Modification Of Pharmacological Activity Of Nebivolol Biology Essay


Lactose, a reducing sugar, is widely used as a filler or filler-binder in the manufacture of pharmaceutical solid dosage forms. The general properties of lactose that contribute to its popularity as an excipient are its lower cost, easy availability, bland taste, low hygroscopicity, excellent physical and chemical stability and water solubility). However, lactose is also notorious for its incompatibility with primary and secondary amines. Lactose undergoes the Maillard reaction with amine group-containing drugs, leading to the typical browning of the dosage forms on storage3). This interaction can be represented as a condensation reaction (yielding Schiff bases, i.e., imines) arising from anhydrosynthesis between an amino group of the drug moiety and the aldehyde group of the open chain formof the glucose part in lactose 4. There are many reports about primary amine drugs 4-13) and secondary amine drugs) undergoing the Maillard reaction in the presence of lactose, leading to instability of the dosage forms of these drugs. Recent reports suggest that the Maillard reaction products may possess their own pharmacological potential16). However, whether the formation of a Maillard product in amine drug formulations affects the pharmacological activities of these drugs has not been studied systematically.

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Nebivolol (NEB) is a unique type of cardioselective and antihypertensive β-blocker that contains four chiral centers and can exist as 16 different enantiomers 17). Chemically it is α,α′-[Iminobis (methylene)]bis [6-fluoro-3,4-dihydro-2H-1-benzopyran-2-methanol]18) and contains a secondary amine group. The effect of NEB on the heart rate is exclusively exerted by d-nebivolol. The hypotensive effect of the d-enantiomer is enhanced by the addition of the l-enantiomer, which in itself does not influence the systolic and diastolic blood pressure). Owing to the presence of a secondary amine group and to its unique pharmacological profile involving enantiomer-specific effects, NEB is a potential candidate whose interactions with lactose are to be investigated.

The present study was designed to investigate the interaction between NEB and lactose and the effect of the resultant Maillard product on the pharmacological activity of NEB.


Materials and Methods


NEB was generously supplied as a gift sample by Cadila Pharmaceuticals Ltd., Ahmedabad, India. Lactose monohydrate was purchased from Loba Chemicals, Mumbai, India. All other chemicals were of high-performance liquid chromatography (HPLC) and analytical grade. A programmable environmental test chamber, CHM-10S, manufactured by Remi Instruments, Ltd., Mumbai, India, was used to perform a stability study.


Interaction studies

Interaction between NEB and lactose at neutral and alkaline pH

Samples of NEB and lactose monohydrate were weighed and dissolved in the ratio of 1:6 in water by stirring and using ultrasound. Triethylamine and methanol were added in an equimolar ratio to NEB to improve its solubility. The turbid solution was then refluxed at 60°C in a water bath for 12 hours. The aqueous insolubility of NEB was exploited to separate the NEB-lactose adduct from the water-insoluble NEB. The solution was placed in a flask into which 5 mL of water was added. The resulting slurry was shaken and sonicated intermittently for 10 minutes. Several milliliters of a clear solution could be obtained by filtration. The above-mentioned procedure was carried out in the presence of a borate buffer (pH 9.2) to check the possibility of the Maillard reaction taking place in alkaline solutions. The dried products of the above reactions were dissolved in methanol: water (1:1) and were subjected to HPLC (gradient and isocratic run) and LC-MS analysis. The intensity of brown color in the above mixtures was determined spectrophotometrically.

The NEB solution, lactose aqueous solution and aqueous mixtures of NEB and lactose with and without pH adjustments were similarly refluxed to monitor the interaction.

Analytical methods

UV-visible spectrophotometry

The UV-visible spectra of NEB and the NEB-lactose adduct were recorded on a double beam UV-visible spectrophotometer (UV-1700, Shimadzu, Japan).

Fourier-transform infrared spectroscopy (FTIR) spectroscopy

The FTIR spectra of NEB, lactose, a NEB-lactose physical mixture and the NEB-lactose adduct were recorded. The spectra were obtained using the diffuse reflectance scan method using KBr on an FTIR spectrophotometer (IR Affinity 1, Shimadzu, Japan). The scanning range was 400-4000 cm-1. Each sample was scanned 45 times consecutively to obtain FTIR spectrum.


