U.S. patent application number 14/904048 was filed with the patent office on 2016-07-07 for methods for treatment of and prophylaxis against inflammatory disorders.
The applicant listed for this patent is THE UNIVERSITY OF UTAH RESEARCH FOUNDATION. Invention is credited to Andrew S. Weyrich, Christian Con Yost, Guy A. Zimmerman.
Application Number | 20160194365 14/904048 |
Document ID | / |
Family ID | 52280493 |
Filed Date | 2016-07-07 |
United States Patent
Application |
20160194365 |
Kind Code |
A1 |
Yost; Christian Con ; et
al. |
July 7, 2016 |
METHODS FOR TREATMENT OF AND PROPHYLAXIS AGAINST INFLAMMATORY
DISORDERS
Abstract
An isolated and purified peptide, neonatal NET-inhibitory Factor
(nNIF), is disclosed. Methods for treatment of and prophylaxis
against inflammatory disorders are also disclosed, including
methods of treatment of and prophylaxis against inflammatory
disorders comprising administering NET-inhibitory peptides (NIPs),
which may be a nNIF, a pharmaceutically acceptable salt of a nNIF,
a nNIF analog, a pharmaceutically acceptable salt of a nNIF analog,
a nNIF-Related Peptide (nNRP), including the nNRP,
Cancer-Associated SCM-Recognition, Immune Defense Suppression, and
Serine Protease Protection Peptide (CRISPP), a pharmaceutically
acceptable salt of a nNRP, a nNRP analog, or a pharmaceutically
acceptable salt of a nNRP analog, to an individual.
Inventors: |
Yost; Christian Con; (Nortn
Salt Lake, UT) ; Zimmerman; Guy A.; (Salt Lake City,
UT) ; Weyrich; Andrew S.; (Salt Lake City,
UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF UTAH RESEARCH FOUNDATION |
Salt Lake City |
UT |
US |
|
|
Family ID: |
52280493 |
Appl. No.: |
14/904048 |
Filed: |
July 7, 2014 |
PCT Filed: |
July 7, 2014 |
PCT NO: |
PCT/US14/45597 |
371 Date: |
January 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61843618 |
Jul 8, 2013 |
|
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|
Current U.S.
Class: |
514/1.4 ;
514/1.5; 514/1.6; 514/1.8; 514/14.9; 514/16.6; 514/19.3; 514/20.9;
530/322 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 38/57 20130101; A61P 29/00 20180101; A61P 11/00 20180101; A61K
38/00 20130101; Y02A 50/30 20180101; A61K 38/1709 20130101; C07K
14/4703 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47; A61K 47/48 20060101 A61K047/48 |
Claims
1. A method for treating a patient having, or at risk of
developing, an inflammatory disorder, comprising: administering to
the patient an effective amount of a pharmaceutical composition
comprising a NET-Inhibitory Peptide (NIP) and a pharmaceutically
acceptable carrier to reduce a pathological effect or symptom of
the inflammatory disorder, or to reduce the risk of developing the
inflammatory disorder.
2. The method of claim 1, wherein the NIP is one of a neonatal
NET-Inhibitory Factor (nNIF), a pharmaceutically acceptable salt of
a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF
analog, a nNIF-Related Peptide (nNRP), a pharmaceutically
acceptable salt of a nNRP, a nNRP analog, or a pharmaceutically
acceptable salt of a nNRP analog.
3. The method of claim 1, wherein the inflammatory disorder is
selected from the group consisting of an acute inflammatory
disorder, a chronic inflammatory disorder, and an immune
disorder.
4. The method for claim 1, wherein the inflammatory disorder is an
autoimmunity disorder.
5. The method of claim 1, wherein the inflammatory disorder is
selected from at least one of acute respiratory distress syndrome
(ARDS), bronchopulmonary dysplasia (BPD), chronic obstructive
pulmonary disease (COPD), cystic fibrosis, inflammation in cancer,
inflammatory bowel disease (IBD), inflammatory lung disease (ILD),
influenza-induced pneumonitis, necrotizing enterocolitis (NEC),
neonatal chronic lung disease (CLD), periodontitis, pre-eclampsia,
retinopathy of prematurity (ROP), sepsis, systemic inflammatory
response syndrome (SIRS), thrombosis, transfusion-related acute
lung injury (TRALI), vasculitis, rheumatoid arthritis (RA),
systemic lupus erythematosus (SLE), Wegener's granulomatosis (WG),
or a disorder of nonresolved inflammation.
6. The method of claim 5, wherein the thrombosis is venous
thrombosis.
7. The method of claim 5, wherein the thrombosis in arterial
thrombosis.
8. The method of claim 1, wherein the pharmaceutical composition
substantially inhibits NET-mediated inflammatory tissue damage.
9. The method of claim 1, wherein the patient is human.
10. A method of treating a patient having, or at risk of
developing, a complication of prematurity, comprising:
administering to the patient an effective amount of a
pharmaceutical composition comprising a NET-Inhibitory Peptide
(NIP) and a pharmaceutically acceptable carrier to reduce a
pathological effect or symptom of the complication of prematurity,
or to reduce the risk of developing the complication of
prematurity.
11. The method of claim 10, wherein the NIP is one of a neonatal
NET-Inhibitory Factor (nNIF), a pharmaceutically acceptable salt of
a nNIF, a nNIF analog, a pharmaceutically acceptable salt of a nNIF
analog, a nNIF-Related Peptide (nNRP), a pharmaceutically
acceptable salt of a nNRP, a nNRP analog, or a pharmaceutically
acceptable salt of a nNRP analog
12. The method of claim 10, wherein the complication of prematurity
is selected from at least one of necrotizing enterocolitis (NEC),
respiratory distress syndrome (RDS), pneumonia, bronchopulmonary
dysplasia (BPD), neonatal chronic lung disease (CLD),
neurodevelopmental delay, retinopathy of prematurity (ROP), or
sepsis.
13. The method of claim 10, wherein the pharmaceutical composition
substantially inhibits NET-mediated inflammatory tissue damage.
14. The method of claim 10, wherein the patient is human.
15. A pharmaceutical composition, comprising: a NET-Inhibitory
Peptide (NIP); and a pharmaceutically acceptable carrier.
16. The pharmaceutical composition of claim 15, wherein the NIP is
one of a neonatal NET-Inhibitory Factor (nNIF), a pharmaceutically
acceptable salt of a nNIF, a nNIF analog, a pharmaceutically
acceptable salt of a nNIF analog, a nNIF-Related Peptide (nNRP), a
pharmaceutically acceptable salt of a nNRP, a nNRP analog, or a
pharmaceutically acceptable salt of a nNRP analog.
17. The pharmaceutical composition of claim 16, wherein the
pharmaceutical composition comprises a nNIF, or the salt thereof,
and wherein the nNIF, or the salt thereof, comprises the amino acid
sequence (SEQ ID NO: 1):
KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGK VVNPTQK.
18. The pharmaceutical composition of claim 16, wherein at least
one amino acid of the nNIF, the salt of the nNIF, the nNIF analog,
the salt of the nNIF analog, the nNRP, the salt of the nNRP, the
nNRP analog, or the salt of the nNRP analog, is bound to a chemical
modifier, and wherein the chemical modifier is selected from at
least one of a lipid, a polyethylene glycol (PEG), or a
saccharide.
19. The pharmaceutical composition of claim 16, wherein the nNIF,
the salt of the nNIF, the nNIF analog, the salt of the nNIF analog,
the nNRP, the salt of the nNRP, the nNRP analog, or the salt of the
nNRP analog, is present in an amount effective to substantially
inhibit damage selected from at least one of inflammatory tissue
injury, or inflammatory vascular injury.
20. The pharmaceutical composition of claim 16, wherein the nNIF,
the salt of the nNIF, the nNIF analog, the salt of the nNIF analog,
the nNRP, the salt of the nNRP, the nNRP analog, or the salt of the
nNRP analog, does not globally depress polymorphonuclear leukocyte
(PMN) function.
21. The pharmaceutical composition of claim 16, wherein the nNIF,
the salt of the nNIF, the nNIF analog, the salt of the nNIF analog,
the nNRP, the salt of the nNRP, the nNRP analog, or the salt of the
nNRP analog, does not substantially inhibit one or more activities
of a polymorphonuclear leukocyte (PMN) selected from the group
consisting of chemotaxis, chemokine synthesis and secretion,
cytokine synthesis and secretion, extracellular bacterial killing,
intracellular bacterial killing, phagocytosis, and reactive oxygen
species (ROS) generation.
22. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is chemotaxis.
23. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is chemokine synthesis and secretion.
24. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is cytokine synthesis and secretion.
25. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is extracellular bacterial killing.
26. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is intracellular bacterial killing.
27. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is phagocytosis.
28. The pharmaceutical composition of claim 21, wherein the
activity of the PMN is ROS generation.
29. The pharmaceutical composition of claim 16, wherein the
pharmaceutical composition comprises a nNIF analog, a salt of a
nNIF analog, a nNRP analog, or a salt of a nNRP analog, and wherein
the analog or the salt thereof is not a naturally occurring analog
or salt thereof.
30. The pharmaceutical composition of claim 16, wherein the nNIF,
the salt of the nNIF, the nNIF analog, the salt of the nNIF analog,
the nNRP, the salt of the nNRP, the nNRP analog, or the salt of the
nNRP analog, is present in an amount effective to substantially
inhibit neutrophil extracellular trap (NET) formation.
31. The pharmaceutical composition of claim 30, wherein the NET
formation is stimulated by at least one of a bacterium, a fungus, a
parasite, or a virus.
32. The pharmaceutical composition of claim 31, wherein the virus
is a hemorrhagic fever virus.
33. The pharmaceutical composition of claim 31, wherein the virus
is a filovirus.
34. The pharmaceutical composition of claim 33, wherein the
filovirus is Ebola virus.
35. The pharmaceutical composition of claim 33, wherein the
filovirus is Marburg virus.
36. The pharmaceutical composition of claim 31, wherein the virus
is an arenavirus.
37. The pharmaceutical composition of claim 36, wherein the
arenavirus is Lassa virus.
38. The pharmaceutical composition of claim 31, wherein the virus
is a hantavirus.
39. The pharmaceutical composition of claim 31, wherein the virus
is a flavivirus.
40. The pharmaceutical composition of claim 39, wherein the
flavivirus is dengue virus.
41. The pharmaceutical composition of claim 39, wherein the
flavivirus is yellow fever virus.
42. The pharmaceutical composition of claim 30, wherein the NET
formation is stimulated by at least one of a Bacillus species, an
Escherichia species, a Francisella species, a Staphylococcus
species, a Streptococcus species, or a Yersinia species.
43. The pharmaceutical composition of claim 42, wherein the
Bacillus species is Bacillus anthracis.
44. The pharmaceutical composition of claim 42, wherein the
Escherichia species is Escherichia coli.
45. The pharmaceutical composition of claim 42, wherein the
Francisella species is Francisella tularensis.
46. The pharmaceutical composition of claim 42, wherein the
Staphylococcus species is Staphylococcus aureus.
47. The pharmaceutical composition of claim 30, wherein the NET
formation is stimulated by at least one of beta-defensin 1, HIV-1,
lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or
Staphylococcus aureus alpha-toxin.
48. The pharmaceutical composition of claim 16, wherein the
pharmaceutical composition comprises a nNRP or the salt thereof,
and wherein the nNRP or the salt thereof is selected from at least
one of a Cancer-Associated SCM-Recognition, Immune Defense
Suppression, and Serine Protease Protection Peptide (CRISPP), or a
CRISPP analog.
