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Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps 5 6 3 4 7 8 9 10 11 12 1 2 4 7 15 16 17 18 19 20 21 22 23 24 25 26

Preclinical evaluation of dual action intranasal formulation intended for postoperative/cancer associated therapies Doaa Ahmed El-Setouhy a,⇑, Sami Ahmed a, Alia Abd El-Latif Badawi a, Mohamed Ahmed El-Nabarawi a, Nada Sallam b a b

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Egypt Department of Pharmacology and Toxicology, Faculty of Pharmacy, Cairo University, Egypt

a r t i c l e

i n f o

Article history: Received 9 January 2015 Received in revised form 31 March 2015 Accepted 20 April 2015 Available online xxxx Keywords: Granisetron Ketorolac Nasal drug delivery Mucosal drug delivery Thermal gels

a b s t r a c t Granisetron hydrochloride is a potent antiemetic yet experiencing first pass metabolism. Ketorolac tromethamine is a potent analgesic NSAID that is known to cause gastrointestinal complications. The purpose of this study is to prepare combined in situ nasal copolymer thermal gel combining both drugs for the management of postoperative and cancer associated nausea, vomiting and pain while avoiding the problems associated with their therapy. In situ gelling nasal formulations with/without different mucoadhesive polymers were prepared and evaluated. Viscosity of different formulations was measured and correlated to in-vitro drug release. Selected formulae were evaluated for in-vivo mucociliary transit time. Based on in-vitro release pattern and mucociliary transit time, the selected formula F4 was evaluated for chemical and thermal anti-nociception activity in rats following intranasal or intraperitoneal administration. Only the intra-nasal administration of the selected formulation F4 showed significant analgesia against chemical nociception during both the early and late phases. Also, intranasal administration of the selected formulation F4 showed significant analgesia against thermal nociception. F4 intranasal formulation may offer higher therapeutic value than oral administration as it may not only avoid granisetron first pass metabolism but may also minimize ketorolac gastrointestinal adverse effects as well. Ó 2015 Published by Elsevier B.V.

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1. Introduction Granisetron hydrochloride and ketorolac tromethamine are two drugs used for postoperative and cancer associated therapies. Granisetron is a highly selective 5-hydroxytryptamine-3 (5HT3) receptor antagonist, it is used as a potent antiemetic in postoperative nausea and vomiting and in acute and delayed emesis in cancer chemotherapy (Aapro, 2004). Ketorolac is a potent analgesic NSAID used in severe to moderate postoperative pain (Buckley and Brogden, 1990). Ketorolac has also been found effective in the treatment of trauma-related pain as well as pain associated with cancer (Angeles, 2009; Joishy and Walsh, 1998; Mercadante et al., 2002). Granisetron suffers from reduced oral bioavailability (60%) due to hepatic metabolism (Sweetman, 2002). Ketorolac can cause gastrointestinal complaints associated with all NSAIDs such as gastrointestinal bleeding, perforation and peptic ulceration (Gillis and Brogden, 1997). ⇑ Corresponding author at: Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562, Egypt. E-mail address: [email protected] (D.A. El-Setouhy).

The use of the nasal cavity as a route for drug delivery has been a growing area of great interest. Intranasal administration offers a simple, practical, noninvasive, convenient, cost effective, and an alternative route for rapid drug delivery to systemic route. Other advantages include avoidance of liver or gastrointestinal metabolism, avoidance of the gastrointestinal irritation, and enhanced patient compliance by self-medication (Costantino et al., 2007). Also, it is an attractive alternative for patients not able to take medications orally, or who are experiencing nausea or vomiting (Singla et al., 2010). Intranasal administration may provide a more convenient form of drug delivery for ambulatory patients when administration by intravenous and intramuscular injection cannot be continued after the patient is discharged (Singla et al., 2010). Since a rapid onset of action is required and because of problems and side effects associated with granisetron and ketorolac administration; intranasal route seems to be a promising alternative to oral and parenteral route {granisetron is currently available as oral, intravenous and transdermal formulations, while ketorolac is available as oral, parenteral and nasal (spray) formulations}. Liquid nasal formulations (solutions and sprays) are easily and accurately instilled in the nasal cavity but they suffer from rapid

http://dx.doi.org/10.1016/j.ejps.2015.04.015 0928-0987/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: El-Setouhy, D.A., et al. Preclinical evaluation of dual action intranasal formulation intended for postoperative/cancer associated therapies. Eur. J. Pharm. Sci. (2015), http://dx.doi.org/10.1016/j.ejps.2015.04.015

