Therapeutic uses of BPI protein products for human meningococcemia

Methods and materials for the treatment of human meningococcemia are provided in which therapeutically effective amounts of BPI protein products are administered.

BACKGROUND OF THE INVENTION 
The present invention relates generally to methods and materials for 
treating humans suffering from meningococcemia by administration of 
bactericidal/permeability-increasing (BPI) protein products. 
Meningococcemia is an infectious disease caused by Neisseria meningitidis 
(also known as meningococcus) in which the bacteria and their products are 
found in the systemic circulation. Its clinical course varies from a 
relatively mild process to a severe, fulminant infection of sudden onset 
and extremely rapid progression, with the time from first fever until 
death spanning as little as 12 hours. The latter, dramatic form of the 
disease occurs in about 10% of patients infected with N. meningitdis. 
Patients may present with normal mental status and symptoms only of fever 
and petechiae, but may rapidly experience hemodynamic collapse, loss of 
the airway, and coma, along with severe coagulopathy, intravascular 
thrombosis, and organ failure. Alternatively, in late stages of the 
disease, patients may be unconscious and unresponsive at the time of 
presentation. 
The mortality rate for acute meningococcal disease has not changed 
significantly over the last few decades despite technological advances in 
antibiotics and intensive care facilities. One retrospective study found 
that the mortality rate from meningococcal infection had not changed 
significantly over 30 years, even after adjusting for disease severity 
[Havens et al., Pediatr. Infect. Dis. J., 8:8-11 (1989)]. Another 
prospective study of meningococcal infections [Powars et al., Clin. 
Infect. Dis., 17:254-261 (1993)] in the years 1986 through 1991 reported 
that 113 patients with bacteriologically proven N. meningitdis infection 
were observed, of whom 15 (13%) died. This mortality rate of 13% had not 
changed appreciably from the mortality rate of 16% reported five decades 
earlier in a Chilean epidemic. An "epidemic" is defined as an increased 
frequency of disease due to a single bacterial clone spread through a 
population. Although epidemics of meningococcemia are widespread in the 
developing world, no national epidemic has occurred in the United States 
since the 1940's. However, a significant increase in the endemic 
occurrence of meningococcemia, along with localized epidemics has occurred 
in the mid-1990s. The disease continues to be seasonal, with peak 
incidence in the late winter and early spring. Between 60% and 90% of all 
cases occur in children, with the peak incidence in children under age 2. 
N. meningitidis is an encapsulated gram-negative coccus, typically 
occurring in pairs (diplococci), which is responsible for a spectrum of 
severe diseases, including meningococcemia. Meningococci are divided into 
nine serogroups on the basis of their capsular polysaccharides, with 
serogroups A, B, C, Y, and W135 accounting for the majority of clinical 
disease. These serogroups are further subdivided into antigenically 
distinct serotypes on the basis of expression of outer membrane proteins. 
Specific clones within each serogroup can be further delineated by protein 
electrophoretic patterns. The outer membrane of meningococci also contains 
a form of lipopolyaccharide (LPS), i.e., "lipooligosaccharide" (LOS), 
which is a common component of the outer membrane of gram-negative 
bacteria. 
The meningococcus is known to colonize the nasopharynx of 5-15% of 
individuals; however, only a small fraction of those colonized will 
experience invasive disease. The transition from colonization to invasive 
disease is multifactorial and incompletely understood. The presence of 
viral upper-respiratory infections, which also peak during the late winter 
and spring, may damage the nasopharyngeal epithelium and permit bacterial 
translocation across an altered barrier. In children under 2 years of age, 
inadequate development of antibodies directed against the meningococcal 
polysaccharide capsule is thought to account for the high attack rate in 
this population. 
The spectrum of disease caused by the meningococcus includes meningitis, 
arthritis, pericarditis, endocarditis, conjunctivitis, endophthalmitis, 
respiratory tract infections, abdominal and pelvic infections, urethritis, 
and a chronic bacteremic syndrome. The predominant clinical syndromes 
requiring pediatric intensive care unit (PICU) admission are meningitis 
and meningococcemia (with or without meningitis). The clinical 
presentation depends on the compartment of the body in which the infection 
and its inflammatory sequelae are primarily localized. 
In contrast to meningococcemia, meningitis is a disease in which the 
bacteria are localized to the meningeal compartment, with signs consistent 
with meningeal irritation. Clinically, meningococcal meningitis is 
dramatically different from meningococcemia, however, it may be 
indistinguishable from other forms of meningitis, and only differentiated 
by culture or immunologic assays. Systemic hemodynamic signs, severe 
coagulopathy and intravascular thrombosis are notably absent. If properly 
treated, mortality is rare and neurologic sequelae, including sensineural 
hearing loss, is uncommon. The approach to diagnosis and treatment of 
meningococcal meningitis is the same as with other forms of bacterial 
meningitis. 
If patients are examined early in the course of their disease when only 
petechiae and mild constitutional symptoms are evident, the diagnosis of 
meningococcemia may be complicated by the number of diseases which present 
with fever and petechiae in children, including, for example, infections 
by enterovirus, rotavirus, respiratory syncytial virus, Haemophilus 
influenzae, or Streptococcus pneumoniae; streptococcal pharyngitis; Rocky 
Mountain spotted fever, Henoch-Schoenlein purpura; or malignancy. However, 
since the outcome of meningococcal disease is highly dependent on rapid 
diagnosis and institution of antibiotics, the suspicion of meningococcemia 
must be aggressively pursued and treatment instituted, particularly since 
H. influenza meningitis has markedly decreased in the United States due to 
use of the vaccine against the bacteria. 
Like other gram negative infections, the pathogenesis of severe 
meningococcemia is initiated by the endotoxin on, associated with or 
released from the bacteria. This bacterial endotoxin activates the 
pro-inflammatory cytokine cascade. In severe meningococcemia, the levels 
of bacterial endotoxin detected in the circulation by the LAL assay have 
been documented to be as much as 50-100 fold greater than levels 
documented in other gram negative infections. The complement cascade is 
also activated by bacteria and their endotoxin in the systemic 
circulation, producing anaphylotoxins which may mediate early hypotension 
and capilary leak. 
In studies thus far, plasma levels of endotoxin [Brandtzaeg et al., J. 
Infec. Dis., 159:195-204 (1989)], TNF [Van Deuren et al., J. Infect. Dis., 
172:433-439 (1995)], IL-6 [Van Deuren et al., supra], and fibrinogen, as 
well as prothrombin time (PTT) [McManus et al., Critical Care Med., 
21:706-711 (1993)] in meningococcemia patients have been correlated with 
the severity and outcome of disease, although the correlation is 
imprecise. It has been suggested that combining ranked values for 
endotoxin, TNF, IL-1 and IL-6 can achieve a score that accurately reflects 
patient outcome [Bone, Critical Care Med., 22:S8-S11 (1994)]. 
Severe coagulopathy and intravascular thrombosis may be rapidly progressive 
and lead to ischemic injury of extremities and vital organs in 
meningococcemia patients. Respiratory failure, renal failure, adrenal 
failure and coma may develop. Petechiae and purpura may be extensive and 
become confluent, in which case the term "purpura fulminans" has been 
applied. In meningococcemic patients with severe disease, significant 
reductions in the coagulation inhibitors antithrombin III, activated 
protein C, and protein S have also been documented. These reductions may 
reflect a relative imbalance of anti-coagulant factors compared to 
pro-coagulants, but may also reflect the general consumption of all 
classes of factors. Quantitative deficiencies may also reflect 
hemodilution and capillary leak of proteins. 
Severe cardiac dysfunction is often present on admission, or may develop 
within the first 24 hours. Ejection fractions of 20% or less are freuent. 
Cardiac dysfunction may be secondary to a number of factors, including: 1) 
myocarditis, which is present to varying degrees in a majority of autopsy 
specimens; 2) myocardial depressant substances; 3) intravascular 
thrombosis and subsequent myocardial ischemia; 4) myocardial interstitial 
edema, resulting in a non-compliant ventricle; 5) hypoxic myocardial 
injury; and 6) metabolic abnormalities. 
Hypotension and circulatory insufficiency are multifactorial, with 
significant contributions from intravascular volume depletion, capillary 
leak, profound vasodilation (secondary to anaphylotoxins, nitric oxide, 
histamine, and other mediators), and depressed myocardial performance. 
Organ damage secondary to hypotension, intravascular thrombosis, and 
direct inflammatory damage may be evident at presentation. 
