Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel DOI: https://doi.org/10.31351/vol31issSuppl.pp32-44 32 Formulation Variables Effect on Gelation Temperature of Nefopam Hydrochloride intranasal in Situ Gel (Conference Paper) # Ammar A. Alabdly*,1 and Hanan J. Kassab ** # 10th scientific conference sponsored by College of Pharmacy, University of Baghdad 2-3 June 2022 * Department of Pharmaceutics, College of Pharmacy, Anbar University, Anbar, Iraq ** Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract Nefopam (NF) HCl is a non-narcotic centrally-acting, non-opioid benzoxazocine analgesic used to relieve acute and chronic pain. It exhibits low bioavailability (about 36%) due to its first-pass degradation in the liver. Intranasal administration has been proposed as a new route for targeting active brain sites and enhancing the bioavailability of NF HCl by bypassing hepatic metabolism. In situ gel of NF HCl was prepared by the cold method using different concentrations of Poloxamer 407 (PoloX 407), Poloxamer 188 (PoloX 188), Hydroxypropylmethyl cellulose (HPMC K4M), Carbapol 934 (Car934), Methylcellulose (MC) and Hyaluronic acid(H.A) polymers. The results showed that identification tests including differential scanning calorimetry (DSC), Fourier Transform Infra-Red Spectroscopy (FTIR), and spectrophotometric λ max determination are superimposed with references, solubility study shows that NF HCl is suitable to be administered intranasally; Compatibility studies reveal incompatibility of N.F HCl with HPMC K4M and Car934; meanwhile, no interaction with MC and H.A. In conclusion, the obtained results revealed the feasibility of the produced NF HCl intranasal in situ gel. Keywords: Nefopam Hydrochloride, Thermosensitive in situ gel, Poloxamer 407, Poloxamer 188. موقعي داخل االنف لعالج النيفوبام تأثير بعض متغيرات الصياغة على درجة حرارة تكون الهالم ال # مؤتمر( )بحث هايدروكلورايد ** جالل كساب وحنان 1*،العبدلي دعبد المجيعمار 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق. االنبار، االنبار، جامعة الصيدلة، كلية الصيدالنيات، فرع * العراق. بغداد، بغداد، جامعة الصيدلة، كلية الصيدالنيات، فرع ** الخالصة األمد، فئتها الكيميائية البنزوكسازوسين، والطويلينتمي عالج النيفوبام الى فئة االدوية غير المخدرة من مسكنات االآلم لالستخدام القصير .تجاوز هذه المشاكلل نف لالللتحضير كهالم موقعي لذلك يرشح .الكبدتكسره في ل %( 36)يبلغ ف يواجه مشكلة في توافره الحيوي و و هيدروكسي بروبيل مثيل 188و بولوكزامر 407تم تحضيير الصيغة باستخدام الطريقة الباردة عن طريق مزج تراكيز مختلفة من بولوكزامر و أظهرت دراسة الذوبانية المصادر، أظهرت النتائج تطابق المواد المستخدمة مع ، و مثيل سليلوز. 934 سليلوز ، حمض الهايلورونك ، كاربوبول اما التوافقية فأظهرت عدم توافق المزيج الذي يحتوي هايدروكسي بروبيل مثيل سليلوز و الكاربوبول بينما الهيئة،قابلية استخدام النيفوبام على هذه يستنتج من ذلك ان النتائج المستحصلة كشفت عن القدرة لتصييغ النيفوبام كهالم موقعي الهايلورونك و المثيل سليلوز. أظهرت توافق استخدام حمض داخل االنف لتحسين توافره الحيوي. . 188، بولوكزامر 407الكلمات المفتاحية : نيفوبام هايدروكلورايد ، هالم موقعي متحسس للحرارة ، بولوكزامر Introduction A direct nose to brain delivery system is an excellent way to increase absorption to the central nervous system via the olfactory zone positioned at the top of the nasal cavities crossing the blood-brain barrier to reach the brain. Nose-to-brain delivery offers many advantages including the absence of stomach and pancreatic enzymes, mucus's pH neutrality in the nasal cavity, less dilution by the gastrointestinal tract contents, and the mucosa are more permeable to drugs than the gastrointestinal system. Additionally, It diminishes the therapeutic dosage and associated adverse effects(1). This mode of administration may be advantageous for treating serious brain diseases like tumors or deteriorating neural illnesses such as Parkinson and Alzheimer (2) or as migraine therapy(3). N.F HCl is a non-narcotic centrally-acting, non-opioid benzoxazocine analgesic used to relieve acute and chronic pain (4) and chemical name of 5- methyl -1 -phenyl -1,3,4,6- tetrahydro- 2,5-benzoxazocine; hydrochloride as seen in Figure 1 and molecular weight of 289.8 log P ~ 3.4(5). 