - Cardboard outer packaging and labels themselves can become substrates for microbial attack under humid conditions; therefore preservatives are often included to reduce their risk of damage. Mahmoud Alkawareek, PhD 38 19  11-Oct-23 Factors Affecting Microbial Spoilage of Pharmaceuticals Protection of m.o. within Pharmaceutical Products - The survival of microorganisms in particular environments is influenced by the presence of various relatively inert materials. - Microbes can be more resistant to heat or desiccation in the presence of some polymers such as starch, acacia or gelatin. - Adsorption onto naturally occurring particulate material may aid establishment and survival in some environments. - The presence of some surfactants, suspending agents and proteins can increase the resistance of microorganisms to preservatives, over and above their direct inactivating effect on the agents. Mahmoud Alkawareek, PhD 39 Hazard to Health Contaminated pharmaceutical products may present a potential health hazard to the patient. Contamination with pathogenic bacteria (e.g. Salmonella spp) forms a special risk since they can cause infections in a wide range of patients. The presence of opportunists with limited pathogenicity also present significant challenge to compromised patients. Mahmoud Alkawareek, PhD 40 20  11-Oct-23 Hazard to Health The outcome of using contaminated products vary from patient to patient depending on the type & degree of contamination & how the product to be used - The most serious effects are expected to be with contaminated injections as generalized bacteraemic shock & sometimes death is reported - Wound or sore in broken skin may become locally infected which may extend the hospital bed occupancy Most medicament related infections are difficult to be recognized by health practitioners which causes the spread of infection over several months Mahmoud Alkawareek, PhD 41 Hazard to Health - Examples Gram -ve bacteria: - G-ve bacteria were responsible for numerous outbreaks. - Pseudomonas spp have simple nutritional requirements & multiply significantly in aqueous products Cornea when scratched or damaged by irritant chemicals offers little resistance to Pseudomonas and hence contaminated ophthalmic solutions have resulted in frequent cases of infections; some leading to loss of sight. Pseudomonas contaminating antiseptic solutions caused skin infections in burnt patients resulting in failure of skin grafts & death. Infections of eczematous skin & respiratory infections in neonates were caused by contaminated ointments & creams. Mahmoud Alkawareek, PhD 42 21  11-Oct-23 Hazard to Health - Examples Gram -ve bacteria: - Infections by Salmonella spp were reported & the m.o. was isolated from products contaminated with it (tablets, pancreatin, thyroid extracts). - Oral mixtures & antacid suspensions can support the growth of Gram -ve bacteria & resulted in serious effects in immunocompromised patients (e.g. as a result of antineoplastic chemotherapy) - Bladder wash out solutions contaminated with Gram -ve bacteria caused painful infections Mahmoud Alkawareek, PhD 43 Hazard to Health - Examples Other cases: - HIV contaminated factor VIII products made from pooled human blood was reported - Creutzfeldt-Jackob disease infection from contaminated injections of human growth hormone derived from human pituitary Mahmoud Alkawareek, PhD 44 22  11-Oct-23 Hazard to Health Microbial toxins: - G-ve bacteria contain endotoxins (LPS) which can remain active in products even after cell death & some can survive moist heat sterilization - Endotoxins are inactive via oral route but if they enter blood stream via contaminated infusion fluids (even in ng level) or via diffusion across membranes from contaminated haemodialysis solution they can induce serious physiological effects. - Endotoxins cause fever, activation of the cytokine system, endothelial cell damage & these all lead to septic & often febrile shock. Mahmoud Alkawareek, PhD 45 Factors Determining the Outcome of a Medicament Borne Infection Clinical rxns may range from local infections of wounds or broken skin (contaminated topical prep) to GI infections (contaminated oral products) to serious widespread infections such as bacteraemia or septicemia possibly resulting in death (contaminated infusion) Clinical rxns resulting from the use of contaminated medicament may be evident in one patient but not in another one depending on many factors, among which are: 1. Type & Degree of Microbial Contamination 2. Route of Administration 3. Resistance