49. The pharmaceutical composition of claim 16, wherein the
pharmaceutical composition comprises a nNRP, and wherein the nNRP
is an isolated and purified component of umbilical cord blood.
50. A composition for inhibiting NET formation in a mammal,
comprising: a NET-Inhibitory Peptide (NIP); and a pharmaceutically
acceptable carrier.
51. The composition of claim 50, wherein the NIP is one of a
neonatal NET-Inhibitory Factor (nNIF), a pharmaceutically
acceptable salt of a nNIF, a nNIF analog, a pharmaceutically
acceptable salt of a nNIF analog, a nNIF-Related Peptide (nNRP), a
pharmaceutically acceptable salt of a nNRP, a nNRP analog, or a
pharmaceutically acceptable salt of a nNRP analog,
52. The composition of claim 50, wherein the mammal is human.
53. An isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein comprising six or more contiguous amino acids of SEQ ID NO:
1.
54. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 53 comprising at least twelve contiguous amino
acids of SEQ ID NO: 1.
55. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 53 comprising at least twenty-four contiguous
amino acids of SEQ ID NO: 1.
56. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 53 comprising SEQ ID NO:1.
57. An isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein, wherein a sequence of the nNIF protein is at least twenty
percent identical to SEQ ID NO: 1.
58. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 57, wherein the sequence is at least forty percent
identical to SEQ ID NO: 1.
59. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 57, wherein the sequence is at least sixty percent
identical to SEQ ID NO: 1.
60. The isolated and purified neonatal NET-Inhibitory Factor (nNIF)
protein of claim 57, wherein the sequence is at least eighty
percent identical to SEQ ID NO: 1.
Description
TECHNICAL FIELD
[0001] The present disclosure is directed to methods for treatment
of and prophylaxis against inflammatory disorders.
BACKGROUND
[0002] Neutrophil extracellular traps (NETs) are extracellular
lattices of decondensed chromatin decorated with antimicrobial
proteins extruded by polymorphonuclear leukocytes (PMNs,
neutrophils) to trap and kill microbes. Although NETs aid in
trapping bacteria and other pathogens, their presence also leads to
inflammatory tissue damage. Indeed, NET formation contributes to
the pathology of several inflammatory disorders including acute
lung injury resulting from influenza or blood transfusions, sepsis,
small vessel vasculitis, systemic inflammatory response syndrome
(SIRS), and chronic autoimmune diseases like systemic lupus
erythematosus. Furthermore, NET formation and, in particular, the
release of NET-associated histones into the extracellular space
directly induces both epithelial and endothelial cell death in
culture.
[0003] An active cell death process distinct from necrosis and
apoptosis, frequently termed "NETosis," leads to formation of three
dimensional lattices studded with granule enzymes and host defense
peptides that bind to nuclear chromatin before extrusion from the
neutrophil. Histones, which have antimicrobial activities, are also
abundant in NETs. NET formation is conserved in many species.
[0004] Deficient NET formation is a mechanism of immunodeficiency
and impaired human host defense. A variety of pathogens induce NET
formation. In addition to Escherichia coli, Staphylococcus,
Streptococcus, Yersinia, and other gram positive and negative
bacteria, fungi, parasites, and viruses trigger generation of NETs
by human PMNs. Lipopolysaccharides (LPS) also induce NETosis,
suggesting that microbial toxins may broadly have this activity.
Endogenous host mediators, including interleukin-8 (IL-8), platelet
activating factor (PAF), and complement factor C5a induce NET
formation directly or after "priming" by other mediators. LPS
stimulated mouse platelets, activated human platelets, and
platelets in a murine model of transfusion-related acute lung
injury (TRALI) that involves LPS priming also trigger NET
formation. H1N1-infected alveolar epithelial cells induce NET
formation by murine neutrophils in vitro. Thus, multiple
interactions between neutrophils and microbes, host cells, and/or
host mediators signal NETosis and NET formation.
[0005] NETs are also major biologic instruments of extravascular
microbial containment and killing in vitro and in vivo, thus
limiting the spread of pathogens. Certain pathogens, however,
express endonucleases that cleave the DNA lattice or inhibitors
that block antimicrobial peptides; these mechanisms act as
virulence factors that limit killing and provide mechanisms for
bacterial escape.
[0006] Failed NET formation appears to be a previously-unrecognized
innate immune deficit that results in severe infections. Patients
with chronic granulomatous disease (CGD), who are deficient in
reactive oxygen species (ROS) generation and acquire recurrent,
often life threatening bacterial and fungal infections, also
demonstrate a defect in NET formation. Gene therapy for this immune
deficiency can restore NET formation and control refractory
pulmonary aspergillosis in patients with CGD. This suggests that
NETs can also demonstrate protective functions in cystic fibrosis
and pneumonia.
[0007] While the intracellular signaling pathways that regulate NET
formation by PMNs remain largely unknown, ROS generation is
considered a key event. Studies in human HL-60 myeloid leukocytes
and genetically-altered mice indicate that activity of
peptidylarginine deiminase 4 (PAD 4), an enzyme responsible for
chromatin decondensation, is also required.
[0008] Additionally, NET formation may require enzymatic activity
of neutrophil elastase (NE) to initiate degradation of core
histones leading to chromatin decondensation prior to plasma
membrane rupture. Alpha 1 anti-trypsin (A1AT) is a serine protease
inhibitor that inactivates NE in plasma; it is, however, not
expressed by human PMNs. A related serine protease inhibitor,
serpin B1, which is expressed as a cytoplasmic protein by human
PMNs, has been shown to restrict NET formation by mouse and human
PMNs. Furthermore, treatment with recombinant serpin B1 inhibits
NET formation in human PMNs stimulated with phorbol 12-myristate
acetate (PMA), a robust inducer of NET formation in vitro.
Recombinant A1AT, however, does not inhibit NET formation.
Inhibition of specific serine proteases such as NE may effectively
inhibit NET formation by human PMNs.
[0009] Although NET formation is a critical innate antimicrobial
function of PMNs, there is now clear evidence that it is a
mechanism of inflammatory tissue injury and thrombosis if
inappropriately triggered and/or dysregulated. See Saffarzadeh and
Preissner, Curr Opin Hematol, 2013, 20: 3-9; and Brinkmann and
Zychlinsky, J Cell Biol, 2012, 198(5): 773-783. NETs mediate
inflammatory damage in multiple models of sterile and infectious
challenge. For example, NET formation may be a key mechanism in the
systemic vasculopathy that is central to the pathogenesis of the
acute, pro-inflammatory phase of sepsis. Bacteria including
Staphylococci and E. coli are major causes of severe sepsis, alone
or as polymicrobial infections, depending on the populations
studied.
[0010] Experiments utilizing human endothelial cells (EC) and
neutrophils in vitro, and an in vivo model of endotoxemia in which
mice were challenged with LPS, indicate that NETs cause endothelial
and liver damage, potentially mediated by neutrophil proteases
associated with NETs. NE and other granule enzymes from neutrophils
can potently injure endothelium and many extravascular cell types.
EC activation, as occurs in sepsis, can enhance NET generation. In
addition to granule enzymes, histones associated with NETs are
previously-unrecognized agonists for endothelial injury in sepsis,
based on experimental models and human samples.
[0011] There is also evidence that NETs and NET components are
potent procoagulants, and that NET components induce thrombosis--a
central pathogenetic feature of sepsis. NET components modify
fibrin stability and fibrinolysis. Thus, while formation of NETs
may be critical for bacterial capture and containment in the early
phases of bacteremia and sepsis based on murine models,
observations to date indicate that NET formation also causes damage
to the host (for example, in acute septic syndromes).
[0012] Activated vascular endothelium may induce NET formation by
human PMNs and lead to endothelial cell damage in vitro. Also,
cellular damage may occur when human endothelial cells are
incubated with activated platelets and PMNs, leading to NET
formation, and liver injury may occur in vivo following NET
formation. Finally, placentas from mothers with severe
pre-eclampsia, a syndrome of pregnancy commonly leading to
premature infant delivery, show exuberant NET formation. Thus,
while essential in preventing severe infections, inappropriate NET
formation appears to also be a mechanism of inflammatory vascular
and tissue injury.
[0013] Inflammatory and infectious pulmonary syndromes provide
another example of NET-mediated tissue injury. NETs form and
contribute to vascular and alveolar dysfunction in animal models of
acute lung injury and adult respiratory distress syndrome (ARDS).
ARDS is a major complication of human systemic and pulmonary
infection and inflammation. NETs are associated with acute lung
injury in models of influenza-induced pneumonitis, a common and
lethal infectious trigger for ARDS. In vitro studies indicate that
NET histones may be critical mediators of alveolar endothelial and
epithelial cell death.
[0014] In addition to sepsis and pulmonary injury, vasculitic
syndromes are yet another example in which NETs play pathogenetic
roles. As in sepsis, NETs may mediate both vascular inflammation
and thrombosis in vasculitis. A variety of inflammatory stimuli and
infectious agents can cause vasculitis.
[0015] Dysregulated inflammation has also been found to contribute
to the pathogenesis of all the major complications of prematurity:
necrotizing enterocolitis (NEC), respiratory distress syndrome
(RDS), pneumonia, bronchopulmonary dysplasia (BPD), neonatal
chronic lung disease (CLD), neurodevelopmental delay, retinopathy
of prematurity (ROP), and sepsis. Neonatal CLD causes significant
morbidity and mortality in the U.S. CLD is a complication of
preterm birth that results from prolonged mechanical ventilation
required for chronic respiratory failure. It occurs in up to 70% of
mechanically ventilated extremely low birth weight infants (ELBW)
with respiratory distress. While surfactant therapy and pre- or
postnatal steroids have decreased the severity of CLD, this
significant morbidity associated with preterm birth remains common
in at-risk infants, with about 8,000 to 10,000 new cases occurring
annually in the U.S. The mortality rate due to CLD in ELBW infants
remains high from 15-60%. Furthermore, CLD remains the most common
cause of long-term hospitalization in neonates and is also
associated with developmental delay.
[0016] Additional current evidence strongly supports the conclusion
that NETs are involved in tissue damage and thrombosis in a variety
of inflammatory syndromes. Consequently, a few therapeutic
strategies to blunt or interrupt NET formation or the activities of
NET components have been investigated. All such strategies
identified to date have major limitations, however. Global
inhibition of neutrophil function and/or specific inhibitors of
oxygen radical generation or key molecular checkpoints such as
HIF-1.alpha. depress other PMN functions, including migration,
phagocytosis, and/or intracellular microbial killing, causing
parallel potential for microbial evasion and iatrogenic
infections.
[0017] Disruption of NETs with DNases, potentially together with
inhibition of NET-associated histones and enzymes as a combination
strategy, represents a therapeutic approach based on experimental
models. Nevertheless, more than twenty different NET-associated
proteins have been identified, making this impractical.
Furthermore, enzymatic disruption of NETs as an intervention may
lead to dissemination of microbes, depending on its timing.
Pharmacologic disruption of NETs in the vasculature also has the
potential to spread histones and toxic neutrophil enzymes to other
vascular beds, initiating or amplifying multiple organ injury.
Finally, heparins inhibit NET formation under some conditions, but
have bleeding as a well-known complication.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The embodiments disclosed herein will become more fully
apparent from the following description and appended claims, taken
in conjunction with the accompanying drawings. These drawings
depict only typical embodiments, which will be described with
additional specificity and detail through use of the accompanying
drawings in which:
[0019] FIG. 1A is an image of adult human PMNs extruding NETs (thin
arrows) to trap and kill bacteria, S. aureus (wide arrows).
