623707
research-article2015
IJLXXX10.1177/1534734615623707The International Journal of Lower Extremity WoundsVella et al.
Clinical and Translational Research
Factors Affecting Penetration of
Ciprofloxacin in Lower Extremity
Ischemic Tissues
The International Journal of Lower
Extremity Wounds
1­–6
© The Author(s) 2015
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DOI: 10.1177/1534734615623707
ijl.sagepub.com
Janis Vella, BPharm, MSc, PhD1, Maria Vella, BSc, Pharm Sci1,
Kevin Cassar, MD, FRCS, MMed, MD, FRCS1, Liberato Camilleri, BEd, MSc, PhD1,
Anthony Serracino-Inglott, B Pharm, Pharm D, MACCP, MR Pharm S1,
Lilian M Azzopardi, B Pharm, MPhil, PhD, MR Pharm S1,
and Godfrey LaFerla, MD, PhD, MRCS, LRCP, FRCS, FRCS, FEBS1
Abstract
The aims of this study were to evaluate factors influencing the distribution of ciprofloxacin in tissue of patients suffering
from varying degrees of peripheral arterial disease (PAD). Blood and tissue samples were collected from patients undergoing
debridement or amputation procedures and the amount of ciprofloxacin in them was determined using high-performance
liquid chromatography. All patients were administered a 200-mg dose of intravenous ciprofloxacin prior to the debridement
or amputation procedure. Data, including patient gender, age, type of diabetes, presence of neuropathy, medications taken,
and severity of PAD were collected. These data were then analyzed to determine factors influencing the concentrations of
ciprofloxacin in tissue of the lower limbs. The Kruskal-Wallis test, Spearman correlation, and chi-square test were used to
relate covariates and fixed factors with the concentration of ciprofloxacin in tissue. Following bivariate analysis, a 3-predictor
regression model was fitted to predict tissue concentrations of ciprofloxacin given information about these predictors.
Blood and tissue samples were collected from 50 patients having an average age of 68 years. Thirty-three patients were
males and 35 patients suffered from type 2 diabetes. The average number of medications that these patients were taking was
10. The majority of patients (n = 35) were suffering from severe PAD. Tissue concentrations of ciprofloxacin were mainly
related to plasma concentrations of ciprofloxacin, number of medications that the patients were taking and severity of PAD.
Keywords
ciprofloxacin, peripheral arterial disease, diabetes, concentration in tissue, debridement and amputation procedures
Peripheral arterial disease (PAD) is a chronic and debilitating condition mainly affecting the lower limbs.1-3 PAD is
associated with a high risk of morbidity and mortality4 and
patients suffering from it are likely to experience cardiovascular events regardless of whether the PAD is symptomatic
or asymptomatic.5,6
Predisposing factors for developing the condition include
age, male gender, cigarette smoking, diabetes, hypertension,
and dyslipidemia7-10 with cigarette smoking and diabetes
being the most important risk factors.11,12 Patients suffering
from both PAD and diabetes are at an even greater risk of
morbidity and mortality due to cardiovascular disease.13,14
Diabetes mellitus is a major risk factor for both PAD as
well as peripheral neuropathy. Both of these, alone or in
combination, significantly increase the risks of patients for
developing foot ulceration as well as the risk of limb loss.15-17
The risk of lower limb amputation is greater in those
patients suffering from PAD.18-20 It is important that the correct dose of antibacterial is present at the site of infection at
concentrations that are high enough to eradicate the causative organism to avoid the need of surgery in such patients
having compromised circulation.
Among the different types of antibiotics that are given to
treat infected diabetic foot ulcers is ciprofloxacin. The
broad-spectrum fluoroquinolone is active against a wide
range of infections caused by various bacteria, especially
Gram-negative bacteria.21,22
A number of studies documenting the determination of
ciprofloxacin in various types of tissue have been published.23-27 Brunner et al,28 studied the distribution of ciprofloxacin in subcutaneous adipose tissue and skeletal muscle
tissue of healthy individuals. It was found that ciprofloxacin
1
University of Malta, Msida, Malta
Corresponding Author:
Janis Vella, Medicine and Surgery, University of Malta, Msida, MSD 2080,
Malta.