1Corresponding author E-mail: amar.abdulmajeed1200a@copharm.uobaghdad.edu.iq Received: 28/ 6/ 2022 Accepted: 5/9 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31issSuppl.pp32-44 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 33 Figure 1. Chemical structure of Nefopam HCl Orally administration is associated with low bioavailability (about 36%) due to its first-pass degradation in the (6) and shows several common side effects related to peripheral action of nefopam hydrochloride, such anticholinergic side effects include urinary retention, dry mouth, and nausea (7). N.F HCl has a pKa of 8.98, s0877t65dc vo it will be mainly in an ionized form in nasal mucosa pH (4.5-6.5) (8) mnhuitrewerjkgoi9 /and a molecular weight lower than 300,allow a better penetration via passive diffusion (9). Also, the log P is higher than 35 therefore, penetration is predicted to be over 80% (10). In situ gel is a solution that undergoes sol-gel transformation by a specific physiological trigger such as body temperature in the case of thermosensitive in situ gel. 3 Dalia M.N. Abouhussein et al. develop rivastigmine nose- to-brain targeted poloxamer based thermosinsitive mucoadhesive in situ gel(11). Accordingly, the current study aims to explore the factors affecting on formulation and gelation temperature of polxamer 407 based intranasal in situ gel of nefopam. Materials and Methods Materials Nefopam (NF) HCl, Hyaluronic acid (H.A), and HPMC K4M were acquired from (Baoji Guokang,Ltd–China),Car934,PoloX 407, and PoloX 188 were purchased from ( Eastman Chemical company – USA), Benzalkonium chloride (BNZ) was obtained as a gift from ( The State Company for Drug Industry and Medical Appliances, SDI – Iraq), Every other substance, including reagents, was of analytical grade. Methods Characterization of NF HCl Identification tests to confirm the active pharmaceutical ingredients (API) and excipients' identities and purities. Determination of the melting point and differential scanning calorimetry (DSC) of NF HCl The melting point was determined using the capillary tube approach (12), and the thermal behavior was evaluated using a DSC- 60 (Shimadzu 60 plus Japan). A small quantity of NF HCl. was sealed in ordinary aluminum pans and then heated at rate of 10°C every min. over a range of temperatures 50°C to 300°C. Fourier Transform Infra-Red Spectroscopy (FTIR) FTIR is effective method for identifying pure compounds and compatibility studies between the active pharmaceutical ingredient (API) and the excipient(s) (13,14). FTIR was performed using Lambda 7600 Australia instrument for NF HCl, HPMC K4M, Car934, H.A, MC, and physical mixture. The FTIR spectrum of N.F HCl intranasal gel of the selected formulas and the excipients used were recorded using FTIR spectrometer between spectral bands 4000 - 400 cm-1. The sample was mixed in a mortar with potassium bromide (KBr), then compressed into a tiny disc using a hydraulic press. Standard calibration curves of NF HCl The Standard calibration curves of NF HCl in double distilled water (D.D.W), simulated nasal fluid (SNF, CaCl2.2 H2O 0.32 g/L , NaCl 7.45 g/L, and KCl 1.29 g/L.)