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Tetracyclines and fluoroquinolones are documented to produce chelates with multivalent ions. Administration of these drugs with drugs containing multivalent ions, calcium supplements, and milk is contraindicated. There is no indication on the use of these drugs with foods containing/fortified with calcium like fruit juices. This study aims to analyze the effects such foods have on intestinal absorption with these drugs.
Caco2 and MDCK cell monolayers simulated the transepithelial transport of the drugs across a 37Â°C diffusion chamber sampled every 15 minutes for 90 minutes. Transport was examined in juice and buffer with/without fortification with calcium. Transport was determined by reverse phase HPLC using a Jupiter C18 column. The mobile phase contained 0.1M citrate buffer: acetonitrile: methanol 76:14:10(v/v/v) for ciprofloxacin and 70:20:10(v/v/v) for doxycycline and a flow rate of 1.5ml/min. Effluents were analyzed using UV at 280nm.
Addition of calcium to a media decreased the transport across the monolayer with respect to that media. The apparent permeability coefficient for ciprofloxacin and doxycycline in media containing varying amounts of calcium is highest in assay buffer II, without calcium, resulting in a permeability of 35.35Â±2.6x10-5cm/sec and 24.25Â±1.2x10-5cm/sec for ciprofloxacin and doxycycline respectively. Permeability is also significantly lower in calcium fortified orange juice compared to regular orange juice.
Concomitant use of products containing/fortified with calcium ions can significantly affect the bioavailability of tetracyclines and fluoroquinolones, reduce therapeutic efficacy thereof, or even result in development of bacterial resistance. Therefore patients should be warned against use of fortified products to avoid potential drug-food interactions.
Doxycycline, Ciprofloxacin, Calcium, Permeability, Caco2
Tetracyclines and fluoroquinolones collectively constitute an important part of the antibiotic regimen widely prescribed by physicians all over the world. In general, these drugs are highly effective against a wide variety of both gram negative and gram positive bacteria. Tetracyclines have the broadest spectrum of antimicrobial activity among all the classes of antibiotics. They exert their action by inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex by binding to the 30S ribosomal subunit in the mRNA translation complex (1). On the other hand fluoroquinolones act by inhibiting Topoisomerase II (DNA gyrase) in gram negative bacteria and topoisomerase IV in gram positive bacteria (2).
Both of the above mentioned classes of antibiotics are known to interact with multivalent anions like calcium, magnesium and zinc etc. The concomitant administration of fluoroquinolones and tetracyclines with multivalent metals has been shown to lead to chelation of the drug, precipitation of the chelate, and reduced absorption in gastrointestinal tract thus reducing overall bioavailability. This reduction in bioavailability can result in failure of antibiotic therapy and even development of resistance to the antibiotic (3-11). Therefore, it is indicated to have a time period of at least 2 - 6 hours between the administration of these drugs and other drugs containing multivalent ions like antacids, sucralfate, bismuth, iron, and calcium supplements.
Although the US Food and Drug Administration (FDA) does recognize the effects of calcium on the usefulness of these classes of drugs current labeling only has contraindications against administration with calcium supplements, antacids, and dairy products (although they are not contraindicated when taken with food). In the modern food industry with a high increase in products fortified with vitamins, minerals and calcium these products at times meet or even exceed the amount of divalent ions present in supplements and with patients being given little or no warning by the prescriber or the pharmacist against the use of such dietary products concurrently with drugs like tetracyclines and fluoroquinolones, it becomes more essential to study and characterize the significance of such food and drug interactions affecting the absorption of these drugs through the gastrointestinal tract. Especially given children, for whom the dosage level is most critical, are often given juices with which to take their medication. Previous clinical trials have also shown that calcium fortified orange juice reduces the bioequivalence of fluoroquinolones to significantly lower than that of the drugs delivered with water (12-14).
Studying and predicting the in vivo intestinal absorption or transport of compounds through epithelial barriers has become easy due to the advent of various cell culture models based on human cell lines (15, 16). Transport of antibiotics and antihypertensive drugs has been earlier studied using Caco-2 (human colonic carcinoma) cells (17, 18). Madin Darby Canine Kidney (MDCK) cells are used as models to study the transport of drugs across the blood brain barrier as they express P-glycoprotein and also express the tight junction proteins Claudin 1,4 and occludin which are important to form a paracellular barrier with tight junctions (19).
