Tolerability and Pharmacokinetic Evaluation of Inhaled Dry Powder Tobramycin Free Base in Non-Cystic Fibrosis Bronchiectasis Patients

Rationale Bronchiectasis is a condition characterised by dilated and thick-walled bronchi. The presence of Pseudomonas aeruginosa in bronchiectasis is associated with a higher hospitalisation frequency and a reduced quality of life, requiring frequent and adequate treatment with antibiotics. Objectives To assess local tolerability and the pharmacokinetic parameters of inhaled excipient free dry powder tobramycin as free base administered with the Cyclops dry powder inhaler to participants with non-cystic fibrosis bronchiectasis. The free base and absence of excipients reduces the inhaled powder dose. Methods Eight participants in the study were trained in handling the device and inhaling correctly. During drug administration the inspiratory flow curve was recorded. Local tolerability was assessed by spirometry and recording adverse events. Serum samples were collected before, and 15, 30, 45, 60, 75, 90, 105, 120 min; 4, 8 and 12 h after inhalation. Results and Discussion Dry powder tobramycin base was well tolerated and mild tobramycin-related cough was reported only once. A good drug dose-serum concentration correlation was obtained. Relatively small inhaled volumes were computed from the recorded flow curves, resulting in presumably substantial deposition in the central airways—i.e., at the site of infection. Conclusions In this first study of inhaled dry powder tobramycin free base in non-cystic fibrosis bronchiectasis patients, the free base of tobramycin and the administration with the Cyclops dry powder device were well tolerated. Our data support further clinical studies to evaluate safety and efficacy of this compound in this population.

of Pseudomonas aeruginosa is an unfavorable prognostic indicator and is associated with increased sputum production, more extensive bronchiectasis on HR-CT of the thorax, more hospitalizations and reduced quality of life. Until now, most patients with non-CF bronchiectasis who are colonized with P. aeruginosa receive inhaled tobramycin every other month, by use of a nebulizer. However, this delivery system has several disadvantages, like a low lung deposition and pollution with tobramycin in the surrounding environment. With an efficient dry powder inhaler (DPI), a three to six fold higher lung deposition compared to a nebulizer can be obtained. Therapy with a DPI is also less time consuming compared to nebulisation. Nebulised tobramycin is used most in routine care; there is also one, rather poorly characterized DPI for tobramycin available, though this DPI is not registrered for non-CF bronchiectasis. We will investigate dry powder tobramycin (DP tobramycin) in a novel device in patients with non-CF bronchiectasis colonized with P. aeruginosa.

Objective:
The main objectives are to investigate the pharmacokinetic properties of DP tobramycin at different dosages together with the local tolerability of DP tobramycin via the Cyclops® at different dosages.
Study design: single center, single ascending, single dose, response study.
Local tolerability of DP tobramycin is determined by scoring adverse events, specifically coughing, and lung function measurement.
Nature and extent of the burden and risks associated with participation, benefit and group relatedness: All participants included in this study are patients recruited from the outpatient department of pulmonology. Before using the DPI they will receive instructions and their inspiratory flow will be tested. Before each test dose an infusion needle will be inserted and after each test dose blood will be collected. To investigate safety, lung function tests will be performed and the occurrence of adverse events will be scored.

Tobramycin inhalation by dry powder
Dry powder inhalation of tobramycin in higher dosages was first described by Geller et al. baseline to day 28 of cycle 3 were similar between groups. The mean reduction in sputum P.
aeruginosa density (log10 CFU/g) on day 28 of cycle 3 was also comparable between groups. As expected, administration time was significantly less for TIP. Treatment for cystic fibrosis. It is now marketed and reimbursed in the Netherlands.

