Characterisation Of Mucoadhesive Nasal Gels Of Midazolam Hydrochloride Biology Essay

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Nasal drug delivery prepared from natural materials is gaining importance in the arena of pharmaceutical technology. Mucilage isolated from Linum usitatissimum L. seeds was reported to be an effective natural mucoadhesive agent. The present study deals with comparision of various characteristics of nasal gels prepared from mucoadhesive agent extracted from Linum usitatissimum L. seeds and synthetic polymers like HPMC and Carbopol 934 in terms of texture profile analysis, mucoadhesive strength and in vivo drug absorption profiles. It was observed that gels formulated with the natural mucilage showed better results in all aspects than the synthetic gels. The absolute bioavailability of midazolam hydrochloride from the natural gel was 97.55% whereas that of synthetic gels were 57.33% and 76.81% respectively.

Uniterms: Linum usitatissimum L./pharmacognosy. Linum usitatissimum L./ natural mucoadhesive agent. Natural Mucoadhesive agent/bioavailability. Natural mucilage.


Mucoadhesive nasal gels provide a firm platform of drug delivery to the nasal cavity than the other types of nasal formulations like solutions, sprays, insufflations, since the mucoadhesive agents make a better contact with the nasal mucosa which helps in enhancing drug bioavailability. Various synthetic agents are available like hydroxy propyl methy cellulose (HPMC), Carbopol 934, sodium alginate and so on. Nowadays, natural mucilages isolated from various plant parts and are used as mucoadhesive agents. It has been observed that natural materials are biocompatible and biodegradable and hence they are more preferred than the synthetic polymers. In our previous work, mucoadhesive nasal gels of midazolam were prepared from mucilages isolated from Linum usitatissimum L. seeds and it was observed that they gave better results than synthetic polymers in terms of viscosity and in vitro release profiles (Basu et al, 2009).

Linum usitatissimum L. mucilage is a water soluble heterogeneous polysaccharide composed of xylose, arabinose, glucose, galactose, galacturonic acid, rhamnose and fructose (Cui et al, 1994; Erskine et al, 1957; Hunt et al, 1962; Muralikrishna et al, 1987). It has good water-holding capacities, owing to its marked swelling capacity and high viscosity in aqueous solution (Basu et al, 2009). It has been reported that presence of many oligo- and polysaccharides in many substances impart mucoadhesive properties (Hunt et al, 1962). Since, this mucilage is a rich source of polysaccharides and has remarkable swelling capacity and high viscosity, it was selected to prepare nasal gels of midazolam.

Midazolam is a fast acting benzodiazepine with a short elimination half-life (1.8 6.4 hr). It is therefore a very useful drug to use for short minor procedures such as dental extraction. However, due to very poor bioavailability of this drug from gastro-intestinal system (≈ 36%), it is administered through intramuscular injection only where bioavailability is over 90% (wikipedia). Nasal route will be one of the best alternative route of administration of this drug to intramuscular injections which is one of the most hazardous route of drug administration as well as most difficult to manufacturer due to stringent rules and regulations.

Hence, an attempt was made to prepare mucoadhesive nasal gels of midazolam which may replace the conventional midazolam injection and widen the scope of novel drug delivery system.

The present study focuses on comparision of texture profile analyses, mucoadhesive strengths and in vivo drug absorption profiles from nasal gels prepared with mucilage isolated from Linum usitatissimum L. seeds and synthetic polymers like HPMC and Carbopol 934.



Midazolam hydrochloride was obtained as a gift from Sun Pharmaceutical Industries Ltd., Gujarat, India. Linum usitatissimum L. seeds were purchased from local market. HPMC, Carbopol 934 and sodium taurocholate were purchased from Loba Chemie Pvt. Ltd., Mumbai, India. All other reagents and chemicals used were of analytical grade.


Preparation of nasal gel containing midazolam

Mucoadhesive nasal gels were prepared by dissolving midazolam hydrochloride in nasal solution (0.65% NaCl, 0.04% KH2PO4, 0.09% K2HPO4 and 0.02% benzalkonium chloride) (pH 6) in a constant stirring condition. Required amounts of LUM and synthetic polymers (HPMC and Carbopol 934) were added to the solution and stirred on a magnetic stirrer until a uniform solution was obtained which was kept at 4 °C overnight to allow complete swelling so that a homogenous gel was formed. Penetration enhancer like sodium taurocholate, and sodium thioglycollate were also used in the formulations at a concentration of 0.50 % (w/v).

In our previous work it was shown that sodium taurocholate produced better drug release profiles than sodium thioglycollate (Basu et al, 2009) and hence, for the in vivo study, nasal gels containing 0.5 % w/v sodium taurocholate were administered to the rabbits. Composition of nasal gels used in the present study is provided in Table I.