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mucociliary clearance that limits the time for effective drug uptake (Ugwoke et al., 1999). Bioadhesive powders and gels have been studied to increase the residence time of the drug in the nasal cavity as well as to facilitate permeation of the drug through the mucosa by loosening the tight junctions between the epithelial cells (Callens et al., 2003). Accurate dosing of conventional bioadhesive gels is problematic due to their high viscosity; on the other hand, bioadhesive powders can cause irritation to the nasal cavity and require sophisticated delivery devices for ideal deposition and accurate dosing. (Li et al., 2014). Nowadays, in situ gels are being preferred widely because they are liquid at room temperature and hence can be easily administered into the nasal cavity as drops that will form a firm gel at the temperature of the nasal cavity. Such types of gels provide accuracy in dose administration which is difficult in case of conventional gels (Hu et al., 2009; Zaki et al., 2007). The aim of this study is to prepare dual effect mucoadhesive copolymer in situ nasal gel containing both drugs to help management of postoperative and cancer associated nausea, vomiting and pain nevertheless avoiding most of problems associated with their therapy. The nasal gel aimed to be of gelation temperature suitable for nasal cavity, adequate pH and release properties, and suitable in-vivo mucociliary transit time. The work also aimed to preclinically evaluate the selected formula (e) for analgesic effect.

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2. Materials and methods

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2.1. Materials

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Granisetron hydrochloride and ketorolac tromethamine (kind gifts from Amriya for Pharmaceutical Industries, Alexandria, Egypt). Pemulen™ TR-2 (P TR-2, gift from Luna Pharmaceutical Co., Cairo, Egypt; it is a polymer of acrylic acid, modified by long chain (C10–C30) alkyl acrylates, and crosslinked with allylpentaerythritol). CarbopolÒ 974P (CP 974P, Lubrizol Advanced Materials Inc., USA). Hydroxypropylmethylcellulose (HPMC K15M, Colorcon, England). Calcium chloride dihydrate, formaldehyde solution (Formalin, 34–38%), Sodium chloride, potassium chloride and triethanolamine (El-Nasr Pharmaceutical, Chemical Co., Egypt). Pluronic F127 (P F127, Sigma–Aldrich Inc., Germany). Benzalkonium chloride (Sigma Chemical Co., USA). Urethane (Sigma–Aldrich Inc., USA). Amaranth (standardcon, Pvt. Ltd., India). Normal saline (Sodium chloride Intravenous Infusion 0.9% w/v, B.P, Ateco Pharma, Egypt).

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2.2. Derivative spectral characteristics of granisetron and ketorolac in simulated nasal electrolyte solution (SNES) pH 5.5 Scanning in the UV range 200–400 nm for granisetron and ketorolac was carried out in SNES pH 5.5 (SNES is composed of 7.45 mg/ml NaCl, 1.29 mg/ml KCl and 0.32 mg/ml CaCl22H2O) (Cheng et al., 2002; Callens et al., 2003). A zero order, first derivative and second derivative spectra were computed for each drug. The first derivative and second derivative of the ratio spectrum were calculated and the zero crossing method was used to detect the suitable wave lengths for each drug (Shimadzu UV-1601PC UV–Vis Double beam Spectrophotometer, Koyo, Japan). 2.3. Recovery study of granisetron and ketorolac mixtures in SNES pH 5.5 Mixtures of known concentrations of granisetron and ketorolac in SNES pH 5.5 were prepared. The amplitude of each drug in SNES at the predetermined wave length was measured, and then the

concentration was back calculated. The recovery percentage (R%) was calculated for each mixture.

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2.4. Compatibility Study of Granisetron, Ketorolac and Different Pharmaceutical excipients using infrared spectroscopy (IR)

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Samples of individual drugs, excipients and physical mixtures weighing about 2–3 mg were mixed with about 400 mg of dry potassium bromide powder in micronized IR grade using pestle and mortar. The powder was compressed into discs under pressure of 10,000–15,000 psi. The infrared spectra of the samples were recorded over a wave number range of 4000–500 cm1 (Genesis II, TM, Mattson Instruments, USA).

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2.5. Preparation of in situ nasal gels

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2.5.1. Preparation of non mucoadhesive in situ nasal gels containing different concentrations of Pluronic F127 The cold method described by Schmolka (1972), was applied. P F127 gels were prepared at different concentrations (16%, 17%, 18%, 19% and 20% w/w) with or without granisetron (0.5% w/w) and ketorolac (5% w/w) to determine the lowest possible concentration that exhibits thermoreversible property between 29 and 34 °C. Formulations were prepared on weight basis, the drugs (medicated gels) were completely dissolved in distilled water, the solutions were cooled down to 4 °C and then a weighed amount of P F127 was added slowly with continuous stirring. The dispersions were then stored in a refrigerator until clear solutions were obtained.