Novel dry powder inhaler (the Cyclops®)
Good performance of a dry powder inhaler (DPI) depends on the balance achieved between three types of forces, the interparticulate forces in the powder, the de-agglomeration forces during inhalation, and the deposition forces in the human lungs (see fig. 1).
The DPI used in this study was developed at the department of Pharmaceutical Technology and Biopharmacy of the University of Groningen. It was specifically designed to deliver high doses of dry powder drugs, for example antibiotics. Its dispersion principle is based on patented air classifier technology, which enables a reduction of the influence of the patient's breathing behavior on drug delivery (see fig. 2).
Background and justification over dry powder inhalation, especially with aminoglycosides, will be explained further in paragraph 5.6.3

Tobramycin
The tobramycin formulation used consists of tobramycin free-base, whereas tobramycin sulphate is used in TOBI ® nebulizer solution. Switching from one salt to another, or as in this case, to the unassociated form, is allowed when bioavailablity, pharmacokinetics and toxicological profile are equivalent. Both the free base and the sulphate are freely soluble in water. Therefore, we believe the free base will behave similarly when it enters the airways.
Upon dissolution in the humid environment of the airways, tobramycin sulphate dissociates immediately, and tobramycin becomes available as free base. Presenting tobramycin free base directly or after immediate dissociation, presumably does not affect its pharmacokinetics. To assess pharmacokinetics and to rule out any chance on giving a dose that might induce unacceptable side effects, a safety and dose escalation regime is chosen for the pilot study, with sufficient time for complete washout prior to the administration of the next dose. However, as we are most interested in clinical and microbiological response, an efficacy and safety study will be planned after the current porposal. That study will compare TOBI ® inhalation solution (the current routine treatment) and dry powder inhalation of tobramycin via the Cyclops® in different treatment strategies. The treatment regimen of DP tobramycin using the Cyclops® will be chosen based on the PK parameters of this study

OBJECTIVES
As we described previously, within the population of patients with bronchiectasis, colonization with P. aeruginosa is associated with increased sputum production, more extensive bronchiectasis on HR-CT of the thorax, more hospitalizations and reduced quality of life. In current guidelines non-CF bronchiectasis patients colonized with P. aeruginosa need to be treated with antibiotics, for example nebulized tobramycin. Occasionally, other organisms as well as mixed infections result in similar problems, and these patients may also benefit from inhaled tobramycin.
The objective of this study is to document pharmacokinetics and local tolerability of dry powder tobramycin inhaled by patients with non-CF bronchiectasis by administering 4 different doses of tobramycin: 30, 60, 120 and 240 mg.

Primary objectives
To establish both pharmacokinetics and local tolerability of DP tobramycin using the Cyclops, in the target population.

STUDY DESIGN
The study will be a single centre, single ascending, single dose study of inhaled dry powder tobramycin as a first step to investigate the pharmacokinetics of four doses with no more local intolerability than when tobramycin is administered by nebulisation. The study includes 8 patients with non-CF bronchiectasis who meet the in-and exclusion criteria. These 8 patients will be recruited from the outpatient department of pulmonology.

Population (base)
All participants will be patients with non-CF bronchiectasis and will be recruited via the outdoor department of the department of pulmonary diseases of the UMCG. Due to the low number of study participants needed to perform this study we expect to reach the target number of patients needed for this study within several months.

Name and description of investigational product(s)
Dry powder (DP) tobramycin consists of pure spray dried tobramycin free base. It is a white to off-white, hygroscopic powder with a particle size distribution appropriate for pulmonary administration. One dose of 30 mg tobramycin is sealed in an aluminium blister. Coarse crystalline α-lactose monohydrate particles are added to the blister (not blended with the tobramycin) to break up any agglomerate that forms during storage.

Summary of findings from non-clinical studies
Preclinical data show that target organs for tobramycin toxicity are the kidneys and the vestibular/cochlear functions. This toxicity was shown at systemic tobramycin levels that were higher than can be achieved by inhalation of the recommended clinical dose given by nebulisation (Summary of Product Characteristics (SPC) TOBI ® , SPC Bramitob ® ).
Administration of inhaled tobramycin during up to 28 consecutive days might give rise to modest and unspecific signs of irritation of the respiratory tract, that were fully reversible on therapy discontinuation (SPC Bramitob ® ).
Subcutaneous administration at doses of 100 mg/kg/day in rats and 20 mg/kg/day in rabbits were not teratogenic during organogenesis. Higher parenteral doses induced maternal toxicity and abortion in rabbits and were not relevant to be assessed on teratogenicity (SPC TOBI ® , SPC Bramitob ® ).

