clindamycin and tetracycline antibiotics

Question 1

synthesis of clindamycin

Answer 1

Clindamycin is synthesized from the naturally occurring antibiotic lincomycin by treatment with chlorine and triphenylphosphine in acetonitrile. The reaction proceeds with inversion of configuration. Lincomycin has antibiotic activity but it is no longer used because of its toxicity.

Question 2

clindamycin MOA

Answer 2

The mechanism of action of clindamycin is similar to that of the macrolide antibiotics like erythromycin. It inhibits protein synthesis by binding to the bacterial 50S ribosome. It binds to the same site as erythromycin. Antagonism and cross-resistance between clindamycin and erythromycin have been reported.

Question 3

clincal use of clindamycin

Answer 3

Clindamycin is most effective against:
1) Aerobic Gram-(+) cocci, including some members of the Staphylococcus and Streptococcus genera.
2) Anaerobic Gram-(–) bacilli, including some members of the Bacteroides and Fusobacterium genera.
Clindamycin may be used systemically to treat bone infections with Staphylococcus aureus, or topically to treat severe acne. It is available as a vaginal cream for treatment of bacterial vaginosis.
Clindamycin has replaced penicillin for treatment of lung abscesses and anaerobic lung and pleural space infections. It is also used to treat MRSA.
Clindamycin is administered IV with pyrimethamine and leucovorin to treat AIDS patients with encephalitis caused by Toxoplasma gondii.
Note: The relatively high incidence of pseudomembranous colitis and diarrhea limit the use of clindamycin to infections in which it is clearly the superior agent.

Question 4

clindamycin dosage forms

Answer 4

Clindamycin preparations for oral administration include capsules and oral suspensions. It is also available for intravenous injection as clindamycin phosphate. Topical foams or solutions contain either clindamycin hydrochloride or clindamycin phosphate.

Question 5

clindamycin metabolism

Answer 5

Clindamycin is extensively metabolized by cytochrome P450 enzymes in the liver to the sulfoxide and the N-demethylated derivative. The metabolites are pharmacologically inactive**.

Question 6

clindamycin PKs

Answer 6

Approximately 90% of the administered dose is absorbed from the GI tract. It is widely distributed and penetrates the CNS in high enough concentrations** to be useful in the treatment of cerebral toxoplasmosis in human immunodeficiency virus-infected patients.
Clindamycin and its metabolites are mainly excreted in the urine and bile. The elimination half life is 1.5-5 hours. Accumulation of clindamycin can occur in patients with hepatic failure, and adjustment of the dosage may be required**.

Question 7

clindamycin AEs

Answer 7

Common adverse reactions include diarrhea, pseudomembranous colitis, nausea, vomiting, abdominal cramps, and rash. Topical application may cause contact dermatitis.
Pseudomembranous colitis is a potentially lethal condition** commonly associated with clindamycin. It may affect up to 2–10% of patients treated with clindamycin. Overgrowth of Clostridium difficile, which is inherently resistant to clindamycin, results in the production of a toxin that causes a range of adverse effects ranging from diarrhea to colitis and toxic megacolon. This should be recognized and treated promptly with metronidazole or vancomycin*.

Question 8

chemical properties of tetracyclines

Answer 8

chelation, epimerization, dehydration and cleavage in base

Question 9

chelation of tetracyclines

Answer 9

Chelation. Tetracyclines form stable chelates with polyvalent metal ions such as Ca++, Al+++, Cu++, and Mg++.
The ability of tetracyclines to form insoluble chelates* has a number of consequences:
1) Tetracyclines should not* be administered with foods that are rich in calcium because the insoluble calcium chelates are not absorbed from the GI tract. They should not* be administered with antacids that contain multivalent metals (e.g. TUMS), or with hematinics containing iron.
When concomitant therapy with tetracyclines and multivalent metals cannot be avoided, the metals should be administered 1 hour before* or 2 hours after** the tetracycline.
In spite of the chelation problem, the preferred route of administration is oral, since oral absorption is adequate in the absence of multivalent metal ions in the GI tract.
2) Tetracyclines chelate calcium during formation of teeth, resulting in permanently brown or gray teeth. Consequently, tetracyclines should not be administered to children when they are forming their permanent teeth. The discoloration of the teeth becomes worse with time as a result of a photooxidation reaction
3) The pain on injection* caused by tetracyclines has been attributed to the formation of insoluble calcium complexes. To minimize this, injectable formulations contain EDTA to chelate the calcium, and they are buffered to acidic pH where chelation is suppressed.

