Inclusion complexes of chloramphenicol with β-cyclodextrin and aminoacids as a way to increase drug solubility and modulate ROS production

Article abstract of DOI:10.1016/j.carbpol.2014.11.017

The aim of this study was to improve the solubility of chloramphenicol and reduce the production of reactive oxygen species (ROS) in leucocytes induced by this drug, using complexation. Multicomponent complexes were prepared by the addition of β-cyclodextrin with glycine or cysteine. Nuclear magnetic resonance and phase solubility studies provided information at the molecular level on the structure of the complexes and their association binding constants, respectively. In the solid state, all systems were extensively characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy, thermal analysis and X-ray powder diffraction. Antimicrobial activity of inclusion complexes was investigated by agar diffusion methods. Finally ROS determination by chemiluminescence was used to investigate the effect of complex formation on the potential toxicity in human leucocytes. These studies revealed that multicomponent complexes can increase the aqueous solubility of chloramphenicol as well as reducing the stress by ROS production in leucocytes and maintaining its microbiological activity.

Full text of DOI:10.1016/j.carbpol.2014.11.017

Accepted Manuscript
Title: Inclusion Complexes Of Chloramphenicol With
␤-Cyclodextrin And Aminoacids As A Way To Increase Drug
Solubility And Modulate Ros Production
´
Author: Virginia Aiassa Ariana Zoppi Ines Albesa Marcela R.
Longhi
PII:
S0144-8617(14)01122-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2014.11.017
CARP 9445
To appear in:
Received date:
Revised date:
Accepted date:
22-7-2014
1-11-2014
1-11-2014
Please cite this article as: Aiassa, V., Zoppi, A., Albesa, I., and Longhi, M. R.,Inclusion
ComplexesOfChloramphenicolWithrmbeta-CyclodextrinAndAminoacidsAsAWay
To Increase Drug Solubility And Modulate Ros Production, Carbohydrate Polymers
(2014), http://dx.doi.org/10.1016/j.carbpol.2014.11.017
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INCLUSION COMPLEXES OF CHLORAMPHENICOL WITH -CYCLODEXTRIN
AND AMINOACIDS AS A WAY TO INCREASE DRUG SOLUBILITY AND
MODULATE ROS PRODUCTION
Virginia Aiassa a,b, Ariana Zoppi a,b, Inés Albesa a, and Marcela R. Longhi a,b*
a Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba, Córdoba, Argentina.
b Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA),
CONICET, Universidad Nacional de Córdoba, Córdoba, Argentina.
aiassa@fcq.unc.edu.ar, ariana@fcq.unc.edu.ar, inesalbesa@fcq.unc.edu.ar,
10 mrlcor@fcq.unc.edu.ar.
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*Corresponding author. Departamento de Farmacia, Facultad de Ciencias Químicas,
Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina.
Tel.: 54-0351-5353865 fax: 54-0351-5353865 line 53364. E-mail:
mrlcor@fcq.unc.edu.ar
Abbreviations used: ROS: reactive oxygen species, CP: chloramphenicol, CPMC:
chloramphenicol multicomponent complexes, Cys: Cysteine, Gly: Glycine, CD:
cyclodextrins, CL: chemiluminescence, PM: physical mixture, FD: freeze-drying, FT-
IR: fourier-transform infrared spectroscopy, SEM: scanning electron microscopy, DSC:
Differential scanning calorimetry, TG: thermogravimetric analysis, PXRD: Powder X-
ray Diffraction, AA amino acid.
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1. INTRODUCTION
Chloramphenicol (CP, Figure 1A) is an antibacterial drug having a wide spectrum of
activity against Gram positive and Gram negative cocci and bacilli, including anaerobes
(Clinical Pharmacology , 2013; Sweetman, 2009). An idiosyncratic reaction resulting in
aplastic anaemia can occur in predisposed patients that are unpredictable, irreversible,
and frequently fatal, with an incidence of one case in 24,000 to 40,000 courses of
therapy. This is thought to be due to the production by the gut flora of a nitro-reduction
derivative of CP that can induce DNA damage in replicating haematopoietic stem cells,
resulting in marrow hypocellularity and progressive pancytopaenia (Laporte, Vidal,
Ballarin & Ibanez, 1998). However, the most common presentation is a predictable
dose-dependent toxicity resulting in an anaemia that is reversible upon drug withdrawal,
which usually occurs when serum CP levels exceed 25 mg/l for prolonged periods of
time. This is associated with the inhibition of mitochondrial protein synthesis, and is
characterized by mild marrow hypocellularity, anaemia, neutropaenia and
thrombocytopaenia (Walker et al., 1998). In addition as non-idiosyncratic aplastic
anaemia and grey baby syndrome are dose-dependent complications of CP usage, these
are non-hematopoietic toxicities of CP that, if left unrecognized and untreated, often
result in cardiovascular collapse and death (Lam et al., 2002; Wiest, Cochran &
Tecklenburg, 2012).
