Search In this Thesis
   Search In this Thesis  
العنوان
Preparation and Evaluation of Medicated Intra-articular
Delivery Systems /
المؤلف
Abou El-Nour, May El-Zanaty Abdel Motalleb Ali.
هيئة الاعداد
باحث / May El-Zanaty Abdel Motalleb Ali Abou El-Nour
مشرف / Ahmed Shawky Geneidy
مشرف / Mahmoud Eid Soliman
مناقش / Rania Aziz Ishak
تاريخ النشر
2019.
عدد الصفحات
337 P. :
اللغة
الإنجليزية
الدرجة
ماجستير
التخصص
الصيدلة ، علم السموم والصيدلانيات (المتنوعة)
تاريخ الإجازة
1/1/2019
مكان الإجازة
جامعة عين شمس - كلية الصيدلة - قسم الصيدلانيات والصيدلة الصناعية
الفهرس
Only 14 pages are availabe for public view

from 337

from 337

Abstract

Rheumatoid arthritis (RA) is an auto-immune disorder that results in chronic, systemic inflammatory conditions. It affects many organs and tissues all over the body, but principally attacks the flexible synovial joints. RA is mainly manifested by an irreversible destruction of the synovium, causing a painful swelling, which can eventually leads to loss of the joint space, bone erosion and joint deformity.
Triamcinolone acetonide (TA) is a synthetic, long-acting corticosteroid, commonly used in RA treatment due to its anti-inflammatory and immune-modulatory effect. It belongs to class IV biopharmaceutical classification system (BCS), therefore TA exhibits formulation-related constraints, in addition to its severe side effects accompanying its systemic administration. Therefore, localizing TA into the target joint via intra- articular (IA) administration can overcome these problems. However, rapid drug efflux from the joint cavity into the systemic circulation can occur following IA injection as a result of the leaky ultra-structure nature of the synovium. Therefore, frequent injections are required to reach the desired therapeutic drug effect, which may lead to patient discomfort, a high risk of infection and a possibility to develop joint injury.
Hence, the aim of work in this thesis was to develop novel delivery systems loaded with TA, acquiring appropriate characteristics for effective IA delivery, targeting to prolong its residence time within the affected joint cavity for efficient RA treatment.

Therefore the work in this thesis is divided into three chapters:
Chapter One: Preparation and Optimization of TA-Loaded MPs.
Chapter Two: Preparation and Evaluation of Thermo-responsive Hydrogels
Loaded with Optimized TA MPs.
Chapter three: In Vivo Evaluation of the selected TA-Loaded
Formulations.
Chapter I: Preparation and Optimization of TA-Loaded MPs.
In this chapter, methoxy-polyethylene glycol-poly-δ-decalactone (PEG-PDL), a novel sustainable copolymer, was prepared from the green δ- decalactone monomer using ring opening polymerization (ROP) technique. Six PEG-PDL copolymers were synthesized with different PDL molecular weights. Being renewable, the synthesized copolymer as well as poly-lactide (PLA) polymer were employed as a blend in the fabrication of TA-loaded microparticles (MPs). Hansen’s solubility parameter approach was applied to determine the compatibility between the drug and the used polymers. This was considered essential to increase the stability of the encapsulated drug and sustain its release from MP matrix.
TA-loaded MPs were successfully prepared using o/w emulsion- solvent evaporation technique, applying 33 full factorial design for the evaluation of the effect of three formulation variables, namely; polymer to drug (P:D) ratio, total polymer concentration and percentage of PEG- PDL1700 copolymer (with respect to the total polymer weight). The tested responses were limited to entrapment efficiency percentage (EE%), particle size (PS) and span index (SI). The three response models were statistically analyzed using analysis of variance (ANOVA) and the mathematical modeling of each response was expressed using polynomial mathematical
equations. Optimization of the prepared MPs was performed using Expert Design® software’s desirability function (D). The optimized formulations were also characterized using scanning electron microscope (SEM), powder X-ray diffraction, differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR) and in vitro release studies.
from the obtained results, it was found that:
1. Six different PEG-PDL copolymers were synthesized using ROP method.
The Mw of the mPEG moiety was fixed to 1.9 kDa in all copolymers, while the Mw of the PDL moiety was found to be 24, 17.5, 9.5, 3, 1.7 and 0.14 kDa for PEG-PDL24000, PEG-PDL17500, PEG-PDL9500, PEG- PDL3000, PEG-PDL1700 and PEG-PDL140, respectively.