The current study was aimed to evaluate the effect of calcium fortified fruit juices on the intestinal absorption of the drugs doxycycline and ciprofloxacin. Various models are available as predictive tools to estimate the intestinal absorption of the drugs and to study the drug - drug and drug - food interactions causing impaired absorption. For this study we have chosen to use a model based upon transport across cellular monolayers grown on permeable membranes. Caco-2 cell monolayers cultured on permeable membrane systems are one such model widely used for this purpose owing to its large resemblance to the normal intestinal membrane (17, 18). Caco2 cells are derived from human colorectal carcinoma and grown on micro porous membranes to produce monolayer of cells which undergo epithelial differentiation. The Caco2 cell line was employed for the study; additionally MDCK cells were used for comparative analysis (19).
MATERIALS & METHODS
Chemicals & Reagents
The drugs ciprofloxacin and doxycycline were purchased from PCCA. Regular and fortified juice was purchased from No Frills grocery store. Methanol, acetonitrile, cell culture media and supplements were purchased from Fisher Scientific. Other chemical reagents used to prepare assay buffers which include sodium chloride, potassium chloride, HEPES, glucose, potassium phosphate, and calcium chloride were purchased from Fisher, Sigma and Spectrum.
Caco2 and MDCK cells were purchased from the American Type Culture Collection (ATCC) and were cultured using a slight modification of the ATCC standard culturing protocol using Minimal Essential Medium with 20% fetal bovine serum and Dulbecco's modified Eagle's medium with 10% fetal bovine serum respectively. Each growth media was also supplemented to 0.1mM nonessential amino acids, 1mM sodium pyruvate, 100U/ml penicillin/streptomycin, and 1mM L-glutamine. Cells were cultured at 37Â°C in a humidified incubator under 5% CO2.
For intestinal absorption studies, following trypsinization and formation of a single cell suspension, cells were seeded onto polycarbonate transwell filter plates (Corning, Costar) at approximately 50,000 cells per ml, and cultured until confluent monolayers were produced. Media was changed every 2-3 days and transepithelial electrical resistance (TEER) values were obtained using the Millipore Millicell-ers TEER meter. Monolayer readiness was determined based upon a stabilized TEER value of 350(Î©/cm2) or higher for Caco2 cells and 1500 (Î©/cm2) or higher for MDCK cells. As well as the status of a co-seeded 6 well culture plate, was used to help estimate the health and condition of the cells in the opaque transwell plate.
Preparation of Buffers and diffusion media
Assay buffer II (Sodium chloride 7.13 g/l, Potassium chloride 0.73g/l, Sodium bicarbonate 2.10 g/l, HEPES 2.38 g/l, Glucose 1.80 g/l, Potassium phosphate 0.07 g/l) without divalent ions was used as the transport buffer. Addition of divalent ions was avoided to prevent the precipitation of drugs. The buffer was prepared by dissolving the above mentioned chemicals in deionized water with stirring. The final pH of the buffer was adjusted to 7.4 by adding hydrochloric acid. The orange juice samples were centrifuged for 20 minutes at 10000 rpm (Sorvall Discovery 90se) in order to remove the fibrous matter. The supernatant collected was adjusted to pH 7.4 using sodium hydroxide prior to use.
Transepithelial transport studies
Transport studies were performed using Permegere side by side jacketed diffusion chambers with an N of three chambers/samples per variable. Prior to each use chambers were thoroughly cleaned, rinsed with deionized water followed by 70% isopropanol, and allowed to dry completely. The temperature was maintained at 37Â°C by circulating water from a heated water bath through the external jacket of the chambers. Polycarbonate membranes containing confluent Caco2 or MDCK cell monolayers were aseptically removed from the transwell plates using a scalpel and forceps. The membranes were then placed between the donor and recipient chamber with apical surface of the membrane facing the donor chamber. The donor and receiver chambers were each filled with 3ml of either assay buffer II containing no divalent ions, assay buffer II supplemented with calcium chloride (0.5g/L), orange juice advertised as being without added calcium (~2% recommended daily allowance), or orange juice advertised as having added calcium (~30% recommended daily allowance) and allowed to equilibrate for 10 minutes. The donor chambers were then emptied of the equilibration solution and filled with 3ml of the respective solution containing a calculated 100ïg/ml doxycycline or ciprofloxacin. The total volume (3 ml) from the receiver chamber was withdrawn and replaced with fresh solution every 15 minutes for a total duration of 90 minutes. Samples from the donor chambers were also taken at zero and 90 minutes. The cumulative transport of the drug across the cell monolayer was then calculated by summing concentrations as determined by high performance liquid chromatography (HPLC) at consecutive time points. Apparent permeability (Papp) was calculated for each drug in each respective transport media using the following equation (20):
Papp = Î”Q/Î”tCoA
Where Co is the initial drug concentration in the donor chamber (by HPLC in ïg), A is the exposed surface area of the membrane contained in the diffusion chamber (in cm2), Î”Q is the amount of drug transported (by HPLC in ïg), and Î”t is the time elapsed (in seconds). This flux of drug across the monolayer is represented by the slope of the amount of drug in the receiver chamber versus time curve.
