Download effect of five alive juice on the dissolution and

EFFECT OF FIVE ALIVE® FRUIT JUICE ON THE
DISSOLUTION AND ABSORPTION PROFILES OF
CIPROFLOXACIN
*Akinleye
M.O., Coker H.A.B., Chuckwuani C.M. and Adeoye A.W.
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, College of
Medicine, University of Lagos, Idi-araba, Lagos.
*Corresponding author, e-mail address: [email protected]
Key Words: Ciprofloxacin, Five Alive Citrus fruit and interaction
1
Abstract
Objective: To determine the effect of interaction of five alive fruit juice® on the
dissolution and absorption profiles of ciprofloxacin tablets using urinary excretion.
Methods: In-vitro dissolution of ciprofloxacin 500mg tablets was studied using US
Pharmacopoeia dissolution apparatus II. This was conducted using 0.1N HCl and
equal volumes of the fruit juice and 0.1N HCL. Samples were collected at
predetermined time intervals for two hours. For the in-vivo study, eleven (5 males and
6 females) healthy volunteers participated in an open, single dose, cross-over
randomized trial. After an overnight fasting, each volunteer either ingested a 500mg
ciprofloxacin tablet with 250ml table water or with the fruit juice. Total urine voided
was collected for 72hrs after each dose of ciprofloxacin. Dissolution and urine
samples were assayed using a controlled and validated UV spectrophotometric
analysis.
Results: A lag time of 5 minutes was observed in dissolution profile of ciprofloxacin
in the juice. The percent dissolved of ciprofloxacin in 0.1NHCl and fruit juice were
found to be dissimilar (f2 =18.2) using similarity factor. There was statistical
difference between the Kel(elimination constant), Ke(excretion rate) and Xu
(cumulative amount excreted unchanged) of subjects on water (0.1759±0.0144;
12.81±1.36; 375.5±41.2) and that of fruit juice (0.1250±0.0161; 7.6±1.07;
241.6±34.0). However, there was no difference between the ti/2 of the subjects
(4.2±0.3; 5.5±0.8). There was a decrease in relative availability of the drug by
35.75%.
Conclusion: We conclude that the absorption of ciprofloxacin can be reduced by
concomitant ingestion of the juice containing calcium carbonate and grape. Therefore
to avoid drug therapeutic failures and subsequent bacterial resistance as a result of
subtherapeutic level of the drug in the systemic circulation, ingestion of the juice with
ciprofloxacin should be discouraged.
2
INTRODUCTION
Ciprofloxacin is a fluoroquinolones with a broad spectrum antibacterial activity. It
has become one of the most commonly prescribed drugs for the treatment of a
plethora of bacterial infections including pseudomonas aeruginosa, Neisseria spp and
staphylococci. It has also been found useful in the treatment of enteric fever, fever of
unknown origin (FUO) including anthrax treatment during biologic warfare 1. It is
rapidly and well absorbed from the gastrointestinal tract, widely distributed in the
body with good tissue penetration2 .
Since its introduction into clinical practice, several pharmacokinetic and
pharmacodynamic interactions have been reported 3. Polyvalent cations such as
aluminium, calcium, copper, iron, magnesium, manganese and zinc found in dairy
products (milk, cheese, and yogurt), antacids and mineral supplements can bind
ciprofloxacin, greatly reducing the absorption of the drug 4. Calcium supplements are
known to interfere with the absorption of ciprofloxacin. The same interaction has
been shown to occur when calcium-fortified orange juice is taken at the same time as
ciprofloxacin 5. The general mechanism by which the interactions occur has been
through formation of insoluble metallic ion complex with ciprofloxacin.
Ciprofloxacin has also been reported to increase serum concentrations of some drugs
that are substrates of cytochrome P450 1A2 and 3A4 by inhibition of these metabolic
enzymes 6.
Grape juice also interacts with several drugs particularly those that are substrates of
cytochrome P450 3A4 e.g. dihydropyrines (nifedipine 7,felodipine 8), cyclosporine
9,midazolam 10 etc.
This isoenzyme is found in the liver and in the enterocytes. Some
drugs are thus metabolized as they are absorbed by the enterocytes. Drugs can also be
3
pumped back into the intestinal lumen from the gut wall by a P-glycoprotein
transporter (P-gp). P-gp and CYP3A4 may therefore act in tandem as a barrier to
drugs moving from the gut to the systemic circulation 11. Grape juice appears to
selectively inhibit intestinal CYP3A4 isoenzyme expression 12. Inhibition of either or
both systems can increase the bioavailability of a drug.