2. Hansen’s solubility parameter approach proved the compatibility and miscibility between TA and all the tested polymers used for MP fabrication. According to the values of Flory-Huggin’s interaction parameter (Xsp) and the total solubility difference between drug and polymer (∆δt), the following polymers were arranged in an ascending order: PDL < PEG-PDL < PEG < PLA. For expedient MP formulation, PEG-PDL copolymer was chosen to be blended with PLA to formulate TA-loaded MPs convenient for IA administration.
3. The preliminary studies applied showed that only MPs fabricated using PEG-PDL1700 copolymer were appropriate for the preparation of TA- loaded MPs to be delivered via IA route. They showed reasonable EE% (49.68 ± 1.66%), PS (14.12 ± 3.95 µm), SI (2.29 ± 0.44) and a good water re-dispersibility.
4. According to the experimental design adopted, the prepared TA-loaded
MPs showed variable drug EE%, LE%, PS, and SI values, ranging from
1.00 ± 1.88 to 86.94 ± 0.36%, 0.10 ± 0.04 to 7.90 ± 0.03%, 8.05 ± 1.14 to
22.72 ± 2.49 µm, and 1.29 ± 0.12 to 2.89 ± 1.65, respectively.
5. Based on the full factorial design implemented:
a) Box-Cox power transformation was recommended for the three tested responses, with suggested best powers of 1.6, -0.54 and 0.18 for EE%, PS and SI, respectively. After power transformation, the response models were re-analyzed by ANOVA test and found to be quadratic, with p-values less than or equal to 0.0001 confirming the significance of the three models.
b) The “lack of fit” was non-significant with respect to the pure error for the three responses. There were high correlations between the experimental and the predicted results for the three models, confirmed by a relatively high coefficient of determination (R²) values of 0.9770,
0.8730 and a moderate R2 of 0.7668 for EE%, PS, and SI, respectively.
The adequate precision values were 33.188, 11.883 and 7.838 for EE%, PS, and SI, respectively; indicating an adequate signal, which confirmed that these models can be used to navigate the design space.
c) Based on the ANOVA results, only P:D ratio and total polymer concentration had significant effects on EE% response ( p<0.05), however both total polymer concentration and percentage of PEG- PDL copolymer only showed significant effects on PS and SI (p<0.05).
d) The numerical optimization using desirability function ‘D’ confirmed the suitability of F-7, F-8 and F-9 for IA delivery, with D- values of 1, 1 and 0.938, respectively, being composed of 10:1 P:D ratio, 3% total polymer concentration and variable percentages of PEG-PDL1700 copolymer, namely; 0, 10 and 20% for F-7, F-8 and F-
9, respectively.
6. SEM images of the three optimized MPs revealed their spherical shape s, showing smooth surfaces and no evidence of any roughness or irregularities. The images of copolymer-based MPs (F-8 and F-9) showed the presence of TA/PEG-PDL co-crystals adhered onto or found just beneath the surface of MPs.
7. The X-ray diffraction pattern of F-7 showed only very few tiny peaks confirming the amorphous nature of these MPs. However, F-8 and F-9
MPs displayed multiple peaks confirming the higher crystallinity of loaded matrices prepared in the presence of copolymer. Relative to pure drug and copolymer, the formation of co-crystal between TA and PEG- PDL copolymer was confirmed.
8. Thermal analysis showed the disappearance of drug endothermic peak in all the optimized loaded MPs indicating a successful drug entrapment within the prepared MPs. Comparing the thermal transitions of the drug- copolymer mixture, the co-crystal formation between TA and PEG-PDL was also confirmed using DSC technique.
9. The FT-IR spectra further confirmed the formation of co-crystal between the drug’s –OH group and the –CH3 groups of the PEG-PDL1700 copolymer.
10. The in vitro drug release study detected the lowering in initial burst release when drug was incorporated in copolymer-based MPs (F-8 and F-9). Also, the sustainment of TA release from 20% copolymer-based MPs (F-9) relative to the drug suspension form and PLA-based MPs (F-
7) was observed. TA release from F-9 MPs was found to be non- Fickian, anomalous transport. Because of the highest drug release sustainment, F-9 MPs was chosen as the optimum formulation to be used for further studies.
