What do you need to consider in antiviral drug development?
Expense vs need vs resistance
Burden of disease and clinical need
Understanding virology: what to target
Drug development: screening compounds or drug design
Clinical trial (Phases 1-3 etc) and impact
Resistance and monitoring
What is the first step of viral replication?
Viral replication has to occur within a cell as viruses have no protein synthesising “machinery” of their own, so instead hijack cells. There are a few main steps that are involved in the viral replication cycle:
Binding and adsorption of the virus occurs to the host cell, facilitated by the action of the haemagglutinin glycoprotein surface molecule of the virus.
What happens inside the cell?
The virus is then endocytosed into the cell, with two steps required for uncoating and successful transcription of viral RNA.
- Firstly, there must be ATP-driven entry into the endosome to allow fusion of the viral membrane to the internal endosomal membrane.
- Secondly, protons must gain entry into the virus itself via an M2-ion channel, and the subsequent fall in pH within the virus allows for the viral coat of the nucelocapsid to breakdown.
The viral RNA is then synthesised and replicated to form new RNA. The virus can then be assembled within the cell.
How is the virus released from the cell?
Following assembly, the virus is released via budding from the cell. Neuraminidase is a glycoprotein antigen found in the viral membrane and acts to prevent the adhesion of viruses upon release.
Describe the burden of Influenza in the UK including Influenza-related complications
Typically 15-25,000 deaths per year can be ascribed to influenza alone. The primary viral infection also leads to secondary infection requiring antibiotic treatment.
High risk groups include elderly, airways and cardiac disease, diabetes and renal
Most complications occur in otherwise healthy persons
- Bronchitis, pneumonia
- Exacerbating of underlying disease
Approximately 60-80% of patients with complications receive antibiotics
Antibiotics prescribed for approximately 30-45% of patients presenting with influenza or influenza-like illness.
What are the three types of Influenza? Explain Antigenic Shift and Drift
Of these Type A is potentially the most serious as they have multiple host species (e.g. bird flu, swine flue) and exhibit antigenic shift and drift. This makes new vaccine development necessary to treat the pre-absorbed virion when it is vulnerable to the pre-vaccinated immune system. Type A is capable of seasonal epidemic and pandemic potential.
- Antigenic Shift: the process by which two or more different strains of a virus, or strains of two or more different viruses, combine to form a new subtype.
- Antigenic Drift: mechanism for variation in viruses that involves the accumulation of mutations within the genes that code for antibody-binding sites.
Influenza B has no animal host and as a lower severity compared to Influenza A. Seasonal epidemic potential.
Influenza C “common-cold like”. Disease burden is not measured but likely mostly in children (less developed immune systems)
NB: Flu Vaccine, made up of 3 different viruses, cause antibodies to develop after triggering an immune response.
What are M2 Ion Channel Blockers and how do they work?
- To gain entry into a host cell, the invading virion first attaches to neuraminic or sialic acid residue on a membrane glycoprotein. The complex then allows the virus to gain entry by endocytosis.
- Following uptake of the virion into a host cell by endocytosis, there are two steps that must precede uncoating and successful transcription of viral RNA.
- The first step involves ATP driven proton entry into the endosome to allow fusion of the viral membrane with the internal endosomal membrane.
- The second step involves entry of protons into the virus itself via a viral Ion channel known as M2. The low pH inside the virus then results in breakdown of the viral coat of the nucleocapsid. The RNA can then escape out into the host cell cytoplasm.
Amantadine and Rimantadine are examples of two M2 Ion channel blockers that act to inhibit the above the step. These drugs were developed from Tricyclic Amines. They originally showed anti-parkinsonism activity but also inhibit viral uncoating, preventing viral replication.
Why is the use of M2 Channel blockers limited? Describe resistance and ADRs.
The use of M2 Ion channel blockers is limited to Influenza Type A (but are active against non-human subtypes). Unfortunately their widespread utility may be limited by a rapid emergence of M2 mutations in H5N1 viruses that appeared in 2003.
Single point mutation in M2 gene: S31N
High-level, rapid emergence resistance
Retained transmissible + infective ability
H5N1 isolates 2003-present: resistant
H3N2 (>60% Asia 2003-4, >90% US 2005-6)
M2 Ion Channel Blockers: ADRs
- Amantadine has more marked ADR risk than Rimantidine of about 5-10%.
