Ciprofloxacin: Antibiotic Interaction with a Topoisomerase Complex
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Lower respiratory tract infections (LRTI) are infections of the trachea, primary bronchi, and lungs caused by bacteria, fungi, or viruses. These are most commonly seen as pneumonia, bronchitis, and influenza and are characterized by shortness of breath, weakness, coughing, high fever, and fatigue. LRTI's can affect people of any age, but are most commonly seen in the pediatric and geriatric patient populations due to their propensity for less-than-optimal functioning immune systems. Bacterial pneumonia should be treated with antibiotics; the selection of which is guided by the characteristics and type of bacteria causing the disease.1
A 68 year-old male presented to the urgent care unit with symptoms of shortness of breath, wheezing, cough, discolored sputum, and a fever. After microbiologic tests were performed to determine the pathogen as streptococcus pneumoniae, the physician wrote a prescription for oral ciprofloxacin, 500mg every 12 hours for 14 days. One week later the patient returned to the urgent care unit with symptoms of severe drowsiness, fatigue, and low blood pressure. During a profile review, the pharmacist noted the concomitant use of tizanidine, as needed for muscle spasms. The pharmacist informed the physician that the patient's tizanidine may be interacting with the ciprofloxacin and was likely the causation of the patient's symptoms.
Pharmacologically, the concomitant use of ciprofloxacin with tizanidine may increase the serum concentration of tizanidine. This occurs due to an interaction at the Cytochrome P450 enzyme CYP1A2. Ciprofloxacin is an inhibitor of CYP1A2, and decreases its ability to perform the necessary metabolic functions for breakdown of other compounds in the body. When CYP1A2 cannot exert its optimal activity, tizanidine cannot be metabolized as quickly, leading to a higher serum concentration of the drug. Consequently, this may lead to adverse drug reactions in the patient such as those seen in this case.
Ciprofloxacin, a second generation fluoroquinolone, interrupts DNA replication by inhibiting both topoisomerase II and IV. Topoisomerase II is an enzyme that reduces the amount of supercoiling of the DNA double-stranded helix during the replication process, while the unlinking of the two daughter strands of DNA is a result of topoisomerase IV. Therefore, Ciprofloxacin has bactericidal and bacteriostatic properties against both Gram-negative and Gram-positive bacterial pathogens. Common Gram-negative pathogens that quinolones work against include Haemophilus, Salmonella, Pseudomonas and Enterobacter; Gram-positive infections include Staphylococci and Streptococci.3 It is commonly used in cases involving infections of the respiratory tract, gastrointestinal tract, urinary tract and abdomen. The action of ciprofloxacin is concentration-dependent; at low concentrations it reduces only the action of topoisomerase II. However, if it is given at higher doses, it is also able to inhibit topoisomerase IV.4 The primary target of this drug is also dependent on the type of pathogen it is being used against. If the infection is caused by Gram-negative bacteria, then topoisomerase II becomes the primary target for the drug; if the pathogen is a Gram-positive bacterium, the primary target becomes topoisomerase IV. In the case of Gram-negative organisms, ciprofloxacin exerts a long post-antibiotic effect (PAE), meaning the drug remains active even after the patient stops taking it.5
Cipro (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-piperzino-quinoline-3-carboxylic acid) began clinical trials in the early 1980s and received patent approval in 1987. The original patent awarded to the drug company, Bayer AG, expired in 1998.3 Ciprofloxacin is differentiated from the quinolone class of antibiotics by the fluorinated carbon atom located at C6 in the aromatic ring.6 This substitution helps increase the specificity of the drug for topoisomerase by a factor of ten.6
Ciprofloxacin is classified as a quinolone antibiotic; a class of broad-spectrum anti-bacterial drugs. Generally, these antibiotics selectively target topoisomerases, enzymes responsible for reducing the supercoiling of the DNA helix prior to replication. Quinolone interaction with DNA was primarily thought to be through Van der Waals forces and p-p stacking.7 However, an alternate binding method has recently been uncovered leading to the revision of this initial mechanism of action.
It has been determined that magnesium ions, which are inherently present, are capable of facilitating the interaction between the antibiotic and its protein target. This interaction includes the magnesium ion and water molecule interacting with amino acid residues. The interaction is thought to be mostly through Van der Waals forces; with the carbonyl at position C4 and the acid at C3 as crucial participants in the binding mechanism.7 Because of the critical interactions that occur at position C4 and C3, large variations in substituents are not seen as they would affect the position of the magnesium ion and the subsequent coordination complex. Positions 1 and 7 possess large, bulky substituent groups that allow the drug to act as a 'wedge' to disfigure the DNA and inhibit topoisomerase binding. The carbon located at C7 allows for the most variation in regards to the substituents that may be substituted within the molecule.6 The magnesium ion interaction also demonstrates the importance of the keto-acid group within the quinolone structure.7
The magnesium ion forms an octahedral coordination sphere with two oxygen atoms from the quinolone, and four water molecules.7 Two of these water molecules can then form hydrogen bonds with Ser84 and Glu88 residues on topoisomerase, binding the antibiotic, and allowing disruption of the enzyme's normal mechanism of action.