These serious side effects and even fatal consequences limit the successful use of this
drug with in vitro and in vivo results indicating that the toxicity of diverse
chemotherapeutic drugs involves an increased production of reactive oxygen species
(ROS) and oxidative stress (Butterfield , 2005). Research into the toxicology of CP
indicates that its propensity can cause damage to blood-forming organs which might be
related to its potential for nitro-reduction and subsequent production of nitric oxide.
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These observations suggest that the para-nitro group of CP could be the cause of
haematotoxicity in susceptible people (Holt & Bajoria, 1999).
In addition, CP is slightly soluble in water and has a bitter taste, Shi & Zhou (2011)
demonstrated that CP complexation with cyclodextrins (CD) enhances its solubility and
covers up its bitter taste. CD are in fact water-soluble cyclic oligosaccharides composed
of 6 (-CD), 7 (-CD, Figure 1B) or 8 (-CD) D-(+)-glucopyranose units arranged in a
truncated cone-shaped structure. Different molecules can penetrate into the relatively
hydrophobic cavity and form non-covalent inclusion complexes, thus modifying their
physical, chemical and biological properties (Loftsson & Brewster, 2010; 2012).
Different researchers have reported the preparation and characterization of CP
complexes with cyclodextrins (Hirayama, Usami, Kimura & Uekama, 1997; Mashhood
Ali, Asmat & Maheshwari, 2004; Li, Luo & Liu, 2005; Fatiha, Leila, Eddine & Leila,
2013; Ramos et al., 2013) and demonstrated that the inclusion complexes are an
effective way to increase the solubility of CP (Zuorro, Fidaleo & Lavecchia, 2010, Shi
& Zhou, 2011).
In addition, in recent years the use of a formation of multicomponent complexes with
cyclodextrin and a third auxiliary substance has become more frequent in order to
facilitate the therapeutic application of different drugs (Palma et al., 2009; Mora, Longhi
& Granero, 2010; Garnero & Longhi, 2010; De Melo et al., 2013; Taupitz, Dressman,
Buchanan & Klein 2013; Wang et al., 2013; Chadha et al., 2014). This approach
permits a lower dose of cyclodextrins to be used and increases the complexation
efficiency. Moreover, by specifically selecting of the auxiliary substance added to the
multicomponent complex, it is possible to reduce other problematic behaviours of the
drugs such as their toxicological properties.
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It is known that thiol compounds such as cysteine (Cys, Figure 1C) and reduced
glutathione (GSH) play an important role in human aging and age related diseases. In
fact, Cys supplementation has been shown by antioxidant actions to ameliorate several
parameters which are known to degenerate during human aging. The decline in
Cys efflux during aging observed in erythrocytes is an important factor in the
development of oxidative stress (Kumar & Maurya, 2013). Also, a protective role has
been suggested for glycine (Gly, Figure 1D) action against the anoxic and oxidative
stress produced by toxic agents (Qu, Ikejima, Zhong, Waalkes & Thurman, 2002).
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Fig 1. Chemical structure and proton atom numbering scheme of: (A) CP, (B) βCD, (C)
Gly and (D) Cys.
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The present work was carried out with the purpose of evaluating the effect of CP-
CD-antioxidant complexation on CP aqueous solubility, antimicrobial activity and
oxidative stress production in human leucocytes. CP multicomponent complexes with
CD and antioxidants (Cys or Gly) were prepared in solution and in solid state with the
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90 effects of complexation on drug solubility, affinity constant (KC) and stoichiometry for
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the complexes being determined in aqueous solution, by means of phase-solubility
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studies and by H NMR and 2D NMR spectroscopy. In the solid state, the
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multicomponent systems, prepared by means of a simple physical mixture (PM) or by
freeze-drying (FD) methods, were studied by fourier-transform infrared spectroscopy
(FT-IR), scanning electron microscopy (SEM), thermogravimetric analysis (TG) and
Powder X-ray Diffraction (PXRD). The antimicrobial activity of inclusion complexes
was investigated by the agar diffusion methods against the Gram-positive species
Staphylococcus aureus and the Gram-negative species Escherichia coli and
Pseudomonas aeruginosa. Finally, the potential toxic effects on human leucocytes were
evaluated by using assays of ROS determination by chemiluminescence (CL).