[0020] FIG. 1B compares the decoration of NETs (thin arrow) with
neutrophil elastase (NE) (thick arrow) formed by adult human PMNs
to neonatal PMNs which do express NE but do not form NETs.
[0021] FIG. 1C is a graph indicating that neonatal PMNs demonstrate
significantly decreased total and NET-mediated bacterial killing as
compared to adult PMNs. * p<0.05, ** p<0.001.
[0022] FIG. 2A is an image of NET formation by human PMNs
stimulated with LPS (1 hour time point).
[0023] FIG. 2B is an image of human PMNs incubated in control
plasma (1 hour time point).
[0024] FIG. 2C is an image of NET formation by human PMNs incubated
in plasma isolated from patients with S. aureus sepsis (1 hour time
point).
[0025] FIG. 3A is a series of images showing LPS-stimulated preterm
PMNs isolated from the same preterm infant. PMNs were isolated from
cord blood, at day of life 3, and day of life 14, as indicated.
[0026] FIG. 3B is a series of images (left to right) showing the
results of pre-incubation experiments: day of life 28 PMNs
pre-incubated in autologous plasma, day of life 28 PMNs
pre-incubated with cord blood plasma, adult PMNs pre-incubated with
autologous plasma, and adult PMNs pre-incubated with cord blood
plasma. NET formation was then induced with LPS stimulation.
[0027] FIG. 3C is a graph depicting extracellular histone H.sub.3
release. Extracellular histone content (fold change over baseline)
is represented on the y-axis. Data from one preterm infant's PMNs
through day of life 28 and one adult's PMNs from the plasma switch
experiments of FIG. 3B are represented on the x-axis.
[0028] FIG. 4A is a list of nNIF candidate proteins, expected
molecular mass, and protein score.
[0029] FIG. 4B is a western blot using a polyclonal antibody
against the carboxy-terminus of A1AT comparing nNIF (.apprxeq.6 kD)
expression in cord blood and adult plasma.
[0030] FIG. 4C is a comparison of the mass spectroscopy obtained
sequences of nNIF, CRISPP, and the A1ATm.sup.358 cleavage fragment
of full length A1AT.
[0031] FIG. 5A is an image of untreated control adult PMNs.
[0032] FIG. 5B is an image of adult PMNs with LPS treatment.
[0033] FIG. 5C is an image of adult PMNs with LPS treatment
following pre-incubation with untreated cord blood plasma.
[0034] FIG. 5D is an image of adult PMNs with LPS treatment
following pre-incubation with nNIF-depleted cord blood plasma.
[0035] FIG. 5E is an image of adult PMNs with LPS treatment
following pre-incubation with affinity purified nNIF from cord
blood plasma.
[0036] FIG. 5F is an image of adult PMNs with LPS treatment
following pre-incubation with full length rA1AT.
[0037] FIG. 6A is a series of images (clockwise from top left)
showing adult human PMN NET formation in: control PMNs,
PMA-stimulated (20 nM) PMNs, PMA-stimulated (20 nM) PMNs with
scrambled peptide (1 nM) pre-incubation, and PMA-stimulated (20 nM)
PMNs with CRISPP (1 nM) pre-incubation.
[0038] FIG. 6B is a graph showing the results of a human histone
H.sub.3 release assay to quantify NET formation. Extracellular
histone content (fold change over baseline) is represented on the
y-axis. Data from human PMNs stimulated under indicated conditions
are represented on the x-axis.
[0039] FIG. 7A is a series of images (clockwise from top left)
showing human PMN NET formation by: no treatment, S. aureus
incubation, S. aureus incubation and scrambled control peptide (1
nM) pre-incubation, and S. aureus incubation and CRISPP (1 nM)
pre-incubation.
[0040] FIG. 7B is a graph showing the results of a human histone
H.sub.3 release assay to quantify NET formation (n=2).
Extracellular histone content (fold change over baseline) is
represented on the y-axis. Data from human PMNs stimulated under
indicated conditions are represented on the x-axis.
[0041] FIG. 8A is a series of images (left to right) showing NET
formation in human PMNs incubated with: LPS (100 ng/mL), dengue
virus media without infection, and dengue virus (0.05 MOI).
[0042] FIG. 8B is a series of images showing (clockwise from top
left) human PMNs incubated with: dengue virus media without
infection and pretreated with scrambled control peptide (1 nM),
dengue virus media without infection and pretreated with CRISPP (1
nM), dengue virus and pretreated with CRISPP (1 nM), and dengue
virus and pretreated with scrambled control peptide (1 nM).
[0043] FIG. 9 is a graph showing total, phagocytotic, and
NET-mediated extracellular bacterial killing of a pathogenic strain
of E. coli by human PMNs with or without LPS stimulation (100
ng/mL). PMNs were also pretreated with CRISPP (1 nM), or a
scrambled peptide control (1 nM). * denotes statistical
significance (p<0.05).
[0044] FIG. 10A is a graph tracking survival of C57BL6 mice in
various treatment arms following intraperitoneal injection of LPS
(20 mg/kg). CRISPP-treated mice (10 mcg/kg/dose) received 2 doses
intraperitoneally; one given 1 hour prior to infection and one dose
given 6 hours after infection. The same dose, delivery route, and
schedule were followed for the scrambled peptide control mice. Six
mice were assessed in both groups. The LPS plus CRISPP group
survival was statistically greater than the LPS plus scrambled
peptide group (p=0.02).
[0045] FIG. 10B is a graph tracking survival of outbred Swiss mice
in various treatment arms following intraperitoneal injection of E.
coli (4.times.10.sup.7 bacteria). As in FIG. 10A, CRISPP-treated
mice (10 mcg/kg/dose) received 2 doses intraperitoneally; one given
1 hour prior to infection and one dose given 6 hours after
infection. The same dose, delivery route, and schedule were
followed for the scrambled peptide control mice. Here, 10 mice were
assessed in each group: no treatment, E. coli plus CRISPP, and E.
coli plus scrambled peptide. The E. coli plus CRISPP group survival
was statistically greater than the E. coli plus scrambled peptide
group (p<0.0001).
[0046] FIG. 11 is a graph tracking survival of outbred Swiss mice
in various treatment arms of a murine CL/P model. CRISPP treated
mice (10 mcg/kg) received 2 doses intraperitoneally; one given 1
hour prior to surgery or sham surgery and one dose given 6 hours
after surgery. The same dose, delivery route, and schedule were
followed for the scrambled peptide control mice. Ten mice were
assessed in each of the CL/P groups. The CL/P plus CRISPP group
survival approached statistical significance compared with the
CL/P--control group (p=0.06).
[0047] FIG. 12A is an image showing NET formation in
paraffin-embedded human lung tissue obtained at autopsy of neonates
who died with CLD. Extracellular, alveolar histone H.sub.3
accumulation consistent with NET formation is indicated by the
white arrow.
[0048] FIG. 12B is a series of images assessing NET formation in
paraffin-embedded preterm lamb lung tissue in a sheep model of CLD.
Referring to the top two images, extracellular, alveolar NET
formation is indicated by the white arrows. Referring to the bottom
two images, H&E staining demonstrates other hallmarks of
neonatal CLD including hypercellularity and alveolar
simplification.
[0049] FIG. 13A is a series of images showing PMNs isolated from
preterm lamb cord blood and from mature ewes. Control samples, and
samples stimulated with LPS (100 ng/mL) for 1 hour are shown, as
indicated.
[0050] FIG. 13B is a series of images showing mature ewe PMNs
pretreated with CRISPP (1 nM) or a scrambled control peptide (1 nM)
for 1 hour prior to LPS-stimulation (100 ng/mL), as indicated.
[0051] FIG. 14A is a series of images showing LPS-stimulated
preterm PMNs isolated from the same preterm infant. PMNs were
isolated at day of life 0, day of life 3, day of life 14, and day
of life 28, as indicated.
[0052] FIG. 14B is a graph depicting extracellular histone H.sub.3
release. Extracellular histone content (fold change over baseline)
is represented on the y-axis. Data from 7 preterm infant's PMNs
through day of life 28 are represented on the x-axis. * denotes
p<0.05 and ** denotes p<0.01 compared to the control (dashed
line), arbitrarily set at 1.
[0053] FIG. 14C is a graph depicting extracellular histone H.sub.3
release. Extracellular histone content (fold change over baseline)
is represented on the y-axis. Data from five preterm infant's PMNs
on day of life 28 and five adult's PMNs from the plasma switch
experiments of FIG. 3B are represented on the x-axis. * denotes
p<0.05 LPS/Adult versus LPS/Preterm, ** denotes p<0.01
LPS/Preterm versus cord blood LPS/Preterm, and .dagger. denotes
p<0.001 LPS/Adult versus cord blood LPS/Adult.
[0054] FIG. 15A is a western blot using a polyclonal antibody
against the carboxy-terminus of alpha 1-antitrypsin (AAT, also
referred to herein as A1AT) comparing nNIF (.apprxeq.4-6 kD)
expression in cord blood (CB) and adult plasma (A).
[0055] FIG. 15B is a graph quantitating the results of FIG. 15A. *
denotes p<0.05.
[0056] FIG. 15C is a series of images (left to right) showing adult
human PMN NET formation in: control PMNs, LPS-stimulated PMNs, AAT
pre-incubated LPS-stimulated PMNs, nNIF pre-incubated
LPS-stimulated PMNs, and scrambled control peptide (SCR)
pre-incubated LPS-stimulated PMNs.
[0057] FIG. 16A is a series of images (clockwise from top left)
showing NET formation in: control PMNs, LPS-stimulated (100 ng/mL)
PMNs, nNIF (1 nM) pre-incubated LPS-stimulated PMNs, CRISPP (1 nM)
pre-incubated LPS-stimulated PMNs, and SCR (1 nM) pre-incubated
LPS-stimulated PMNs.
[0058] FIG. 16B is a graph depicting extracellular histone H.sub.3
content (fold change over baseline) on the y-axis. Data from human
PMNs under indicated conditions are represented on the x-axis. The
dashed line represents the control values, arbitrarily set at 1. **
denotes p<0.05 for LPS and SCR/LPS compared to control, and
.dagger. denotes p<0.05 for CRISPP/LPS and nNIF/LPS compared to
both the LPS and SCR/LPS groups.
[0059] FIG. 16C is a graph depicting extracellular histone H.sub.3
content (fold change over baseline) on the y-axis. Data from human
PMNs under indicated conditions are represented on the x-axis. The
dashed line represents the control values, arbitrarily set at 1. *
denotes p<0.05 for CRISPP/PMA compared to PMA and SCR/PMA
groups.
[0060] FIG. 16D is a graph showing the results of a human histone
H.sub.3 release assay to quantify NET formation (n=2).
Extracellular histone content (fold change over baseline) is
represented on the y-axis. Data from human PMNs stimulated under
indicated conditions are represented on the x-axis.
[0061] FIG. 17A is a graph depicting mononuclear cell counts in
peritoneal fluid under indicated conditions. * denotes p<0.05
for CTL versus all other groups.
[0062] FIG. 17B is a graph depicting neutrophil cell counts in
peritoneal fluid under indicated conditions. * denotes p<0.05
for CTL versus all other groups and .dagger. denotes p<0.05 for
CRISPP/E. coli versus SCR/E. coli and E. coli groups.
[0063] FIG. 17C is a graph depicting E. coli colony-forming units
(cfu) in peritoneal fluid under indicated conditions. * denotes
p.ltoreq.0.05.
