lec 5- Anti-fungal agents

Question 1

Infections caused by yeast

Answer 1

• Candida albicans causes infections of mucous membranes (thrush) and blood stream
• Cryptococcus Neoformans causes pneumonia and meningitis

Question 2

Infections caused by moulds

Answer 2

• Filamentous fungi (dermatophytes)
• Trichophyton rubrum

Question 3

Human ringworm

Answer 3

• Tinea corporis
• Classic circular ringworm inflammatory lesion- spreading outwards/healing in the centre
• Trichophyton sp.
• Athletes foot (Tinea pedis)
• Broken skin and inflammation lesions between toes
• Topical: Azole or Terbinafine
• Allylamine= C=C bond with CH2 then amine

Question 4

Targets for Antifungal drug development

Answer 4

• Mannoproteins
• Caspofungin
• b-(1,6)-glucan
• b-(1,3)-glucan
• Chitin
• Phospholipid bilayer- Ergosterol- polyenes
• DNA/RNA synthesis- 5-FC
• Ergosterol synthesis pathway- imidazoles, triazoles
• Squalene- terbinafine

Question 5

Ergosterol/Cholesterol antifungal drugs

Answer 5

• Work by exploiting differences between mammalian and fungal cells to kill the fungal organism without dangerous effects on the host
• Both fungi and humans eukaryotes
• Difficult to find or design drugs that target fungi without affecting human cells (side effects)
• Fungal cell membranes have unique sterol (ergo)- which replaces cholesterol in the mammalian cell membrane

Question 6

Sterol chemical structures

Answer 6

Question 7

Cholesterol/Ergosterol- Similarities and Differences

Answer 7

Similarities

• Ring A/B/C/D tetracyclic
• Template and side chain
• Secondary alcohol

Differences

• The double bond in ring B (diene)
• A side chain Me, double bond
• These Structural differences are key to antifungal chemotherapy

Question 8

Amphotericin B

Answer 8

• Target = Ergosterol
• Broad spectrum polyene macrolide antibiotic
• Large ring cyclic ester
• Most potent antifungal agent for systemic mycosis, in clinical use since 1960
• Fungicidal drug at higher concentrations and static at lower levels

Produced by Streptomyses nodosum natural product

• CSF conc= 2-3% of blood conc
• Highest concentration in liver, spleen, bone marrow with less in kidneys and lungs

Question 9

Mechanism of action of polyene antifungal

Answer 9

• Amphotericin B
• Binds to ergosterol
• Intercalation of cell membrane (makes pores/holes into the membrane)
• allows ions (Na, K, Ca) as well as proteins to flow out of the cell
• low conc = fungistatic
• High conc= fungicidal

Question 10

Mechanism of action

Answer 10

• Binds to fungal cell membrane (ergosterol) =>
• Forms pores =>
• Alters permeability and transport =>
• Cell death

Question 11

Clinical Use

Answer 11

• Treatment of nearly all life threatening mycotic infections
• For systemic disease: slow IV
• Local: topical application
• Cholesterol, present in host cell membranes, closely resembles fungal ergosterol
• Result: High toxicity of AM-B in humans
• Highlight- Fungal arthritis = Local injection

Question 12

Liposomal amphotericin B

Answer 12

• New lipid formulations
• Amphotericin B is incorporated into lipid formulations to reduce toxicity and enhance efficacy + bioavailability
• This allows higher dose to be used without increasing the toxicity
• Much more expensive than ordinary AM-B

Question 13

Target: Cell wall- antifungal agents

Answer 13

• Cyclic lipo-peptide
• Caspofungin (Cancidas, Merck): life-threatening systemic mycoses but not cryptococcal meningitis
• Structure: A semi-synthetic echinocandin Cyclic lipoprotein
• Mechanism: inhibition of synthesis of 1,3,-b-D-glucan in fungal cell walls
• Anidulafungin (Ecalta, Pfizer): similar to caspofungin

Question 14

Caspofungin- structure

Answer 14

• cyclic
• Amide bonds

Question 15

Echinocandins

Answer 15

• The newest class of antifungal
• Active against candida and aspergillus (mould + yeast)

