The effect of natural sunlight on ciprofloxacin ophthalmic solution (1).

Ciprofloxacin (0.3% preparation) is available on the market for topical ophthalmic use. The marketed eye drop samples, as well as reference ciprofloxacin in aqueous solution were found to degrade to an ethylenediamine derivative of ciprofloxacin when exposed to natural sunlight. The degradation product was identified by comparison to a reference standard as well as by LC-MS, M[S.sup.2] and M[S.sup.3]. A model compound, phenylpiperazine, was found to decompose to a N-phenylethylenediamine after sunlight exposure at a much slower rate compared to ciprofloxacin. When tested against Bacillus pumillus, the sunlight exposed ophthalmic solution of ciprofloxacin showed reduction by 2.6% of antimicrobial potency in 24 hrs compared to properly stored solution. However, ciprofloxacin ophthalmic drop containers stored in original cartons, showed no HPLC detectable photolytic degradation.

Keywords: Ciprofloxacin ophthalmic solution, sunlight, photodegradation, phenylpiperazine, electrospray mass spectrometry 

**********

Ciprofloxacin is the generic international name for the most common and widely used fluoroquinolone antibacterial agent originally introduced by Bayer Pharmaceuticals. In general, ciprofloxacin has good activity against both gram-negative and gram-positive bacteria. It is effective against a number of different types of bacteria that cause severe infections in humans, including Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Proteus mirabillis, Klebsiella pneumoniae and Pseudomonas aeruginosa. This antibiotic has now been released as ophthalmic drops and is useful against eye infections. When used topically, corneal toxicity and retinal toxicity rarely occur. Corneal precipitation occurs with ciprofloxacin but does not appear to interfere with healing (Smith et al., 2001).

Ciprofloxacin solution (0.3%) is currently available for topical ophthalmic use. Ciprofloxacin and ofloxacin are recognized as being safe to use, but there have been reports of skin reddening and rashes in patients taking these antibiotics after sun exposure, symptoms common to photosensitivity (The Fluoroquinolone Toxicity Research Foundation, Webpage). Research at Southmead Hospital Bristol on the stability of ciprofloxacin and ofloxacin eye drops showed that loss of antibiotic potency occurred when ciprofloxacin was irradiated at 320 nm and then tested against Escherichia coli (Tobin, Webpage; Phillips et al., 1990). On the other hand, Sunderland et al. in 1999 reported that the photo-degradation products of ofloxacin were effective in killing bacteria. In addition, initial experiments suggest that the photodegradation products may cause toxicity. There is an extensive report (Sunderland et al., 2001) on the antibiotic activity of photo-decomposed products of ofloxacin, levofloxacin, ciprofloxacin and moxifloxacin. The antibiotic activities were compared by parallel-line bioassays using Escherichia coli, Enterobacter cloacae and Klebsiella oxytoca. With ofloxacin and levofloxacin, the zone size for the control solution was significantly less than that of the irradiated solutions. More than 15% photodegradation in at least two of the indicator organisms indicated that the photo-degradation products possess antimicrobial activity. There is, however, no difference seen with ciprofloxacin at any level of photodegradation with any of the indicator organisms, nor with moxifloxacin at 30 and 54% photodegradation. This conflicts with the earlier findings (Phillips et al., 1990). A significant difference was observed with E. cloacae only, at 83% photodegradation. On the other hand, the main degradation product of ciprofloxacin, after both artificial and daylight exposure, was reported as 7-amino-1-cyclopropyl-6-fluroro-1,4-dihydro-4-oxo-3-quinolone carboxylic acid, commonly known as ethylenediamine derivative of ciprofloxacin (Torniainen et al., 1997). Although the antimicrobial activity of photodegradation products of some fluoroquinolones including ciprofloxacin has been investigated, the general pathways in forming the degradation products have not been evaluated.

The objective of this investigation is to identify the major degradation products of ciprofloxacin in eye drop preparations when placed in natural sunlight. The antibiotic activity was investigated using different samples including controlled, photo-decomposed and reference samples. In addition, experiments were performed using a simple model compound to obtain preliminary information on the mechanism of this photodegradation.

