Antifungal agents (DONE)

Question 1

What are fungi?

Answer 1

Fungi are eukaryotic cells which have few differences from human cells
Fungi have relatively few unique and essential targets for anti-fungal agents:
- unique cell wall using chitin as the main structural polymer
- cell cytoplasmic membrane contains unique fungal ergosterol
- some unique cytoplasmic membrane enzymes in some species

Question 2

Azoles bioavailability

Answer 2

The most important category of antifungals
Imidazoles such as clotrimazole, ticonazole and miconazole show very good anti-fungal activity when topically applied
Their activity when given orally or IV is less:
- susceptible to metabolic inactivation
- very lipophilic which leads to high levels of binding to plasma proteins and low plasma levels of drug

Question 3

Imidazole and triazoles

Answer 3

Imidazoles: ketoconazole is an improvement on early azoles:
-less metabolic instability
-less lipophilic and so higher plasma levels
-still metabolised (<1% unchanged drug excreted in urine)
- still bound to plasma proteins (<1% in unbound state)
Triazoles: fluconazole has a half life of 30h in plamsa:
-80% of orally administered fluconazole is excreted unchanged in the urine
-low protein binding (approx. 12%)
-licensed in 1991 (generic fluconazole in 2003)

Question 4

Voriconazole

Answer 4

Active against yeasts and moulds, oral and IV preparations, well absorbed, 96% bioavailability, approved for treatment of aspergillosis, extensive hepatic metabolism, licensed in 2002

• case reports of success with fluconazole resistant strains
• survival benefit and superior outcome vs amphotericin B in invasive aspergillosis
• acceptable overall safety
• generally better tolerated than amphotericin
• manageable drug interactions

Question 5

Azoles mechanism of action

Answer 5

Inhibit a cytochrome P450 which catalyses the 14-demethylation of lanosterol
Inhibition of C-14 demethylase
The azoles complex to the iron atom of the cytochrome P450 preventing substrate binding

Question 6

Azoles effects on membrane structure

Answer 6

Leads to accumulation of 14a methyl steroids
14a methyl steroids are much more bulky and the extra methyl destroys the planarity of the sterol ring and affects the ability of the steroids to interact with hydrophobic phospholipids

Question 7

Azoles selectivity

Answer 7

Human cells contain a sterol C14 demethylase which is involved in the biosynthesis of cholesterol
Azoles are selectively active against fungal C14 demethylase
The reason for selectivity is differences in binding pockets of the human and fungal enzymes
May affect CYP450 iso-enzymes which may account for liver toxicity

Question 8

Fugal resistance

Answer 8

Fungal resistance has occurred in HIV infected people who are on long term fluconazole treatment
Estimated that 30% of AIDS patients in UK have fluconazole resistant C. albicans
Cross resistance to other azoles

Question 9

Factors contributing to resistance

Answer 9

Azoles are fungistatic rather than fungicidal, C. albicans lives on mucosal membranes as a biofilm
Mutations in active site- mutations which give loss in activity of enzyme, mutations which reduce inhibitor binding but retain substrate binding
Gene amplification- over expression of the sterol synthesis genes
Decreased accumulation of azoles- thought to be cause by active efflux mechanism

Question 10

Mechanisms in resistance

Answer 10

ABC pumps (ATP-binding cassette)
MDR proteins (multi-drug resistance)
In resistant fungi the efflux pumps are over expressed

Question 11

Amphotericin B

Answer 11

Polyene antifungal
The only polyene which can be used for the treatment of systemic fungal infections, not absorbed orally so given IV, liposomal preparations are increasingly popular
Nystatin is too toxic for systemic use, but can be used topically and for intestinal fungal infections as it is not absorbed

Question 12

Amphotericin B- mechanism of action

Answer 12

Molecules are a similar length to phospholipid molecules (2.1nm)
Molecules of amphotericin B aggregate within the plasma membrane to form either a half channel or a full channel
Once the channel is formed it allows leakage of ions, probably protons and to some extent potassium ions

Question 13

Amphotericin B structure

Answer 13

Rigid hydrophobic face binds to ergosterol
Hydrophilic face forms aqueous pore or ion channel
Polar head group oriented outside membrane

Question 14

Amphotericin B selectivity/toxicity

Answer 14

Amphotericin B has a stronger binding constant with ergosterol (the principle sterol in fungi) than cholesterol (the principle sterol in humans)

Question 15

Amphotericin B- lipid preparations

Answer 15

Incorporated into liposomes has a reduced toxicity, presumably due to slow release of amphotericin B
Amphotericin B deoxycholate: au natural, heat treated, 24hr infusions, mixed with intralipid, licensed in the 1960s
Lipid preparations: liposomal, amphotericin B lipid complex, amphotericin B colloidal dispersion, licensed in 1996

Question 16

Advantages and disadvantages of lipid preparations

Answer 16

Increases selectivity for fungal cell
Uptake into phagocytes delivers drug to site of infection
Markedly reduces toxicity
Higher dosages possible
Better therapeutic index
Very expensive

