Lecture 3: Parenteral Drugs and Sterility (1.28.25)

As far as the formulation of a parenteral, what are some properties that we should be concerned about that we’re lsee so for oral admin?

• pH

• Particulates

• Precipitation upon injection

• Water solubility

• Sterility

Drug recalls

• Contains undeclared drug ingredients

• Visible particulates

• Unapproved drug

• Lack of sterility assurance

• Bacterial contamination

• Incorrect ingredient level

• Contains wrong ingredients

• Accelerated or delayed release of active ingredient

• Costly for manufacturer

• Costly for patients

• Costly for manufacturer

• Materials costs

• Investigations

• Clean-up

• Manufacturing downtime

• Fines from regulatory agencies

• Lost market share

• Reputation

• Costly for patients

• Drug shortages

• Adverse events

• May require switch to another therapeutic regime

Sterility differences:

Injectable preparations vs oral

Sterile definition:

• Not able to produce children or young

Free from bacteria or other living organisms

Parenteral products should be sterile

• Must meet sterility standards

• Must not exceed endotoxin limits

Sterility is critical for parenteral products

• IV products are injected directly into the bloodstream

• Administration of non-sterile products that are intended to be sterile can result in life-threatening infections

• Depending on route of administration, possible infections can be systemic or localized

• Lack of sterility a greater concern for immunocompromised patients

• Need to also sterilize body surfaces and injection/infusion equipment

Microorganisms (primarily fungi and bacteria)

• Contain degradative enzymes that can act much like mammalian enzymes (phosphatases, proteases, peptidases, CYP450s, etc)

• Potential for local or systemic infections

• Can cause toxicity

• Major cause of product recalls

• Contain degradative enzymes that can act much like mammalian enzymes (phosphatases, proteases, peptidases, CYP450s, etc)

• Can change structure or break down structures of APIs and excipients

• May impact therapeutic properties or patient acceptance of formulation

• Potential for local or systemic infections

Immunocompromised or with preexisting infections at greater risk for adverse effects

• Can cause toxicity

Production of toxins

Production of toxins:

Mycotoxins, lipopolysaccharides

Fungi

• Molds, yeasts

• Uni- or multicellular organisms

• Produces spores to reproduce

• Examples

• Relatively large (~2-50 μm)

• Produce mycotoxins

• Route

• Examples:

Aspergillus, Penicillium, Candida, Rhizopus, and Fusarium

• Produce mycotoxins

• Primarily from Aspergillus flavus and Aspergillus parasiticus

• Including highly toxic aflatoxins and fumonisins (LD₅₀ 0.5-10 mg/kg)

• Route:

Inhalation of spores, topical administration

Viruses

• Non-living organisms

• Many different structures

• Very small (~20-200 nm)

• Can be difficult to detect

• Commonly attributed to animal-derived raw materials

• Non-living organisms

• Require a host organism for survival

• Cannot reproduce by themselves

• Use host cell machinery to replicate

• Many different structures

• Less similar to human cells than bacteria

• RNA or DNA genome

• Capsid-protein shell

• Envelope

• Capsid-protein shell

Nucleoproteins

• Envelope

Membrane that not all viruses have

• Can be difficult to detect

Sometimes affects the host cells; sometimes detectable only by molecular methods

• Commonly attributed to animal-derived raw materials

But not always

Bacteria

• Living, single-celled organisms

• Bear some similarity to human cells

• Examples

• Relatively large (~1 μm) compared to proteins, but much smaller than human cells (~8-20 μm)

• Reproduce through binary fission

• Some bacteria also produce spores

• Examples:

Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes

• Reproduce through binary fission

• Asexual reproduction where a bacteria divides and generates an identical copy

• E. coli can divide every 20 minutes

• Growth of E. coli, S. aureus, S. pyogenes fastest at 37°C

• Some bacteria also produce spores

• Dormant state of the bacteria

• Very resistant to thermal and chemical disinfection

The bare bones of microbiology terminology

• Gram-positive bacteria

• Gram-negative bacteria

• Gram-positive bacteria

• Single plasma membrane

• Thick peptidoglycan cell walls

• Lipoteichoic acid (LTA)

• Examples

• Examples:

Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes

• Gram-negative bacteria

• Have both inner and outer membrane

• Thin peptidoglycan layer

• Examples

• Examples:

Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa

Most drug recalls have been due to…

Gram (-) bacteria, rather than gram (+) bacteria

Bacterial secretions

• Lipopolysaccharide (LPS)

• Endotoxins

• ‘Exotoxins’

• Lipoteichoic acid (LTA)

• Lipopolysaccharide (LPS)

• Components associated with outer membrane of gram-negative bacteria

• Amphiphilic

• Hydrophobic with a net negative charge

• Amphiphilic:

Comprised of lipid, polysaccharide, and O-antigen

• Endotoxins =

Lipopolysaccharides

• Endotoxins = Lipopolysaccharides

• Integral part of the cell wall

• Shed by gram (-) bacteria during death and lysis

• ~10-20 kDa molecular weight

• Hard to remove

• Water soluble, but tend to absorb onto surfaces

• Shed by gram (-) bacteria during death and lysis

Lots per bacteria - 2 million LPS per E. coli

• Hard to remove:

