الفهرس 
Preparation and characterization of Fast Dissolving MTK Loaded Spray Dried Agglomerated MicroparticlesOnly 14 pages are availabe for public view
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Abstract
Direct drug targeting to its site of action offers always numerous advantages over traditional routes. Targeting drugs to lungs was recently considered as a promising route of administration of drugs for both local action in lungs or to the systemic circulation.
The pulmonary route offers large surface area for drug absorption, with little or no enzymatic activity. Thus avoidance the first pass effect presented lungs as an alternative to the systemic route for administration of proteins, hormones and other macromolecules with high systemic bioavailability. For drug exerting their action in lungs for treatment of asthma, fibrosis or lungs infections, targeting allows the use of smaller drug doses with little or no systemic side effects.
Dry powder platform is delivered to the lungs using dry powder inhalers, offering long term stability being in a dry state. The powder should have suitable mass median aerodynamic diameter to allow for deep deposition in alveoli, where high surface area and thin non ciliated wall offering no barriers for the deposited particles. It is to be noted that the freely roaming macrophages in alveoli can engulf and digest the deposited particles. So, after deposition, fast dissolution of particles (if soluble) or fast deaggregation to their payload of nanoparticles is a crucial step to skip these macrophages, unless macrophage targeting was an aim.
Montelukast (MTK) is a novel antiasthma drug of the leukotriene inhibitor group. It has been used for the management of asthma specially in children, when avoidance of corticosteroids therapy is a must. The aim of this work was to develop MTK dry powder formulations for both fast and prolonged drug action.
Accordingly, the work was divided to three chapters as follows:
• Chapter I: Preparation and characterization of Fast Dissolving MTK Loaded Spray Dried Agglomerated Microparticles.
• Chapter II: Preparation and characterization of Agglomerated Spray Dried MTK Loaded PLGA Nano-In-Microparticles.
• Chapter III: In-Vivo Pharmacologic Efficacy of MTK loaded Spray Dried Powders Formulae.
Chapter I: Preparation and characterization of Fast Dissolving MTK Loaded Spray Dried Agglomerated Microparticles.
This chapter was concerned with the preparation of agglomerated fast dissolving MTK loaded microparticles using the spray drying technique. Two types of matrix formers were studied in this chapter, mannitol and ovalbumin. For each type of soft agglomerates, varying ratios of different excipients and drug were prepared and characterized viz: powder yield, drug content, flow parameters, particle size, aerosolization properties, particle size analysis. X ray powder diffraction, Differential scanning calorimetry, Fourier transform infrared spectroscopy were used also to evaluate the prepared powder formulae. The developed agglomerates were visualized using scanning electron microscopy, deagglomeration study was also performed to evaluate the ability of the soft agglomerates to deagglomerate under reduced pressures. Finally, in-vitro release study and long term stability study were performed on the selected formula.
The results of this chapter could be summarized in the following points:
1. Non polymer based MTK loaded agglomerated microparticles were successfully prepared with the spray drying technique using mannitol and ovalbumin as matrix formers.
2. Spray drying of drug with mannitol alone or in combination with leucine showed an acceptable powder yield. The spray dried drug alone or in combination with high albumin content showed very low yields and sticking of the dried powder to the cyclone wall. This indicated the need of suitable excipients in order to formulate MTK in a stable dry powder platform with acceptable properties.
3. The association efficiency of all formulae was more than 90%, indicating the suitability of the method of preparation and spray drying conditions, allowing for no drug loss.
4. Leucine proved to be an essential additive for producing powder with acceptable flow properties. Increasing its percent in the final formulae yielded powders with good aerosolization properties and high fine particle fraction.
5. X Ray diffractogram showed that the drug was successfully amorphized after spray drying, allowing for rapid drug dissolution after deposition. Residual excipients crystallinity was also evident with both mannitol and leucine, offering more chemical and physical stability than the completely amorphous powders. This residual crystallinity was not detected in ovalbumin as additive, and the residual crystallinity in ovalbumin based formulae was originated from leucine only.
6. Agglomeration was successfully achieved using 5% lecithin as soft binder. This agglomeration was confirmed by particle size measurements and SEM imaging of both types of agglomerates. The agglomerates showed rough surface with mannitol, in albumin based formulae, corrugated surface was obtained.
7. The mass median aerodynamic diameter of all formulae lied between 1 and 5 µm, indicating the successful production of inhalable powders suitable for pulmonary delivery.
8. The produced agglomerates were soft enough to restore their original size by applying different air pressures during dry particle size measurement.
9. The produced microparticles were fast dissolving, as proved in the in-vitro release study. where more than 95% of MTK was released from the tested soft agglomerates within the first 5 minutes. Allowing to skip uptake by the freely roaming macrophages in alveoli, as this engulfment starts within 30 minutes after particles deposition.
10. The use of twin stage impinger proved the production of inhalable powders with expected suitable deposition in the deep alveoli when administered via the pulmonary route. Formulae containing 50% leucine (namely M4 and O4) showed best fine particle fraction and aerosolization properties.
11. Mannitol based soft agglomerates offered drug loading flexibility as evidenced in the in-vitro pulmonary deposition. where increasing drug loading was found to have no negative impact on the aerosolization properties of the prepared powders. On the contrary, for albumin based soft agglomerates, high drug loading adversely affected powder deposition.
12. The selected formula (M4) showed long term stability for up to six months at normal storage conditions. This might be attributed to both, its low residual moisture content, and the residual excipients crystallinity which protected the drug payload from any degradation upon storage.
13. The successfully agglomerated formula M4 (D5:M40:L50: S5), with best yield value (87.23), best powder flow properties, highest stage 2 deposition and fastest dissolution profile, drug loading flexibility and low residual moisture content, was selected for further in-vivo characterization.