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2. MATERIALS AND METHODS
2.1. Chemicals and reagents
CP base and Gly were obtained from Parafarm (Argentina). βCD (MW = 1135) was
kindly supplied by Roquette (France). Cys, Ficoll-Hypaque (Histopaque-1077), dextran,
luminol (4-amino-2,3-dihydro-1,4-phthalazine-dione) and the D2O 99.9 atom % D used
in spectroscopic studies were purchased from Sigma®. All other materials and solvents
were of analytical reagent grade. A Milli-Q Water Purification System (Millipore, USA)
generated the water used in these studies.
2.2. Phase solubility analysis
Experiments were carried out at 25°C in stoppered glass tubes containing an excess
amount of CP (100 mg) with Cys or Gly 10 mM, and 5 ml of βCD with increasing
concentrations (0–13.2 mM), according to the method reported by Higuchi & Connors
(1965). The tubes were placed in a 25.0 (±0.1) ºC thermostatized water bath (Circulators
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HAAKE F3-K, Germany) for 72 h, and the resulting suspensions were vortexed 15 seg
twice a day to achieve equilibrium. Each sample suspension was filtered through a 0.45
m membrane, and the filtrate was appropriately diluted for quantitative analysis of CP
in each multicomponent complex by the HPLC method. The HPLC equipment
consisted of an Agilent S1100 system with UV detection at 278 nm, using a Luna C8
(4.6 x 150 mm, 5 µm, Phenomenex, USA) reversed-phase column with pre-column. The
mobile phase was acetonitrile used with a pH 3.0 potassium phosphate buffer (10 mM)
at a 50: 50 mixture, and a flow rate of 1.0 ml/min. Assays were performed at 25 ºC, by
injecting 10 µl of solution in each chromatographic run. Under these conditions, the
retention time for CP was 4.4 min. Each experiment was repeated at least three times,
125 and the results reported are the mean values. The stability constant KC values for the
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corresponding complexes were calculated from the slope of the phase-solubility
diagrams and S0, according to Eq. (1):
KC= slope / S0 (1- slope)
Eq. (1)
The stability of the drug was determined in water at 25 ºC, with no drug degradation
being found after 72 h of incubation.
2.3. NMR studies
The NMR spectra of multicomponent systems were recorded on a Bruker Avance II
High Resolution Spectrometer equipped with a Broad Band Inverse probe (BBI) and a
134 Variable Temperature Unit (VTU). The spectra were measured at 298 K. D2O was used
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as solvent and the resonance at 4.8 ppm due to the residual solvent (HDO) was used as
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the internal reference. Induced changes in the H chemical shifts for CP, CD, Gly and
Cys (Δδ), which originated due to their complexations, were calculated using the
following equation:
Eq. (2)
∆δ = δcomplex - δfree
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The 2D-ROESY spectra of the multicomponent systems were recorded on the same
spectrometer, with a relaxation delay of 2 s and 32 scans.
2.4. Preparation of complexes in solid state
The preparation of solid CP multicomponent systems with a 1:1:1 molar ratio was
carried out using the freeze-drying (FD) method. Solutions of CP with βCD and Cys or
Gly were prepared in water, frozen at – 40 °C and then freeze dried (Freeze Dri 4.5
Labconco Corp., Kansas City, MI). Physical mixtures (PM) were prepared by mixing
147 CP with βCD and Cys or Gly powders, uniformly in a mortar at a 1:1:1 molar ratio.
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2.5. Fourier-transform infrared spectroscopy (FT-IR)
FT-IR spectra of CP, βCD, Cys, Gly and their complexes (KBr disks) were recorded
on a Nicolet Avatar 360 FT-IR spectrometer. The spectra of the complexes were
compared with those of the corresponding 1:1:1 molar ratio physical mixture and also
with the pure CP, βCD, Cys and Gly. All spectra were obtained and processed using EZ
OMNIC E.S.P v.5.1 software.