- ADRs include dizziness, GI disturbance and hypotension - renal excretion is affected
- More serious are confusion and insomnia and hallucination which can be problematic in the elderly, in whom these symptoms may be present and further exacerbated. For this reason Rimantidine is usually preferred over Amantadine (less side effects).
- Can cause brain neurotoxicity in patients without Parkinson’s.
Describe how M2 Blockers actually inhibit the channel
The structure of a possible target, or binding of an existing compound can be used to evaluate and design new drugs
Gene sequencing of protein
Nuclear Magnetic Resonance
Originally thought M2 inhibitors blocked the ion channel but now thought the drugs stick to the bottom and alter the conformation of the channel, thereby blocking it. This pathway is easily subject to mutations – resistance can easily develop. 1 single amino acid substitution is sufficient to prevent drug binding.
How do Neuraminidase inhibitors work?
Neuramidase Inhibitors act by blocking virion release from the host cell membrane
Neuramidase is one of three transmembrane viral proteins, which enables newly formed virions to escape from their host cell. It is essential for replication – the surface of influenza is highly variable but the neuraminidase active site is conserved across subtypes
- Human and non-human influenza A
- Influenza B
- M2 resistant viruses
- Avian strains including H5N1
- Reconstructed 1918 pandemic H1N1
As the newly formed virus egresses from its host cell, many re-attach to the sialic acid membrane glycoprotein residues on the cell membrane.
To get released and infect other host cells they need to break this bond which is carried out by the viral neuramidase.
New sialic acid analogues were developed ‘from scratch’ with very high binding affinities (Ki~0.1 nM) and potency (ED50~30 nM). These bind more strongly with the Neuramidase than the competing endogenous sialic acid.
Both Influenza Types A and B can be treated by Neuramidase Inhibitors
Describe the Pharmacokinetics of Neuraminidase inhibitors and associated ADRs
Neuramidase Inhibitors Pharmacokinetics: Zanamivir and Oseltamivir (or Tamiflu).
- Zanamivir is given as a dry powder aerosol and has low oral bioavailability and can only be used for treatment. It remains detectable in sputum up to 24 hours post dosing and is really excreted.
- Oseltamivir (Tamiflu) is a prodrug and by contrast is well absorbed, with 80% bioavailability. This enables it to be given orally for both treatment and prophylaxis.
Neuramidase Inhibitor ADRs
- Generally not serious – GI disturbance – vomiting, abdominal pain, headache, nosebleed (epistaxis)
- Rarely respiratory depression, bronchospasm
- No drug-related serious adverse events
- Low rates of discontinuation in studies
Explain how the results of Neuraminidase inhibitors in clinical trials have helped inform clinical therapy dosing strategy
Phase III Clinical Trials have directly informed therapeutic strategy. Trial outcomes are discussed with respect to: severity of symptom related to dose; timing of initiation of treatment and illness duration; mortality; prophylaxis
- US and rest of world clinical trials (N = 1355)
- Adult 18-65 years with febrile (38 C or greater) RTI (respiratory tract infection)
- At least 1 respiratory and systemic symptom
- Cough, sore throat or nasal symptoms
- Myalgia, chills, malaise, fatigue, headache
- Influenza present in community
- Treatment within 36 hours of symptom onset
- Oseltamivir 75mg or 150mg bd (5 day) vs placebo
Explain how clinical trials have shown difference in symptom severity and dose reduction results
Symptom Severity and Dose Reduction in symptom severity in Placebo:
75mg:150mg treatment groups showed no difference in outcome between the two active groups with decrease in symptoms score of about 40% vs Placebo.
Explain how results have shown early interventions results in maximal clinical benefits
The earlier treatment is started after symptom onset the shorter the duration of symptoms; earlier treatment maximizes clinical benefits
- Prospective, open-label, non-controlled multicentre study investigated the relationship between time to intervention and maximum treatment benefit
- Patients aged 12-70 years – within 48 hours of sudden onset of symptoms
- Oseltamivir 75 mg bd for 5 days
The time window for significant reduction goes up to 48 hours
Little benefit accrues past this time.
