PHAR 506 Exam III Lecture Review

(3/12+14) Jaki Lecture: Anti-Mycobacterial Agents I Tuberculosis is a disease found globally. It has higher prevalence in tropical climates, and affects over 2 billion world-wide. Though, not many present with the disease. Treatment proves to be very difficult due to the duration/adherence

Drug-Sensitive Multi-Drug Resistant (MDR) Excessive Drug Resistance (XDR)

Infected 2 billion 70 million ~630k Resistance N/A Isoniazid, Rifampin Isoniazid, Rifampin, FQ Injectables Tx Duration 6mo 12-18mo 24+mo

Mycobacteria: A slow-growing ubiquitous bacteria that are difficult to culture on a plate. They are best identified by performing Acid-fast tests, staining a pinkish-purple. Mycobacteria typically present as respiratory infections

- Frank Pathogens: Capable of being the primary cause of Tuberculosis o M. tuberculosis, M. bovis, M. leprae, M. ulcerans

- Opportunistic Pathogens: Responsible for producing secondary disease o Slow-growing (Very slow): M. avaium, M intracellulare, M. kansasii, M. scrofulaceum, M. marinum o Rapid-Growing (still slow): M. fortuitum, M. chelonae, M. abscessus

- Mycobacterium tuberculosis (MTB): The ancient infective pathogen, very slow growth, but much resistance o Cell Wall: MTB has 4000 genes, most of which are dedicated to constructing the complex cell wall

§ Similar to gram+ bacteria, thick peptidoglycan layer with no outer membrane § Mycolic Acid layer, with polar sugar adducts superficial to the peptidoglycan layer § Acid-Fast Stain (AFB) can be used to help identify cultured M. tuberculosis.

o Life Cycle: Anaerobic: Dormant Stage Intracellular: Within Macrophages Aerobic: Growth Stage, can be found in calcified TB cavities

o Not ubiquitous, No reservoir: Can be eradicated. - Non-tuberculosis mycobacteria (NTM): We do not need to be aware of these.

o Slow Growing: M. bovis, M. avium-intracellulare, M. kansasii (bovis used for attenuated BCG vaccine) o Rapid Growing: Mycobacterium fortuitum, Mycobacterium chelonae (Rapid, grow < 7 days)

Tuberculosis (TB) - Definition: An infectious process predominately restricted to the lungs characterized by an insidious onset,

however, it may involve any organ. Over time, the lung tissue becomes necrotic, presenting as hemoptysis - Sx: Nonspecific symptoms such as malaise, weight loss, night sweats, and cough become more severe over time

o Hallmark: First true sign of TB is Hemoptysis (coughing up blood). Usually a long-term cough precedes - Pathophysiology: TB is very contagious in the active form, however is not infective in the dormant state. Overall,

only 10% of infected individuals will develop the active disease, and 10% of immunocompromised individuals in the dormant state will activate each year.

o Infection: The immune response is cell-mediated, whereupon the bacterium is taken up by macrophages. Yet, some macrophages have difficulty killing the bacterium, so it then sits and persists, forming a granuloma. A granuloma is an accumulation of macrophages, immune cells, and bacterium.

o Latent Infection (LTBI): Asymptomatic, the bacteria simply lie dormant. Positive (+) Skin TB Test o Active Disease: When the macrophages cannot maintain TB anymore, the agent converts to the active

phase and begins to grow, liquefying the granuloma, and spreading to other organs. Symptoms present. - Dx: Skin Test è Blood Test è Symptoms è Chest X-Ray è Finalize with definitive culture

- Tuberculin Skin Test (TST): Inject PPD (cell wall material) intradermal. Measure induration at 48h, which is the size of the bump. Pretty much is a test to see if you’re vaccinated

- QuantiFERON: Blood Test, more specific than TST, capable of discriminating between MTB and BCG using Ag. It compares the interferon release rate, which occurs when T-Cells interact with macrophages

- Rapid Detection: BACTECT, tests for active bacteria by pressure associated with oxygen consumption - Xpert MTB/RIF: A small instrument that is like a mini PCR, taking 2 hours at 92% sensitivity for TB Dx

