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1. INFECTIONS > emma (L25) > Flashcards

emma (L25) Flashcards

1
Q

Properties of antivirals

A

a) Specificity and potency in vitro
b) Good selective index:
50% toxic concentration / 50% virus inhibitory concentration (in vitro)
c) Good therapeutic index:
Minimum toxic dose/therapeutic dose (in vivo)
d) Good oral bioavailability if possible

((Properties such as acid stability and resistance to digestive enzymes required))

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2
Q

pharmacokinetics and what influences it

A

Pharmacokinetics: the variation in the circulating blood concentration of a drug under a particular dose regime.

Influenced by: ADME

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? Excretion - how quickly is it excreted from the body?

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3
Q

define bioavailability

A

Bioavailability is the fraction of administered drug that makes it into the circulating bloodstream.

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4
Q

Discovery of new compounds

A

High throughput screening of small molecules
for virus growth inhibition in cell culture
for enzyme inhibition in vitro

Molecular modelling
- using known 3D structures of proteins, substrates, interactions to design inhibitors

Structure-activity relationships
modifications to enhance activity or pharmacokinetics of lead compounds

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5
Q

drug development

A

In vitro studies of antiviral effect and cytotoxicity
–> good selective index

Animal models for safety and activity
–> good therapeutic index

Clinical trials in humans - phases I to IV
10 years, £400M

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6
Q

drug development phase 1

A

Also known as first-in-man trials.

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)

2006 Northwick Park trial resulted in new guidelines.

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7
Q

drug development phase 2

A

Small number (50-100) of patients.

IIa - confirm metabolism is same in patients as in healthy volunteers

IIb - compare with placebo for efficacy. Usually double-
blinded.

Another 30% of drugs fail phase II.

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8
Q

drug development phase 3

A

Large numbers (1000s) of patients.

Randomised double-blind trial versus placebo and existing treatments.

Spectrum of therapeutic benefit:risk

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9
Q

drug development phase 4

A

After approval for marketing.

Large scale, broader patient population.

Monitored for long term effectiveness and long term side effects. Eg Vioxx.

Further studies may test the drug in new age groups or patient types, and in new formulations.

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10
Q

examples of antivirals

A

Herpesviruses - The first antivirals; Next generation drugs; Influenza drugs Ribavirin; Interferons

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11
Q

herpes virus structure

A

Large DNA viruses, encoding many proteins.

Cause chronic, persistent infections. Life-long latency with periodic reactivations.
Human herpesviruses include:
Herpes simplex (HSV) - cold sores, genital herpes Varicella zoster (VZV) - chicken pox, shingles
Epstein-Barr (EBV) - glandular fever, Burkitt’s lymphoma Cytomegalovirus (CMV) - asymptomatic in adults but life

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12
Q

what were the first antivirals

A

Drugs targeting DNA replication in herpesviruses were the first antivirals.
Development started in 1960s using nucleoside analogues (thymidine, idoxuridine and trifluridine)

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13
Q

role of viral dna polymerase

A

Viral DNA polymerase catalyses the addition of dTTP, dCTP, dATP or dGTP to the growing strand of viral DNA.
Pyrophosphate (PPi) is released.

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14
Q

how are idoxuridine triphosphate and trifluridine triphosphate incorporated into DNA?

A
  • Incorporated into viral DNA by HSV DNA polymerase. DNA prone to strand breakage, and faulty transcription (because it doesn’t base pair).
    [[ it works because idoxuridine is converted to the triphosphate form in the cell by viral and cellular kinases ]]
  • idoxuridine triphosphate and trifluridine triphosphate can also be incorporated into DNA by the host cell DNA polymerase (Toxic to cells; low selectivity)
    [[ No use as systemic antivirals, but still used topically, for herpesvirus keratitis and conjunctivitis. ]]
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15
Q

Next generation herpesvirus drugs

A

Guanosine analogues

  • aciclovir
  • ganciclovir
  • penciclovir
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16
Q

ACICLOVIR (ZOVIRAX)

A

ACV is activated only in virus-infected cells = high selectivity
ACVtri-p competes with dGTP as a substrate for DNA polymerase. Causes chain termination.
ACVtri-p has 100x higher affinity for HSV DNA pol than cellular DNA pol = high selectivity

Highly selective, low toxicity.
Used worldwide in topical, oral and intravenous formulations.
Effective against HSV, VZV, EBV infections, but not against CMV.

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17
Q

GANCICLOVIR

A

Active against CMV. Used in transplant and AIDS patients.

CMV protein UL97 converts ganciclovir to GCV- monophosphate, cellular enzymes convert to GCV- triphosphate.

