PHYSICOCHEMICAL CHARACTERIZATION OF
BROMHEXINE HYDROCHLORIDE, LECITHIN AND
WATER IN BINARY AND TERNARY SYSTEMS
M. A. Schubert, C. C. Müller-Goymann
Technische Universität Carolo-Wilhelmina zu Braunschweig, Institut für Pharmazeutische Technologie, Mendelssohnstr. 1, D-38106 Braunschweig
INTRODUCTION
Bromhexine hydrochloride (Br-HCl) is a potent mucolytic
agent that acts by itself and its metabolites e.g. ambroxol. Due
to the low solubility of Br-HCl in water (4.54 mg/g) and a
partition coefficient of 10.60 for n-octanol / buffer of pH 7.4
[1], an investigation of the interactions between the drug and
colloidal drug delivery systems (DDS) seems to be appropriate.
As colloidal DDS model aqueous lecithin dispersions were
chosen.
The aim of the present work is to achieve a better understand-
ing of the mode of interactions between the drug and colloidal
lecithin associates in aqueous systems and to develop a model
that takes into account the structural changes that are observed
by the interactions between drug and carrier.
EXPERIMENTAL METHODS
• Materials
- Bromhexine hydrochloride (Br-HCl) was supplied by
Thomae (D-Biberach/Riß).
- Phospholipon® 90G (P 90G) is a highly purified
sojabean lecithin that contains at least 90% phosphati-
dylcholine. P 90G was supplied by Rhône-Poulenc-
Rorer (D-Köln).
- Water was used in bidistilled quality.
- Deuterium oxide (D2O) was purchased from Aldrich
(D-Steinheim).
• Preparation of binary and ternary systems
All components depending on the composition of the systems
were weighed in sealed containers up to a lecithin concentra-
tion of 20% (m/m). The mixtures were stirred with a Teflon
coated magnet for 20 minutes at 333 K. Stirring was continued
until room temperature was reached.
• Methods
High Performance Liquid Chromatography (HPLC)
Br-HCl analyzis was performed by reversed phase chromatog-
raphy using a column of Hypersil® ODS, 125·4 mm (Grom, D-
Herrenberg). The mobile phase consisted of acetonitrile and
phosphate buffer of pH 7.0 (80:20) with a flow rate 1.5 ml/min
using a spectroflow 400 pump (Kratos, D-Weiterstadt). Peaks
were detected with a Beckman System Gold Detector Module
166 (Beckman, D-München) at a wave length of 240 nm, peak
identification and integration was carried out by Beckman
System Gold Software Version 6.01 (Beckman, D-München).
Calibration was performed within a range of 10 ng/ml to 50
ng/ml with a correlation coefficient of 0.999.
Transmission electron microscopy (TEM)
Binary and ternary systems with a lecithin content up to 20 %
(m/m) were shock-frozen in melting nitrogen at 63 K between
two flat gold holders. The frozen samples were fractured at 173
K in a BAF 400 (Balzers, D-Wiesbaden). Samples were
shadowed with platinum/carbon (2 nm thickness) at 45° and
with pure carbon (20 nm) at 90° for replica preparation. A
chloroform-methanol mixture (1:1 (v/v)) was used for cleaning
the replicas. The replicas on uncoated grids were viewed using
a transmission electron microscope EM 300 (Philips, D-
Kassel).
Small-Angle X-ray Diffractometry (SAXD)
SAXD studies were performed in a compact small-angle
system connected to a PW 2213/20 X-ray tube (Philips, D-
Kassel) with a copper anode. A nickel foil served as Kβ-filter.
The tube voltage was 40 kV and the anode current 25 mA. An
OED 50 position sensitive detector (Braun, D-München) was
linked to a Canberra MCA 8100 multichannel analyser
(Canberra Electronics, D-Frankfurt).
31
P Nuclear Magnetic Resonance (31P NMR)
31
P Nuclear Magnetic Resonance spectra were obtained on a
AC Bruker 200MHz spectrometer (Bruker, D-Rheinstetten) at
293 K with orthophosphoric acid as external reference. The
various systems were prepared as described above and
afterwards sonicated with a Soniprep 150 (MSE Scientific
Instruments, GB-Crawley) for 10 cycles with 60 seconds on
and 60 seconds off each.
