Lecture 10. Antiviral Therapy

Question 1

What are viruses insensitive to?

Answer 1

Antibiotics

Question 2

What do antivirals need to be when compared with antibiotics?

Answer 2

Much more specific (no broad spectrum drugs)

Question 3

What types of infections do most antivirals target?

Answer 3

Chronic, persistent or latent viral infections (e.g HIV, herpesviruses and hepatitis C due to the window of opportunity to treat them)

Question 4

What else is important for success in viral infections?

Answer 4

An intact immune system is important for success in treating viral infections
Antivirals don’t tend to destroy viral particles, just inhibit them

Question 5

What are the properties that antivirals need to have?

Answer 5

Specificity and potency in vitro
Good selective index
Good therapeutic index
Good oral bioavailability if possible

Question 6

What does it means for a drug to have good specificity and potency in vitro?

Answer 6

Specificity = drug needs to be specific at inhibiting enzyme or virus without being toxic to own cells
Potency = how effective it is at clearing/inhibiting viral replication

Question 7

What is an example of what we mean by specificity and potency in vitro?

Answer 7

Compound X inhibits viral enzyme Y at a concentration of 100 nM in vitro. At this concentration, compound X does not affect cells

Question 8

What does a good selective index mean?

Answer 8

Calculating 50% toxic concentration/50% virus inhibitory concentration (in vitro)

Question 9

What is an example of what we mean by good selective index mean?

Answer 9

Compound X is toxic to cells at a concentration of 5 mM, but it inhibits the growth of virus in cells by 50% at a concentration of 200 nM
Selective index = 5x10⁻³/2x10⁻⁷ = 2.5x10⁴ / 25000
Range of x10³ - x10⁶ what we’re aiming for

Question 10

What does a good therapeutic index mean?

Answer 10

Minimum toxic dose/therapeutic dose (in vivo)

Question 11

What is an example of what we mean by good therapeutic index mean?

Answer 11

Compound X is toxic to mice at 10 mM but clears virus from the mice at 20μM
Therapeutic index = 1x10⁻²/2x10⁻⁵ = 5x10³ (5000)

Question 12

Why is the therapeutic index different to the selective index?

Answer 12

Selective index is measured in vitro whilst the therapeutic index is measured in vivo

Question 13

What is pharmacokinetics?

Answer 13

The variation in the circulating blood concentration of a drug under a particular dose regime

Question 14

What is pharmacokinetics influenced by?

Answer 14

Absorption - how well does the drug get into circulation?
Distribution - does it get into the right tissues?
Metabolism - how quickly is it broken down in the body? (also depends on how drug is taken)
Excretion - how quickly is it excreted from the body?

Question 15

What is bioavailability?

Answer 15

The fraction of administered drug that makes it into the circulating bloodstream

Question 16

What does good oral bioavailability mean?

Answer 16

If you take something by mouth (e.g tablet), how much will end up in the bloodstream
Also increases drug compliance

Question 17

What properties influence oral bioavailability?

Answer 17

Properties such as acid stability and resistance to digestive enzymes required

Question 18

What two ways can new antiviral compounds be discoevered?

Answer 18

Directly or random

Question 19

What is the random way that new antiviral compounds can be discovered?

Answer 19

High throughput screening of small molecules for virus growth inhibition in cell culture or for enzyme inhibition in vitro (worth noting not all viruses grown in cell culture or can be purified for assay)

Question 20

What is the directed way that new antiviral compounds can be discovered?

Answer 20

Molecular modelling - using known 3D structures of proteins, substrates, interactions to design inhibitors to make antivirals that inhibit active sites etc.

Question 21

What is structure-activity relationship testing?

Answer 21

When you make modifications to enhance the activity or pharmacokinetics of lead compounds (multiple rounds of this)
Starts relatively random in vitro until moving on to work in animals to see effects and enhance pharmacokinetics

Question 22

What do in vitro studies of antiviral effect and cytotoxicity provide?

Answer 22

Good selective index

Question 23

What do animal models for safety and activity provide?

Answer 23

Good therapeutic index

Question 24

How many phases of clinical trials in humans are there, how often do they last, and how much do they cost?

Answer 24

4 phases (phases I to IV)
Normally takes 10 years
Costs £400M

Question 25

What happens in Phase I of clinical trials (also known as first-in-man trials)?

Answer 25

Small number (10-50) of healthy volunteers Single small dose increasing to higher multiple doses Monitor for adverse effects and pharmacokinetics (40% fail phase I)

Question 26

Why do ~40% of antivirals fail Phase I?

Answer 26

Not usually because of safety, but pharmacokinetics (all excreted for example)

Question 27

What is a Phase 0 clinical trial?

Answer 27

Only used by some companies, looks at the bioavailability of the antiviral

Question 28

What happens in Phase II of clinical trials?

Answer 28

Small number (50-100) of patients (not healthy volunteers) IIa - confirm metabolism is same as healthy volunteers IIb - compare with placebo for efficacy. Usually double-blinded (these days, standard treatments are more commonly compared with new antiviral instead of placebos) Another 30% of drugs fail phase II (again normally due to efficacy instead of health reasons)

Question 29

What happens in Phase III of clinical trials?