Email: janis.vella@um.edu.mt
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The International Journal of Lower Extremity Wounds 
concentrations attained in the tissue were sub-inhibitory
despite adequate concentrations present in blood.28 In
another study, the same authors stated that there were differences in the pharmacokinetics at the tissue target site when
2 bioequivalent dosage forms of ciprofloxacin were administered to healthy volunteers.29 Licitra et al30 measured ciprofloxacin concentrations in different tissue, including
tissue from the leg and foot. Penetration of ciprofloxacin
into the infected tissue was reported to be excellent.30 In
another study performed on healthy volunteers, Joukhadar
et al31 reported that local warming of the skin resulted in
more ciprofloxacin reaching the lower limbs due to
increased vasodilation indicating that the drug probably has
a greater penetration in tissue that is inflamed than healthy
tissue. This is not consistent with findings made by Müller
et al32 who studied the penetration of ciprofloxacin in
infected diabetic foot ulcers. The authors claimed that there
was no significant difference between the concentrations of
ciprofloxacin in healthy and inflamed tissue.32 The sample
size in these studies, however, was small.
Inlight of this it was decided to measure ciprofloxacin concentrations in the tissue of patients with infected foot ulcers
who are suffering from varying degrees of PAD where tissue
penetration of anti-infective agents might be predicted to be
less due to the presence of the condition. There have been similar studies documenting the determination of other anti-infective agents in the tissue of patients suffering from PAD.33,34
However, the distribution of ciprofloxacin in patients suffering
from diabetic foot ulcers with varying degrees of PAD is not
known and is being investigated in this study.
The aims of the study were to determine concentrations
of ciprofloxacin in plasma and tissue of patients suffering
from varying degrees of PAD. The study also aimed to identify factors influencing the distribution of ciprofloxacin in
the tissue.
Patients and Methods
Degree of Peripheral Arterial Disease
The presence or absence and the severity of PAD were
assessed in the clinical setting. The ankle brachial pressure index (ABPI) and the spectral waveform at the ankle
were recorded. Patients having triphasic waveforms with
an ABPI of 0.91 to 1.29 were considered to have no PAD.
Those having biphasic waveforms and/or an ABPI of 0.51
to 0.90 were suffering from mild to moderate PAD.
Patients suffering from severe PAD were those having
monophasic waveforms and an ABPI lying between 0.00
and 0.50.
Drug Administration
A 200-mg intravenous dose of ciprofloxacin was administered to the patient 30 minutes prior to the surgical intervention via infusion, which lasted about 20 minutes.
Blood and Tissue Sampling
A blood sample ranging between 3 to 5 mL was taken by
the anesthetist present during the debridement or amputation procedure. The sample was collected about 30 minutes after the administration of ciprofloxacin. Blood was
collected peripherally from the region of the cubital fossa
and stored in a red-top Vaccutainer. Following collection,
the blood was allowed to clot, the tube centrifuged and
the upper serum level decanted and stored at −20°C until
analysis.
Tissue samples were taken from the wound by the
surgeon carrying out the debridement or amputation procedure. The average amputation or debridement procedure took between 15 to 30 minutes to be complete.
Each sample was rinsed with sterile saline solution and
stored in sterile plastic containers at −20°C until
analysis.
Patients
Analytical Method
Ethics approval was obtained from the University Research
and Ethics Committee of the University of Malta. Informed
consent was obtained from patients participating in the
study. Inclusion criteria included patients undergoing a
debridement of a foot ulcer or amputation procedure who
were receiving intravenous ciprofloxacin. Exclusion criteria included pregnancy or lactation and those who were
younger than 18 years. Patients who did not give any consent to participate in the study as well as those suffering
from ascites were excluded from the study. A patient profile
form was used to record the patients’ gender, age, weight,
type of diabetes, and presence of neuropathy. The smoking
status, types of comorbidities, number of medications taken,
and creatinine clearance were also recorded on this form.