TABLE I- Composition of nasal gels alongwith formulation codes

Formulation Midazolam HCl LUM HPMC Carbopol 934 Sodium taurocholate

code (%w/v) (%w/v) (%w/v) (%w/v) (%w/v)

F1 5.0 3.0 - - 0.5

F2 5.0 4.0 - - 0.5

F3 5.0 5.0 - - 0.5

F4 5.0 - 3.0 - 0.5











- 4.0 - 0.5

- 5.0 - 0.5

- - 3.0 0.5

- - 4.0 0.5

- - 5.0 0.5

Characterisation of gels

Determination of texture profile analysis

Texture profile analyses of the gels were evaluated using QTS-25 Texture Analyser (Brookfield Engineering Labs., USA) to determine the mechanical parameters like hardness, cohesiveness and adhesiveness. An analytical probe of diameter 1.2 cm was depressed twice into each sample to a fixed depth (15 mm), at a defined rate (30 mm/min), with a defined recovery period (15 s), between the end of the first compression and the beginning of the second. A trigger force of 4 g was applied. At least six replicate analyses of each sample were performed at 37±1°C. Data collection and evaluation were done by Texture pro software, version 2.1 (Cevher et al, 2008).

TABLE II - Values of texture profile analysis and mucoadhesive strength of conventional gels.

Data represent mean ± SD (n=6)

Formulation Hardness (g) Adhesiveness (gs) Cohesiveness Mucoadhesive

code strength (g)










28.00 ± 1.11

38.33 ± 0.69

55.00 ± 0.38

11.00 ± 0.50

14.00 ± 0.48

18.00 ± 0.36

15.00 ± 0. 51

21.00 ± 0.36

27.05 ± 0.32

-89.00 ± 7.12

-95.00 ± 2.65

-112.08 ± 5.89

- 30.60 ± 3.03

- 40.62 ± 4.88

- 51.26 ± 6.45

-45.03 ± 5.02

- 69.36 ± 8.56

-80.36 ± 7.98

1.01 ± 0.05 16.26 ± 0.98

0.99 ± 0.02 19.76 ± 0.85

0.97 ± 0.03 22.45 ± 1.02

1.15 ± 0.01 8.00 ± 0.85

1.02 ± 0.05 9.50 ± 0.36

1.00 ± 0.03 11.95 ± 0.56

1.10 ± 0.05 9.63 ± 1.02

1.06 ± 0.04 13.22 ± 1.05

0.96 ± 0.03 16.39 ± 0.89

Evaluation of mucoadhesive strength

Mucoadhesive strengths of the gels were determined by measuring force required to detach nasal mucous membrane from the gel using the same texture analyser. Freshly excised goat nasal membrane was attached to the upper probe of the instrument, and fixed amount of gel was kept below that. The upper probe was then lowered at a speed of 10 mm/min to touch the surface of the gel. A force of 0.1 N was applied for 5 min to ensure intimate contact between the membrane and the gel. The surface area of exposed mucous membrane was 1.13 cm2.

Calibration curve of midazolam in rabbit plasma

Stock solutions of midazolam were prepared in a mixture of 10 mM phosphate buffer (pH 6.0): acetonitrile (80:20) to give final concentrations of 1 mg/ml. Aliquots of 0.1, 0.5, 1.0, 2.0, 6.0, 8.0 and 10 µg/ml of midazolam by appropriate dilutions of the reference solution with the solvent mixture. Plasma standards for calibration curves were prepared by spiking 1.0 ml aliquots of pooled drug free plasma with 100 µl of the above mentioned working solutions, to make midazolam plasma standards ranging from 10 to 1000 ng/ml.

TABLE III- Interday and intraday accuracy and precision data for quantitation of midazolam in rabbit plasma

Amount of drug added (ng/ml)

Concentration in plasma Accuracy Precision (% CV)


Intraday Interday Intraday Interday Intraday Interday





9.91±0.24 9.55±0.38 99.10 95.50 2.42 3.97

199.05±2.15 195.99±4.51 99.53 98.46 1.08 2.30

599.30±2.65 596.08±6.58 99.88 99.34 0.44 1.10

998.05±3.95 994.09±5.01 99.80 99.41 0.39 0.50

In vivo drug absorption study

Selection of animals

In vivo drug absorption study was conducted with prior approval of the Institutional Animal Ethics Committee and it was conducted according to the institutional guidelines of Animal Ethics Committee of Dr. B. C. Roy College of Pharmacy and Allied Health Sciences, West Bengal University of Technology, as recognized by the Committee for the Purpose of Control and Supervision on Experiments on Animals, India.