[0064] FIG. 17D are images depicting NET formation in peritoneal
fluid following E. coli injection.+-.pre-injection of CRISPP (10
mg/kg) or SCR (10 mg/kg), as indicated.
[0065] FIG. 17E is a graph depicting histone H.sub.3 release to
assess NET formation. Extracellular histone content (fold change
over baseline) is represented on the y-axis. The dashed line
represents the control values, arbitrarily set at 1. * denotes
p<0.05 for CRISPP/E. coli and nNIF/E. coli groups compared to E.
coli and SCR/E. coli groups.
[0066] FIG. 17F are images depicting NET formation on peritoneal
tissue following E. coli injection.+-.pre-injection of CRISPP (10
mg/kg) or SCR (10 mg/kg), as indicated.
[0067] FIG. 17G is a graph depicting quantitation of NET formation
using IMAGEJ software and a standardized grid in 5 randomly
selected visual fields for each sample on confocal microscopy. The
graph represents the number of NETs crossing the standardized grid
lines shown on the y-axis and the experimental groups shown on the
x-axis. * denotes p<0.05 for LPS v. Control, ** denotes
p<0.01 for SCR/LPS group v. Control, and .dagger. denotes
p<0.05 for CRISPP/LPS and nNIF/LPS groups v. SCR/LPS.
[0068] FIG. 18A is a graph depicting chemotaxis induced by IL-8 (2
ng/mL) for PMNs isolated from healthy adults following
treatment.+-.nNIF, CRISPP, or SCR peptide control, as indicated.
The ability of nNIF and CRISPP to induce chemotaxis on their own
was also measured (CRISPP-BLW, NIF-BLW, and SCR-BLW columns). The
y-axis shows fold change over baseline of PMN
chemotaxis.+-.standard error of the mean (SEM).
[0069] FIG. 18B is a graph depicting phagocytosis by PMNs isolated
from healthy adults following a four hour incubation with
fluorescently labelled E. coli BIOPARTICLES (6.times.10.sup.6
particles/mL). The y-axis depicts the percent of PMNs positive for
intracellular E. coli.+-.SEM as assessed via confocal microscopy
with multi-plane assessment. Cytochalasin B and D (10 .mu.M)
pretreatment was used as a control for phagocytosis inhibition. *
denotes p<0.05.
[0070] FIG. 18C is a graph depicting respiratory burst activity in
LPS-stimulated PMNs (100 ng/mL).+-.pre-incubation for 1 hour with
CRISPP (1 nM) or SCR (1 nM). ROS generation was measured using a
dihydrorhodamine assay. The columns indicate ROS generation shown
as percent gated events.+-.SEM.
[0071] FIG. 18D is a graph depicting total, phagocytic, and
extracellular NET-mediated bacterial killing by LPS-stimulated
human PMNs (100 ng/mL).+-.pretreatment with CRISPP (1 nM) or SCR (1
nM). * denotes p<0.05 and ** denotes p<0.01.
[0072] FIG. 18E is a graph depicting platelet p-selectin expression
following pre-incubation with CRISPP (1 nM) or SCR (1 nM) prior to
thrombin stimulation (0.1 IU/mL), as indicated. The dashed line
indicates the level of platelet p-selectin expression at baseline.
.dagger. denotes p<0.001 for CRISPP and SCR groups compared with
all three thrombin-stimulated groups and baseline.
[0073] FIG. 18F is a graph depicting LPS-stimulated PMN aggregation
with thrombin (0.1 IU/mL) activated platelets.+-.CRISPP (10 nM) or
SCR (10 nM) pre-incubation. The dashed line represents the LPS
control group. * denotes p<0.05 for CRISPP/Factor IIa and
SCR/Factor IIa groups compared to baseline.
[0074] FIG. 18G is a series of images depicting CRISPP-FLAG Tagged
(1 nM) and SCR-FLAG Tagged (1 nM) uptake by PMNs, co-incubated
platelets and PMNs, and platelets, as indicated.
[0075] FIG. 19A is a series of images depicting the nuclear area of
PMNs pre-incubated with CRISPP (1 nM), SCR (1 nM), or Sivelestat
(200 nM) prior to stimulation with PMA (20 nM), as indicated. White
arrowheads highlight decondensed nuclei.
[0076] FIG. 19B is a graph quantifying the results of the assays
depicted in FIG. 19A. ** denotes p<0.01 and *** denotes
p<0.001.
[0077] FIG. 19C is a series of images showing FLAG-tagged CRISPP
and SCR peptide cellular localization, as indicated.
[0078] FIG. 19D is a series of images depicting neutrophil elastase
and CRISPP-F peptide co-localization. The control image (Co)
depicts PMNs treated with both antibodies but without FLAG-tagged
peptide treatment.
[0079] FIG. 20 is a graph depicting an assessment of ten mice in
each of four groups: no treatment, E. coli, CRISPP-Post/E. coli,
and SCR-Post/E. coli.
[0080] FIG. 21A is a series of images showing NET formation
following LPS-stimulation.+-.pretreatment with CRISPP, CRISPP-F, or
SCR-F, as indicated.
[0081] FIG. 21B is a graph showing the results of a histone H.sub.3
release assay to quantify NET formation in response to LPS
stimulation following pre-incubation with SCR, CRISPP, or
CRISPP-FLAG. Extracellular histone content (fold change over
baseline.+-.SEM) is represented on the y-axis. * denotes
p<0.05.
DETAILED DESCRIPTION
[0082] This disclosure is related to methods for treatment of and
prophylaxis against inflammatory disorders. It will be readily
understood that the embodiments, as generally described herein, are
exemplary. The following more detailed description of various
embodiments is not intended to limit the scope of the present
disclosure, but is merely representative of various embodiments.
Moreover, the order of the steps or actions of the methods
disclosed herein may be changed by those skilled in the art without
departing from the scope of the present disclosure. In other words,
unless a specific order of steps or actions is required for proper
operation of the embodiment, the order or use of specific steps or
actions may be modified.
[0083] Each and every patent, report, and other reference recited
herein is incorporated by reference in its entirety.
DEFINITIONS
[0084] A "NET-Inhibitory Peptide (NIP)" is an anti-inflammatory
agent that inhibits neutrophil extracellular trap (NET) formation.
Examples of NIPs include, but are not limited to: a neonatal
NET-Inhibitory Factor (nNIF); a pharmaceutically acceptable salt of
a nNIF; an analog of a naturally occurring form of nNIF, which nNIF
analog inhibits NETosis and/or the formation of NETs and is
structurally altered, relative to a given human nNIF, by at least
one amino acid addition, deletion, substitution, or by
incorporation of one or more amino acids with a blocking group; a
pharmaceutically acceptable salt of a nNIF analog; a nNIF-Related
Peptide (nNRP); a pharmaceutically acceptable salt of a nNRP; a
nNRP analog; or a pharmaceutically acceptable salt of a nNRP
analog.
[0085] A "neonatal Neutrophil Inhibitory Factor peptide" or "nNIF
peptide" is defined herein as a nNIF which is naturally occurring
in mammals.
[0086] A "neonatal NIF-related peptide" or "nNRP" is defined herein
as a Cancer-Associated SCM-Recognition, Immune Defense Suppression,
and Serine Protease Protection Peptide (CRISPP) which is naturally
occurring in humans; A1ATm.sup.358, which has been shown to inhibit
NET formation; and other nNIF-related peptides; and as analogs of
naturally occurring forms of nNRPs that inhibit NETosis and/or the
formation of NETs and are structurally altered, relative to a given
human nNRP, by at least one amino acid addition, deletion,
substitution, or by incorporation of one or more amino acids with a
blocking group.
[0087] "Inflammatory disorders" are defined herein as disorders
characterized by pathological inflammation. Inflammatory disorders
include, but are not limited to, conditions associated with
infection, autoimmunity, and allergy. Inflammatory disorders as
defined herein may include, but are not limited to, acute
respiratory distress syndrome (ARDS), bronchopulmonary dysplasia
(BPD), chronic obstructive pulmonary disease (COPD), cystic
fibrosis, inflammation in cancer and its complications,
inflammatory bowel disease (IBD), inflammatory lung disease (ILD),
influenza-induced pneumonitis, necrotizing enterocolitis (NEC),
neonatal chronic lung disease (CLD), periodontitis, pre-eclampsia,
retinopathy of prematurity (ROP), sepsis, systemic inflammatory
response syndrome (SIRS), thrombosis, transfusion-related acute
lung injury (TRALI), vasculitis, autoimmune syndromes including,
but not limited to, rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), and Wegener's granulomatosis (WG), and
disorders of nonresolved inflammation. There are other inflammatory
disorders not listed herein but known to those skilled in the art.
For example, see Kumar et al., Robbins and Cotran Pathologic Basis
of Disease, pp. 43-77, 8th Edition, 2010, Saunders Elsevier,
Philadelphia, Pa.; Nathan, Nature, 2002, 420: 846-852; and Amulic
et al., Annu Rev Immunol, 2012, 30: 459-489.
[0088] The phrase "does not globally depress polymorphonuclear
leukocyte (PMN) function," when used in connection with a NIP,
means that although the NIP may inhibit or substantially inhibit
NETosis, the NIP does not inhibit or substantially inhibit other
PMN functions. Other PMN functions include, but are not limited to,
chemotaxis, chemokine synthesis and secretion, cytokine synthesis
and secretion, extracellular bacterial killing, intracellular
bacterial killing, phagocytosis, and/or reactive oxygen species
(ROS) generation. Methods of assaying these functions are known in
the art. For example, Example 11 describes methods of assaying
phagocytic bacterial killing.
[0089] Methods
[0090] This disclosure relates to therapeutic and related uses of
NET Inhibitory Peptides (NIPs), neonatal NET-Inhibitory Factors
(nNIFs), nNIF analogs, nNIF-Related Peptides (nNRPs), and nNRP
analogs, particularly for inhibiting NETosis and/or the formation
of neutrophil extracellular traps (NETs).
[0091] A first aspect of the disclosure relates to methods of
treating inflammatory disorders.
[0092] In certain embodiments, this disclosure provides methods of
treating a patient having an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a NIP, or a pharmaceutically
acceptable salt of a NIP, and a pharmaceutically acceptable carrier
to reduce a pathological effect or symptom of the inflammatory
disorder. The pathological effects or symptoms may include one or
more of the following: pain, heat, redness, swelling and/or edema,
hypotension, fibrosis and/or post-inflammatory fibrosis, end organ
failure (i.e., renal, cardiac, hepatic), tissue damage, and/or loss
of function.
[0093] In some embodiments, this disclosure provides methods of
treating a patient having an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF, or a pharmaceutically
acceptable salt of a nNIF, and a pharmaceutically acceptable
carrier to reduce a pathological effect or symptom of the
inflammatory disorder.
[0094] In other embodiments, the disclosure provides methods of
treating a patient having an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF analog, or a
pharmaceutically acceptable salt of a nNIF analog, and a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the inflammatory disorder.
[0095] In yet other embodiments, the disclosure provides methods of
treating a patient having an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP, or a pharmaceutically
acceptable salt of a nNRP, and a pharmaceutically acceptable
carrier to reduce a pathological effect or symptom of the
inflammatory disorder.
[0096] In still other embodiments, the disclosure provides methods
of treating a patient having an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising an nNRP analog, or a
pharmaceutically acceptable salt of a nNRP analog, and a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the inflammatory disorder.