Question 10

Epimerization of tetracyclines

Answer 10

The hydrogen on the amine-bearing carbon atom is acidic, resulting in enolization and epimerization. The epitetracycline product is pharmacologically inactive. This can occur in the solid state (in capsules) as well as in solution. Old tetracycline preparations can loose approximately half of their potencies in this way. Epimerization is slow in the solid state and most rapid in solution at pH 4.

Question 11

dehydration of tetracyclines

Answer 11

The tertiary, benzylic hydroxyl group at C-6 has an antiperiplanar relationship with the proton at C-5a, so it is “set up” for elimination. Note that the C-12a hydroxyl group is tertiary, but it is not next to an antiperiplanar hydrogen and is also deactivated by being next to a carbonyl group, so it is stable.
Discolored, old tetracycline samples should be thrown out. Not only is 4-epianhydrotetracycline inactive as an antibiotic, it is also toxic to the kidneys and can produce a Fanconi-like syndrome (failure of the reabsorption mechanism in the proximal convoluted tubules) that can be fatal. Therefore, commercial samples of tetracycline are closely monitored for 4- epianhydrotetracycline. Some tetracyclines, such as minocycline and doxycycline, lack a C-6 hydroxyl group and are therefore completely free of this potential for toxicity.

Question 12

cleavage in base of tetracyclines

Answer 12

Tetracyclines undergo cleavage in base at pH values of 8.5 or above. The lactone product in inactive.

Question 13

tetracyclines MOA

Answer 13

Tetracyclines bind to the 30S ribosomal subunit and inhibit bacterial protein synthesis by blocking the attachment of the aminoacyl-tRNA to the A site of the ribosome, resulting in termination of peptide chain growth. More precisely, they are inhibitors of the codon-anticodon interaction. The tetracycline binding sites do not overlap with the erythromycin binding site. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required for toxicity because eukaryotic cells, in contrast to bacteria, do not have a tetracycline uptake mechanism.
Tetracycline binds to the small ribosomal subunit in six different locations. The Tet1 site has highest occupancy and is located near the site where the aminoacyl-t-RNA docks in the A site of the ribosome. The functional significance of each site is not known.

Question 14

tetracyclines therapeutic use

Answer 14

The tetracyclines are broad-spectrum antibiotics. Their most common use is for treatment of acne. They remain the treatment of choice for infections caused by chlamydia (trachoma, psittacosis, salpingitis, urethritis, and lymphogranuloma venereum), Rickettsia (typhus, Rocky mountain spotted fever), brucellosis, and spirochetal infections (borreliosis, syphilis, and Lyme disease). They are also used to treat anthrax, plague, tularemia, and Legionnaires’ disease.

Question 15

tetracycline

Answer 15

1) Tetracycline is the classical antibiotic of the tetracycline class. It is produced by fermentation of Streptomyces aureofaciens. It is generic and relatively inexpensive.
2) Food and milk lower oral absorption by about 50%.

Question 16

demeclocycline

Answer 16

1) Demeclocycline is produced by a genetically altered strain of Streptomyces aureofaciens.
2) It has a secondary hydroxyl group at C-6 instead of the tertiary hydroxyl group. Demeclocycline therefore dehydrates more slowly than tetracycline because the secondary cation intermediate formed from demeclocycline in the dehydration reaction is less stable (higher energy) than the tertiary cation intermediate (lower energy) formed from tetracycline.
3) Food and milk lower oral absorption by about 50%

Question 17

minocycline

Answer 17

1) Minocycline lacks a C-6 hydroxyl group and therefore does not undergo acid-catalyzed dehydration. It therefore has no potential for 4-epianhydrotetracycline-mediated toxicity.
2) It is synthesized from demeclocycline.
3) Minocycline has 90-100% oral bioavailability.
4) The absorption of minocycline is lowered by about 20% when taken with food or milk.
5) Minocycline has vestibular toxicities** (vertigo, ataxia, nausea) not shared with other tetracyclines.