Chapter II: Preparation and Evaluation of Thermo-responsive
Hydrogels Loaded with Optimized TA MPs.
In this chapter, the optimized MP formulation (F-9) was loaded into a thermo-responsive hydrogel, with a low critical solution temperature (LCST) near the physiological temperature. This allows the thermo-responsive preparations to be in the liquid state at room temperature, thus facilitating its injection, while converted into gel state at physiological temperature, providing a controlled release of the loaded drug. Additionally, the bio- adhesive property of these thermo-responsive hydrogels is crucial aiming to extend the drug residence inside the joint and hence improve the therapeutic effect of the intra-articularly injected drug.
For this purpose, free radical polymerization (FRP) technique was successfully used to synthesize star-shaped, thermo-responsive poly(polyethylene glycol methacrylate) [poly(PEGMA)] copolymers. In order to obtain polymers with gelation temperatures close to the physiological temperature, copolymerization of 16 and 10% (w/w) of the more hydrophilic PEGMA475–ME was performed with the PEGMA188–
ME and PEGMA246–EE, forming the copolymers coded G-1 and G-2, respectively. Rheometrical analysis, determination of hydrophilic-lipophilic balance (HLB), hydrophilic surface area and cloud point temperatures (Cpt) were preformed to characterize the synthesized poly(PEGMA) copolymers. The gelation temperatures of these copolymers were determined using the tube inversion technique, after being loaded with 3% (w/v) F-9 MPs. Different concentrations of the prepared copolymer solutions were tested with respect to their gelation temperatures.
The optimized MP-hydrogel dispersions were characterized using SEM imaging, viscosity determination, in vitro bio-adhesion and in vitro drug release studies.
from the obtained results, it was found that:
1. Two poly(PEGMA) thermo-responsive polymers were prepared using FRP technique, namely; poly(PEGMA188-ME-co-PEGMA475-ME) and poly(PEGMA246-EE-co-PEGMA475-ME), coded G-1 and G-2, respectively. Both copolymers showed viscoelastic properties, possessing both elastic/storage modulus (G´) and viscous/loss modulus (G´´). Coil- to-globule transition occurred when G´ > G´´ as an indication of thermo- gelation.
2. The HLB values of PEGMA188-ME, PEGMA246-EE and PEGMA475- ME monomers were determined and found to be 11.43, 12.27 and 17.61, respectively. Also, the hydrophilic surface areas of those respective monomers were calculated reaching 7.34 × 109, 10.35 × 109 and 25.68 ×
109 cm2/mol. This confirms the higher hydrophilicity of PEGMA475-ME
monomer, while PEGMA188-ME was the least hydrophilic. Thus, it could be assumed that G-1 copolymer was less hydrophilic than G-2.
3. Both G-1 and G-2 copolymers exhibited Cpt values of 32 and 31 ˚C, respectively, recorded as a result of the partial breaking of the formed hydrogen bonds at temperatures exceeding the copolymer’s LCST.
4. The sol-to-gel transitions of the MP-hydrogel dispersions were dependent on the copolymer concentration. Irreversible aggregates were obtained upon increasing temperatures when dispersing F-9 MPs in 2, 5 and 10% (w/v) of either G-1 or G-2 solutions. However, gel formation occurred at
20 and 30% (w/v) of either copolymer dispersions. The optimum MP- hydrogel dispersions showed gelation temperatures at 33 and 37 ˚C recorded after dispersing MPs in 20% G-1 (G-19) and 30% G-2 (G-29), respectively.
5. SEM images of plain lyophilized hydrogels revealed the porous microstructure of the formed gel networks, while loaded hydrogels showed the adsorption of the gel networks onto the surface of the flocculated MPs.
6. The viscosity of both G-19 and G-29 preparations decreased significantly
(p<0.05) upon increasing the shear rate applied from 0.5 to 100 rpm, at
37˚ C, which confirms the pseudoplastic (shear-thinning) behavior of both copolymer-based hydrogels. G-19 showed significantly higher viscosity compared to G-29 (p<0.05) due to its relatively higher hydrophobicity.
7. The in vitro bio-adhesion study confirmed the significantly higher force of detachment value and hence, bio-adhesive property of G-19
formulation relative to G-29 (p<0.05), recording 6778.64 ± 865.92 and
3666.32 ± 1176.08 dyne/cm2, respectively, related to the higher hydrophobicity of the former gel. However, there was no significant difference between the force of detachment values of plain and MPs - loaded hydrogels (p>0.05).