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2.5.2. Preparation of mucoadhesive in situ nasal gels The method of preparation is the same as that mentioned under the previous section except that benzalkonium chloride and the mucoadhesive polymer were accurately weighed and stirred in the calculated quantity of distilled water together with granisetron and ketorolac prior to cooling and subsequent addition of P F127. Various prepared mucoadhesive in situ nasal gel formulations are given in Table 1.

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2.6. Characterization of different prepared in situ nasal gels

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2.6.1. Measurement of gelation temperature An aliquot of 10 ml of each formulation was put into a beaker (25 ml) placed on thermostatically controlled magnetic stirrer at room temperature (Model SB 162, Thermolyne Corporation, USA). A thermometer was immersed in the sample solution. The solution was gradually heated under continuous stirring using a magnetic bar. The temperature at which the magnetic bar stopped moving

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Table 1 Composition of the different prepared mucoadhesive in situ nasal gels. Formula

Pluronic F127 (%w/w)

HPMC (%w/w)

Pemulen™ TR-2 (%w/w)

CarbopolÒ 974P (%w/w)

F1 F2 F3 F4 F5 F6 F7 F8 F9

17 17 17 17 17 17 17 17 17

0.5 1 1.5 – –

–

– – – – –

– – –

– 0.05 0.1 0.2 – – –

0.1 0.2 0.3

Each Formula Contains 0.001% w/w Benzalkonium Chloride, 5% w/w Ketorolac Tromethamine and 0.5% w/w Granisetron Hydrochloride. Non medicated non mucoadhesive nasal gels were coded as P; medicated non mucoadhesive nasal gels were coded as M throughout the manuscript.


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due to gelation was reported as the gelation temperature (Ravi et al., 2013). Each formulation was measured in triplicate. 2.6.2. Determination of drug content The prepared formulae were assayed individually for the content of the drugs as follows: one gram of each sample was dissolved in 100 ml SNES pH 5.5. The drugs content of each sample was assayed spectrophotometrically at kmax 304 nm (second derivative) and 342 (first derivative) for granisetron and ketorolac respectively after proper dilution using SNES pH 5.5 as a blank. Each formulation was measured in duplicate. 2.6.3. Measurement of steady shear viscosity The rheological properties of in situ gels were studied using cone and plate viscometer (Viscometer DVT-I, RV Model, Brookfield, USA). A sample (0.5 ml) of the prepared formula was applied to the lower plate of the viscometer using a spatula. The measurements were made at 34 °C using spindle 52 at a shear rate ranging from 0.5 to 100 rpm. The shear rate (c) in s1 and the viscosity (g) in centipoises (cps) were determined from the instrument readings and fincitted to the power law constitutive equation (Tung, 1994):

g ¼ mc

n1

ð1Þ

The two dimensionless quantities: the consistency index (m) and the flow index (n) characteristic for each formulation were obtained by plotting log viscosity versus log shear rate to obtain straight line equation where intercept (a) = log m which means that m = 10(a) and slope = n 1. 2.6.4. In-vitro drug release from selected in situ nasal gels The drug release from selected formulations (M 17% P F127, F1, F4, F5, F7 and F8) was carried out using a dissolution apparatus according to USP method type II (paddle) (Vision G2 Classic 6™, Hanson Research, USA). The prepared formula was filled into small, circular plastic container (2.5 cm inner diameter and 1.2 cm in depth) lined externally with equally sized stainless steel wire screen, 100 mm mesh size. This assembly was placed at the bottom of a USP dissolution vessel containing 200 ml of SNES pH 5.5 at 34 ± 0.5 °C and the paddle speed was at 50 rpm. Samples (3 ml) were withdrawn at predetermined time intervals (5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 300 & 360 min) and the volume was replaced with fresh medium. The samples were filtered through membrane filter and analyzed by UV spectrophotometry at kmax 304 nm and 342 nm for granisetron and ketorolac respectively. The release studies were conducted in triplicate and the average values were plotted versus time. 2.6.5. In-vivo mucociliary transit time (MTT) of the selected mucoadhesive in situ nasal gels The in-vivo MTT previously reported by Lale et al. (1998), was adopted with slight modification, using dye amaranth instead of indigo carmine. Male albino rats, weighing 200–300 g were used after being anaesthetized by an intraperitoneal injection of urethane solution. Each 100 g body weight of the animal received a 1 ml freshly prepared urethane solution (1300 mg/10 ml). Aliquots (100 ll) of the in situ gel containing the dye amaranth (3 mg/ml) were instilled 0.5 cm deep into the right nostril of the rat, using a micropipette. In order to record the time between placement in the nose and appearance of the dye in the pharyngeal cavity; the pharyngeal cavity was swabbed with moistened cotton-tipped applicators every 2 min for the following 20 min and then every 5 min for the next 60 min (or until the appearance of the dye). As a first control, 100 ll of dye solution (prepared in normal saline), and as a