Adverse effects

• Extremely well tolerated- specific to fungi cell wall
• Minor GI side effects
• Flushing
• Elevated Liver enzymes (Caspofungin + Cyclosporine)
• Histamine release during IV infusion

Question 16

Mechanism of action

Answer 16

• Inhibit the synthesis of b-glycan in the fungal cell wall =>
• Disruption of the fungal cell wall and cell death

Question 17

Molecular target DNA

Answer 17

FLUCYTOSINE 5-FC

• 5-fluorocytosine
• pyrimidine
• Compare 5-FU anticancer agent

Question 18

Flucytosine (5-FC)

Answer 18

• Pyrimidine Anti-metabolite, narrow-spectrum fungistatic activity
• Water-soluble- good BBB penetration
• Oral only
• poor protein binding
• CSF conc- 75% serum concentration- Excellent CSF
• Treatment of fungal meningitis- BBB penetration

Question 19

5-fluorocytosine inhibits DNA synthesis in fungi

Answer 19

• 5-Fluorocytosin =(fungal deaminase)=>
• 5-Fluorouracil ==>
• 5F-dUMP => blocks conversion to dTMP by thmidylate synthetase, DNA synthesis inhibited
• 5-FU = anti-cancer

Question 20

Mode of action

Answer 20

• It is converted only in fungus by fungal deaminase to 5-FU- desamination
• 5-FU is then converted to 5-fluorodeoxyuridine monophosphate (FdUMP)- glycosylation
• And then fluorouridine triphosphate (FUTP), phosphorylation/ kinase
• Which then inhibit DNA and RNA synthesis (enzyme inhibitor)
• BONUS: Flucytosine is taken up by fungal cells via the enzyme cytosine permease- Selective uptake
  
  • Not in humans

Question 21

Why doesn’t the drug act on human cells

Answer 21

• Human cells are unable to convert the parent drug to its active metabolites

Question 22

Adverse effects

Answer 22

• Bone marrow toxicity with anemia, leukopenia thrombocytopenia
• Mammalian bone marrow cell have the capacity to covert 5-FC => 5-FU
• GI disturbance
• Mild and reversible liver dysfunction

Question 23

Griseofulvin

Answer 23

• A very insoluble molecule, poor bioavailability of drug (need for new formulations)
• Fungistatic- derived from a species of Penicillium, isolated natural product
• Better absorption when given with fatty foods
• It is deposited in the newly forming skin where it binds to keratin, protecting the skin from new infection
• Interferes with spindle formation in dividing cells and therefore with mitosis, unique mode of action
• Spiro compound
• Chiral
• natural product- benzofuran

Question 24

Overview: antifungal agents

Answer 24

1. Griseofulvin- oral treatment of dermatophytes
2. 5-fluorocytosin (5-FC)- life-threatening systemic mycoses (candidiasis, cryptococcal meningitis)
3. Polyenes (amphotericin, nystatin): Life-threatening systemic mycoses (candidiasis, aspergillosis, cryptococcal meningitis)
4. Imidazole (miconazole, ketoconazole)
5. Triazoles (Fluconazole, intraconazole): candidiasis superficial
6. Candidal infection (thrush): topical mycoses
7. Allylamines (terbinafine)- dermatophytes (skin, nails)
8. Echinocandins- newest class, act on cell wall

Question 25

From metronidazole to imidazole lead structure

Answer 25

• Metronidazole active: anaerobic organisms (Trichomonas vaginalis, clostridia, Bacteroides species)
• Converted to active radical of the nitro group inside the cells which causes DNA strand breakage

Question 26

Azoles- TARGET= Ergosterol synthesis

Answer 26

1. Imidazoles
   
   • Ketoconazole
   • Miconazole
   • Clotrimazole
2. Triazoles
   
   • Intraconazole
   • Fluconazole
   • Voriconazole
   • Posaconazole

Question 27

Triazoles

Answer 27

• High CSF: Serum conc ration is very high for fluconazole meaning it can be used for fungal meningitis
• Higher half-life means OD administration
  *