Ciprofloxacin was analyzed by HPLC methods modified and previously published by our lab (21). Doxycycline was analyzed by a slightly modified version of the ciprofloxacin method. The amount of drug transported was calculated by measuring the concentration of samples withdrawn using reverse phase HPLC. A C18 Jupiter column (Phenomonex) was used. The mobile phase comprised of 0.1M citrate buffer/acetonitrile/methanol (76/14/10 for ciprofloxacin and 70/20/10 for doxycycline) adjusted to pH 2.4 using perchloric acid. The flow rate was set at 1.5 ml/minute, and was carried out at room temp. The samples were analyzed using by HPLC - UV detection at a detection wavelength of 280 nm for both the drugs. The total run time for ciprofloxacin was 10 min while the run time for doxycycline was 15 min. Ciprofloxacin was eluted at 4.2 min while doxycycline was eluted at 11.8 minutes.
The amount of drug transported across the cell layers at each time point was determined by HPLC-UV detection at 280 nm. There was no interference from the components of diffusion media or the mobile phase at the retention time of both the drugs. Ciprofloxacin showed a linear response (R2 >0.999) in the range of 0.5 to 25 Âµg/ml, and doxycycline showed a linear response from 0.39 to 100 ïg/ml.
The transport of ciprofloxacin and doxycycline from the apical to basal side of the membrane in paired media containing varying amounts of calcium was studied in 15 minute increments over a total duration of 90 minutes. Figure 1 shows the percent transport of doxycycline (a) and ciprofloxacin (b) across a MDCK cell monolayer over time. Figure 2 indicates the percent transport of doxycycline (a) and ciprofloxacin (b) across Caco2 cell monolayers over time. In all but two paired groups (of 8) the final percent transport in calcium fortified solutions was significantly different (P<0.05 by 2 tailed t-test) than in solutions containing no or minimal calcium.
The permeability of the drugs (doxycycline and ciprofloxacin) was determined across Caco2 and MDCK cell monolayers in assay buffer II with and without calcium, regular orange juice, and orange juice fortified with calcium. The calculated apparent permeability coefficients for both ciprofloxacin and doxycycline under treatments across Caco2 and MDCK cell monolayers are listed in table I. In all solutions the addition (or fortification) with calcium results in a significantly (P<0.05 by 2 tailed t-test) different Papp for both drugs. In all but one of the 8 pairings the addition of calcium resulted in a lowered Papp as much as 9.3 times lower permeability (in the case of doxycycline in orange juice substrate across a Caco2 monolayer). These permeability coefficients are expressed graphically by drug in figure 3.
In the case of transport across Caco2 monolayers (Figure 2) the reduction of calcium ions (within a media) reliably results in an increased percent absorption, as well as an increase in permeability (Figure 3). This trend, however, does not directly relate to the calcium content between different transport media (assay buffer II vs. orange juice). The Papp and percent transport for doxycycline and the Papp for ciprofloxacin in orange juice fortified with calcium, which contains ~30% RDA of calcium (approximately 1.25mg/ml), is significantly higher (P<0.05 by 2 tailed t-test) than the respective Papp with assay buffer II containing only 0.18 mg/ml. In the case of ciprofloxacin transport in MDCK cells this trend is less apparent by Papp or percent transport. However, when viewed in the context of total amount transported we find this trend re-emerges (Figure 4). This suggests that the divalent calcium ions likely resulted in chelation and precipitation of T0 drug, resulting in an artificially enlarged Papp or percent transport.