(15), and aqueous phosphate buffer saline (PBS) at pH 7.4, were made by controlled dilutions of the stock solution (1mg/mL) at various concentrations (50–350 g/ml). Each solution's absorbance was measured at the N.F HCl λmax. Plotting was done between the measured absorbance and the corresponding concentration. Solubility study of NF HCl The test tube method was used to measure the solubility of drugs in an aqueous buffer, simulated nasal fluid, and deionized water by adding an excess amount of NF HCl in 10 ml of solvents mentioned above in a glass tube, then stored at 34 oC takes 72 hours to establish an equilibrium state in a shaker water bath. After that, a solution centrifuged for 15 minutes at 3000 rpm ; then, the solution is filtered using 0.45 μm filter paper and a final concentration of NF HCl measured after a suitable dilution to be read by UV-Vis (16). this test is checked in triplicate manner. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 34 Preparation of NF HCl intranasal in situ gel The cold approach was applied to formulate different compositions of mucoadhesive thermosensitive in situ gel as detailed in Table 1. Different concentrations of Poloxamer 407 (PoloX 407) (17-20%) were blended with or without Poloxamer 188 (PoloX 188) (3-4%) in cold double distilled water (D.D.W) immersed in ice with continuous stirring for 4-6 hours. The obtained cloudy mixture was stored in the refrigerator at 4 oC for 24 hours to get a clear solution. The gelation temperature was determined in triplicate for each formula; only compositions with a gelation temperature in the range of 30-36 oC (17) were selected for further addition to the final formula. Table 1. Compositions of intranasal in situ gel polymer mixture (P). P No. PoloX 407 (%w/v) PoloX 188 (%w/v) D.D.W q.s ml 1 17 10 2 18 10 3 19 10 4 20 10 5 17 4 10 6 18 4 10 7 19 4 10 8 20 4 10 9 17 3 10 10 18 3 10 11 19 3 10 12 20 3 10 After that, different concentrations of different mucoadhesive polymers (HPMC K4M, Car934,H.A, and M.C) as shown in Table 2. were added slowly to a selected polymer mixture with continuous stirring for 30 min at 50 rpm. The slow stirring speed was applied to prevent foam formation that can affect the mucoadhesive polymer solubility in poloxamer solution. Then N.F HCl and BNZ at a concentration (20 mg/ml) and (0.01 % w/v), respectively, were added slowly with continuous slow stirring for the same reason mentioned above, and complete the volume with double distilled water up to 10 ml. Finally, the mixture was stored for 24 hours at 4 oC to eliminate a formed foam. Table 2. Compositions of the formulated intranasal in situ gel with mucoadhesive polymers (P.I.). P.I. No. NF mg PoloX 407(%) PoloX 188(%) HPMC K4M (%) H.A (%) Car934 (%) M.C (%) 1 200 17 1 2 200 19 4 1 3 200 18 3 1 4 200 19 3 1 5 200 17 0.75 6 200 19 4 0.75 7 200 18 3 0.75 8 200 19 3 0.75 9 200 17 0.5 10 200 19 4 0.5 11 200 18 3 0.5 12 200 19 3 0.5 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 35 Continued table 2. P.I. No. NF mg PoloX 407(%) PoloX 188(%) HPMC K4M (%) H.A (%) Car934 (%) M.C (%) 13 200 17 0.75 14 200 19 4 0.75 15 200 18 3 0.75 16 200 19 3 0.75 17 200 17 0.5 18 200 19 4 0.5 19 200 18 3 0.5 20 200 19 3 0.5 21 200 17 0.25 22 200 19 4 0.25 23 200 18 3 0.25 24 200 19 3 0.25 25 200 17 0.3 26 200 19 4 0.3 27 200 18 3 0.3 28 200 19 3 0.3 29 200 17 0.2 30 200 19 4 0.2 31 200 18 3 0.2 32 200 19 3 0.2 33 200 17 0.1 34 200 19 4 0.1 35 200 18 3 0.1 36 200 19 3 0.1 37 200 17 0.3 38 200 19 4 0.3 39 200 18 3 0.3 40 200 19 3 0.3 41 200 17 0.2 42 200 19 4 0.2 43 200 18 3 0.2 44 200 19 3 0.2 45 200 17 0.1 46 200 19 4 0.1 47 200 18 3 0.1 48 200 19 3 0.1 Gelation temperature The gelation temperature is determined in triplicate by visual inspection of the glass tube when it is tilted at 90 degrees. Gelation temperature recorded when no movement of solution occurred as a result of conversion into a gel. Each glass tube that contained 2 ml of the formulation was kept in the water bath at 25 oC; then, the temperature raised gradually at a rate of 1 oC / min, and at the end of the minute, a glass tube titled at 90 0 in a horizontal position to show if the solution is converted into a gel (18). Appearance Visual inspection of selected formulas against white and black paper in triplicate were conducted to examine the occurrence of any incompatibility due to an interaction between API with excipients or among excipients themselves. A change in color or precipitation was considered as a sign of incompatibility (19). Compatibility study FTIR was carried out on several separated formulas observed by appearance test to compare its FTIR spectra with the original spectra of drug and polymers. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 36 FTIR was performed with the same method mentioned previously in FTIR section. Gelling capacity By adding 1 ml of the system to a vial containing 2 ml of freshly prepared and equilibrated at 34° C simulated nasal fluid, and visually observing the gel formation and noting the time for gelation every 5 sec. and the time taken for the gel formed to dissolve, noting desolvation every 15 min., the gelling capacity was determined in triplicate(20). pH of NF HCl intranasal in situ gel The pH of the selected P.I formulas was measured by using HI98107 pH meter, Hanna, Italy, in triplicate at room temperature using pH meter. pH 4 and pH 7 standard buffer solutions were utilized to calibrate the equipment (21). The osmolarity of NF HCl intranasal in situ gel Osmotic pressure was measured by using a OSMOMAT 030 cryoscopic osmometer. The osmometer was zeroed by purified water and then 300 mOsmol NaCl (1%NaCl) standard solution for calibration. Then, 50 µL of the P.I formulas were used in a clean, dry microtube. after checking for bubbles of air , the measurement performed. (22), and the results were recorded in triplicate. Mucoadhesive strength The weight needed to separate a preparation from nasal mucosa of sheep using a modified pan balance for P.I formulae yielded the mucoadhesive detachment force. Within 30 minutes of the sheep's sacrifice, we were able to retrieve a sheep nose from the slaughterhouse. The sheep's nasal mucosa was preserved in 0.9% NaCl. It was prepared for usage after blood and bony cartilage were taken out of the mucosal membrane. A glass vial was used to hold the mucosal side. Before they were connected to the equipment, these vials were held at 34 °C for 5 minutes. One side included a modified syringe vial that was inverted and held 1 mL of formula, and the other side contained a small plastic beaker that had previously been weighted. The syringe held specific amounts of each formulation (1 mL). By permitting two minutes of contact time, it was possible to confirm that tissue glued to a petri dish underneath the syringe was in intimate contact with the formula. The weight increased in gradually in the plastic pan on the other side until the formula separated from the tissue. The bioadhesive force, which represents the minimal weight necessary for tissues to detach from the surface for any formulation, is determined using the equation; Mucoadhesive force (dynes/cm2 ) = MG / A where M is the smallest weight in grams needed to separate two vials, G is the acceleration gravity which is (980 cm/s2), and A is the exposed area of the tissue. For each measurement, the newly formed nasal mucosa was employed (21). Results and Discussion Melting point determination The temperature at which NF HCl melted by the method mentioned above was found to be 260 °C, which coincides with the reported melting point (23). Differential Scanning Calorimetry (DSC) The DSC of NF HCl, shown in Figure 2 , agrees with the reference that it exhibits an endothermic peak at 259.8 oC (24) both melting point and DSC give good evidence for the purity of crystalline drug powder. Figure 2. DSC thermogram of N.F HCl Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 37 Fourier Transform Infra-Red Spectroscopy (FTIR) FTIR was carried out on drug and polymers powder, as shown