Fulminant disease may be associated with adrenal hemorrhage, adrenal 
cortical necrosis, and rapid demise (Waterhouse Friederickson Syndrome). 
Even extensive adrenal hemorrhages, however, do not necessarily denote 
adrenal insufficiency, since normal or even elevated systemic cortisol 
levels have been documented in such patients. In a minority of patients 
with rapidly progressive disease, adrenal hemorrhages are associated with 
serum cortisol levels which are normal or subnormal (in a setting where 
elevated levels are expected). Other metabolic derangements such as 
metabolic acidosis, hypoglycemia, hypocalcemia, and hypomagnesemia are 
also frequently present. 
Patients with severe disease are at highest risk of mortality. If they 
survive, they often experience severe morbidities, including extensive 
tissue and bone destruction that requires debridement and/or amputation 
followed by skin grafting procedures. In one study [Powars et al., supra], 
among the 28 patients with purpura fulminans, the hallmark of severe 
meningococcemia, 14 patients (50%) died. Of the 14 surviving patients who 
had purpura fulminans, 10 suffered soft tissue gangrene with deforming 
autoamputation. In another report [Genoff et al., Plastic Reconstructive 
Surg., 89:878-881 (1992)], six patients with meningococcemia and purpura 
fulminans were followed, of whom four patients required severe amputations 
(wrist or above for the upper limbs, or ankle or above for the lower 
limbs). Genoff et al. note that even after the life-threatening acute 
phase of the disease has passed, complications continue and require 
revisions to a higher level of amputation and multiple grafting 
procedures. Sheridan et al., Burns, 22:53-56 (1996), confirms that 
meningococcernia with purpura fulminans has a reported mortality rate of 
50%, with high rates of major amputations in survivors. In their 
experience, surviving patients are often left with full thickness wounds 
involving the skin, subcutaneous tissue and often underlying muscle and 
bone; half of the surviving patients require major amputations. 
Patients with meningococcal disease may also develop neurologic sequelae, 
including electroencephalogram (EEG) abnormalities, computerized 
tomography (CT) scan abnormalities, hearing impairment and 
neuropsychological testing deficits. In one study, 99 consecutive children 
and adult patients with acute, bacteriologically confirmed meningococcal 
disease were followed and tested for neurologic sequelae one year after 
their illness. [Naess et al., Acta Neuron. Scand., 89:139-142 (1994).] In 
the category of patients suffering from meningococcemia with hypotension 
and/or ecchymoses, but without signs of meningitis, neurologic sequelae 
were observed in 5 of the 12 patients. In the category of patients 
suffering from meningococcemia with hypotension and/or ecchymoses, and 
with signs of meningitis, neurologic sequelae were observed in 7 of 13 
patients. 
Clinical outcome can be reasonably predicted by scoring of risk factors 
originally identified in large cohorts of meningococcemia patients. In 
1966, Stiehm and Damrosh, J. Pediatrics, 68:457-467 (1966), reviewed 63 
cases of meningococcal infection and identified clinical features 
associated with poor outcome. Poor prognostic factors included: onset of 
petechiae within 12 hours prior to admission, absence of meningitis 
(cerebrospinal fluid (CSF) WBC&lt;20), shock (systolic blood pressure&lt;70), 
normal or low white blood count (WBC &lt;10,000), and normal or low 
erythrocyte sedimentation rate (&lt;10 mm/hr). The presence of 3 or more of 
these criteria was associated with poor outcome. Niklasson et al., Scand. 
J. Infect. Dis., 3:17-25 (1971), substantiated these risk factors in 1971, 
and added temperature&gt;40.degree. C. and thrombocytopenia to the list of 
poor prognostic signs. The specific predictive abilities of the Stiehm and 
Damrosh criteria and the Niklasson criteria have been challenged in a 
series from McManus [McManus et al., supra] in 1993.In this series, 
mortality was significantly less than predicted by earlier criteria and 
was more likely related to the presence or absence of coagulopathy. 
The most widely used meningococcal sepsis scoring system was published in 
1987 by Sinclair et al., Lancet, 2:38 (1987), and has become known as the 
Glasgow Meningococcal Septicemia Prognostic Score (Glasgow score). Its 
utility stems from its reliance on bedside clinical indicators, which 
facilitates triage in the field or during transport. Points are given on a 
rated scale for seven parameters as follows: (1) BP&lt;75 mm Hg systolic, 
age&lt;4 years or BP&lt;85 mm Hg systolic, age&gt;4 years (3 points); (2) 
skin/rectal temperature difference&gt;3.degree. C. (3 points); (3) modified 
coma scale&lt;8, or deterioration of 3 or more points in 1 hour (3 points); 
(4) deterioration in hour before scoring (2 points); (5) absence of 
meningism (2 points); (6) extending purpura or widespread ecchymoses (1 
point); and (7) base deficit (capillary or arterial)&gt;8 (1 point). The 
maximum Glasgow score is therefore 15 points. 
Since meningococcemia is frequently characterized by rapid and fuliniant 
deterioration, vigilant monitoring is mandated. The great majority of 
patients should be admitted directly to the intensive care unit, where 
invasive monitoring can be instituted, and supportive therapy provided. 
Specific additions to monitoring and laboratory evaluation may include 
obtaining samples from CSF, blood cultures, skin lesions and throat swabs. 
However, CSF should be obtained only if the patient's clinical condition 
is stable enough to tolerate the procedure. Blood cultures should be 
obtained, but are positive in only 50% of untreated patients. Bacteria can 
also be detected in up to 70% of cases by Gram stain and culture of 
aspirated (or biopsied) hemorrhagic skin lesions. Examination of skin 
lesions is especially important for cases in which antibiotics have been 
administered prior to obtaining blood cultures. Throat swabs, if carefully 
obtained and rapidly plated, may also yield meningococci and support a 
presumptive diagnosis of meningococcemia. Alternatively, CSF may be 
obtained for detection of meningococcal antigens. If an organism is 
obtained, it should be serotyped and forwarded to a reference laboratory 
for additional subtyping. Epidemic control through immunization can only 
occur if the specific organisms responsible for disease are identified. In 
the unusual circumstance in which blood cultures cannot be obtained, 
antibiotics should still be administered without delay; microbiologic 
investigation can be accomplished at a later time by alternate methods. 
Management of children with meningococcemia relies on intensive, aggressive 
monitoring and therapy. In particular, early protection of the airway, 
aggressive volume replacement, and appropriate institution of vasoactive 
agents, e.g., epinephrine, dopamine and dobutamine, are critical to 
restore tissue perfusion and oxygen delivery. A few specific issues in the 
treatment of meningococcemia, including treatment with antibiotics, 
steroids, fresh frozen plasma (FFP) replacement, heparin, and several new 
agents are briefly highlighted below. 
An ongoing debate continues concerning whether antibiotics should be 
administered as soon as the diagnosis is suspected or after a period of 
stabilization. Although not resolved by randomized trials, the 
preponderance of evidence suggests that antibiotics should be administered 
immediately, while other supportive therapies are being instituted. 
Speculations regarding a post-antibiotic release of bacterial endotoxin in 
meningococcemia have not been substantiated by human data. Serial 
quantitation of bacterial endotoxin levels in plasma samples from humans 
with meningococcemia have failed to demonstrate a post-antibiotic surge in 
plasma endotoxin levels. 
Initial therapy of suspected cases currently is typically recommended to be 
a third generation cephalosporin (e.g. Ceftriaxone) until other causes of 
severe infectious purpura with shock have been ruled out (H. influenza, S. 
pneumoniae, other gram negative bacteria). Therapy can then be switched to 
parenteral penicillin or ampicillin. 
To date, there are currently no randomized, placebo controlled data to 
support the routine use of corticosteroids in patients with 
meningococcemia. However, data have demonstrated that a minority of 
patients with severe disease and adrenal hemorrhage exhibit normal or 
subnormal levels of plasma cortisol (in a situation during which elevated 
levels are expected). Although the lack of data precludes an affirmative 
or negative recommendation, the physician should consider administering 
adrenal replacement steroids (hydrocortisone 1-2 mg/kg i.v.) in a clinical 
situation of rapidly progressive shock that is unresponsive to fluids and 
inotropes. 