Majority of clinically relevant food-drug or drug-drug interactions are caused by
food/drug-induced changes in the bioavailability of the drug. Since the bioavailability
and clinical effect of most drugs are correlated 13, the bioavailability is then an
important pharmacokinetic parameter. However, in order to evaluate the clinical
relevance of a food-drug interaction, the impact of food intake on the pharmacokinetic
and clinical effects of the drug has to be quantified.
It is common place for patients to take drugs with drinks other than water for various
reasons, one of which is to mask the unpleasant taste already associated with some
drugs and secondly, fruit juice is often part of the menu for breakfast in some homes
when medications are taken. Moreover it’s also being used to administer drugs to
children for better compliance. Some of these drinks include fruit juice, milk,
beverages and yogurt. It is not impossible that ciprofloxacin can auto inhibit these
enzymes (cytochrome P450 3A4 & 1A2) and paves way for it entrance into the
systemic circulation, we therefore hypothesized that there may be a relevant
interaction between grape juice; calcium content of the juice and the drug. Of what
effect is the presence of these counteracting components to the pharmacokinetics of
ciprofloxacin? This study was therefore carried out to determine if the in vitro
dissolution release and systemic availability of ciprofloxacin would be altered when
administered with Five alive® citrus fruit juice which contains among others, grape
juice and fortified with calcium.
4
MATERIALS AND METHODS
Drugs, Chemicals and Equipment
Ciprofloxacin standard (Fig. 1) was provided by Fidson Health Care Nigeria Limited
while ciprofloxacin (Ciprotab®) tablets were purchased from a registered
pharmaceutical chemist. 0.1N Hydrochloric acid (HCl) and deionized water were
obtained from Swiss Pharma Nigeria limited. Sontax AT 7 vessel dissolution
apparatus USP II (Bassel, Switzerland) and Agilent 8453 UV/Visible system with
ChemStation software coupled with Hp Computer system; universal paper pH
indicator were used. Five Alive® Citrus Burst fruit Juice(FACBF) (Batch ADJ5107, Reg. 01-5715) and Eva® table water, products of Nigerian Bottling Company
under license and authority of Coca-cola Company, Nigeria were procured from
registered premises. The FACBF contains 43% Lemon, Orange, Lime, Grape juice,
tangerine; calcium phosphate, calcium Lactate (120mg/100ml) with no flavours,
colours or preservative.
IN-VITRO STUDY
Pre-sample tablets analysis was done by evaluating two physicochemical
characteristics vis-à-vis disintegration test and assay in order to ascertain the
suitability of the ciprofloxacin tablets for the studies according to the official
specifications.
Dissolution Testing
A 7-compartment dissolution US Pharmacopoeia apparatus II was maintained at 37±
0.5oC with a degree of agitation of 50revolution/minute and 900ml of dissolution
medium per vessel was used. The dissolution experiment was conducted in two
phases; one involved the dissolution of 500mg ciprofloxacin tablet using 0.1NHCl as
dissolution medium while the other contained equal 450mls of 0.1N HCl and FACBF
juice. Sample (2.0ml) was collected at pre-determined time intervals (5, 10, 20, 30,
5
40, 50, 60, 80 and 120minute). 0.1ml of the sample diluted to 10mls with 0.1NHCl
was filtered with 0.45µm Millipore filter and assayed by UV spectrophotometric
method at a wavelength of 277nm.
Graph of absorbance versus concentration was plotted for the ciprofloxacin standard
solutions, thus the concentration of each sample was determined using the equation of
the calibration. Provision was made in the calculation for each sample volume taken
without replacement.
IN-VIVO STUDY
The study was a randomized, open and cross-over study design in two phases of an
interval of one week wash-out period. Written consent was obtained from 12
volunteers (6 men, 6 women) aged from 21 to 41 years (mean age, 27.4± 7.9 years)
with a mean weight of 63.3±5.6 kg (ranged 57 – 78kg) were enlisted into the study.
The study was approved by Research and Ethics Committee, Lagos University
Teaching Hospital (LUTH). Volunteers were excluded from the study if they had any
known gastrointestinal problem, such as peptic ulcer disease, colitis, nausea,
vomiting, and diarrhea or malabsorption syndromes. No alcohol or citrus fruits or
juices were to be ingested in any form three days pre-treatment day. Volunteers who
were taking any drug or had a history of hypersensitivity to quinolones and fruit juices
were also excluded from the study.