Summary of findings from clinical studies
Several studies in the CF population with inhaled tobramycin were done in the late eighties and early nineties of the last century with promising results 19 . However not all therapies that are effective in CF are effective in non-CF bronchiectasis 20 .
Later, studies with tobramycin solution for inhalation were performed in patients with non-CF bronchiectasis. Barker  to bronchospasm despite intense bronchodilator treatment. One patient in group B died due to respiratory failure after 300 days of follow up. No differences in lung function or arterial blood gas analysis were seen between the two groups. There was only one hospital admission in group A and 7 patients who had to be admitted in group B. In both groups P. aeruginosa was still present. They did not study sputum density 21 .
Couch et al studied 74 patients with non-CF bronchiectasis colonized with P. aeruginosa.
Patients were evenly divided in two groups, one group is receiving tobramycin inhalation therapy and the other group receives placebo. The treatment group received tobramycin for 4 weeks 300 mg twice daily. This group had a significant reduction in sputum P.
aeruginosa density while the placebo group showed no change. The incidence of dyspnoea, wheezing and chest tightness was again significantly greater in the tobramycin group 22 . In controlled clinical trials with TOBI®, dysphonia and tinnitus were the only unwanted effects reported in significantly more patients treated with TOBI®. In controlled clinical trials with Bramitob, most common adverse events were those concerning the respiratory tract (cough, dyspnoea, increased expectoration, FEV1 reduction). Furthermore, nausea is reported as common adverse event (occurrence >1/100).

Benefits
Intermittent use of nebulized tobramycin is indicated for the long-term management of chronic pulmonary infection due to P. aeruginosa in CF patients aged 6 years and older (SPC TOBI ® ). Several studies (see paragraph 6.3) showed that treatment with inhaled tobramycin might also be beneficial to patients with non-CF bronchiectasis whom are colonized by P. aeruginosa. Long-term intermittent administration appears to be safe and lessen disease severity.
Benefits of administering tobramycin by use of the DPI instead of by nebulisation are a less time consuming and easier drug administration that is less of a burden on the patient's (social) life and no pollution of the environment. Furthermore, because it is a single-use device there is no need for hygiene control or maintenance of the equipment and no risk of device contamination and patient (re)infection.

Description and justification of route of administration and dosage
The way of administration will be as dry powder using a DPI. Further information about the technical aspects of the DPI see section The dry powder inhaler in the Introduction and rationale. This device can generate high volumes of dry powder, needed for a substance and with an aim like tobramycin.

Dosages, dosage modifications and method of administration
In a study with CF patients 112 mg of dry powder tobramycin using the Podhaler was comparable with 300 mg of tobramycin via nebulisation, which is the recommended clinical dose in this population [13][14][15] . However for patients with non-CF bronchiectasis no optimal dosage has been studied.
Based on the in vitro data analysis, we expect that a lower dose of DP tobramycin using the Cyclops® will result in similar deposition characteristics with similar pharmacokinetic parameters compared to DP tobramycin using the Podhaler. Since, the Cyclops® has a higher inhaler resistance than the Podhaler®, the airflow through the Cyclops® (at the same pressure drop) will be lower and this will result in a more efficient lung deposition and thus, we expect that a lower tobramycin dose will result in the same plasma levels as

Dosages:
We will do an ascending dose response study with 4 different doses (30, 60, 120 and 240 mg). The doses will be fixed rising to react to possible adverse events and a possible end of the study for the participant.

Method of administration:
Tobramycin will be administered by inhalation using a DPI, called the Cyclops®. The DPI has been developed at the department of Pharmaceutical Technology and Biopharmacy of the University of Groningen.

