Drug Mech: Antibiotics 5

• Rifamycins
• Quinolones

• 1) Nalidixic Acid
• 2) Cinoxacin

The Fluoroquinolones, which include:

• 1) Ciprofloxacin
• 2) Enoxacin
• 3) Lomefloxacin
• 4) Norfloxacin
• 5) …etc

Quinolones are broad spectrum antibiotics.

Quinolones are bactericidal at the clinical dose.

Compared to the First Generation quinolones, the Fluoroquinolones exhibit enhanced potency, extended spectrum of activity, and improved distribution properties (they are widely distributed in body tissues).

• Gram Negative Bacteria
• DNA Gyrase is the primary quinolone target in Gram-negative bacteria. Quinolones inhibit DNA synthesis by inhibiting bacterial DNA Gyrase, an enzyme responsible for introducing negative supercoils into circular duplex DNA.

Negative supercoils separate the two strands of DNA and allow replication and transcription to occur.

Gram Postive Bacteria

Bacterial Topoisomerase IV, which separates interlinked daughter DNA molecules that are the product of DNA replication, is the primary quinolone target for inhibition in Gram-positive bacteria.

Mammalian cells contain a type II DNA Topoisomerase enzyme similar to the bacterial DNA Gyrase. Quinolone serum concentrations 100- to 1000-fold higher than the bactericidal plasma concentration are required to inhibit the mammalian topoisomerase. Therefore, Quinolones are selective at the clinical dose.

Target Modification Expression of an altered DNA Gyrase or Topoisomerase IV with low binding affinity to the drug as a result of mutation in the genes responsible for encoding these two enzymes.

Active efflux (energy-dependent) of the drug out of bacterial cells.

Quinolones bind to polyvalent cations. As a result, antacids, iron supplements, multivitamins/minerals supplements, calcium supplements, and dairy products can decrease the oral bioavailability of quinolones by as much as 90%.

In addition, it has been shown that drugs which contain divalent or trivalent cations, such as Sucralfate (contains aluminum ions) and Didanosine tablets (contain buffers with aluminum and magnesium ions), reduce oral bioavailability of quinolones during concomitant administration.

Quinolones are inhibitors of cytochrome P450 enzymes in the liver and have been reported to increase blood levels of Theophylline, Digoxin, Caffeine, Warfarin, and Cyclosporine.

Quinolones can cause cartilage malformations in immature laboratory animals at doses several-fold greater than the clinical dose. As a result, quinolones have the potential to cause Arthropathy, a chronic degenerative disease of the joints, and are not recommended for use in pregnant women and children under the age of 16.

Tendinitis or tendon rupture may occur rarely with systemic use of any fluoroquinolone, either while the drug is being taken or for up to several months afterwards. The risk is higher for patients > 60 years old and for those taking corticosteroids.

• Phototoxicity is more common with Sparfloxacin
• and Lomefloxacin than with other fluoroquinolones. Patients should avoid sunlight and artificial UV-light.

• Trovafloxacin, excreted primarily in the bile,
• causes serious liver injury. It should only be used to treat life-threatening infections (its use has been restricted by the FDA). [Note: these restrictions are only with this specific Quinolone.]

• In Summary, the five general properties of Quinolones, a very important class are:
• 1) They chelate polycations (just like tetracyclines), so avoid Calcium, dairy, antacids, etc.
• 2) phototoxic (just like tetracyclines), so avoid sun!
• 3) They inhibit cartilage, so they are for an "adult audience only."
• 4) They can cause tendonitis (rupture of tendons).
• 5) They are inhibitors of cytochrome P450 enzymes.

• Polypeptides
• Cyclic Lipopeptides

• 1) Polymixins
• 2) Gramicidin

They are bactericidal at the clinical dose.

Large molecules (mostly cyclic).

They frequently contain D-amino acids and/or ‘unnatural’ amino acids not found in higher plants or animals.

They can be acidic, basic, zwitterionic, or neutral depending on the number of free carboxylic acid and amino or guanidino groups in their structures (polymyxins are basic; gramicidins are neutral).

They are not absorbed from the GI Tract. Therefore, must be administered parenterally...yet...

Nephrotoxic and neurotoxic when used systemically.

• 1) Polymyxin B
• 2) Polymyxin E (Colistin)

• Active mainly against Gram-negative bacilli, including Pseudomonas aeruginosa, E. coli, H.
• influenzae, and Klebsiella pneumoniae.

They are not active against Gram-positive bacteria, Gram-negative cocci (N. gonorrhea, N. meningitides), and fungi.

They are not absorbed from the GI Tract. They do not penetrate into the CSF, synovial fluid, aqueous humor of the eye, or across the placenta.