Tuberculin Skin Test (TST) QuantiFERON-TB Blood

CXR

Symptoms

Physical Findings

Latent TB Infection

+,+ Negative None None

Pulmonary TB Infection

+,+ ~Abnormal Fever, cough, night sweats, weight loss, fatigue, hemoptysis

Respiratory specimens may (+)

Treatment of Active TB It takes a very long time “Do not worry about doses” - Intensive Phase: 2 Months of Daily Therapy

o Isoniazid (INH): 300mg Qdaily o Rifampin (RIF): 600mg Qdaily o Pyrazinamide (PZA): 1-2g Qdaily o If >4% in community, add-on Ethambutol (EMB) 2-5g Qdaily OR Streptomycin 1g Qdaily

- Continuation Phase: 4 Months of Daily Therapy o Isoniazid (INH): 300mg Qdaily o Rifampin (RIF): 600mg Qdaily

Therapy Activity: Each component of therapy serves a specific role in treating TB - Hidden in Granulomas (Active): INH, RIF, ±EMB

o Within the granuloma cavities are the largest subpopulation of MTB. To ensure activity, 2, if not 3 agents are used to outcompete resistance. Within 2-3 months, all of these active bacteria should be eliminated.

- Waiting in Macrophages (Active): INH, RIF o Macrophages that take up MTB are considered infected. Therapy can

eliminate them in 1.5-2 months - Hidden in Granulomas (Dormant): PZA, RIF(50%)

o The dormant bacteria that inhabit the cavities are difficult to kill – taking up to 6 months. This helps stress the importance of adherence to therapy. While PZA is effective against the entirety of the dormant stage, RIF only acts upon the In-and-Out part.

Mechanism of Resistance - Step 1: Spontaneous mutations in MTB are capable of eliciting drug-resistance. Mutations in the drug-target, the

TB enzymes responsible for activating our prodrugs, the efficiency of efflux pumps, and the cell wall can all significantly contribute to resistance at the cost of fitness. Natural mutants like this always exist.

- Step 2: Exposure to antibiotic therapies will impose a selective pressure on the bacterial population, leaving only survivors. The survivors whose mutations are least reducing of fitness will proliferate and disseminate

Strategies to Prevent Development of Drug Resistance - Multiple Drug Therapy

o In a single caseous lesion, there are 108-1010 bacteria. Some of our therapies are more prone to natural mutation frequencies than others. 1 in 106 bacteria will have resistance to INH, which means as monotherapy, each lesion may have 100-10,000 bacteria resistance to INH. If we add RIF, which has a mutation frequency for resistance of 1 in 108, our multiple drug regimen now has a 1 in 1014 rate of resistance to both INH and RIF. As a result, it is unlikely in a lesion that any bacteria are resistant.

o Primary Resistance = The frequency of resistance to the cocktail therapy. Previous example, 1 in 1014 - DOTS: Directly Observed Therapy, Short-course (6mo)

o Program initiated by the World Health Organization (WHO) to make the patient feel more responsible for taking the daily dose.

- Monitoring: Response to therapy can be monitored with AFB sputum smears, culturing sputum, CXR o Failure to achieve culture negativity after 2 months suggests drug-resistance, which should be addressed

by repeating drug-susceptibility testing and extending treatment course - Rule: Never add 1 drug to a failing regimen. This is essentially initiating monotherapy, because the other drugs

are not working. AND, we know that monotherapy is minimally effective - Fixed-Dose Combinations Drug-Susceptibility Testing

Treatment Algorithms - Latent TB (LTBI): For LTBI, monotherapy may be used as the bacterial load will likely be low enough to ignore

the risk of resistance occurring. o Option 1: INH alone for 9 months o Option 2: RMP alone for 4 months o Option 3: INH + Rifapentine (weekly) for 3 months

- Active TB: Multi-drug therapy for at least 6 months o Considerations: Location of infection (pulmonary, or extrapulmonary), meningitis, resistance o TB Meningitis: INH, PZA, EMB, CS o RIFR: Replace with FQ and extend therapy to 12 months o MDR-TB: Use FQ and other 2nd line drugs, extending therapy to 18-24 months o HIV-infected: Use Rifabutin with an adjusted dosage, or adjust the dose the pt’s PI/NNRTI therapy