2.6. Differential scanning calorimetry (DSC) and thermogravimetric analysis
(TG)
The DSC curves of the CP, βCD, Cys, Gly and their complexes were produced using
a DSC TA 2920, and the TG curves were recorded on a TG TA 2920 (TA Instruments,
Inc., New Castle, DE). The samples were placed in aluminium hermetic pans, with the
experiments being carried out under a nitrogen gas flow, at a heating rate of 10 °C/min,
and over a temperature range of 25- 300 °C and 25- 350 °C for the DSC and TG studies,
respectively. In both cases, data were obtained and processed using TA Instruments
Universal Analysis 2000 software.
2.7. Powder X-ray diffraction studies
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The powder X-ray diffraction patterns were obtained with a Philips X’Pert PRO
PAN analytical powder diffractometer (Philips®, The Netherlands), using Ni-filtered
CuKα radiation at 45 kW and 30 mA. The diffractograms were recorded from 2º to 40º
(2θ), with a step size of 0.02° and a scan step time of 2 s.
2.8. Scanning electron microscopy studies (SEM)
Microscopic morphological structures of the solid samples were investigated and
photographed using a Carl Zeiss Σigma scanning electron microscope, at the
Laboratorio de Microscopía y Análisis por Rayos X (LAMARX) of the National Uni-
versity of Córdoba. The samples were fixed on a brass stub using double-sided
aluminium tape, and to improve the conductivity, they were gold/palladium-coated
under vacuum employing a sputter coater Quorum 150. The magnification selected was
sufficient to appreciate in detail the general morphology of the samples under study.
2.9. Antimicrobial activity assay
Antimicrobial susceptibility tests were performed by the agar diffusion method with
some modifications (Zuorro, Fidaleo & Lavecchia, 2010). Briefly, American Type
Culture Collection (ATCC®) bacterial cells of Staphylococcus aureus ATCC® 25923,
180 Escherichia coli ATCC® 25922 or Pseudomonas aeruginosa ATCC® 27853 from an
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exponential-phase culture obtained from a single colony were spread on the surface of
agar plates using a sterile swab soaked in the bacterial suspension diluted to 106 colony
forming units per millilitres (CFU/ml). A disk of 6 mm of sterile Whatman N°1
membrane filters resting on an agar culture medium was inoculated with 10 μl of a
solution containing the free or complexed CP (30 g). Aqueous solutions of pure CD,
Cys and Gly were used as a placebo control. After overnight incubation was performed
at 37 °C, the plates were examined and the diameters of the inhibition zones measured.
Measurements were made in triplicate and the results were averaged.
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2.10. Leukocyte preparation from human blood
Leucocytes were isolated by a combined dextran (Sigma, average MW= 78.000)
/Ficoll-Hypaque (Histopaque-1077, Sigma) sedimentation procedure. Sedimentation in
6 % dextran solution was performed before carrying out gradient centrifugation. A
mixture of Ficoll-Hypaque was then used to isolate the mononuclear cells from the
remaining haematic cells. After sedimentation, hypotonic lyses of the erythrocytes were
performed. The leukocyte layer was washed twice and suspended in Hanks’ balanced
197 salt solution. Cell preparation was adjusted to 106/ml leucocytes for the assay.
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2.11. ROS determination by the chemiluminescence (CL) assay
CL was measured at room temperature using a luminometer (Bio-Orbit 1253, Turku,
Finland) with disposable polypropylene tubes. The basal value of the leucocytes CL was
measured in the presence of luminol (4-amino-2,3-dihydro-1,4-phthalazine-dione,
Sigma). A volume of 0.2 ml of 106/ml leucocytes was incubated with 0.2 ml of 3.4 μM
luminol and: 1) 0.2 ml of CP (0.3 mM), 2) 0.2 ml of βCD (0.3 mM), 3) 0.2 ml of
CP:βCD (0.3 mM of CP, 0.3 mM of βCD), 4) 0.2 ml of CP:βCD:Gly (0.3 mM of CP,
0.3 mM of βCD, 10 mM of Gly) or 5) 0.2 ml of CP:βCD:Cys (0.3 mM of CP, 0.3 mM
206 of βCD, 10 mM of Cys).
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The CL background of each vial was checked before being used and the light
emission was measured for 105 s, at 5 s intervals. Results were expressed in relative
light units (RLU) at different times, with the background value being subtracted
(Caldefie-Chezet et al., 2002).
