Although inhibition of Mcm occurs at ciprofloxacin concentrations higher than its normal therapeutic range also see below , our results suggest that some of the side effects seen with this and other fluoroquinolones may be because of inhibition of DNA replication. Fluoroquinolones serve as potent antibiotics due to their strong inhibition of the prokaryotic DNA gyrase.
Although eukaryotes are relatively resistant to ciprofloxacin at normal therapeutic levels, cytotoxicity is noted at high drug concentrations reviewed in [ 24 ]. The eukaryotic Topo II enzyme is a target for fluoroquinolones such as ciprofloxacin, as the drug inhibits Topo II in vitro [ 28 ], and mutants in Topo II have been isolated with increased in vitro fluoroquinolone resistance [ 29 ].
Moreover, cells exposed to cytotoxic levels of fluoroquinolones arrest in G2 and demonstrate chromosomal breaks consistent with the known role of topoisomerase II in mitosis [ 30 ]. However, it should be noted that these are also relatively common phenotypes of various known DNA replication mutants e.
Both our in vitro and cell-based studies strongly support Mcm as a new eukaryotic target for fluoroquinolones. Our finding that the Mcm mcm4chaos3 mutant has significantly increased ciprofloxacin resistance provides evidence that at least part of fluoroquinolone cytotoxicity is likely due to defects in DNA replication. Although chemically reactive amino acid modifying agents are too unstable, non-specific and irreversible to assist in studies of Mcm in vivo , there is considerable precedence for using modifying reagents in vitro to determine a mode of action in complex systems [ 4 ].
For example, DNA replication requires a large number of nucleotide hydrolases e. This property could make PG an experimentally useful reagent in vitro if Mcm activity needs to be specifically ablated. Our results suggest that most of the studied inhibitors likely interfere with the ATPase active sites of the helicases. However, if fluoro quinolones act as inhibitors of ATPase active sites, how can the relatively minor inhibition of ATP hydrolysis be explained?
There are mutations that cause substantial reductions in ATP hydrolysis but have only minor effects on in vitro DNA unwinding e. The remaining active sites, although clearly essential, hydrolyse ATP poorly. These data suggest that occupancy and turnover at these sites correspond predominately to a regulatory role rather than a direct contribution to helicase function. If the fluoro quinolone inhibitors preferentially target the regulatory rather than catalytic sites, only a modest change in ATP hydrolysis might be observed.
Alternatively, the inhibitors may function to poison the helicase. By binding to a single active site, the inhibitor might uncouple ATP hydrolysis from DNA unwinding by altering the ability of adjacent active sites to communicate. This model also explains the effect of these inhibitors on TAg, a homohexameric helicase that contains identical ATPase active sites that coordinately unwind DNA during SV40 replication [ 23 ].
Finally, the fluoroquinolones could inhibit helicase activity by blocking ssDNA binding; however, this interpretation is difficult to reconcile with our observations that elevated levels of ATP restore Mcm helicase activity in the presence of most of the examined fluoroquinolones Figure 3 B. Helicases are abundant in eukaryotes. In addition to Mcm, many human helicases e. Given the paucity of available helicase inhibitors and our observations that different fluoroquinolones differentially inhibit a variety of helicases Supplementary Figure S3 , fluoroquinolones may provide a general and malleable molecular scaffold for the development of efficient helicase inhibitors with tailored specificities.
Further development of fluoroquinolones provides a useful route to develop Mcmspecific inhibitors of the therapeutic value, as Mcm overexpression correlates with cancer, and multiple studies indicate that the Mcm subunits are potential targets [ 14 , 36 ].
Several of the inhibitors that we examined ciprofloxacin, and , demonstrate at least partial selectively for Mcm over a host of other helicases tested and ciprofloxacin appears to target Mcm in yeast. As ciprofloxacin and related fluoroquinolones are common and approved human antibiotics [ 37 ],this molecular scaffold has proven pharmaceutical utility.
Although our inhibitors only act at concentrations that exceed typical therapeutic use, this situation has precedence. For example, high doses of sodium phenylbutyrate are used in the treatment of malignant tumours, in which plasma concentrations of the compound are well over 1 mM [ 38 ].
Given the degree of selectivity that we observe with an off-the-shelf pharmaceutical designed for an entirely different application, our limited screen of ciprofloxacin-related compounds has identified several chemicals with improved properties, validating the likelihood that additional structural refinement using ciprofloxacin as a starting point will yield molecules with enhanced potency and specificity.