Question 17

Griseofulvin

Answer 17

Isolated from Penicillum griseofulvum
Active against dermatophytes
Fungistatic rather than fungicidal
Mechanism of action: primary site of action is thought to be binding to RNA, manifestations include effects on cell wall synthesis and microtubule and spindle synthesis
Toxicity: toxic effects in animals at high doses, teratogenic and mutagenic

Question 18

5-Fluorocytosine

Answer 18

First developed as an anti-cancer agent
Flucytosine licensed in 1975
Good bioavailability and penetration into cerebrospinal fluid
Only useable on susceptible species, narrow range of activity- systemic candidiasis and cryptococcal meningitis
Often used in conjunction with amphotericin B for yeast induced meningitis
Resistance can develop during therapy

Question 19

5-Fluorocytosine mode of action

Answer 19

Activity requires deamination by cytosine deaminase: inhibits thymidylate synthase (synthesis of thymidine), incorporated into RNA (inhibits protein synthesis)
Selectivity: susceptible fungal cells have cytosine deaminase activity, human cells have little or no cytosine deaminase activity

Question 20

Caspofungin (Echinocandins)

Answer 20

Casposfungin licensed since 2002
Semi-synthetic lipopeptide from fermentation broth
Bioavailability: poor oral bioavailability in animals, plasma half life of 9-11 hours in man
Metabolic fate: no oxidative metabolism (chemical degradation and hydrolysis), not a substrate for or an inhibitor of P450, high protein binding (97%)

Question 21

Caspofungin activity

Answer 21

Use in systemic candidosis and aspergillosis- highly active against all Candida, except C. parapsilosis and C. guilliermondii including isolates resistant to fluconazole, amphotericin B or flucytosine
No activity against Cryptococcus
Biofilm activity: potent activity against C. albicans biofilm associated infections, susceptibility of antimicrobial therapy against fungal biofilms- fluconazole and amphotericin
Cellular morphology and overall biofilm architecture significantly affected

Question 22

Caspofungin mode of action

Answer 22

Blocks the synthesis of a major fungal cell wall component, 1-3-beta-glucan, presumably via inhibition of 1,3-beta-glucan synthase

Question 23

Costing: antifungal therapies

Answer 23

Antifungal drugs are currently recorded in four categories- hospital prescription, gynaecological, dermatological and topical/OTC. This makes accurate data collection difficult
Oral drugs are less expensive, and used for much more variable lengths of time. For example, vaginal thrush is treated with a single dose or two doses in most instances, but may be treated for 3-6 months in the few women with persistent problems

Question 24

Role of test laboratories- microscopy and culture of nail clippings

Answer 24

Fungal nail infections require therapy for 3-6 months
Often testing of the nail is not undertaken and therapy is prescribed empirically
If the diagnosis is incorrect, or if the fungus is not sensitive to the antifungal drug used, the therapeutic course is wasted and exposes the patient to unnecessary potential toxicity

Question 25

Role of test laboratories- appropriate therapy and diagnosis

Answer 25

Cryptococcoal antigen: takes 2-4 days, and may take more than a week in some cases, cryptococcal meningitis is fatal if not diagnosed properly
Aspergillus antigen: neutropenic leukaemic patients with invasive aspergillosis several days earlier than clinical features and x-ray abnormalities appear, early diagnosis has a major impact on survival, critical in optimising therapy, and allows discontinuation of ineffective drugs

Question 26

Role of test laboratories- appropriate therapy and fungal resistance

Answer 26

Sensitivity testing of Candida to fluconazole:
The majority of isolates of candida are sensitive to fluconazole, but up to 15% may be resistant or of intermediate susceptibility
Many units faced with this rate of resistance will use either caspofungin or an amphotericin B preparation, rather than risk therapeutic failure with fluconazole
Fluconazole is much less expensive, is available orally and is associated with little toxicity so is a better choice in many patients, especially if they can take oral therapy

Question 27

Identification and antifungal sensitivity testing of moulds

Answer 27

A wide range of filamentous fungi cause invasive infection, even though most cases are due to A. fumigatus
Itraconazole resistance has been identified in A. fumigatus and amphotericin B resistance in A. terreus. Many other fungi are resistant to one or more antifungal agents

Question 28

Flucytosine therapeutic monitoring

Answer 28

Flucytosine is toxic to the liver and bone marrow at high concentrations. It is excreted by the kidneys and is often given in combination with amphotericin B which can impair kidney function
Monitoring is therefore crucial, especially in patients with renal impairment and in premature neonates

Question 29

Appropriate therapy- minimise toxicity

Answer 29

Azole therapeutic monitoring:
-Itraconazole and voriconazole are compounds with substantial inter-patient variation in bioavailability
-Both are also associated with numerous drug interactions
-The bioavailability of oral itraconazole is poor, and a threshold for protection in prophylaxis or treatment has been defined
Voriconazole plasma concentrations are often very low in children and dose escalation is required for optimal therapy