Antifungal

Below, which is supplied as topical and systemic? A. Ketoconazole B. Fluconazole C. Nystatin D. Caspofungin

*A. Ketoconazole: This is the answer* B. Fluconazole: Oral C. Nystatin: Topical D. Caspofungin: IV

Interference of Ergosterol Synthesis

1. Azoles ➢ Inhibits 14-α-demethylase 2. Allylamines ➢ Inhibit squalene 2,3 epoxidase ❖ Both these enzymes are required for ergosterol synthesis → Makes a good target that doesn't bother the host

Disruption of the Fungal Cell Wall

1. Echinocandins 2. Macrolide Polyenes

Antifungal Drug Classes

1. Polyenes 2. Azoles 3. Pyrimidine analogs 4. Echinocandins 5. Allylamines 6. Inhibitor of tubulin dynamic

Which of the following is unique to fungal cell walls? A. 7-dehydrocholesterol B. 1, 3 B-D-glucans C. Mycolic acid D. Arabinogalactans

Answer B: 1, 3 B-D-glucans ➢ Mycolic acid and arabinogalactans are in TB ➢ 7-dehydrocholesterol is a vitamin D precursor

Which is correctly matched? A. Nystatin → Nephrotoxicity B. Itraconazole → Decreased inotropy C. Flucytosine→ Suppression of bone marrow D. Caspofungin→ Decrease in visual acuity

Answer C: Suppression of bone marrow ➢ Nystatin: When taking systemically, can cause nephrotoxicity → Can't take systemically because it's too toxic ➢ Itraconazole increases inotropy (contractility)

Which of the following antifungal drugs significantly differs from the others below? A. Terbinafine B. Butenafine C. Tolnaftate D. Flucytosine

Answer D: Flucytosine

Interference of Ergosterol Synthesis: Azoles

Inhibit 14α-demthylase and prevent fungal cell from making ergosterol

Interference of Ergosterol Synthesis: Allylamines

Inhibit squalene 2,3 epoxidase

Review Question: Which anti-Tb antibiotic inhibits trans-translation?

Pyrazinamide

Azoles: Characteristics

❖ *Know the blue* ❖ Both fluconazole and voriconazole have a fluoride and will go into the CNS ➢ ex. Would give these 2 for fungal meningitis ❖ Most are protein bound in circulation except for fluconazole ❖ Drug interactions are due to inhibition of CYPs ➢ ex. Get an Rx for fluconazole that she got from systemic glucocorticoids → Doesn't take statin for 2 weeks ❖ Teratogen: Pregnancy risk C ➢ So it is possible

Polyene antifungals: Amphotericin B (AMB)

❖ *MOA*: Binds to fungal cell membrane ergosterol ➢ Incorporates into the membrane ➢ Forms a pore → Ions and other small molecules leak out of the cell eventually causing cell death. ❖ Picture: ➢ Pink: Ergosterol ➢ Dark blue side = Hydrophobic ▬▬ Align with lipid bilayer in fungal cell membrane ▬▬ Very comfortable near ergosterol ➢ White side = Hydrophilic ▬▬ There are two hydrophilic sides coming from two molecules of AMB → Face each other towards the inside ➢ All of this forms a pore

Macrolide Polyenes

❖ *Nystatin*: Topical Only ➢ "Nystatin swish": take a mouthful, swish and swallow it; seen in hospital setting ▬▬ This treats fungi in your mouth, your esophagus and gut ▬▬ ❖ *Amphotericin B*: Systemic ➢ Polyene: Many double bonds ➢ Lactone Ring ➢ IV ➢ Shown in picture ▬▬ Top side is very polar and water soluble because of the OH's (key in MOA) ▬▬ Bottom is our "ene" side and is not polar ❖ *Neither one of these is absorbed in the GI tract*

Echinocandins: ADME

❖ Administered: IV ❖ Metabolized by hydrolysis and N-acetylation in the liver. ➢ No CYPs (good thing) ❖ Metabolites are excreted via urine and feces. ❖ Main side effects are reaction at injection site and *elevation of liver enzymes* ➢ However, long list of possibles. ❖ *Poor CNS penetration* → Not for meningitis

