Lecture 11 Pyrimidine and Polyenes

Question 1

Name the one example of a pyrimidine

Answer 1

5-flurocytosine

Question 2

By which enzyme is 5-flurocytosine taken up by fungi?

Answer 2

Cytosine permease

Question 3

Explain the mode of action of 5-flyrocytosine

Answer 3

inhibits 2 of the following pathways:
5-flurocytosine → Flurouridine monophosphate (FUMP) → flurouridine triphosphate (FUTP) → incorporation in RNA → disrupts translocation

5-flurocytosine → flurodeoxyuridine monophosphate → inhibition of thymidylate synthetase → inhibition of DNA synthesis

Question 4

Which fungi is 5-flurocytosine active against?

Answer 4

Generally yeast: cryptococcus neoformans, most candida, some dematiaceous (brown) moulds

Question 5

Which fungi is 5-flurocytosine inactive against?

Answer 5

Candida krusei
Aspergillus spp.
Histoplama capulatum
Most moulds

Question 6

Why is 5-flurocytosine used in combination with amphotericin B/fluconozole?

Answer 6

Risk of resistance developing quickly in monotherapy

Question 7

What uses is 5-flurocytosine licenced for?

Answer 7

Treatment of systemic fungal infections caused by candidosis, cryptococcosis and chromoblastomycosis (brown mould)
Main use with amphoterinin B in cryptococcal meningitis

Question 8

Describe the study that explains the efficacy of amphotericin B and flurocytosine by Bennett et al, 1979

Answer 8

27 patients treated with either amphotericin B alone and 24 treated with amphotericin B + flurocytosine
Mortality rate found to be similar
Combination therapy showed: more rapid CFS sterilisation, lower rate of relapse

Question 9

Describe the study that explains the efficacy of flurocytosine monotherapy vs combined by Day et al, 2013)

Answer 9

Cyptococcal meningitis in HIV-AIDS
3 groups: Amp B only, Amp B + flurocytosine, Amp B + fluconozole
Mortality at 2 weeks as follows:
Amp B only 25/99, Amp B + flurocytosine 15/100, Amp B + fluconozole 22/99
Therefore combination therapies were more effective

Question 10

Explain the absorption of 5-flurocytosine

Answer 10

readily absorbed
wide distribution in tissues and body fluids e.g. almost the same levels in CFS as in the blood
Minimally absorbed by gut flora due to lack of deaminase

Question 11

What is the half life of 5-flurocytosine?

Answer 11

3-6 hours

Question 12

What is the normal dose of 5-flurocytosine given?

Answer 12

3-4 doses a day

Question 13

How is 5-flurocytosine excreted?

Answer 13

mainly via urine

Therefore good choice for UTIs as a monotherapy - though there is still a risk of resistance

Question 14

What are the side effects associated with 5-flurocytosine?

Answer 14

> 100mg/L for 2 weeks = risk of bone marrow supression - leucopenia, thrombocytopenia, aplastic anaemia
Rare: allergic reactions, liver toxicity

Question 15

What drugs are known to interact with 5-flurocytosine?

Answer 15

Brivudine (antiviral) - inhibits dihydropyrimidine dehydrogenase which normally degrades flurouracil = can lead to fluoruracil toxicity

Phenytoin - higher levels of phenytoin may occur

Question 16

Which fungi are particularly vulnerable to developing resistance to 5-flurocytosine?

Answer 16

can develop quickly for candida and cryptococcus

Question 17

Explain the mechanism of action of resistance to 5-flurocytosine

Answer 17

Most mutations occur to the uracil phosphoribosyltransferase - therefore probably the more important pathway during drug activation
Most mutations also occur to the cytosine permease and cytosine deaminase (see slide for pathway)

Question 18

What are the 2 examples of polyene antifugals?

Answer 18

Nystatin

Amphotericin B

Question 19

Where is the source of polyene antifungals?

Answer 19

natural products of streptomyces species

Question 20

What is the mode of action of polyene antifungals?

Answer 20

damage to cells by increasing cell permeability = K+ release
Bind to ergosterol via hydrophobic side of macrolide rings = distortion of membrane bilayers = pore for leakage to occur
Also theory of damage through auto-oxidation of amphotericin B though the exact mechanism is unknown

Question 21

Explain the spectrum of activity for amphotericin B

Answer 21

Broad spectrum
Most yeasts/moulds are sensitive
The parasite Leishmania
Exceptions: Aspergillus terreus, most scedosporium and lomentospora are resistant

Question 22

What was the problem associated with amphotericin B deoxylate and how was this overcome?

