Janne Raula1, Ville Vartiainen1, Esko I. Kauppinen1, Jouni Hirvonen2 & Luis M. Bimbo2

1) Aalto University, School of Science, Espoo, Finland2) University of Helsinki, Faculty of Pharmacy , Helsinki, Finland

Aerosolization, Drug Permeation and Cellular Interaction of Leucine Coated Combination DPI powders

Flowable and dispersible inhalable

powder

Aqueous drug suspension with the amino acid leucine

B

ED (mg/dose) EDeff (%) CVED

FPF (%, d<5µm)

2 kPa 4 kPa 2 kPa 4 kPa 2 kPa 4 kPa 2 kPa 4 kPa

Twister 3.6 4.2 71.8 83.8 0.11 0.06 40 40

Easyhaler 3.5 3.6 - - 0.13 0.11 47 49

C

SUMMARYWe investigated the effect of L-leucine coating on particle integrity,aerosolization properties, cellular interaction, viability and drug permeationproperties of combination drug powder particles for dry powder inhalation.The powder was prepared using an aerosol flow reactor method to combine awater-soluble β-agonist drug, salbutamol sulphate, and corticosteroid,beclomethasone dipropionate, into fine particles which were subsequentlyencapsulated and coated with L-leucine nanocrystals. Fine particle fraction(FPF) was slightly higher (47-49%) from Easyhaler® whereas the emitted dose(3.6-4.2 mg/dose) was higher from Twister™. The powder’s dose repeatibility(CV<0.13) and aerosolization independency on applied pressure drop wereexcellent. Dissolution profiles showed a faster dissolution of beclomethasonedipropionate, while leaving the salbutamol dissolution unhampered whencompared with their physical mixture. Permeation of beclomethasonedipropionate across a differentiated Calu-3 cellmonolayer was increased butalso time-dependent recrystallization of the drug on top of the Calu-3 cellmonolayer was observed. The particles were further investigated forcytocompatibility in three different pulmonary(Calu-3, A549 and BEAS-2B) andone human macrophage (THP-1) cell lines showing excellent tolerability. Theyalso elicited low reactive oxygen species generation in pulmonary BEAS-2Band macrophage THP-1 cell lines. The inhalable drug powders coated withleucine offer thus an interesting alternative for the delivery of poorly solubledrugs to the lung.

Fig. 1. A) SEM image, B) schematic view of partic le s tructure and C) the aerosolization results of the BSL partic les .

Fig. 2. Dissolution profiles of beclomethasone dipropionate (blue) and salbutamol sulphate (red) from the BSL powder (circles) and its phys ical mixture (squares) in phosphate buffer pH 7.4 and 37 °C. Dissolution medium was 0.2 M KH2PO4, 0.1 M NaOH. Error bars represent SD (n ≥ 3).

Excellent aerosolization due to a rough crystalline leucine coating

Dissolutions of the poorly soluble and freely soluble drugs

Nanos-in-Micros dry powder inhalation (DPI) formulations for in vitro drug transport studies

Janne-Juhani Raula, PhD 13 September 2013

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Fig. 2 Deposition and drug permeation of Nano-in-Micros particle assemblies across a Calu-3 cell monolayer.

5. Importance of the research

Current marketed DPI systems encounter dosing problems which are due to very cohesive nature of micronized fine drug particles. Current commercial inhalation powder contains 1-5 % micronized drug powder blended 95-99 % of coarse carrier particles. IN this fashion, drug particles are difficult to detach from the carriers and to be delivered to the alveoli area of lungs. The loss of drug increases the costs and patient inconvenience. Cohesiveness of micronized powders is due to large contact area between other surfaces. The efficiency of drug delivery is commonly very low, only 5-10%. Using our method, the contact area can be reduced very much with rounded surface having small, hard crystals on top. We have shown that drug delivery without any carrier particles is improved notably being around 40-60 % having only 2-10 % of leucine coating, and the rest is drug.