With the drug bound to the topoisomerase enzyme, it is close enough to interact with the DNA of the replicating cell. In order to achieve contact with the DNA, the antibiotic forms a ParC55 closed homodimer with ParE30 (TOPRIM, metal-binding domain) monomers on each side.4 In ParC55, the a1 helix forms a long chain-like segment that holds the ParE domain close to the external side of the ParC55 domain.4 This linkage is critical as it is most likely the one responsible for directing the subsequent interactions between the domains of the topoisomerase enzyme. The N-terminus of the ParC dimer structure is part of the CAP-like DNA binding domain, also known as the Winged Helix Domain. It is the helix-turn-helix structure that contains Arg117 and Tyr118 within its binding site and where the antibiotic ligand is able to bind and intercalate between base pairs.4,7 By inserting here, the antibiotic is able to induce double-stranded breaks, which ultimately leads to the death of the replicating cell.7 In contrast to most intercalating molecules, fluoroquinolones have regions of non-planar geometry. At positions 1, 7, and 8, the functional groups are non-planar, making the drug wedge-like, as previously mentioned, rather than the typical flat shape that most antibiotics of the same class share.7
Arg117 and Tyr118 view 2Additionally, this new interaction between the drug and the magnesium ion may provide an explanation as to why mutations involving these residues in topoisomerase II and IV are closely related to microorganism resistance to quinolone and fluoroquinolone antibiotics.7 Resistance to these antibiotics may be related to the new conformation that the antibiotic forms when binding with water molecules that are now present with the involvement of the magnesium ion. After nicking of the DNA strand, two ciprofloxacin molecules intercalate between nucleotides on either side of a 4bp 'sticky end' at positions -1 and +1 on the DNA. The cyclopropane ring of the drug is close to ParC residues Ser79 and Asp83. If either residue is mutated, resistance to the drug is seen. The most common mutations that lead to ciprofloxacin resistance involve S79F and S79Y. When these two Serine residues are mutated, they are able to facilitate the insertion of the large C7-bound side chain into the binding site on topoisomerase that would otherwise normally be occupied by the antibiotic. This can potentially affect allosteric drug binding.4
DNA replication places a substantial portion of dependence on the proper functioning of topoisomerase II and topoisomerase IV to help uncoil the double helix structure of the DNA. As an inhibitor of these enzymes, ciprofloxacin is able to effectively disrupt replication, ultimately leading to apoptosis of the replicating cell. Based on the patient's illness, this antibiotic was prescribed at a therapeutic dosage that would achieve maximal inhibition of these topoisomerase enzymes.
Active ciprofloxacin molecules bind two sites within the
topoisomerase and are held in place through Van der Waals forces between the drug's ketone group and a magnesium ion. The presence of this magnesium ion allows for the formation of an octahedral coordination sphere at the carbonyl and carboxylic acid groups attached to C3 and C4 respectively, as well as hydrogen bonding to two amino acid residues that attach the drug to the topoisomerase.7
Once bound, the drug acts like a physical wedge within topoisomerase and prevents it from binding DNA and exerting its effects during replication. However, the use of this antibiotic may cause unintentional drug-drug interactions (DDIs) when used in combination with other medications due to its metabolism by the Cytochrome P450 (CYP450) family of enzymes.
Overall Ciprofloxacin InteractionAs previously mentioned, ciprofloxacin is an inhibitor of the CYP1A2 enzyme; the same enzyme necessary for the metabolism of the muscle-relaxant, tizanidine. As a centrally acting alpha-2 adrenergic agonist, tizanidine suppresses activity in the sympathetic nervous system and may lead to effects such as decreased heart rate and reduced blood pressure in addition to other central nervous system effects such as drowsiness, fatigue, and GI upset. Due to the inhibition of CYP1A2, tizanidine is metabolized more slowly and with subsequent dosing, may accumulate in the blood. It is this accumulation that increases the risk of occurrence for serious side effects such as hypotension and sedation as seen with the patient.
Due to the appearance of these serious side effects, it became necessary to change this patient's therapy in order to treat the bacterial infection while avoiding any potential harm as the result of the DDI. In addition, abrupt discontinuation of tizanidine may result in negative cardiovascular effects such as rebound hypertension, tachycardia, and hypertonia. For this reason it is suggested that tizanidine be tapered down as opposed to discontinued abruptly.9
After a brief discussion with the physician, the patient was administered isotonic intravenous fluids to restore a normotensive state and was able to speak to the pharmacist about the cause of the new symptoms they had been experiencing. Therapeutically, the patient was switched to diazepam (Valium™) 5mg tablets with directions to take one tablet, by mouth, 3-4 times daily10 as needed for muscle spasms. This medication was given to replace their previous tizanidine regimen. The patient was strongly advised against alcohol use in combination with diazepam and was educated on the potential side effects including trouble sleeping, headache, and dizziness.11 The patient was instructed to gradually reduce their daily dose of tizanidine by 2-4mg every day. By instituting this 'taper', the patient would be able to fully discontinue the tizanidine after 3 days and avoid the negative cardiovascular effects that are common when abruptly stopping a2-agonists. This new treatment regimen was developed through inter-professional cooperation utilizing pharmacodynamic profiles9, 10, 11, 12, 13 and drug interaction reports based on the patient's current need for infrequent muscle spasm relief as well as the need to treat the active LRTI. Under the new therapeutic regimen both of the patient's chief complaints were addressed while reducing the overall risk for adverse effects due to DDIs.
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9. Tizanidine. In: Micromedex [database online]. Truven Health Analytics; 2014. http://www-micromedexsolutions-com.proxy.lib.aurora.org/micromedex2/ librarian/ND_T/evidencexpert/ND_PR/evidencexpert/CS/CD2221/ND_AppProduct/evidencexpert/DUPLICATIONSHIELDSYNC/E17D1B/ND_PG/evidencexpert/ND_B/evidencexpert/ND_P/evidencexpert/PFActionId/evidencexpert.DisplayDrugdexDocument?docId=924129&contentSetId=100&title=Tizanidine+Hydrochloride&servicesTitle=Tizanidine+Hydrochloride. Accessed November 27, 2014.
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