Tetracyclines and fluoroquinolones are excellent antibiotic drugs. They offer various advantages over other classes of anti-microbial agents, not only do they have a wide spectrum, but their ease of administration, dosing schedule, excellent oral administration, prolonged half life makes them the most commonly prescribed antibiotics despite ever-growing problems with bacterial resistance to these drug classes. In general these classes of drugs are considered safe to be administered with food. FDA requirements make it mandatory for the drug to be tested with a high calorie and high fat diet for it to be labeled as compatible with food. However, recently there has been an increasing trend of food items being fortified with calcium, minerals and vitamins in order to provide a more nutritious diet, but these products can lead to potential drug - food interactions such as chelates which need to be characterized and studied more thoroughly. As previous works have shown reduced bioequivalence in fluoroquinolones co-administered with fortified orange juice (12-14), the above study was aimed to identify the effects of fortified orange juice on the transepithelial transport of tetracyclines and fluoroquinolones. The fortified orange juice used in this study contained approximately 30% of the daily dietary calcium requirement (~1.25mg/ml) which is more than the amount of calcium present in the regular orange juice (~0.08mg/ml) and slightly more than the calcium content in non-fortified milk (~1.1-1.2mg/ml) (7). The assay buffer II with added calcium used in this study contained 0.18mg/ml (~4.4% RDA/240ml).
Fortification of orange juice and the addition of calcium to the assay buffer significantly (P<0.05 by 2 tailed t-test) reduced the apparent permeability coefficient of both the drugs across Caco2 cell monolayer. The percentage drug transport of ciprofloxacin across Caco2 cell layer over a period of 90 minutes was reduced from 54.86Â±3.1% when absolutely no calcium was present (assay buffer II without calcium) to only 24.44Â±6.3% with fortified orange juice which had the highest calcium content (Figure 3B). The total amount of transport with regular orange juice decreased to 35.88Â±1.7% but still it was significantly (P<0.05 by 2 tailed t-test) more as compared to the transport with fortified orange juice. Similar results were observed with doxycycline in Caco2 cells. The percentage transport decreased from 34.62Â±0.7% in assay buffer with no calcium to 23.93Â±0.3% with regular orange juice and 14.16Â±0.6% with fortified orange juice. However, the assay buffer II with added calcium, though it had similar calcium content to the regular orange juice, showed the lowest transport (quantity and percent) producing only 7.7% and 1.9% transport in 90 minutes for ciprofloxacin and doxycycline respectively. This suggests a system that although highly influenced by even small amounts of calcium, is not entirely dependent upon calcium content for these effects.
Similar reductions in the amount of drug transport in the presence of fortified orange juice were observed in MDCK cells; from 10.67Â±0.4 and 19.45Â±3.1% in assay buffer with no calcium, 27.00Â±0.2 and 3.60Â±0.05% with regular orange juice, and 19.56Â±1.7 and 0.78Â±0.05% with fortified orange juice for doxycycline and ciprofloxacin respectively. The assay buffer II with added calcium produced non-detectable transport levels for doxycycline and 44% (of 3.9mg calculated free calcium (Figure 4)) for ciprofloxacin. MDCK cells are more tightly packed compared to Caco2 cells and thus offer less paracellular space. This could explain the relatively lower transport with assay buffer between the two cell lines. However, as the transport of doxycycline in orange juice is relatively unaffected by the cell line (P>0.05) it suggests that perhaps transport occurs in a different manner between transport media as well (Figures 1A and 2A).
The morphology of Caco2 cell layers resembles highly to that of intestinal mucosa and thus they have been widely employed to study the in vitro absorption of drugs. The transport pattern across Caco2 monolayers can be correlated to the intestinal absorption of a drug taken orally. A decrease in the total amount of drug crossing the membrane is an indicator of a reduction in systemic availability of the drug. This reduction in systemic availability can even lead to failure of therapy if the drug concentration fails to reach the minimum inhibitory concentration, which could be fatal.
The study demonstrated that exposure to fortified orange juice or products containing free calcium can significantly decrease the transepithelial transport of ciprofloxacin and doxycycline, and thus the intestinal absorption. Suggesting that the previously observed lack of bioequivalence is likely due to the production of insoluble chelates. Although the event does not appear to be entirely calcium concentration mediated calcium does play a major role. It could be expected to observe similar results when exposing these drugs or other drugs in the same class to other calcium fortified products.
The presence of calcium or additional calcium, with all other things being equal, can significantly reduce the drug available for absorption, the permeability of, and ultimately the bioavailability of the drugs ciprofloxacin and doxycycline. Although there is an apparent inverse relationship between calcium content and transepithelial transport, this also appears to be dependent on the content of the calcium containing media and may produce a greater effect in different media. Though the current contraindication for administering these drugs with milk does help prevent restriction of transport, this study furthers evidence that administration of these drugs with products with even a small amount of calcium may significantly affect bioavailability, and that administration with products either high in or fortified with calcium is likely to result in reductions in bioavailability of a magnitude that they may render the drug ineffective. Patients should be warned against the use of any calcium containing products with tetracyclines and fluoroquinolones to avoid potential drug food interaction.