Justification of the new dry powder inhalation device, the Cylops ®
Aminoglycosides (like tobramycin) are very hygroscopic. The delivery with re-usable capsule inhalers can cause particles to retain within the inhaler, which can absorb moisture from the air and stick firmly to the inhaler walls and this can influence following inhalations.
The Cyclops ® is designed as a disposable device with a high inhaler resistance to reduce the flow rate, and thus, the speed of entry of the tobramycin particles. This will significantly reduce the mouth and throat deposition, and increase the dose fraction available for deposition in the lungs.
To reach the highest deposition in the peripheral airways (94% of total lung volume is in the generations 17 to 23), the total dose has to enter the airways within the first 0.5 to 1 L of air inhaled. When the emission time is longer than two seconds at a flow rate of 30 L/min, more than 1 L of air has to be inhaled to take the entire dose. The emission time from the Cyclops ® is less than 2 seconds. Table 3 shows that the dispersion efficiency of the Cyclops ® .
The formulation used in the Cyclops ® consists of 100 per cent tobramycin. Because this inhaler was specifically designed for tobramycin, no excipients are needed to disperse the formulation. Moreover, because no excipients are needed, a single blister can contain a much higher fraction of the total drug dose. In conclusion, the specific design of the Cyclops ® with the high inhaler resistance (that prevents high mouth and throat deposition), and the patented air classifier technology, will result in an efficient dispersion, and thus probably in a more efficient therapy.

Preparation:
A solution of 50 mg/mL tobramycin in water for injections was spray dried with a Büchi Mini B-290 spray dryer. The spray dried product was transferred to a freeze-dryer (Christ, Epsilon 2-4) to remove residual water. Tobramycin is very hygroscopic and water can cause the formation of agglomerates, which can negatively affect powder dispersion properties.

Filling:
Aluminum blisters were filled with 30 mg of tobramycin and 10 mg of α-lactose monohydrate sweeper crystals (Ph.Eur. quality; DMV Fonterra Excipients, Goch, Germany) to obtain the final DP Tobramycin product.

Sealing:
The blisters are sealed thermally with standard sealing equipment. The seal is formed between a PVC coating on the aluminum blister and a heat lacquer (suitable for PVC) on the aluminum foil strip by heating to around 200 °C for a few seconds.

Labelling:
Labels will contain the following information: a) name, address and telephone number of the investigator; b) pharmaceutical dosage form, route of administration, quantity of dosage units; c) the batch number; d) a trial reference code; e) "for clinical trial use only" or similar wording; f) the storage conditions; g) expiry date.

Drug accountability
Drug accountability will be performed according GCP guidelines and performed by the investigator and the Hospital Pharmacy.

Main study parameter/endpoint
The following pharmacokinetic parameters will be calculated:  Adverse events

Pharmacokinetic analysis
Pharmacokinetic analysis is studied using software suitable for population pharmacokinetic modeling (i.e. MW/Pharm NonMem) 23 .

Actual inhaled dose
After inhalation the inhaler will be tested for the amount of drug remaining in the device.
The device will be rinsed with water and the remaining tobramycin will be determined using a modification of the 2,4,6-Trinitrobenzene Sulfonic Acid (TNBSA) assay. The actual inhaled dose of tobramycin will be calculated by subtracting the amount of drug remaining in the device after inhalation from the total dose weighed into the device.

Blood sampling and analysis:
Venous blood samples will be collected after inhalation of tobramycin from an infusion needle. Serum samples will be collected directly before and after 15 minutes, 30 minutes, will be centrifuged immediately and the serum will be stored at -20°C during the sampling day (UMCG, Haren). After shipment to the UMCG (Groningen) samples will be stored at -80C until analysis.

Gastro-intestinal absorption of tobramycin
From the literature it is expected that the gastro-intestinal absorption of tobramycin is far less than 1%. Therefore, we will not test the gastro-intestinal absorption in this group of subjects.

Serum creatinin analysis
Serum creatinin will be determined in the routine UMCG central lab.

Creatinin clearance
Will be calculated using the formula of Cockroft-Gault

Adverse effects
Spirometry will be used to test for adverse broncoconstrictive reactions due to the inhalation, immediately before, 15, 30 and 90 minutes after inhalation. It will be performed by standardized procedure according to the ERS guidelines and reference values. A 10% or more decrease in FEV1 (in mL) will be considered clinically relevant.
Other adverse effects will be asked by the investigator for spontaneous reporting. Special attention will be for cough and dyspnea.

X-ray of the chest
Before the start of the study a X-ray of the chest will be made of the patients who haven't had one the last year before screening for this study. This is both to find no existing abnormalities before start of the study and to have a reference in case an adverse event has happened.