First-Line Agents Second-Line Agents Third-Line Agents Isoniazid Rifampin

Pyrazinamide Ethambutol

Old Streptomycin Cycloserine

p-Aminosalicylic acid Ethionamide

Amikacin/Kanamycin Capreomycin

New Fluoroquinolones

Linezolid Bedaquiline

Imipenem Clofazimine

Clarithromycin Thioridazine

----FIRST LINE Isoniazid (INH) Isonicotinylhydrazide ‘Most efficient anti-TB drug’ Cmax: 7µg/mL

- Structure: An isonicotinic acid, with a pyridine ring and a hydrazide group substitution - Bioactivation: INH is a prodrug that is bioactivated by catalase-peroxidase katG - MoA: Both INH and ETH inhibit mycolic acid synthesis by forming NAD-Adducts. The

NAD-adduct blocks the InhA coenzyme binding site, preventing cell wall synthesis o InhA = Enoyl-ACP reductase, a component of fatty acid synthase complex (FAS II)

- Resistance Mechanisms [4% primary, 11% acquired] o katG: missense or large deletions in catalase-peroxidase (most common) o inhA: Mutations in the NADH binding site

- AE: INH therapy can damage nerve tissue, leading to peripheral neuropathy. Patients should receive Vitamin B6 (pyridoxine) supplementation to protect the tissue. Also, hepatotoxicity risk

Rifampin (RIF): Natural product derived from Rifamycins Cmax: 8-10µg/mL - Structure: Polyketide rifamycin with a naphthoquinone ring and aliphatic ansa chain

o Target H-bonding: (1) 4x-OH of Ansa bridge, (2) Napthol ring o Naphthoquinic chromophore gives rifampin its orange color (Counseling)

- MoA: Inhibits RNA Synthesis. Rifampin binds to b-subunit of RNA polymerase (rpoB), preventing the binding of nucleotides and therefore inhibiting the initiation of transcription. This is a broad spectrum bactericidal feature

- Resistance Mechanisms [0.2% primary, 2.5% acquired] - Much lower than INH! o Single AA substitution in a hotspot of rpoB

- AE: Hepatotoxic, Induction of liver enzymes, Thrombocytopenia, slow blood clotting and easy bruising. GI Upset, and orange urine

o Induction of liver enzymes reduces the T1/2 of steroids, anticoagulant agents, antimicrobials (macrolides/imidazoles), and protease inhibitors (NNRTIs). For AIDs patients, Rifampin should be swapped out for Rifabutin, so as to continue TB therapy and decrease toxicity.

- Rifabutin: The rifamycin of choice for HIV/TB co-infection when using protease inhibitors due to less activation of CYP450

o Additional Benefit: It is active against some RIF-resistant strains of MTB - Rifapentine: Boasting a longer T1/2 than RIF, some studies have shown its intermittent

dosing, twice weekly in initial phase and once weekly in continuation phase) can be more effective than RIF. However, it is not effective against RIF-resistant strains.

Pyrazinamide (PZA): A TB-specific drug that shortens treatment Cmax: 45µg/mL - Structure: - MoA: Though the mechanism of action is uncertain, it requires activation by MTB-specific

enzymes such as pyrazinamidase (pncA) to form pyrazinoic acid. The drug is only active at low pH (<6) and MTB do not have sufficient efflux pumps to combat its accumulation

- Resistance Mechanisms: pncA mutation - AE: Significant hepatotoxicity, monitor ALT AST. PZA may also precipitate arthralgia and/or arthritis

Ethambutol (EMB): Requires high doses! Cmax: 2-5µg/mL - Structure: 2 butanols, 2 amines, and a central ethyl – super simple! - MoA: Inhibits cell wall synthesis. Inhibits arabinosyl transferase, thereby preventing the

synthesis of arabinogalactan and lipoarabinomannan for the MTB cell wall. - Resistance Mechanisms [0.1% primary, 1.8% acquired]

o Overexpression of embA, embB, embC or mutations in embB - AE: Optic neuritis is a concern, producing vision problems (changes in color vision) that may be permanent