in the Figures 3 and 4, r Figure 3. FTIR of NF HCl Spectra is compatible with the characteristics absorption peaks of N.F HCl FTIR spectra, at 3440 cm-1 ,3049 cm-1 , 2947 cm-1 , 2440 cm-1 , 1464 cm-1 , 1437 cm-1 , 1357 cm-1, 1107 cm-1 , 1028 cm-1 , and 760 cm-1 ,correlates to C-O-C of ring, substituted aromatic ring, C-H of alkane(s), resonance of amine salt NH+ , C=C of substituted aromatic ring, C3-N, C-O stretch of cyclic ether, o-disubstituted aromatic ring, and mono-substituted and o-disubstituted aromatic rings, respectively. These findings are superimposed with reference (25). Figure 4. FTIR for HPMC K4M (A), H.A (B), MC (C), Car934 (D), PoloX 407 (E), and PoloX 188 (F). Spectra show characteristics peaks of HPMC at 3650-2800 cm-1 broad peak, 1653 cm-1, 1450 cm-1, and 1026 cm-1, that are corresponded to a broad range of hydroxyl H- bond that overlap and cover sp3 C-H stretching of alkane, cellulose nitrate ester group that converted from cellulose hydroxyl groups, spectra of poly-(vinyl alcohol) O-H deformation spectra of a secondary alcohol, and C-O-C spectra, respectively. Spectra coincide with reference (26). H.A peaks at 3500-3000 cm-1,2891 cm-1, 1603 cm-1, and 1030 cm-1 are indicated for broad spectra of H-bond of hydroxyl of acidic, alcoholic, and amine groups of hyaluronate, C- H stretching of ring moieties, COOH group of an acidic group, and C-O-C ether group. That agrees with reference (27). MC peaks at 3600-3050 cm-1,2926 cm-1, 1640 cm-1, 1456 cm-1, and 1046 cm-1; these are spectra for broad H-bond spectra of acidic and alcoholic -O.H. group that overlap weak C-H stretching peak at 3000-3100 cm-1, C-H stretching of cyclic alkane, cellulose nitrate ester group that converted from cellulose hydroxyl groups, poly- (vinyl alcohol), and C- O-C peak, respectively. These peaks are compatible with the reference peaks(28). Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 38 Car934 peaks at 3500-2500 cm-1, 1711 cm-1, and 1165 cm-1, correlate to carboxylic dimer H-bond COOH that hide sp3 C-H stretching peak, C=O, and C-O stretching mode, respectively. That is match the reference(29). PoloX 407 , and PoloX 188 has two characteristic peaks appear at 2868 cm-1 and 1100 cm-1, representing the reading of R-CH3 and C-O-C peaks, respectively. These are agreed with the reference(30,31). Standard calibration curves NF HCl shows a maximum absorption peak at 266 nm. That is compatible with the results of references (32,33). Accordingly, standard calibration (STD) curves Figure 5 were used to calculate saturated solubility of NF in different media. Figure 5. Standard calibration curves of NF HCl in different solvents Solubility The solubility of NF HCl in aqueous buffer saline at pH 7.4, simulated nasal fluid pH 6.4, and deionized water pH 7, shown in Table 3. Table 3. Saturated solubility of NF HCl in different solvents. Solvent Saturated solubility (mg/ml) (Mean±SD, n=3) Aqueous buffer 21.957 ± 0.05 SNF 13.2 ± 0.11 Deionized water 21.679 ± 0.07 The fact that only (100 – 250 µl ) can be instilled in a human nose (34) makes a potent drug with high solubility essential for intranasal formulations. Drugs with poor water solubility at high strengths are challenging to synthesize without employing significant amounts of solubilizing agents, which might possibly harmful excipients. (35). Gelation temperature Initially sol-gel temperature was measured for polymer mixtures that composed of only poloxamer 407 and poloxamer 188 as shown in Table 4. Table 4. Temperature of gelation for intranasal in situ gel polymer mixture (P). P No. PoloX 407 (%w/v) PoloX 188 (%w/v) D.D.W q.s ml Sol-Gel Temp. (Mean±SD, n=3) 1 17 10 32.1 ± 0.653 2 18 10 29.5 ± 0.430 3 19 10 27.3 ± 0.120 4 20 10 23.8 ± 0.115 5 17 4 10 42.4 ± 0.346 6 18 4 10 38.5 ± 0.152 7 19 4 10 35.1 ± 0.247 8 20 4 10 28.4 ± 0.432 9 17 3 10 40.6 ± 0.461 10 18 3 10 36.0 ± 0.154 11 19 3 10 32.3 ± 0.280 12 20 3 10 27.8 ± 0.316 Only P formula with gelation temperature 30-36 oC 15 were selected for further addition of mucoadhesive polymers and N.F HCl. Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 39 Formulations inclusion criteria and exclusion criteria Gelation occurs at room temperature if the temperature is lower than 32oC, which makes it difficult to manufacture, handle, and administer. The formulation will remain liquid at body temperature if the gelation temperature is higher than 34oC, resulting in nasal drainage. Drug and additives may increase or decrease gelation temperature. As a result, the formulation's gelation temperature must fall between the following range: 30-36ºC for P formula to allow study the effect of drug and additives on gelation temperature. And the P.I formulations of Table 5 must have gelation temperature 32-34 oC. For the same reasons mentioned above, any formula above 36 oC or below 30 oC for P formula , and any P.I formula above 34 oC or below 32 oC are excluded from further studies. Results from Table 4 indicated that PoloX 407 or PoloX 188 alone could not provide a suitable gelation temperature. Only formula ( P No. 1 ) with (17% PoloX 407) show gelation temperature at suitable range. In cases of mixtures of PoloX 407 and PoloX188, several formulations ( P No. 7,10,and 11) are gelled at the included temperature.Table 5 show that (P.I No. 1, 4, 5, 8, 9, 12 ,17 ,20, 21, 24, 28, 29, 32,33,36,41,44,and 45) are gelled within included temperature. Table 4 and 5 show that as the concentration of PoloX 407 increase, the gelation temperature decreases due to aggregation and formation of critical gel concentration (cgc),which become more easily as the concentration increases, and as the concentration of PoloX188 , HPMC K4M, H.A, M.C, and Car934 increases, the Sol-Gel temperature increase because these polymers will build up inside a gel texture of PoloX407 and inhibit gel formation so that the critical gel temperature will be higher(36). Table 5. Gelation temperature(S-G) of the P.I formula. P.I 1 2 3 4 5 6 7 8 S-G Temp (Mean±SD, n=3) 33.9 ± 0.113 40.1 ± 0.425 38.2 ± 0.236 34 ± 0.364 33.2 ± 0.168 39.4 ± 0.536 40.2 ± 0.325 33.1 ± 0.364 P.I 9 10 11 12 13 14 15 16 S-G Temp (Mean±SD, n=3) 33.2 ± 0.424 40.2 ± 0.244 41.3 ± 0.461 32.2 ± 0.145 34.1 ± 0.295 41.2 ± 0.196 42.1 ± 0.325 34.2 ± 0.212 P.I 17 18 19 20 21 22 23 24 S-G Temp (Mean±SD, n=3) 33.2 ± 0.315 40.4 ± 0.213 41.5 ± 0.116 34 ± 0.187 34 ± 0.285 41.9 ± 0.314 39.8 ± 0.412 33.9 ± 0.440 P.I 25 26 27 28 29 30 31 32 S-G Temp (Mean±SD, n=3) 34.8 ± 0.173 41.7 ± 0.238 42 ± 0.315 34 ± 0.164 34 ± 0.253 42.1 ± 0.426 40.2 ± 0.154 33.1 ± 0.332 P.I 33 34 35 36 37 38 39 40 S-G Temp (Mean±SD, n=3) 33.9 ± 0.227 37.8 ± 0.463 38.9 ± 0.352 33.1 ± 0.109 35.9 ± 0.267 39 ± 0.315 40.7 ± 0.573 38.8 ± 0.012 P.I 41 42 43 44 45 46 47 48 S-G Temp (Mean±SD, n=3) 34 ± 0.173 38.5 ± 0.532 38.9 ± 0.425 34 ± 0.316 33.1 ± 0.305 35.7 ± 0.311 36.9 ± 0.130 35.8 ± 0.189 Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 40 Appearance Appearance and clarity test reveal formation of cloudy and precipitate at the bottom of container for formulas that contain HPMC K4M and Car 934 in all concentrations. Table 6. shows that P.I formula No. (1-12) and (25-36) are cloudy with precipitate, and one selected precipitated cloudy formula for HPMC K4M (P.I No.4) and Car934 (P.I No.26) are subjected to FTIR scan to check its compatibility and reason of separation. Table 6. Appearance (P.A) results (Cloudy -, Very Cloudy --, Clear +, Glassy ++, Precipitate (ppt.)) P.I 1 2 3 4 5 6 7 8 P.A - ppt. - ppt. - ppt. -- ppt. - ppt. - ppt. - ppt. - ppt. P.I 9 10 11 12 13 14 15 16 P.A - ppt. - ppt. - ppt. - ppt. ++ + + + P.I 17 18 19 20 21 22 23 24 P.A ++ + ++ + ++ ++ ++ ++ P.I 