الفهرس 
Abstract
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Abstract
The main goal of dosage form design is to achieve a proper therapeutic response to the drug involved in a suitable formulation. Non steroidal anti-inflammatory drugs (NSAIDs) are the major drugs to relive the pain and / or inflammation in musculoskeletal diseases such as rheumatic arthritis and osteoarthritis. There are several adverse effects commonly associated with the oral route of administration of NSAIDs such as gastrointestinal complications, hepatic and kidney disorders.
Topical applications of NSAIDs have been known as an alternative route of administration to the oral route and so they could be used for a long term therapy. Naproxen is NSAIDs that produces analgesic and antipyretic effects and is currently used for the treatment of rheumatoid arthritis, osteoarthritis and traumatic disorders. However, it has been associated with gastrointestinal side effects. Because of the pharmaceutical advantages of topically applied dosage form, it is worthy to study the formulation of naproxen in gel formulations to be available for use in the treatment rheumatic arthritis and osteoarthritis.
Naproxen is characterized by having poor aqueous solubility (27 mg /l) and thus could show minimal percutaneous absorption. In addition, the protective barrier exhibited by stratum corneum could render the topical route of administration ineffective. It seems to be worthy to enhance naproxen solubility to facilitate its formulation into proper topical dosage forms. So the present thesis comprises the following chapters:-
• Chapter One: Solubility Studies of Naproxen.
• Chapter Two: Preparation and Evaluation of Naproxen Topical Gels.
In–vivo evaluation of naproxen in the selected
formulations.
Chapter One
Solubility Studies of Naproxen
● Assessment of the Equilibrium Solubility:
In this study the equilibrium solubility of naproxen in water , acetate buffer at pH 5.8 ,citrate buffer at pH 5.8 ,phosphate buffer at both pH 7.4 and 5.8 was investigated by equilibrium solubility technique at 37 ºC over 24 hrs at predetermined time intervals and assayed spectro- photometrically at λ max 272 nm. The results obtained showed that the equilibrium solubility in all the tested media was attained after two hours and the equilibrium solubility of naproxen in acetate buffer was 207.78 ± 0.26 mcg/ml while in citrate buffer was 191.47 ± 0.15 mcg /ml, phosphate buffer at pH 7.4 was 411.14 ± 4.54 mcg/ml and phosphate buffer at pH 5.8 was 186.32 ± 0.26 mcg /ml.
●Assessment of the Solubility of Naproxen in Presence of Co-solvents:
The effect of different concentrations (0 -30 % v/v) of co-solvents on the solubility of naproxen in water at 37 ºC was determined. The selected co-solvents were PEGs, PG, glycerol, DMF, DMSO, DMA, ethanol and isopropanol. The results showed that the solubility of naproxen was improved in presence of the tested co-solvents and this effect can be ranked in the following order according to their solubility power at 30 % co-solvent concentrations (w/v) or (v/v) as DMA >DMF >PEG 600 >PEG 400 > PEG 200 > Isopropyl alcohol > DMSO > ethyl alcohol > PG > glycerol.
●Assessment of the Solubility of Naproxen in presence of surfactants:
The effect of different concentrations (0 -10 % w/v) of hyDROPhilic surfactants; Tween 20, Tween 40 and Tween 80 on the solubility of naproxen in water at 37 ºC was determined. Further, the solubility of NPX was determined in 30% PEG 400 water system in the presence of Tween 80 (0-10 %).
The results showed that there was a marked improvement in the solubility of the drug in the presence of tested surfactants, and this improvement was increased by increasing the surfactants concentrations. Tween 80 exhibited the highest solubilizing effect 2524.92 ± 49.35 mcg /ml and Tween 20 showed the lowest solubilizing effect 1880.54 ± 7.59 mcg/ml.