[0097] In some embodiments, the patient may be a mammal. In certain
embodiments the patient may be a human. Any patient or subject
requiring inhibition of NETosis and/or NET formation may
potentially be a candidate for treatment with a NIP, a
pharmaceutically acceptable salt of a NIP, a nNIF, a
pharmaceutically acceptable salt of a nNIF, a nNIF analog, a
pharmaceutically acceptable salt of a nNIF analog, a nNRP, a
pharmaceutically acceptable salt of a nNRP, a nNRP analog, and/or a
pharmaceutically acceptable salt of a nNRP analog.
[0098] In some embodiments, the inflammatory disorder may at least
partially involve or be at partially caused by neutrophil
extracellular trap (NET) formation and/or NETosis. In some
embodiments, the inflammatory disorder may be an acute inflammatory
disorder, a chronic inflammatory disorder, and/or an immune
disorder. In other embodiments, the inflammatory disorder may be an
autoimmunity disorder. In yet other embodiments, the inflammatory
disorder may be a disorder of coagulation.
[0099] In some embodiments, the inflammatory disorder may be one or
more of, but not limited to, acute respiratory distress syndrome
(ARDS), bronchopulmonary dysplasia (BPD), chronic obstructive
pulmonary disease (COPD), cystic fibrosis, inflammation in cancer
and its complications, inflammatory bowel disease (IBD),
inflammatory lung disease (ILD), influenza-induced pneumonitis,
necrotizing enterocolitis (NEC), neonatal chronic lung disease
(CLD), periodontitis, pre-eclampsia, retinopathy of prematurity
(ROP), sepsis, systemic inflammatory response syndrome (SIRS),
thrombosis, transfusion-related acute lung injury (TRALI),
vasculitis, autoimmune syndromes including, but not limited to,
rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and
Wegener's granulomatosis (WG), and disorders of nonresolved
inflammation. There are other inflammatory disorders not listed
herein but known to those skilled in the art. For example, see
Kumar et al., Robbins and Cotran Pathologic Basis of Disease, pp.
43-77, 8th Edition, 2010, Saunders Elsevier, Philadelphia, Pa.;
Nathan, Nature, 2002, 420: 846-852; and Amulic et al., Annu Rev
Immunol, 2012, 30: 459-489.
[0100] In some embodiments, the pharmaceutical composition may
substantially inhibit NET formation and/or NETosis. In other
embodiments, the pharmaceutical composition may inhibit or
substantially inhibit NET-mediated inflammatory tissue damage.
[0101] The particular form of NIP, nNIF, nNIF analog, nNRP, nNRP
analog, and/or salt thereof selected for inhibiting NETosis and/or
NET formation can be prepared by a variety of techniques known for
generating peptide products. For example, vertebrate forms of nNIF
and nNRP can be obtained by extraction from the natural source,
using an appropriate combination of protein isolation techniques.
Other techniques are also within the scope of this disclosure.
[0102] In certain embodiments, NIPs, nNIFs, nNIF analogs, nNRPs,
nNRP analogs, and/or salts thereof can be synthesized using
standard techniques of peptide chemistry and can be assessed for
inhibition of NETosis and/or NET formation activity. With respect
to synthesis, the selected NIP, nNIF, nNIF analog, nNRP, nNRP
analog, and/or salt thereof can be prepared by a variety of
techniques for generating peptide products. Those NIPs, nNIFs, nNIF
analogs, nNRPs, nNRP analogs, and/or salts thereof that incorporate
only L-amino acids can be produced in commercial quantities by
application of recombinant DNA technology. For this purpose, DNA
coding for the desired NIP, nNIF, nNIF analog, nNRP, and/or nNRP
analog is incorporated into an expression vector and transformed
into a host cell (e.g., yeast, bacteria, or a mammalian cell) which
is then cultured under conditions appropriate for NIP, nNIF, nNIF
analog, nNRP, and/or nNRP analog expression. A variety of gene
expression systems have been adapted for this purpose, and
typically drive expression of the desired gene from expression
regulatory elements used naturally by the chosen host.
[0103] In an approach applicable to the production of a selected
NIP, nNIF, nNIF analog, nNRP, and/or nNRP analog, and one that may
be used to produce a NIP, nNIF, nNIF analog, nNRP, and/or nNRP
analog that incorporates non-genetically encoded amino acids and N-
and C-terminally derivatized forms, the techniques of automated
peptide synthesis may be employed, general descriptions of which
appear, for example, in Stewart and Young, Solid Phase Peptide
Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford,
Ill.; Bodanszky and Bodanszky, The Practice of Peptide Synthesis,
1984, Springer-Verlag, New York, N.Y.; and Applied Biosystems 430A
Users Manual, 1987, ABI Inc., Foster City, Calif. In these
techniques, a NIP, nNIF, nNIF analog, nNRP, and/or nNRP analog is
grown from its C-terminal, resin-conjugated residue by the
sequential addition of appropriately protected amino acids, using
either the 9-fluoroenylmethyloxycarbonyl (Fmoc) or
tert-butyloxycarbonyl (t-Boc) protocols, as described for instance
by Orskov et al., FEBS Lett, 1989, 247(2): 193-196.
[0104] Once the desired NIP, nNIF, nNIF analog, nNRP, and/or nNRP
analog has been synthesized, cleaved from the resin and fully
deprotected, the peptide may then be purified to ensure the
recovery of a single oligopeptide having the selected amino acid
sequence. Purification may be achieved using any of the standard
approaches, which include, but are not limited to, reversed-phase
high-pressure liquid chromatography (RP-HPLC) on alkylated silica
columns (e.g., C4-, C8-, or C18-silica). Such column fractionation
is generally accomplished by running linear gradients (e.g.,
10-90%) of increasing percent organic solvent (e.g., acetonitrile,
in aqueous buffer) usually containing a small amount (e.g., 0.1%)
of pairing agent such as trifluoroacetic acid (TFA) or
triethanolamine (TEA). Alternatively, ion-exchange HPLC can be
employed to separate peptide species on the basis of their charge
characteristics. Column fractions are collected, and those
containing peptide of the desired and/or required purity are
optionally pooled. In one embodiment of the invention, the NIP,
nNIF, nNIF analog, nNRP, and/or nNRP analog may then be treated in
the established manner to exchange the cleavage acid (e.g., TFA)
with a pharmaceutically acceptable acid, such as acetic,
hydrochloric, phosphoric, maleic, tartaric, succinic, and the like,
to generate a pharmaceutically acceptable acid addition salt of the
peptide.
[0105] Analogs of human NIPs, nNIFs, and/or nNRPs can be generated
using standard techniques of peptide chemistry and can be assessed
for inhibition of NETosis and/or NET formation activity, all
according to the guidance provided herein. Particularly preferred
analogs of the invention are those based upon the sequences of
human nNIF (SEQ ID NO: 1) and/or CRISPP (SEQ ID NO: 2), as follows
(wherein X can be any naturally occurring amino acid):
TABLE-US-00001 SEQ ID NO: 1
KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPL FMGKVVNPTQK SEQ
ID NO: 2 MXIPPEVKFNKPFVFLMIDQNTKVPLFMGK
[0106] Any substitution, addition, or deletion of an amino acid or
amino acids of a NIP, nNIF, and/or nNRP that does not destroy the
NET-inhibitory activity of the NIP, nNIF, and/or nNRP may be
usefully employed in this disclosure. In certain embodiments, the
NIP, nNIF, and/or nNRP analogs are at least as active as the native
human NIP, nNIF, and/or nNRP. NET-inhibitory activity may be
determined in vitro as described in this disclosure. In other
embodiments, the NIP, nNIF, and/or nNRP analog has one or more
enhanced properties compared with the native human NIP, nNIF,
and/or nNRP. For example, such analogs may exhibit enhanced serum
stability, enhanced receptor binding and enhanced
signal-transducing activity. Other modifications to NIPs, nNIFs,
nNIF analogs, nNRPs, and/or nNRP analogs that may usefully be
employed in this disclosure are those which render the molecule
resistant to oxidation.
[0107] A researcher may determine whether a particular NIP, nNIF,
nNIF analog, nNRP, nNRP analog, and/or salt thereof may be used to
treat an inflammatory disorder by administering the peptide or
analog to individuals who have the inflammatory disorder. The
researcher may then determine, using diagnostic biomarkers, whether
the individuals thus treated show decreased inflammation and
improvement of the inflammatory condition.
[0108] The disclosure also encompasses non-conservative
substitutions of amino acids in any vertebrate NIP, nNIF, and/or
nNRP sequence, provided that the non-conservative substitutions
occur at amino acid positions known to vary in NIPs, nNIFs, and/or
nNRPs isolated from different species. Non-conserved residue
positions are readily determined by aligning known vertebrate NIP,
nNIF, and/or nNRP sequences.
[0109] For administration to patients, the NIP, nNIF, nNIF analog,
nNRP, nNRP peptide, and/or salt thereof, may be provided in
pharmaceutically acceptable form (e.g., as a preparation that is
sterile-filtered, e.g., through a 0.22.mu. filter, and
substantially pyrogen-free). It may be desired that the NIP, nNIF,
and/or nNRP peptide to be formulated migrates as a single or
individualized peak on HPLC, exhibits uniform and authentic amino
acid composition and sequence upon analysis thereof, and otherwise
meets standards set by the various national bodies which regulate
quality of pharmaceutical products.
[0110] The aqueous carrier or vehicle may be supplemented for use
as an injectable with an amount of gelatin that serves to depot the
NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof at
or near the site of injection, for its slow release to the desired
site of action. Concentrations of gelatin effective to achieve the
depot effect are expected to lie in the range from 10-20%.
Alternative gelling agents, such as hyaluronic acid (HA), may also
be useful as depoting agents.
[0111] The NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs, and/or
salts thereof of the present disclosure may also be formulated as
slow release implantation formulations for extended and sustained
administration of the NIP, nNIF, nNIF analog, nNRP, nNRP analog,
and/or salt thereof. Examples of such sustained release
formulations include composites of biocompatible polymers, such as
poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose,
hyaluronic acid, collagen, and the like. The structure, selection,
and use of degradable polymers in drug delivery vehicles have been
reviewed in several publications, including, Domb et al., Polym
Advan Technol, 1992, 3: 279-292. Additional guidance in selecting
and using polymers in pharmaceutical formulations can be found in
the text by Chasin and Langer (eds.), Biodegradable Polymers as
Drug Delivery Systems, Vol. 45 of Dekker, Drugs and the
Pharmaceutical Sciences, 1990, New York, N.Y. Liposomes may also be
used to provide for the sustained release of a nNIF, nNIF analog,
nNRP, nNRP analog, and/or salt thereof. Details concerning how to
use and make liposomal formulations of drugs of interest can be
found in, among other places, U.S. Pat. Nos. 4,944,948; 5,008,050;
4,921,706; 4,927,637; 4,452,747; 4,016,100; 4,311,712; 4,370,349;
4,372,949; 4,529,561; 5,009,956; 4,725,442; 4,737,323; and
4,920,016. Sustained release formulations may be of particular
interest when it is desirable to provide a high local concentration
of a NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or salt thereof
(e.g., near the site of inflammation to inhibit NETosis and/or NET
formation, etc.).
[0112] The NIPs, nNIFs, nNIF analogs, nNRPs, nNRP analogs, and/or
salts thereof of the present disclosure may also be incorporated
into a device or devices, both implanted or topical, for extended
and sustained administration of the NIP, nNIF, nNIF analog, nNRP,
nNRP analog, and/or salt thereof.