Amphotericin B: Formulation

❖ Available in 4 formulations ❖ Not really very water soluble, so you have to create a vehicle where you can get the amphotericin in a dosage form that can be given IV *1. Amphotericin B (AmB)* ➢ Put bile salts together with amphotericin = AmB ➢ Forms a colloid, not a solution *2. Ambisome (LAmB)* ➢ L = liposomal ➢ Form a unilamellar liposome ➢ This formulation ↓toxicity ➢ Very expensive → $950 a dose, so you could give them AmB instead *3. ABLC* ➢ Another group of lipids that can be used to solubilize this group of compounds ➢ Ribbon-like sheet → Don't get same blood levels that you get with AmB but you do get less toxicity

Flucytosine

❖ Classified as an anti-metabolite ❖ Has a fluoride group ❖ Cytosine is the base ❖ Why this works selectively for particular fungi? ➢ It has permease in it its membrane → If fungi doesn't have that, then it will be resistant to the drug ▬▬ *Without permease, drug won't work at all* ➢ This fungal cytosine deaminase is different enough from ours that makes the drug more selective for fungus once fungus takes it up ▬▬ *Without the cytosine deaminase or if there is a mutation* → There may not be activity for the drug, but there a couple of bugs for which this is a good agent ❖ Taking up Flucytosine → Undergoes cytosine deaminase → When that happens by virtue of the enzyme, end up with 5-fluorouracil (common for skin cancer drug) → But we need dUMP for this reaction, so add a phosphate group with the enzyme: uPRTase → Get 5-FUMP → So take ribonucleotide reductase and reduce that ribose to get *5-fluoro-dUMP* (this is the main metabolite that we'll see that limits fungal growth) → that will participate in aforementioned reaction and limit the thymidylate (dTMP) that we can make ➢ That complex of the 3 (dUMP, thymidylate synthase, and THF) → Donated a carbon to get thymidylate → What happens with fluoride group? → It makes this reaction *very stabe* so the 3 will stay together instead of coming apart → So we can't have anymore dihydrofolate → Can't regenerate and reuse thymidylate synthase ➢ *Main idea: Fluorine inhibits the complex from coming apart so we can't regenerate and reuse* ❖ Other Mechanisms: ➢ 5-fluoro-UMP: Put 2 more phosphate groups on that and it goes into RNA → Get dysfunctional mRNA ➢ We now limit the amount of dTMP that we can make→ We need dTMP to make dTDP and dTTP→ If we don't have any of those, the body will try to put uracil (UTP) into DNA. But UTP doesn't belong to DNA→ We're gonna have dysfunctional DNA as well ➢ *Main MOA*: Making that complex become so stable that we no longer have the components readily available to keep making dTMP

Echinocandins

❖ Cyclic lipopeptide ❖ *MOA*: Inhibits the synthesis of cell wall component *1, 3 β-D-glucan* ➢ Thought to interfere with fungal enzymes1 *1-3, β-D-glucan synthase* ❖ Rather rapid destruction of the cell wall ➢ Microscopically, blebs in the cell wall; hyphae swell. ➢ Doesn't take months and months to kill fungi like other drugs ❖ *MOR*: Mutate the enzyme 1,3 B-D-glucan synthase

Azoles: Drugs

❖ Divided into 2 groups: Imidazoles and Triazoles ➢ Don't have to know for test ❖ *Note that ketoconazole is both systemic and topical* ❖ A lot of these are OTC

Echinocandins: Drugs Available

❖ Drugs: End in -fungin ❖ All 3 are IV ❖ *Caspofungin* ➢ Typically used in *esophageal candidiasis* (thrush) ▬▬ It isn't given for thrush 1st, usually we will try topical before this ▬▬ People get thrush when they don't rinse and spit after using steroid inhalers ▬▬ Nystatin won't work because this bug is resistant to that (talking about the more entrenched infection) ➢ *Pneumocystis jirovecii* ▬▬ In a hospital setting where there are a lot of sick people ▬▬ When people got HIV infection, they'd get this infection for a long time if they're immunosuppressed ➢ *Invasive aspergilosis* ❖ *Micafungin* ➢ Some people will use this prophylactically if they're doing a bone marrow transplant until they get enough WBCs ❖ *Anidulafungin*