In vivo studies were conducted on 12 New Zealand albino male rabbits weighing between 2 and 2.5 kg. Based on results of texture profile analyses, mucoadhesive strengths and in vitro drug release pattern (Basu et al, 2009), nasal gels containing 3% w/v LUM, 5% w/v Carbopol 934 and 5 % w/v HPMC along with 0.5% sodium taurocholate were selected for in vivo study. The animals were kept in individual metal cages and maintained at 25°C for 10 days prior to the experiment. They were provided with standard diet and water ad libitum. The rabbits were kept in fasting condition for 24 h before the experiment commenced. The rabbits were grouped into four (group I, II, III and IV), each group containing three rabbits. Group I was administered intravenous bolus injection of midazolam. Groups II, III and IV were administered nasal gels of midazolam prepared with LUM, Carbopol 934 and HPMC. Single dose of midazolam (2 mg/kg body weight of rabbit) was administrated intravenously to compare the pharmacokinetic parameters. No anaesthesia was used for the intravenous study. Midazolam was injected through cannulated marginal ear vein. After every 20 min, each rabbit was administered one-third of the initial dose of xylazine and ketamine intramuscularly to maintain a light plane of anaesthesia. In case of nasal gels, the dose of midazolam that was administered was also 2 mg/kg body weight of rabbit.

Collection of blood samples

Two millilitres of blood samples were collected before intravenous injection and then at 5-, 10-, 15-, 20-, 30-, 45-, 60-, 90-, 120-, 180-, 240- and 300-min intervals in eppendorffs containing heparin sodium (100 U/ml). Immediately after each blood sample collection, the catheter was flushed with 0.2 ml of a 10% (v/v) heparin/normal saline solution to prevent blood clotting inside the catheter. The blood samples were kept on ice and centrifuged at 3,000 rpm for 10 min immediately after collection to separate the plasma and stored at −20°C until the time of analysis.

HPLC analysis

Reverse phase HPLC was used to quantitate midazolam in plasma samples. Midazolam was extracted with 3 ml of cyclohexane/diethyl ether (3:7) after the addition of 10 μl of 2% sodium hydroxide (Shih et al, 2002). The organic phase was removed and evaporated to dryness under nitrogen, and the residue was reconstituted in 200 μl of the mobile phase (10 mM phosphate buffer (pH 6.0)/acetonitrile, 80:20). From the mixture, 100 μl was injected for chromatographic analysis. The mobile phase consistedof phosphate buffer/acetonitrile (80:20) v/v. The mobile phase was delivered into the HPLC apparatus at a flow rate of 1 ml/min (isocratic pump, Model LC-10AS, Jasco, Japan). The detection wavelength was 218 nm (ultraviolet variable wavelength detector, Model SPD-10A), and a C18 column was used. All assays were performed at ambient temperature.

Pharmacokinetic analysis

Pharmacokinetic parameters like peak plasma concentration (Cmax), time to reach peak plasma concentration (tmax), area under the (concentration-time) curve (AUC), mean residence time (MRT), elimination half-life (t1/2) and total body clearance (CL) were calculated following non-compartment model by Kinetica 4.4, PK/PD Analysis, Thermoelectron Corporation. All the parameters were calculated for i.v. bolus injection of midazolam and in situ nasal gels. Fraction of dose absorbed (F) was calculated by the following equation:

F = AUC(nasal) X Dose(iv)

AUC(i.v.) X Dose(nasal)

where Dose(iv)=dose of midazolam given as i.v. solution, Dose(nasal)=dose of midazolam in nasal gels, AUC(i.v.)=AUC after i.v. administration of midazolam and AUC(nasal)=AUC after nasal administration of midazolam.

TABLE IV - Comparative pharmacokinetic parameters of midazolam hydrochloride following administration of intravenous and nasal gels in rabbits (dose=2 mg/kg)

Pharmacokinetic Intravenous HPMC CP LUM

parameters solution

Cmax (ng/ml) 573.64 ± 5.23 151.47 ± 7.22 164.07 ± 6.85 181.12 ± 6.21

Tmax (min) - 60.00 ± 5.68 45.00 ± 3.66 30.00 ± 4.75

AUClast 32671.50 ± 90.56 18034.30 ± 101.45 22074.10 ± 96.72 6168.80 ± 123.67

AUC extra 865.87 ± 37.25 1191.83 ± 45.26 3684.71± 60.58 5546.92 ± 48.76

AUCtotal 33537.40 ± 101.25 9226.10 ± 97.25 25758.90 ± 95.46 32715.72 ± 98.77

MRT (min) 71.61 ± 5.05 130.16 ± 6.58 162.50 ± 7.25 190.07 ± 8.05

T1/2 (min) 58.78 ± 6.32 68.44 ± 5.28 99.68 ± 4.89 122.55 ± 5.87

Clearence X 10-5 5.79 ± 6.95 10.11 ± 3.66 7.53 ± 2.89 6.67 ± 4.22

(mg/ Kg*min/(ng/ml))


The present study determines the various mechanical properties as well as mucoadhesive strengths of the gels with the help of QTS - 25 Texture Analyser. Results of the texture profile analysis and mucoadhesive strengths are tabulated in Table I. Hardness of LUM gels ranges from 28.00 ± 1.11g to 55.00 ± 0.38g. In case of HPMC gels, it ranges from 11.00 ± 0.50g to 18.00 ± 0.36g and for Carbopol 934 it varies from 15.00 ± 0.51g to 27.05 ± 0.32g. Values of adhesiveness of LUM, HPMC and Carbopol 934 gels varies from -89.00 ± 7.12gs to -112.08 ± 5.89gs, -30.60 ± 3.03gs to -51.26 ± 6.45 gs and -45.03 ± 5.02gs to -80.36 ± 7.98 gs, respectively.