Our discovery of Mcm inhibitors has utility in other areas. First, they may function as a useful research tool both in vitro and in vivo. As each of the six Mcm subunits are individually essential, analysis of the role of the replicative helicase has largely focused on model systems such as S. Such inhibitors also have potential utility for biochemical studies, especially using systems e. Secondly, the discovery that fluoroquinolones can inhibit the eukaryotic helicase may explain some of the cytotoxic effects observed with ciprofloxacin and other fluoroquinolones [ 40 ].
Our finding that the mcm4chaos3 allele confers resistance to ciprofloxacin supports our hypothesis that the Mcm complex is a ciprofloxacin target in cells and suggests that it could also be contributing to the deleterious side effects seen with this class of compounds.
Nicholas Simon and Matthew Bochman performed the experiments and analysed the data. Sandlin Seguin performed the human cell culture assays. W illiam L. Seibel assembled and provided the small molecule library and verified the purity of the individual compounds. Anthony Schwacha and J effrey L. Brodsky designed experiments and supervised the work.
Bochman and Anthony Schwacha wrote the paper. We thank P. Sign In or Create an Account. Advanced Search. Sign In. Skip Nav Destination Article Navigation.
Close mobile search navigation Article navigation. Volume 33, Issue 5. Previous Article Next Article. All Issues. Cover Image Cover Image. Article Navigation. Research Article October 07 This Site. Google Scholar. Matthew L. Bochman ; Matthew L. Bochman 1. Sandlin Seguin ; Sandlin Seguin. Jeffrey L. Brodsky ; Jeffrey L.
William L. Seibel ; William L. Anthony Schwacha Anthony Schwacha 2. Biosci Rep 33 5 : e Article history Received:. Ciprofloxacin resistance varies significantly from country to country with the highest resistance reported in developing countries Fasugba et al. Due to an increase in resistance, Infectious Diseases Society of America IDSA recommends to limit the use of fluoroquinolones to infections, where other antibiotics cannot be used due to reasons including associated side effects or causative organisms are found to be resistance to alternative antibiotics.
There could be a number of reasons behind the observed increase in bacterial resistance to ciprofloxacin. One reason could be the overuse or misuse of ciprofloxacin likely to be due to wide spread availability of generic versions of ciprofloxacin. Overuse or misuse of antibiotics is known to promote bacterial resistance and is likely to limit the effectiveness of ciprofloxaxin.
After the introduction of generic ciprofloxacin in the market, an increase in consumption of ciprofloxacin was observed. A study in Denmark showed that total consumption of oral ciprofloxacin increased significantly from 0. In another study, it was reported that the resistance of isolated E.
Similarly, a wide spread use of ciprofloxacin was thought to be responsible for a significant rise in ciprofloxacin resistance over time Fasugba et al. As the driving force for the prevalence of antibiotic resistance is the extent of drug use, there is a positive correlation between consumptions of quinolones and antibiotic resistance Jensen et al.
In certain parts of the densely populated developing countries such as Brazil, Indonesia, Pakistan, India and China, there are hot spots for emergence and spread of antibiotic resistance Huynh et al. It was speculated that South Asia is the reservoir for the global spread of ciprofloxacin-resistant infections caused by various types of bacteria including S.
Mulder et al. The frequent use of prescription drugs in such communities has contributed to increase bacterial resistance Morgan et al. Another reason for the observed increase in bacterial resistance to ciprofloxacin could be due to easy accessibility to substandard and spurious formulations of ciprofloxacin.
Substandard and spurious generic versions of drugs are therapeutically ineffective when used clinically Agudelo and Vesga, As a consequence, treatment fails and this potentially enhances the selection of bacterial resistance Rodriguez et al. Weir et al. In addition, a number of formulations had the concentration of ciprofloxacin sufficiently lower to have negative impact on clinical outcome leading to increased risk of bacterial resistance. Prazuck et al.
One collected formulation in fact expired 11 months ago. This study highlighted the availability of poor quality of ciprofloxacin formulations in India. Previous studies have also shown that the production of substandard drugs is common in Southeast Asia Newton et al. Bate et al. They collected 50 and 53 ciprofloxacin treatment packs from Delhi and Chennai pharmacies respectively. The quality was determined based on the content of ciprofloxacin and disintegration test.