Stable to heat, too small to filter out using sterile filtration

• ‘Exotoxins’ -

Secreted toxins

• ‘Exotoxins’ - secreted toxins

Usually secreted by gram (+) bacteria

• Lipoteichoic acid (LTA)

Cell wall of gram (+) bacteria

Pyrogens

• Greek ‘pyros’

• Something that causes fever

• Induction of inflammatory response, potentially leading to fever, shock, hypotension, organ failure, and death

• Greek ‘pyros’ =

Fire

• Something that causes fever

• Includes bacterial endotoxins from gram (-) bacteria

• Gram (+) bacteria, yeast, molds, viruses

• Also endogenous inflammatory markers released upon vural bacgterial infection

• Sometimes syntheic materials

Includes bacterial endotoxins from gram (-) bacteria

As little as 0.1 ng endotoxin/kg body weight can act as a pyrogen

• Also endogenous inflammatory markers released upon vural bacgterial infection

TNF-alpha, IL-1beta, IL-6

How to make things Sterile

• Steam

• Dry heat

• Filtration

• Gas

• Ionizing radiation

Steam

• Water boild at 100°C (212°F)

• Ideal gas law

• Autoclave

• Good for moisture-insensitive and temperature insensitive formulations

• Water boild at 100°C (212°F) -

Can’t make steam hotter unless we raise the pressure

• Ideal gas law

If volume (V) is fixed, increasing pressure (P) will increase temperature (T)

• Autoclave =

Steam under pressure

• Autoclave = Steam under pressure

• Example: +1 atm pressure (15 lbs) raises temp. to 121°C (250°F)

• Higher pressure, less time required

• Higher pressure, less time required

• 15 lb pressure, 250°F, 20 minutes

• 20 lb pressure, 260°F, 15 minutes

• Good for moisture-insensitive and temperature insensitive formulations

• Sealed ampules containing aqueous formulations

• Bulk solutions

• Laboratory ware

Dry Heat

• Mechanism: Dehydration and slow oxidation of the microbial cell

• Denaturation and coagulation of proteins

• Longer times at lower temperatures can be used for more sensitive products

• Good for oils, heat stable powders

Gas

• Ethykene oxide (EtO)

• Used for temperature/moisture sensative materials

• Mechanism: Interferes with bacterial metabolism

• Four parameters

• Relatively expensive

• Good penetration for materias

• Can be hazardous to use and needs to be destroyed prior to environmental release

• Mechanism: Interferes with bacterial metabolism

Alkylation of protein, DNA, and RNA

• Four parameters:

Gas concentration, temperature, humidity, and exposure time

• Four parameters: Gas concentration, temperature, humidity, and exposure time

Doesn’t require heating

Doesn’t require heating

Although effectiveness can be increased with temperature and humidity

• Can be hazardous to use and needs to be destroyed prior to environmental release:

• Explosive gas

• Toxic

• Designated as a human carcinogen by the EPA in 2016

Ionizing Radiation

• Energy is inversely proportional to wavelength

• Causes the ionization of water yielding the production of highly reactive free radicals

• Good for heat-sensitive drug products

• Energy is inversely proportional to wavelength

X-rays and gamma rays have shorter wavelengths, hence more energy, than visible light

• Causes the ionization of water yielding the production of highly reactive free radicals

These reactive species are capable of killing microorganisms

• Good for heat-sensitive drug products

But, best to carry out on dry solids as the presence of water and oxygen can promote undesired chemical reactions

Endotoxin Units

• The endotoxin levels in a compounded formulation are additive

• Everything is toxic if you have enough of it

• We’re probably not going to be able to get rid of all endotoxins

• The maximum allowable endotoxin levels for a given drug are listed in the USP-NF

• The endotoxin limit (EL) is determined by the following equation: EL = K / M

• To determine EU levels, we need to test for sterility using either in vitro or in vivo methods

• Two methods

• These methods measure the response relative to the amount of endotoxin present

• We’re probably not going to be able to get rid of all endotoxins

• But, we can tolerate certain levels of them

• Limits are given in Endotoxin Units (EU)

• The tolerable amount depends on the dose of the drug and route of administration

• The maximum allowable endotoxin levels for a given drug are listed in the USP-NF

• Examples:

  • Dextrose injection: contains not more than 0.5 USP EU/mL for injections containing < 5% dextrose

  • 0.5-0.9% sodium chloride: contains not more than 0.5 USP EU/mL

  • Gentamicin injection: contains not more than 0.71 USP EU/mg of gentamicin

• Recall that the endotoxin levels in a compounded formulation are additive

• The endotoxin limit (EL) is determined by the following equation: EL = K / M

• EL is the endotoxin concentration that must not be met or exceeded to release product for sale

• K = Threshold human pyrogenic dose of endotoxin/kg of body weight

• M = Maximum human dose of drug that would be administered/kg of body weight in a single hour

• EU/kg (K) is divided by (mg drug)/kg (M) yields EU/mg drug (EL)

• K = Threshold human pyrogenic dose of endotoxin/kg of body weight

• Threshold pyrogenic dose (K) of 5 EU/kg for human (and rabbit) parenteral formulations

• For non-inthreecal formulations, > 5 EU/kg deemed pyrogenic; < 5 EU/kg deemed non-pyrogenic

• Threshold pyrogenic dose (K) of 5 EU/kg for human (and rabbit) parenteral formulations

Except for intrathecal (CSF) which is 0.2 EU/kg

• M = Maximum human dose of drug that would be administered/kg of body weight in a single hour

• Depends on drug, formulation, disease state, etc.