8. The in vitro drug release confirmed the sustainment of TA release from G-19 and G-29 preparations compared to F-9 MPs. A non-Fickian anomalous transport mechanism of TA from MPs-gel systems was confirmed. Due to the sustainment of drug release from both G-19 and G-29 hydrogel preparations, they were chosen for further in vivo studies.
Chapter III: In Vivo Evaluation of the selected TA-Loaded
Formulations.
In this chapter, the adjuvant-induced monoarthritis (AIMA) was adopted in the knee joints of male albino rats, aiming to mimic the inflammatory condition associated with human RA and further examine the in vivo anti-inflammatory effects of the optimized TA-loaded formulations after IA delivery compared to TA suspension and TA marketed injection product (Kenacort®).
Complete Freund’s adjuvant (CFA) was injected into the left knee joints of forty-five rats to induce arthritis, the right knee joints were considered the negative controls. Three days post induction, treatment injection was conducted as a first dose, followed by another one at the seventh day of injecting the treatments. The whole treatment duration lasted for 14 days and the drug dose was maintained at 250 μg/100 μl in all the used treatment. The forty-five rats were divided into nine groups, five
animals in each. group I (G-I) represented the positive control group. Groups II (G-II) and III (G-III) were injected intra-articularly with plain PLA MPs and plain 20% PEG-PDL1700-containing MPs, respectively. Groups IV (G-IV) and V (G-V) were injected with TA suspension and Kenacort®, respectively. Additionally, groups VI (G-VI) and VII (G-VII) were injected intra-articularly with F-7 and F-9 MPs, respectively. Finally, groups VIII (G-VIII) and IX (G-IX) were injected intra-articularly with G-
19 and G-29, respectively.
To assess the degree of inflammation in each treatment group, knee diameters were measured daily, starting from the day of treatment till the end of the experiment, expressed as the % inhibition of inflammation. Also, the area under the curve (AUC) was calculated for each drug-treatment group to evaluate the effect of different drug treatments. Additionally, histopathological examinations were performed on treated knee joints at the 7th and 14th day of treatment. The arthritis severity of the prepared histopathological joint samples was evaluated using a scoring system.
from the obtained results, it was found that:
1. AIMA was adopted in all animal groups just few hours after injecting CFA, visualized as edema and redness of the left knee joint, reaching maximum swelling after three days of induction.
2. All the groups treated with TA exhibited a discrepancy in % inhibition of inflammation in terms of extent and rate, though TA was administered intra-articularly at equivalent doses.
3. G-I (positive control) showed persistent inflammation all over the study period.
4. G-II and G-III showed almost zero % inhibition of inflammation values at both the 7th and 14th day of injecting treatments, with non- significant difference between both groups (p>0.05), confirming the lack of any therapeutic activity of both carriers.
5. G-VI and G-VII injected with F-7 and F-9, respectively, revealed a significant improvement in % inhibition of inflammation relative to the drug suspension (G-IV) and marketed product (G-V) (p<0.05). F-9 MPs maintained its % inhibition till the 14th day of treatment, however a non- lasting inhibition of inflammation was obvious in case of F-7 MPs.
6. G-VIII and G-IX injected with G-19 and G-29 preparations showed significant reduction in inflammation (p<0.05) compared with TA suspension, Kenacort®, F-7 MPs and F-9 MPs (G-IV to G-VII) as well.
7. The AUC values, calculated from the % inhibition of inflammation
versus time curve, of drug suspension, Kenacort®, F-7 MPs, F-9 MPs, G-
19 and G-29 were 144.58 ± 18.36, 297.72 ± 14.08, 354.16 ± 7.40, 450.58
± 5.91, 554.52 ± 5.03 and 514.50 ± 28.01 %.day, respectively. These data confirm the superiority of G-19 formulation for suppressing inflammation, being significantly different from drug suspension, Kenacort®, F-7 MPs and F-9 MPs (p<0.05).
8. The histopathological examinations and scores confirmed the superiority of G-19 and G-29 preparations in alleviating AIMA relative to all of the other treatments.
9. All of the findings obtained confirmed that both G-19 and G-29 formulations are promising treatments in alleviating RA.