Question 18

oxytetracycline

Answer 18

1) Oxytetracycline is produced by fermentation of Streptomyces rimosis.
2) It is the most hydrophilic tetracycline and has been largely replaced by its semisynthetic descendants.
3) It is available with polymyxin B as an opthalmic ointment.

Question 19

doxycycline

Answer 19

1) Doxycycline is synthesized from oxytetracycline.
2) It lacks a C-6 hydroxyl group and therefore does not undergo acid-catalyzed dehydration. It therefore has no potential for 4-epianhydrotetracycline-mediated toxicity.
3) Because it has no potential for 4-epianhydrotetracycline-mediated toxicity and produces fewer GI symptoms, it is considered to be the tetracycline of choice by many physicians.
4) It has 90-100% oral bioavailability.
5) The absorption of doxycycline is lowered by about 20% when taken with food or milk.
6) It has a half-life (18-22 hours) that permits once a day dosing.

Question 20

Tigecycline

Answer 20

Tigecycline. A glycylcycline antibiotic derivative of minocycline. Active against a variety of gram-positive and gram-negative bacteria. Parenteral, administered by slow IV infusion.

1) Tigecycline lacks a C-6 hydroxyl group and therefore does not undergo acid-catalyzed dehydration. It therefore has no potential for 4-epianhydrotetracycline-mediated toxicity.
2) It is synthesized from minocycline.
3) It is administered by slow IV infusion. No oral form is available.
4) Hepatotoxicity (rare), pancreatitis, and anaphylactoid reactions may occur.
5) Inhibits protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A site of the ribosome. This prevents incorporation of amino acid residues into elongating peptide chains.
6) Tigecycline is protected from resistance development due to efflux pump induction and also due to ribosomal protection proteins.

Question 21

Chloramphenicol MOA

Answer 21

Chloramphenicol binds reversibly to the 50S ribosomal subunit at a site that is near the site for erythromycin and clindamycin (competitive binding interactions occur among these drugs). It inhibits the peptidyl transferase activity of the ribosome and thus blocks peptide bond formation between the P site and the A site.

Question 22

Chloramphenicol therapeutic use

Answer 22

Chloramphenicol sodium succinate is a prodrug for IV or IM administration that is hydrolyzed to chloramphenicol in the liver. Chloromycetin succinate is specifically indicated for bacterial meningitis, typhoid fever, rickettsial infections, intraocular infections and other serious infections where bacteriological evidence or clinical judgment indicates that chloramphenicol is an appropriate antibiotic.
Chloramphenicol is lipid soluble, and it remains relatively unbound to plasma proteins. It penetrates effectively into all tissues of the body, including the brain.

Question 23

chloramphenicol MOR

Answer 23

Resistance to chloramphenicol results from 1) reduced membrane permeability; 2) mutation of the 50S ribosomal subunit, and 3) elaboration of chloramphenicol acetyltransferase, which acetylates one or both of the hydroxy groups to form metabolites that do not bind to the 50S ribosomal subunit.

Question 24

Cloramphenicol metabolism

Answer 24

Chloramphenicol is metabolized to its glucuronide in the liver. The glucuronide is pharmacologically inactive and is readily excreted by the kidneys. The reaction involves nucleophilic attack of the less hindered primary alcohol on UDPGA, catalyzed by glucuronyl transferase.
The dose of chloramphenicol must therefore be reduced if hepatic function is impaired. There is no standard dose reduction for chloramphenicol in liver impairment, and the dose should be adjusted according to measured plasma concentrations. Neonates cannot metabolize chloramphenicol and should never receive it.
Chloramphenicol is also metabolized by reduction of the nitro group to an amino. The metabolite is less active than chloramphenicol.