The concentration of ciprofloxacin was determined from
each patient sample. Ciprofloxacin concentrations were
determined in patient plasma using a previously developed and validated method involving high-performance
liquid chromatography (HPLC).35 The concentration of
ciprofloxacin in tissue was determined using a developed
and validated method, which is similar to that used to
determine the antibiotic in plasma. Five hundred milligrams of patient subcutaneous tissue was accurately
weighed and homogenized. Protein precipitation was carried out to separate the antibiotic and analysis was carried
out on a Varian HPLC unit using the same chromatographic conditions as those used to determine ciprofloxacin in patient plasma.
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Vella et al.
Data Analysis
Table 1. Most Common Patient Comorbidities (N = 50).
Data about PAD severity, the concentrations of ciprofloxacin in plasma and tissue and that collected from the patient
profile form were processed using SPSS statistics 22. Data
were analyzed for linear regressions and also for descriptive
statistics. The Kruskal-Wallis test, Spearman correlation,
and chi-square test were used to correlate covariates and
fixed factors with the concentration of ciprofloxacin in tissue. Parameters which could affect the concentration of ciprofloxacin in patient tissue were determined by means of
the parsimonious model. The parameters that were considered for the model were age, gender, weight, HbA1c levels,
creatinine clearance, number of medications that the patient
was taking, smoking status, the type of diabetes that the
patient was suffering from, and presence or absence of neuropathy. Other parameters which were considered were the
type of wound and severity of PAD. Backward elimination
was then carried out to exclude factors which were considered to be insignificant due to the large P value attained and
a 3-parameter regression model was developed.
From the patient samples included in the study, results
from 10 of these patients were randomly selected to test the
developed regression model. Data from the most significant
3 predictors were inputted to work out the expected tissue
concentration using this model. The calculated value for ciprofloxacin tissue concentration obtained from the regression
model was then compared with the already calculated ciprofloxacin tissue concentration (which was previously determined by HPLC).
Comorbidity
Results
Patient Characteristics
Blood and tissue samples from a total of 50 patients were used
in the study. Thirty-three of these patients were male and 17
female. The patients’ age ranged from 28 to 92 years with a
mean age of 68 years. The patients’ weight ranged from 59 to
165 kg with a mean weight of 80 kg. The majority of patients
(n = 38) had never smoked. Eight patients were nonsmokers but
had smoked in the previous 10 years. Four patients were current
smokers. The average number of medications that each patient
was taking was 10 (range 1-18 different types of medication per
patient). The most common comorbidity that these patients
were suffering from was diabetes (n = 49). Table 1 shows the
most common comorbidities in this group of patients.
Fourteen patients were suffering from type 1 diabetes
and 35 patients were suffering from type 2 diabetes. Only 1
patient did not suffer from diabetes. Out of those suffering
from diabetes, 3 were on diet alone while 32 were on oral
hypoglycemic agents. Twenty-six patients were using insulin while 13 patients were controlling diabetes using both
oral hypoglycemic agents and insulin.
Diabetes mellitus
Hypertension
Ischemic heart disease
Congestive heart failure
Renal impairment
Hypercholesterolemia
Hyperlipidemia
Hypothyroidism
Prostatic hyperplasia
Cataracts
Gout
Anxiety and depression
No. of Patients Suffering
49
40
29
16
9
7
5
4
3
3
2
2
The average HbA1c level for this population of
patients was 8% (range 5% to 14%) and the majority of
patients (n = 44) were suffering from neuropathy. In this
population of patients, the mean creatinine clearance,
which was measured on admission was 68 mL/min (range
10-177 mL/min). Thirty patients had no renal impairment, 13 patients had mild renal impairment, 3 patients
had significant renal impairment while 4 patients suffered from severe renal impairment.