There have also been to date no randomized, placebo controlled data to 
determine whether, or to what degree, biochemical coagulopathy should be 
treated with FFP. Although correction of biochemical abnormalities may 
appear logical, administration of FFP has been viewed by many as "fueling 
the fire" of coagulopathy. In a case-control trial of 336 patients in 
Norway, treatment with plasma or blood products (as opposed to albumin or 
plasma substitutes) was independently associated with poorer outcome. A 
surge in plasma endotoxin was also documented in a C6 deficient human 
following FFP administration during treatment for meningococcemia. These 
data suggest that administration of FFP may be harmful in some situations 
and therefore should be done carefully and only when there are compelling 
clinical indications. 
Although small retrospective reports advocate the use of heparin as a 
treatment for purpura fulminans, the preponderance of data (small 
randomized trials and large case-control studies) do not indicate a 
beneficial effect of heparin therapy. There is currently no evidence to 
support the routine use of heparin in the treatment of meningococcemia. A 
large scale, double-blind, placebo-controlled Phase III trial of a 
monoclonal anti-lipid A antibody (HA-1A) in meningococcemia has been 
conducted in Europe. No results have been published to date. 
In addition, a number of other biological agents are candidates for 
treatment of severe coagulopathy and intravascular thrombosis. These 
agents include: antithrombin III, protein C, and tissue factor pathway 
inhibitor. Anecdotal experiences with protein C and antithrombin m have 
already been published pending definitive trials. Other clinical 
interventions have been reported but have not been systematically tested, 
including: plasma and whole blood exchange, leukaplasmapheresis, 
continuous caudal blockade to relieve lower extremity ischemia, and 
topical application of nitroglycerin to vasodilate the peripheral vascular 
bed. 
BPI is a protein isolated from the granules of mammalian polymorphonuclear 
leukocytes PMNs or neutrophils), which are blood cells essential in the 
defense against invading microorganisms. Human BPI protein has been 
isolated from PMNs by acid extraction combined with either ion exchange 
chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli 
affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI 
obtained in such a manner is referred to herein as natural BPI and has 
been shown to have potent bactericidal activity against a broad spectrum 
of gram-negative bacteria. The molecular weight of human BPI is 
approximately 55,000 daltons (55 kD). The amino acid sequence of the 
entire human BPI protein and the nucleic acid sequence of DNA encoding the 
protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 
264:9505 (1989), incorporated herein by reference. The Gray et al. amino 
acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat. No. 5,198,541 
discloses recombinant genes encoding and methods for expression of BPI 
proteins, including BPI holoprotein and fragments of BPI. 
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for 
the high net positive charge; the C-terminal half of the molecule has a 
net charge of -3.[Elsbach and Weiss (1981), supra.] A proteolytic 
N-termninal fragment of BPI having a molecular weight of about 25 kD 
possesses essentially all the anti-bacterial efficacy of the 
naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 
262: 14891-14894 (1987)]. In contrast to the N-terninal portion, the 
C-terminal region of the isolated human BPI protein displays only slightly 
detectable anti-bacterial activity against gram-negative organisms. [Ooi 
et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of 
approximately 23 kD, referred to as "rBPI.sub.23, " has been produced by 
recombinant means and also retains anti-bacterial activity against 
gram-negative organisms. Gazzano-Santoro et al., Infect. Immun. 
60:4754-4761 (1992). 
The bactericidal effect of BPI has been reported to be highly specific to 
gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic 
Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven 
Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative 
bacteria is not yet completely elucidated, but it is believed that BPI 
must first bind to the surface of the bacteria through electrostatic and 
hydrophobic interactions between the cationic BPI protein and negatively 
charged sites on LPS. In susceptible gram-negative bacteria, BPI binding 
is thought to disrupt LPS structure, leading to activation of bacterial 
enzymes that degrade phospholipids and peptidoglycans, altering the 
permeability of the cell's outer membrane, and initiating events that 
ultimately lead to cell death. [Esbach and Weiss (1992), supra]. LPS has 
been referred to as "endotoxin" because of the potent inflammatory 
response that it stimulates, i.e., the release of mediators by host 
inflammatory cells which may ultimately result in irreversible endotoxic 
shock. BPI binds to lipid A, reported to be the most toxic and most 
biologically active component of LPS. 
BPI has never been used previously for the treatment of subjects infected 
with N. meningitidis, including subjects suffering from meningococcemia. 
In co-owned, co-pending U.S. application Ser. Nos. 08/378,228, filed Jan. 
24, 1995, 08/291,112, filed Aug. 16, 1994, and 08/188,221, filed Jan. 24, 
1994, incorporated herein by reference, the administration of BPI protein 
product to humans with endotoxin in circulation was described. [See also, 
von der Mohlen et al., J. Infect. Dis. 172:144-151 (1995); von der Mohlen 
et al., Blood 85:3437-3443 (1995); de Winter et al., J. Inflam. 45:193-206 
(1995)]. Thornton et al., FASEB J., 8(4):A137, 1994, report that BPI 
inhibited the release of TNF in vitro by human inflammatory cells in 
response to LOS derived from two Neisseria species, N. meningitidis and N. 
gonorrhea; and the report in International Application Publication No. WO 
94/25476 published Nov. 10, 1994, of methods of treating endotoxin-related 
disorders, including Gram-negative meningitis. 
In spite of treatment with antibiotics and state-of-the-art medical 
intensive care therapy, the mortality and morbidities associated with 
human meningococcemia remain significant and unresolved by current 
therapies. New therapeutic methods are needed that could reduce or 
ameliorate the adverse events and improve the clinical outcome of human 
meningococcemia, including, for example, reducing mortality, amputations, 
grating procedures, permanent neurologic impairment and improving 
pediatric outcome scores. 
SUMMARY OF TIHE INVENTION 
The present invention provides novel methods for treatment of humans with 
meningococcemia involving the administration of BPI protein products to 
provide clinically verifiable alleviation of the adverse effects of, or 
complications associated with, this human disease, including mortality and 
morbidities. 
According to the invention, BPI protein products such as rBPI.sub.21 are 
administered to humans suffering from meningococcemia in amounts 
sufficient to prevent mortality and/or to reduce the number or severity of 
morbidities, including but not limited to amputations, grafting procedures 
and/or permanent neurologic impairment. 
Numerous additional aspects and advantages of the invention will become 
apparent to those skilled in the art upon consideration of the following 
detailed description of the invention which describes presently preferred 
embodiments thereof.

DETAILED DESCRIPTION 
Human meningococcemia is an increasingly prevalent, life-threatening, 
debilitating disease for which conventional antibiotics and intensive care 
are inadequate. In particular, significant mortality and severe 
morbidities have remained in spite of state-of-the-art medical intensive 
care. It has now been unexpectedly found that the administration of BPI 
protein products to humans with meningococcemia has effectively decreased 
mortality and reduced the number and severity of morbidities, including 
amputations, debridement of dead tissue followed by extensive grafting 
procedures, and/or permanent neurologic impairment resulting in 
significant and long-term impairment of neurologic function (e.g., 
cerebrovascular accidents, cerebral atrophy, or seizures requiring 
medication). These unexpected effects on the mortality and morbidities 
associated with and resulting from meningococcemia demonstrate that BPI 
protein products have effectively interfered with or blocked a number of 
the multiple poorly-understood pathophysiologic processes that have led to 
poor outcomes in this human disease. 
BPI protein products are expected to provide other beneficial effects for 
meningococcemia patients, such as reduced number of episodes of 
hypotension or cardiac arrhythmia or arrest, reduced length of time on 
ventilatory support and inotropic (vasoactive) therapy, reduced duration 
and severity of associated coagulopathy, reduced stay in the ICU, and 
reduced incidence of complications such as respiratory failure, renal 
failure, coma, adrenal cortical necrosis, pericarditis, endocarditis, 
cardiomyopathy, endophthalmitis, and arthritis. 
BPI protein products have been demonstrated to have a bactericidal effect 
in vitro against serogroups A, B, C and W135 of the gram-negative 
bacteria, Neisseria meningitidis, that causes meningococcemia. BPI protein 
products may exert their effect in human meningococcemia through such 
direct bactericidal action, or through enhancing the effectiveness of 
antibiotic therapy as described in co-owned, co-pending U.S. application 
Ser. No. 08/311,611 filed Sep. 22, 1994, which issued U.S. Pat. No. 
5,523,288 on Jun. 4, 1996, and which is incorporated herein by reference. 
