Drug Mech: Antifungals

Fungal infections in humans have increased in incidence and severity in recent years.

The increase in the number of patients who are at risk for fungal infections has been attributed mainly to the overuse of broad-spectrum antimicrobials and the HIV epidemic.

mycotic infection

The rise in the number of patients at risk for mycotic infections, as well as the emergence of fungal resistance to the azole antifungal drugs, has created new challenges and stimulated an interest in reconsidering combination antifungal therapy.

• Systemic Antifungals for Systemic Infections
• Systemic Antifungals for Mucocutaneous Infections
• Topical Antifungals

• Mucocutaneous refers to the three keratinized tissues in the body:
• 1) skin
• 2) hair
• 3) nails

• Polyenes
• Flucytosine
• Azoles (Imidazoles & Triazoles)
• Echinocandins

• Griseofulvin
• Allylamines (Terbinafine)

• "ene"
• Therefore, polyenes are characterized by several double bonds.

• Amphotericin B
• Nystatin

Only amphotericin B is used parenterally to treat systemic fungal infections. Nystatin is too toxic for parenteral administration; it is used topically and orally.

• not absorbed from the skin
• not absorbed from the mucous membranes
• not absorbed from the the GI Tract
• active against most candida species (yeast)
• most commonly used for the treatment of local candida infections
• used orally to treat intestinal candidiasis
• less active than Amphotericin B

Nystatin is less active than Amphotericin B

Amphotericin B remains the antifungal agent with the broadest spectrum of activity.

It is not absorbed from the GI Tract

No. Neither Nystatin nor Amphotericin B are absorbed from the GI Tract.

Polyenes are large water-insoluble, amphoteric (can react in an acid as well as a base) molecules.

They are very sensitive to UV-light (which isomerizes the double bonds from the active trans to the inactive cis conformation).

The presence of conjugated trans double bonds in these molecules has been associated with increased potency and decreased toxicity to humans (ie, increased selectivity against fungal cells).

Amphotericin B, which has more conjugated trans double bonds than nystatin, is 10 times more potent (and is less toxic) than nystatin.

They are fungicidal.

They are active against yeast infections (in addition to their activity against fungi).

Polyenes are selective in their fungicidal effect because they exploit the difference in lipid composition of fungal and mammalian cell membranes.

Ergosterol, a cell membrane sterol, is found in the cell membrane of fungi, whereas the predominant sterol in the cell membrane of mammalian cells is cholesterol.

Amphotericin B has more double bonds than Nystatin. Therefore, Amphotericin B is more selective in its bonding.

Conformation is very important in double bonds. The double bonds must be in the trans conformation to be active. The cis conformation is inactive.

If you expose the polyene molecules to UV light, the active trans conformation will be converted to inactive cis conformation, and therefore the drug molecules will be rendered inactive.

Ergosterol, a cell membrane sterol, is found in the cell membrane of fungi, whereas the predominant sterol in the cell membrane of mammalian cells is cholesterol.

First, the polyene molecule combines with the ergosterol molecules along the double bond-rich side of the polyene structure and associates with water molecules along the hydroxyl-rich side.

After the polyenes bind to ergosterol, they alter the permeability of the fungal cell by forming polyene-associated pores in the cell membrane.

The pores allows the leakage of intracellular ions and macromolecules, leading to cell death.

In other words, the drug molecule binds to ergosterol, the predominant sterol molecule in fungal cells and pokes holes in the cell membrane. These holes are called "pores." This causes the permeability of the cell to go haywire, so the components of the cell start leaking out...and the cell dies.

The polyene’s amphipathic characteristic facilitates pore formation by multiple polyene molecules, with the lipophilic portions around the outside of the pore and the hydrophilic regions lining the inside.

The affinity of the polyenes for binding to fungal ergosterol is greater than their affinity for binding to cholesterol in human cell membranes.

Amphotericin B is much more selective in its binding to fungal ergosterol than nystatin. Therefore nystatin is more toxic than amphotericin b.

