Effect of intrapulmonary administration of recombinant human activated protein C on local coagulation and inflammation after bronchial instillation of lipopolysaccharide in humans

Recombinant human Activated Protein C (rhAPC; drotrecogin alfa (activated)) has
been shown to reduce the mortality of patients with severe sepsis. The
biological effects of APC are pleiotropic, and can be roughly divided in
anticoagulant and cytoprotective effects. Lung infection and inflammation are
associated with reduced bronchoalveolar levels of endogenous APC. Recent
evidence derived from animal studies indicates that local administration of
rhAPC into the lungs exerts anti-inflammatory and anticoagulant effects in the
pulmonary compartment. In this study we propose to study the potential of
locally administered APC, within a lung subsegment, to inhibit
lipopolysaccharide (LPS) induced lung inflammation and coagulation, thereby
avoiding systemic APC effects, in humans. Inhaled APC could be a new
therapeutic approach in inflammatory lung diseases such as asthma and acute
lung injury/acute respiratory distress syndrome, disorders in which systemic
effects of APC are not required and/or desired.

The primary objective is to determine the effect of locally administered rhAPC
on LPS-induced lung inflammation and coagulation. By using measurements on
cells harvested from bronchoalveolar lavage (BAL)-fluid and in BAL-fluid
supernatants, we will seek to obtain insight into neutrophil responses, the
response of alveolar macrophages, activation of the cytokine network,
activation of the chemokine network and activation of coagulation and
fibrinolysis.

First a dose-escalation study will be performed (N=4-16): healthy volunteers
will be challenged with LPS (4 ng/kg) into a subsegment of both lungs; in one
lung segment LPS will be combined with rhAPC, whereas in the contralateral lung
segment LPS will be combined with normal saline. Six hours later a bilateral
bronchoalveolar lavage (BAL) will be done in order to obtain BAL-fluid and
cells. Since rhAPC has not been administered into the lungs of humans before we
will conduct a dose-escalation study with rhAPC administered in a lung
subsegment by bronchoscope (N = 4 per dose, 5-fold escalation after each dose).

Thereafter a follow-up study will be performed (N=36): This will be done in 24
subjects who will be challenged with LPS in one lung subsegment and with normal
saline in a contralateral lung subsegment; in 12 of these subjects LPS will be
combined with rhAPC (dose determined in the dose-escalation study), in the
other 12 subjects LPS will be combined with saline. In addition, 12 subjects
will receive saline in both lung subsegments (not LPS), combined on one side
with either rhAPC (N=6) or saline (N=6).

Local intrabronchial administration of rhAPC after challenge with
intrabronchial LPS.

Bronchoscopy, segmental instillation and BAL:
The protocol for segmental instillation by bronchoscopy followed by a second
bronchoscopy has been described extensively in the international scientific
literature. This protocol has been mainly used to study local effects of
purified allergens, and more recently also to study local inflammation on
purified bacterial antigens, such as LPS (references 12, 21, 22). Both
bronchoscopies will be done by experienced pulmonologsts. The first
bronchoscopy (for segmental instillation) will take less than one minute. The
second (for bronchoalveolar lavage) takes about five minutes. The most
important discomfort experienced during a bronchoscopy is a dry cough and pain
in the nose on introduction of the scope. This can be prevented by local
anaesthetics of the mucosa with lidocaine. In our two recent studies in healthy
subjects, volunteers shortly experienced the complaints described above and
recovered quickly and completely within minutes. Hence, in our own experience
the procedure of both the bronchoscopy and BAL is tolerated very well by
healthy non-smoking subjects.

Signs and symptoms induced by LPS:
LPS at the dose given in this study has been administered into a lung segment
by bronchoscope previously in other centers (references 12, 21) and in the
Academic Medical Center (reference 22). This procedure did not induce clinicals
signs or symptoms besides a modest rise in body temperature not exceeding 37.3
degrees Celcius. However, this LPS-dosage did induce detectable inflammatory
and procoagulant reactions in the instilled lung subsegment, especially influx
of neutophiles, cytokine release, coagulation activation and inhibitiion of
fibrinolysis (references 12, 21, 22). This study will be the first to
administer LPS in two contralateral lung subsegments of the same individual.
Considering the total absence of clinical findings after LPS administration in
a single lung subsegment and considering that only a subsegment of each lung is
challenged, significant clinical symptoms are not expected.

Intrapulmonary administration of rhAPC
RhAPC has not been administered directly in the human lung before. Animal
studies, discussed above (see research protocol pages 3 and 4) have revealed
that APC exerts anticoagulant and anti-inflammatory effects in the
bronchoalveolar space upon intrapulmonary delivery. If rhAPC is to be used for
direct intrapulmonary delivery in humans in the future, it is likely that this
compound will be administered by inhalation of nebulized product. In the
current study we seek to obtain proof of concept that direct administration of
rhAPC into the lung inhibits coagulation and inflammation in the human lung
after challenge with LPS. We chose not to conduct studies using inhalation of
nebulized APC since this would expose both (entire) lungs of patients to a
compound that has thus far not been used for nebulisation or inhalation in
humans. By administering APC via a bronchoscope only a subsegment of a lung
will be exposed. The study will start with a dose-escalation study, using an
initial APC based on our previous study in which the same drug was infused
intravenously in healthy humans (references 12 and 13). The risks regarding
local administration of recombinant human APC are thereby minimized.