Lecture 11 Pyrimidine and Polyenes

Question 1

Name the one example of a pyrimidine

Answer 1

5-fluorocytosine

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-fluorocytosine → Fluorouridine monophosphate (FUMP) → fluorouridine triphosphate (FUTP) → incorporation in RNA → disrupts translocation

5-fluorocytosine → fluorodeoxyuridine monophosphate (FdUMP) → inhibition of thymidylate synthetase → inhibition of DNA synthesis

Question 4

Which fungi is 5-fluorocytosine active against?

Answer 4

Generally yeast: Cryptococcus neoformans, most candida, some dematiaceous (brown) molds

Question 5

Which fungi is 5-fluorocytosine inactive against?

Answer 5

Candida krusei
Aspergillus spp.
Histoplasma capsulatum
Most molds

Question 6

Why is 5-fluorocytosine used in combination with amphotericin B/fluconazole?

Answer 6

Risk of resistance developing quickly in monotherapy

Question 7

What uses is 5-fluorocytosine licensed for?

Answer 7

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

Question 8

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

Answer 8

27 patients treated with amphotericin B alone and 24 treated with amphotericin B + fluorocytosine
Mortality rate found to be similar
Combination therapy showed: more rapid CFS sterilization, a lower rate of relapse.

Question 9

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

Answer 9

Cryptococcal meningitis in HIV-AIDS
3 groups: Amp B only, Amp B + fluorocytosine, Amp B + fluconazole
Mortality at 2 weeks as follows:
Amp B 25%, Amp B + fluorocytosine 15%, Amp B + fluconazole 22%
Therefore combination therapies were more effective

Question 10

Explain the absorption of 5-fluorocytosine

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-fluorocytosine?

Answer 11

3-6 hours

Question 12

What is the normal dose of 5-fluorocytosine given?

Answer 12

3-4 doses a day

Question 13

How is 5-fluorocytosine excreted?

Answer 13

mainly via urine

Therefore a 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-fluorocytosine?

Answer 14

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

Question 15

What drugs are known to interact with 5-fluorocytosine?

Answer 15

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

Phenytoin - higher levels of phenytoin may occur

Question 16

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

Answer 16

Candida sp. and Cryptococcus

Question 17

Explain the mechanism of action of resistance to 5-fluorocytosine

Answer 17

1) Most mutations occur to the uracil phosphoribosyltransferase (UPRT) → inhibition of protein synthesis.
2) Mutations also occur to the cytosine permease and cytosine deaminase →inhibition of thymidylate
synthetase ∴ disruption of DNA synthesis

Question 18

What are the 2 examples of polyene antifungals?

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 the hydrophobic side of macrolide rings → distortion of membrane bilayers = leakage
Also theory of damage through auto-oxidation of amphotericin B but the exact mechanism is unknown

Question 21

Explain the spectrum of activity for amphotericin B

Answer 21

Broad spectrum
Most yeasts/molds are sensitive
Visceral Leishmania
Exceptions: Aspergillus terreus, most Scedosporium, and Lomentospora are resistant

Question 22

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

Answer 22

Deoxycholate was associated with infusion-related kidney toxicity - now rarely used
Ambisome produced - a lipid-based formulation that has fewer side effects

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
Occasional 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 an empirical treatment?

Answer 26

Neutropenia | 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 an amphotericin B for persistent febrile neutropenia despite antibacterial therapy - 50% had leukemia Amphotericin B deoxycholate = 49.4% success Ambisome 50.1% success Therefore no difference in success but fewer side effects with Ambisome

Question 29

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

Answer 29

Ambisome vs virconazole: Ambisome = 31% success Virconazole = 26% success Ambisome vs caspofungin: Ambisome = 34% Caspofungin = 34%

Question 30

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

Answer 30

``` 355 acute lymphocytic leukemia 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 lung and kidney, low in heart and brain

Question 32

What is the half-life of ambisome?

Answer 32

initially 24h | later up to 15 days

Question 33

How is ambisome eliminated?

Answer 33

43% in bile | 21% in urine

Question 34

Does ambisome 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 deoxycholate?

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 the effects of muscle relaxants Fluorocytosine: nephrotoxicity may reduce fluorocytosine 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 a high minimum inhibitory concentration 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

Because the main target of the drug is 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 involved in ergosterol synthesis = increase in minimum inhibitory concentration Increased catalase activity may reduce oxidative damage