Amphotericin b has increased affinity for ergosterol and decreased affinity for cholesterol. However, amphotericin b is still able to bind to cholesterol which is why there is some toxicity associated with these drugs.

Some binding to the cell membrane sterols of human cells does occur, which accounts for the prominent toxicity of the amphotericin B.

Some binding to the cell membrane sterols of human cells does occur, which accounts for the prominent toxicity of the polyenes.

Nystatin is used orally and topically. It is not absorbed to a significant degree from skin, mucous membranes, or the GI Tract. As a result, it has little toxicity, though oral use is often limited by the unpleasant taste.

• The most significant toxic reaction to amphotericin therapy is a dose-dependent nephrotoxicity; the irreversible form of its nephrotoxicity usually occurs as a result of prolonged administration
• (> 4 g cumulative dose).

Therapy with amphotericin B is often limited by toxicity.

The use of lipid formulations of amphotericin B (liposomal amphotericin B preparations) allows for a reduction of toxicity without sacrificing efficacy and permits the use of larger doses.

In these liposomal formulations, the drug is packaged in lipid delivery vehicles. Amphotericin B binds to the lipids in these vehicles with an affinity between that for fungal ergosterol and that for human cholesterol (ie, its affinity for binding to the lipids in the formulation is lower than its affinity for binding to fungal ergosterol, but it is greater than its affinity for binding to human cholesterol).

The lipid vehicle then serves as an amphotericin B reservoir, reducing nonspecific binding to human cell membranes. This preferential binding permits the use of effective doses of the drug with lower toxicity.

Some fungi produce lipases that can liberate free amphotericin B from the lipid vehicles directly at the site of infection.

Because the lipid preparations of amphotericin B are much more expensive than conventional formulations, their use is usually restricted to patients who are intolerant or not responding to conventional amphotericin B treatment.

• Fungal resistance to the polyenes occurs if binding to ergosterol is inhibited, either by:
• 1) Down Regulation of biosynthetic pathway
• The cell decreases the concentration of ergosterol in the membrane. In other words, the cell decreases its production of ergosterol which alters the permeability of the cell, which causes less pore formation in the cell. Note that the fungal cell still does suffer, but its better for the fungal cell to suffer than to die (at least in the fungal cells point of view).
• 2) Target Modification
• The cell will alter the chemistry of the ergosterol molecule to reduce its affinity for binding to the polyene.

Flucytosine (or 5-FC) is a water-soluble pyrimidine analog related to the anticancer drug Fluorouracil (5-FU).

5-FC has a much narrower antifungal spectrum of activity than that of amphotericin B.

5-FC is fungistatic at the clinical dose.

• 5-FC is used only in combination therapy,
• with amphotericin B or the azole antifungals,
• because of its demonstrated synergy with other antifungals and to avoid the emergence of resistance. It is given orally.

The amphotericin b drills holes in the cell membrane, which allows flucytosine access inside where it works. Therefore, amphotericin b increases the uptake of 5-FC.

• 1) 5-FC is converted intracellularly first to 5-FU by fungal cytosine deaminase enzyme.
• 2) 5-FU is then converted to 5-fluorodeoxyuridine monophosphate (5-FdUMP) and 5-fluorouridine triphosphate (5-FUTP).
• 3) 5-FdUMP inhibits DNA synthesis, while 5-FUTP is an inhibitor of RNA synthesis.
• 4) The overall effect of 5-FC is fungistatic.

Human cells are unable to convert 5-FC to its active metabolites (selectivity).

In other words, 5-FC penetrates both human cells and fungal cells. However, 5-FC needs to be activated to 5-FU. Therefore, 5-FC is selective because 5-FC can be converted to 5-FU in fungal cells but not in human cells. Why? Because cytosine deaminase enzyme is only found in fungal cells.

5-FC penetrates both human cells and fungal cells. However, 5-FC needs to be activated to 5-FU. Therefore, 5-FC is selective because 5-FC can be converted to 5-FU in fungal cells but not in human cells. Why? Because cytosine deaminase enzyme is only found in fungal cells.