Inhalation flow measurement
Prior to inhalation the patients will receive inhalation instructions and the opportunity to practice the inhalation manoeuvre. This will be done by using an empty inhaler connected to an electronic inspiratory flow measurement device (University of Groningen). With this device inhalation flow curves will be recorded and as soon as a series of consistent maneuvers is obtained, the Cyclops® containing tobramycin is given to the patient for administration of the drug. During the inhalation of tobramycin the inspiratory flow will be measured as well.

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. The investigator can decide to withdraw a subject from the study for urgent medical reasons.

Specific criteria for withdrawal
Special criteria for withdrawal include an allergic or anaphylactic reaction to tobramycin, and drop in FEV 1 > 10%, cough or dyspnoea in direct relation to the inhalation given the rising dose administration.

Replacement of individual subjects after withdrawal
If a subject withdraws him or herself, he or she will be replaced in order to reach the number of subjects needed for this study.

Follow-up of subjects withdrawn from treatment
Follow-up will be done by their chest physician or their general practitioner

Premature termination of the study
The criteria for terminating the study prematurely are a high percentage of adverse events like allergic reactions, and a high number of withdrawals for whatever reason.

Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspension would jeopardise the subjects' health. The investigator will take care that all subjects are kept informed.

Adverse events (AEs)
Adverse events are defined as any undesirable experience occurring to a subject during the study, whether or not considered related to [the investigational product / the experimental interventiont]. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.

Serious adverse events (SAEs)
A serious adverse event is any untoward medical occurrence or effect that at any dose: results in death; is life threatening (at the time of the event); requires hospitalisation or prolongation of existing inpatients' hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; -Any other important medical event that may not result in death, be life threatening, or require hospitalization, may be considered a serious adverse experience when, based upon appropriate medical judgement, the event may jeopardize the subject or may require an intervention to prevent one of the outcomes listed above.
The sponsor will report the SAEs through the web portal ToetsingOnline to the accredited METC that approved the protocol, within 15 days after the sponsor has first knowledge of the serious adverse reactions.
SAEs that result in death or are life threatening should be reported expedited. The expedited reporting will occur not later than 7 days after the responsible investigator has first knowledge of the adverse reaction. This is for a preliminary report with another 8 days for completion of the report.

Suspected unexpected serious adverse reactions (SUSARs)
Adverse reactions are all untoward and unintended responses to an investigational product related to any dose administered.
Unexpected adverse reactions are SUSARs if the following three conditions are met: 1. the event must be serious (see chapter 9.2.2); 2. there must be a certain degree of probability that the event is a harmful and an undesirable reaction to the medicinal product under investigation, regardless of the administered dose; 3. the adverse reaction must be unexpected, that is to say, the nature and severity of the adverse reaction are not in agreement with the product information as recorded in: -Summary of Product Characteristics (SPC) for an authorised medicinal product; -Investigator's Brochure for an unauthorised medicinal product.
The sponsor will report expedited the following SUSARs through the web portal The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as notification to the competent authority.
The sponsor will report expedited all SUSARs to the competent authorities in other Member States, according to the requirements of the Member States. The expedited reporting will occur not later than 15 days after the sponsor has first knowledge of the adverse reactions. For fatal or life threatening cases the term will be maximal 7 days for a preliminary report with another 8 days for completion of the report.

Annual safety report
As we expect to end this study within one year, a safety report will be submitted after the study is ended to the accredited METC, competent authority, Medicine Evaluation Board and competent authorities of the concerned Member States.
This safety report consists of:  a list of all suspected (unexpected or expected) serious adverse reactions, along with an aggregated summary table of all reported serious adverse reactions, ordered by organ system, per study;  a report concerning the safety of the subjects, consisting of a complete safety analysis and an evaluation of the balance between the efficacy and the harmfulness of the medicine under investigation.

Follow-up of adverse events
All AEs will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.
SAEs need to be reported till end of study within the Netherlands, as defined in the protocol

Data Safety Monitoring Board (DSMB) / Safety Committee
Will not be established for this very short time, 8 subject study of a new device but with a well known drug.

