International Journal of Research Studies in Science, Engineering and Technology Volume 3, Issue 7, July 2016, PP 7-14 ISSN 2349-4751 (Print) & ISSN 2349-476X (Online) International Journal of Research Studies in Science, Engineering and Technology [IJRSSET] 7 In Vitro Cytotoxicity of Dry Powder Inhaler Medical Devices Efstathia Giannopoulou 1 , Georgia Efstratiadou 1, 2 , Stavroula Rozou 3 , George Ismailos 3 , Konstantinos Theofanopoulos 3 , Eirini Panoelia 2 , Haralabos P. Kalofonos 1 , *Gregory Sivolapenko* 2 1 Clinical Oncology Laboratory, Division of Oncology, Department of Medicine, University of Patras, Rio 26504, Greece 2 Pharmacokinetics Laboratory, Department of Pharmacy, University of Patras, Rio 26504, Greece 3 ELPEN Pharmaceutical Co. Inc., 95, Marathonos Ave., 19009 Pikermi, Greece *gsivolap@upatras.gr Abstract: Dry powder inhalers (DPIs) are widely accepted inhaled delivery dosage forms that are currently used by a large number of patients for the delivery of medications to treat asthma and chronic obstructive pulmonary disease (COPD). The cytotoxicity test of DPIs is necessary since the mouthpiece of the device is in contact with the user on a daily basis for a long period of time. In the current study, we evaluated the cytotoxicity of the mouthpiece of 4 marketed DPIs: Aerolizer ® (Novartis), Diskus ® (GlaxoSmithKline), Elpenhaler ® (Elpen Pharma) and Turbuhaler ® (AstraZeneca). The experimental procedure was designed according to the guidelines of the International Organization for Standarization (ISO), the Food and Drug Administration (FDA) and the United States Pharmacopoeia (USP). Tetrazolium salt (MTT) reduction assay and cell morphology observation are recommended by the three organizations and the results were supplemented and compared with the lactate dehydrogenase (LDH) test, an alternative cytotoxicity assay. The experiments were performed in L-929 cells using elution test. None of the tested DPI mouthpieces showed any cytotoxicity effect using the current assays, 48 and 72h after extracts application in cells, compared to negative control. Surprisingly, an increase in cytotoxic response of the negative control was observed at 72h using MTT and LDH tests. All DPI mouthpieces are equivalently safe for long term use. The methods presented in the current study offer an easy and sensitive way for the test of cytotoxicity of a biomaterial and can be applied to other medical devices. Keywords: Biocompatibility, Cell morphology, In vitro test, MTT assay, Dry Powder Inhalers 1. INTRODUCTION Asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory lung diseases. In both cases, inflammation is associated with structural alterations at large and small airway levels, which may relate to phenotypic overlaps occurring in these diseases 1-6 . According to the latest World Health Organization (WHO) estimates, currently 235 million people suffer from asthma and 64 million people have COPD worldwide. During the years 2010-2012 more than 380,000 people died of asthma annually, and even though COPD is less frequent than asthma, it is estimated that, for the same period of time, more than 3 million people died of COPD. WHO predicts that COPD will become the third leading cause of death worldwide by 2030 4-8 . The treatment of asthma and COPD includes the inhalation of medication to the site of the disease process. Inhalers are the principle vehicles for the effective administration of medication. They allow high lung deposition of the drug and minimize systemic bioavailability, thus reducing possible systemic adverse drug reactions 5,9-14 . Dry powder inhalers (DPIs), pressurized metered dose inhalers (pMDIs) and nebulizers are the devices used for respiratory drug delivery 10,11,15,16 . Inhalers are often being used both by adult and paediatric patients for long term therapy of asthma or COPD, hence the biological safety of the inhaler medical device is of paramount importance to the user. It must be ensured that the mouthpiece in particular, causes no toxicity whatsoever following the usual direct, frequent and daily exposure to long treatment periods. In the USA, a number of tests have been proposed by the Technical Committee of International Organization for Standarization (ISO) 10,11,17 , by the Food and Drug Administration (FDA) 18 , and by the United States Pharmacopoeia (USP) 19-22 , for screening biocompatibility of materials intended for use in medical devices. The choice of a biomaterial is based on the outcome of in vitro and in vivo cytotoxicity tests. ISO describes a set of standards, designed as ISO 10993 series, of which Part 5 focuses on the in vitro