3

second control, non-mucoadhesive 17% P F127 in situ gel were introduced by a micropipette and observed similarly. The experiment was conducted using four rats for each formulation.

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2.7. Pharmacological study

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Granisetron exhibits both antiemetic and analgesic activities; both activities are related to peripheral and central 5-HT3 receptors antagonism (Christidis et al., 2005; Haus et al., 2004; Martin et al., 1998; Thompson and Lummis, 2007). Also, ketorolac is a potent analgesic. Therefore, the selected formulation (F4) was evaluated for analgesic activity using the formalin test and hot plate test and compared to individual drugs.

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2.7.1. Animals Rats were obtained from the institute of ophthalmological research, Giza, Egypt. The animal care and experimental protocol was reviewed and approved by the institutional ethical committee, Faculty of Pharmacy, Cairo University (NO. PI (264)). The rats were randomly allocated in 3 main groups: Group A received the treatments intra-nasally (i.n) and underwent the formalin test. Group B received the treatments intra-peritoneally (i.p) and underwent the formalin test. Group C received F4 and saline intra-nasally and underwent the hot plate test.

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2.7.2. The formalin test To test chemical nociception, the formalin test was used according to the method of Dubuisson and Dennis, 1977. The inspection glass box is 30 cm wide, 40 cm long and 30 cm high with a glass floor and a mirror at a 45° angle under the floor to allow clear observation of the rats’ paws. Each rat was acclimatized to the inspection box for 15 min, and then 50 ll of formalin solution (1% v/v) was injected into the dorsal surface of its right hind paw. The rat was replaced in the inspection box and was observed for the number of licks and bites it performed to the injected paw during the 0–5 min and 10–60 min post formalin injection by a blinded observer. Group A rats were divided into 4 subgroups of six rats each that received the selected formulation (F4, A1), granisetron (10 mg/kg, A2), ketorolac (80 mg/kg, A3) or saline (0.9% w/v, A4) intranasally (100 ll in each nostril) 15 min before formalin injection. The selected formulation was prepared as follows: 17% w/w P F127 + 0.05% w/w P TR-2 + 0.5% w/w granisetron + 5% w/w ketorolac. Granisetron was prepared as follows: 16% w/w P F127 + 0.05% w/w P TR-2 + 0.5% w/w granisetron (This formulation was evaluated for gelation temperature and it showed gelation temperature of 29.50 °C which is within the desirable range 29–34 °C). Ketorolac was prepared as follows: 17% w/w P F127 + 0.05% w/w P TR-2 + 5% w/w ketorolac. Group B rats were divided into 3 subgroups of five rats each that received the combination of granisetron and ketorolac ‘‘B1’’ (10 mg/kg and 80 mg/kg respectively), granisetron (10 mg/kg, B2), or saline intra-peritoneally (1 ml/kg body weight, B3) 15 min before formalin injection. Granisetron and ketorolac were dissolved in saline.

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2.7.3. Hot plate test To test thermal nociception, rats were placed on hot plate (Hot Plate Analgesy Meter, MK-350D, Muromachi Kikai Co., Ltd., Japan) maintained at 51 ± 1 °C. The time taken by each rat to begin licking or biting its forepaw was recorded and called the reaction time (Wiesenfeld-Hallin et al., 1991). A cut-off limit of 25 s was applied to avoid tissue damage. The hot plate test was carried out at 0, 15,

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30, 45 and 60 min post intra-nasal administration (100 ll in each nostril) of the selected formulation (F4, n = 6) or saline (n = 6). 2.7.4. Statistical analysis Data were displayed as mean ± standard error (S.E.). Statistical analysis was performed using Prism version 5.0 (GraphPad software, California, USA). Results were compared using one-way analysis of variance (ANOVA) followed by Bonferroni post-test. The statistical significant difference was set at P < 0.05.

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3. Results

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The prepared in situ nasal gels were found to be clear at a pH range of 4.5–6.5 which is considered as nasal physiological pH range (Bhandwalkar and Avachat, 2013).