Descriptive statistics
Descriptive statistics will be used for calculation (geometric) mean and (interquartile) range of observed age, weight, length, dose, clinical parameters, (unbound) AUC 0-24h , and Cmax/MIC ratio, (unbound) AUC 0-24h /MIC, T max , C max , T 1/2 will be calculated with a 95% confidence interval. The other end-point will be proportions of subjects reporting adverse effects on all treatment days and / or percentage of drop of FEV1 of more than 10%.

Inferential statistics
Univariate analysis will be used to test if co-variables, including sex, ethnicity, comorbidity, medical history, dose and duration of (TB) and co medication have an effect on PK parameters like T max , C max and the (unbound) AUC 0-24h of tobramycin. Differences in clinical parameters between baseline and end of study will be calculated. When not normally distributed, non-parametric tests were used, i.e. Mann-Whitney U test and Wilcoxon rank sum test for ordinal data and Chi square tests will be used for nominal data. Correlation coefficients (Spearman's or Pearson's depending on distribution) will be used to test

Multivariate analysis
Multivariate analyses will be used to test relations between AUC 0-24h and the tobramycin dose corrected for bodyweight and potential other confounders.

Regulation statement
This study will be conducted according to the principles of the Declaration of Helsinki (version 9, October 2008, www.wma.net) and in accordance with the Medical Research Involving Human Subjects Act (WMO).

Recruitment and consent
Participants will be recruited from the outpatient clinic of pulmonology. First, patients will be contacted by their physician. If patients are willing to participate, they are referred by their treating physician to the investigator. Their treating physician gives the intended participant the subject information brochure of this study. On day 1 of the contact between patient and investigator (see flow diagram page 15), in-and exclusion criteria will be checked, and the -study participants will be informed about the nature and goals of the study, as well as the advantages and disadvantages of study participation. Questions will be answered and sufficient time (2 weeks) will be offered. If participants so wish they will be offered the opportunity to see an independent physician. Both the participant and the investigator will sign the informed consent if the patient is willing to participate.
For the informed consent and the subject information letter see attachments.

Benefits and risks assessment, group relatedness
No direct benefits to participants are expected. The risks are deemed relatively minor; there could be transient cough or dyspnea reactions immediately following inhalation.

Compensation for injury
The UMCG has a liability insurance that is in accordance with article 7, subsection 6 of the WMO.
The UMCG (also) has an insurance which is in accordance with the legal requirements in The insurance applies to the damage that becomes apparent during the study or within 4 years after the end of the study.

Incentives
Subjects whom participating the study after inclusion will receive 100 euro in gift

Handling and storage of data and documents
Handling of the personal data in this observational study will comply with the Dutch Personal Data Protection Act. Data will be handled confidentially and anonymously. To trace the data of individual patients, the study will use a subject identification code list that is linked to the date of the participating patients. The code will not be based on the patient initials and birth date, but on number of enrolment into the study. At the end of the study, all data will be kept for of 20 years

Monitoring and Quality Assurance
Not applicable

Amendments
A 'substantial amendment' is defined as an amendment to the terms of the METC application, or to the protocol or any other supporting documentation, that is likely to affect to a significant degree: the safety or physical or mental integrity of the subjects of the trial; the scientific value of the trial; the conduct or management of the trial; or the quality or safety of any intervention used in the trial.
All substantial amendments will be notified to the METC and to the competent authority.
Non-substantial amendments will not be notified to the accredited METC and the competent authority, but will be recorded and filed by the sponsor.

Annual progress report
As we expect to end this study within one year, the sponsor/investigator will submit a final report of the trial to the accredited METC after terminating the study. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments.

End of study report
The sponsor will notify the accredited METC and the competent authority of the end of the study within a period of 90 days. The end of the study is defined as the last patient's last visit.
In case the study is ended prematurely, the sponsor will notify the accredited METC and the competent authority within 15 days, including the reasons for the premature termination.
Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC and the Competent Authority.

Public disclosure and publication policy
All investigators and sponsor are part of the UMCG and/or Rijksuniversiteit Groningen.
They have agreed between them that results will be submitted for publication with joint authorship. The trial will be added in a public registry at www.Clinicaltrials.gov.