Answer 22

Deoxylate was associated with infusion related kidney toxicity - now rarely used
Ambisome produced - a lipid based formulation that has less side effects (see slide for structure of molecule)

Question 23

In what form is nystatin given?

Answer 23

topical
not orally absorbed
too toxic for IV

Question 24

What is Amphotericin B indicated for?

Answer 24

Empirical treatment for suspected fungal infections in immunocompromised patients
Treatment of a wide range of fungal infections including candidosis, aspergillosis, zygomycosis and cryptococcosis
Treatment of visceral leishmaniasis
Occassional topical use e.g. eyes, mouth, ears

Question 25

What is Nystatin indicated for?

Answer 25

treatment of oral or vaginal candidosis

Question 26

What signs/symptoms would a patient present with that would prompt a clinician to give amphotericin B as empirical treatment?

Answer 26

Neuropenia | Pyrexia

Question 27

Explain the evidence for the use of amphotericin B as empirical therapy

Answer 27

early studies comparing amphotericin and placebo Improvement in survival from fungal infections No overall increase in survival from empirical therapy Many problems regarding side effects seen

Question 28

Explain the study that compares Ambisome and amphotericin deoxycholate by Walsh et al, 1999

Answer 28

687 patients on either Ambisome or a amphotericin B for persistant febrile neutropenia despite antibacterial therapy - 50% had leukaemia Amphotericin B deoxylate = 49.4% success Ambisome 50.1% success Therefore no difference in success but less side effects with Ambisome

Question 29

Explain the study that compares Ambisome and other agents for empirical therapy

Answer 29

Ambisome vs virconazole: Ambisome = 30.6% success Virconazole = 26% success Ambisome vs caspofungin: Ambisome = 33.7% Caspofungin = 33.9%

Question 30

Explain the study that looks into the use of amphotericin as a prophylactic drug

Answer 30

355 acute lymphocytic leukaemia patients Ambisome vs placebo Rates of invasive fungal disease (change not significant): Placebo = 45% Ambisome = 48% Therefore no evidence for efficacy as prophylaxis

Question 31

Explain the tissue distribution of Ambisome

Answer 31

95% protein bound | High accumulation in liver and spleen, medium in lunch and kidney, low in heart and brain

Question 32

What is the half life of abisome?

Answer 32

initially 24h | later up to 15 days

Question 33

How is abisome eliminated?

Answer 33

43% in bile | 21% in urine

Question 34

Does abisome need drug monitoring?

Answer 34

No

Question 35

What are the adverse effects of ambisome use?

Answer 35

Acute infusion related toxicity: fever, chills and rigors, nausea, vomiting, headaches, hypotension Nephrotoxicity: raised serum creatinine levels, hypokalemia

Question 36

Which side effects have significantly reduced from the use of ambisome compared to amphotericin B deoxylate?

Answer 36

Fever, chills and rigors, vomiting, hypotension | raised creatinine levels

Question 37

What drugs does Amphotericin B interact with?

Answer 37

Other nephrotoxic agents: ciclosporin, aminoglycosides, antibiotics, some anti-neoplastic agents Corticosteroids, diuretics: increases risk of hypokalaemia Skeletal muscle relaxants - effects of hypokalaemia may heighten effects of muscle relaxants Flurocytosine: nephrotoxicity may reduce flurocytosine clearance and result in high serum levels = bone marrow suppression

Question 38

Name species that are resistant to Amphotericin B

Answer 38

Candida krusei and C. glabrata may have high minimum inhibitory concentrations C. lusitaniae can develop resistance in vitro Aspergillus terreus intrinsically resistant Scedosporium, Lomentospora and Fusarium are often resistant

Question 39

Explain why there is not a simple mutation-resistance relationship

Answer 39

Due to the main target of the drug being ergosterol which is not a protein

Question 40

Explain the mechanisms of action of resistance to Amphotericin B

Answer 40

C. lusitaniae - reduced ergosterol content of membranes (Peyron et al, 2002) C. glabrata - mutation in the ERG2 gene coding for an isomerase invoved in ergosterol synthesis = increase in minimum inhibitory concentration Increased catalase activity may reduce oxidative damage caused