Explain how results have shown mortality is reduced with intervention
It appears that Oseltamivir could offer ~70% reduction in risk of mortality in one Canadian study.
Rates of lab-confirmed influenza in patients admitted to hospital for respiratory illness during influenza season – viral testing not routine, under diagnosis, many cases untreated
Importantly, this was achieved even when dosing was delayed as long as 64 hours after symptom onset.
Mean time onset of symptoms was 43 hours, mean time from onset of symptoms to treatment was 64 hours
Describe results regarding seasonal prophylaxis
Randomised, placebo-controlled, double-blind
- Occurrence of influenza-like illness (ILI)
- Recorded by patients onto diary cards
Treatment for six weeks with 75 mg significantly reduced incidence of flu in both healthy adults and frail elderly subjects.
Appreciate how emergence of resistance of different viral strains to Neuramidase Inhibitors will affect therapy
The Neuramidase Enzyme also appears to be evolutionary conservative, which is a great advantage for any antimicrobial therapeutic although reports of resistance in various HA and NA surface glycoprotein antigens subtypes have been reported.
In the small number of those treated for H5N1 bird flu, resistance appears to be high.
Emergence of N1 resistance 2007
- Countries noted oseltamivir H1N1 resistance (H274Y mutation around active site).
- First identified in Norway in Dec 2007
- Highly variable rates of resistance globally
- Has spread to South Africa and Australia in 2008
- No known reason: not drug related
- Resistance now persistent in N1 virus
- Viruses generally remain zanamivir sensitive
Zanamivir easily fits into the active site – mutations around the site do not affect binding
Oseltamivir has a longer stalk. Tight fit into a pocket of active site
N1 has a smaller pocket than N2
Mutations around the site close it
Describe resistance in swine H1N1 and resistance surveillance
Sporadic case reports of oeseltamivir resistant swine H1N1 isolated from post treatment samples
One case of pre-treatment oseltamivir resistance
No increase in baseline or transmitted resistance as yet
Structural analysis of swine N1 suggest more like N2 structure – less susceptible to resistance.
- Influenza WHO system
- Neuraminidase Inhibitor Susceptibility Network (NISN)
- National and European surveillance
- Detailed evaluation of post treatment isolates
What are the two good reasons for antimicrobial use?
Antimicrobial agents are used to target solely the microbial biochemistry; a perfect antibiotic would affect the bacteria solely and have no effect on the body itself, causing no drug-drug interactions or ADRs. However, antibiotics do come with their risks and drawbacks.
There are 2 good reasons for using antibiotics: prevention of infections and therapy of significant bacterial infections
Prophylaxis of bacterial infections: people at increased risk of infection
- Peri-operative: prevention of surgical site infections e.g. orthopaedic surgeons using cement beads of antibiotic => high concentrations of antibiotic locally, prevents systomic toxicity and emergence of resistance
- Short term: meningitis contacts
- Long term: asplenia, immunodeficiency (any immunocompromised individual)
Therapy of significant bacterial infections
- Treatment of culture proven infection
- Empirical treatment of suspected infection
Treating against significant bacterial infections normally consists of commencing empirical treatment initially (based on demographics, individual factors etc) and then starting on revised treatment following culture.
What are the three main ways antibiotics can act? Give examples
Antibiotics can be classified based on their mechanism of action, chemical structure or spectrum of activity, yet all act by bactericidal or bacteriostatic action.
There are 3 main ways in which antibiotics can act
- Affect on DNA synthesis, which include
Quinolones (such as ciprofloxacin) act by preventing bacterial DNA replication; they are broad-spectrum antibiotics
Folic acid antagonists (such as trimethoprim or sulphonamides) prevent DNA precursors from being produced
Affect on protein synthesis, which include
Aminoglycosides (such as gentamicin or streptomycin) act by binding to the 30S ribosomal subunit
Macrolides (such as erythromycin) act by preventing peptidyltransferase from adding the peptidyl attached to tRNA to the next amino acid.
Tetracyclines e.g. doxycycline act by inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex.