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3.1. Spectrophotometric analysis of granisetron and ketorolac in SNES pH 5.5

3.4.1. Effect of P F127 concentration Gelation temperatures for plain P F127 in situ gels were found to decrease upon increasing concentration of P F127 (28.50 ± 0.50, 26.50 ± 0.87, 23.33 ± 0.58, 21.5 ± 0.50 and 19.83 ± 0.29 °C for 16% through 20% w/w respectively).

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3.4.2. Effect of drug addition Fig. 1a showed that, loading of 5% ketorolac and 0.5% granisetron into P F127 solutions increased gelation temperature. Similar results were obtained by Zaki et al. (2007) and Bhandwalkar and Avachat (2013), who observed that loading 10% w/w metoclopramide HCl and 21.09% w/w venlafaxine HCl into P F127 solutions increased the gelation temperature of 18% P F127 gels. The increase in gelation temperature of the non mucoadhesive medicated in situ gel (M, 17% P F127 w/w) was still within the target range (29–34 °C) for in situ gelling at the nasal cavity and hence, it was selected for further study.

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3.4.3. Effect of mucoadhesive polymers The mucoadhesive polymers lowered the gelation temperature of medicated P F127 solution (M, 17% P F127 w/w) depending on the concentration of the polymer, Fig. 1b. In situ gelling systems, F1, F4, F5, F7 and F8 showed optimum results in terms of gelation temperature from 29 °C to 34 °C (nasal cavity temperature). Hence, they were selected for further study.

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3.5. Determination of drug content

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The drug content of selected mucoadhesive in situ gelling systems is shown in Table 2. The drug content is found to be in acceptable range for all formulations indicating uniform distribution of the drugs.

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For simultaneous determination of granisetron and ketorolac in SNES, the overlaying of absorption curves of granisetron and ketorolac was obtained for zero, first and second derivative orders. Zero order spectra showed interference between absorbance of granisetron and ketorolac; therefore a derivative of absorbance was calculated. A wavelength at 342 nm (first derivative) was suitable for measuring ketorolac without interference from granisetron. On the other hand, it was not appropriate to measure granisetron from first derivative spectra, therefore second derivative spectra was applied and a wavelength at 304 nm was suitable for measuring granisetron without interference from ketorolac.

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3.2. Recovery study of granisetron and ketorolac mixtures in SNES

3.6. Measurement of steady shear viscosity

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The accuracy of the assay method ranges from 98.30% to 106.21% and from 97.80% to 104.76% for granisetron and ketorolac respectively. The results revealed the suitability of the adopted UV spectrophotometric method for simultaneous determination of granisetron and ketorolac in SNES pH 5.5.

Table 2 reveals that the steady-shear behavior of P F127 based formulations is influenced by addition of drugs, mucoadhesive polymers and concentration of mucoadhesive polymers.

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3.3. Compatibility study of granisetron, ketorolac and different pharmaceutical excipients using infrared spectroscopy (IR)

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The IR scan of granisetron showed characteristic bands at 3232 cm1 due to indazole ring and at 2937 cm1 due to the alkene group. It also showed bands at 2447 cm1 and 1645 cm1 characteristic to protonated tertiary amine group and C@O stretch respectively (Late and Banga, 2008). The IR scan of ketorolac showed characteristic bands at 3448 cm1 due to COOH stretching and at 3348 cm1 due to NH stretching. It also showed bands at 1595 cm1 and 1274 cm1 characteristic to AC@O stretching and ACAN stretching respectively (Rao et al., 2009). There were no considerable changes in the IR peaks of both drugs in the different examined physical mixtures, indicating the absence of chemical interaction.

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3.4. Measurement of gelation temperature

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The Mean nasal mucosal temperature was reported to be in the range of 30.2 °C to 34.4 °C (Lindemann et al., 2002). Our aim is to develop nasal thermogel(s) of gelation temperature between 29 °C to 34 °C.

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Fig. 1. Effect of drugs (a) and mucoadhesive polymers (b) added on gelation temperature of different P F127 solutions. P: Plain P F127 and M: Medicated P F127.


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a *

Formula

% GH contenta (w/w)

% KT contenta (w/w)

m*

n

P (17%PF127) M (17%PF127) F1 F4 F5 F7 F8

– 99.10 ± 0.03 98.69 ± 3.69 97.40 ± 0.09 97.95 ± 1.13 101.48 ± 1.85 98.91 ± 1.49

– 101.5 ± 2.99 100.1 ± 4.58 99.29 ± 1.31 99.25 ± 2.45 101.42 ± 3.48 98.39 ± 1.52

273,400 129,003 252,348 155,632 184,884 218,776 238,726

0.0944 0.0724 0.0774 0.1209 0.1039 0.1171 0.0995

Each value represents the mean ± SD (n = 2). P; Plain, M; Medicated. Significant difference at p 6 0.05.