Cohesiveness of LUM gels ranges from 1.01 ± 0.05 to 0.97± 0.03. For HPMC gels, it varies from 1.15 ± 0.01 to 1.00 ± 0.03 and for Carbopol 934 gels, it varies from 1.10 ± 0.05 to 0.96 ± 0.03.

Values of mucoadhesive strengths of the gels are also displayed in Table I which shows that with increase in concentration of the gels from 3% to 5%, mucoadhesive strengths of LUM gels varies from 16.26 ± 0.98g to 22.45 ± 1.02g. For HPMC gels and Carbopol 934 gels, it ranges from 8.00 ± 0.85g to 11.95 ± 0.56 g and from 9.63± 1.02g to 16.39 ± 0.89grespectively.

Calibration curve of midazolam hydrochloride prepared in rabbit plasma was found to be linear over the concentration range of 10-1,000 ng/ml (r2=0.9999). The experiment was repeated six times a day and for six consecutive days. Interday and intraday accuracy and precision values are displayed in Table II.

Plasma concentration-time profiles of midazolam after administration of i.v. solution and the nasal gels are shown in Figure 1. Pharmacokinetic parameters were displayed in Table III. Cmax values of i.v. injection, LUM, HPMC and Carbopol 934 gels were 573.64±5.23, 135.03 ± 10.25, 101.99 ± 4.59 and 110.52 ± 5.02 ng/ml, respectively. tmax values of i.v. injection, FCM, HPMC and CP gels were 0.00, 30.00 ± 9.76, 60.00 ± 6.25 and 45.00 ± 7.88 min, respectively. t1/2 values of i.v. injection, FCM, HPMC and CP gels were 58.78±6.32, 163.76 ± 7.05, 79.04 ± 3.96 and 138.78 ± 8.36 min, respectively.

FIGURE 1 - Plasma concentration-time profiles of midazolam hydrochloride after administration of intravenous solution and the nasal gels in rabbits. Data represent mean ± SD (n=3).


Results of texture profile analysis reveal that with increase in concentration of the mucoadhesive agent, values of hardness and adhesiveness also increased whereas cohesiveness was found to decrease. Hardness of a gel determines the drug release pattern from the gel. From the results it can be said that gel containing 3% LUM showed optimum hardness. Adhesiveness determines proper gel contact and retention at the site of application, thereby leading to enhanced bioavailability of the drug. In the present study, adhesiveness of gels was enhanced significantly with increase in amount of mucoadhesive agent. Cohesiveness was observed to be reduced with increase in amount of mucoadhesive agents. This happens because with increase in amount of dispersed solids, that is the mucoadhesive agents, semisolid nature of the gels increased, which caused the gel to become less coherent (Cevher et al, 2008)

Mucoadhesive strength of LUM gels was found to be higher than those prepared with synthetic mucoadhesive polymers and it increased with corresponding increase in concentration of mucoadhesive agent used. This may be due to presence of certain functional groups in the mucilage were able to establish a more intimate contact with mucin of the mucosa (Basu et al, 2009).

Higher plasma concentration of midazolam hydrochloride is observed in case of LUM gels in comparison to HPMC and Carbopol 934 gels (Figure 1), and accordingly, absolute bioavailabilities of midazolam from LUM, HPMC and Carbopol gels were reported to be 97.55%, 57.33% and 76.81%, respectively. From the in vivo study we can conclude that bioavailability of midazolam from nasal gels prepared from LUM was better than those prepared from synthetic polymers. This confirms that LUM can be effectively used as a mucoadhesive agent instead of synthetic polymers for delivery of midazolam via nasal route.


From the above study it is confirmed that mucoadhesive nasal gels of midazolam hydrochloride prepared with mucilage isolated from Linum usitatissimum L. seeds exhibited better mechanical properties as well as in vivo drug absorption pattern. Also, bioavailability of midazolam was higher from the gels prepared with this natural mucilage than from synthetic gels. Moreover, in our previous work, histopathological study confirmed that LUM can be used safely as a mucoadhesive agent. Hence, this new dosage form of midazolam with a natural mucilage is a safe and cost effective form of nasal drug delivery system.