Chapter Two
Preparation and Evaluation of Naproxen Topical Gels
1. Preparation of Naproxen Topical Gel Formulations:
Preliminary trials were conducted to establish the conditions that ensure the formation of a transparent aqueous gel taken in consideration the low aqueous solubility of NPX. These trials revealed that a good transparent carbapol 940 gel could be obtained by including co-solvents namely PG and PEG 600 to ensure complete solubility of the drug in the gel.
2. Evaluation of Naproxen Gels:
2.1. Physicochemical Evaluation of Different Prepared Gel
Formulations:
The physical parameters of the different prepared gels and emulgels were evaluated. from the results, it was found that all the formulations showed good homogeneity except two non-aqueous emulgel formulations which were excluded from further evaluation .The prepared gels were translucent in appearance and non gritty in texture .
The pH values of the hydroalcoholic gel formulations were in the range of 4.61 - 4.75, those of non aqueous emulgels were in the range of 5.02 - 5.24 and carbopol gels formulations were ranging from 5 - 5.52 which lie in the normal pH range of the skin that would not produce any skin irritation.
The viscosity values of each type of the tested formulations were in the range of 2500± 500 cps to 37500 ± 500 cps. Viscosity is an important physical property of topical formulations, which affects the rate of drug permeation; in general it was found that an increase of the viscosity of vehicles would cause a more rigid structure with a consequent decrease of the rate of drug permeation.
2.2. Permeation of Naproxen from Carbapol 940 Gels through Cellophane Membrane:
Carbopol 940 gel bases containing different ratios of PG and PEG 600 were prepared and the permeation of naproxen from these formulations was evaluated. The data revealed that the optimum permeation profile was attained from formulation containing PG and PEG 600 at a ratio of 5:3 respectively and so this formulation was considered as a control gel base.
2.2.1. Effect of Different Enhancers on the Permeation Pattern of Naproxen from the Control Gel Base:-
The effect of enhancer type and its respective concentration on the in –vitro permeation profile was studied. Enhancers used in this study include the following:-
2.2.1.1. DimethylSulfoxide (DMSO):
We preferred to study the effect of low concentrations of DMSO because we were concerned with its toxicity at higher concentrations. The concentrations tested were 3%, 5% and 10% (w/w). The results revealed that as the concentration of DMSO increased, the release of naproxen was found to be decreased. This was well correlated to the increase in the viscosity of the vehicle of the prepared gel by increasing the concentration of DMSO.
2.2.1.2. Isopropyl Myristate (IPM):
It was studied at concentration levels of 3%, 5% and 10% (w/w). The results showed a lower release of NPX from gel containing isopropyl myristate compared to that obtained from the control one. Also there was an inverse proportionality between the isopropoyl myristate concentrations and the cumulative amount of NPX permeated.
2.2.1.3. Terpenes:
Menthol is a terpene compound containing alcohols which have been widely used as skin penetration enhancers for a variety of compounds. Menthol was selected for our studies because it is also considered as fragrant agent that induces a strong cooling sensation when applied on the skin and numbs the sensation of pain. For this reason, it may provide an advantage for analgesic topical formulations. Menthol was used at two concentrations 3% and 5% (w/w) and for comparative purposes we choose thymol at the same concentrations.
The results showed that the cumulative amount of NPX permeated from carbapol gel formulations containing menthol, was inversely proportional to the menthol concentrations. This may be due to the increase in the viscosity value between the two tested monoterpene formulations which led to an increase in the resistance of naproxen permeation from the vehicle. In addition it was noticed that the presence of thymol at varying concentrations did not facilitate the permeation of NPX from carbapol gel formulations through the cellophane membrane. It is unclear whether the terpenes affect the drug partioning or not. The drug affinity to the vehicle may be enhanced during the presence of the oily terpenes and this in turn retarded the NPX flux with respect to the control base gel formulation.
2.2.1.4. Surfactants:
The influence of hyDROPhilic functional group of non ionic surfactants on the skin permeability was studied. Sorbitan monolaurate (Span 20) and polysorbate 20 (Tween 20) was selected in this study because they have the same lipophilic portion C12 alkyl chain (laurate) and they differ mainly in their hyDROPhilic group. Polysorbate 20(Tween 20) is characterized by having a more hyDROPhilic group than Span 20. Tween 20 and Span 20 were used at 1%, 3% and 5% (w/w) concentrations.