Review Question: Add a 2nd line antibiotic for MDR-TB

❖ Fluoroquinolone ❖ Levofloxacin ❖ Cycloserine ❖ Ethionamide

Yeast and Fungal Infections: T-Cells

❖ Fungal infections (TB also) are fought by the immune system mainly using T-cells ➢ We'll still see leukocytes and neutrophils, but primarily t-cell ❖ If we wipe out the T cells we'll wipe out the ability to fight fungi, viruses, and TB

Amphotericin B: Antifungal Spectrum

❖ Generally considered a broad spectrum, effective antifungal agent ❖ Most fungi, specifically: ➢ *Aspergillus sp** ➢ Sporothrix sp ➢ Blastomyces sp ➢ Mucor sp ➢ *Candida sp** ▬▬ This is basically everywhere, doesn't really bother us normally unless you're asthmatic against fungi (can be found on bread) ➢ * → May harbor resistance ➢ Learn bolded ones

Thymidine Synthesis: Reminder

❖ In order to make DNA, need thymidine, which is a pyrimidine ❖ If we want to make dTMP, we need a few things 1. Reduced tetrahydroflorate (N5, N10- methylenetetrahydrofolate) ▬▬ To recycle dihydrofolate, we need dihydrofolate reductase ▬▬ Amino acid (Serine) donates a carbon and become Glycine → We need a carbon donor) ▬▬ N5, N10-methylenetetrahydrofolate functions as a carbon donor 2. The other thing that needs to be in the reaction is deoxyuridylate (*dUMP*) 3. Enzyme: *Thymidylate synthetase* ❖ The 3 things come together and when they're in that little complex, THF donates a carbon group to deoxyuridylate (dUMP) and enzyme (Thymidylate synthetase) facilitates that reaction → When finished, we get thymidylate → End up with dihydrofolate→ Cycle repeats ➢ Ignore the dihydrofolate reductase part for now

Amphotericin B: Nephrotoxicity

❖ Increased renal vascular resistance → Decreased GFR. ➢ *Azotemia* → Accumulation of metabolic nitrogen → Indicative of renal dysfunction ▬▬ Also accumulates in the renal tubules so it's toxic to the cells where it's accumulating ❖ Nephrotoxicity is usually reversible with discontinue. ➢ However, some permanent changes persist. ▬▬ Although the kidney's function has returned, you can still see persistent changes ❖ May decrease the ability to concentrate urine in the collecting ducts. ❖ Loss of K⁺, Na⁺, Mg⁺⁺ ❖ The dose has to be limited because of the nephrotoxicity

Yeast and Fungal Infections: Who are at greatest risk for a serious fungal infection?

❖ Innate and acquired immunity are critical ➢ Normal physical barriers (burn patient, step on thorn) ➢ Cancer chemotherapy (hard to kill cancer without hurting yourself) ▬▬ There are a lot of target for bacteria and fewer target for yeast, even less targets for virus because viruses use our machinery ▬▬ The chemotherapy we have now is not very selective ➢ Immunosuppression ➢ HIV ➢ Normal flora ▬▬ If we're on a lot of antibiotics, these fungal infections can overgrow → Yeast infection or other infections that's hard to get rid of ➢ Transplant ❖ Metabolic aberrations ➢ Diabetes ▬▬ Metabolism is different ▬▬ Don't have the best circulation ➢ Patient age ▬▬ Newborns: Don't have a fully developed immune system ▬▬ Elderly: As we age our immune system isn't what it used to be

Azoles: Adverse Reactions

❖ Ketoconazole and adrenal corticosteroid synthesis. ➢ Inhibits the adrenal steroid synthesis (if used at high enough doses) ❖ Itraconazole may increase inotropy in the heart ❖ Fluconazole: QTc prolongation ❖ Voriconazole: *Broader spectrum*, elevated liver enzymes, prolongs QTc interval, ADR, *changes to vision in 18-30 % of patients*