Question 28

Differences AZOLES

Answer 28

• BROAD SPECTRUM OF ACTIVITY
• Differences in water solubility
• Absorption, membrane penetration
• Half-life/ elimination
• ADME not MOA
• Formulation, route of administration

Question 29

Feature

Answer 29

• Fluconazole
• High water solubility
• High membrane penetration
• Half-life
• Imidazole, triazole, extra nitrogen atom, N polar
• Small, low MW
• F atom in para position blocks metabolism, hydroxylation

Question 30

Adverse effects

Answer 30

• Relatively nontoxic- allow slight toxicity because they are only acute treatment
• Minor GI upset
• Abnormalities in liver enzymes- Inhibit cytochrome P450 enzymes
• Very rarely, clinical hepatitis

Question 31

Mechanism of Action

Answer 31

• Inhibition of fungal cytochrome P450 enzymes ==>
• Reduction of ergosterol synthesis

Question 32

ChE synthesis

Answer 32

• Lanosterol-Demethylase= P450 enzyme

Question 33

Ergosterol synthesis

Answer 33

Question 34

AZOLES

Answer 34

• Inhibition of biosynthesis, ergosterol
  
  • In humans, lanosterol gets converted into ChE
  • In micro-organism, it gets converted into ergosterol
• P450, oxidising enzymes, catalyse the oxidation
• Special case, demethylation, 14-alpha-demethylase
• Newly formed double bond, in conjugation, to double bond

Question 35

Ketoconazole

Answer 35

• Older Imidazole, more toxic, replaced by itraconazole, but less costly
• The first oral AZOLE introduced into clinical use
• It is less selective for fungal P450 than are the newer azoles
• Absorption variable (better in acidic medium)
• Penetration in brain & CSF is poor
• In high doses inhibits adrenocortical steroids and testosterone synthesis, resulting in gynecomastia in some males

Question 36

Itraconazole

Answer 36

• Triazole
• Broad-spectrum antifungal with fungistatic action
• MOA: inhibits fungal ergosterol synthesis like other azoles
• Drug absorption is increased by good and by low gastric pH
• Pentration of drug in brain& CSF in poor
• Much more selective than ketoconazole

Question 37

Posaconazole

Answer 37

• The newest triazole
• It is the broadest spectrum member of the azole family
• Selectivity: it is the only azole with significant activity against the agents of zygomycosis and mucormycosis

Question 38

Fluconazole

Answer 38

• Broad-spectrum fungicidal drug
• It is also effective against some Gram-positive & anaerobic bacteria
• Good oral bioavailability, 94% is absorbed
• Penetration in brain & CSF is good, hence used for cryptococcal meningitis
  *

Question 39

Topical AZOLES

Answer 39

• Clotrimazole, miconazole
• Vulvovaginal candidiasis, oral thrush, dermatophyte infections, including tinea corporis, tinea pedis and tinea cruris
• Absorption is negligible, and adverse effects are rare
• Topical and shampoo forms of ketoconazole for seborrheic dermatitis and pityriasis Versicolor

Question 40

Topical Allylamines

Answer 40

• Terbinafine & Naftifine
• Naftifine: Synthetic allylamine
• Both are effective for treatment of tinea cruris and tinea corporis
• Terbinafine concentrates in skin and especially at nail beds, making it quite useful for fungal nail infections
• Inhibition biosynthesis ergosterol
• MOA: Terbinafine inhibits squalene epoxidase in the early stage of ergosterol synthesis in fungi
• Accumulation of intracellular squalene (toxic to the organism) & deficient ergosterol synthesis subsequent fungal cell death
• Keratophillic, fungicidal
• Dermatophytoses, Human ring worm

Question 41

MOA of allylamines

Answer 41

• Like the azole drugs, allylamines interfere with ergosterol biosynthesis
• But rather than interacting with the P450 system/ 14-Alpha-demethylase
• Terbinafine inhibits the fungal enzyme squalene epoxidase (similar structure to squalene)
• This leads additionally to the accumulation of the sterol Squalene which is toxic to the organism

Question 42

Overview biosynthesis ergosterol

Answer 42

Question 43

Targets for antifungal drug development