Nasal LPS Challenge in Healthy Volunteers

STUDY DESIGN AND METHODOLOGY This is a single-centre study which will be carried out at the Imperial Clinical Respiratory Research Unit (ICRRU) at St Mary's Hospital in London Paddington.

RECRUITMENT The study will recruit a total of 15 healthy participants.

STUDY VISITS The study will involve a total of 11 outpatients visits. The screening visit is the initial visit where the study is discussed with the participant and written informed consent is obtained by the PI or his designee. This is done prior to performing any study related procedures. The participant will have had the study information sheet for at least 24 hours prior to the visit.

The visit will include the collection of demographic data, height and weight, physical examination by a doctor, medical history, vital signs, body temperature, 12 lead ECG, laboratory assessments (safety bloods) urine drugs of abuse test and pregnancy tests for females. The participant will also be asked to indicate the presence of any nasal symptoms by completing a modified total nasal symptoms score sheet and perform a peak nasal inspiratory flow (PNIF) test.

If eligible, participants will be asked to return for further study visits. Participants will be given 4 different doses of LPS and a placebo in a randomised manner.

There are a total of 5 treatment periods. Each treatment period consists of a 11h visit the first day and a 20 minute visit the following day. There is a total of 12-28 days interval between each treatment period visit.

During the treatment period visits, the study participants will undergo the following study procedures:

• Nasal lavage to both nostrils and discard with 5ml of 0.9% saline in order to clean the nose prior to nasal LPS challenge.
• Nasal challenge with LPS (placebo, 1,10, 30 or 100µg per nostril)
• Nasosorption (right nostril) at -30m, +30m, +1h, then hourly to 10h and 24h
• Nasal lavage (left nostril) at -30m, +2h, +4h, +8h, +24h
• Nasal scrape (left nostril) at -30m, +3h, +6h, +24h
• Modified total nasal symptom score (TNSS) at -30m, +30m, +1h, then hourly to 10h and 24h
• Peak nasal inspiratory flow (PNIF) rate at -30m, +2h, +4h, +8h, 24h
• Systemic adverse events
• Temperature (oral) at -30m, +2h, +4h, +8h
• Blood pressure and heart rate at -30m
• Females of child bearing potential will have a pregnancy test performed.

NASAL PROCEDURES IN THE STUDY

• Nasal washing (nasal lavage): Nasal washings in the nose are carried out by passing a small volume of salt water (saline) fluid into the nose. A nasal adapter shaped like an olive is placed inside the nostril. The participant is then asked to sit forward and the salty water solution (saline) is used to flush in and out of the nose over a one minute period. This procedure is carried out on both nostrils to clean up the nose on the initial study visit and then repeated only on the left nostril on each treatment visit.
• Nasal lining fluid absorption (nasosorption or SAM): A small strip of absorbent material, that looks and feels like soft tissue paper, will be used to absorb moisture from the inside surface of the nostril. The special absorptive paper will be placed inside the nostril and left for a period of 2 minutes to absorb the nasal lining fluid before being removed, gently sucking up fluid like blotting paper. Putting the paper into the nose can tickle, and cause your eyes to water a little. However, the nasosorption does not hurt and the investigators method has proved to be well tolerated in babies, children and adults. The paper absorbs the nasal fluid and many substances produced by the nasal cells can then be extracted from the paper and measured in the laboratory.
• Nasal scrape (nasal curettage or Rhinoprobe): A small sterile disposable plastic probe will be inserted into the nose and will be gently pressed against the inside surface lining of the nostril. The 3 inch long probe has a tiny scoop on the end, which can barely be see. 24 tiny samples from each nostril (a pinhead, 2mm, of tissue) will be taken from a part of the nose that has a reduced nerve supply to limit any discomfort. Taking this sample does not cause bleeding, but may cause some mild discomfort, and may make the eyes water slightly. The probe will collect nasal cells from the surface of the nostril which will then be sent for analysis. This method has been performed on many adults, children, and babies, and has been very well tolerated.

STATISTICAL ANALYSIS Each of the measures will be summarised descriptively by dose and time using appropriate summary statistics. If appropriate, response variables will be transformed (e.g. log-transformed). Gene expression data will be normalised by house-keeper gene. The TNSS will be calculated by summing the scores of nasal blockage, nasal discharge/rhinorrhoea, nasal burning/itching/pain sensation, and sneezing at each timepoint. Weighted means of symptom scores will be calculated over the time interval 0 to 10h.

TNSS, nasal sniff pressures, nasosorption measures and gene expression data from the nasal scrapes will each be plotted against time, with a separate plot for each subject and measure, and with LPS dose and placebo represented by separate lines on the plots. Summary statistics (e.g. mean +/- 95% confidence intervals or median and interquartile range) for nasal lavage and gene expression data will be plotted by dose against time.

Correlations will be explored using scatter plots for the following measures:

• Weighted means TNSS vs. maximum TNF-α
• Weighted means TNSS vs. maximum IL-8
• Weighted means TNSS vs. maximum s-ICAM-1
• Maximum ICAM-1 expression vs. maximum TNF-α

Reproducibility of the nasal LPS challenge for ICAM-1 expression and TNF-α will be explored using the following subsets of data:

• Predose data for each period to explore intra-subject variability. Parameter vs. period number will be plotted with a separate line for each subject to explore whether there is a period effect. Box and whisker plots will also be used to summarise the parameter by period.
• Placebo period data - Parameter vs time will be plotted with a separate line for each subject to explore whether there is a time-course effect. Box and whisker plots will also be used to summarise the parameter over time.

Box and whisker plots for ICAM-1 expression and TNF-α and dose and time will be presented to investigate whether these measures return to baseline after 24h.

Additional exploratory analyses may be performed to further characterize the expression profile of ICAM following nasal LPS challenge.

Inclusion criteria

• Males and females aged 18 to 60 years
• Current non-smokers for last 6 months (<5 cigs per week), with a smoking history of <5 pack years
• BMI 20-30 kg/m2

Exclusion criteria

• History of allergy
• Upper airway infection in 2 weeks before screening
• Lower respiratory tract infection in past 3 months
• Treatment with local or systemic corticosteroids during previous 2 months
• Signs or symptoms of chronic rhinitis, hypertrophy of turbinates, major septum deviation, nasal polyposis or recurrent sinusitis
• Previous nasal or sinus surgery
• Clinically significant cardiovascular, hepatic, GIT, renal, endocrine, infective, haematological, neurological, dermatological, neoplastic conditions, gastro-oesophageal reflux, depression, TB
• Participation in a therapeutic drug trial in the prior 30 days.
• Medical therapy other than that permitted for contraception.
• Positive pregnancy test
• Inability or unwillingness to use contraception if the patient is a female of child-bearing age.
• History of drug abuse or urine test showing evidence of recreational drug abuse