Answer 29

Large numbers (1000s) of patients Randomised double-blind trial versus placebo and existing treatments Much larger numbers so you an get a better statistical power with the efficacy - pick up on problems that don't appear with smaller sample sizes (interactions with other diseases and/or drugs)

Question 30

What happens in Phase IV of clinical trials?

Answer 30

After approval for marketing Large scale, broader patient population Monitored for long term effectiveness and long term side effects (side effects that have appeared years after treatment) Further studies may test the drug in new age groups or patient types, and in new formulations

Question 31

What are examples of antivirals used against HIV?

Answer 31

Nucleoside reverse transcriptase inhibitors Non-nucleoside reverse transcriptase inhibitors Protease inhibitors Integrase inhibitors Fusion inhibitors Resistance and combination therapy

Question 32

What are the targets for anti-HIV therapy?

Answer 32

The viral enzymes or the viral processess

Question 33

What viral enzymes are targeted by anti-HIV therapy?

Answer 33

Reverse transcriptase - makes dsDNA copy of HIV RNA genome Protease - cleaves Gag and Gag/Pol polyproteins into structural and enzymatic viral components Integrase - catalyses insertion of dsDNA copy of viral genome into host cell chromosome

Question 34

What viral processes are targeted by anti-HIV therapy?

Answer 34

Cell attachment and entry - HIV envelope proteins bind to cell surface receptors and cause membrane fusion

Question 35

What are nucleoside reverse transcriptase inhibitors (NRTIs)?

Answer 35

Phosphorylated by cellular enzymes to the triphosphate form Bind the active site of RT preferentially, incorporated into viral DNA causing chain termination

Question 36

What was the first anti-HIV drug?

Answer 36

Zidovudine (AZT) my beloved Thymidine analogue Incorporated into DNA instead of TTP

Question 37

What are examples of current NRTI frontline drugs?

Answer 37

Lamivudine (cytidine analogue) Emtricitabine (cytidine analogue) Abacavir (guanidine analogue) Tenofovir (adenosine analogue)

Question 38

What are non-nucleoside reverse transcriptase inhibitors (NNRTIs)?

Answer 38

Diverse chemical structures. Bind to an allosteric site on RT (known as the NNRTI pocket) Changes the conformation of the reverse transcriptase so it cannot catalyse reactions Very specific – active against HIV-1 but not HIV-2

Question 39

What was the first NNRTI and what is the current frontline NNRTI used?

Answer 39

First: Nevirapine Frontline: Efavirenz

Question 40

What are protease inhibitors?

Answer 40

Antivirals that cleave the protease cleavage site of HIV, cutting between the Phe and Pro in the following sequence Leu-Asn-Phe-Pro-Ile

Question 41

What was the first protease inhibitor and how did it function?

Answer 41

Saquinavir - 1995 Transition state analogue of the proteolytic cleavage reaction Has some of the same amino acids that bind into the active site, blocks substrate from getting into the active site

Question 42

What are examples of current frontline protease inhibitors?

Answer 42

Indinavir Atazanavir Fosamprenavir

Question 43

What are integrase inhibitors?

Answer 43

Inhibits strand transfer between the viral DNA ends and cellular DNA

Question 44

What was the first integrase inhibitor and when was it approved?

Answer 44

Raltegravir - approved Oct 2007

Question 45

What was the first fusion/cell entry inhibitor?

Answer 45

First entry inhibitor was enfuvirtide (T20) Prevents hairpin formation so HIV and host cell membranes not brought together for fusion 36 amino acid peptide that binds HIV envelope protein (gp41) and prevents the conformational change required for fusion

Question 46

When are fusion/cell entry inhibitors approved for patients?

Answer 46

Approved for patients in whom other drugs have failed

Question 47

What are the downsides of using fusion/cell entry inhibitors?

Answer 47

Very expensive; difficult regimen (self-injection); 98% skin reactions

Question 48

What do the next generation of entry inhibitors target?

Answer 48

Target cell surface receptors used by HIV for entry: Maraviroc (Aug 2007) - blocks CCR5 Ibalizumab (March 2018) - monoclonal antibody against CD4 and also non-immunosuppressive; successful against multi-drug resistant HIV

Question 49

How does antiviral resistance form?

Answer 49

All current HIV drugs select resistant mutants. These mutant viruses then grow to dominate the population Resistance is due to amino acid substitutions in the viral protein target of the drug. Protease and integrase mutants arise less rapidly than reverse transcriptase mutants

Question 50

In which groups of antivirals is cross-resistance seen in particular?

Answer 50

NNRTIs Any mutations that occur allow the virus to become resistant to anti-viral drug, rapid spread in the population until mutation becomes dominant

Question 51

What is the standard therapy for HIV?

Answer 51

Combination therapy: Virus replication is suppressed to a minimum, less chances to mutate Virus takes much longer to develop resistance to 3 different drugs Any resistant viruses that do evolve are likely to be less virulent Can target multiple cell types and mechanisms of distribution Combination makes it harder for virus to mutate/spread

Question 52

What is the current treatment regime for HIV?

Answer 52

Highly Active Antiretroviral Therapy (HAART): 2 NRTIs + 1 NNRTI or 2 NRTIs + 1 PI (protease inhibitor)

Question 53

What can some triple combination treatments come in now?

Answer 53

The same pill eg. Atripla (200 mg emtricitabine, 300 mg tenofovir, 600 mg efavirenz) Increases adherence to treatment regime – less likely to develop resistance