[0113] For therapeutic use, the chosen NIP, nNIF, nNIF analog,
nNRP, nNRP analog, and/or salt thereof may be formulated with a
carrier that is pharmaceutically acceptable and is appropriate for
delivering the peptide by the chosen route of administration.
Suitable pharmaceutically acceptable carriers are those used
conventionally with peptide-based drugs, such as diluents,
excipients and the like. Reference may be made to Remington: The
Science and Practice of Pharmacy, 22nd Edition, 2012,
Pharmaceutical Press, London, UK. In one embodiment of the
invention, the compounds may be formulated for administration by
infusion or by injection (e.g., subcutaneously, intramuscularly, or
intravenously), and may be accordingly utilized as aqueous
solutions in sterile and pyrogen-free form and optionally buffered
to physiologically tolerable pH (e.g., a slightly acidic or
physiological pH). Thus, the compounds may be administered in a
vehicle such as distilled water, saline, phosphate buffered saline,
or 5% dextrose solution. Water solubility of the NIP, nNIF, nNIF
analog, nNRP, nNRP analog, and/or salt thereof may be enhanced, if
desired, by incorporating a solubility enhancer, such as acetic
acid.
[0114] Another aspect of the disclosure relates to methods of
treating complications of prematurity.
[0115] In embodiments, this disclosure provides for methods of
treating a patient having a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a NIP, or a pharmaceutically
acceptable salt of a NIP, and a pharmaceutically acceptable carrier
to reduce a pathological effect or symptom of the complication of
prematurity, such as the prolonged need for oxygen support
associated with neonatal chronic lung disease or the need for
surgical intervention or prolonged total parenteral nutrition in
infants that develop necrotizing enterocolitis.
[0116] In some embodiments, this disclosure provides for methods of
treating a patient having a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF, or a pharmaceutically
acceptable salt of a nNIF, and a pharmaceutically acceptable
carrier to reduce a pathological effect or symptom of the
complication of prematurity.
[0117] In other embodiments, the disclosure provides methods of
treating a patient having a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF analog, or a
pharmaceutically acceptable salt of a nNIF analog, and a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the complication of prematurity.
[0118] In yet other embodiments, the disclosure provides methods of
treating a patient having a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP, or a pharmaceutically
acceptable salt of a nNRP, and a pharmaceutically acceptable
carrier to reduce a pathological effect or symptom of the
complication of prematurity.
[0119] In still other embodiments, the disclosure provides methods
of treating a patient having a complication of prematurity
comprising administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP analog, or a
pharmaceutically acceptable salt of a nNRP analog, and a
pharmaceutically acceptable carrier to reduce a pathological effect
or symptom of the complication of prematurity.
[0120] In some embodiments, the patient may be a mammal. In certain
embodiments the patient may be a human.
[0121] In some embodiments, the complication of prematurity may at
least partially involve or be at least partially caused by
neutrophil extracellular trap (NET) formation and/or NETosis. In
certain embodiments, the pharmaceutical composition may
substantially inhibit NET formation and/or NETosis. In other
embodiments, the pharmaceutical composition may inhibit or
substantially inhibit NET-mediated inflammatory tissue damage.
[0122] In some embodiments, the complication of prematurity may be
one or more of, but not limited to, necrotizing enterocolitis
(NEC), respiratory distress syndrome (RDS), pneumonia,
bronchopulmonary dysplasia (BPD), neonatal chronic lung disease
(CLD), neurodevelopmental delay, retinopathy of prematurity (ROP),
and/or sepsis.
[0123] A further aspect of the disclosure relates to methods of
prophylaxis against inflammatory disorders.
[0124] In some embodiments, the disclosure provides for methods of
prophylaxis against an inflammatory disorder in a patient at risk
of developing an inflammatory disorder, comprising administering to
the patient an effective amount of a pharmaceutical composition
comprising a NIP, or a pharmaceutically acceptable salt of a NIP,
and a pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the inflammatory
disorder.
[0125] In some embodiments, the disclosure provides for methods of
prophylaxis against an inflammatory disorder in a patient at risk
of developing an inflammatory disorder, comprising administering to
the patient an effective amount of a pharmaceutical composition
comprising a nNIF, or a pharmaceutically acceptable salt of a nNIF,
and a pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the inflammatory
disorder.
[0126] In other embodiments, the disclosure provides for methods of
prophylaxis against an inflammatory disorder in a patient at risk
of developing an inflammatory disorder comprising administering to
the patient an effective amount of a pharmaceutical composition
comprising a nNIF analog, or a pharmaceutically acceptable salt of
a nNIF analog, and a pharmaceutically acceptable carrier to reduce
the risk of developing a pathological effect or symptom of the
inflammatory disorder.
[0127] In yet other embodiments, the disclosure provides for
methods of prophylaxis against an inflammatory disorder in a
patient at risk of developing an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP, or a pharmaceutically
acceptable salt of a nNRP, and a pharmaceutically acceptable
carrier to reduce the risk of developing a pathological effect or
symptom of the inflammatory disorder.
[0128] In still other embodiments, the disclosure provides for
methods of prophylaxis against an inflammatory disorder in a
patient at risk of developing an inflammatory disorder comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP analog, or a
pharmaceutically acceptable salt of the nNRP analog, and a
pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the inflammatory
disorder.
[0129] In some embodiments, the patient may be a mammal. In other
embodiments the patient may be a human.
[0130] In some embodiments, the inflammatory disorder may at least
partially involve or be at partially caused by neutrophil
extracellular trap (NET) formation and/or NETosis. In some
embodiments, the inflammatory disorder may be an acute inflammatory
disorder. In other embodiments, the inflammatory disorder may be a
chronic inflammatory disorder. In other embodiments, the
inflammatory disorder may be an autoimmunity disorder. In yet other
embodiments, the inflammatory disorder may be a disorder of
coagulation.
[0131] In some embodiments, the inflammatory disorder may be one or
more of, but not limited to, the inflammatory disorders defined
and/or listed above.
[0132] In some embodiments, the pharmaceutical composition may
substantially inhibit NET formation and/or NETosis. In other
embodiments, the pharmaceutical composition may inhibit or
substantially inhibit NET-mediated inflammatory tissue damage.
[0133] Another aspect of the disclosure relates to methods of
prophylaxis against complications of prematurity.
[0134] In embodiments, this disclosure provides methods of
prophylaxis against complications of prematurity in a patient at
risk of developing a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a neonatal NIP, or a
pharmaceutically acceptable salt of a NIP, and a pharmaceutically
acceptable carrier to reduce the risk of developing a pathological
effect or symptom of the complication of prematurity.
[0135] In some embodiments, this disclosure provides methods of
prophylaxis against complications of prematurity in a patient at
risk of developing a complication of prematurity comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a neonatal nNIF, or a
pharmaceutically acceptable salt of a nNIF, and a pharmaceutically
acceptable carrier to reduce the risk of developing a pathological
effect or symptom of the complication of prematurity.
[0136] In certain embodiments, the disclosure provides methods of
prophylaxis against complications of prematurity in a patient at
risk of developing a complication of prematurity, comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF analog, or a
pharmaceutically acceptable salt of a nNIF analog, and a
pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the complication of
prematurity.
[0137] In yet other embodiments, the disclosure provides methods of
prophylaxis against complications of prematurity in a patient at
risk of developing a complication of prematurity, comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNIF-Related Peptide
(nNRP), or a pharmaceutically acceptable salt of a nNRP, and a
pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the complication of
prematurity.
[0138] In still other embodiments, the disclosure provides methods
of prophylaxis against complications of prematurity in a patient at
risk of developing a complication of prematurity, comprising
administering to the patient an effective amount of a
pharmaceutical composition comprising a nNRP analog, or a
pharmaceutically acceptable salt of a nNRP analog, and a
pharmaceutically acceptable carrier to reduce the risk of
developing a pathological effect or symptom of the complication of
prematurity.
[0139] In some embodiments, the patient may be a mammal. In other
embodiments the patient may be a human.
[0140] In some embodiments, the complication of prematurity may at
least partially involve or be at least partially caused by
neutrophil extracellular trap (NET) formation and/or NETosis. In
other embodiments, the pharmaceutical composition may substantially
inhibit NET formation and/or NETosis. In yet other embodiments, the
pharmaceutical composition may inhibit or substantially inhibit
NET-mediated inflammatory tissue damage.
[0141] In other embodiments, the complication of prematurity may be
one or more of, but not limited to, necrotizing enterocolitis
(NEC), respiratory distress syndrome (RDS), pneumonia,
bronchopulmonary dysplasia (BPD), neonatal chronic lung disease
(CLD), neurodevelopmental delay, retinopathy of prematurity (ROP),
and/or sepsis.
[0142] Pharmaceutical Compositions
[0143] In a further aspect, this disclosure relates to
pharmaceutical compositions comprising NIPs.
[0144] In some embodiments, the pharmaceutical composition may
comprise a neonatal NET-Inhibitory Factor (nNIF), or a
pharmaceutically acceptable salt of a nNIF, and a pharmaceutically
acceptable carrier. In another embodiment, the pharmaceutical
composition may comprise a nNIF analog, or a pharmaceutically
acceptable salt of a nNIF analog, and a pharmaceutically acceptable
carrier. In yet other embodiments, the pharmaceutical composition
may comprise a nNIF-Related Peptide (nNRP), or a pharmaceutically
acceptable salt of a nNRP, and a pharmaceutically acceptable
carrier. In still other embodiments, the pharmaceutical composition
may comprise a nNRP analog, or a pharmaceutically acceptable salt
of a nNRP analog, and a pharmaceutically acceptable carrier.
[0145] In certain embodiments, the pharmaceutical composition may
comprise nNIF, or the salt thereof, and the nNIF, or the salt
thereof, may comprise the amino acid sequence:
TABLE-US-00002 SEQ ID NO: 1:
KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPL FMGKVVNPTQK
[0146] In other embodiments, at least one amino acid of nNIF, the
salt of the nNIF, the nNIF analog, the salt of the nNIF analog, the
nNRP, the salt of the nNRP, the nNRP analog, or the salt of the
nNRP analog, may be bound to a chemical modifier. In some
embodiments, the chemical modifier may be selected from at least
one of a lipid, a polyethylene glycol (PEG), a saccharide, or any
other suitable molecule. Other chemical modifications of the
pharmaceutical composition, for example, cationization,
methylization, and cyclization, are also within the scope of this
disclosure.
[0147] Attachment of a lipid to the peptide (lipidization) may
increase lipophilicity of the pharmaceutical composition.
[0148] Attachment of a PEG to the peptide (PEGylation) increases
the molecular weight of the peptide. In some embodiments,
PEGylation may improve solubility of the pharmaceutical
composition. In other embodiments, PEGylation may reduce dosage
frequency and/or toxicity of the pharmaceutical composition. In
other embodiments, PEGylation may extend circulating life of the
pharmaceutical composition, and/or extend stability of the
pharmaceutical composition, and/or may enhance protection of the
pharmaceutical composition from proteolytic degradation. PEGylation
may also reduce immunogenicity and/or antigenicity of the
pharmaceutical composition.
[0149] Attachment of one or more saccharides to the peptide
(glycosylation) may serve a variety of functional and/or structural
roles in the pharmaceutical composition. Glycosylation may improve
delivery of the pharmaceutical composition to a target or to
targets of choice. Glycosylation may also reduce the toxicity of
the pharmaceutical composition.
[0150] In some embodiments, the pharmaceutical composition
comprising nNIF, the salt of the nNIF, the nNIF analog, the salt of
the nNIF analog, the nNRP, the salt of the nNRP, the nNRP analog,
or the salt of the nNRP analog, may be present in an amount
effective to inhibit, or to substantially inhibit, damage selected
from at least one of inflammatory tissue injury and/or inflammatory
vascular injury.