Amphotericin B: ADME

❖ Little to no absorption in the GI tract ❖ Given IV (Unless fungal infection is in the GI tract) ❖ *Circulates highly protein bound* ❖ Distribution to tissues: Varies, not for CNS ➢ Intrathecal administration is possible → Catheter drip directly into spinal cord ❖ Metabolism/excretion: Liver/renal ➢ Some studies say it's broken down then excreted renally ➢ Some studies show it's excreted in liver as well ❖ Accumulates in tissues: T½ = 15 days ❖ We won't be using Amphotericin unless it's super serious. ➢ Should try azoles first. ➢ Usually see a bad infection for us to use it in hospital ➢ It's broad spectrum so it's used for the resistant organisms

Antifungal Spectrum of Flucytosine

❖ May also compete with uracil in RNA synthesis ❖ Spectrum: Used to treat *yeast meningitis* ➢ Cryptococcus neoformans ➢ Candida sp. ▬▬ Some Candida sp. are always resistant ❖ Does not have activity for Aspergillus sp. , Sporothrix sp., Blastomyces sp. , Histoplasma sp. , Coccidiodies sp. (FYI) ❖ Don't need to learn the name of the two yeasts, just know it is used to treat yeast meningitis

Amphotericin B: Anemia

❖ Most patients will have a decrease in their hematocrit ➢ Normochromic, normocytic anemia ▬▬ RBC under microscope are normal size & have same amount of hemoglobin in each one so they look really similar to normal RBC, but there are *fewer* of them ❖ Less Erythropoietin ➢ Erythropoietin is made in the kidney → In kidney dysfunction we will have less erythropoietin ➢ Not a deal breaker, can give recombinant erythropoietin product ▬▬ This product costs a lot ($5,000/year) & some dosage forms of amphotericin are expensive too ❖ *Reversible* ➢ Usually does not require transfusion of packed RBC. ❖ Other serious but rare side effects: Cardiac arrhythmias, anaphylaxis ➢ Fungal infections in hospitals are pretty serious so use this as a resort, but look at risks and benefits ➢ It is a product of another fungus, so you can have anaphylaxis, but everything can cause anaphylaxis ➢ *Main thing: Anemia, hematocrit, erythropoietin, K+, Mg2+, Na+*

Mechanism of resistance of the azoles

❖ Mutation in 14α-demthylase enzyme ❖ Upregulation of drug efflux pumps ❖ Confers cross-resistance to all azoles ➢ Resistant to one azole, resistant to all of them

Flucytosine: ADME

❖ Oral dosage form, well absorbed, well distributed. ➢ High concentraiton in CNS so it's good for yeast meningitis ❖ Mainly excreted unchanged in the urine ❖ Adverse reactions ➢ Nausea and vomiting ➢ Diarrhea ➢ Activates chemotrigger zone ▬▬ High concentration in CNS → So this drug can trigger chemotrigger zone in brainstem → Thus causes nausea/vomiting ➢ Bone marrow suppression (at high enough doses) ▬▬ We have to make TMP's and we use the same kind of enzyme ▬▬ It is seen in the bone marrow because bone marrow divides all the time → If there's a lot of DNA synthesis happening in a particular tissue and drug is an anti-metabolite for pyrimidine or purine bases, we will see some activity in that normal tissue just by virtue the fact that these cells are replicating ▬▬ GI cells and skin cells are always replicating as well → Think of someone on chemotherapy → Nausea, vomiting, diarrhea → Bone marrow suppression → Hair loss → These are the places that normal cells are suffering from toxicity of chemotherapy

Nystatin

❖ Similar macrolide, polyene structure and MOA ❖ *Topical only* ➢ Oral formulations but used to treat oral mucocandidiasis or infection of the GI tract ❖ Even if you swallow it, it's not being absorbed, it is just coating surfaces of GI tract ❖ This is more toxic than AMB so it can't be give IV

Allylamines: Adverse Reactions

❖ Skin ➢ Rash, pruritis (itchy) ❖ Exanthematous pustulosis: Pustular rash ➢ More serious