Question 25

Chloramphenicol toxicity

Answer 25

The most serious toxicity of chloramphenicol is aplastic anemia. The effect is rare and generally fatal. The effect usually becomes apparent weeks or months after chloramphenicol treatment has been stopped. The highest risk is with oral chloramphenicol (affecting 1 in 24,000–40,000) and the lowest risk occurs with eye drops (affecting less than 1 in 224,716 prescriptions). It is recommended that blood levels be monitored to keep chloramphenicol concentrations less than 25 mcg/mL.
Bone marrow suppression is common, and is due to impairment of mitochondrial function resulting from inhibition of protein synthesis. This effect is completely reversible once the drug is stopped, and does not predict future development of aplastic anemia. The effect occurs quite predictably once a cumulative dose of 20 g has been given.
There is an increased risk of childhood leukemia and the risk increases with length of treatment.
Nausea, vomiting and diarrhea occur occasionally in adults and rarely in children.

Question 26

Chloramphenicol drug interactions

Answer 26

Chloramphenicol inhibits cytochrome P450 so drug interactions can be expected with drugs that are metabolized by cytochrome P450.

Question 27

Chloramphenicol distribution

Answer 27

The concentration achieved in brain and CSF is about 30 to 50% that of the plasma even when the meninges are not inflamed; this increases to as high as 89% when the meninges are inflamed.

Question 28

1st gen quinolone chemistry

Answer 28

The first generation quinolones were developed because of their activity against Gram-(–) bacteria. They have limited activity vs. Gram-(+) bacteria. They do not achieve useful systemic concentrations and are only useful for treatment of lower urinary tract infections. Both oxolinic acid and nalidixic acid have been discontinued.

Question 29

second generation quinolones chemistry

Answer 29

The second generation agents have a fluorine substituent at C-6 and a heterocyclic ring (usually piperazine) at C-7. They have a broader spectrum of bactericidal activity and are more potent. Ciprofloxacin is the most potent fluoroquinolone showing MIC = 0.01 to 1 µg/mL for Gram-(–) organisms. These drugs have extended activity against Gram-(+) organisms and Mycoplasma.

Question 30

third and fourth generation quinolone chemistry

Answer 30

The third and fourth generation quinolones have improved activity against Gram-(+) organisms, particularly Streptococcus pneumoniae, and none of them are as potent as ciprofloxacin against Gram-(–) organisms. Levofloxacin shows a 40-100 fold increase in potency over nalidixic acid against most Gram-(–) species including Pseudomonas sp, with ranges of values for MIC = 0.06 to 4 µg/mL. Moxifloxacin is considered to be a “drug of last resort” because of its severe side effects

