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Pulmonary delivery of dry powders to rats: the limits of an intra-tracheal administration model J. 1: 2 Montharu , A. 1 Guillon , V. 2 Schubnel , G. 2 Roseau , L. 3 Vecellio , J. 4 Guillemain , P. 1 Diot , F. 1 Gauthier , M. de 1 Monte Unité INSERM U618 « Protéases et Vectorisation Pulmonaires », 2: PPF Animaleries, Université François Rabelais, Faculté de Médecine, Tours. 3: AERODRUG, Faculté de Médecine, Tours. 4: SESAME, Tours. Introduction Rodent models to deliver locally-acting or systemic therapies to the lung were based on methods using inhalation chambers and nose-only apparatus, resulting in limited lung deposition. Intubation of rats with endotracheal probes generating liquid aerosols allows administering precise and controlled large quantities of drug directly into the lungs. However, preliminary data related to intratracheal dry powder administration procedures have demonstrated some artefacts which were related to precise quantification of administered powder and to lung tolerability. The aim of the study was to evaluate the reproducibility and the lung safety of a method based on the intratracheal administration of a dry powder in a rat model. Method The Microsprayer® is a delivery tube connected to a holding chamber (aerosol generator). Number and volume of air puffs necessary to empty the chamber from the loaded powders were determined in vitro. In each case, air puffs were administered by a plastic syringe or by to the AP-1 Air Pump (PennCentury) connected to the chamber (Fig. 2). Twelve groups of 6 female Sprague Dawley rats were subjected to single administrations of air alone (2, 2X2 or 5X2 mL), lactose (10 mg/rat) or magnesium stearate (2 or 10 mg/rat), using a DP-4 Microsprayer® (PennCentury, U.S.A.) - (Fig. 1) Administration procedure Rats were anesthetized with Isoflurane at a 4% vaporizer output concentration, Aerosol generator or holding chamber contained loaded powders and placed in a supine position, suspended by their incisors, on a work stand allowing a 45° angle. Tongue was pulled out of the mouth, in order to get a clear view of the trachea. Air Pump-1 (0-5mL capacity) air puffs pushed powders into the delivery tube An otoscope was used to keep the mouth open and to immobilize the tongue at its position outside the mouth. Fig.1: Intratracheal administration of an aérosol to a rat, using a Microsprayer® Endotracheal metal 2’’ delivery tube tip located close to the carina in order to obtain optimal lung deposition Microsprayer® was inserted into the trachea, through larynx, and air or powders were administered to the lungs. Fig.2: Dry Powder-4 (DP-4) Microsprayer® connected to Air Pump-1 (AP-1) Results Fig. 3: Percentages/ranges of delivered powder according to compound/dose Actual volume of powder administered (by weighting device before and after dosing) 100 6 rats / groupe 90 7 - 100 80 The actual volume of powder given to each rat varied statistically according to compounds and/or doses administered (Fig. 3). 2mg MS - Syringe (2mL air) 2mg MS - Air pump (2mL air) 70 14 - 85 10mg MS - Syringe (2X2mL air) 60 10mg MS - Air pump (2X2mL air) 11 - 80 50 10mg L - Syringe (5X2mL air) 10mg L - Air pump (5X2mL air) 40 The total amount of loaded powder could rarely be administered whatever compound or doses. 30 2 - 71 8 - 58 20 2 – 97 10 0 Clinical response related to administration Lactose 1 Magnesium stearate Short duration respiratory arrests (~ 4-5s) were observed during 13/72 administration procedures (12/13 with magnesium stearate, 1/13 with lactose). Only one death occurred. This was not observed during administration of air alone. About 1/3 powder dosing procedures resulted in respiratory failures. Fig. 4: Percentages of rats according to number/gravity of lung lesions after treatment - 6rats / group Lung gross pathology 100% 90% 80% 83% and 56% of the lungs were impaired in animals respectively administered a powder or air alone. Presence/absence of lung impairment varied statistically according to air and compound/dose administered i.e. the number of rats with lung impairment increased with insufflations of a higher air volume and/or with presence of powder (Fig. 4). 70% 60% 50% 40% 30% 20% 10% No lung impairment Lung with slight impairment 0% 2mL seringue 2mL air pump 2X2mL 2X2mL air 5X2mL 5X2mL air seringue pump seringue pump Stéarate 2mgseringue Stéarate 2mg-Air Pump Administration of air alone Stéarate 10mgseringue Stéarate 10mg-Air Pump Lactose 10mgseringue Lactose 10mg-Air Pump Lungs with severe impairment Generalized lung trauma Administration of powders Gravity of lesions varied in the same manner i.e. lung lesions were more severe in rats receiving powders than in rats administered air alone, but generalized trauma, could also be induced by delivering air alone (Fig. 4, 5). Air alone or powder spraying using air as a vehicle could induce lung lesions due to high pressure and/or high volume traumatism Lung with “slight impairment” i.e. one lobe with edema, hemorrhage and atelectasia Lung with “severe impairment” i.e. > 1 lobe with edema, hemorrhage or atelectasia Lungs with “generalized trauma” i.e. all lobes with edema, hemorrhage or atelectasia Fig. 5: Lung gross pathology, each lung being classified in one category according to the severity of external pulmonary impairment The severity of lung lesion was statistically linked to respiratory arrest (9/13 respiratory failures were observed in rats demonstrating lungs impairment with “generalized trauma”). Conclusion This model for powder administration suffers from artefacts which may limit its relevance. These restrictions are of particular importance, especially in the frame of regulatory toxicity studies . Protéases et Vectorisation Pulmonaires INSERM U-618 Pulmonary delivery of dry powders to macaques: set up of an intra-tracheal administration model A. 1: 1 Guillon , J. 2 Montharu , V. 2 Schubnel , G. 2 Roseau , 3 Vecellio , L. J. 4 Guillemain , P. 1 Diot , F. 1 Gauthier , M. de 1 Monte Unité INSERM U618 « Protéases et Vectorisation Pulmonaires », 2: PPF Animaleries, Université François Rabelais, Faculté de Médecine, Tours. 3: AERODRUG, Faculté de Médecine, Tours. 4: SESAME, Tours. Introduction The inhaled route to deliver both locally-acting and systemic therapies is developing. Rodent models are widely used to evaluate the pulmonary safety of dry power administration. However, regulatory toxicity studies required several animal species. Monkeys, that more closely replicate human disease, require specialized management. Repeated aerosol drug administration to anaesthetized monkey is probably not free of consequences, even with inactive drug aerosol (lactose). Thus, the aim of the study was the subacute tolerance of repeated lactose powder administration by intra tracheal route, in monkeys. Method Two male cynomolgus monkeys (5.3 and 7.5 kg) were administered lactose 7 times in 15 days (2.5mg/kg every 48 hours), using a Microsprayer® (Pencentury, USA). Clinical, laboratory investigations (haematology, biochemistry), and histopathology of the lung and trachea were performed. The Microsprayer® is a delivery tube connected to a holding chamber (aerosol generator). Air puffs were administered by a plastic syringe connected to the chamber. Administration procedure: Monkeys were sedated with an intramuscular injection of ketamine. Anaesthesia was induced with isoflurane at a 5% vaporizer output concentration in oxygen via a facial mask. Tracheal intubation was performed under direct laryngoscopy. Microsprayer® (Fig. 1) was inserted into the trachea probe, and lactose powder were administered to the lungs. Fig.1: Dry Powder-4 (DP-4) Microsprayer® Plastic seringe air puffs pushed powders into the delivery tube Aerosol generator or holding chamber contained loaded powders Anaesthesia Sedation Endotracheal metal 11.8’’ delivery tube tip located close to the carina in order to obtain optimal lung deposition Monotoring procedure: Animals kept spontaneous ventilation during anaesthesia. Monitoring included pulse-oximetry and heart rate recording. Monitoring Blood sampling Administration Intubation Blood sampling: Toxicokinetic studies were performed on the first and the last days (7 blood sample of 1.0 mL in 24h). ml Results Clinical evaluation No clinical sign related to administration of lactose was recorded, no body weight loss was observed. Laboratory investigations Hyperleucocytosis, hepatic cytolysis and rhabdomyolysis were recorded at the end of the study period (Table 1 - median values). Hepatic cytolysis could be explained by the repeated fasting periods. Rhabdomyolysis was probably due to prolonged anaesthesia and rise in the compartment pressure. Table 1 Before treatment: -10 days After treatment: Day 14 UREA CREAT GLU mmol/L µmol/L mmol/L 5.15 89 3.45* 81.5* GGT IU/L ALAT ASAT IU/L IU/L 4.15 116.5 33.5 3.7 44 ALP IU/L CK IU/L 566 603 101.5 65* 106* 481 CA++ PHOS Na+ K+ Clmmol/L mmol/L mmol/L mmol/L mmol/L 2.5 1.63 ALB g/L 145 3.9 106 41.5 3536* 2.61 0.91* 146 4.2 106 42.5 PROT BIL g/L µmol/L 81 4.25 WBC g/L N % 7.45 38 HGB g/dL HCT % 14.15 42.05 PLT g/L PT s FIB g/L APTT s 348 11.45 2.21 22.95 10.9 1.99 21.35 79.5 4.05 12.7* 66.5* 13.55 40.85 336.5 CREAT: creatinin; GLU: glucose, GGT: Gamma glutamyltransferase; ALAT: Alanine aminitransferase; ASAT: Aspartate aminotransferase; ALP: Alkaline phosphatase; CK: Creatinine kinase; PHOS: Phosphorus, ALB: Albumin; PROT: Protein; BIL: Bilirubin; WBC: White blood cells; N: Neutrophil, HGB: Heamoglobin; HCT: Haematocrit; PLT: Platelet; PT: Prothrombin time; FIB: Fibrinogen; APTT: Activate dprothrombin time; *: « Modified » values (no statistical test due to low number of animals) Lung and trachea histopathology No relevant histological effects were induced by powdered lactose administration Conclusion Repeated aerosol of lactose did not have local consequences after macroscopic and histological examination. This result suggests that this model is suitable to evaluate sub acute local toxicity after aerosol drug administration. Lactose aerosol administration can be used as a negative control. However, experimental procedures need to be improved in order to limit consequences of repeated anaesthesia. Protéases et Vectorisation Pulmonaires INSERM U-618