Saprophytic Infections of the Skin and Nails: Dermatophytes

❖ Skin, hair and nails. ➢ Eating the dead stuff ❖ Contact with infected individuals, animals or inanimate objects, contaminated with spores. ➢ Group of "dermatophytes" are causative agents. ❖ Tend to grow in moist conditions ➢ Skin folds (Tinea corporis) ▬▬ Tinea corporis/pedis → Ring worms ➢ Sweaty feet (Athlete's foot, Tinea pedis) ❖ Treat with topical anti-fungals (OTC) ❖ Superficial infections: They don't reach our barriers, but can be annoying

Griseofulvin

❖ Tablets, suspension → Ultramicronized ➢ Very insoluble in water. ❖ Primarily *dermatophytes* ➢ *So hair, skin, and nails* ❖ Not effective for Candida sp. Aspergillus sp. ➢ Not effective for yeasts and fungi that cause deep tissue infections ❖ Binds to *fungal tubulin* molecules and inhibits fungal mitosis ➢ Tubulin is different from human tubulin, so is somewhat selective. ❖ Accumulates in nails, hair, skin but only new protein is resistant to fungus. ➢ Only new protein that is just being made is resistant to fungus→ In other words, if you have fungal infection in your nail, it's going to take a while for you to see your nail free of fungus because it takes a while for the nails to turn over→ This is one of the disadvantages of the drug ▬▬ This isn't a problem with terbinafine because it accumulates there and diffuses thru nails→ Clean the infection rather quickly ❖ Really old drug

Systemic Allylamines

❖ Terbinafine ❖ Used for systemic drug delivery as well as a topical. ❖ Oral dosing: Well absorbed ❖ In circulation: ➢ Extensively protein bound ➢ Significant first pass effect. ❖ Accumulates in the nail for a very long time ➢ Two weeks of drug → 3 months of activity ❖ Also accumulates in adipose tissue ❖ Metabolized in the liver by oxidation and hydrolysis ➢ By-products are excreted in the kidneys ❖ *MOR* ➢ The main resistance of mechanism here is a mutation in squalene 2,3 epoxidase such that drug won't bind or an increase in efflux pumps, ➢ Mentions that amphotericin resistance is the fungus → Alters the cell membrane so that it doesn't need ergosterol ▬▬ I find this confusing → Amphotericin has a mechanism that involves making a pore inside the wall, but doesn't seem to deal directly with ergosterol → But terbinafine does

Allylamines: Drugs

❖ Terbinafine ➢ Systemic (Rx) and topical (OTC for athlete's foot) ➢ Rarely see it in systemic infections ➢ Lamisil (tablets) = Systemic ➢ Will most often be used for other fungal nail infections → runners who have sweaty shoes, nails will get thick ❖ Butenafine and Naftifine are both topical ➢ Both can be used for *cutaneous infections* ➢ Terbinafine can't be used for cutaneous infections

Miscellaneous topical anti-fungals: Ciclopirox

❖ Topical, for superficial fungal infections ➢ Dermatophyte infections ❖ Interferes with bacterial as well as fungal membrane selective transport. ❖ Different dosage forms including a shampoo and nail lacquer.

Ambisome: Liposomal Formulation

❖ Unilamellar liposome ❖ Lyophilized; liposomes form when water is added. ❖ Distribution ➢ Liver ➢ Spleen ❖ Due to a reduced nephrotoxicity, a higher dose can be used

Therapeutic Targets

❖ We could identify some spots in the bacteria where the bacteria are significantly different because they're prokaryotes and we're eukaryotes ❖ Now we're talking about eukaryotic cell because eukaryotes are same in mammalian and yeast. People research genes in yeast because they're eukaryotes and you can do experiment quickly and then put it into cell model. ❖ Many things are the same ➢ Nuclear membrane ➢ 80s ribosomes ❖ Therapeutic targets ➢ Our cell membrane uses cholesterol to increase fluidity but fungi use *ergosterol* ➢ We don't have a *cell wall* but bacteria do

Basic Fungal Cell Wall Structure

❖ β-glucans → Target → Instead of a peptidoglycan fungi have this ❖ Chitin → Target → There are some new drugs that are being developed that may go after chitin, so one day will see this ❖ Cell membrane (ergosterol) → Another target