There could be a number of reasons behind the availability of substandard ciprofloxacin worldwide. However, one of the common reasons could be the lack of regulatory policies in relation to both appropriate manufacturing practice and quality assurance procedures. Currently, approaches to determine bioavailability and bioequivalence of pharmaceutical products has been largely standardized.
In United States, the sale of generic drugs is approved by Food and Drug Administration when they meet all the regulatory requirements provided in the Code of Federal Regulations Kaushal et al.
The regulatory environment of the country of marketing is important to assure the assessment of bioequivalence of drug products. WHO has made tremendous progress in developing international consensus for standardizing and harmonizing the regulatory requirement, mainly for manufacturing control, safety and efficacy of new drugs and assessing bioequivalence of generics.
Different resources, expertise and stringent regulation and enforcement are required to ensure proper implementation to ensure generic formulations are safe and effective Sarker et al.
Moreover, post-marketing surveillance is also necessary as the use of higher strength drug than the labeled value can result in toxic effects due to overdose whereas taking substandard agents can result in treatment failure. Similarly, strict monitoring of the production of ciprofloxacin formulations by drug regulatory agencies as well as post-marketing quality control studies are required to prevent and identify the substandard and spurious brands of oral ciprofloxacin in order to prevent the risk of treatment failure and antibiotic resistance.
The substandard quality ciprofloxacin is potentially the driving force for ciprofloxacin resistance. The availability and use of substandard quality ciprofloxacin would jeorpadise the clincial efficacy of this broad spectrum antibitoic.
Drug quality assurance and strict laws and regulations for manufacturing, importation, distribution, and sale of ciprofloxacin are required to prevent the availability and use of substandard ciprofloxacin formulations, which in turn will minimize the risk of treatment failure and antibiotic resistance. An international comprehensive policy for addressing the resistance of ciprofloxacin and other fluroquinonoles in general is much needed.
All authors contributed toward revising the paper and agreed to be accountable for all aspects of the work. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Agudelo, M. Therapeutic equivalence requires pharmaceutical, pharmacokinetic, and pharmacodynamic identities: true bioequivalence of a generic product of intravenous metronidazole. Agents Chemother. Aldred, K. Mechanism of quinolone action and resistance. Biochemistry 53, — Azad, M. Bioequivalence and pharmacokinetic study of two oral formulations of ciprofloxacin tablets in healthy male volunteers.
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Cuadrado, A. Philippine studies showed that the most frequent isolates of necrotizing fasciitis included E. In addition, results of further investigations show that evaluating polar surface area is a highly efficient method of predicting oral absorption, intestinal absorption, membrane permeation, and drug transport properties [ 10 , 11 ]. Clearly E. This study demonstrates that the modification of structural substituents of the broad-spectrum antibacterial agent ciprofloxacin will result in antibacterial agents having molecular properties beneficial for inhibiting the proliferation of E.
Chemicals and reagents were analytical grade and obtained from Aldrich Chemical Company P. All glassware utilized in synthesis was washed thoroughly in distilled water and baked to dryness. Molecular modeling and molecular properties were determined by utilizing ChemSketch v. Environmental Protection Agency. For each of drugs 2, 3, and 4 derivatives of ciprofloxacin, a mass of 90 milligrams of ciprofloxacin was measured out into a large test tube.
The thionyl chloride is placed first in a dry test tube followed by the addition of solid ciprofloxacin. The thionyl chloride reacts with the carboxyl group found within the structure of ciprofloxacin. The mixture is then subjected to microwave excitation for a total of three to five minutes which heats the mixture combination and must be monitored due to fluctuations in energy of microwave instrument the mixture must be monitored for excess heat that induces degradation of the reagents.
The reactant mixture is allowed to cool and 0. The formation of the ester reaction is immediate with excess alcohol or amine being removed by evaporation using vacuum pump drying. Formation of ester derivatives is confirmed by chemical test. Each drug was diluted in Luria-Bertani media with the addition of E. An overnight growing plasmid-induced ampicillin-resistant E. The five iterations of drugs with bacteria were as follows: ciprofloxacin alone; mixture of ciprofloxacin 15 milligrams plus drug 2 34 milligrams ; 3 mixture of ciprofloxacin 20 milligrams plus drug 3 49 milligrams ; 4 mixture of ciprofloxacin 24 milligrams plus drug 4 63 milligrams ; and 5 mixture of ciprofloxacin 33 milligrams plus drug 2 33 milligrams plus drug 3 29 milligrams plus drug 4 34 milligrams.