MATERIALS AND METHODS

Ciprofloxacin ophthalmic solutions. Ciprofloxacin ophthalmic solution manufactured in Bangladesh (Ciprocin, trade name, manufactured by Square Pharmaceuticals Ltd.) and a commercially available ophthalmic solution manufactured in USA (Ciloxan, trade name, manufactured by Alcon Manufacturing Ltd. and ciprofloxacin eye drop manufactured by Falcon Pharmaceuticals) were used in this study. The natural sunlight-stability experiments were conducted by keeping samples in an original container as well as in different plastic containers such as translucent (light can interact with the contents of these type of containers), amber-colored (usually used to protect the contents of the container from exposure of light), and with carton or without carton in sunlight exposure from 10 AM to 4 PM. The reference samples for ciprofloxacin and the ethylenediamine derivative of ciprofloxacin [1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-ethylenedimino)-3-quinoline carboxylic acid or desethylene ciprofloxacin] were USP Reference Standards having catalogue no. 13433 and catalogue no. 13432, respectively.

High Performance Liquid Chromatographic (HPLC) analysis. Reversed phase high performance liquid chromatography analysis of ciprofloxacin and its different ophthalmic solutions including sunlight-exposed samples of eye drops were carried out using a Shimadzu LC-10 as pump, a Shimadzu SPD-10A UV detector, a Shimadzu C-R 6A integrator, and a Rhedyne-7125 injector. A reversed phase column (Zorbax ODS, 4.6mm id X 250 mm) was used for analysis. The mobile phase consisted of 0.025 M phosphoric acid solution (pH 3 [+ or -] 0.1 adjusted with triethylamine) and acetonitrile in the ratio of 87:13 v/v (United States Pharmacopoeia, XXV). The chromatographic conditions are mentioned in Figure 1.

Electrospray MS and Liquid Chromatography-Mass Spectrometric (LC-MS) analysis. The electrospray mass spectrometric analysis was carried out on a Finnigan LCQ ion trap mass spectrometer using direct injection of samples in 0.1% formic acid in acetonitrile. LC-MS was carried out on a BAS HPLC interfaced with the mass spectrometer with 5% of the effluent flowing to the mass spectrometer. A reverse phase C-18 column (Econosphere) and conditions of HPLC analysis described above were used in the separation of degraded products of ciprofloxacin ophthalmic solution.

Bioassay. The sunlight exposed samples as well as the unexposed samples of ophthalmic solutions were bioassayed with USP agar 1. Bacillus pumillus (NCTC 8241) was used (2 mcg/ml suspension) as the indicator organism. The diameter of the zones of growth inhibition around the wells were measured and plotted against the logarithm of parent ciprofloxacin concentration.

RESULTS

The opthalmic solutions kept in direct sunlight for 3 months were analyzed by HPLC. The identification as well as quantification of the major degradation product, the ethylenediamine derivative of ciprofloxacin, was done using HPLC by comparison with the properties of a reference compound. Typical HPLC chromatograms representing both sunlight unexposed and exposed samples of ciprofloxacin ophthalmic solution are shown in Figure 1. The percent loss of potency resulting from exposure of ciprofloxacin ophthalmic solution to sunlight in different packaging conditions is shown in Table 1.

The identity of the ethylenediamine derivative of ciprofloxacin was also confirmed by multiple stage mass spectral fragmentation and deuterium exchange followed by mass spectral fragmentation. This electrospray mass spectrometric identification technique can be used where a reference compound representing a ethylenediamine derivative of ciprofloxacin is not available. The electrospray LC-MS of sunlight-exposed ciprofloxacin ophthalmic solution or sunlight-exposed ciprofloxacin in aqueous solution produced a molecular ion (M + H)[.sup.+] at m/z 306 for the first peak (photodegradation product) in Figure 1(B) which on further fragmentation (M[S.sup.2] of m/z 306) yielded ions at m/z 288 by loss of [H.sub.2]O. The mass fragmentation pattern of this photodegradation product, i.e., an ethylenediamine derivative of ciprofloxacin is shown in Figure 2. The peak representing the ethylenediamine derivative of ciprofloxacin was then collected in a test tube and mixed with [D.sub.2]O. The deuterated sample, when injected directly into the mass spectrometer, resulted in deuterium scrambling the mass spectrum (Figure 3). On further fragmentation, the individual deuterated ions also showed the loss of [D.sub.2]O, HDO, or [H.sub.2]O in a scrambled manner.