Chapter II: Preparation and characterization of Agglomerated Spray Dried MTK Loaded PLGA Nano-In-Microparticles.
The objective in this chapter was to prepare prolonged release MTK loaded formulae in a dry powder platform. Entrapping drug in PLGA nanoparticles was performed using the double emulsion solvent evaporation method. The developed NPs were then agglomerated in microparticle size using the spray drying technique with the aid of matrix formers in the external aqueous phase. selected nano-in-MPs formula was then subjected to agglomeration study using 5% lecithin as binder. this agglomeration was performed aiming for more powder stability, improvement of flow properties and fines control.
The developed formulae were characterized based on their original NPs size and drug entrapment efficiency. The yield value, flow parameters, particle size and aerosolization properties and SEM imaging were used to evaluate the nano-in-MPs. The ability of the MPs to restore their original NPs size was also a crucial study, fast recovery is supposed to offer higher chance to skip macrophages uptake if the recovered NPs size was less than 260 nm within 30 min.
The agglomerated powders were also characterized; their ability to deagglomerate under reduced pressures was an important concern. The selected formula was then studied based on X-ray powder diffraction, Differential scanning calorimetry, Fourier transform infrared spectroscopy, TEM imaging, drug release study and long term stability study.
The results of this chapter could be summarized in the following points:
1. MTK loaded PLGA nanoparticles were successfully prepared using double emulsion solvent evaporation method.
2. The presence of DPPC was found to be essential in nanoparticles preparation, in order to obtain NPs of suitable PS (below 300 nm).
3. High entrapment efficiencies exceeding 80% were obtained in formulae with both high PVA content (in both aqueous phases) and a matrix former (mannitol or leucine) in the external aqueous phase.
4. NPs particle size of less than 300 nm was achieved when using 0.5% PVA and L as excipient owing to their antiaggregating activities (formulae P19, P10, P11 & P12).
5. Fast NPs recovery from MPs within 15-30 minutes was achieved in selected formula, indicating the probable ability to escape the macrophages uptake of the deposited MPs in lungs.
6. These selected nano-in-MPs formulae showing high EE and the best flow properties were successfully agglomerated to larger agglomerates using lecithin and proved by size measurements, SEM images and deagglomeration studies.
7. The in-vitro lung deposition showed a stage 2 deposition reaching 30% and good inhalation indices with the best formula were achieved, indicating the suitability of the prepared powder for the pulmonary administration.
8. The selected agglomerated formula P11 was included In further studies. The X-ray diffractogram showed complete drug amorphization which is important for fast drug dissolution after release. Residual leucine crystallinity was also observed, this is offering an advantage for optimum formula stability upon storage than the completely amorphous powders.
9. TEM imaging was performed on the selected P11 formulae, it showed the original nanoparticles before spray drying and the recovered nanoparticles. This confirmed the recovery study results.
10. Prolonged montelukast sodium release profile was achieved over four weeks period, this could help in eliminating the need of daily MTK dosing for asthma symptoms control.
11. Formula P11, with best entrapment efficiency, acceptable yield and powder flow properties, good NPs size and successful agglomeration, good NPs recovery and prolonged MTK release was selected and included in further in-vivo studies.
Chapter III: In-Vivo Pharmacologic Efficacy of MTK loaded Spray Dried Powders Formulae.
In this chapter we aimed to evaluate the pharmacologic efficacy of the prepared selected formulae in the treatment of experimentally induced asthma. For this purpose, formula M4 prepared in chapter one and formula P11 prepared in chapter two were both included in this study.
Asthma was successfully induced using toluene di-isocyanate as sensitizer following one month asthma induction protocol. The asthma progression was followed at different time intervals based on two main markers. First was the total and differential leukocytes count in the bronchoalveolar lavage of the sacrified rats which are correlated with the asthma progression. Second was the histopathologic examination of rats lungs to show the structural changes and airway remodelling in asthmatic animals. These findings were compared to positive asthmatic and negative non-asthmatic groups. A group of rats were receiving the brand marketed product was included for comparison purpose also.
The results of this chapter could be summarized in the following points:
1. Asthma was successfully induced in rats using toluene-di-isocyanate (TDI) as sensitizer over one month sensitization protocol period as proved by the significant increase in the total and differential leukocytes count compared to their count in the normal non asthmatic rats. This asthma progression was also evidenced in histopathologic examination of rats’ lungs
2. The fast dissolving MTK loaded SA (M4) succeeded to protect rats from the experimentally induced asthma, with efficacy comparable to the brand oral marketed product, at one tenth the oral dose only for one day period. This was proved by both bronchoalveolar total leukocytic count and its differentials and histopathologic examination.
3. Targeting the drug to its site of action (lungs) decreased the drug dose needed for pharmacologic action to one tenth the oral dose. This localization is supposed to decrease also the systemic side effects.
4. The prolonged release formula (P11) showed pharmacologic efficacy for up to one week in the total leukocytic count and its differentials. On the other hand, the histopathologic examination showed non asthmatic airways for up to 5 days only when given at one tenth the oral reported dose.
These results showed that the fast dissolving selected formula (M4) showed pharmacologic efficacy comparable to the brand product. It also offered an additional advantage concerning the decrease in the needed drug dose to only one tenth the reported dose, with possible decrease in the associated systemic side effects reported with the oral drug regimen.
Montelukast entrapment in PLGA nanoparticles created a prolonged release drug delivery system, able to deliver its drug payload slowly after deposition, eliminating the need of once daily treatment regimen. This is supposed to offer more patient adherence to therapy with a regimen every 5 to 7 days.