It was found that there was a significant decrease in the permeation profile of NPX from gel preparations containing Span 20. In addition the cumulative amount of NPX permeated from the selected gel formulations was directly proportional to the concentration of Tween. This could be explained on the basis that the solubility of NPX in the presence of the surfactant increased as its concentration increased.
It was evident that the permeation profiles from gel formulations containing different concentrations of Tween 20 were more enhanced than those obtained in presence of different concentrations of Span 20 .The enhancing effect of polysorbates 20 on the permeation of NPX from the tested formulations could be explained on the basis that a part of NPX will be partitioned into the micellear core of the tested polysobates resulting in an increase in the total amount of NPX solubilized in the presence of Tween 20. On the other hand, the micellar core of Span 20 is hyDROPhilic in nature and a small fraction of NPX, lipophilic , will be entrapped and its aqueous solubility will not be significantly increased and this leads to a reduction of its permeation .
2.2.2. Permeation of Naproxen from Carbapol 940 Hydroalcoholic Gels through Cellophane Membrane:
The in -vitro permeation study of naproxen was carried out using a mixture of water and ethyl or isopropyl alcohol on the ratio 3:7 respectively. The observed data revealed that the best permeation profile was attained from the formulation containing ethyl alcohol and water. The difference in the permeation pattern may be due to the difference in the molecular weight between the two tested alcohols which affect the permeation from both formulations. The molecular weight of ethyl alcohol is (46.07) and that of isopropyl alcohol is (60.1). The large isopropyl molecules are more likely to be delayed compared to smaller one of ethyl alcohol.
2.2.3. Permeation of Naproxen from Non aqueous Carbapol 940 Emulgels through Cellophane Membrane:
Different formulations were prepared using glycerol, instead of water, olive oil as an oily phase for the preparation of the emulsion and carbopol 940 as gelling agent. The effect of different concentrations of olive oil at 5 and 7.5 % were studied. Also, different concentrations of mixture of Span 20 and Tween 20 at 2%, 4% and 6% were studied.
The higher permeation of NPX was obtained from F6 containing 5% of olive oil and 4% of total surfactant mixture .This could be explained on the basis that, the concentration of the drug in the external aqueous phase of the emulgel is inversely proportional to the concentration of olive oil as internal phase .
2.3. Kinetic Analysis of the Permeation Data of NPX from Different Gel Formulations through Cellophane Membrane:
The permeation data of the drug from different gel formulations were subjected to mathematical treatment according to zero, first order and Higuchi diffusion model mechanisms. The obtained data analysis revealed that the permeation of NPX from various formulations follows to a great extent zero order model as the highest regression coefficient was obtained.
2.4. Permeation Studies of Naproxen from Selected Gel Formulations through Abdominal Rat Skin:-
2.4.1. Permeation Studies of NPX from the Control Gel Base Containing 5:3 PG and PEG 600:
The solubility of the drugs in the glycols is the most important factor because it determines to what extent this effect influences drug transport. PG acts by solvating the keratin structure of the cells and therefore, PG could be involved in transcellular diffusion enhancement.
2.4.1.1. Effect of Different Enhancers on the Permeation Pattern of Naproxen from the Control Gel Base through Abdominal Rat Skin:-
2.4.1.1.1. DMSO:
from the in-vitro permeation study of naproxen from carbapol gels containing 1% ,5% and 10% DMSO., The results revealed that the gel formulations containing DMSO at 10 % concentrations increased the flux of naproxen ( 61.312 µg/cm2h ) with regard to the control base gel ( 60.704 µg/cm2h ) . On the other hand, formulations containing DMSO at 1% and 5% did not enhance the flux of NPX from gel formulations. The significant increase in the amount permeated of NPX from the control base gel at higher DMSO concentrations, indicated that DMSO permeation enhancement activity was concentration dependant.