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The optical rotation of the samples was determined using a polarimeter with a sodium vapour lamp as a light source with a 1.0 decimeter sample tube. The Polarimeter (220 m. m.) RSP-1, manufactured by Rajdhani Scientific Instruments Co., New Delhi, India was used to study optical rotation. The instrument was calibrated with a sucrose solution (10%, v/v) before sample analysis 21).

Differential scanning calorimetry (DSC)

Thermal analysis of NEB and the NEB-lactose adduct was performed by DSC using a TA 6000 SIIO thermal analyzer. Individual samples as well as the Maillard adduct (about 5 mg) were weighed in the DSC aluminium pan and were scanned in the temperature range of 25-300°C temperature range. A heating rate of 20°C per minute was used. The thermograms were reviewed for evidence of interaction. HPLC analysis

The HPLC system used for analysis consisted of Agilent Technologies 1200 series equipment a G1315D quaternary pump, a G1315D diode array detector and a rheodyne injector fitted with a 20-μL loop. Data were recorded and evaluated using the EZChrome Elite software package. Compounds were separated on a Phenomenex C 8 column (250 - 4.6 mm i.d. - 5 μm). The mobile phase was water:methanol (90:10, v/v) with a flow rate of 0.9 mL per minute. Detection was performed at 291 nm.

LC-MS analysis

The was performed using Varian 500 MH mass spectrometer with Varian C18 column (50 - 2 mm i.d. - 3 µm) eluted at a flow rate of 0.2 mL per minute, at ambient temperature in positive ion mode. Elution was performed with water:methanol (90:10, v/v) as the mobile phase. The mass spectrometer consists of electrospray ionization technique for sample ionization. The analysis of data was performed using control system variants.

Pharmacological activity

The rats used for this study were obtained from the institutional animal house facility, which is registered with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA; registration number 651/02/c/CPSEA). Approval to conduct the experiment described below was obtained from the institutional animal ethical committee, constituted as per CPCSEA guidelines (approval letter number: RCPCOP/IAEC/23-2009, dated 25 June 2009).

Animal preparation and haemodynamic measurements

The effects of NEB and the other study samples on blood pressure and heart rate were determined following the procedure of Sacco et al 20). A total of 18 Wistar rats aged 9-10 weeks were used. On the day of the experiment, each rat was anaesthetized by intraperitoneal injection of 60 mg/kg ketamine and 2.5 mg/kg diazepam. After induction of anaesthesia, the rat was placed on a homoeothermic blanket system to maintain the body temperature at 37°C throughout the experiment. The trachea was exposed, and a polyethylene cannula was inserted to allow adequate ventilation.

The blood pressure was measured by means of a polyethylene cannula (PE 50) filled with heparinized saline (25 IU/mL) inserted into the left carotid artery. The cannula was connected to a pressure transducer (physiological pressure transducer SP 844, MEMSCAP AS, Norway) and the signal was amplified using a bridge amplifier (Power Lab 8/30, AD Instruments, Australia). The heart rate was simultaneously recorded using ECG probes. The acquired data were analyzed using the ChartTM5 Pro software package (AD Instruments, Australia). Both the blood pressure and heart rate signals were continually recorded for up to 1 hour following administration of a test dose.

Study design

The effects of NEB and the NEB-lactose adduct were noted on six rats each. The NEB-treated animals received NEB solution in the vehicle (lactose with 0.075% (w/v) β-cyclodextrin in sterile water) at 1 mg/kg through the cannulated jugular vein. This dose was selected on the basis of an earlier report 20). The NEB-lactose adduct was administered in the same manner. The remaining six rats received the vehicle at the same dose volume (1mL/kg). The blood pressure and heart rate were recorded for 1 hour after administration of NEB, the NEB-lactose adduct or the vehicle.