BPI protein products may also exert their effect in human meningococcemia 
through neutralizing LOS endotoxin that has been released from or remains 
in association with the bacteria and bacterial fragments. The effects of 
BPI protein products in humans with endotoxin in circulation, including 
effects on TNF, IL-6 and endotoxin is described in co-owned, co-pending 
U.S. application Ser. No. 08/378,228, filed Jan. 24, 1995, which in turn 
is a continuation-in-part application of U.S. Ser. No. 08/291,112, filed 
Aug. 16, 1994, which in turn is a continuation-in-part application of U.S. 
Ser. No. 08/188,221, filed Jan. 24, 1994, all of which are incorporated 
herein by reference. BPI protein products exhibit both anticoagulant and 
fibrinolytic effects, as described in co-owned, co-pending U.S. 
application Ser. No. 08/644,290 filed concurrently herewith, which is 
incorporated herein by reference. BPI protein products may act on other 
pathologic processes that accompany meningococcemia including, for 
example, coagulopathies. 
Therapeutic compositions comprising BPI protein product may be administered 
systemically or topically. Systemic routes of administration include oral, 
intravenous, intramuscular or subcutaneous injection (including into a 
depot for long-term release), intraocular and retrobulbar, intrathecal, 
intraperitoneal (e.g. by intraperitoneal lavage), intrapulmonary using 
aerosolized or nebulized drug, or transdermal. The preferred route is 
intravenous administration. When given parenterally, BPI protein product 
compositions are generally injected in doses ranging from 1 .mu.g/kg to 
100 mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 20 mg/kg 
per day, more preferably at doses ranging from 1 to 20 mg/kg/day and most 
preferably at doses ranging from 2 to 10 mg/kg/day. The treatment may 
continue by continuous infusion or intermittent injection or infusion, at 
the same, reduced or increased dose per day for, e.g., 1 to 3 days, and 
additionally as determined by the treating physician. BPI protein products 
are preferably administered intravenously by an initial bolus followed by 
a continuous infusion. The preferred regimen is a 1 to 20 mg/kg 
intravenous bolus of BPI protein product followed by intravenous infusion 
at a dose of 1 to 20 mg/kg/day, continuing for up to one week. The most 
preferred dosing regimen is a 2 to 10 mg/kg initial bolus followed by 
intravenous infusion at a dose of 2 to 10 mg/kg/day, continuing for up to 
72 hours. Topical routes include administration in the form of salves, 
ophthalmic drops, ear drops, irrigation fluids (for, e.g., irrigation of 
wounds) or medicated shampoos. For example, for topical administration in 
drop form, about 10 to 200 .mu.L of a BPI protein product composition may 
be applied one or more times per day as determined by the treating 
physician. Those skilled in the art can readily optimize effective dosages 
and administration regimens for therapeutic compositions comprising BPI 
protein product, as determined by good medical practice and the clinical 
condition of the individual patient. 
As used herein, "BPI protein product" includes naturally and recombinantly 
produced BPI protein; natural, synthetic, and recombinant biologically 
active polypeptide fragments of BPI protein; biologically active 
polypeptide variants of BPI protein or fragments thereof, including hybrid 
fusion proteins and dimers; biologically active polypeptide analogs of BPI 
protein or fragments or variants thereof, including cysteine-substituted 
analogs; and BPI-derived peptides. The BPI protein products administered 
according to this invention may be generated and/or isolated by any means 
known in the art. U.S. Pat. No. 5,198,541, the disclosure of which is 
incorporated herein by reference, discloses recombinant genes encoding and 
methods for expression of BPI proteins including recombinant BPI 
holoprotein, referred to as rBPI.sub.50 (or rBPI) and recombinant 
fragments of BPI. Co-owned, copending U.S. patent application Ser. No. 
07/885,501 and a continuation-in-part thereof, U.S. patent application 
Ser. No. 08/072,063 filed May 19, 1993 and corresponding PCT application 
Ser. No. 93/04752 filed May 19, 1993, which are all incorporated herein by 
reference, disclose novel methods for the purification of recombinant BPI 
protein products expressed in and secreted from genetically transformed 
mammalian host cells in culture and discloses how one may produce large 
quantities of recombinant BPI products suitable for incorporation into 
stable, homogeneous pharmaceutical preparations. 
Biologically active fragments of BPI (BPI fragments) include biologically 
active molecules that have the same or similar amino acid sequence as a 
natural human BPI holoprotein, except that the fragment molecule lacks 
amino-terminal amino acids, internal amino acids, and/or carboxy-terminal 
amino acids of the holoprotein. Nonlimiting examples of such fragments 
include a N-terminal fragment of natural human BPI of approximately 25 kD, 
described in Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant 
expression product of DNA encoding N-terninal amino acids from 1 to about 
193 or 199 of natural human BPI, described in Gazzano-Santoro et al., 
Infect. Immun. 60:4754-4761 (1992), and referred to as rBPI.sub.23. In 
that publication, an expression vector was used as a source of DNA 
encoding a recombinant expression product (rBPI.sub.23) having the 
31-residue signal sequence and the first 199 amino acids of the N-terminus 
of the mature human BPI, as set out in FIG. 1 of Gray et al., supra, 
except that valine at position 151 is specified by GTG rather than GTC and 
residue 185 is glutamic acid (specified by GAG) rather than lysine 
(specified by AAG). Recombinant holoprotein (rBPI.sub.50) has also been 
produced having the sequence (SEQ ID NOS: 1 and 2) set out in FIG. 1 of 
Gray et al., supra, with the exceptions noted for rBPI.sub.23 and with the 
exception that residue 417 is alanine (specified by GCT) rather than 
valine (specified by GTT). Other examples include dimeric forms of BPI 
fragments, as described in co-owned and co-pending U.S. patent application 
Ser. No. 08/212,132, filed Mar. 11, 1994, and corresponding PCT 
application Ser. No. PCT/US95/03125, the disclosures of which are 
incorporated herein by reference. Preferred dimeric products include 
dimeric BPI protein products wherein the monomers are amino-terminal BPI 
fragments having the N-terminal residues from about 1 to 175 to about 1 to 
199 of BPI holoprotein. A particularly preferred dimeric product is the 
dimeric form of the BPI fragment having N-terminal residues 1 through 193, 
designated rBPI.sub.42 dimer. 
Biologically active variants of BPI (BPI variants) include but are not 
limited to recombinant hybrid fusion proteins, comprising BPI holoprotein 
or biologically active fragment thereof and at least a portion of at least 
one other polypeptide, and dimeric forms of BPI variants. Examples of such 
hybrid fusion proteins and dimeric forms are described by Theofan et al. 
in co-owned, copending U.S. patent application Ser. No. 07/885,911, and a 
continuation-in-part application thereof, U.S. patent application Ser. No. 
08/064,693 filed May 19, 1993 and corresponding PCT application Ser. No. 
US93/04754 filed May 19, 1993, which are all incorporated herein by 
reference and include hybrid fusion proteins comprising, at the 
amino-terminal end, a BPI protein or a biologically active fragment 
thereof and, at the carboxy-terminal end, at least one constant domain of 
an immunoglobulin heavy chain or allelic variant thereof. Similarly 
configured hybrid fusion proteins involving part or all Lipopolysaccharide 
Binding Protein (LBP) are also contemplated for use in the present 
invention. 
Biologically active analogs of BPI (BPI analogs) include but are not 
limited to BPI protein products wherein one or more amino acid residues 
have been replaced by a different amino acid. For example, co-owned, 
copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993 
and corresponding PCT application Ser. No. US94/01235 filed Feb. 2, 1994, 
the disclosures of which are incorporated herein by reference, discloses 
polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is 
replaced by a different amino acid. A preferred BPI protein product 
described by this application is the expression product of DNA encoding 
from amino acid 1 to approximately 193 or 199 of the N-terminal amino 
acids of BPI holoprotein, but wherein the cysteine at residue number 132 
is substituted with alanine and is designated rBPI.sub.21 .DELTA.cys or 
rBPI.sub.21. Other examples include dimeric forms of BPI analogs; e.g. 
co-owned and co-pending U.S. patent application Ser. No. 08/212,132 filed 
Mar. 11, 1994, and corresponding PCT application Ser. No. PCT/US95/03125, 
the disclosures of which are incorporated herein by reference. 
Other BPI protein products useful according to the methods of the invention 
are peptides derived from or based on BPI produced by recombinant or 
synthetic means (BPI-derived peptides), such as those described in 
co-owned and co-pending U.S. patent application Ser. No. 08/504,841 filed 
Jul. 20, 1995 and in co-owned and copending PCT application Ser. No. 