5

profiles up to 60 min as follows: (60 min release time was used for comparison because the performed pharmacological studies were designed throughout a time course of 60 min)

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(i) Concerning granisetron release (Fig. 2b), all mucoadhesive in situ gels showed comparable release profiles and only F4 showed 100% release by the end of the 60 min (similar to non mucoadhesive medicated gel). (ii) Concerning ketorolac release (Fig. 2d), F5 and F8 showed significantly (p 6 0.05) lower release rate than other mucoadhesive nasal formulae {ketorolac dissolution efficiency at 60 min ‘‘DE 60’’ is ranked as follows: F4 (51.29 ± 6.55) > F1 (40.63 ± 3.22) > F7 (32.92 ± 0.51) > F5 (22.89 ± 3.07) F8 (21.53 ± 1.14).

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Concerning the effect of the drug, the value of consistency index (m) is 2.11 times lower for medicated non mucoadhesive P F127 in situ nasal gel (M, 17% w/w P F127) compared to the plain non mucoadhesive 17% w/w P F127 in situ gel (P, 17% w/w P F127), indicating the viscosity-lowering effect of granisetron and ketorolac. This finding is consistent with those obtained by Zaki et al. (2007), who reported that addition of metclopramide HCl had lowering effect on steady-shear behavior of 18% w/w P F127 in situ gel. Regarding the effect of the mucoadhesive polymer, addition of mucoadhesive polymers increased the viscosity of the medicated non mucoadhesive P F127 in situ nasal gel (17% w/w P F127). This is revealed by an increase in (m) values of F1 through F8 compared to the in situ gel containing no mucoadhesive polymer. In addition, increase in the polymer concentration reinforced the viscosity of medicated P F127 (M, 17% P F127). For F4 (17% P F127/0.05% P TR-2) and F5 (17% P F127/0.1% P TR-2), 1.21 times (129003 to 155632) and 1.43 times (129003 to 184884) higher (m) values were observed for F4 and F5 respectively compared to that of P F127 (17% w/w) in situ nasal gel. Similar findings are also obtained for in situ gels containing CP 974P, increasing polymer concentration reinforced the viscosity of non mucoadhesive medicated P F127 in situ nasal gel. This is revealed by 1.69 times and 1.85 times higher (m) values for F7 and F8 respectively compared to that of non mucoadhesive medicated P F127 in situ nasal gel (M, 17% w/w P F 127). Flow indices for all the selected in situ nasal gel formulations are found to be less than one referring to non-Newtonian flow.

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3.7. In-vitro drug release from different in situ nasal gels

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In-vitro release of various formulations prepared is graphically illustrated in Fig. 2. The in situ nasal gel formulated with HPMC as a mucoadhesive polymer (F1) showed almost 100% granisetron and 90% ketorolac release after 4 h and 6 h respectively, compared to non mucoadhesive medicated in situ nasal gel (M, 17% P127) which exhibits 100% granisetron and 100% ketorolac release after 1 h and 3 h respectively. Obviously, inclusion of HPMC in F1 retarded both drugs release rates compared to the in situ nasal gel (M, 17% P127). F4 (17% P F127/0.05% P TR-2) showed 100% granisetron and 100% ketorolac release after 1 h and 4 h respectively, whereas F5 (17% P F127/0.1% P TR-2) exhibited 100% granisetron and 91.19% ketorolac release after 2 h and 6 h respectively. Similarly, F7 (0.1% CP 974P) showed 100% granisetron and 91.45% ketorolac release after 4 h and 6 h respectively, while F8 (0.2% CP 974P) showed 100% granisetron and 83.26% ketorolac release both after 6 h. It can be concluded that inclusion of mucoadhesive polymers retarded the overall granisetron and ketorolac release rates (Fig. 2a and c). For simplicity of comparison the selection of mucoadhesive in situ gels for subsequent studies was based upon data of release

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Accordingly, non-mucoadhesive (M, 17% P F127) and mucoadhesive (F4 & F7) in situ nasal gels were chosen for in-vivo mucociliary transit time. Despite that DE 60 of F1 > F7, the expected higher mucoadhesive power of CP974P (poly acrylic acid derivative) upon which F7 is based favored its selection over F1 (neutral HPMC based).