After the subjects fasted overnight, each volunteer then swallowed a single 500mg tablet
of ciprofloxacin (as the hydrochloride salt) with 250ml of either water or FABCF juice
simultaneously at 0900. Zero hour urine was collected. In order to promote adequate
urine flow during the 12 hours of the first day, the subjects were allowed to take water but
debarred from taking breakfast the morning of the study until four hour post drug
administration. The total urine voided was collected, the pH and volume measured and
6
recorded. 20ml aliquots of the urine was taken and stored in refrigerator at 0 to -100C
until analyzed
Analysis of urine samples
A known concentration (10µg/ml) of CP standard was prepared in O.1N HCl and scanned
in UV spectrophotometer to obtain the λmax of absorption of the drug.
Ciprofloxacin stock (5-60µl) solution of 5mg/ml was spiked in 1ml of blank urine to give
a concentration range of 25-350µg/ml. 0.4ml of concentrations (25-350µg/ml) was
further diluted to 10ml with 0.1N HCl to give a concentration range of 1-12µg/ml for the
calibration standard plot (n in triplicate). The regression coefficient, r2 of the calibrator
was determined prior to analysis of the samples.
For the first 24hr urine samples, I in 250 dilutions in 0.1N HCl was used while 48-72hrs
samples were diluted 25 times. Individual zero hour urine was used as blank for analysis
of CP in subject samples. On the spot % recovery was evaluated by comparing the
absorbance of CP spiked in urine with that prepared in 0.1N HCL using UV
spectrophotometer at the scanned wavelength 277nm.
Data Analysis
The concentrations of CP measured in urine were multiplied by the respective urinary
volume to obtain the amount excreted in the sampling time. The dilution of the samples
was taken into account for calculation of the drug concentration. The rate plot (urinary
excretion rate plot versus time) for both CP in juice and water did not yield a straight line
rather the resulting curve was biexponential. The elimination rate constant (Kel) was
calculated by regression of the terminal portion of the log-linear excretion rate-mid time
curve. The ti/2 of elimination was the evaluated from expression 0.693/Kel. The
7
cumulative amount excreted up to 72hrs time (Xu) was obtained by summing the amount
excreted unchanged in each time interval up to that time. The excretion rate constants
(Ke) and Xu were also evaluated using the Microsoft Excel Tools.
Statistical Analysis
Kel, t1/2, Ke were compared by student’s t test of subjects taking ciprofloxacin with water
and FABCF juice. In all cases, a confidence level of 95% (α = 0.05) was chosen for all
statistical test and a value of p≤0.05 was considered significant. Graph pad prism version
4.0 software program and Microsoft Excel Analytical Tools were applied. The dissolution
data were also analyzed by estimation of similarity factor, f2 using dissolution time points
(5 to 40mins) in 0.1N HCl as reference and presented in the following equation.
F2 = 50log{[1 + 1/n∑(Rt – Tt)2]-0.5 x 100}
where Rt and Tt are percent dissolved at
each time point for reference and test respectively. Using the f2 values, dissolution
profiles were considered dissimilar if these values were less than 50 14,15.
RESULTS
The assay and average disintegration time were found to be 104.5 ± 1.2% and 3.0 ±
0.1 minutes respectively. The percent ciprofloxacin released from dissolution in 0.1N
HCl and fruit juice was presented in Fig.II. The results of mathematical comparison
of dissolution time points profile in water and the juice using similarity factor, f2 was
found to be 18.8.
Eleven subjects, 21-42 years of age (mean ± SD., 27.2 ± 8.2 years) with a mean
weight of 63.6 ± 5.7 (ranged 57-78kg) completed the study. Figure IIIa & b
represent the UV spectra of blank urine and spiked drug standard (1-12µg/ml) in urine
at λmax wavelength 277nm. There was an average % recovery of 88.9 ± 11.8 of CP in
8
urine compared to the drug in 0.1N HCl solution at 5 different concentrations. Fig.
IIIc represents the overlaid sample spectra for two subjects.
The mean biexponentials curves of Log excretion rate versus the sampling time of
subjects using water and juice are represented in Figure IV. Figure V represents
average cumulative amount of unchanged CP excreted Plots versus mid-time of both
subjects (water/juice). The statistical student’s t test parametric comparison of urinary
excretion date of the drug taken with water versus juice following oral administration
are shown in Table I . There were significant level of difference between the two
treatment in Kel. Ke and Xu while no significant difference was obtained in elimination
t1/2 . The bioavailability of CP given concurrently with the juice relative to that
administered with table water was 0.64.