PCT/US94/10427 filed Sep. 15, 1994, which corresponds to U.S. patent 
application Ser. No. 08/306,473 filed Sep. 15, 1994, and PCT application 
Ser. No. US94/02465 filed Mar. 11, 1994, which corresponds to U.S. patent 
application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a 
continuation-in-part of U.S. patent application Ser. No. 08/183,222, filed 
Jan. 14, 1994, which is a continuation-in-part of U.S. patent application 
Ser. No. 08/093,202 filed Jul. 15, 1993 (for which the corresponding 
international application is PCT application Ser. No. US94/02401 filed 
Mar. 11, 1994), which is a continuation-in-part of U.S. patent application 
Ser. No. 08/030,644 filed Mar. 12, 1993, the disclosures of all of which 
are incorporated herein by reference. 
Presently preferred BPI protein products include recombinantly-produced 
N-terminal fragments of BPI, especially those having a molecular weight of 
approximately between 21 to 25 kD such as rBPI.sub.23 or rBPI.sub.21, or 
dimeric forms of these N-terminal fragments (e.g., rBPI.sub.42 dimer). 
Additionally, preferred BPI protein products include rBPI,.sub.50 and 
BPI-derived peptides. 
The administration of BPI protein products is preferably accomplished with 
a pharmaceutical composition comprising a BPI protein product and a 
pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein 
product may be administered without or in conjunction with known 
surfactants, other chemotherapeutic agents or additional known 
anti-microbial agents. One pharmaceutical composition containing BPI 
protein products (e.g., rBPI.sub.50, rBPI.sub.23) comprises the BPI 
protein product at a concentration of 1 mg/ml in citrate buffered saline 
(5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of 
poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and 0.002% 
by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, 
Del.). Another pharmaceutical composition containing BPI protein products 
(e.g., rBPI.sub.21, ) comprises the BPI protein product at a concentration 
of 2 mg/mL in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188 and 0.002% 
polysorbate 80.Such combinations are described in co-owned, co-pending PCT 
application Ser. No. US94/01239 filed Feb. 2, 1994, which corresponds to 
U.S. patent application Ser. No. 08/190,869 filed Feb. 2, 1994 and U.S. 
patent application Ser. No. 08/012,360 filed Feb. 2, 1993, the disclosures 
of all of which are incorporated herein by reference. 
Other aspects and advantages of the present invention will be understood 
upon consideration of the following illustrative examples. Example 1 
addresses the effect of BPI protein product administration on mortality 
associated with meningococcemia. Example 2 addresses the effect of BPI 
protein product administration on morbidities associated with 
meningococcemia. Examples 3 and 4 describe the effect of BPI protein 
product administration on the course of meningococcemia in two particular 
individuals. 
EXAMPLE 1 
Clinical Study Protocol--Effect of BPI Protein Product on Mortality 
A human clinical study was designed to examine the effect of an exemplary 
BPI protein product, rBPI.sub.21, on clinical outcome in pediatric 
patients suffering from severe systemic meningococcal disease. Clinical 
outcomes (mortality, amputations, grafts, permanent neurologic impairment) 
were assessed through study day 28 or discharge, whichever occurred first. 
Additionally, the safety, pharmacokinetics and hemodynamic effects of the 
BPI protein product were assessed. 
Thus, a Phase I open-label multicenter study of the effects of BPI protein 
product on pediatric patients with severe meningococcemia receiving 
standard care was implemented. Patients who met eligibility criteria were 
enrolled following informed consent of the parent or legal guardian. The 
eligibility criteria were such that the patients enrolled had a 90% 
predicted rate of severe adverse outcome, defined as death, stroke, 
amputation, or skin grafting. All patients received comprehensive 
pediatric intensive care consistent with the usual standard of care, and 
received their first dose of antibiotics no more than 8 hours prior to the 
beginning of BPI protein product administration. 
The first four patients received an infusion of 0.5 mg/kg rBPI.sub.21 over 
30 minutes, followed immediately by a continuous infusion of rBPI.sub.21, 
at a rate of 0.5 mg/kg/day for 24 hours. The next six patients received an 
infusion of 1.0 mg/kg rBPI.sub.21, over 30 minutes, followed immediately 
by a continuous infusion of rBPI.sub.21 at a rate of 1.0 mg/kg/day for 24 
hours. The remaining patients received an infusion of 2.0 mg/kg 
rBPI.sub.21 over 30 minutes, followed immediately by a continuous infusion 
of rBPI.sub.21 at a rate of 2.0 mg/kg/day for 24 hours. All study centers 
escalated to the higher dose levels at the same time. 
The pharmacokinetics of the BPI protein product and circulating endotoxin 
levels were assessed by serial monitoring of plasma for rBPI.sub.21, and 
endotoxin by limulus amoebocyte lysate (LAL) assay. Any acute hemodynamic 
effects associated with administration of rBPI.sub.21, were described by 
recording standard hemodynamic parameters, including: heart rate, invasive 
systemic arterial blood pressure, electrocardiogram, oxygen saturation, 
and invasive hemodynamic measurements obtained from a pulmonary artery 
catheter. No new invasive devices were placed for the purposes of the 
study; the placement of medical devices was at the sole discretion of the 
attending physician and his/her staff, and only for the purpose of 
monitoring the patient consistent with normal standards of care. 
Safety was monitored by continuous measurements of vital signs and 
hemodynamics, physical examinations and pre- and post-treatment safety 
laboratory assessments. Patients were followed for safety until death, 
hospital discharge or study day 28, whichever occurred first. 
Patients with severe meningococcemia were selected for enrollment in the 
study if they met the following inclusion and exclusion criteria. 
Inclusion criteria were: (1) age 1 year to 18 years inclusive; (2) 
presumptive diagnosis of meningococcemia, based on any or all of the 
following: (a) petechiae or purpura, fever, and hemodynamic instability in 
a clinical context consistent with the diagnosis of meningococcemia, (b) 
demonstration of gram-negative diplococci in blood, cerebrospinal fluid, 
or skin lesions in a clinical context consistent with the diagnosis of 
meningococcemia, and/or (c) demonstration of meningococcal antigens by 
immunologic determination in a clinical context consistent with the 
diagnosis of meningococcemia; (3) Glasgow Meningococcal Septicemia 
Prognostic Score of 8 or greater [Sinclair et al., supra]; (4) patient 
history of having received the first dose of antibiotics no more than 8 
hours prior to beginning BPI protein product administration; (5) negative 
pregnancy test for pubertal or post-pubertal females; (6) written informed 
consent obtained from the parent or legal guardian; and (7) collection of 
confidential patient follow-up information. Exclusion criteria were: (1) 
insufficient vascular access to administer BPI protein product without 
compromising routine ICU care; (2) exposure to investigational agents 
during the last 30 days prior to study entry; and (3) any condition that 
in the attending physician's judgment would make the patient unsuitable 
for participation in the study, including imminent mortality. 
The following were performed within 24 hours prior to enrollment in the 
study: (1) medical history, (2) complete physical examination, (3) chest 
x-ray, (4) laboratory evaluation: Hematology: CBC, differential; 
Coagulation: PT, PTT, fibrinogen, D-Dimers; Microbiology: cultures, Gram 
stains, serology as indicated; Chemistries: sodium, potassium, chloride, 
bicarbonate, glucose, BUN, creatinine, ionized calcium, phosphorus, 
magnesium, bilirubin, AST, ALT, CPK (with isoenzymes), LDH; Arterial Blood 
Gases; Urinalysis: chemistry and microscopic; and (5) procurement of 
written informed consent and collection of confidential follow-up 
information. 
The rBPI.sub.21, was supplied as a clear, colorless, sterile non-pyrogenic 
solution in 10 mL single use glass vials at a concentration of 2 mg/mL in 
5 mM sodium citrate/0.15 M sodium chloride buffer, pH 5.0 with 0.2% 
poloxamer 188 and 0.002% polysorbate 80 containing no preservative. For 
storage, the rBPI.sub.21 vial was refrigerated at 2-8.degree. C. at all 
times prior to administration. The product was brought to room temperature 
prior to infusion, and was administered via a central vein or other 
suitable vein. Suitability of intravenous access was determined by easy 
withdrawal of blood from the access, as well as easy infusion of 
intravenous fluids without infiltration. rBPI.sub.21, was the sole agent 
administered in the chosen port during the course of the infusion 
protocol. The venous access port was not heparinized, but was flushed as 
necessary with physiologic saline. 