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3.8. In-vivo mucociliary transit time (MTT) of the selected in situ nasal gels

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Fig. 3, illustrates MTT of amaranth dye solution, non mucoadhesive and mucoadhesive in situ nasal gels. MTT of the non-mucoadhesive M, 17% P F127 in situ gel was 2.36-times higher (P < 0.05) than that of control (13 min and 5.5 min respectively). In case of the mucoadhesive in situ gels a further increase in the MTT was observed as compared to the nonmucoadhesive in situ gel and control dye solution. The increase in MTT is significant for all mucoadhesive polymers (P < 0.05), however, the highest increase of MTT was presented by CP 974P containing in situ gel (F7) which showed 3.46-times and 8.18-times increase in MTT as compared to the non-mucoadhesive gel and the control dye solution respectively. The result of M, 17% P F127 is consistent with the previously reported by Zhou and Donovan (1996), who demonstrated that P F127 gels, had significantly prolonged residence time in the rat nasal cavity. M, 17% P F127 exhibits in situ gelling behavior therefore; it can be retained at the site of administration for longer period of time than control dye solution.

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3.9. Pharmacological study

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3.9.1. The formalin test Effects of all treatments administered intra-nasally on formalin-induced nociception are graphically illustrated in Fig. 4 (panels a & b). Both in the early and late phases, rats of subgroups A1, A2 and A3 treated with F4i.n, granisetroni.n, and ketorolaci.n respectively bit and licked their injected paws significantly less than the saline treated group (A4) indicating significant analgesia following the intra-nasal administration. Following intraperitoneal injection, only subgroup B1 which received the selected formulation F4i.p. showed significant analgesia during the early phase compared to the control group, as shown in Fig. 5, panel a. Worth noting that, the analgesic effect of F4i.p. was significantly higher than granisetroni.p. Comparing the effects of the different treatments following intranasal and intraperitoneal administration, Fig. 5 (panels a & b); it is clear that only the intra-nasal administration of the selected formulation F4 showed significant analgesia during both the early and late phases indicating its potential therapeutic value.

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Fig. 2. In-vitro release profiles of granisetron and ketorolac from different in situ nasal gels, complete profiles (a and c respectively) and during 60 min time course (b and d respectively). GH, Granisetron; KT, Ketorolac.

Fig. 3. In-vivo MTT of different in-situ nasal gels. ⁄Significant difference from controls at p < 0.05.

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3.9.2. Hot plate test Results of hot plate test are graphically illustrated in Fig. 6. Similar to the results of the formalin test, intranasal administration of the selected formulation F4i.n showed significant analgesia indicated by longer reaction time, compared to the control group at 30, 45 and 60 min post administration. The peak effect of analgesia was observed 30 min post administration.

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P F127 was selected as the base for developing in situ gelling systems because it has excellent thermoreversible gelling property, low toxicity and irritation ‘‘good tolerability’’, good drug release and compatibility with different chemicals and has been found useful in nasal formulations (Cho et al., 2011; Park et al., 2002; Zaki et al., 2007; Zhang et al., 2002). The thermosensitive gelling property is known to result from the change in micellar number with temperature. With increasing temperature, the number of micelles formed increases as a consequence of the negative coefficient of solubility of block copolymer micelles. Eventually the micelles become so tightly packed that the solution becomes immobile and gel is formed (Kabanov et al., 2002).

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Fig. 4. Effect of group ‘‘A’’ treated via intranasal route on early (a) and late (b) phases of formalin-induced nociceptive response.


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Fig. 5. Effect of different groups treated via intranasal and intraperitoneal routes on early (a) and late (b) Phases of formalin-induced nociceptive response.

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Evaluating gelation temperature, the results showed dependence of gelation temperature on P F127 concentration, such dependence was previously reported (Alexandridis and Alan, 1995; Edsman et al., 1998; Wei et al., 2002). The investigators suggested that as P F127 concentration is increased, the intermicellar distance and degree of micellar swelling necessary for P F127 to interact are reduced leading to a decrease in gelation temperature. Also, drug addition positively affected gelation temperature. This may be due to water soluble nature of both drugs which may cause modification of the process of micellar association of P F127 gels thereby increasing their gelation temperature (Gilbert et al., 1987). The observed lowering effect of mucoadhesive polymer on gelation temperature could be explained by their ability to bind to polyethylene oxide (PEO) chains present in the P F127 molecules promoting dehydration and causing an increase in entanglement of

Fig. 6. Analgesic effect of the selected formula (F4i.n) compared to Controli.n using hot plate method. ⁄p < 0.05, statistically different from the control solution.