of the Patient to Microbial Infections Mahmoud Alkawareek, PhD 46 23  11-Oct-23 Factors Determining the Outcome of a Medicament Borne Infection 1. Type & Degree of Microbial Contamination - Microorganisms contaminating medicaments are classified into true pathogens or opportunistic pathogens. - Pathogens rarely occur in products but if present they cause serious problems, examples: Clostridium tetani: caused wound infections & cases of neonatal death resulted from use of contaminated talcum powder Salmonella spp: caused outbreaks of salmonellosis due to ingestion of contaminated thyroid & pancreatic powders Mahmoud Alkawareek, PhD 47 Factors Determining the Outcome of a Medicament Borne Infection 1. Type & Degree of Microbial Contamination - Opportunists like P. aeruginosa, Klebsiella spp. and Serratia spp are more frequently isolated from medicinal products. The main concern with these organism is that their simple nutritional requirements enable them to survive in a wide range of pharmaceuticals, thus they present in high numbres 106-107 CFU/g or ml, although the product itself may not show visible sign of contamination. Compromised patients are considered at risk from infection with these m.o. - The critical dose of m.o. that will initiate an infection is highly variable (i.e. varies between spp & within spp.) Mahmoud Alkawareek, PhD 48 24  11-Oct-23 Factors Determining the Outcome of a Medicament Borne Infection 2. Route of Administration - Ophthalmic & parenteral route: Contaminated products injected directly into blood stream or instilled into the eye cause the most serious problems. Injectable & ophthalmic solutions are often simple & provide sufficient nutrient for G-ve opportunists. If contaminated; the product may end up with a bioburden as high as 106 CFU/ml in addition to the potential production of endotoxins Total parenteral nutrition fluids provide even more nutritional support for contaminants Intrathecal & epidural injections are potentially hazardous procedures, and thus in practice they are given through bacterial filters. P. aeruginosa (contaminant of eye drops) has caused serious ophthalmic infections, including loss of sight. The problem is compounded when the eye is damaged by improper use of contact lenses or scratched by fingernails or cosmetic applicators. Mahmoud Alkawareek, PhD 49 Factors Determining the Outcome of a Medicament Borne Infection 2. Route of Administration - Contaminated orally ingested products have different fate. This depends on whether the drug was taken on full or empty stomach as stomach acidity provides a barrier. - Contaminants in topical products may cause little harm if applied on intact skin, why? because intact skin provides a mechanical barrier & normal flora competes with the few contaminants. however, damaged skin (sores, burns, surgery, wounds) may be rapidly colonized & infected by opportunists potentially causing serious problems Mahmoud Alkawareek, PhD 50 25  11-Oct-23 Factors Determining the Outcome of a Medicament Borne Infection 3. Resistance of the Patient to Microbial Infections - Very important in determining the outcome of a medicament borne infection - Hospitalized patients are more exposed and susceptible to infection than those treated in the general community - Neonates, the elderly, diabetics and traumatized patients (by surgery, accidents…) are at special risk because they may have impaired defence mechanisms - Immunocompromised people (patients with leukaemia, HIV or treated with immunosuppressants) are most prone to infections. That’s why it is better to provide them with all medicines in a sterile form! Mahmoud Alkawareek, PhD 51 Preservation of Medicines Using Antimicrobial Agents Why to add a preservative: - To kill any anticipated low levels of contaminants, where from? a. Remaining in a non-sterile medicine after manufacturing b. Entering during storage c. Introduced during usage especially the repeated withdrawal of doses from a multi-dose container - To further reduce the risk of spoilage and health hazard Mahmoud Alkawareek, PhD 52 26  11-Oct-23 Preservation of Medicines Using Antimicrobial Agents If a medicine is unlikely to encourage growth or survival of contaminants and the infective risk is low, then a preservative might be unnecessary - Examples: tablets, capsules and dry powders Preservatives should not be added to deal with failures in poorly controlled manufacturing processes. Mahmoud Alkawareek, PhD 53 Preservation of Medicines Using Antimicrobial Agents Properties of an ideal preservative: - Broad