• Because they have a very high affinity for
• cell membranes (both bacterial and mammalian), there is little systemic absorption of the polymyxins following topical administration (even when applied to open wounds).

They are administered parenterally (IM, IV, or Intrathecal) to treat severe systemic infections caused by susceptible Gram-negative bacteria.

They are nephrotoxic and neurotoxic when administered parenterally. They should not be used with other nephrotoxic or neurotoxic agents.

They are rarely if ever used systemically due to toxicity and the availability of less toxic antibiotics.

The bottom line is Polymixins are used as a LAST RESORT when literally all else has failed. This is because of their extreme toxicity to humans!

Polymyxins are relatively simple, highly basic peptides with molecular weights of about 1000 daltons.

• They are amphipathic, surface-active
• agents (containing both lipophilic and hydrophilic groups within the molecule) that act as ‘cationic detergents’.

In other words, they have a hydrophobic head (which serves as the FUEL of the molecule because it has Carbons and Hydrogen molecules) and the hydrophilic tail, which is very polar and water soluble because of the many amino groups on it. Therefore, this hydrophobic tail allows the molecule to be positively charged in a physiologic pH, and therefore the molecule can act like soap, a cationic detergent.

They are capable of penetrating the outer membrane (cell wall) of Gram-negative bacteria through porin channels to act on the inner cell membrane (The much thicker cell wall of Gram-positive bacteria is an effective barrier to penetration by the polymyxins). So Polymixins are active against Gram Negative and NOT Gram Positive).

They bind ionically to phospholipids (recall that on the phospholipid bilayer you have negatively charged phosphate groups on the outside that will attract the positively charged Polymixin molecule) and penetrate into the structure of cell membranes. As a result, they disrupt cell membrane permeability.

They also bind to phospholipids of the outer membrane of Gram-negative bacteria, which contributes to the overall sensitivity of these organisms to the polymyxins.

• The cell wall of certain resistant Gram-negative species (Proteus and Serratia) may prevent access of the polymyxins to the cell membrane.
• Cell membrane permeability changes immediately on contact with the polymyxins, resulting in the loss of intracellular components that are essential to the survival of the cell.

In vivo activity of the polymyxins is decreased by the presence of divalent cations such as calcium, which is believed to interfere with the binding of the drug to the cell membrane.

Daptomycin is a bactericidal lipopeptidolactone antibiotic.

It is indicated for the treatment of serious Gram-positive bacterial infections.

It is administered as an IV infusion.

Its mechanism of action is distinct from any other antibiotic; because of its unique mode of action, daptomycin retains potency against Gram-positive bacteria that are resistant to other antibiotics including methicillin, vancomycin, and linezolid.

Daptomycin inhibits the biosynthesis of the lipoteichoic acid component of the cell wall of Gram-positive bacteria.

Daptomycin, in complex with calcium ions, disrupts cell membrane permeability through a membrane-seeking, surface-active behavior (similar to the mode of action of the polymyxins).

In addition, daptomycin binds to bacterial cell membranes (like the polymixins) and causes a rapid depolarization of membrane potential (unlike the polymixins); this leads to inhibition of bacterial protein, DNA, and RNA synthesis.

Major toxicities of daptomycin include superinfection (e.g., pseudomembranous colitis, Candida infections) and peripheral or cranial neuropathy.

• 1) Sulfonamides
• 2) Trimethoprim

• 1) Sulfanilimide
• 2) Sulfadiazine
• 3) Sulfamethoxazole
• 4) Sulfisoxazole

They are bacteriostatic at the clinical dose.

• They inhibit:
• 1) dihydrofolic acid
• 2) tetrahydrofolic acid

Tetrahydrofolic acid is what both human cells and bacterial cells use to make purines and DNA.

When you inhibit Tetrahydrofolic acid, you will inhibit the biosynthesis of DNA.

• Sulfonamides are readily absorbed from the GI Tract and well distributed throughout the
• body (including the CSF). They pass through the placenta and reach the fetal circulation.

The usefulness of the sulfonamides in treating systemic infections, however, has diminished considerably due to resistance and toxicity.

To overcome resistance, sulfonamides are rarely used as single agents to treat infections.

The sulfonamides may cause crystalluria, which is crystallization of a sulfonamide in the urine.

In other words, the drug molecule will come out of solution in the urine to become a solid particle. This causes problems because the solid causes friction (by rubbing against the tissues) and can cause kidney damage.

Sulfonamides are acidic molecules, so they have positive protons in their chemistry. These protons can be lost at certain pH levels (such as in the pH of urine, which is between 5 or 6). This would then cause crystalluria.

Recall the pK_a is a measure of how strong or weak an acid is, and therefore, how likely a molecule is to give up a proton. Because of their chemistry, sulfonamides are likely to give up a proton, and therefore crystallize into a solid in the pH of urine.