These mixtures 1 from 4 to placed into tissue culture with penicillin are resistant E. Necrotizing soft-tissue infection is highly lethal and leaves patients, even those receiving prompt and adequate care, requiring reconstructive intervention and rehabilitation. Some studies have concluded that time leading to operative intervention is the most important determinant of mortality [ 2 ].
Similarly, other observations conclude that optimal treatment is attained with early diagnosis, radical surgical debridement of all necrotic tissue, application of broad-spectrum antibiotics, and aggressive nutritional support [ 12 ].
Administration of broad-spectrum antibiotics must be initiated at the earliest sign. Ciprofloxacin is a fluoroquinolone type of antimicrobial agent that inhibits bacterial enzyme DNA gyrase needed for replication of DNA.
It is effective with both oral or intravenous administration and demonstrates potent antibacterial activity against most gram-negative bacteria and many gram-positive bacteria having particularly good activity against gram-negative bacteria [ 13 ].
Ciprofloxacin reaches concentrations in most tissues and body fluids sufficient to inhibit the majority of susceptible pathogens [ 13 ].
Ciprofloxacin is an effective treatment for those infections of the skin and soft-tissues [ 13 ]. Ciprofloxacin is effective for both Streptococcus pyogenes and Staphylococcus aureus [ 14 ] and is prescribed for polymicrobial anaerobic infections that involve clostridia [ 15 ].
Ciprofloxacin is clearly a versatile and potent antibacterial agent that was utilized in this study along with three aliphatic ester derivatives of ciprofloxacin. The molecular structures of parent ciprofloxacin drug 1 , n-propyl ester drug 2 , n-pentyl ester drug 3 , and n-octyl ester drug 4 are presented in Figure 1 for comparison.
Note that the original carboxyl group —C O OH of the parent ciprofloxacin is replaced by ester functional groups —C O OR in subsequent derivatives drug 2, drug 3, and drug 4. All other structural features of ciprofloxacin are preserved in derivatives. Various molecular properties were determined for these agents with descriptors included in Table 1. All agents show zero violations of the Rule of 5 which is a favorable outcome when evaluating drug likeness [ 16 ]. The Rule of 5 serves as a guideline for screening potential drug candidates for effective absorption or permeation.
Subsequently ciprofloxacin and all derivatives are expected to be effective and orally active with favorable permeation and absorption. Note that as the length of the ester functional group increases from drug 2 n-propyl to drug 4 n-octyl , the number of —OH, —NH n , oxygens, and nitrogen remains constant thus restraining the PSA to nominal The decreasing hydrophilic property of the derivatives e.
Growth inhibition of penicillin-resistant E. The outcome of in vitro titration of drugs 1 through 4 showed extremely strong bacterial inhibition even to concentration level below one microgram per milliliter see Figure 2. These numerical values for decreased bacterial survival plotted in Figure 2 indicate clearly that the combination of ciprofloxacin with various ester derivatives expresses very strong bacterial growth inhibition.
In addition, the enhanced tissue penetration by derivatives of ciprofloxacin is then anticipated to improve antibacterial activity. Necrotizing fasciitis can be classified according to the depth of infection as well as microbial origination [ 2 ].
Necrotizing fasciitis is an infection of the deeper layers of skin, notably the hypodermis layer referred to as superficial fascia, subcutis, and subcutaneous layer , that spreads across the fascial plane within the subcutaneous tissue [ 2 , 3 ].
The dermal permeability coefficient is the most used and effective for predicting percutaneous penetration of drugs in quantitative structure property activity relationships and basis predictions on the physiochemical properties of the penetrant drug [ 17 ]. The physical and chemical properties of a drug or carrier vehicle have a decisive effect on the drugs permeation through the dermal tissues [ 17 ].
Previous studies have confirmed that is a successful model for estimating drug proliferation based on molecular properties [ 18 ]. The rate of drug permeation in centimeter per hour is determined from the following equation MW; molecular weight [ 19 ]:. Values of for drugs 1 through 4 are shown in Table 3 as rates expressed in centimeter cm per hour. Pharmacists can review the antibiogram and verify the dosing and duration of ciprofloxacin. Nursing can counsel the patient on taking the medication, monitoring patient compliance and therapeutic effectiveness, answering any questions, and reporting any concerns to the prescriber.
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