UV-visible absorption spectroscopy

The intensity of brown color was measured spectrophotometrically at 420 and 490 nm22). The UV-visible absorption spectrum of the NEB-lactose adduct shows an increase in absorption in the visible range as compared with NEB with methanol as the solvent. The increased absorption of the NEB-lactose adduct in the visible region is due to an extension of the chromophoric system, which confirms the formation of the NEB-lactose adduct.


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The FTIR absorption patterns of NEB, lactose, the NEB-lactose physical mixture immediately after mixing and the NEB-lactose adduct are shown in Fig. 1. With the help of FTIR, it is easy to detect the interaction between amine and carbonyl group which shows imine band formation. For imine C=N stretching appears at 1630-1690 cm-1 region23-27). The band at 1667 cm-1 for imine was present in the FTIR spectrum of the NEB-lactose adduct, whereas it is absent in that of the freshly prepared physical mixture of NEB and lactose. The N-H stretching band of secondary amine appears at 3452 cm-1, and the peak for the lactose O-H appears at 3529 cm-1 in the infrared spectra of NEB and lactose. The peaks for N-H and O-H stretching appear in the spectrum of the physical mixture, but the peak for N-H disappears in the spectrum of the adduct. This may indicate the reaction of the amine with the reducing sugar, or it may due to overlapping of peak of N-H stretch with that of O-H stretching. The peaks for N-H bending at 1495 cm-1 and N-H wagging near 800 cm-1 were observed in the infrared spectrum of NEB and not in the spectrum of the NEB-lactose adduct.

The FTIR spectra of NEB, lactose and the NEB-lactose adduct suggest an interaction between NEB and lactose leading to the formation of a Maillard product.


The specific optical rotation value for NEB in THF/water (4:1, 1% w/v concentration) was found to be -7317). Lactose is insoluble in the THF/water solvent system, and hence methanol:water (1:1) was used as the solvent for further studies. A decrease in the optical rotation value of the NEB-lactose adduct (-6.8) was observed compared with that of NEB (-7.2) with a concentration of 1% w/v. The control samples of lactose and the physical mixture of NEB with lactose were also analyzed.


The Fig. 2 shows the DSC scans of NEB, lactose and the NEB-lactose adduct. The lactose shows peaks at 153.8°C and 222°C due to dehydration and melting of NEB. The melting peak of the lactose at 218°C is characteristic of a monohydrate α lactose form15). The disappearance of NEB melting peak in NEB-lactose adduct sample confirms NEB-lactose interaction.

The peak in the thermogram of the NEB-lactose adduct and pure lactose monohydrate starts at 110°C or nearer but ends differently at a later time. This shows the broad peak appeared in thermogram of NEB-lactose adduct is different from that the peak in lactose. The DSC peak of adduct is broaden may be due to merging of another peak with it. This study significantly confirms maillard reaction between amine and carbonyl group.


Different HPLC methods have been used in NEB identification 28-30); however, none of the reported methods was adequate to confirm the formation of an adduct between NEB and lactose because of the difference in their solubilities. We initially performed a gradient run of water and methanol, given the preliminary information regarding the unknown peaks 22) shown in Fig. 3 for NEB, the NEB-lactose adduct and lactose in borate buffer and neutral solution. The mobile phase optimized to separate the NEB-lactose adduct was water: methanol (90:10, v/v) with a flow rate 0.9 mL per minute. The NEB-lactose adduct elutes at 3.4 minutes. Control samples of NEB with and without lactose were also prepared for method selectivity.

Adduct HPLC study

The nominal NEB concentration (100 µg/mL) was prepared by dissolving NEB-lactose adduct in methanol:water (1:1) mixture. Control samples were prepared to use heated NEB and lactose monohydrate in borate and neutral media. Compared with the control, isocratic HPLC analysis of the NEB-lactose adduct revealed one extra peak (Fig. 4) that eluted before NEB. Performing analysis under same chromatographic parameters, no anther peak was observed in heated sample of lactose compared to NEB-lactose adduct. HPLC analysis of a sample maintained at 40°C and 75% RH for 15 days also shown one extra peak for the NEB-lactose adduct as shown in Fig. 4.