2.12. Statistical analysis
Data from the ROS determination were statistically assessed by a one-way ANOVA.
Differences were considered to be significant at p < 0.05.
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3. RESULTS AND DISCUSSION
3.1. Behaviour of the multicomponent systems in aqueous solution
3.1.1. Phase solubility analysis
The phase solubility profiles of CP with βCD and Gly or Cys (data not shown) can
219 be classified as AL being of the type according to Higuchi & Connors (1965), and
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showed a linear increase of drug solubility, which is indicative of the formation of
soluble complexes.
The corresponding stability constant values calculated from each phase-solubility
223 diagram were 107 ± 4 and 101 ± 8 M-1 for the systems with Gly and Cys, respectively.
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In both cases, the increase in drug solubility was 1.5 fold. From these values, it can be
deduced that these were similar interactions between the drug and the βCD with each
226 amino acid. The stability constant of the binary complex CP:βCD was also calculated
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for comparison, obtaining a value of 180 ± 12 M-1, which is comparable to that found
for this system for Hirayama et al. (1997). So, it is possible to suggest that the
differences found for the stability constants of the multicomponent complexes regarding
with the binary one may be due to the amino acid that competes with the drug on the
formation of hydrogen bonds with the macromolecule.
3.1.2. NMR Studies
Table 1 presents the assignments of the CP, βCD, Gly and Cys peaks and the
chemical shift deviations due to complexation (proton numbering in Figure 1). By
236 comparing the chemical shifts of the CP:βCD:AA (AA= amino acid) systems with those
237 of the pure components, it is possible to observe significant changes that are indicative
238 of the complex formation between CP and βCD in the presence of each amino acids.
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Firstly, the CP signals show a considerable deshielding as a result of the interaction
with βCD and/or each amino acid. Secondly, the βCD signals are also affected,
241 revealing in this case a marked shielding of the signals corresponding to the H3 and H5
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protons, thus suggesting the insertion of the aromatic ring of the drug into the inside the
macromolecule.
244 Table 1: Chemical shifts displacements for the protons CP, βCD, Gly and Cys in
245 complex forms.
System CP protons
βCD protons
Δδ (ppm)
AA protons Δδ (ppm)
Δδ (ppm)
H1
-
-
H1
-0.0508
-0.0361
H2
H2
H3
H4
H5
H6
H1
H2
H3
H4
H5
H6
H1
H2
H3
H4
H5
H6
-0.0366
-0.1068
-0.0419
-0.184
0.1023
overlapped
0.0962
CP:βCD
H3
H4
H5-H8
H7-H6
H1
0.0891
-0.0429
0.3831
0.0052
-0.077
-0.0489
0.0098
Ha
0.0097
H2
0.1325
overlapped
0.1272
CP:βCD:Gly
H3
H4
-0.0006
-0.1196
0.0009
-0.0067
0.0031
-0.0612
-0.0016
-0.1010
0.0023
H5-H8
H7-H6
H1
0.1270
- 0.0019
0.0149
Ha
Hb
0.0176
H2
0.1094
- 0.1297
CP:βCD:Cys
H3
overlapped
0.1055
H4
H5-H8
H7-H6
0.1060
0.0061
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In order to try to confirm the interaction mode of these systems, the ROESY spectra
(Figure 2) were examined, with intermolecular cross-peaks being observed in these
assays between the aromatic protons of CP (H5-H8 and H7-H6) and the protons located
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250 inside the βCD cavity (H3 and H5). From these results, we can confirm the formation of
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inclusion complexes for the two systems studied, which had similar conformations in
aqueous solution, characterized by a deep insertion of the aromatic ring of CP into the
hydrophobic cavity of βCD. Finally, the results obtained for the binary system CP:βCD
(Table 1) were compared with those of the ternary ones, with differences being
observed in the displacement of the chemical shifts in the presence of the third
component. Additionally, is important to note that the protons of both amino acids also
257 experienced shifts in the CP:βCD:AA complexes. All these observation, allows us to
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hypothesize that Gly and Cys are interacting with the inclusion complex, but without
substantially modifying the CP insertion mode.