Potential issues of concern
a. Level of knowledge about mechanism of action Tobramycin is an antibiotic drug from the pharmacotherapeutic group of aminoglycoside antibacterials which are useful in infections caused by aerobic Gram-negative bacteria, including P. aeruginosa. It is derived from the aminoglycoside antibiotic complex nebramycin produced by a strain of Streptomyces tenebrarius.
The exact mechanism of action of aminoglycosides is not known. Binding to the bacterial 30S (also 50S) ribosomal subunit, inhibiting the translocation of the peptidyl-tRNA from the A-site to the P-site, as well as misreading of mRNA have been reported, which leaves the bacterium unable to synthesize proteins that are vital to its growth 24  c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human cell material?
No, the main study parameters/endpoints are AUC0-12 (area under the curve from 0 -12 h), Cmax (maximum plasma concentration), Tmax (time to maximum plasma concentration), Ka (absorption rate constant), T1/2 el (terminal elimination half-life), CL/F (clearance following pulmonary administration (F= bioavailability)). Furtermore, local tolerability of DP tobramycin will be determined by lung function measurement and questioning for adverse events. These data can only be obtained by a study in patients.  The patients treated with tobramycin had a significant reduction in sputum P. aeruginosa density at all time points of the study. The placebo group had a negligible change in sputum density at all time points. Unfortunately, the incidence of dyspnoea, chest pain and wheezing was significantly greater in the tobramycin group 1 .
f. Pharmacokinetic considerations DP tobramycin has not been evaluated in clinical studies yet. The clinical pharmacokinetics and safety profile of nebulised tobramycin and parenterally administered tobramycin is knowledge which is available in the public domain. In addition, the TOBI ® Podhaler ® with a dry powder tobramycin formulation is already market approved. The pharmacokinetics and local tolerability of DP tobramycin are the objectives of this presented clinical study.

g. Study population
Patients with non-CF bronchiectasis, with recurrent bacterial exacerbations (i.e., ≥2 in previous year)with or without Pseudomonas aeruginosa isolated from sputum.

h. Interaction with other products
The risk for nephrotoxicity is particularly increased by concurrent therapy with cyclosporin, cisplatin, and amfotericin B. This risk may also be increased when tobramycin is combined with certain cephalosporins, polymyxins, vancomycin and NSAIDs. Use of these products should be ceased if possible (NSAIDs), otherwise the patient cannot participate in the study.
i. Predictability of effect The pharmacokinetic data gained can be compared with data from literature. Tobramycin has been used for decades to treat bacterial infections.
The efficacy of DP tobramycin will not be investigated during this study, only pharmacokinetic data will be gathered to be able to estimate the systemic toxicity of DP tobramycin.
j. Can effects be managed?
The study is designed in such a way that the risks are as low as possible (dose response).
During this study DP Tobramycin will only be administered in a hospital setting, so in case of an emergency adequate action can be taken (e.g. salbutamol for bronchoconstriction).

Synthesis
The study design is chosen as such to minimize the chance on the occurrence of an adverse event. The starting dose is much lower than the dose given with TOBI Podhaler®, because we expect that the fraction of the dose deposited in the lungs is higher for our device than for the TOBI Podhaler®. The doses will be administered in a rising order to be able to react to possible adverse events and to possibly end the study prematurely for the participant if necessary. During this study DP Tobramycin will only be administered in a hospital setting, so in case of an emergency adequate action can be taken (e.g. salbutamol for bronchoconstriction).
DP Tobramycin contains only tobramycin and lactose, both compounds that are approved for pulmonary administration. Furthermore, tobramycin has been used for long-term therapy for decades already. The long term risks for tobramycin, predominantly nephrotoxicity and ototoxicity, are due to prolonged exposure to and cumulation of the compound in these organs. In this study, a patient receives four individual doses in four weeks, and no chronic therapy. To reduce the risk of interaction with other drugs, we will screen for cyclosporin, cisplatin, amfotericin B, cephalosporins, polymyxins, vancomycin and NSAIDs. Use of these products should be ceased if possible (NSAIDs), otherwise the patient cannot participate in the study. Therefore, we conclude that the long term risks are negligible.