After BPI protein product infusion had started, patients were bserved for 
the possible development of adverse events. Plasma samples for 
determination of rBPI.sub.21 levels were collected immediately prior to 
the start of the infusion (time zero) and at the following times after the 
start of the infusion: 30 min., 90 min., 240 min., 720 min., just prior to 
termination of infusion at 24 hours 30 min., 24 hours 37 min., 24 hours 45 
min., 25 hours, 25 hours 30 min., 26 hours 30 min., 27 hours 30 min., and 
48 hours. Plasma samples for determination of endotoxin levels were drawn 
immediately prior to the onset of the infusion (time zero) and at the 
following times after the start of the infusion: 30 min., 90 min., 240 
min., 720 min. and at 48 hours. Serum ionized calcium concentrations were 
determined immediately prior to the onset of the infusion (time zero) and 
at the following times after the start of infusion: 30 min., 2 hours, 6 
hours, 12 hours, and 24 hours. Monitoring the ionized calcium 
concentrations is the usual standard of care in meningococcemia and 
normally occurs every 4 hours. All samples were obtained via a line not 
used to infuse BPI protein product. 
The following vital signs were recorded every 5 min. for thirty min. prior 
to beginning the infusion, every 5 min. during the 30-min. loading dose, 
and every 30 min. thereafter for 24 hours: (a) heart rate; (b) systemic 
arterial blood pressures: systolic, diastolic, and mean; and (c) 
respiratory rate (if the patient was spontaneously breathing). In addition 
to the manual collection as outlined above, data was digitally recorded 
and stored every minute within the bedside monitor during the ICU stay. 
Once the patient left the ICU, vital signs were collected daily until 
hospital discharge. 
The following invasive hemodynamic parameters were recorded every 10 min. 
for the 30 min. prior to beginning the infusion, every 10 min. during the 
30-min. loading dose, and every 2 hours thereafter for 24 hours: (a) mixed 
venous oxygen saturation (oximetric catheters only); (b) pulmonary artery 
wedge pressure; (c) pulmonary artery pressures: systolic, diastolic, mean; 
(d) cardiac index (CI); (e) systemic vascular resistance index (SVRI); (f) 
pulmonary vascular resistance index (PVRI); and (g) stroke volume index 
(SVI). A complete profile of medications and vasoactive infusions was 
recorded through hospital discharge. The following were documented with a 
frequency determined by the primary care physician consistent with 
standard management of severe meningococcal disease: (a) arterial blood 
gases, (b) venous blood gases, (c) oxygen delivery (DO.sub.2) (d) oxygen 
consumption (VO.sub.2), (e) hematology, (f) coagulation, and (g) blood 
chemistries. The PRISM Score (Pediatric Risk of Mortality Score) was also 
calculated and recorded at the end of the first hospital day. Table 1 
below shows the factors with the corresponding number of points used to 
calculate the PRISM score (Pollack et al., "The pediatric risk of 
mortality (PRISM) score, " Critical Care Medicine 16:1110, 1988). 
TABLE 1 
______________________________________ 
Pediatric Risk of Mortality Score 
(PRISM Score) 
AGE RESTRICTION AND RANGES 
Infants Children 
FACTORS only only All Ages Pts 
______________________________________ 
Systolic BP 
130-160 150-200 2 
(mm/Hg) 55-65 65-75 2 
&gt;160 &gt;200 6 
40-54 50-64 6 
&lt;40 &lt;50 7 
Diastolic BP &gt;110 6 
(mm Hg) 
Heart Rate &gt;160 &gt;150 4 
(beats/min) 
&lt;90 &lt;70 4 
Respiratory Rate 
61-90 51-70 1 
(breaths/min) 
&gt;90 &gt;70 5 
APNEA APNEA 5 
PaO.sub.2 /Fi0.sub.2 200-300 2 
&lt;200 3 
PaCO.sub.2 (mm Hg) 51-65 1 
&gt;65 5 
Glasgow score &lt;8 6 
Pupillary Unequal 4 
Reactions or dilated 
Fixed and 
10 
dilated 
PT/PTT &gt;1.5 .times. 
2 
Control 
Total Bilirubin &gt;3.5 
(mg/dl) at age &gt;1 
6 
month 
Potassium 3.0-3.5 1 
(meq/l) 6.5-7.5 1 
&lt;3.0 5 
&gt;7.5 5 
Calcium 7.0-8.0 2 
(mg/dl) 12.0-15.0 
2 
&lt;7.0 6 
&gt;15.0 6 
Glucose 40-60 4 
(mg/dl) 250-400 4 
&lt;40 8 
&gt;400 8 
Bicarbonate &lt;16 3 
(meq/l) &gt;32 3 
______________________________________ 
At the end of the study (i.e., study day 28 or at time of discharge, 
whichever occurred first), a physical exam, including vital signs, and a 
review of any adverse events were performed. The following clinical 
outcomes were also assessed: (a) mortality; (b) amputations; (c) ging 
procedures; (d) permanent neurologic impairment including but not limited 
to cerebrovascular accidents, cerebral atrophy, and seizures requiring 
medication that manifested as impaired neurologic function; and (e) 
pediatric outcome scores (based on the Pediatric Cerebral Performance 
Category Scale and/or the Pediatric Overall Performance Category Scale, as 
described by Fiser, "Assessing the outcome of pediatric intensive care," 
J. Pediatncs 121:1 68-74, 1992). 
A review was conducted of the medical records of patients admitted to one 
participating clinical center, Study Center 1, during the two years 
immediately prior to the initiation of the BPI study. From these records, 
14 children were selected for comparative analysis because they met the 
first three above-described inclusion criteria regarding age, presumptive 
meningococcemia diagnosis and Glasgow score. Six of these 14 "historical 
control" children died. This high mortality rate was expected, considering 
that the inclusion criteria required a Glasgow score of 8 or greater. A 
Glasgow score of 8 or greater always indicates severe disease. 
In striking contrast, none of the 10 patients in the BPI study at Study 
Center 1 died. Thus, the administration of BPI protein product 
dramatically reduced the mortality rate of severe pediatric 
meningococcemia at Study Center 1 from 43% to 0%. When results from all of 
the participating clinical centers are included, of the 14 total patients 
enrolled in the BPI study so far, only one patient has died--a mortality 
rate of only 7%. This low mortality rate is particularly remarkable 
considering that 12 of the 14 patients had a Glasgow score of 10 or 
greater when they entered the study. Multiple analyses that calculated 
expected mortality for the 14 patients in the BPI study, based on 
indicators (levels of endotoxin, TNF, IL-6 and fibrinogen, and FM) that 
have been shown to correlate with disease severity and outcome in various 
studies [Brandtzaeg et al., supra, Van Deuren et al., supra, McManus et 
al., supra, and Bone, supra] predicted a mortality rate ranging from about 
20% to about 50% for this population. Endotoxin, TNF and IL-6 levels for 
the initial 10 patients in the BPI study are shown in FIGS. 1-6. 
EXAMPLE 2 
Effect of BPI Protein Product on Morbidity 
The clinical outcomes of patients treated in accordance with the BPI 
protein product study protocol described in Example 1 above are summarized 
in Table 2 below and compared with the clinical outcomes of the 14 
"historical control" children at Study Center 1 who would have met the 
study's inclusion criteria during the two years immediately prior to the 
initiation of the study. The natural history of the clinical course of 
meningococcemia would have been largely similar; previously healthy 
children underwent a 12-24 hour flu-like prodrome, developed purpura, and 
then died or became moribund within 4-6 hours. Typically, such 
meningococcemia patients continue to become sicker in the PICU, at least 
for the first 12 hours, and often succumb to irreversibly progressive 
shock. There were no known differences in the standard of care provided to 
the patients enrolled in the BPI study compared to the 14 previous 
"historical control" patients at Study Center 1, with the exception of BPI 
protein product administration. 
TABLE 2 
______________________________________ 
"Historical 
Control" 
BPI Study 
Children at 
Children at 
BPI Study 
Study Study Children at 
Center 1 
Center 1 All Centers 
______________________________________ 
No. Satisfying Inclusion 
14 
Criteria or Enrolled in BPI 
10 14 
Study 
No. of Deaths 6 (43%) 0 (0%) 1 (7%) 
Morbidities 
No. of Survivors 
2/8 1/10 1/13 
With Severe (25%) (10%) (8%) 
Amputations 
No. of Survivors 
2/8 0/10 0/13 
With Permanent (25%) (0%) (0%) 
Neurologic 
Impairment 
Total Morbidity Events 
4/8* 1/10 1/13 
(severe amputation or 
(50%) (10%) (8%) 
permanent neurologic 
impairment) 
______________________________________ 
*One patient experienced both severe amputations and permanent neurologic 
impairment. 