The majority of patients (n = 39) underwent surgery
involving a toe amputation, 5 underwent surgery involving
trans-metatarsal amputation, and 6 patients underwent
debridement of a foot ulcer. Only 3 patients had nil or borderline PAD. The rest of the patients were suffering from
mild to moderate (n = 12) and severe PAD (n = 35). None of
the patients in this group were suffering from chronic
venous insufficiency.
A positive correlation was seen between the presence
and severity of PAD and gender (P = .05). One male
patient and 2 female patients had nil/borderline PAD.
There were more male patients (n = 12) than female
patients (n = 1) suffering from mild/moderate PAD and
20 male patients and 14 female patients were suffering
from severe PAD. One patient had nil/borderline PAD
and did not suffer from diabetes. The largest number of
patients (n = 23) suffering from severe PAD had type 2
diabetes. Eleven and 3 patients suffering from type 1 diabetes had severe PAD and mild/moderate PAD,
respectively.
Concentration of Ciprofloxacin
Patient plasma ciprofloxacin concentrations were higher
than those present in tissue. The average concentration of
ciprofloxacin in plasma was that of 2.14 µg/mL (range 0.008.00 µg/mL) and that in tissue was 0.28 µg/mL (range 0.002.06 µg/mL).
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The International Journal of Lower Extremity Wounds 
Table 2. Testing the Regression Model.
Patient
Code
3.
7.
12.
15.
21.
25.
26.
28.
40.
43.
No. of
Medications
Conc. of
Ciprofloxacin in
Plasma (µg/mL)
10
13
7
9
9
10
3
7
7
12
2.07
1.78
3.41
1.95
0.99
1.95
1.60
3.96
2.80
0.10
Severity of PAD
Conc. of Ciprofloxacin
in Tissue Calculated
by Model (µg/mL)
Conc. of Ciprofloxacin
in Tissue Determined
by HPLC (µg/mL)
Severe
Severe
Severe
Severe
Severe
Severe
Severe
Mild/moderate
Mild/moderate
Mild/moderate
0.12
0.11
0.11
0.12
0.14
0.11
0.18
0.32
0.34
0.34
0.13
0.13
0.11
0.11
0.11
0.11
0.18
0.37
0.35
0.33
Abbreviations: Conc., concentration; HPLC, high-performance liquid chromatography; PAD, peripheral arterial disease.
Pharmacokinetic Models
Testing the Regression Model
The parsimonious model for ciprofloxacin tissue concentration includes 3 predictors: serum ciprofloxacin concentration, number of medications administered to patients, and
the severity of PAD.
Peripheral arterial disease severity was the best predictor,
followed by the number of medications and the concentration
in patient plasma. The coefficient for the concentration in
plasma (0.0175) indicates that for every 1 unit increase in
plasma ciprofloxacin concentration the concentration in tissue
is expected to increase by 0.0175 units. The coefficient for the
number of medications (−0.0093) indicates that for every 1
unit increase in the number of medications the concentration
in tissue is expected to decrease by 0.0093 units. The regression coefficient for nil/borderline PAD (1.3416) indicates that
the concentration in tissue for this category is expected to be
1.3416 units higher compared with the group of patients suffering from severe PAD. The coefficient for mild to moderate
PAD (0.215) indicates that the concentration of ciprofloxacin
in tissue in this category is 0.215 units higher than in the one
for severe PAD given that other effects are kept constant.
The regression model relating concentration in tissue to
PAD severity, plasma ciprofloxacin concentration and number of medications is
Table 2 shows that the values calculated by HPLC analysis
were comparable to those estimated by the regression model
and differences were not significant. The average results for
the concentration of ciprofloxacin in tissue calculated by
the regression model and by HPLC were 0.125 µg/mL
(±0.007), 0.12 µg/mL (±0.014), 0.11 µg/mL (±0.000), 0.115
µg/mL (±0.007), 0.125 µg/mL (±0.021), 0.11 µg/mL
(±0.000), 0.18 µg/mL (±0.000), 0.345 µg/mL (±0.035),
0.345 µg/mL (±0.007), and 0.335 µg/mL (±0.007).