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Fig 2. Partial contour plot of the 2D ROESY spectrum of: (A) CP:βCD:Gly system and
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3.2. Behaviour of the multicomponent systems in solid state
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A comprehensive multicomponent system characterization, in solid state, was
performed by applying different analytical techniques, including: FT-IR (Fig. 3), DSC
and TG (Fig. 4), PXRD (Fig. 5) and SEM (Fig. 6). In addition, the behavior of the pure
components was compared with the multicomponent systems obtained by using the PM
or FD methods. Below, we sum up the most relevant results obtained by this
characterization.
Notably, for all of the analytical techniques used, it can be concluded that in the PM
systems the natural structure of each pure component remained revealing that no
interactions took place among them. In contrast, the first evidence of complex
formations in the FD systems prepared with Gly or Cys was obtained by the FT-IR
studies, with the most interesting feature being the modification of the CP bands at 1686
cm-1 (assigned to C=O vibration) and 1559 cm-1 (corresponding to NO2 group). In the
CP:βCD:Gly system, the band at 1686 cm-1 was shifted to 1689 cm-1, while in the
CP:βCD:Cys system the bands at 1686 and 1559 cm-1 decreased notably. The indicating
of these bands may suggest the establishment of intermolecular interactions involving
these groups, thus confirming the formation of a multicomponent complex.
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282 Fig 3. IR spectra of: (A) CP, βCD, CP:βCD:Gly PM system and CP:βCD:Gly FD
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system, (B) CP, βCD, CP:βCD:Cys PM system and CP:βCD:Cys FD system.
Fig. 4 shows the TG and DSC curves of the pure components and multicomponent
systems. The TG results for the FD systems, in both cases, showed that the onset
temperature for thermal decomposition of CP shifted to a lower temperature, probably
due to complex formation. In addition, the DSC curves of pure CP presented a
characteristic melting endothermic peak at 152 °C, whereas the FD systems did not
show any endothermic peak at this temperature, indicating that amorphous solids were
obtained in both these cases.
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Fig 4. TG and DSC curves of: (A) a) CP, b) βCD, c) Gly, d) CP:βCD:Gly FD system
and e) CP:βCD:Gly PM system, (B) a) CP, b) βCD, c) Cys, d) CP:βCD:Cys FD systems
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297 and e) CP:βCD:Cys PM system.
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These thermal analysis results are supported by findings from the PXRD
measurements (Fig. 5), since the extremely low crystalinity of the FD multicomponent
systems suggests the complete amorphization of CP.
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303 Fig 5. X-ray diffractograms of: (A) a) CP, b) βCD, c) Gly, d) CP:βCD:Gly FD system
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and e) CP:βCD:Gly PM system, (B) a) CP, b) βCD, c) Cys, d) CP:βCD:Cys FD systems
305 and e) CP:βCD:Cys PM system.
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Finally, the SEM images of the FD samples (Fig. 6) showed similar morphologies,
which were completely different to those of the pure components. Taken together, all
these experiments confirm the formation of amorphous multicomponent complexes
between CP and βCD, when using Gly or Cys as a third component, for systems
prepared by the FD method.
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Fig 6. Scanning electron microphotographs of: a) CP, b) βCD, c) Gly, d) CP:βCD:Gly
314 FD system, e) CP:βCD:Gly PM system, f) Cys, g) CP:βCD:Cys FD system, h)
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CP:βCD:Cys PM system.
3.3. Antimicrobial activity
The antimicrobial activity of the multicomponent systems was studied in order to
assess the ability of βCD to release the drug from the inclusion complex, taking into
account that a decrease in the drug release capability would in turn cause a reduction in
the antimicrobial potency of the CP present in a given pharmaceutical formulation. The
322 antimicrobial activity of CP multicomponent complex against S. aureus, E. coli and P.
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aeruginosa was investigated by the agar diffusion methods and compared with that of
pure CP. These assays showed that there were no significant differences between the
pure and complexed inhibition zones of CP, for the three species under study. The
inhibition zones were 24.5 + 0.7 mm, 25 + 1 mm, 24.5 + 0.7 mm, 25.5 + 0.5 mm for
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CP, CP:CD, CP:CD:Cys and CP:CD:Gly respectively in S. aureus, 25 + 1 mm for
CP, CP:CD and both multicomponent complexes in E. coli and 25.6 + 0.6 mm, 26 + 1
mm, 26 + 1 mm and 28 + 1 mm for CP, CP:CD, CP:CD:Cys and CP:CD:Gly
330 respectively in P. aeruginosa. These results are indicating that drug complexation did
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not interfere with the microbiological activity of CP and allow us to postulate that the
interactions between the components in the complex were sufficient to improve the
solubility of the drug though not so strong as to reduce its microbiological activity.