• Synergy with amphotericin B is attributed to enhanced penetration of 5-FC through
• the fungal cell membrane that was damaged by the antifungal action of amphotericin B.

Synergy with the azole antifungals has also been reported, although the mechanism is unclear.

Fungal resistance to 5-FC is mediated through altered metabolism of 5-FC.

Resistance develops rapidly in the course of 5-FC monotherapy.

• The most common adverse effects of 5-FC are Bone marrow toxicity with:
• anemia
• leucopenia
• thrombocytopenia

Toxicity of 5-FC results from its metabolism by intestinal microflora to the toxic 5-FU.

• Imidazoles
• Triazoles

• Azoles are classified into Imidazoles and Triazoles, depending on the number of nitrogen atoms in the five-membered azole ring which is present in their chemical structures.
• 

• Imidazoles have two nitrogens in ring
• Triazoles have three nitrogens in ring

• Ketoconozole
• Miconazole
• Clotrimazole

• Itraconazole
• Fluconazole
• Voriconazole
• Posaconazole

The azoles are broad-spectrum antifungal agents.

Imidazoles and triazoles inhibit the biosynthesis of fungal ergosterol by inhibiting fungal cytochrome P450 14a-demethylase enzyme.

They are also inhibitors of the mammalian (human) cytochrome P450 14a-demethylase enzyme; however, they have greater affinity for the fungal enzyme.

The overall effect is fungistatic.

Azoles bind to the catalytic site of the 14 alpha-demethylase enzyme. This is the "heme" portion of the enzyme, or the Fe in the middle (see figure below).

The 14 alpha-demethylase enzyme is important in the biosynthesis of ergosterol because it converts lanosterol to 14 demethylianosterol.

Therefore, it can be said that azoles inhibit the second-to-last step in the biosynthesis of ergosterol of the fungal cell membrane.

The triazoles are better than the imidazoles because they are more selective and less toxic.

There are a lot of adverse effects with the imidazoles because they are potent inhibitors of cytochrome P450 enzyme in the liver, whereas triazoles are weak inhibitors of these enzymes.

Ergosterol

Ergosterol will decrease permeability of cell membrane. Therefore, decreased ergosterol will increase permeability of the cell membrane. This is why many drugs will attack some step in the biosynthesis of the ergosterol because if they can decrease or stop the production of ergosterol, they can gain access to the cell to destroy it because there will be nothing to hold the cell together. The fungal cell's guts fall out without ergosterol.

• 1) Squalene is converted to Squalene Epoxide via Squalene Epoxidase enzyme
• 2) Squalene Epoxide is converted to Lanosterol via Squalene Epoxide cyclase
• 3) Lanosterol is converted to 14-Demethyllanosterol via Lanosterol 14 alpha demthylase enzyme
• 

Inhibition of the biosynthesis of fungal ergosterol results in decreased sterol content of the fungal cell membrane, leading to altered physical and chemical properties of the fungal cell, increased cell permeability, and malfunction of membrane proteins.

The imidazoles are less selective than the triazoles in their binding to fungal cytochrome P450 14a-demethylase enzyme (ie, the imidazoles have a higher affinity for binding to the human P450 enzyme than the triazoles); this accounts for the higher incidence of drug interactions and side effects associated with the use of the imidazoles.

The imidazoles are less selective than the triazolesin their binding to fungal cytochrome P450 14a-demethylase enzyme (ie, the imidazoles have a higher affinity for binding to the human P450 enzyme than the triazoles); this accounts for the higher incidence of drug interactions and side effects associated with the use of the imidazoles.

The azoles are relatively nontoxic. The most common adverse reaction is GI upset.

Fungal resistance to the azoles occurs via a number of mechanisms, including the production of an altered fungal P450 14a-demethylase (which has a very low affinity for binding to the azoles) and active efflux of the azoles from fungal cells.

All azoles cause abnormalities in liver enzymes and, very rarely, clinical hepatitis.

Azoles can, very rarely, cause clinical hepatitis.

All azoles inhibit human cytochrome P450 enzymes in the liver, particularly CYP3A4.