In order to gain insight into the mechanism of this photolytic degradation of the ciprofloxacin molecule, a model compound, phenylpiperazine in aqueous solution (0.3%), was exposed to natural sunlight alongside a solution of ciprofloxacin solution (0.3%) in water. The degradation of the model compound occurred at the piperazinyl ring in a similar fashion as observed for ciprofloxacin (Figure 4). The photodegradation product of phenylpiperazine has been identified as N-phenylethylenediamine (or ethylenediaminobenzene) from mass spectrometric data. The mass spectral data showed [M+H][.sup.+] at m/z 137, which on further fragmentation yielded m/z 120 by the loss of neutral ammonia (m/z 17). However, the photodegradation of phenylpiperazine is very slow in comparison to that of ciprofloxacin.

The sunlight-exposed ciprofloxacin ophthalmic solution samples at different time intervals were assessed for their antimicrobial activity against Bacillus pumillus and the results are shown in Figure 5. The antimicrobial activity of sunlight-exposed samples against Bacillus pumillus decreased gradually with the sunlight exposure time. About 3% reduction in antimicrobial activity was found over 24 hours sunlight exposure when compared to that of sunlight-unexposed sample. On 100 hours sunlight exposure, the sample had about 92% antimicrobial activity when compared to the activity of sunlight-unexposed, properly stored sample.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

DISCUSSION

Fluoroquinolones are photodegradable compounds that produce various photodegradation products (Tiefenbacher et al., 1994). However, a systematic qualitative and quantitative evaluation of all the flouoroquinolones has not been published. The British Pharmacopoeia has listed 5 different impurities of ciprofloxacin. The UV irradiation of rufloxacin, a fluoroquinolone antibacterial that shows photosensitizing properties toward biological substrates, leads to formation of two main photoproducts characterized by a decarboxylation process and an opening of the piperazinyl ring, respectively (Condorelli et al., 1999). The photochemical reactivity of four fluoroquinolone antibiotics has been examined (Fasani et al., 1998). For norfloxacin, enoxacin and lomefloxacin, the process occurring is defluorination. Ofloxacin is less light sensitive and undergoes, in part, reactions different from defluorination. Interestingly, photodegradation by natural sunlight of ciprofloxacin in eye drops preparation produces mostly an ethylenediamine derivative of ciprofloxacin by opening of the piperzinyl ring (Figure 4). This impurity is also mentioned in both the British Pharmacopoeia and the United States Pharmacopoeia.

In this study, the structure of the degradation product of ciprofloxacin exposed to natural sunlight was also confirmed by electrospray mass spectrometric analysis. The ciprofloxacin structure contains a piperazine group that is the site of photodegradation. The photolytic breakdown of the piperazine ring in ciprofloxacin produces the ethylenediamine derivative. The model compound, phenylpiperazine, was also found to decompose in a similar fashion producing phenylethylenediamine. However, the breakdown of ciprofloxacin occurred more rapidly compared to that of phenylpiperazine (Figure 4). The strong inductive effect of ortho-fluorine in ciprofloxacin may enhance the light-catalyzed cleavage of the piperazine ring, although further studies would be required to establish the actual mechanism of the reaction.

The results from sunlight-exposed samples of ciprofloxacin eye drop kept for a 3 month period indicated 20-30% loss of potency of ciprofloxacin whereas there was no HPLC detectable degradation if the eye drop container were kept inside its carton (Table 1). On the other hand, sunlight-exposed samples showed slightly lesser antimicrobial activity than the control sample against Bacillus pumillus (Figure 5).

Pharmaceutical products, especially eye drops are exposed to light while being:

* manufactured as solution.

* held in pharmacy retail shops or in hospitals pending use.

* held by consumers pending use or while in use over the period of treatment.

* exposed to sunlight if one goes into sun immediately after instillation of drops.

The marketed samples examined by HPLC did not show any degradation at the beginning which means that no degradation had taken place during manufacturing or as long as the product remained in the carton after manufacturing. It was also found that no photodegradation occurred in eye drop products exposed to sunlight while covered by packaging container. Thus, as long as the containers are inside the carton there is little or no photodegradation. However, if the containers are translucent, the eye drop containers should not be exposed to sunlight. It is also advisable not to go out in sun immediately after instillation of ciprofloxacin eye drops without sunglasses. The use of amber colored containers or light-reflecting containers could be an alternative solution for photosensitive eye drop preparations.