Expected Ctissue = 0.2366 + 1.3416 ( PAD1 )
+ 0.215 ( PAD 2 ) − 0.0093 ( Meds )
+ 0.0175Cplasma
where Ctissue= tissue concentration (µg/g), PAD1 = 1 for
patients with nil/borderline PAD, PAD1 = 0 for patients
with mild/moderate or severe PAD, PAD2 = 1 for patients
with mild/moderate PAD, PAD2 = 0 for patients with nil/
borderline and severe PAD, Meds = number of medications
taken and Cplasma = plasma concentration (µg/g).
Discussion
The concentration of ciprofloxacin in the subcutaneous tissue of this group of patients is related to the concentration
of ciprofloxacin in plasma, the number of medications that
the patients are taking and also on the severity of PAD. In a
similar study conducted by Zammit et al34 using gentamicin
as the anti-infective agent of interest, gentamicin tissue concentrations were found to mostly depend on gender and
degree of PAD severity.
Ciprofloxacin plasma concentrations were found to be
higher in the plasma than in tissue at the time of sampling.
These findings agree with those made in previous pharmacokinetic studies where ciprofloxacin plasma concentrations were higher than those in tissue after 30 to 60 minutes
from administration.28,29,32
Ciprofloxacin tissue concentrations were found to be
lower in patients suffering from more severe forms of PAD.
These expected results indicate that poor peripheral perfusion in patients suffering from more severe forms of the disease was probably the reason why ciprofloxacin
concentrations were lower at the site of interest. In cases
where inadequate concentrations of anti-infective agents
are reached in the affected area, dose adjustment would be
necessary to help achieve concentrations which are above
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Vella et al.
the minimum inhibitory concentration for most bacteria
causing the infection.
Patients taking a greater number of medications to treat
their comorbidities were found to have lower ciprofloxacin
tissue concentrations. It is unlikely that drug interactions
due to enzyme metabolism and competitive inhibition affect
concentrations of ciprofloxacin present in tissue.
Ciprofloxacin itself is mostly excreted unchanged with
about 10% to 20% of the administered dose being
metabolized.36(p79),37 Ciprofloxacin is an inhibitor of
CYP1A2 and therefore affects the metabolism of other
drugs being metabolized by that same enzyme.38
A reason that could justify the relationship between tissue concentrations of ciprofloxacin and the number of medications that these patients were taking could be due to
comorbidities present. On observing the comorbidities
present in this group of patients, it could be noted that the
most common ones were diabetes, hypertension, ischemic
heart disease, and congestive heart failure. These 4 conditions are often related to vascular rarefaction.39-43
Rarefaction can be a consequence of vascular remodeling
and is related to peripheral resistance and to vessel inflammation and damage. This peripheral resistance can stem
from vascular dysfunction and this can be with or without
structural changes.43 Rarefaction could therefore be a possible reason why tissue concentrations of ciprofloxacin
were found to be lower in patients taking a greater number
of medications to treat comorbidities.
The predicted values of tissue concentrations estimated
using the parsimonious regression model were very similar
to those obtained using HPLC. This indicates that the model
is effective in predicting what concentration of ciprofloxacin is likely to reach the affected tissue. Keeping the factors
making part of the regression model in mind, will help predict whether the concentration of ciprofloxacin in tissue lies
above the minimum inhibitory concentration for the bacteria causing the infection. The developed model can be
effectively applied in a clinical setting to determine the concentration of ciprofloxacin at the site of interest. By knowing the number of medications that the patient is on to treat
existing comorbidities, the concentration of ciprofloxacin
in the patient’s plasma and the degree of severity of any
PAD present, the concentration of ciprofloxacin in the tissue can be determined. This might help the clinician determine how much of the drug is reaching the affected area and
if this concentration is enough for successful treatment of
the infection. This would enable administration of the
appropriate dose of antibiotic to ensure that the tissue thus
possibly reducing the need for surgical interventions.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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