Question 31

quinolone MOA

Answer 31

It is necessary to review the action of DNA topoisomerases and gyrases in order to understand how the quinolone antibiotics work. During DNA replication, strand separation of the double helix causes the DNA to twist, resulting in tangling and kinking of the DNA in front of the replication fork. Gyrases and topoisomerases have evolved that untangle the DNA by cutting one (topoisomerase I) or two (topoisomerase II) strands, and then allowing strand passage through the break or allowing the DNA to twist.
The topoisomerases and gyrases cleave DNA by carrying out a nucleophilic attack on a phosphodiester linkage, so one part of the strand becomes “free” and the other one becomes enzyme-linked. The nucleophile is the phenolic hydroxyl group of a tyrosine residue, and different topoisomerases cleave the DNA so that either the 3’-hydroxyl or the 5”-hydroxyl of the DNA can become enzyme-linked. The topoisomerase-catalyzed reaction is reversible, and usually the equilibrium is in favor of the uncleaved DNA so that the cleavage complexes are
present in very small amounts.
The proposed mechanism for DNA transport by bacterial type IV topoisomerases and gyrases is shown below. Color scheme: ATPase domains, orange, DNA capture domains, purple; B’ domains corresponding to the C-terminal half of GyrB, blue; CAP regions, green; and the remainder of the A’ domains, red. The N-gate is open in panels a-c and closed in panels d-f. The C-gate is closed in panels a-e and open in panel f. The G-segment and T-segment DNAs are shown as black and gray rods, respectively.
The first step of the reaction is the binding of a G-segment DNA to the high-affinity binding site II located across the tops of the two CAP regions of the dimer (a to b). This requires an open N gate and also an opening between the two B’ regions of the protein. Once the DNA is bound, the CAP regions remain in a closed conformation. Two ATP molecules bind to the ATPase domains, leading to N-gate closure with a T-segment trapped in the DNA capture domain, and then a gate opens in the G-segment DNA (c and d). The T-segment DNA is passed through the gate. The Gsegment
DNA religates and the C-gate opens.
It is likely that a conformational change that is coupled to ATP hydrolysis occurs after DNA cleavage and helps “squeeze” the T-segment through the open gate. The cycle is completed by the rapid closure of the C-gate followed presumably by hydrolysis of the second ATP. The release of the products of ATP hydrolysis opens the N-gate and prepares the enzyme for another cycle. This mechanism operates with bacterial gyrase, bacterial DNA topoisomerase IV, and mammalian topoisomerase II. These enzymes share to following mechanistic features:
1) The dimeric enzyme binds duplex DNA and cleaves both opposing strands with a four-base stagger.
2) Cleavage involves covalent attachment of each subunit of the dimer through a phosphotyrosine linkage to the 5’ end of the DNA (both 3’-hydroxyls are leaving groups).
3) The two DNA ends at the cleavage site are pulled apart by a conformational change of the enzyme to create an opening in the gated (G-segment) DNA. The transported DNA duplex (T-segment) is then passed through the opening.
4) The transported DNA can be from the same molecule (relaxation, knotting, or unknotting) or from a different molecule (catenation and decatenation).
5) All of the type II enzymes can be distinguished by their relative abilities to relax DNA vs. decatenate (or catenate) DNA, and this property probably reflects their roles in the cell.
6) The catalysis requires Mg++ and ATP hydrolysis is involved.
The unwinding of DNA by the strand passage mechanism is shown below. This scheme portrays the DNA unwinding from the perspective of the gross structure of DNA, The steps involved in the unwinding are:
1) Binding of dsDNA that covers up to 140 bases and wraps around the two A-subunits in the dimeric protein.
2) Cleavage of a phosphodiester bond on each strand of DNA by the nucleophilic attack of a tyrosine (protein)-OH group to form two covalent bonds between protein and DNA.
3) The dsDNA is “passed through” the cleavage site and this is dependent upon ATP hydrolysis in the B-subunits to induce a conformational change.
4) The phosphodiester backbone is rejoined (ligated) by nucleophilic displacement of the protein tyrosine residue by the 3’-OH of the cleaved strand.
5) To repeat the cycle, the ATP has to be hydrolyzed.
The quinolone antibiotics bind to the cleavage complex that exists after step 2. The drug molecules are stacked between the base pairs at the cleavage site so that the cleavage complex is stabilized and the religation reaction is inhibited (see below). This blocks the progression of the replication fork and the double-strand breaks eventually lead to apoptosis (bacterial cell death). The type II topoisomerase subunits have been crystallized and their X-ray structures determined
DNA religation is blocked by the quinolones:
S. aureus gyrase has been complexed with DNA and two molecules of ciprofloxacin and the structure has been determined by crystallography.
The type II topoisomerase subunits have been crystallized and their X-ray structures determined. In the following protein structures, the subunits are color-coded to correspond to the “cartoon” of the strand-passage mechanism shown above.
Crystal structure above: the N-terminal half of the E. coli DNA gyrase B-subunit is shown with ATPase domains (monomers colored yellow and gold) oriented above the DNA capture domain (monomers colored magenta and blue). The bound ATP is shown in red ball and stick.