spectrum and rapid activity to rapidly kill all microbial contaminants as they enter the medicine - Non-irritant and non-toxic to the patient - Selective in reacting with contaminants and not the formulation ingredients - Stable and effective throughout the life of the product Mahmoud Alkawareek, PhD 54 27  11-Oct-23 Preservation of Medicines Using Antimicrobial Agents But: - The most active antimicrobial agents are generally non-selective - The remaining preservatives have only modest antimicrobial efficacy - There are no preservatives considered sufficiently non-toxic for use in highly sensitive areas such as CNS and within the eye - Rapid killing of all contaminants may only be possible for relatively simple aqueous solutions, whereas for physicochemically complex systems only inhibition of growth and slow rate of killing may be realistically achieved. Mahmoud Alkawareek, PhD 55 Factors Influencing Antimicrobial Activity Within Medicines Effect of preservative concentration, temperature and size of inoculum: - Changes in the efficacy of preservatives vary exponentially with changes in concentration depending on the type of preservative Remember the concentration exponent (η)! - Changes in product temperature will alter efficacy in proportions, related to different types of preservative Q10! - If concurrent change in temp & conc → more complex scenario Example: if a 0.1% chlorocresol (η = 6, Q10 = 5) solution completely killed a suspension of E. coli at 30°C in 10 minutes, it would require around 90 minutes to achieve a similar effect if the temperature was lowered to 20°C and slight overheating during production had resulted in a 10% loss by vaporization in the chlorocresol concentration (other factors remaining constant) Mahmoud Alkawareek, PhD 56 28  11-Oct-23 Factors Influencing Antimicrobial Activity Within Medicines Effect of preservative concentration, temperature and size of inoculum: - Preservative molecules are used up as they inactivate microorganisms and as they interact non-specifically with the significant quantities of contaminant 'dirt‘ introduced during use. This will result in a progressive and exponential decline in the efficiency of the remaining preservative. - Preservative 'capacity' : describes the cumulative level of contamination that a preserved formulation can cope with before the preservative becomes ineffective. This will vary with preservative type and complexity of the formulation. Mahmoud Alkawareek, PhD 57 Factors Influencing Antimicrobial Activity Within Medicines Most preservatives interact in solution with ingredients of pharmaceutical formulations to varying extents via a number of weak bonding attractions as well as with any microorganisms present. - This can result in unstable equilibrium in which only a small proportion of the total preservative present is 'available' to inactivate m.o., and the resultant rate of killing may be less than anticipated from the performance of simple aqueous solutions. - However, the 'unavailable' preservative may still, contribute to the irritancy and toxicity of the product. When solute concentrations are very high and Aw is appreciably reduced, the efficiency of preservatives is often reduced and may be even inactive at very low Aw. - That’s why it is pointless to include preservatives in very low Aw products such as tablets and capsules. Mahmoud Alkawareek, PhD 58 29  11-Oct-23 Factors Affecting the 'Availability' of Preservatives 1. Effect of product pH - In the weakly acidic preservatives, the unionized molecules are the active ones & they have significant efficacy at pHs where ionization is low. Benzoic and sorbic acids (pKa = 4.2 and 4.75, respectively) have limited preservative usefulness above pH 5. 4(p)-hydroxybenzoate esters with their non-ionizable ester group and poorly ionizable hydroxyl substituent (pKa ca. 8.5) have moderate protective effect even at neutral pH levels. The activity of quaternary ammonium preservatives and chlorhexidine resides with their cations and are effective in products of neutral pH. - Formulation pH can also directly influence the sensitivity of microorganisms to preservatives Mahmoud Alkawareek, PhD 59 Factors Affecting the 'Availability' of Preservatives 2. Efficiency in multiphase systems - In a multiphase formulation, such as an oil-in-water emulsion, preservative molecules distribute themselves in an unstable equilibrium between the bulk aqueous phase and the oil phase by partition the surfactant micelles by solubilisation polymeric suspending agents and other solutes by