Pharmaceutical Microbiology (Note 3)

MCB 525

500 Level

Chloramphenicol

Chloramphenicol (INN) is a bacteriostatic antimicrobial that became available in 1949. It is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines, and as it is both cheap and easy to manufacture it is frequently an antibiotic of choice in the Developing World.

Chloramphenicol, also known as chlornitromycin, is effective against a wide variety of Gram-positive and Gram-negative bacteria, including most anaerobic organisms. Due to resistance and safety concerns, it is no longer a first-line agent for any infection in developed nations, with the notable exception of topical treatment of bacterial conjunctivitis. Nevertheless, the global problem of advancing bacterial resistance to newer drugs has led to renewed interest in its use. In low-income countries, chloramphenicol is still widely used because it is inexpensive and readily available.

The most serious adverse effect associated with chloramphenicol treatment is bone marrow toxicity, which may occur in two distinct forms: bone marrow suppression, which is a direct toxic effect of the drug and is usually reversible, and aplastic anemia, which is idiosyncratic (rare, unpredictable, and unrelated to dose) and generally fatal.

                                                             Pure chloramphenicol

History

Chloramphenicol was originally derived from the bacterium Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice in 1949, under the trade name Chloromycetin. It was the first antibiotic to be manufactured synthetically on a large scale.

Spectrum of activity

Chloramphenicol has a broad spectrum of activity and has been effective in treating ocular infections caused by a number of bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli. Chloramphenicol is not effective against Pseudomonas aeruginosa. The following represents MIC susceptibility data for a few medically significant organisms.

• Escherichia coli: 0.015 μg/mL - 10,000 μg/mL
• Staphylococcus aureus: 0.06 μg/mL - >128 μg/mL
• Streptococcus pneumoniae: 2 μg/mL - 16 μg/mL

Therapeutic uses

The original indication of chloramphenicol was in the treatment of typhoid, but the now almost universal presence of multiple drug-resistant Salmonella typhi has meant it is seldom used for this indication except when the organism is known to be sensitive. Chloramphenicol may be used as a second-line agent in the treatment of tetracycline-resistant cholera.

Because of its excellent blood-brain barrier penetration (far superior to any of the cephalosporins), chloramphenicol remains the first choice treatment for staphylococcal brain abscesses. It is also useful in the treatment of brain abscesses due to mixed organisms or when the causative organism is not known.

Chloramphenicol is active against the three main bacterial causes of meningitis: Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. In the West, chloramphenicol remains the drug of choice in the treatment of meningitis in patients with severe penicillin or cephalosporin allergy and GPs are recommended to carry intravenous chloramphenicol in their bag. In low income countries, the WHO recommend that oily chloramphenicol be used first-line to treat meningitis.

Chloramphenicol has been used in the U.S. in the initial empirical treatment of children with fever and a petechial rash, when the differential diagnosis includes both Neisseria meningitidis septicaemia as well as Rocky Mountain spotted fever, pending the results of diagnostic investigations.

Chloramphenicol is also effective against Enterococcus faecium, which has led to its being considered for treatment of vancomycin-resistant enterococcus.

Although unpublished, recent research suggests chloramphenicol could also be applied to frogs to prevent their widespread destruction from fungal infections. Chloramphenicol has recently been discovered to be a life-saving cure for chytridiomycosis in amphibians. Chytridiomycosis is a fungal disease, blamed for the extinction of one-third of the 120 frog species lost since 1980.

Adverse effects

Aplastic anemia

The most serious side effect of chloramphenicol treatment is aplastic anaemia. This effect is rare and is generally fatal: there is no treatment and no way of predicting who may or may not get this side effect. The effect usually occurs weeks or months after chloramphenicol treatment has been stopped, and there may be a genetic predisposition. It is not known whether monitoring the blood counts of patients can prevent the development of aplastic anaemia, but patients are recommended to have a blood count check twice weekly while on treatment. 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).

Thiamphenicol, a related compound with a similar spectrum of activity, is available in Italy and China for human use, and has never been associated with aplastic anaemia. Thiamphenicol is available in the U.S. and Europe as a veterinary antibiotic, and is not approved for use in humans.

Bone marrow suppression

Chloramphenicol may cause bone marrow suppression during treatment; this is a direct toxic effect of the drug on human mitochondria. This effect manifests first as a fall in hemoglobin levels, which occurs quite predictably once a cumulative dose of 20 g has been given. The anaemia is fully reversible once the drug is stopped and does not predict future development of aplastic anaemia. Studies in mice have suggested that existing marrow damage may compound any marrow damage resulting from the toxic effects of chloramphenicol.

Leukemia

Leukemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells. There is an increased risk of childhood leukemia, as demonstrated in a Chinese case-controlled study, and the risk increases with length of treatment.

Gray baby syndrome

Intravenous chloramphenicol use has been associated with the so-called gray baby syndrome. This phenomenon occurs in newborn infants because they do not yet have fully functional liver enzymes (i.e. UDP-glucuronyl transferase), so chloramphenicol remains unmetabolized in the body. This causes several adverse effects, including hypotension and cyanosis. The condition can be prevented by using the drug at the recommended doses, and monitoring blood levels.

Pharmacokinetics

Chloramphenicol is extremely lipid soluble; it remains relatively unbound to protein and is a small molecule. It has a large apparent volume of distribution of 100 litres, and penetrates effectively into all tissues of the body, including the brain. The concentration achieved in brain and cerebrospinal fluid (CSF) is around 30 to 50%, even when the meninges are not inflamed; this increases to as high as 89% when the meninges are inflamed.

Chloramphenicol increases the absorption of iron.