----SECOND LINE Fluoroquinolones (FQ): Nalidixic Acid derivatives- Moxifloxacin, Gatifloxacin, Levofloxacin are preferred

- MoA: DNA Gyrase inhibitors, preventing the ATP-dependent DNA topoisomerase from catalyzing the negative supercoiling of DNA

- Role in Therapy: Shown to shorten the duration of therapy, showing some activity against the dormant TB

- Resistance: gyrA mutations: Missense mutations in small regions Linezolid: An oxazolidinone bacteriostatic agent that is effective against gram(+) bacteria only

- MoA: Inhibits Protein synthesis - Role in MDR Therapy: Has activity in both MDR and XDR-TB @ 600mg BID - AE: Due to inhibition of human mitochondrial protein synthesis, Linezolid is associated with

neuropathy and myelosuppression Bedaquiline (Diarylquinoline) MIC = 0.06µg/mL

- A drug specifically developed for TB, Bedaquiline is highly potent against non-replicating MTB. Though, due to significant concerns over adverse effects, it is reserved for cases where no other options are available. AE: Significant cardiotoxicity (QT prolongation), Hepatotoxicity

Aminoglycosides: Streptomycin, Kanamycin, Amikacin, Paromomycin - Structure: 6-membered aminocyclitol ring with glycosidic linkage to 2 or more amino-sugar cations. Due to poor

bioavailability, these medications are administered via IM injection - MoA: Bind to the 30s subunit of bacterial ribosomes - Resistance: rpsL (S12 protein) mutations, 16S RNA - Streptomycin Cmax: 25-50µg/mL

o AE: Ototoxicity (Ear damage), Nephrotoxicity (kidney damage), Neuromuscular blockade Ethionamide (ETH) Very similar in structure to INH Cmax: 2-20µg/mL

- Structure: An isonicotinic acid, with an ethyl-substituted pyridine ring and para-thioamide. The C=S bond is usually a ‘No No’ in medicine due to associated toxicity, but in this case risks<benefits

- Bioactivation: ETA is a prodrug that is bioactivated by monooxygenase etA - MoA: Interrupt cell wall synthesis. Both INH and ETH inhibit mycolic acid synthesis by forming NAD-Adducts.

The NAD-adduct blocks the InhA coenzyme binding site, preventing cell wall synthesis o InhA = Enoyl-ACP reductase, a component of fatty acid synthase complex (FAS II)

- Resistance Mechanisms: inhA: Mutations in the NADH binding site D-Cycloserine (CS): Cmax: 10µg/mL

- Indication: TB Meningitis - MoA: Competitive inhibition of D-Ala racemase (alr) and D-alanyl-D-ala synthetase (ddlA) - AE: Able to cross the BBB, cycloserine is prone to inducing neurologic toxicity, including psychosis and seizures - Resistance: One mechanism of resistance is the overexpression of D-Ala Racemase (alrA)

Para-aminosalicylic acid (PAS) Cmax: 75µg/mL - A derivative of salicyclic acid, this antifolate is formulated as granules for better GI tolerance. It is reserved for

resistance cases. It is a bacteriostatic compound requiring heavy doses for efficacy - MoA: Folate antagonist and/or inhibition of mycobactin synthesis from salicylate - AE: GI issues

Capreomycin/Viomycin, Kanamycin Cmax: 30/25-50µg/mL - MoA: Inhibit protein synthesis (translocation) - AE: Nephrotoxicity and ototoxicity - Resistance: There is cross resistance among CAP/VIO/KAN

o A>G mutations in 16sRNA (rrs) Nitroaromatics – Nitroimidazoles

- New prodrugs that are in the pipeline. They are bioactivated by MTB and likely operate via liberation of NO molecules

New drugs for the MDR/XDR-TB have an impact in: - 1: Reducing Treatment Duration, which improves compliance, drug resistance, and the labor associated with

DOTS - 2: Reduce the transmission of MDR-TB - 3: Cure latent TB infection – helping to eliminate the disease reservoir, and hopefully the planet

Moxifloxacin (Avelox)