[0151] In some embodiments, the pharmaceutical composition
comprising nNIF, the salt of the nNIF, the nNIF analog, the salt of
the nNIF analog, the nNRP, the salt of the nNRP, the nNRP analog,
or the salt of the nNRP analog, may not globally depress functions
of polymorphonuclear leukocytes (PMNs). The functions of PMNs
include, but are not limited to, chemotaxis, phagocytosis, reactive
oxygen species (ROS) generation, cytokine/chemokine synthesis and
secretion, NET formation/NETosis, and/or
intracellular/extracellular bacterial killing. In certain
embodiments, the pharmaceutical composition may not inhibit or
substantially inhibit PMN phagocytosis. In other embodiments, the
pharmaceutical composition may not inhibit or substantially inhibit
PMN chemotaxis. In yet other embodiments, the pharmaceutical
composition may not inhibit or substantially inhibit generation of
ROS. In other embodiments, the pharmaceutical composition may not
inhibit or substantially inhibit PMN intracellular bacterial
killing.
[0152] In some embodiments, the pharmaceutical composition may
comprise a nNIF analog, a salt of a nNIF analog, a nNRP analog, or
a salt of a nNRP analog, and the analog or the salt of the analog
may not be a naturally occurring analog or salt of the analog.
[0153] In some embodiments, the pharmaceutical composition may be
present in an amount effective to inhibit, or to substantially
inhibit, NET formation and/or NETosis. In some embodiments, the NET
formation and/or NETosis may be stimulated by a bacterium, a
fungus, a parasite, a virus, and/or any other appropriate
stimulator of NET formation and/or NETosis. In certain embodiments,
the virus may be a hemorrhagic fever virus. Hemorrhagic fever
viruses are described, e.g., in Borio et al., JAMA, 2002, 287(18):
2391-2405 and include, but are not limited to, filoviruses such as
Ebola virus and Marburg virus, arenaviruses such as Lassa virus,
hantaviruses, and flaviviruses such as dengue virus and yellow
fever virus. In other embodiments, the NET formation and/or NETosis
may be stimulated by one or more bacterial species, including, but
not limited to, Bacillus species, Escherichia species, Francisella
species, Streptococcus species, Staphylococcus species, Yersinia
species, and/or any other appropriate gram-negative or
gram-positive bacterium or bacteria. In embodiments, the Bacillus
species may be Bacillus anthracis (anthrax). In embodiments, the
Escherichia species may be Escherichia coli. In embodiments, the
Francisella species may be Francisella tularensis (tularemia). In
embodiments, the Staphylococcus species may be Staphylococcus
aureus.
[0154] In other embodiments, the NET formation and/or NETosis may
be stimulated by beta-defensin 1, HIV-1, lipopolysaccharide (LPS),
phorbol myristate acetate (PMA), and/or Staphylococcus aureus
alpha-toxin.
[0155] In some embodiments, the pharmaceutical composition may
comprise nNRP, Cancer-Associated SCM-Recognition, Immune Defense
Suppression, and Serine Protease Protection Peptide (CRISPP),
and/or a CRISPP analog. In some other embodiments, the
pharmaceutical composition may comprise A1ATm.sup.358, and/or an
A1ATm.sup.358 analog, as A1ATm.sup.358 also inhibits NET formation.
In other embodiments, the pharmaceutical may comprise another nNRP.
In other embodiments, the nNRP may be an isolated and purified
component of umbilical cord blood.
[0156] In an additional aspect, this disclosure relates to
compositions for inhibiting the formation of NETs and/or NETosis in
a mammal.
[0157] In some embodiments, a composition for inhibiting the
formation of NETs and/or NETosis in a mammal may comprise an nNIF,
a pharmaceutically acceptable salt of the nNIF, a nNIF analog, a
pharmaceutically acceptable salt of the nNIF analog, an nNRP, a
pharmaceutically acceptable salt of the nNRP, a nNRP analog, or a
pharmaceutically acceptable salt of the nNRP analog, and a
pharmaceutically acceptable carrier. In certain embodiments the
mammal may be a human.
[0158] Isolated and Purified Peptides
[0159] In a further aspect, this disclosure relates to a
NET-inhibitory peptide (NIP).
[0160] In some embodiments, the NIP may be an isolated and purified
nNIF protein comprising SEQ ID NO: 1. In certain other embodiments,
the isolated and purified nNIF protein may comprise at least
twenty-four contiguous amino acids of SEQ ID NO: 1. In yet other
embodiments, the isolated and purified nNIF protein may comprise at
least twelve contiguous amino acids of SEQ ID NO: 1. In still other
embodiments, the isolated and purified nNIF protein may comprise at
least six contiguous amino acids of SEQ ID NO: 1.
[0161] In some embodiments, the NIP may be an isolated and purified
nNIF protein wherein the sequence may be at least eighty percent
identical to SEQ ID NO: 1. In other embodiments, the isolated and
purified nNIF may be at least sixty percent identical to SEQ ID NO:
1. In yet other embodiments, the isolated and purified nNIF may be
at least forty percent identical to SEQ ID NO: 1. In still other
embodiments, the isolated and purified nNIF may be at least twenty
percent identical to SEQ ID NO: 1.
[0162] In another aspect, this disclosure relates to a nNIF protein
analog. In some embodiments, the nNIF protein analog may be an
isolated and purified nNIF analog.
[0163] An effective dosage and treatment protocol may be determined
by conventional means, e.g., by starting with a low dose in
laboratory animals and then increasing the dosage while monitoring
the effects, and systematically varying the dosage regimen as well.
Numerous factors may be taken into consideration by a clinician
when determining an optimal dosage for a given subject. Primary
among these is whether any NIPs are normally circulating in the
plasma and, if so, the amount of any such NIPs. Additional factors
include the size of the patient, the age of the patient, the
general condition of the patient, the particular disorder being
treated, the severity of the disorder, the presence of other drugs
in the patient, the in vivo activity of the NIP, nNIF, nNIF analog,
nNRP, nNRP analog, salt thereof, and the like. The trial dosages
may be chosen after consideration of the results of animal studies
and the clinical literature.
[0164] There are many specific therapeutic regimens used to assess
whether a molecule has a desired effect. A researcher faced with
the task of determining whether a particular NIP, nNIF, nNIF
analog, nNRP, and/or nNRP analog may be used for inhibition of
NETosis and/or NET formation would choose the appropriate regimen
to make this determination.
[0165] Delivery methods and formulations useful for administering
peptides to individuals are known in the art, and a skilled person
would be able to determine the suitability of any particular method
of delivery of a peptide to an individual for particular
circumstances. For the purposes of illustration only, the following
examples of methods and formulations for administering peptides to
individuals are provided.
[0166] Peptides may be administered to individuals orally; however,
actions of the digestive system may reduce the bioavailability of
the peptide. In order to increase peptide oral bioavailability,
peptides may be administered in formulations containing enzyme
inhibitors, or the peptides may be administered as part of a
micelle, nanoparticle, or emulsion in order to protect the peptide
from digestive activity.
[0167] Peptides may also be administered by means of an injection.
The peptides may be injected subcutaneously, intramuscularly, or
intravenously. Further disclosure regarding methods of
administering peptides through injection is found, e.g., in U.S.
Pat. No. 5,952,301.
[0168] Peptides may further be administered by pulmonary delivery.
A dry powder inhalation system may be used, wherein peptides are
absorbed through the tissue of the lungs, allowing delivery without
injection, while bypassing the potential reduction in
bioavailability seen with oral administration. See Onoue et al.,
Expert Opin Ther Pat, 2008, 18: 429.
[0169] For use in inhibiting NETosis and/or NET formation in a
mammal, including a human, the present disclosure provides in one
of its aspects a package or kit, in the form of a sterile-filled
vial or ampoule, that contains a NETosis and/or NET formation
inhibiting amount of a NIP, nNIF, nNIF analog, nNRP, nNRP analog,
and/or salt thereof in either unit dose or multi-dose amounts,
wherein the package or kit incorporates a label instructing use of
its contents for the inhibition of such NETosis and/or NET
formation. In one embodiment of the invention, the package or kit
contains the NIP, nNIF, nNIF analog, nNRP, nNRP analog, and/or salt
thereof and the desired carrier, as an administration-ready
formulation. Alternatively, and according to another embodiment of
the invention, the package or kit provides the NIP, nNIF, nNIF
analog, nNRP, nNRP analog, and/or salt thereof, in a form, such as
a lyophilized form, suitable for reconstitution in a suitable
carrier, such as phosphate-buffered saline.
[0170] In one embodiment, the package or kit is a sterile-filled
vial or ampoule containing an injectable solution which comprises
an effective, NETosis and/or NET formation inhibiting amount of
NIP, nNIF, nNIF analog, nNRP, nNRP analog and/or salt thereof
dissolved in an aqueous vehicle.
EXAMPLES
[0171] To further illustrate these embodiments, the following
examples are provided. These examples are not intended to limit the
scope of the claimed invention, which should be determined solely
on the basis of the attached claims.
Example 1
Comparing Human Adult and Neonatal PMNs
[0172] NET-formation was assessed in human and neonatal PMNs. In
contrast to adult human PMNs, as shown in FIG. 1B, neonatal PMNs
isolated from cord blood do not form NETs. Further, NET-mediated
bacterial killing was assessed in human adult and neonatal PMNs. As
indicated in FIG. 1C, neonatal PMNs demonstrate significantly
decreased total and NET-mediated bacterial killing as compared to
adult PMNs. * p<0.05, ** p<0.001.
Example 2
Inducing NET Formation by S. aureus Sepsis
[0173] NET-formation was assessed in control and stimulated human
PMNs at a 1 hour time point. Human PMNs were stimulated with either
LPS, control plasma, or with plasma isolated from patients with S.
aureus sepsis. Plasma from patients with S. aureus sepsis induces
NET formation by human PMNs, as shown in FIGS. 2A-2C.
Example 3
Observing Impaired NET Formation at Birth
[0174] PMNs were isolated from the cord blood of infants born at
less than 30 weeks gestation and, subsequently, from serially drawn
peripheral blood from the same infants through the first 60 days of
life. As shown in FIGS. 3A-3C, NET formation was assessed
qualitatively and quantitatively, and impaired NET formation by
PMNs was observed at the time of birth. In contrast, robust NET
formation by PMNs was observed from blood samples isolated on day
of life 3 or later.
Example 4
Demonstrating NET Formation Inhibitor in Cord Blood
[0175] PMNs from healthy adult donors and from 28 day old preterm
infants were pre-incubated for one hour with thawed cord blood
plasma from that particular preterm infant or with autologous
plasma collected that day from either the preterm infant peripheral
blood sample or the blood sample from the healthy adult. NET
formation was then assessed in vitro following LPS-stimulation (100
ng/mL) for 2 hours. Cord blood plasma pre-incubation significantly
decreased NET formation by both neonatal and adult LPS-stimulated
PMNs as compared to PMNs pre-incubated with control plasma (see
FIGS. 3B and 14C).
[0176] As illustrated in FIGS. 3B and 14C, human cord blood
pre-incubation experiments demonstrate that cord blood plasma can
inhibit NET formation by day of life 28 autologous preterm infant
PMNs and by healthy adult donor PMNs, while pre-incubation with
autologous plasma isolated from the respective study subjects on
that day may not. FIG. 3B shows NET-associated, extracellular DNA
and nuclear DNA (60.times. magnification).