competitive displacement of water of solvation particulate and container surfaces by adsorption any microorganisms present Mahmoud Alkawareek, PhD 60 30  11-Oct-23 Factors Affecting the 'Availability' of Preservatives 2. Efficiency in multiphase systems - Generally, the overall preservative efficiency can be related to the small proportion of preservative molecules remaining unbound in the bulk aqueous phase Although as this becomes depleted some slow re- equilibration between the components can be anticipated - The loss of neutral molecules into oil and micellar phases may be favoured over ionized species, although considerable variation in distribution is found between different systems. Mahmoud Alkawareek, PhD 61 Factors Affecting the 'Availability' of Preservatives 3. Effect of container or packaging - Preservative availability may be reduced by interaction with packaging materials. - Examples: The permeation of phenolic preservatives into the rubber closure of multi-dose injection or eye-drop containers and their interaction with flexible nylon tubes for creams. Quaternary ammonium preservative levels in formulations have been significantly reduced by adsorption onto the surfaces of plastic and glass containers. Volatile preservatives such as chloroform are lost by the routine opening and closing of containers Mahmoud Alkawareek, PhD 62 31  11-Oct-23 Quality Assurance & the Control of Microbial Risk in Medicines Mahmoud Alkawareek, PhD 63 Quality Assurance & the Control of Microbial Risk QA forms a scheme of management which includes all the procedures necessary to provide a high probability that a medicine will conform consistently to a specified description of quality, it includes measures taken during: - Formulation design & development (R&D) - Good pharmaceutical manufacturing practice (GPMP) - Quality control (QC) - Post marketing surveillance Mahmoud Alkawareek, PhD 64 32  11-Oct-23 Quality Assurance & the Control of Microbial Risk Risk assessment for each product should be made starting from raw materials, to administration. - Risk assessments are complicated due to uncertainties about the exact contaminant & spoilage expected. - Usually the manufacturers make the worst-case scenario & design strategies to cover it fully, so that lesser problems are also included. Also, it must be assumed that people administering the medicament are not highly skilled & aware of contamination control so that additional detailed information on administration & even training must be provided. Mahmoud Alkawareek, PhD 65 Formulation Design & Development The best way to eliminate the risk of contamination & spoilage is by sterilization, but! - it incurs high cost so sterile products are kept to be used for situations were there is high risk of consequent infection from contamination. In parenteral products, high risk of infection by contamination & presence of concerns of systemic toxicity of preservatives resulted in the production of sterile single-dosage units. However, with eye-drops the risk is lesser & sterile multi- dose products with preservatives to protect against in- use contamination is accepted. – Sterile ophthalmic single-dose units are more common in hospitals where there is increased risk of infection. Mahmoud Alkawareek, PhD 66 33  11-Oct-23 Formulation Design & Development Oral & topical routes of admin present low risk of infection & the emphasis is on the control of microbial content during manufacturing & subsequent protection of the formulation from spoilage. In the design process it is necessary to include features in the formulation & delivery system that provide protection against contamination because preservative use should be only considered when there is clear benefit due to toxicity & irritancy problems. Among these features: - Manipulation of physicochemical properties like Aw - Elimination of certain ingredient - Selection of container & preservative individually and collectively contribute to the microbial stability of the product. Mahmoud Alkawareek, PhD 67 Formulation Design & Development Laboratory tests (preservative effectiveness tests) were designed to challenge the product with artificial bioburden. It should be part of the formulation development & stability trials to ensure that activity is likely to remain throughout shelf life. Problems with these tests: - Does the performance of these tests gives reliable prediction of real in-use efficiency? - Repeated cultivation on microbiological media results in reduced aggressiveness of strains Mahmoud Alkawareek, PhD 