Due to the nano-scale crystalline roughness, the leucine coated inhalable powders show good flowability and dispersibility characteristics. Therefore, these powders can dosed from DPIs without any additional carrier particles; carrier-free powder formulations. Important criteria for aerosolization performance are dose repeatibility and independence on applied inhalation flow (e.g. child vs. adult). The leucine coated powders show not only much higher drug emission with variability from inhaler than that of a commercial powder but also the drug dose is independent on inhalation flow. Also, the lung deposition of powder to alveoli area is 3-4 times higher than commercial powder.

The aerosol method presented here enables easy combination of drugs with different solubilities in the same particle, which is protected against humidity using encapsulation and further coated with a rough and crystalline leucine layer to provide excellent aerosolization properties. These improve therapeutic effect, long-time stability and delivery to the lung alveoli. Moreover, DPI drugs are administered without carrier particles which leads to drastic reduction in the amount of drugs and excipients needed for efficient pulmonary delivery.

Although we have shown that Nanos-in-Micros formulations improve the dissolution of poorly soluble drugs, we still lack the knowledge on how particles and drug release take place on a cellular environment (mucoadhesion and mucopenetration as well as permeation across cell line). With this information we would be able to modify particles in terms of morphology and surface characteristics to optimize delivery properties on lung epithelium. Therefore, the current research project is expected to bring important information to optimize the formulation and to assess particle behavior after deposition. 6. Current phase of the study and schedule

We have produced several new Nano-in-Micros DPI powders including corticosteroids beclomethasone or budesonide and β-agonist salbutamol sulphate with varying ratios. All the particles are encapsulated and coated by leucine and they perform good DPI delivery properties in vitro. In this project we will conduct cellular permation studies at the Division of Pharmaceutical Technology, University of Helsinki under the supervision of Dr. Luis Bimbo. This collaborator has many years of experience conducting cellular permeation studies with different types of micro- and nanoparticles. It is estimated that this project will take 6 months which will promote and accelerate our pulmonary research and bring new knowledge to the field of pulmonary drug delivery.

The proposed research project will be executed by a research assistant to be recruited under the direct

supervision of the applicant (Dr. Janne Raula), and the applied funding will cover the research assistant’s full

salary for the 6 months duration of the project according to the budget on Table 1. together with all materials

Drug permeation through the Calu-3 cell line improved by the powder formulation

Fig. 3. Upper image illustrates the par tic le depos ition on the cells and tra nsport t hroug h t he cellmonolayer into the basal chamber. A ) Permeation profiles of beclomethasone dipropionate f rom BSLpowder (blue solid) and i ts phys ical mixtu re ( red dashed); B ) Permea tion profiles of salbu tamolsulphate from the BSL powder (blue solid) a nd its ph ys ical mixture ( red dashed) across adifferentiated Calu-3 cell monolayer at apical pH of 7.4. Error bars represent SD (n ≥ 3).

Fig. 4. SEM images of the BSL powders in top of a differentiated Calu-3 monolayer at (A and B) 0 min, (C) 30 min and (D) 180 min. Scale bars are 10 µm.

Some beclomethasonecrystallized on the top of the Calu-3 cells

Fig. 5. In tracellular R OS assessment of (A) BE AS- 2B and (B) THP- 1 macrop hage cells incubated wit hdifferent concent rations o f B SL powders for 1 h, 3 h a nd 6 h determi ned wi th a fluorescent DCF -DAassay. All data sets were compared with a negative control of HBSS. Error bars represent ± SD (n ≥ 9).

Nanoscale roughness by leucine crystals

Cellular viability assessment provided an excellent tolerability profile for the BSL formulation

Acknowledgements: We thank Academy of Finland for the funding, Aalto University’s Nanomicroscopy Center for providing microscopy facilities and TeicosPharma for co-developing the technology and powder formulation

Particle drying

Coating by leucine

A