GCV-tri-p competes with dGTP for CMV DNA polymerase.
It is not a chain terminator, but disrupts DNA structure.

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18
Q

PENCICLOVIR

A

Penciclovir is converted to PCV-monophosphate by HSV and VZV thymidine kinase.

Less selective than aciclovir and ganciclovir because it can be converted to PCV-mono-p by cellular enzymes too (although slower than viral tk).

More stable than aciclovir, so persists in the body for longer.

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19
Q

pro-drugs

A

valaciclovir

famciclovir

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20
Q

VALACICLOVIR

A

Aciclovir, ganciclovir and penciclovir are poorly bioavailable by oral uptake (10-20%).

Improved to 50-60% if given as a valine ester:

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21
Q

FAMCICLOVIR

A

Or a diacetyl ester:
penciclovir and famciclovir

Metabolised in vivo to the active drugs.

22
Q

FOSCARNET

A

Pyrophosphate (PPi) analogue.

Binds to PPi-binding site on viral DNA pol at concentrations that don’t affect cellular DNA pol.
= low toxicity.

Active against HSV, VZV, CMV. Has also been used against poxviruses, hepatitis B, HIV.

23
Q

SORIVUDINE

A

Bromine-modified uracil
Potent antiviral activity against VZV; also HSV, EBV.
Approved in Japan despite 3 deaths in clinical trials.
Post-marketing, 16 more deaths = withdrawn.
Fatal interaction with the anti-cancer drug 5-fluorouracil.

24
Q

FIALURIDINE

A

Fluorine modified idoxuridine
Phase II trials, 5 out of 15 patients died, 2 more needed liver transplants. Delayed toxicity - showed up as a large scale trial was about to be launched. No toxicity in animal trials.

25
Q

influenza drugs

A

Influenza viruses are negative strand RNA viruses, causing acute infections.

Small window of opportunity for effective antiviral therapy - prophylactically, or immediately at the onset of symptoms.

Two classes of drugs:

  • Adamantanes (flu A only)
  • Neuraminidase inhibitors (flu A and B)
26
Q

adamantanes

A

Adamantanes - Amantidine and Rimantidine

Block M2 proton channel.
Resistance has developed due to mutations in M2.
Also used to treat Parkinson’s disease as they increase dopamine release.

27
Q

Neuraminidase inhibitors

A

Neuraminidase inhibitors - Zanamivir (Relenza) and Oseltamivir (Tamiflu)

Neuraminidase cleaves terminal neuraminic acid from cell surface carbohydrates to enable virus release.

Inhibitors are transition state analogues of this cleavage reaction.

28
Q

ribavarin

A

Broad spectrum anti-RNA virus compound.

Ribose nucleoside analogue. Cellular enzymes convert it into the triphosphate - inhibits RNA-dependent polymerases.

Also inhibits guanyltransferases, preventing mRNA capping; IMP dehydrogenase, depleting cellular GMP levels.
= toxic, side effects.

Used to treat children with respiratory syncytial virus (RSV can be fatal in infants).

Only known treatment for viral hemorrhagic fevers (Lassa fever, Crimean-Congo fever, Ebola).

Also active against flaviviruses (hepatitis C, west nile virus, dengue fever).

29
Q

type 1 interferons

A

Type I interferons (α/β):

Natural cellular early immune response to viruses.

Used against hepatitis B and C (with ribavirin), clearing virus in 20-30% of cases.

Many of the symptoms of viral illness are caused by the interferon-induced inflammatory response
= ‘flu’-like side effects of this therapy.

30
Q

HIV

A

Human immunodeficiency virus

HIV is a retrovirus: lentivirus genus.
Causative agent of Acquired Immunodeficiency Syndrome (AIDS).

No vaccine available.
Antiviral therapy provides hope for disease management and delay of the onset of AIDS.

31
Q

HIV life cycle

A

DIAGRAM IN L25 S45

32
Q

Targets for anti-HIV therapy

A

Viral enzymes:
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.

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

33
Q

NRTI

A

Nucleoside Reverse Transcriptase Inhibitors

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

Zidovudine (AZT) was the first anti-HIV drug.

34
Q

next generation NRTIs

A

Next generation NRTIs - current front-line drugs

lamivudine
emtricitabine
abacavir
tenofovir

35
Q

NNRTIs

A

Non-nucleoside reverse transcriptase inhibitors
Diverse chemical structures. Bind to an allosteric site on RT (known as the NNRTI pocket).
Very specific – active against HIV-1 but not HIV-2.

