Borage and Echium Seed Oils for Asthma

To evaluate the mechanisms by which borage seed oil and echium seed oil block mediator
generation in asthma, we performed a cross-over trial design to reduce the numbers of
patients needed for the study. Each treatment period was six weeks with a six-week washout
period. For simplicity all power calculations are made assuming a single factor or predictor
variable and a single result or outcome variable. We focused on changes in mediator
generation by effector cells. Calculations below suggest that we have resolution to pick up
clinically meaningful effects of potentially important explanatory variables with acceptable
or high power.

1. Study Design The goals were to examine the mechanisms that underlie the effects of borage
seed oil and echium seed oil on proinflammatory mediator production by effector leukocytes.

In the treatment arm, subjects consumed 4.0 g/day borage seed oil and 7.0 g/day echium seed
oil (containing totals of ~1.6 g/day of GLA and ~0.9 g/day of SDA). The oils were packaged in
capsules each of which contained 1 gram of oil. Therefore subjects will ingest 10 capsules a
day (3 in the morning, 3 in the afternoon, and 4 in the evening) with meals. In the placebo
arm, subjects took matching capsules containing corn oil; three 1 gram capsules in the
morning and afternoon, and four in the evening, with meals.

The study began with a screening visit. All subjects had physician-diagnosed asthma. All
performed a baseline set of lung function measurements. They entered a 2-week run-in period
during which they kept a diary of peak flows, asthma symptoms, and beta agonist use. Subjects
were instructed in peak flow technique and asked to perform three maneuvers each time. The
maneuver was considered technically sufficient if the variation in the values obtained on the
three attempts are with 10% of each other, and the highest value was recorded in a diary.
Subjects whose diaries were > 80% complete entered the study and were randomly assigned to
active treatment or placebo. Spirometry was repeated. Blood was taken for fatty acid
composition of plasma and leukocytes, LT generation, and DNA for genotyping at the LTC4S
locus. Subjects were provided with peak flow diaries, instructed in the appropriate
performance or peak flow maneuvers, and asked to record morning and evening peak flow each
day (reflecting the best of three efforts each time). After three weeks of treatment, diary
cards will be collected and spirometry repeated. After six weeks of treatment, blood was
drawn for safety monitoring, for measurements of fatty acids in plasma and leukocytes, and
for the functional analyses. Spirometry was repeated. Subjects then entered a six-week
washout period. Two weeks before the end of this period they again started to keep diary
cards. At the conclusion of the 6-week washout period they entered the cross-over treatment
phase (six weeks) which followed the same protocol as the first treatment period with
assessment of fatty acid analyses, cellular function studies, safety monitoring, and
spirometry. Pregnancy testing was performed before and after each treatment period. Dairy
cards (with peak flow monitoring results) will be collected at each visit (3 week intervals
during the placebo and active treatment arms of the study).

3. Statistical Analyses

a. Power Calculations - For simplicity all power calculations are made assuming a single
factor or predictor variable and a single result or outcome variable. While a number of
correlated outcomes will be investigated, we will focus on changes in mediator generation and
levels of enzyme expression. Calculations below suggest that we have resolution to pick up
clinically meaningful effects of potentially important explanatory variables with acceptable
or high power.

i. Biochemical Endpoints - Power is based on paired t-tests comparing continuous measures of
response between treatment and placebo. We assume 40 subjects will be available after
allowing for a drop out rate of 20% from our original sample of 50 subjects. For the endpoint
of leukotriene production by leukocytes, the power calculations suggested that we will have
80% power to detect a true within-subject difference of 0.8 of the within-subject SD between
treatment and placebo with an α value of 0/05. On the basis of our previous studies, we
assume a within-subject coefficient of variation (CV) of 13% for LTC4 generation. This will
translate to an approximate detectable difference from 10.5% in these parameters.Using a
paired two-sided t-test and prior mean(standard deviation) estimates for the placebo of
.001464 ( .002268) and for the borage oil of .000289( .000709) , we would need 40subjects for
80-90% power at a 0.05 level of significance to detect a change in LTC4S transcript, protein,
and enzymatic activity of 50% between placebo and borage arms.

b. Analysis Plan - Initially, we will present descriptive statistics (mean, standard
deviation, range) for all endpoints to treatment and placebo groups. We will also present
graphical plots of all measures across the study and for each group within each period. For
the 2x2 crossover study, we will compare the treatment and control on each endpoint
separately by employing a generalized mixed model approach with the predictors:
group(sequence), patient nested within group as a random effect, period(dichotomous),
treatment(dichotomous), and seasonality(dichotomous), with unstructured covariance. We will
utilize mixed model diagnostics such as residual plots to assess goodness of fit.

Inclusion Criteria:

1. Male or female 18 years to 65 years of age

2. Stable physician diagnosed asthma with FEV1 of greater than 50% of predicted but less
than 90% of predicted or less than 90% of known best and an asthma control
questionnaire(ACQ) < 1.5.

Exclusion Criteria:

1. Pregnant or nursing

2. Smoking history of > 10 pack years or active smoking within the past year.

3. Due to possible effects on LT biosynthesis, use of the following asthma treatments
within the preceding month will be exclusion criteria:

- LT modifying drugs (zileuton, montelukast, zafirlukast)

- theophylline

- oral steroids

- dietary supplements with fatty acids or other products that may interfere with LT
generation.

4. Treatment within the previous three months with omalizumab (monoclonal antibody
directed against IgE)

5. Subjects will not be permitted to take non-steroidal anti-inflammatory drugs in the
week prior to any measurements of ex vivo LT generation because of their effects on LT
biosynthesis via inhibition of prostaglandin generation.

6. A history of aspirin-sensitive asthma will be an exclusion criterion as the effects of
GLA/SDA on prostanoid biosynthesis have not been adequately elucidated.

7. Significant abnormalities in CBC, differential white cell count, renal function, and
liver function, or urinalysis.

8. No subjects will have any serious co-morbid medical condition.