2.4.1.1.2. Isopropyl Myristate (IPM):
The influence of IPM concentrations on the permeation of NPX across the rat skin from the control gel base was studied. It could be observed that isopropyl myristate has a significant influence on increasing NPX flux at all the tested concentrations (3%, 5% and 10% (w/w). It was noticed that, the optimum concentration of IPM used to enhance the flux of NPX was 5% (w/w) which gave the significant highest cumulative amount of NPX permeated after 4 hr.
Isopropyl myristate appears to affect the fluidity of the lipid components of the stratum corneum by facilitating the permeation through lipoidal pathways. The reason for liquefying effect may be due to its branched structure. Moreover IPM is able to dissolve considerable amounts of cholesterol, which act as a membrane stabilizer.
2.4.1.1. 3. Terpenes:
No enhancing effects were observed on the permeation of naproxen from formulations containing menthol at 3% and 5% as compared to the control gel base. It was observed that the amount of NPX permeated per unit surface area after 4 hr from the control gel base was (237.804 ± 0.65 mcg /Cm2) while the cumulative amount permeated from formulation containing 5% menthol was 237.269 ±0.67 mcg /Cm2 and from formulation containing 3% menthol was 174.103 ± 0.23 mcg /Cm2 . This was explained on the basis that the presence of menthol produced a dose proportional increase in NPX flux , permeability coefficient and diffusion coefficient, which in turn improved the skin permeation of drugs by increasing drug diffusivity in the skin through modifying the inter cellular packing ,disrupting highly ordered structure of lipids.
The results of the effect of thymol on the permeation of naproxen indicated that the presence of thymol produce a dose proportional increase on NPX flux and permeability coefficient ( P < 0.001) . However the formulation containing 5% thymol produced a cumulative amount permeated per unit surface area of NPX after 4 hr (163.199 ± 0.32 mcg /Cm2 ) with higher diffusion coefficient and higher permeability than that obtained from the formulation containing thymol 3% (152.86 ± 0.37 mcg /Cm2 ) which exhibited lower diffusion and lower permeability. Controversially, there was a non enhancing effect on the permeation of the naproxen from gels containing thymol compared with the control gel base.
It was also found that the thymol was not effective as menthol as a permeation enhancer. Thymol showed a lower flux compared to menthol .This is could be explained as the efficacy of terpenes on the percutaneous absorption of different drugs is not similar, and the percutaneous absorption of the drug may be closely related to the physicochemical nature of the drugs and the terpenes as well as the vehicle into which they are formulated.
2.4.1.1.4. Effect of Surfactant:
The Permeation profiles of NPX from F21 , F22 and F23 containing 1% , 3% and 5% Span 20 and F1 as a control revealed that the amount permeated of NPX after 4 hr per unit surface area from F21 (289.33 ± 0.639 mcg /Cm2 ) and from F22 (468.72 ± 0.93mcg /Cm2 ) which are significantly higher than the amount permeated from F1 (237.804 ± 0.65 mcg /Cm2) while moderate significant from F 23 (271.34 ± 1.295mcg /Cm2). The increase in the cumulative amount of naproxen permeated indicated a synergetic effect between PG and Span 20 for enhancing the drug flux.
Span 20 exhibited an enhancing effect on the skin penetration at 1% and 3% concentrations. This may be attributed to the effect of Span 20 on the intercellular lipids by making them more fluid and thus enhancing the diffusivity of NPX. As known that the lipophilic molecules diffuse through the stratum corneum by solubilizing in the continuous intercellular lipid phase of the stratum corneum (lipophilic pathway).
Regarding to the permeation profiles of NPX from carbapol gel formulations (F24, F25 and F26) containing 1%, 3% and 5% Tween 20 respectively and the control carbapol gels (F1). F25 containing 3% Tween20 exhibited the higher permeation rate of NPX as compared with the other tested concentrations F24 and F26.