25 26 27 28 29 30 31 32 P.A - ppt. -- ppt. -- ppt. -- ppt. - ppt. -- ppt. - ppt. - ppt. P.I 33 34 35 36 37 38 39 40 P.A - ppt. -- ppt. - ppt. - ppt. + + + + P.I 41 42 43 44 45 46 47 48 P.A + + + + ++ + + + Compatibility study Physicochemical compatibility between NF HCl and different excipients was studied using FTIR to examine any interactions between the NF HCl and the excipients used in the P.I formulation. The spectrums were compared to the spectrum of NF HCl alone. FTIR spectra of P.I No. 4 and 26. are shown in Figure 6. Figure 6. FTIR of P.I No. 4 (A) and P.I No. 26 (B). In comparing with FTIR of pure drug Figure 3., we find that a peak has lower intensity with the absence of peaks at 2800 cm-1 – 1460 cm-1 region and absence of a characteristic peak at 1000 cm-1 – 1100 cm-1 of cyclic C-O-C; these might be resulted from the interaction of N.F HCl with HPMC K4M and Car934. Despite the critical need to verify API- excipient compatibility, there is currently no globally acknowledged standard for evaluating such interactions; FTIR and appearance are straightforward methods often used in analytical labs (13). Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 41 Incompatibility is characterized as a change that occurs and an unfavorable product is produced, which may influence the pharmaceutical product's safety, effectiveness, stability, and appearance (19). Gelling capacity Gelation time is an important feature as gelation temperature, because the formula should transform into gel upon instillation but also remain as gel for the period of time sufficient for the drug release. Only clear and un precipitated formulas are checked. Gelation time is described in Table 7. Table 7. Gelation time and gelling capacity of PI formulas Gelling capacity (hr:min) (Mean±SD, n=3) Gelation time (Sec.) (Mean±SD, n=3) P.I Formula No. 6:15 ± 0.00 56.66 ± 2.886 13 6.1 ± 0.086 60 ± 0.00 16 5.2 ± 0.173 46.66 ± 2.886 17 4:15 ± 0.00 48.33 ± 2.886 20 5.1 ± 0.086 46.66 ± 2.886 21 3.63 ± 0.317 40 ± 0.00 24 5.1 ± 0.086 50 ± 0.00 41 3.816 ± 0.317 46.66 ± 2.886 44 5.1 ± 0.173 50 ± 0.00 45 The gelation study was conducted in SNF (pH 6.5). All the formulations on contact with the gelation medium had undergone sol-to-gel transition due to the presence of gel-forming polymers such as poloxamer. Gelation characteristics of the formulations showed that increasing the concentration of poloxamer 407 increased the gelation time, while the combination with poloxamer 188 showed less gelling capacity as seen in Table 7. Also the rise in the concentration of HA and M.C increased the time of gelation for the formulas (3) , meanwhile different concentrations of different polymers show non-significant effect on gelation time. pH of selected NF HCl intranasal in situ gel The degree of ionization of a drug molecule intended to be administered intranasally depends on two factors, the dissociation constant of the drug and the pH value (37) of nasal fluid (4.5-6.5) (38). The pH value of the formula plays a significant role in the ionization process of the molecule so that it will affect its solubility and penetration capacity; the intranasal formulation should have a controlled pH range (4.5-6.5) (38) to be tolerable. Increased pH increases the likelihood of nasal cavity infections by inhibiting the lysozymes responsible for eliminating them, while a decrease in its value will irritate the tissue (2). The pH range of selected formulas as shown in Table 8 were 5.7-6.1 , which are accepted to be used intranasally and compatible with the nasal pH range 4.5 – 6.5 (38) at this pH range, the drug NF HCl will be mainly in ionized form to be completely soluble in the formula. Table 8. The pH value of selected IP formulas. P.I 13 16 17 20 21 24 41 44 45 pH (Mean±SD, n=3) 5.7 ± 0.243 5.8 ± 0.152 5.9 ± 0.216 5.9 ± 0.022 6.1 ± 