Use in special populations

Chloramphenicol is metabolized by the liver to chloramphenicol glucuronate (which is inactive). In liver impairment, the dose of chloramphenicol must therefore be reduced. There is no standard dose reduction for chloramphenicol in liver impairment, and the dose should be adjusted according to measured plasma concentrations.

The majority of the chloramphenicol dose is excreted by the kidneys as the inactive metabolite, chloramphenicol glucuronate. Only a tiny fraction of the chloramphenicol is excreted by the kidneys unchanged. Plasma levels should be monitored in patients with renal impairment, but this is not mandatory. Chloramphenicol succinate ester (an intravenous prodrug form) is readily excreted unchanged by the kidneys, more so than chloramphenicol base, and this is the major reason why levels of chloramphenicol in the blood are much lower when given intravenously than orally.

Chloramphenicol passes into breast milk, so should therefore be avoided during breast feeding, if possible.

Dose monitoring

Plasma levels of chloramphenicol must be monitored in neonates and in patients with abnormal liver function. Plasma levels should be monitored in all children under the age of four, the elderly and patients with renal failure. Peak levels (one hour after the dose is given) should be 15–25 mg/l; trough levels (taken immediately before a dose) should be less than 15 mg/l.

Drug interactions

Administration of chloramphenicol concomitantly with bone marrow depressant drugs is contraindicated, although concerns over aplastic anaemia associated with ocular chloramphenicol have largely been discounted.

Chloramphenicol is a potent inhibitor of the cytochrome P450 isoforms CYP2C19 and CYP3A4 in the liver. Inhibition of CYP2C19 causes decreased metabolism and therefore increased levels of, for example, antidepressants, antiepileptics and proton pump inhibitors if they are given concomitantly. Inhibition of CYP3A4 causes increased levels of, for example, calcium channel blockers, immunosuppressants, chemotherapeutic drugs, benzodiazepines, azole antifungals, tricyclic antidepressants, macrolide antibiotics, SSRIs, statins and PDE5 inhibitors.

Mechanism of action

Chloramphenicol is a bacteriostatic drug that stops bacterial growth by inhibiting protein synthesis. Chloramphenicol prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome. It specifically binds to A2451 and A2452 residues in the 23S rRNA of the 50S ribosomal subunit, preventing peptide bond formation. While chloramphenicol and the macrolide class of antibiotics both interact with ribosomes, chloramphenicol is not a macrolide. It directly interferes with substrate binding, whereas macrolides sterically block the progression of the growing peptide.

Resistance

There are three mechanisms of resistance to chloramphenicol: reduced membrane permeability, mutation of the 50S ribosomal subunit and elaboration of chloramphenicol acetyltransferase. It is easy to select for reduced membrane permeability to chloramphenicol in vitro by serial passage of bacteria, and this is the most common mechanism of low-level chloramphenicol resistance. High-level resistance is conferred by the cat-gene; this gene codes for an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covalently linking one or two acetyl groups, derived from acetyl-S-coenzyme A, to the hydroxyl groups on the chloramphenicol molecule. The acetylation prevents chloramphenicol from binding to the ribosome. Resistance-conferring mutations of the 50S ribosomal subunit are rare.

Chloramphenicol resistance may be carried on a plasmid that also codes for resistance to other drugs. One example is the ACCoT plasmid (A=ampicillin, C=chloramphenicol, Co=co-trimoxazole, T=tetracycline), which mediates multiple-drug resistance in typhoid.

Currently, some Enterococcus faecium and Pseudomonas aeruginosa strains are resistant to chloramphenicol. Some Veillonella app. and Staphylococcus capitis strains have also developed resistance to chloramphenicol to varying degrees.

Formulations

Chloramphenicol is available as 250 mg capsules or as a liquid (125 mg/5 mL). In some countries, it is sold as chloramphenicol palmitate ester (CPE). CPE is inactive, and is hydrolysed to active chloramphenicol in the small intestine. There is no difference in bioavailability between chloramphenicol and CPE.

Manufacture of oral chloramphenicol in the U.S. stopped in 1991, because the vast majority of chloramphenicol-associated cases of aplastic anaemia are associated with the oral preparation. There is now no oral formulation of chloramphenicol available in the U.S.

In molecular biology, chloramphenicol is prepared as 25–50 mg/mL stock in ethanol.

Intravenous

The intravenous (IV) preparation of chloramphenicol is the succinate ester, because pure chloramphenicol does not dissolve in water. This creates a problem: Chloramphenicol succinate ester is an inactive prodrug and must first be hydrolysed to chloramphenicol; however, the hydrolysis process is often incomplete, and 30% of the dose is lost and removed in the urine. Serum concentrations of IV chloramphenicol are only 70% of those achieved when chloramphenicol is given orally. For this reason, the dose needs to be increased to 75 mg/kg/day when administered IV to achieve levels equivalent to the oral dose.

Oily

Oily chloramphenicol (or chloramphenicol oil suspension) is a long-acting preparation of chloramphenicol first introduced by Roussel in 1954; marketed as Tifomycine, it was originally used as a treatment for typhoid. Roussel stopped production of oily chloramphenicol in 1995; the International Dispensary Association has manufactured it since 1998, first in Malta and then in India from December 2004.

Oily chloramphenicol is recommended by the World Health Organization (WHO) as the first-line treatment of meningitis in low-income countries, and appears on the WHO essential drugs list. It was first used to treat meningitis in 1975 and numerous studies since have demonstrated its efficacy.

Eye drops

In the West, chloramphenicol is still widely used in topical preparations (ointments and eye drops) for the treatment of bacterial conjunctivitis. Isolated case reports of aplastic anaemia following use of chloramphenicol eyedrops exist, but the risk is estimated to be less than 1 in 224,716 prescriptions. In Mexico, this is the treatment used prophylactically in newborns.