68 34  11-Oct-23 Good Pharmaceutical Manufacturing Practice (GPMP) GPMP is concerned with control of ingredients, plant construction, process validation, production & cleaning. QC is part of GMP that deals with testing, specs, documentation & assessing conformance to specs. Relying on finished product testing may result in financial loss if non-compliance was detected at this late stage, besides microbiological test methods have poor precision & accuracy, so that end product testing may not detect failures. Mahmoud Alkawareek, PhD 69 Good Pharmaceutical Manufacturing Practice (GPMP) Assurance of overall product quality can only come from detailed specs, control & monitoring of all stages of manufacturing process not just from testing finished product. e.g. real estimate of product microbial quality comes from knowledge of bioburden of starting raw material, temp record of the granules, moisture level of the granules, validation record of the machine cleaning, foil strip packaging & testing of finished tablets. Each batch should meet all its specification, but this does not necessary means that all tests should be performed on finished product. The manufacturer can carry out parametric release. Parametric release: is to provide assurance that the product is of stipulated quality based on evidence of successful validation of the manufacturing process & review of the documentation on process monitoring carried out during manufacturing to provide the desired assurance of product quality. Mahmoud Alkawareek, PhD 70 35  11-Oct-23 Good Pharmaceutical Manufacturing Practice (GPMP) Points to be considered to assure finished product microbial quality: - Raw materials: Raw materials to be free from pathogenic microorganisms & with low bioburden. For ingredients from bovine source, exclude suppliers of materials were BSE is endemic - Manufacturing plant: Identify areas where m.o. can thrive & colonize to treat & clean them. The design & construction should allow for thorough cleaning. (machines are made from materials that can be cleaned & disinfected, some machines are provided with clean-in place systems) - Manufacturing process: Some products may require addition of some steps to reduce bioburden or improve lethal sterilization (e.g. include ultra filtration step rather than conventional sterilization cylcle) - Validation to the cleaning system: To challenge the ability of cleaning system to remove deliberately introduced contaminant Mahmoud Alkawareek, PhD 71 Quality control (QC) The current methods for counting & detecting some microbes in non sterile products have poor accuracy & precision, examples: - low no. m.o. sometimes damage the product but can’t be isolated and hence products with active spoilage yields very low microbial count - m.o. in high no. but it is not the primary spoilage agent nor pathogenic Uneven distribution of m.o present serious sampling problems Mahmoud Alkawareek, PhD 72 36  11-Oct-23 Quality control (QC) Pharmacopeias (Ph Eur, BP & USP) have included quantitative & qualitative standards for non-sterile products. - There are maximum total microbial levels & exclusion of specific m.o. depending on route of administration. - Example: for non-aqueous oral preparations, the count per ml or gram should be: no more than 103 total aerobic microbial count (TAMC) no more than 102 total yeast and mold (fungi) count (TYMC) no E. coli - Higher levels are permissible if the product contains raw material of natural origin. Mahmoud Alkawareek, PhD 73 Mahmoud Alkawareek, PhD 74 37  11-Oct-23 Quality control (QC) Endotoxin Test: - Endotoxin (pyrogen) levels in parenterals must be very low to prevent endotoxic shock. - Formerly this was tested by injecting rabbits & noting any febrile response. - Nowadays the test is performed using Limulus amoebocyte lysate (LAL) test where an amoebocyte lysate from the horseshoe crab Limulus polyphemus reacts specifically with microbial lipopolysaccharides to give a gel or opacity (turbidity) even at very high dilutions. - Tissue culture test are under development where the ability of endotoxins to induce cytokine release is measured directly Mahmoud Alkawareek, PhD 75 Post market surveillance It is impossible to guarantee that a medicine will never fail under real life conditions, but a proper quality assurance system must include procedures to monitor in use performance & respond to customer complaints in order to re-evaluate the constructed & implemented schemes for product safety & check whether they need modification. Mahmoud Alkawareek, PhD 76 38