0.171 6.1 ± 0.142 6 ± 0.415 6 ± 0.104 6.1 ± 0.028 Osmolarity of NF HCl intranasal in situ gel Osmotic pressure is required for all biological activities that involve solute diffusion or fluid transfer across membranes. While hypotonic formulation induces epithelial cells to expand and promote water intake and drug absorption, hypertonic solutions increase cell shrinking and decrease the likelihood of release. The formulation should, however, range from 200 to 600 mOsm/l. to protect the nasal mucosa's since these changes might have a harmful impact during chronic treatment. Finally, hypertonic formulations stimulate mucociliary clearance, Iraqi J Pharm Sci, Vol.31(Suppl.) 2022 Nefopam HCl intranasal in situ gel 42 increasing medication excretion (2). Table 9 shows the osmolarity range 590 – 311 was within an acceptable range; osmolarity is directly proportional to the concentration of thermosensitive and mucoadhesive polymers, with H.A It shows the significant impact on osmolarity. Table 9. Osmolarity value of selected P.I formula P.I 13 16 17 20 21 24 41 44 45 Osmolarity (Mean±SD, n=3) 543 ± 0.081 590 ± 0.102 484 ± 0.119 552 ± 0.258 403 ± 0.317 461 ± 0.163 358 ± 0.401 396 ± 0.259 311 ± 0.066 Mucoadhesive strength The residence time of the P.I formulas in the nose can be prolonged by the mucoadhesive force of the gel. The mucoadhesive force of the P.I formulas should be enough to supply good opposition to the gel mucocilliary clearance. The oligosaccharide sequence of the mucin glycoprotein in the mucus membrane forms a hydrogen bond with the polymer in the formula, producing the mucoadhesive force. Evaluation of the mucoadhesive strength in terms of detachment stress demonstrated (Table 10) that formulations with higher concentrations had improved mucoadhesive qualities. Table 10. shows the mucoadhesive force of the selected P.I formulations Mucoadhesive force (dyne/cm2) Detachment weight (g) (Mean, n=3). P.I Formula No. 8776.31 7.03 ± 0.055 13 9388.03 7.52 ± 0.170 16 8089.68 6.48 ± 0.271 17 9125.86 7.31 ± 0.320 20 7502.93 6.01 ± 0.076 21 8401.78 6.73 ± 0.176 24 3458.09 2.77 ± 0.116 41 4431.85 3.55 ± 0.273 44 3283.31 2.63 ± 0.079 45 Mucoadhesive strength for formulations containing H.A (P.I No. 13,16,17,20,21,and 24) show two- three folds higher mucoadhesive detachment force than that of formulations that containing M.C (P.I No. 41,44,and 45) , due to the HA capacity to include hydrogen bonding , and large molecular weight (39). Also, poloxamers have shown mucoadhesive effect. The faster fluid uptake from the mucus layer that enables the polymer chain to penetrate the mucin network and establish adhesive contacts is thought to be the cause of the higher mucoadhesive strength of the delivery system, which may lead to enhance retention and improved absorption. Strong mucoadhesive force of the P.I can avoid the drainage of the drug from the nose, causing greater absorption through mucosal tissues and extended retention. The nasal mucosal membrane, however, can be harmed by excessive mucoadhesive force (i.e., more than 10,000 dyne/cm2 gel) (40).All formulations does not topped the highest limit and hence appeared as formulations with optimal mucoadhesion properties. Conclusion From these research findings, it was determined that N.F HCl intranasal in situ gel that is prepared by the cold method could be formulated by Poloxamer 407 as a gel-forming polymer with Poloxamer 188 to modify its gelation temperature, along with Hyaluronic acid and Methylcellulose as mucoadhesive polymers, these studies highlighted the impacts of polymer type and concentrations on gelation temperature. It was concluded that N.F HCl P.I formulas (13,16,17,20,21,24,41,44 and 45) could be used to formulate NF HCl intranasal mucoadhesive in situ gel. 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