In complex mixtures such as formulations containing drug and excipients to detect unknowns at very low concentration LC-MS technique is preferred due to its sensitivity31). LC-MS has been used to analyze amine-lactose condensation products). In the positive ion mode with electrospray ionization technique the solutions of NEB-lactose adduct at pH 7.0 and 9.2 were analysed by LC-MS. The MS spectra shows the precursor ions of NEB and adduct were protonated molecules ([M+H]+) of m/z 405.40 and 730.69, respectively (Fig. 5). The Fig. 6 shows proposed structure of NEB-lactose adduct. The NEB-lactose adduct molecular mass is consistent with NEB-lactose condensation product. The loss of one water molecule from parent leads to maillard type condensation product.

Pharmacological activity

The average initial blood pressure and heart rate of the rats were 135.0 ± 3.0 mm Hg and 332.3 ± 4.6 beats per minute. In the vehicle-treated rats, the blood pressure and heart rate remained stable throughout the total observation period of 2 hours. The intravenously administered NEB induced a 10.7 ± 1.3% fall in blood pressure at a 1 mg/kg dose of NEB, and the NEB-lactose adduct, administered at a dose containing an equal amount of NEB (before adduct formation) induced a 7.9 ± 1.9% fall. The difference in these values is not statistically significant as estimated by the unpaired t-test (p = 0.276).

The physical mixture of NEB and lactose induced a 20.0 ± 0.7% fall in the heart rate, whereas the NEB-lactose adduct induced only a 7.9 ± 2.3% fall. The bradycardic effect of the physical mixture of NEB and lactose was significantly more potent than that of the adduct (p < 0.0001). The percentage loss of hypotensive and bradycardic activity of the physical mixture of NEB and lactose and that of the NEB-lactose adduct are shown in Fig. 7. These results clearly indicate that the NEB-lactose adduct has a significantly lower bradycardic effect compared with the physical mixture of NEB and lactose.


The results of the present investigation reveal the formation of the NEB-lactose adduct in neutral and alkaline solutions as well as in the dry physical mixture maintained at 40°C and 75% RH (extreme but environmentally feasible conditions) for 15 days. The FTIR, HPLC and LC-MS analyses confirmed the formation of the Maillard adduct. It is noteworthy that the NEB-lactose adduct was less hypotensive than the physical mixture of NEB and lactose and that it had significantly less bradycardic effects. The hypotensive activity of NEB is because of the combined effect of the d- and l-enantiomers. The significant loss of bradycardic activity observed in the present investigation suggests a loss of d- enantiomer activity. However, to prove this, a separate investigation to determine whether lactose preferentially interacts with any one of the isomers of NEB is needed. The preferential degradation of amino acid isomers in the Maillard reaction has been reported earlier32-34). Reports on the significance of such enantiomer-specific interactions in drugs with lactose are scarce. Nevertheless, the loss of effects on the heart rate and blood pressure indicates that the NEB-lactose adduct is pharmacologically less active compared with NEB.

In spite of numerous studies having confirmed the occurrence of the Maillard reaction in lactose-containing formulations of amines, lactose is still preferred as an excipient in dosage forms of amines. There are few reports on whether the products of the Maillard reaction have any effects on the pharmacological activity of the active components of the dosage forms. In the present study, it was observed that in the presence of lactose, NEB undergoes the Maillard reaction even under conditions that are likely to be encountered in the environment. The formation of the NEB-lactose adduct significantly affects the pharmacological activity of NEB.


The present study proves that NEB undergoes the Maillard reaction in the presence of lactose. The formation of the NEB-lactose adduct leads to a decrease in the hypotensive effect and to a highly significant loss in the bradycardic effect of NEB. In the light of these data, the use of lactose in formulations of NEB, a secondary amine, needs to be reconsidered.


The authors are thankful to the Officer in Charge, Sophisticated Instrumentation Facility, Indian Institute of Technology-Bombay, Mumbai, India for providing the liquid chromatography-mass spectrometer facility for sample analysis. Thanks are due to the principal of H. R. Patel and R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India for providing laboratory facilities.