Consequently, they are prone to drug interactions (they increase the efficacy of many drugs which are metabolized by these enzymes).

Fluconazole (a triazole) has the least inhibitory effect of all the azoles on hepatic cytochrome P450 enzymes; as a result, drug interactions and adverse effects are less common with fluconazole therapy (Fluconazole has the widest therapeutic index of all the azoles).

Ketoconazole (an imidazole) is a more potent inhibitor of mammalian cytochrome P450 enzymes than the triazoles (i.e., it is less selective for fungal P450 than are the triazoles), and it has more adverse effects than the triazoles.

As a result, systemic ketoconazole is no longer a treatment of choice in systemic antifungal therapy.

• Caspofungin
• Micafungin
• Anidulafungin

Echinocandins are large cyclic peptides linked to a long-chain fatty acid.

Echinocandins are fungicidal at the clinical dose.

They are active against yeast infections and fungal (mold) infections.

Echinocandins inhibit fungal cell wall synthesis.

They inhibit the enzyme b(1,3)-glucan synthase which is responsible for the synthesis of (1,3)-b-D glucan (a polysaccharide in the cell wall). As a result, they inhibit the synthesis of (1,3)-b-D-glucan which is an important polysaccharide component of the fungal cell wall. This results in the rupture of the fungal cell wall and cell death.

In other words, echinocandins inhibit the enzyme that makes the polysaccharide. If the polysaccharide is not made, then there is a missing component of the fungal cell wall and the fungal cell will die.

Griseofulvin is a fungistatic antifungal antibiotic.

It is administered orally and used only for the systemic treatment of infections caused by dermatophytes (localized superficial fungal infections of the skin, hair, and nails).

It is not active against yeast infections; it is also not active against systemic fungal infections.

Griseofulvin is an inducer of liver cytochrome P450 enzymes; as a result, it decreases the efficacy of many drugs which are metabolized by these enzymes (drug interactions).

The mechanism of action of griseofulvin at the cellular level is unclear; however, it is deposited in newly forming skin/hair/nail (in keratin precursor cells) where it binds to keratin, protecting the keratinized tissue from new infection.

In other words, griseofulvin has a protective effect on newly formed keratinized cells, therefore, you must wait until healthy new cells are formed to get rid of the infection.

Since its antifungal activity is attributed to its keratophilic properties and to preventing infection of the new skin/hair/nail structures, it must be administered for 2-6 weeks for skin and hair infections to allow the replacement of infected keratin by the resistant structures. Nail infections may require therapy for months to allow regrowth of the new protected nail and is often followed by relapse.

Terbinafine (Lamisil®)

Terbinafine (Lamisil®) is a highly lipophilic, synthetic allylamine derivative. It is given orally.

It is fungicidal against dermatophytes and other filamentous fungi; it is fungistatic against yeasts (candida species).

Like griseofulvin, it is keratophilic; it is indicated for the treatment of onychomycosis of the nail due to dermatophytes. The optimal clinical effect of terbinafine is seen only after several months following curing of the infection and cessation of treatment (a period of time is required for the outgrowth of healthy nails).

It is more effective (cure rate of up to 90%) than griseofulvin or itraconazole for the treatment of onychomycosis.

The most common adverse effects are GI upset and headache.

Terbinafine is an inhibitor of cytochrome P450 2D6 enzyme in the liver and may increase the effect or toxicity of drugs metabolized by this enzyme.

Terbinafine inhibits the biosynthesis of fungal ergosterol by inhibiting squalene epoxidase enzyme, which is a key enzyme in sterol biosynthesis in fungal cells. Inhibition of fungal squalene epoxidase leads to a deficiency in ergosterol and accumulation of the sterol squalene which is toxic to fungal cells.

Terbinafine inhibits the enzyme squalene epoxidase, which inhibits squalene from converting to squalene epoxide. This is the first step in the biosyntheis of the fungal cell membrane.

Terbinafine has no significant effect on cholesterol biosynthesis in human cells (it is a weak inhibitor of the mammalian squalene epoxidase).