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adjacent molecules with more extensive intermolecular hydrogen bonding (Choi et al., 1998; Ryu et al., 1999; Wei et al., 2002). As in the in vitro release study, the observed differences in the overall drug release patterns from different in situ nasal gels correlated well with the differences observed in viscosity study as a function of the polymer concentration. The release rate lowering effect obtained with mucoadhesive polymers addition could be explained by their ability to increase the formulation viscosity as illustrated under the steady shear viscosity measurement which in turn decreases diffusion of the drug through gel matrix, thereby reducing release rate. Furthermore, Ryu et al., 1999, attributed the release retarding effect of mucoadhesive polymers to their possible squeezing effect on the aqueous channels of poloxamer micelles through which the drug diffuses. Increasing the mucoadhesive polymer concentration; P TR-2 concentration from 0.05% (F4) to 0.1% (F5) and CP 974P concentration from 0.1% (F7) to 0.2% (F8), was accompanied by further retardation in granisetron and ketorolac overall release rates. These results agree with their viscosity behavior as F5 and F8 show higher consistency indices (m) than that of F4 and F7 respectively. Such retardation of release may be due to reduction in number and dimensions of the channels in gel structure by increased viscosity of the formulation (Bhalerao et al., 2009). As shown under MTT, addition of mucoadhesive polymer to non mucoadhesive medicated in situ gel helped in retaining the formulation in nasal cavity for longer time period. F4 and F7 (containing P TR-2 and CP 974P respectively) significantly increased (P < 0.05) MTT values (40 min and 45 min for F4 & F7 respectively) compared to aqueous dye solution (5.5 min) and non mucoadhesive medicated in situ gel (13 min). Retention of F4 and F7 in nasal cavity for extended period of time may be due to mucoadhesive polymers which have a very high percentage (52–56% & 58–68% for P TR-2 and CP 974P respectively) of carboxylic groups (US Pharmacopeia 30/NF 25, 2007) that undergo hydrogen bonding with sugar residues in oligosaccharide chains in the mucus membrane, resulting in formation of a strengthened network between polymer and mucus membrane. Thus, these polymers having high density of available hydrogen bonding groups would be able to interact more strongly with mucin glycoproteins (Boyapally et al., 2010). The results of pharmacological study showed that intranasal administration of the selected formulation F4 produced a significant analgesic effect against chemical and thermal nociception as reflected by the results of the formalin test and the hot plate test. These findings indicated the successful absorption of the formulation’s component drugs, granisetron and ketorolac after intranasal administration. Our results are in line with previous studies showing the analgesic effect of ketorolac (Bustamante and Paeile, 1993) and granisetron (Loyd et al., 2012; Moser, 1995; Nikfar et al., 1998; Oyama et al., 1996). The current study also compared the analgesic effect of the selected formulation following intranasal administration to that following intraperitoneal administration. It is clear that intranasal delivery of the selected formulation F4 offered significant advantages over the intraperitoneal injection as it produced significant analgesia during both the early and late phases of the formalin induced nociception, whereas intraperitoneal injection of the selected formulation F4 produced significant analgesia only during the early phase. This difference is probably attributed to the extensive first pass effect granisetron experiences following intraperitoneal injection (Tan et al., 2002) but is avoided by intranasal delivery. It is safe to predict that intranasal delivery of the selected formulation may offer higher therapeutic value than oral administration, as intranasal administration can avoid granisetron first pass effect, and potentially minimizes the gastrointestinal adverse effects associated with ketorolac oral therapy as well.


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Pluronic F127, CarbopolÒ 974P have been previously reported as safe vehicles for nasal delivery systems (Callens et al., 2001; Park et al., 2002). Pemulen™ TR-2 is a toxicologically preferred non benzene carbomer copolymer recently used for nasal delivery (US patent, 2010). Safety of the designed F4 on the nasal mucosa could be predicted yet planned to be performed.

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Dual action granisetron/ketorolac copolymer in-situ nasal thermal gel was successfully prepared (17% w/w P F127/0.05% w/w P TR-2). It showed a pH and gelation temperature (30.83 ± 0.29 °C) suitable for nasal administration with satisfactory in-vitro release pattern (100% and 77% at 1 h of granisetron and ketorolac respectively) and MTT (40 ± 4.08 min). Selected formulation, F4 significantly modulates chemical and thermal nociception indicating bioavailability of the formulation in a superior manner to intraperitoneal injection and probably to oral administration as well. We are recommending bioavailability study in human in the future.

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Appendix A. Supplementary material

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Supplementary data associated with Pleathis article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps. 2015.04.015.

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