Question 32

quinolone therapeutic uses

Answer 32

1) Urinary tract infections. Ciprofloxacin is effective.
2) Prostatitis. Ciprofloxacin and ofloxacin are effective.
3) Sexually transmitted diseases. The quinolones are effective against Neisseria gonorrhoeae (ciprofloxacin), Chlamydia trachomatis (levofloxacin), and Haemophilus ducreyi (ciprofloxacin). Increasing resistance to the quinolones has led to ceftriaxone (a cephalosporin antibiotic) being the first line treatment of Neisseria gonorrhoeae.
4) Gastrointestinal infections. The quinolones are effective for treatment of traveler’s diarrhea (frequently caused by enterotoxigenic E. coli). Ciprofloxacin is effective in treatment of shigellosis.
5) Respiratory tract infections. Many of the newer fluoroquinolones, including moxifloxacin, have excellent activity vs. Streptococcus pneumoniae. Respiratory tract exacerbations in cystic fibrosis patients have responded to fluoroquinolone therapy.
6) Bone, joint, and soft tissue infections. Fluoroquinolones have been employed. Ciprofloxacin as a sole therapy is effective in 50% of diabetic foot infections.
7) The increase in potency observed with ciprofloxacin has opened new opportunities for their therapeutic applications. These drugs have useful activities against intracellular bacteria (requiring high blood levels for treatments) like Chlamydia, Mycoplasma, Legionella, Brucella, and Mycobacterium.

Question 33

quinolone MOR

Answer 33

Increased fluoroquinolone resistance rates are correlated with use.
Fluoroquinolone resistance mechanisms include decreased cellular permeability, efflux pumps, and mutation of the target enzymes.
1) The incidence of resistance is low* relative to other antibacterial agents.
2) Resistant organisms have spontaneously occurring point mutations in the A-subunit of DNA gyrase* which lead to an enzyme with altered binding affinity for the drug that translates into a 16-fold higher MBC for fluoroquinolone drugs.
3) A second type of mutation that occurs less frequently in the B-subunit of the DNA gyrase* results in a lower level of resistance. However, additive effects of mutations in both the A and B-subunits are known to give rise to more highly resistant strains.
4) Fluoroquinolone penetration of Gram negative bacteria is dependent upon diffusion through porin channels. Resistance mutations that increase the expression of genes encoding efflux pumps* in occur in Escherichia coli and Pseudomonas aeuroginosa.
5) Cross resistance* to all the quinolones is encountered with the point mutations. However, these mechanisms do not to appear to result in cross resistance to other classes of antibiotics.
6) Under-dosing should be avoided* to minimize opportunities for selection of resistant organisms.
As with other antibiotics, the emergence of resistant strains is facilitated by overuse and inappropriate prescribing, improper use (the patient stops taking the drug too early), and use in livestock (80% of the antibiotics produced in the US are used in agriculture).

Question 34

quinolone PKs

Answer 34

1) The fluoroquinolones are all readily absorbed orally* and have a high degree of bioavailability.
2) All the fluoroquinolones are widely distributed.
3) Renal and hepatic clearance are important.
4) Interstitial fluid concentrations range from 50-100% of serum concentrations after 2 hours and exceed serum concentrations** from 4 to 24 hours. CSF levels of drug range from 40-90% of serum levels.
5) Quinolones form insoluble chelates* with heavy metals and should therefore not be administered with foods and drugs that contain heavy metals.

Question 35

quinolone metabolism

Answer 35

The major inactive metabolite is the glucuronide at the 3 carboxyl position and this is excreted in the urine

Question 36

quinolone AEs

Answer 36

In general, the fluoroquinolones are very well tolerated. The most common side effect include nausea, vomiting and diarrhea* (3-17% of patients). CNS adverse reactions include h8eadache and dizziness (1-11%). Rarely, hallucinations, delirium and seizures* occur, mainly in patients taking theophylline and nonsteroidal antiinflammatory drugs. Skin rash and abnormal liver function tests have been reported. Quinolones are associated with peripheral neuropathy.
Fluoroquinolones may damage growing cartilage** and cause arthropathy (reversible), and are therefore not normally recommended for treatment of patients under 18 years of age. There is agreement that fluoroquinolones can be used to treat pseudomonal infections in children with cystic fibrosis, where the benefits outweigh the risks.
Tendonitis in adults is a rare complication that is serious because of the risk of tendon rupture.*
Gatifloxacin** has been associated with hyperglycemia and hypoglycemia in diabetic patients**