The results summarized in Table 2 show that administration of BPI protein 
product not only vastly reduced the mortality rate at Study Center 1, but 
also reduced the incidence of severe morbidities from 50% to 10% at Study 
Center 1.When results from all of the participating clinical centers are 
included, the overall severe morbidity rate remains low, at 8%. 
Interpretation of morbidity data from this study is somewhat complicated 
by the fact that BPI protein product treatment had a significant effect on 
reducing the number of mortalities associated with this disease, that is, 
a number of the severely ill patients were rescued who would have 
otherwise succumbed to the disease. No analysis was performed to predict 
the morbidities patients would have experienced had they not died. For 
this morbidity outcome analysis, amputations at the wrist or above, or at 
the ankle or above, were considered to be severe amputations. Neurologic 
abnormalities that resulted in significant and permanent impairment of 
motor, cognitive or sensory function were considered to be permanent 
neurologic impairments. Four additional BPI study patients (all at Study 
Center 1) experienced minor amputations of toes or fmgers (and one partial 
foot amputation). Four additional "historical control" patients at Study 
Center 1 experienced other neurologic abnormalities, including 
cerebrovascular accidents, seizures, CT scan or EEG abnormalities, and 
cranial nerve palsy. One BPI study patient (at Study Center 1) experienced 
a neurologic abnormality (see Example 4). 
EXAMPLE 3 
Clinical Course of One Individual in the BPI Study 
This patient (number 2 in the BPI study) was a seven year old white male 
who was previously healthy. On the night prior to admission to the 
pediatric intensive care unit (PICU), he came to the emergency room with 
symptoms of fever, vomiting, and headache. His white blood cell count 
(WBC) was 17,500, but he was sent home because his exam was not suggestive 
of significant disease. He was seen again the next morning, when a diffuse 
petechial rash was noted. His WBC had dropped to 6,300, and his fever and 
headache had worsened. Meningococcemia was suspected, and after initial 
fluid infusion and antibiotic administration, he was admitted to the 
Pediatric Intensive Care Unit (PICU) at Study Center 1. 
He was intubated and mechanically ventilated, fluid resuscitated, and begun 
on inotropic support with an epinephrine infusion. He was treated with the 
antibiotic cefttiaxone. Before enrollment in the BPI study, his Glasgow 
score was 14/15, his PRISM score was 17, and his physical exam revealed a 
temperature of 38.8.degree. C., a heart rate of 145, a respiratory rate of 
16 on the ventilator, and a blood pressure of 107/46. His PTT value was 
25.2 and his fibrinogen level was 480. Blood cultures revealed N. 
meningitdis serotype C. A pulmonary artery catheter was placed for 
monitoring, and written informed consent was obtained for the BPI protocol 
described above in Example 1. 
An rBPI.sub.21 infusion was started at 14:35 on the day of admission to 
PICU (Day One). He received a dose of 0.5 mg/kg rBPI.sub.21 over 30 
minutes followed by an infusion of 0.5 mg/kg over the following 24 hours. 
He tolerated his infusion well with no hemodynamic changes or other 
complications. His PICU course was relatively uneventful; his inotropic 
support was discontinued on the night of Day Four, after which his 
ventilator was rapidly weaned. He was taken off the ventilator during Day 
Five without any problems. He was transferred to the general pediatric 
wards on the afternoon of Day Six and discharged from the hospital on Day 
Nine without any complications. 
EXAMPLE 4 
Clinical Course of Another Individual in the BPI Study 
This patient (number 6 in the BPI study) was an 18 year-old white male who 
was previously in excellent health. Prior to admission, he experienced a 
two-day history of sore throat and lethargy. On the evening of admission, 
his roommate found him in the corner of his dormitory room, unresponsive 
and covered with a purple petechial rash. He was transported via ambulance 
to the hospital, at which time his temperature was 101.degree. F. and he 
was in fulminant shock. He was treated with fluid resuscitation, 
ceftriaxone antibiotic, and a dopamine infusion to improve circulation. He 
was transported via helicopter to Study Center 1. 
On arrival, he was intubated and ventilated. Before enrollment in the BPI 
study, his Glasgow score was 12/15 and his PRISM score was 16.He was 
moribund. His physical exam was significant for rapidly expanding diff-use 
purpura, a capillary refill greater than eight seconds, and minimally 
detectable pulses. His feet were blue, cold, and pulseless, as were all 
ten fingers. He had a temperature of 37.7.degree. C., a heart rate of 150, 
a respiratory rate of 20 on the ventilator, and a blood pressure of 
133/66. His laboratory evaluation was significant for a WBC of 7,500 with 
a differential of 73% segs and 14% bands. His PT was 26, his PTT was 
greater than 114, his fibrinogen level was 121, and his D-dimers were 
greater than 8.Cerebrospinal fluid cultures revealed N. meningitidis 
serotype C. He had biochemical evidence of multi-organ system failure on 
arrival. Fluid resuscitation continued and an epinephrine infusion was 
begun. Informed consent was obtained and he was enrolled in the BPI study 
according to Example 1 above. 
An rBPI.sub.21, infusion at a dose of 1 mg/kg over 30 minutes was begun at 
07:15 on the day of his admission to PICU (Day One), followed by a dose of 
1 mg/kg over the next 24 hours. The rBPI.sub.21, infusions were well 
tolerated without adverse hemnodynamic effects. He required hemodynamic 
support with inotropic infusions of dopamine, dobutamine and epinephrine. 
He was weaned off of epinephrine and dopamine on the morning of Day Three, 
and rapid weaning of his dobutamine followed. Invasive evaluation of his 
hemodynamic status with a Swan-Ganz catheter revealed a hyperdynamic state 
which did not undergo transition to hypodynamic state, as would be typical 
of such severely ill children on day two of therapy. Although his 
coagulopathy was initially severe and required multiple transfusions of 
fresh frozen plasma, packed cells, and platelets, the coagulopathy had 
rapidly resolved by Day Three of hospitalization. Also by Day Three, his 
initial cold and unperfused feet began to return to a normal pink color, 
with evidence of warmth and circulation being restored. Additional 
antibiotic therapy with vancomycin and tobramycin was begun. On Day Four 
his clinical status had improved to the point that his ventilator was 
weaned. The ventilatory wean continued throughout that day, but this wean 
was interrupted on Day Five by transient pulmonary edema. This pulmonary 
edema was believed due to resorption of third spaced fluid and healing of 
his vascular leak. His ventilator wean was continued later on Day Five. 
His circulation in his lower extremities had markedly improved to the 
point that upon his discharge from PICU on Day Eight, tissue injury was 
only evident in his left heel, which was debrided, and his left second 
toe, which was ultimately amputated. During his hospital course, following 
an episode of transient hypertension, he underwent a CT-scan which 
revealed evidence of a right temporal/parietal cerebrovascular accident 
(CVA) which dated approximately to the date of his admission to PICU. The 
CVA was neurologically silent and did not compromise any motor, cognitive, 
or sensory functions. His post-PICU stay on the pediatric ward consisted 
primarily of physical therapy, occupational therapy, and enhanced 
nutrition. He was discharged on Day Fourteen to a rehabilitation facility 
for further work on strengthening and general rehabilitation. 