DISCUSSION
Pre-sample assay of the ciprofloxacin tablets showed conformity with official
stipulation in the BP 1998 16.
The tablets were found to disintegrate at average time
of 3.0± 0.1 minutes which also fell within the official limit.
The similarity factor, f2 (18.2) confirms dissimilarity between the dissolution of
ciprofloxacin in 0.1N HCl and FACBF juice. The absolute reduction of ciprofloxacin
release in the fruit juice at 5minutes as shown by the lag phase (Fig. II) might be an
indication of complexation between the drug and the calcium ions in the fruit juice.
The manufacturer’s label claim of the Five Alive® citrus fruit juice shows calcium
content of 120mg/100ml while the five fruits (lemon, orange, and lime, tangerine and
grape fruit) constitute 43% of the total juice contents. Out of these ingredients,
9
calcium compounds have been reported 17,18 and extensively implicated in the
interaction with ciprofloxacin both in-vitro and in-vivo. A mean reduction of 30 - 40%
in Cmax and 30 - 51% in plasma AUC and urinary excretion has been reported 19,20,21.
We observed 35.75% reduction in relative availability of the drug in juice. This finding
is in congruence with the earlier reports but however our result could not ascertain if
any contrary effect was contributed by the grape fruit content of the juice. It could well
mean that the calcium carbonate complexation effect overrides the possible grape fruit
inhibition of intestinal enzyme (CytoP4503A4) as reported with some drugs such as
Felodipine, Nifedipine etc. Perhaps, the enzyme inhibition effect may be infinitesimal.
It was also noticed in one of the subject where there was over 90% inhibition of the
drug excreted in the urine using the juice.
There was no gender difference in all the urinary parameters derived except in
excretion rate where the rate tends to be faster in females than the males using the juice
to swallow the drug and this was found to be statistically significant (p≤ 0.006).
We also found out that the rate of absorption in water juice is faster than that of the
juice which explained the decrease in availability of ciprofloxacin in FACBF juice.
Brittan et al. 22, reported a t1/2 of 4.14 hr on oral single dose of 500mg ciprofloxacin
when used with water while t1/2 of 4.2±0.3hr was got in this study.
By way of conclusion, we have confirmed that concomitant administration of
ciprofloxacin in the presence of calcium carbonate and grape fruit in Five Alive® juice
still lead to reduction of bioavailability. The phenomenal increase in bioavailability of
drug co-administered with grape fruit was not noticed. Therefore, the incessant habit of
swallowing drug with fluids other than water should be done with great caution.
10
Acknowledgment
We wish to appreciate the management and staff of Swiss Pharma Nigeria Limited for
making their laboratory space and equipment available for the study.
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11
[9]. Ducharme MP, Provenzano R, Dehoorne-Smith, Edwards DJ. Trough
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13
Table 1
Mean± SEM: Urinary Excretion Data of CP taken with Water versus
Juice
Parameter/
Treatment
Unit
Water
Juice
P-value
N
11
11
t1/2 /hr
4.2±0.3
5.5±0.8
Kel /hr-1
0.1759±0.0144
0.1250±0.0161
* 0.046
Ke /mg/hr
12.81±1.36
7.60±1.07
* 0.007
Xu /mg
375.5±41.2
241.6±34.0
* 0.021
N = number of subject
* = significant
o = not significant
Fig. 1: Structure of ciprofloxacin
O
COOH
F
N
N
N
H
14
o
0.176
Fig. II: Dissolution profile of Ciprofloxacin (500mg) tablets in 0.1N
HCl and Fruit juice as dissolution media
120
% ciprofloxacin released
100
80
60
40
CP500mg in 0.1NHCl
20
CP500mg in juice
0
0
20
40
60
80
-20
time/min
Fig 3a The blank spectrum of urine
15
100
120
Fig. 3b: The processed standard spectra of CP spiked in urine
Fig. 3c: Overlaid sample spectra of recovered CP in two of the
subjects
16
Fig. 4
Mean ± SEM: Average Excretion rate (CP) versus mid-time in
water and juice
Excretion rate (CP),mg/hr
50
40
30
20
10
0
-10
0
20
40
60
mid-time/hr
□ = water
□= juice
17
80
Fig.5: Mean ± SEM. Cumulative amount of ciprofloxacin excreted
unchanged when administered with water and fruit juice
450
400
350
X u/mg
300
250
200
water
150
100
juice
50
0
0
10
20
30
40
50
60
mid-time/hr
18
70
19