2.4.2. Permeation Study of NPX from Hydroalcoholic Carbapol gel Formulations:
The results showed that the highest permeability and diffusion coefficient obtained from hydroalcoholic gel formulations containing 70% ethanol with compared to the control gel base, containing 5:3 PG and PEG 600, respectively. The permeation enhancing effect from ethanol containing gel is much greater than that from PG containing gel alone which indicates that the PG alone as an enhancer didn’t affect the lipid structure of the skin like ethanol did.
2.4.3. Permeation Study of NPX from non-Aqueous Cabapol Emulgel Formulations:
A comparison between the permeation profiles of carbapol gel base (F1) and non aqueous emulgels (F5 and F 6) containing the same concentration of NPX ( 2%) was performed to evaluate the effect of different vehicles on the permeation of NPX. from the obtained results it was found that there was a relative enhancing effect on the permeation observed in F6 containing 5% olive oil and there was an increase in the diffusion coefficient and enhancement in the permeability. This could be attributed to the effect of olive oil on the skin permeability. However olive oil did not bring any effect on the permeation study of naproxen from cellophane membrane while enhanced the permeation through the skin.
2.5. Kinetic Analysis of the Permeation Data of NPX from Different Gel Formulations through Abdominal Rat Skin:-
The results of the work revealed that the drug permeation from all investigated formulations followed the Higuchi diffusion model, as confirmed from the good correlation coefficients ranging from 0.98 to 0.99. This finding indicates that the rate-controlling stage in the permeation process was the diffusion of the dissolved drug through the different skin layers to the external medium
3. In-vivo Evaluation of the Efficacy of Naproxen in the Selected Gel Formulations:-
The in- vivo studies lead to the identification of the design and formulation parameters in gel delivery systems which may be useful in modulating drug action in terms of anti-inflammatory effect, skin irritation tests and histological studies.
3.1. Anti-inflammatory activity study (Carragenan –induced Paw edema):
The carrageenan –induced rat paw odema method was used to evaluate the anti-inflammatory efficacy of naproxen formulated in F15 (5% IPM), F22 (3% Span 20) and compared with the marketed product Ibutop 5%. The results indicated that F15 gave rise to a rapid onset of action and higher activity during the initial periods due to their enhanced absorption rates of the drug from F15. It was found that the nature of topical vehicle plays a vital role in promoting drug release and consequently its absorption through the skin. The maximum reduction of edema was significantly obtained from carbopol gels containing 5% IPM and PG which provided good enhancement effects over the other tested formulations. The synergistic action of PG and IPM at 5% concentrations facilitated the diffusion of the drug through the skin layers and so more potent ant-inflammatory effect was obtained.
4. In-vivo Evaluation of the Safety of Naproxen in The Selected Gel Formulations:-
4.1. Superficial Skin Irritation Test:-
Skin irritation test was done during a period of ten days. The test was applied to the formulations exhibited higher flux of naproxen (F15 and F22). It was concluded that neither naproxen nor its excipients caused any skin irritation and can be incorporated in the gel formulations due to their safety and acceptability for topical route of administration.
4.2. Histological Study of The Skin Treated with The Formulations:-
Histological changes in hairless rat’s skin were examined after pretreatment with F 15, F 22, Placebo F15 and Placebo F22. Application of the formulations and their placebo was done every 12 hour for two weeks. After the first three days three rats of each group were killed and samples of the applied skin area were taken. This sequence was repeated after one week and after two week to evaluate the effect of both drug and vehicle on the histological changes of the skin. The following histological stains were used:
(1) Hematoxylin- eosin stains for general histological examination.
(2) Van Gieson stains for demonstration of collagen fibers.
It could be concluded that there were different effects between the two enhancers in promoting the drug diffusion and penetration. On the other hand, there were no great harmful effects of the two formulations and their vehicles were observed, as normal skin undergone normal skin turnover to replace the dead cells with normal ones so these vehicles were considered safe and suitable for the delivery of NPX. The effect of NPX on the collagen fibers will need further study and examinations and will be our future work.