Furthermore, in the same test, the use of βCD, Cys and Gly alone did not present any
antimicrobial activity against the species under study.
3.4. ROS determination
Determining the capacity of CP to generate aplastic anaemia requires evaluation of
the metabolic alterations that precede this severe affection. Thus, the development of
toxicity assays could be useful in ameliorating this severe risk. Previous studies have
demonstrated that ROS and nitrite production, together with the alteration of antioxidant
enzymes, may explain the leukotoxicity of CP in neutrophils (Páez, Becerra & Albesa,
2008). In fact, is known that cells contain some antioxidant systems in order to protect
themselves from the injury induced by increased intracellular ROS. Several
investigations have reported that the levels of GSH are modified in response to
anaesthetics and other drugs (Wong et al., 2006; Fernández et al., 2004). Consequently,
it could be interesting to determine the effect of exogenous antioxidants on oxidative
stress caused by CP, especially as the effect of CP combined with antioxidants has not
yet been investigated.
Therefore, in the present study the amino acids Gly and Cys were used as a third
component in the multicomponent complexes, taking into consideration that these two
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352
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amino acids have been previously proposed as antioxidants and cytoprotectors (Deters,
Strubelt & Younes, 1997; Kumar & Maurya, 2013). Now, recalling that the
haematological alteration after therapy with CP seemed to be a consequence of
oxidative damage, the results effectively confirm the presence of oxidative stress of
leukocytes produced by CP, since ROS production measured by CL (Figure 7) was
significantly greater in leucocytes treated with CP (100 g/ml) in comparison with the
358 untreated ones (P<0.05). CD alone did not produce an increase in ROS, while the
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CP:CD complex behaved as pure CP and showed a rise in ROS. Finally when the
multicomponent systems were assayed, the production of ROS was significantly lower
than in the untreated samples, suggesting that Gly and Cys acted as potent ROS
inhibitor/scavengers, thereby indicating the advantages of these multicomponent
formulations containing amino acids in order to reduce the harmful effects of CP
without affecting its therapeutic activity.
365
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Fig 7. Reactive oxygen species induced by Chloramphenicol quantification in human
leucocytes by chemiluminescence assay. The graph shows the treatment for 105 sec.
368 with CP (▲), CD (○), CP:CD (), CP:CD:Gly (■) and CP:CD:Cys (●). For both
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ternary systems, P < 0.05, compared to the untreated sample (◊).
4. CONCLUSIONS
This study has shown that the solubility of CP can be improved by inclusion
complexation with CD and amino acids, with the complexation of this antimicrobial
drug not interfering with its microbiological activity. Moreover, the antioxidants Gly
and Cys when used as a third component were able to produce a protective effect
against the increase of ROS induced by CP in leucocytes. Based on this behaviour of
the multicomponent complexes, it can be concluded that they are potentially useful
application in the future formulation of forms of chemotherapeutic drugs that produce
ROS and oxidative stress.
ACKNOWLEDGMENTS
The authors thank the Secretaría de Ciencia y Técnica de la Universidad Nacional
de Córdoba (SECyT) and the Consejo Nacional de Investigaciones Científicas y
Tecnológicas de la Nación (CONICET) for financial support. We also thank Ferromet
385 S.A. (agent of Roquette in Argentina) for its donation of β-cyclodextrin. We are grateful
386 to Dr. Gloria Bonetto for NMR measurements and for her helpful discussions of the 1H
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NMR spectra, and to Dr. Paul Hobson, native English speaker, for revision of the
manuscript.
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391
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Highlights
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• Chloramphenicol multi-component complexes were prepared and characterized.
• The water solubility of chloramphenicol was improved.
• Chloramphenicol production of reactive oxygen species in leucocytes was decrease.
503 • The complexes maintain good antibacterial activities in vitro.
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Graphical Abstract
MULTI-COMPONENT COMPLEX
Chloramphenicol:-cyclodextrin: amino acid
Multicomponent complexes
characterization
Antimicrobial activity
Reactive oxygen species production
Phase solubility analysis
NMR studies
Infrared spectroscopy
X-ray powder diffraction
Scanning electron microscopy
Thermal analysis
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
100
Time (seconds)
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