SAXD measurement of the interlamellar distance d
The effect of Br-HCl on the colloidal microstructure of lecithin
Solubilized amount of Br-HCl [mg]
was evaluated by SAXD. The measurements were performed
50
with binary and ternary systems containing water and P 90G in
40 different concentrations. Ternary systems were loaded with the
maximal amount of Br-HCl. Both binary and ternary systems
30
showed interference patterns that correspond to a lamellar
bilayer microstructure [2]. Furthermore, the incorporation of
20
Br-HCl leads to a significant decrease of the interlamellar
10
distance d from 6.4 nm to 4.2 nm that is independent from the
P 90G concentration. The distance d in the lamellar micro-
structure results from the phospholipid bilayer and interlamel-
0
0 5 10 15 20
larly bound water. Therefore, the decrease of d by the incorpo-
Phospholipon 90G - concentration [%] ration of Br-HCl results from either a reduction of interlamel-
larly bound water or a new arrangement of the fatty acid chains
Figure 1: Influence of the lecithin concentration on the in the bilayer.
maximal solubility of Br-HCl in ternary systems 31
P NMR spectroscopy
31
P NMR was used to locate and characterise the site and mode
of incorporation of Br-HCl in the lecithin bilayer. Both binary
and ternary systems display one relatively narrow and symmet-
rical peak, reflecting isotropic motional averaging and arising
mostly from lateral diffusion and Brownian tumble [3]. As no
shift split is observed interparticular interactions can be
excluded and the incorporation within the bilayer structure
appears to be homogeneous. Based on the small chemical shift
of Br-HCl loaded systems in contrast to drug free systems the
incorporation of Br-HCl seems not to influence the phosphate
group of P 90G. Therefore can be concluded that the incorpo-
ration of Br-HCl takes place in the lipophilic part of the
phospholipid bilayer. Furthermore, Br-HCl reduces the half-
line width (∆ν1/2) of the systems of which a homogeneous and
narrow particle size distribution can be deduced.
Table 1: 31P NMR results of binary and ternary systems
Figure 2: TEM micrograph of a lamellar vesicle disper-
composition [%] Peakposition ∆ν
sion containing 10% lecithin, 1.5% Br-HCl and
88.5% water (bar = 500nm) P90 G D2O Br-HCl [ppm] [ppm]
5 95 - - 0.18412 0.58
5 94 1 - 0.23428 0.33
Binary systems of P90 G and water 10 90 - - 0.1478 0.40
Ternary systems of P90 G, water and Br-HCl
Interlamellar distance d [Å]
70 10 89 1 - 0.19356 0.26
60
50
40 CONCLUSIONS
30
P 90G increases the maximal solubility of Br-HCl in water
20 significantly. Both binary and ternary systems using P 90G
concentrations from 0% to 20% form a lamellar microstructure
10
as TEM and SAXD studies indicate and the incorporation of
0
5 10 15 20 Br-HCl in the phospholipid bilayer reduces the interlamellar
Phospholipon 90G - concentration [%] distance d from 6.4 nm to 4.2 nm. 31P-NMR data suggest that
drug incorporation is homogeneous and that the site of drug
Figure 3: SAXD measurement of the interlamellar incorporation is in the lipophilic part of the bilayer structure
distance d in binary and ternary systems effecting a new arrangement of the fatty acid chains of lecithin
that results in the observed decrease of the interlamellar
distance.
RESULTS AND DISCUSSION ACKNOWLEDGEMENTS
Determination of the maximal solubility of Br-HCl
We like to thank Rhône-Poulenc-Rorer and Thomae for kind
HPLC measurements of the maximal solubility of Br-HCl support with materials.
show that the maximal solubility is higher in ternary systems
than in the aqueous drug solution and that the maximal REFERENCES
solubility increases linearly with growing P 90G concentration.
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[2] Luzatti V., Mustacchi H., Skoulios A., Husson F., La
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solubilization of Br-HCl in binary systems containing lecithin
J. Pharm. Sci. 81 (1992), 777-786
and water. In figure 2 a typical micrograph of a lamellar vesicle
dispersion is shown.