Affect on cell wall synthesis, which include:
Beta-lactams (such as penicillins, cephalosporins and carbapenems) act by inhibiting the action of transpeptidase
Glycopeptides (such as vancomycin) act by binding to amino acids within the cell wall, preventing the addition of new units to the peptidoglycan.
What does antibiotic choice depend on?
Empirical antibiotic use can be done by simply following the local antibiotic formulary. However, there are factors that affect certain antibiotic choice, based on both cause of infection, effectiveness of antibiotics, and safety of antibiotic
Need to also consider cost + efficacy when choosing the best antibiotic!
NB: whilst local formularies can be used, it can sometimes be better to use alternative empirical treatments if other factors suggest doing so.
Describe Cause of Infection factors
Duration of illness
Time of year
Past medical history/personal background
Describe Effectiveness of Antibiotic and Safety of Antibiotic factors
Effectiveness of antibiotics:
- Community or healthcare setting
- Severity of infection
- Rate of resistance
- Immune status of patient
- Consider likely susceptibility and consequences of wrong choice!
- Drug interactions
- Pregnancy/breast feeding
- Organ function
- Administration route and efficacy
Describe the common antibiotic ADRs
The ADRs of antibiotics broadly are toxicities, allergic reactions and some idiosyncratic reactions specific to selected antibiotics, all of which range in their severity.
The common adverse effects are diarrhoea (due to the effect the antibiotics have on the intestinal flora), fever, nausea and vomiting, as well as immediate hypersensitivity reactions (e.g. anaphylaxis) or delayed hypersensitivity (e.g. skin rashes).
E.g. macrolides are gastric motility agonists – can increase gastric emptying.
What is meant by chromosomal mutation and horizontal gene transfer?
v Random mutations can confer resistance
v Horizontal Gene Transfer
- Transformation: many bacterial species incorporate naked DNA into their genome e.g. Streptococcus pneumoniae and Neisseria gonorrhoae incorporate small sections of penicillin-binding protein genes from closely related species to produce a penicillin binding protein that binds penicillin less avidly, so becoming more resistant. Such organisms are still able to synthesize peptidoglycan and maintain their cell walls in the presence of penicillin.
- Conjugation: bacteria contain plasmids. Many genes are carried on plasmids including those that encode metabolic enzymes, virulence determinants and antibiotic resistance. Plasmids can pass from one bacterium to another by conjugation, allowing ‘resistance genes’ to spread rapidly in populations of bacterial species that share the same environment e.g. within the intestine. Combined with antibiotic selective pressures (e.g. in hospitals) that favour the survival of organisms with resistance plasmids, multi-resistant populations may develop.
- Transduction: transposons and integrons are mobile genetic elements able to encode transposition and move between the chromosome and plasmids, and between bacteria. Many functions including antibiotic resistance can be encoded on a transposon. Resistance genes can also be mobilized by bacteriophages (viruses that live in bacteria).
Briefly describe biochemical mechanisms of antibiotic resistance
Intrinsic resistance, where the resistance develops as a result of gene mutations within the bacterial itself
Extrinsic resistance, where the resistance is acquired from another bacterium passing on the information; this can be via 3 mechanisms of transformation (uptake of naked DNA), conjugation (uptake of plasmids), and transduction ((movement of genetic material by bacteriophages).
Resistance can be prevented by controlling and limiting the use of antibiotics; even “recycling” of antibiotics can work due to changes in selection pressures mean previously ineffective antibiotics (due to widespread resistance) can be effective again.
Be aware of scale of emergent patterns of antibiotic resistance and name the main organisms e.g. MRSA, staphylococcus aureus. What are the antibiotic resistrance categories?
The emergence of MRSA highlights how quickly resistance can develop and spread throughout populations of bacterium. Penicillin was introduced in 1941 and by 1942, the first case of penicillin resistant Staphylococcus Aureus was reported.
Methicillin (or flucloxacillin) was introduced for Staphylococcus Aureus infections in 1961 and by the same year MRSA was reported. Vancomycin-Resistance Staphylococcus Aureus (VRSA) has since been identified since 2002, as a result of continued treatment of Staphylococcus Aureus with vancomycin.
Other antibiotic resistant pathogens include Glycopeptide-Intermediare Staphylococcus Aureus (GISA), Glycopeptide-Resistant Enterococci (GRE) and Extensively Drug-Resistant Klebsiella Pneumonia (XDR-KP).