• Example: Maximum dose of cyanocobalamin injection 14.3 mcg/kg

• Depends on drug, formulation, disease state, etc.

Usually ‘bolus’, but can be per one hour if usage is intermittent or continuous

• EU/kg (K) is divided by (mg drug)/kg (M) yields EU/mg drug (EL)

• EL = K / M

• Example: (5 EU/kg) / (14.3 mcg/kg) = 0.35 EU/mcg cyanocobalamin

• To determine EU levels, we need to test for sterility using either in vitro or in vivo methods

• In vivo - in living organism

• In vitro - outside of living organism

• Two methods

• Pyrogen testing

• Limulus amebocyte lysate (LAL) assay

• Pyrogen testing -

Carried out in rabbits

• Limulus amebocyte lysate (LAL) assay -

Carried out in blood extract of the horseshoe crab

• These methods measure the response relative to the amount of endotoxin present

More endotoxins, greater response

USP Pyrogen Test

• In vivo test

• Take the temperature of 3 healthy rabbits

• Inject rabbits in ear with 10 mL/kg solution potentially containing pyrogens

• Check their temperature evero 0.5 hours for 3 hours

• Inject rabbits in ear with 10 mL/kg solution potentially containing pyrogens

Why rabbits? Rabbits have similar tolerance to endotoxins as people

• Check their temperature evero 0.5 hours for 3 hours

• If temp. doesn’t rise by more than 0.5 C, then pyrogen free

• If temp. rises in a single rabbit, repeat on 5 other rabbits

Endotoxin Test

• Limulus amebocyte lysate (LAL) assay

• Gram (-) bacteria induces an immune response in the horseshoe crab (Limulus polyphemus) and causes its blood to coagulate around the bacteria

• An aqueous extract of blood cells (amoebocytes) will also clot in the presence of endotoxins

• An aqueous extract of blood cells (amoebocytes) will also clot in the presence of endotoxins

In vitro test

Pyrogen test vs. LAL Assay

• The LAL assay is more sensitive

• The LAL can only detect LPS

• The pyrogen test detects both LPS and other pyrogens

• Certain drugs interfere with the LAL assay

• Rabbits can build up tolerance to pyrogens upon repeated administration

• Animal welfare considerations

• Expense of pyrogen test and animal maintenance

• The LAL can only detect LPS

Good for gram (-) bacteria, but not other pyrogens from other sources

• The pyrogen test detects both LPS and other pyrogens

But has low sensitivity to certain pyrogens (Example: Legionnaires’ endotoxin)

• Certain drugs interfere with the LAL assay

Heparin (an anticoagulant) prevents LAL coagulation

• Rabbits can build up tolerance to pyrogens upon repeated administration

Lessens sensitivity

In Vitro Virus (IVV) Assay

Samples of raw materials/cell cultures used for product manufacturing are introduced to cell lines that are susceptible to the viruses likely present in the culture

Samples of raw materials/cell cultures used for product manufacturing are introduced to cell lines that are susceptible to the viruses likely present in the culture

• ‘Adventitious’ viruses

• Observe changes in cell behavior in a cell-based biochemical assays

When is sterilization needed?

• To reduce the risk of microbial contamination (lack of sterility), products are manufactured in a clean environment

• Need to sterilize body surfaces and injection/infusion equipment

• To reduce the risk of microbial contamination (lack of sterility), products are manufactured in a clean environment

• Swab testing of equipment and surfaces to determine bioburden

• ‘Bioburden’ refers to the total # of viable microorganisms on a device, container, or component

• It is hard to remove endotoxins, so best to limit the possibility of the bacteria that produce them up front!

• ‘Bioburden’ refers to the total # of viable microorganisms on a device, container, or component

• This includes bacteria, fungi, yeast, and mold

• If the bioburden is known, the ‘dose’ (time, temperature, amount of radiation, etc.) can be chosen to effectively sterilize, but limit possible damage to components

• Need to sterilize body surfaces and injection/infusion equipment

• Application of chemical disinfectants (example: rubbing alcohol) prior to injection

• Chemical disinfection/autoclaving (not for body surfaces!) of equipment can be done on-site

• Gas, dry heat, irradiation typically at the manufacturing end so the equipment arrives sterile

When is sterilization done?

• Sterilization is often done on containers and some raw materials, but these processes can negatively impact the final product

• Aseptic processing

• Terminal sterilization

• Sterilization is often done on containers and some raw materials, but these processes can negatively impact the final product

• Many of the drug degradation processes are a function of heat, moisture, and oxygen

• Sterilization processes should not impact the final product!