36
Q

Protease Inhibitors

A

HIV protease cleavage site: Leu-Asn-Phe-Pro-Ile

  • saquinavir
  • indinavir
  • atazanavir
  • fosamprenavir
37
Q

saquinavir

A

First protease inhibitor (1995). Transition state analogue of the proteolytic cleavage reaction.

Low cytoxicity; high therapeutic index; obtained by structure-activity relationships

38
Q

integrase inhibitors

A

First integrase inhibitor is Raltegravir (approved Oct 2007).
Inhibits strand transfer between the viral DNA ends and cellular DNA.

39
Q

fusion/cell entry inhibitors

A

First entry inhibitor was enfuvirtide (2003).
Approved for patients in whom other drugs have failed. 36 amino acid peptide that binds HIV envelope protein
(gp41) and prevents the conformational change required for fusion.

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

Next generation of entry inhibitors target cell surface receptors used by HIV for entry:

40
Q

types of fusion/cell entry inhibitors

A

Maraviroc (Aug 2007) – blocks CCR5
Ibalizumab (March 2018) – monoclonal antibody against CD4

These drugs target cellular proteins = side effects? But 10% of people lack CCR5 = viable target.
Ibalizumab non-immunosuppressive; successful against multi-drug resistant HIV.

41
Q

resistance

A

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.

Cross-resistance is seen among NNRTIs in particular.

42
Q

combination therapy

A

Combinations of drugs:

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 (different drugs have different pharmachokinetics)

43
Q

current treatment regimes

A

Highly Active Antiretroviral Therapy (HAART):

2 NRTIs + 1 NNRTI
2 NRTIs + 1 PI

Some triple combinations now come in the same pill eg.
Atripla (200 mg emtricitabine, 300 mg tenofovir, 600 mg efavirenz).
Increases adherence to treatment regime – less likely to develop resistance.

44
Q

what does therapy depend on?

A

Therapy depends on:

  • country (developing vs developed nations)
  • age (adults vs children)
  • illness
  • T cell count
  • viral RNA level
  • previous therapy
  • resistance profile
  • side effects
  • drug interactions (other illnesses)
45
Q

gene therapy

A

therapeutic molecules expressed from modified genes
DNA -> RNA -> protein [interfere with viral replication]

Put modified genes into target cells, express RNA or protein-based drug in situ.

Challenge is to deliver the modified genes into the target cells – in this case virus-infected cells.
Example – anti-HIV gene therapy.

46
Q

HIV-1 gene structure and rna species

A

9kb unspliced - Gag, Pol
4kb singly spliced - Env, Nef
2kb doubly spliced - Tat, Rev

47
Q

rna based drugs

A

ribozymes:

  • rna enzymes
  • designed to bind and cleave viral RNA targets

antisense (AS) RNAs

  • bind to complementary mRNAs
  • may result in nuclear retention of the mRNA
  • block translation of the mRNA
  • degradation of the duplex

RNA interference

  • short hairpin (sh) RNAs
  • lead to degradation of mRNAs that contain exactly the same sequence

RNA decoys

  • small RNAs identical to protein-binding sequences
  • sequester viral proteins
48
Q

solutions to mutations

A

Targeting viral RNAs can result in mutation = resistance.

One alternative is to knock down levels of cellular protein eg CCR5.
OR
Combination RNA therapy (in clinical trials): Anti-CCR5 ribozyme
TAR decoy
shRNA against Tat and Rev mRNA

49
Q

protein-based drugs

A

Modified viral proteins
Eg. RevM10 – mutant version of Rev that binds RRE in the viral RNA but unable to function in nuclear export.

Modified cellular proteins
Eg. TRIM5α - 1 amino acid change to this cellular protein prevents HIV infecting the cell.

Intracellular antibodies (intrabodies)

  • bind to their target proteins inside cells
  • prevent them from functioning
  • trigger degradation of their target

Zinc finger nuclease (ZFN) fusion proteins

  • “molecular scissors”
  • combination of a non-specific DNA cleavage protein and a site-specific DNA recognition protein.
  • ZFN binds the target DNA sequence and cleaves the DNA. Repair often introduces deletions and mutations.
50
Q

blood stem cell therapy

A

Delivery of anti-HIV gene therapy agents into white blood cells.
Isolate blood stem cells

51
Q

T cell therapy

A

HIV infects CD4+ T cells and causes their destruction. Leads to depletion of this population of immune cells.

First trials of T cell therapy in advanced stage AIDS patients.
Patients have been followed for one year: Procedure safe.
Viral RNA levels unchanged.
CD4+ T cell count significantly increased even 1 year later. Patients had improved health.

1. INFECTIONS (17 decks)

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