No new antibiotics have been discovered since 1987.
Definitions of antimicrobial resistance:
- MDR (Multi-Drug Resistant): non-susceptibility to at least one agent in three or more antimicrobial categories
- XDR (Extensively Drug Resistant): non-susceptibility to at least one agent in all but two or fewer antimicrobial categories.
- PDR (Pan-Drug Resistant): non-susceptibility to all agents in all antimicrobial categories
What are the main steps in avoiding drug resistance?
To avoid the spread of resistant organisms, simple steps can be used to ensure this via infection control and judicious antibiotic use. Infection control can be achieved by preventing bacterial exposure to antibiotics (via minimizing risk of infection or monitoring and controlling antibiotic prescribing), or by preventing the spread of recognised resistant bacterium (by isolation or cohorting, hand hygiene and decolonisation of patients).
Antimicrobial stewardship: Judicious antibiotic use means only the right antibiotic, at the right dose, frequency, route, time and duration, to prevent unnecessary resistance developing.
What are disc sensitivity and E-tests used to measure?
The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration determine the effectiveness and potency of antibiotics. The antimicrobial agents with the lowest MIC and MBC values become an important factor in deciding which antibiotics to use.
Disc sensitivity testing and E-tests (predefined gradient of antibiotic concentrations on a plastic strip, used to determine MICs) are ways of measuring anti-bacterial activity
Minimum Inhibitory Concentration (MIC) – minimum concentration of antibiotic required to inhibit growth of a bacterium in vitro
- Units are mg/l (or equivalently micrograms/ml)
- Antibiotic and isolate specific
- Broadly speaking big zones are associated with sensitivity, small zones are associated with resistance but need to compare with official guide tables!
- Considers the MIC and antibiotic pharmacokinetics
- Predicts likely response – categories as susceptible, intermediate or resistant
Describe the pharmacokinetics and pharmacodynamics of antibiotics
Pharmacokinetics: determinants of blood and tissue antibiotic concentrations
- The pharmacokinetic aspects of antibiotics involve a few ADME factors, similar to any other drug’s pharmacokinetics. Absorption has varying effects orally due to variable oral bioavailability and metabolism and elimination are commonly hepatic and renal respectively.
Pharmacodynamics: The two major determinants of bacterial killing are time and concentration about the MIC; the area under the curve for antibiotic concentration against time can quantify the amount of exposure of the organism to the antibiotic during any one dosing interval.
What is meant by Time-Dependent and Concentration-Dependent Killing?
Time dependent killing: For certain antibiotics, the major killing effect against an organism is how long the drug is bound to the receptor sites for and is known as time-dependant killing. E.g. certain antibiotics such as penicillins, cephalosporins, glycopeptides or macrolides can be effective due to the extensive amount of time the antibiotic binds to the microorganism and the pharmacodynamics parameter is simply how long they spend above the MIC. Successful treatment requires prolonged antibiotic presence at site of infection but not high concentration. So the Time > MIC parameter is very important.
Concentration dependent killing: For other antibiotics, the concentration they act on a cell at determine how effective they are and is known as concentration-dependant killing. Examples of concentration-dependant antibiotics are aminoglycosides, quinolones or metronidazole. Successful treatment requires high antibiotic concentration at site of infection but not for long. The pharmacodynamics parameter is the Cmax to MIC ratio.
Why is Therapeutic Monitoring important?
Therapeutic monitoring is required for a few antibiotics (such as aminoglycosides or vancomycin) to check that adequate doses are being achieved, but more importantly to ensure non-toxic doses are maintained.
Monitoring markers of potential toxicity
- Daptomycin is useful for treatment of gram positive infections such as Staphlococcus Aureus including MRSA but side effects include muscle breakdown and allergy so need to monitor creatine kinase and eosinophils
- When prescribing linezolid, chloramphenicol need to monitor full blood count
- Need to monitor renal function and auditory function when prescribing aminoglycosides such as gentamicin – can be nephrotoxic and also toxic to the vestibulocochlear nerve.
- When using most antibacterials in hospital, need to do a stool chart to spot early signs of Clostridium difficile.