ACKNOWLEDGMENTS

This research was supported in part by grant RR16477 from the National Center for Research Resources awarded to the West Virginia Biomedical Research Infrastructure Network (under which Faculty summer fellowship awarded to Jiben Roy while he was a faculty at Salem International University). The authors acknowledge the support of Square Pharmaceuticals Ltd, Dhaka, Bangladesh for allowing us to conduct part of this research work.

LITERATURE CITED

Condorelli, G., G.D. Guidi, S. Giulfrido, S. Sortino, R. Chilleni, and S. Sciuto. 1999. Molecular mechanisms of photosensitization induced by drugs XII Photochemistry and photosensitization of rufloxacin: An unusual photodegradation path for the antibacterials containing a fluoroquinolone-like chromophore. Photochem. & Photobiol. 70: 280-286.

Fasani, E., A. Profumo, and A. Albini. 1998. Structure and medium-dependent photodecomposition of fluoroquinolone antibiotics. Photochem. & Photobiol. 68:666-674.

Phillips, G., B.E. Johnson, and J. Ferguson J. 1990. The loss of antibiotic activity of ciprofloxacin by photodegradation. J. Antimicrobial Chemother. 26:783-789.

Smith, A., P.M. Pennefather, S.B. Kaye, and C.A. Hart. 2001. Fluoroquinolones-place in ocular therapy. Drugs. 61(6):747-761.

Sunderland, J., C.M. Tobin, A.J. Hedges, P. Alasdair, A.P. MacGowan, and L.O. White. 2001. Antimicrobial activity of fluoroquinolone photodegradation products determined by parallel-line bioassay and high performance liquid chromatography. J. Antimicrobial. Chemother. 47:271-275.

Sunderland, J., C.M. Tobin, L.O. White, A.P. MacGowan, and A.J. Hedges. 1999. Ofloxacin photodegradation products posses antimicrobial activity. Drugs. 58 (Suppl2):171-172.

The Fluoroquinolone Toxicity Research Foundation, Webpage: http://fqresearch.org/m23.htm; last entered on 10/28/2004)

Tiefenbacher, E., E. Haen, B. Przybilla, and H. Kurz. 1994. Photodegradation of fluoroquinolones used as antimicrobial therapies. J. Pharm Sci. 81:463-467.

Tobin, C. Sunlight, eyes and light-sensitive antibiotics. Southmead Hospital Bristol, University of Bristol, Webpage (Iris fund home page, http://www.irisfund.org.uk/sunlight.htm; last entered on 9/02/2004).

Torniainen, K., J. Mattinen, C.P. Askolin, and S. Tammilehto. 1997. Structure elucidation of a photodegradation product of ciprofloxacin. J. Pharm. Biomed. Anal. 15:887-894.

United States Pharmacopoeia, XXV, page 424.

Jiben Roy (2), Diaa M. Shakleya (3), Patrick S. Callery (3), Dilip K. Sarker (4), Anwar H. Miah (4), and Subodh C. Das (4)

(2) Division of Science & Math, Mississippi University for Women, Columbus, MS 39701; (3) School of Pharmacy, West Virginia University, Morgantown, WV 26506; and (4) Product Development Department, Square Pharmaceuticals Ltd, Pabna, Bangladesh

(1) Presented as a poster in MAS 69th Annual Meeting, Feb 17, 2005 Oxford, Mississippi.

(2) Author for Correspondence. Department of Chemistry; Phone: [phone]; Email: [e-mail]

Table 1. Natural sunlight induced (3 months) loss of potency of
ciprofloxacin in different packaging conditions.

                                                   Loss of potency of
                                                   Ciprofloxacin (%)
Products                  Packaging conditions     N = 3

Sample A: (Ciprofloxacin  Translucent plastic      28.5 [+ or -] 1.4
eye drop manufactured in    container without
Bangladesh)                 carton
Sample B: (Ciprofloxacin  Translucent plastic      19.2 [+ or -] 1.1
eye drop manufactured in    container without
USA)                        carton
Sample A                  Amber colored plastic    8.4 [+ or -] 0.5
                            container without
                            carton
Sample A                  Translucent plastic      No loss detected by
                            container with carton    HPLC
Sample A                  Amber colored plastic    No loss detected by
                            container with carton    HPLC