• 1) Use Sulfanilamides with lower pKa values (a pK_a value closer to the pH of urine). All sulfonamides that are used clinically today have pK_a
• values between 5 and 8.

2) Increase the pH of urine by administering oral sodium bicarbonate.

• 3) Increase urine flow by drinking more fluids.
• Water is the best think to drink. Avoid acidic drinks such as soda or fruit juices.

4) Avoid any decrease in the pH of urine. As you decrease the pH of urine, you bring the pK_a value further from the the pK_a of solubility.

They are bacteriostatic.

Sulfonamides are structural analogs and competitive antagonists of p-aminobenzoic acid (PABA), thus preventing normal bacterial utilization of PABA for the synthesis of folic acid.



They are competitive inhibitors of Dihydropteroate Synthase, the bacterial enzyme responsible for the incorporation of PABA into dihydropteroic acid, the immediate precursor of folic acid.



Sulfonamides do not affect mammalian cells by this mechanism. Mammalian cells utilize preformed folic acid from the diet (they do not synthesize folic acid from PABA).

Resistance against the sulfonamides originates by random mutation and selection or by transfer of R-Factors.

Known resistance mechanisms include:

Target Modification via expression of an altered dihydropteroate synthase that has lower affinity for the sulfonamides.

Decreased bacterial permeability or active efflux of the drug.

Developing an alternative metabolic pathway for the synthesis of THF (e.g., the use of preformed folic acid from the host to make THF).

Increased production of PABA (e.g., some resistant Staph. strains may synthesize as much as 70 times the amount of PABA produced by the sensitive parent strains). Notice in the picture below the very similar chemistry between a Sulfanilamide and PABA. Thus, there is COMPETITION and inhibition is CONCENTRATION DEPENDENT. Therefore, some bacterial cells resist by increasing PABA concentration.

Cross-resistance among the sulfonamides is 100%.

Hypersensitivity reactions

Hemolytic anemia (in individuals with a genetic deficiency in glucose-6-phosphate dehydrogenase enzyme)

Aplastic anemia (bone marrow does not produce sufficient amount of red blood cells)

Agranulocytosis diminshed numbers of neutrophils

Crystalluria crystallization of drug molecule that damages kidneys by friction

The Sulfamethoxazole-Trimethoprimcombination is bactericidal.

For the greatest number of bacterial species, the most synergistic concentration ratio of the two drugs in the combination is 20:1 (sulfamethoxazole:trimethoprim).

• Selective toxicity against bacterial cells is achieved
• as follows:

• 1) Mammalian cells do not synthesize folic acid from PABA. They utilize preformed folic
• acid from the diet.

• 2) Trimethoprim is a highly selective inhibitor
• of bacterial DHFR. Its affinity to the bacterial DHFR is 100,000-fold higher than its affinity to the mammalian DHFR.

Trimethoprim is a potent and selective competitive inhibitor of microbial Dihydrofolate Reductase (DHFR), the enzyme that reduces dihydrofolic acid to tetrahydrofolic acid (THF).



Combining Trimethoprim and a Sulfonamide (sulfamethoxazole) introduces sequential blocks in the pathway by which microorganisms synthesize THF from precursor molecules.

Since the two drugs act on sequential steps in an obligate enzymatic pathway in bacteria, their combination is Synergistic. In vitro and in vivo studies have confirmed the synergistic nature of the combination.

Bacterial resistance to trimethoprim sulfamethoxazole combination is a rapidly increasing problem, although resistance is lower than it is to either of the agents alone.

Target Modification Resistance is often due to the acquisition of a plasmid that encodes for an altered DHFR.

Trimethoprim (in Bactrim®) can cause leukopenia, megaloblastosis, and thrombocytopenia in patients with folate deficiency due to inhibition of human DHFR.

Yes! Anti-TB agents must be used in combination therapy.

Typically a patient would receive 3 or 4 drugs for long term treatment (at least 6 months...sometimes greater than a year)

If microbial resistance is detected during treatment, the patient is switched to the second-line of therapy.

Isoniazid

Rifampin

Rifabutin

Ethambutol

Pyrazinamide

Streptomycin

p-Aminosalicylic Acid

Ethionamide

Cycloserine

Capreomycin

Kanamycin

Amikacin

Moxifloxacin

Linezolid

p-Aminosalicylic Acid (bacteriostatic): inhibits folic acid synthesis

Isoniazid (bactericidal): inhibits cell wall synthesis

Ethambutol (bacteriostatic): inhibits cell wall synthesis

Pyrazinamide (bactericidal): unknown

Ethionamide (bacteriostatic): inhibits bacterial protein synthesis

Capreomycin (bacteriostatic): unknown