Numerous modifications and variations of the above-described invention are 
expected to occur to those of skill in the art. Accordingly, only such 
limitations as appear in the appended claims should be placed thereon. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 2 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 1813 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 31..1491 
- (ix) FEATURE: 
(A) NAME/KEY: mat.sub.-- - #peptide 
(B) LOCATION: 124..1491 
- (ix) FEATURE: 
(A) NAME/KEY: misc.sub.-- - #feature 
#"rBPI" (D) OTHER INFORMATION: 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC AT - #G GCC AGG GGC 
54 
#Glu Asn Met Ala Arg Gly 
25 
- CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG AT - #G GTG CTC GTC GCC ATA 
102 
Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Me - #t Val Leu Val Ala Ile 
10 
- GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GG - #C GTC GTG GTC AGG ATC 
150 
Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gl - #y Val Val Val Arg Ile 
# 5 1 
- TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CA - #G GGG ACG GCC GCT CTG 
198 
Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gl - #n Gly Thr Ala Ala Leu 
# 25 
- CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GA - #C TAC TCA GAC AGC TTT 
246 
Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro As - #p Tyr Ser Asp Ser Phe 
# 40 
- AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AG - #C TTC TAC AGC ATG GAC 
294 
Lys Ile Lys His Leu Gly Lys Gly His Tyr Se - #r Phe Tyr Ser Met Asp 
# 55 
- ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG AT - #A AGC ATG GTG CCC AAT 
342 
Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Il - #e Ser Met Val Pro Asn 
# 70 
- GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AA - #T ATC AAG ATC AGC GGG 
390 
Val Gly Leu Lys Phe Ser Ile Ser Asn Ala As - #n Ile Lys Ile Ser Gly 
# 85 
- AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA AT - #G AGC GGC AAT TTT GAC 
438 
Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Me - #t Ser Gly Asn Phe Asp 
#105 
- CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GA - #T CTG AAG CTG GGC AGT 
486 
Leu Ser Ile Glu Gly Met Ser Ile Ser Ala As - #p Leu Lys Leu Gly Ser 
# 120 
- AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TG - #C TCC AGC TGC AGC AGC 
534 
Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cy - #s Ser Ser Cys Ser Ser 
# 135 
- CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AA - #G AGC AAA GTC GGG TGG 
582 
His Ile Asn Ser Val His Val His Ile Ser Ly - #s Ser Lys Val Gly Trp 
# 150 
- CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TC - #T GCG CTT CGA AAC AAG 
630 
Leu Ile Gln Leu Phe His Lys Lys Ile Glu Se - #r Ala Leu Arg Asn Lys 
# 165 
- ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AA - #T TCT GTA TCC TCC AAG 
678 
Met Asn Ser Gln Val Cys Glu Lys Val Thr As - #n Ser Val Ser Ser Lys 
170 1 - #75 1 - #80 1 - 
#85 
- CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA AT - #G ACC AAA ATA GAT TCT 
726 
Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Me - #t Thr Lys Ile Asp Ser 
# 200 
- GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CC - #T CCA GCA ACC ACG GCT 
774 
Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pr - #o Pro Ala Thr Thr Ala 
# 215 
- GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TT - #T TAC AGT GAG AAC CAC 
822 
Glu Thr Leu Asp Val Gln Met Lys Gly Glu Ph - #e Tyr Ser Glu Asn His 
# 230 
- CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG AT - #G GAG TTT CCC GCT GCC 
870 
His Asn Pro Pro Pro Phe Ala Pro Pro Val Me - #t Glu Phe Pro Ala Ala 
# 245 
- CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GA - #C TAC TTC TTC AAC ACA 
918 
His Asp Arg Met Val Tyr Leu Gly Leu Ser As - #p Tyr Phe Phe Asn Thr 
250 2 - #55 2 - #60 2 - 
#65 
- GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TT - #G AAG ATG ACC CTT AGA 
966 
Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Le - #u Lys Met Thr Leu Arg 
# 280 
- GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CG - #A CTG ACA ACC AAG TTC 
1014 
Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Ar - #g Leu Thr Thr Lys Phe 
# 295 
- TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AA - #G TTT CCC AAC ATG AAG 
1062 
Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Ly - #s Phe Pro Asn Met Lys 
# 310 
- ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CC - #A CAC CTG TCT GTG CAG 
1110 
Ile Gln Ile His Val Ser Ala Ser Thr Pro Pr - #o His Leu Ser Val Gln 
# 325 
- CCC ACC GGC CTT ACC TTC TAC CCT GCC GTG GA - #T GTC CAG GCC TTT GCC 
1158 
Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val As - #p Val Gln Ala Phe Ala 
330 3 - #35 3 - #40 3 - 
#45 
- GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TT - #C CTG ATT GGC ATG CAC 
1206 
Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Ph - #e Leu Ile Gly Met His 
# 360 
- ACA ACT GGT TCC ATG GAG GTC AGC GCC GAG TC - #C AAC AGG CTT GTT GGA 
1254 
Thr Thr Gly Ser Met Glu Val Ser Ala Glu Se - #r Asn Arg Leu Val Gly 
# 375 
- GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CT - #G AAG CAC TCA AAT ATT 
1302 
Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Le - #u Lys His Ser Asn Ile 
# 390 
- GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT AT - #C ATG AAC TAC ATT GTA 
1350 
Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Il - #e Met Asn Tyr Ile Val 
# 405 
- CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AA - #A CTA CAG AAA GGC TTC 
1398 
Pro Ile Leu Val Leu Pro Arg Val Asn Glu Ly - #s Leu Gln Lys Gly Phe 
410 4 - #15 4 - #20 4 - 
#25 
- CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TA - #C AAC GTA GTG CTT CAG 
1446 
Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Ty - #r Asn Val Val Leu Gln 
# 440 
- CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GA - #C GTT GTC TAT AAA 
1491 
Pro His Gln Asn Phe Leu Leu Phe Gly Ala As - #p Val Val Tyr Lys 
# 455 
- TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GC - #TGTGGGGC 
1551 
- ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CT - #TGCAAACT 
1611 
- TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TG - #GAAAAGTG 
1671 
- CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AG - #AGATATTT 
1731 
- CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CA - #TGAAAAAA 
1791 
# 1813ATG TG 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 487 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Met Arg Glu Asn Met Ala Arg Gly Pro Cys As - #n Ala Pro Arg Trp Val 
20 
- Ser Leu Met Val Leu Val Ala Ile Gly Thr Al - #a Val Thr Ala Ala Val 
# 1 
- Asn Pro Gly Val Val Val Arg Ile Ser Gln Ly - #s Gly Leu Asp Tyr Ala 
# 15 
- Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Gl - #u Leu Lys Arg Ile Lys 
# 30 
- Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Ly - #s His Leu Gly Lys Gly 
# 45 
- His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Gl - #u Phe Gln Leu Pro Ser 
# 65 
- Ser Gln Ile Ser Met Val Pro Asn Val Gly Le - #u Lys Phe Ser Ile Ser 
# 80 
- Asn Ala Asn Ile Lys Ile Ser Gly Lys Trp Ly - #s Ala Gln Lys Arg Phe 
# 95 
- Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Il - #e Glu Gly Met Ser Ile 
# 110 
- Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Th - #r Ser Gly Lys Pro Thr 
# 125 
- Ile Thr Cys Ser Ser Cys Ser Ser His Ile As - #n Ser Val His Val His 
130 1 - #35 1 - #40 1 - 
#45 
- Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gl - #n Leu Phe His Lys Lys 
# 160 
- Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Se - #r Gln Val Cys Glu Lys 
# 175 
- Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pr - #o Tyr Phe Gln Thr Leu 
# 190 
- Pro Val Met Thr Lys Ile Asp Ser Val Ala Gl - #y Ile Asn Tyr Gly Leu 
# 205 
- Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Le - #u Asp Val Gln Met Lys 
210 2 - #15 2 - #20 2 - 
#25 
- Gly Glu Phe Tyr Ser Glu Asn His His Asn Pr - #o Pro Pro Phe Ala Pro 
# 240 
- Pro Val Met Glu Phe Pro Ala Ala His Asp Ar - #g Met Val Tyr Leu Gly 
# 255 
- Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Le - #u Val Tyr Gln Glu Ala 
# 270 
- Gly Val Leu Lys Met Thr Leu Arg Asp Asp Me - #t Ile Pro Lys Glu Ser 
# 285 
- Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Th - #r Phe Leu Pro Glu Val 
290 2 - #95 3 - #00 3 - 
#05 
- Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Il - #e His Val Ser Ala Ser 
# 320 
- Thr Pro Pro His Leu Ser Val Gln Pro Thr Gl - #y Leu Thr Phe Tyr Pro 
# 335 
- Ala Val Asp Val Gln Ala Phe Ala Val Leu Pr - #o Asn Ser Ser Leu Ala 
# 350 
- Ser Leu Phe Leu Ile Gly Met His Thr Thr Gl - #y Ser Met Glu Val Ser 
# 365 
- Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Ly - #s Leu Asp Arg Leu Leu 
370 3 - #75 3 - #80 3 - 
#85 
- Leu Glu Leu Lys His Ser Asn Ile Gly Pro Ph - #e Pro Val Glu Leu Leu 
# 400 
- Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Le - #u Val Leu Pro Arg Val 
# 415 
- Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pr - #o Thr Pro Ala Arg Val 
# 430 
- Gln Leu Tyr Asn Val Val Leu Gln Pro His Gl - #n Asn Phe Leu Leu Phe 
# 445 
- Gly Ala Asp Val Val Tyr Lys 
450 4 - #55 
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