[0177] Extracellular histone H.sub.3 release was used as a
quantifiable surrogate for NET formation as depicted in FIG. 14C.
As illustrated, extracellular histone content (fold change over
baseline) is represented on the y-axis, and data from five preterm
infant's PMNs on day of life 28 and five adult's PMNs, from the
above-described plasma switch experiments, are represented on the
x-axis. * denotes p<0.05 LPS/Adult versus LPS/Preterm, **
denotes p<0.01 LPS/Preterm versus cord blood LPS/Preterm, and
.dagger. denotes p<0.001 LPS/Adult versus cord blood LPS/Adult.
The one-way ANOVA statistical tool with Tukey's post-hoc testing
was employed.
[0178] Cord blood and peripheral blood plasma switch experiments
demonstrated that autologous cord blood plasma inhibits NET
formation by NET-competent PMNs isolated from the same infant on
day of life 28, and also by PMNs isolated from healthy adults,
indicating that an inhibitory factor that blocks PMN NET formation
is present in cord blood, but that activity of the factor
disappears or is dramatically reduced after birth (see FIGS. 3B and
3C).
Example 5
Identifying Neonatal NET-Inhibitory Factor (nNIF)
[0179] Proteomic techniques were used to compare the cord blood
plasma proteome with that of plasma isolated from the same preterm
infant at 28 days of age (see FIGS. 3B and 14C). Following abundant
protein removal, parallel 2-dimensional gel electrophoresis was
performed on each plasma sample, separating proteins first by
isoelectric focusing and then by molecular weight. Six protein
spots were noted to be differentially present upon comparison of
the two gels. Four protein clusters were present in the gel of cord
blood plasma but not in the 28 day plasma gel; two protein clusters
were present in the 28 day plasma gel but not the cord blood plasma
gel. Each of these protein clusters were cut out of the respective
gels for protein identification. The oligopeptide sequences
following trypsin-digest and tandem mass spectroscopy were compared
to the NCBI Human trypsin-specific database (current through May
13, 2011). A partial list of proteins identified by mass
spectroscopy on one of the protein clusters present on the cord
blood gel but not on the 28 day plasma gel is included in FIG. 4A.
Of the proteins identified in this protein cluster, two related
peptides with predicted molecular weights of .apprxeq.4 kD and
protein scores of >100 became candidate peptides for
NET-inhibitory activity. The novel peptide was named neonatal
NET-inhibitory Factor (nNIF). A nNIF-related peptide (NRP) was
known in the literature: Cancer-Associated SCM-Recognition,
Immunedefense Suppression, and Serine Protease Protection Peptide
(CRISPP). Next, it was determined that nNIF and CRISPP have
significant homology to the carboxy terminus of full length alpha
1-antitrypsin (AAT, also referred to herein as A1AT) (see FIG. 4C).
Using a polyclonal antibody generated against the last 18 amino
acids of AAT, it was demonstrated using western blotting that nNIF
is present in increased amounts in cord blood plasma as compared to
healthy adult plasma (see FIGS. 15A and 15B).
[0180] As discussed, protein expression in cord blood plasma and
day of life 28 plasma from the same preterm infant was compared
using 2D-gel electrophoresis, and a protein spot present in cord
blood but not in day of life 28 plasma was detected. A list of nNIF
candidate proteins from that protein spot following mass
spectroscopy is outlined with expected molecular masses and protein
scores in FIG. 4A. The list includes full length AAT and two 29
amino-acid peptides with significant homology to the
carboxy-terminus of AAT: nNIF and CRISPP. As illustrated in FIG.
4C, nNIF and AAT share significant carboxy terminus homology. The
sequences obtained from mass spectroscopy are shown and compared,
and the published sequence of CRISPP is also compared. Western
blotting using a polyclonal antibody against the carboxy-terminus
of AAT is shown in FIG. 15A, demonstrating increased expression of
nNIF (.apprxeq.4-6 kD) in cord blood as compared to adult plasma.
As shown in FIG. 15B, these results were quantified using relative
fluorescence and a statistically significant difference was found
between four cord blood plasma nNIF levels (CB) and those of four
healthy adult controls (A). * denotes p<0.05. The Student's
t-test statistical tool was used.
[0181] In summary, protein expression in cord blood and day of life
28 plasma from the same preterm infant was compared using 2D-gel
electrophoresis. A protein spot present in cord blood but not in
day of life 28 plasma was detected. An endogenous peptide
(.apprxeq.4-6 kD) with significant NET inhibitory activity was
identified. This peptide was provisionally named neonatal
NET-inhibitory factor (nNIF).
Example 6
Characterizing the nNIF Peptide
[0182] Mass spectroscopy was used to further characterize the nNIF
peptide of Example 5 and to resolve major portions of its sequence.
A list of nNIF candidate proteins from the protein spot of Example
5 following mass spectroscopy is outlined with expected molecular
mass and protein score in FIG. 4A. As shown in FIG. 4C, nNIF was
found to have significant homology to a carboxy-terminus cleavage
fragment of A1AT. An antibody against the carboxy terminus of A1AT
was used to detect a 4-6 kD peptide in cord blood plasma that was
only minimally present in adult plasma (see FIG. 4B).
Example 7
Further Characterization of the nNIF Peptide
[0183] Cord blood plasma was depleted of all full length and
fragmented A1AT using affinity purification (AP) with the A1AT
carboxy-terminus antibody. As shown in FIGS. 5A-5F, cord blood
plasma depleted of nNIF failed to inhibit NET formation, while
pretreatment with the AP eluted proteins inhibited NET formation by
LPS-stimulated PMNs. Addition of recombinant full length A1AT to
the depleted plasma did not inhibit NET formation. These data
suggest that nNIF is a cleavage fragment of A1AT or a similar
peptide and not the full length protein.
Example 8
Identifying the nNIF-Related Peptide (nNRP), CRISPP
[0184] Significant homology was identified between nNIF and
Cancer-Associated SCM-Recognition, and Serine Protease Protection
Peptide (CRISPP) (see FIG. 4C). CRISPP peptide was synthesized and
it was observed that CRISPP blocked NET formation by LPS-stimulated
PMNs in a concentration dependent manner. A scrambled control
peptide did not block NET formation by LPS-stimulated PMNs.
Example 9
Inhibiting NET Formation with CRISPP Treatment
[0185] NET formation by PMA-stimulated adult PMNs with or without
CRISPP pre-incubation was assessed. As shown in FIGS. 6A and 6B,
CRISPP pretreatment (1 nM) for 1 hour prior to stimulation
inhibited NET formation while pretreatment with a scrambled control
peptide (1 nM) did not. CRISPP was observed to block NET formation
induced by PMA.
Example 10
Analyzing NET Formation in Multiple In Vitro Infection Models
[0186] A human histone H.sub.3 release assay was used to quantify
NET formation (n=2). NET-inhibitory activity of CRISPP was tested
using in vitro models of two human infectious diseases:
Staphylococcus aureus bacteremia and dengue fever. S. aureus has
previously been shown to induce NET formation by PMNs from human
adults. Pretreatment with CRISPP completely blocked NET formation
by human PMNs incubated with live S. aureus (see FIGS. 7A and 7B).
In contrast, incubation with a scrambled control peptide did not
block NET formation by human PMNs.
[0187] As shown in FIGS. 8A and 8B, human PMNs were incubated with
dengue virus (0.05 MOI) and qualitatively assessed for NET
formation. Mock incubation using dengue virus media without
infection served as a control. Dengue virus induced NET formation
by PMNs from human adults. Pretreatment with CRISPP (1 nM) for 1
hour prior to stimulation completely blocked NET formation by human
PMNs incubated with dengue virus. In contrast, incubation with a
scrambled control peptide (1 nM) did not block NET formation by
human PMNs.
Example 11
Determining Effect of CRISPP Treatment on PMN Functions
[0188] Total, phagocytotic, and NET-mediated extracellular
bacterial killing of a pathogenic strain of E. coli by human
PMNs.+-.LPS stimulation (100 ng/mL) was assessed, and the results
are summarized in FIG. 9. PMNs were also pretreated with CRISPP (1
nM), or a scrambled control peptide control (1 nM). Phagocytotic
killing was inhibited with cytochalasin B and D pretreatment (10
.mu.M). NET-mediated killing was inhibited using DNase treatment to
dismantle NETs prior to addition of the bacteria. CRISPP
pretreatment significantly decreased total and NET-mediated
bacterial killing of E. coli, but did not alter phagocytic killing,
as measured using the one-way ANOVA statistical tool with
Newman-Keuls Multiple Comparison Test post-hoc analysis.
Example 12
Assessing Effect of CRISPP Treatment in Murine Models
[0189] The ability of CRISPP to inhibit NET formation in vivo was
assessed using murine models of E. coli peritonitis. Outbred
Swiss-Webster mice were injected intraperitoneally with
4.5.times.10.sup.7 colony-forming units (cfu) of E. coli.+-.nNIF,
CRISPP, or scrambled control peptide (SCR) (10 mg/kg, IP injection)
one hour prior to E. coli infection. At the 3 hour time point after
infection, 3 mice were sacrificed and the peritoneal fluid and
peritoneal tissue were collected and analyzed for leukocyte
accumulation, bacteriology, and in vivo NET formation. Total
mononuclear and polymorphonuclear leukocyte counts were determined
in the peritoneal fluid and demonstrated a statistically
significant increase in peritoneal PMN accumulation in all
experimental groups compared to control (see FIG. 17A). Mice
pretreated with CRISPP demonstrated a significant increase in PMN
accumulation compared to E. coli alone or SCR pretreated mice (see
FIGS. 17A and 17B). This result appears consistent with a
CRISPP-mediated decrease in neutrophil cell death through
inhibition of NETosis. See Fuchs et al., J Cell Biol, 2007, 176:
231-241. The number of E. coli cfu/mL of peritoneal fluid in CRISPP
pretreated mice was also determined compared to SCR mice (see FIG.
17C). A significant increase in peritoneal fluid bacterial
concentration was detected in CRISPP pretreated animals compared to
SCR, indicating that the inhibition of NETosis and NET formation
may lead to decreased bacterial killing and higher peritoneal fluid
concentrations of bacteria.
[0190] Survival of outbred Swiss mice was tracked in various
treatment arms following intraperitoneal injection of either LPS
(20 mg/kg) or E. coli (4.times.10.sup.7 bacteria), as shown in
FIGS. 10A and 10B. CRISPP treated mice (10 mcg/kg/dose) received 2
doses intraperitoneally; one given 1 hour prior to infection and
one dose given 6 hours after infection. The same dose, delivery
route, and schedule were followed for the scrambled control peptide
control mice. Six mice were assessed in both groups: LPS+CRISPP and
LPS+scrambled control peptide. The LPS+CRISPP group survival was
statistically greater than the LPS+scrambled control peptide group
(p=0.02). Ten mice were assessed in each group: no treatment, E.
coli+CRISPP, and E. coli+scrambled control peptide. No antibiotics
were given. No deaths occurred in the no treatment group. The E.
coli+CRISPP group survival was statistically greater than the E.
coli+scrambled control peptide group (p<0.0001). Referring to
FIG. 11, CRISPP was also evaluated in a murine model of
polymicrobial infection caused by cecal ligation and puncture
(CL/P). CL/P was also performed without concomitant antibiotic
treatment. Ten mice were assessed in each of the CL/P groups. The
CL/P+CRISPP group survival approached statistical significance
compared with the CL/P control group (p=0.06). FIG. 20 is a graph
depicting the assessment of ten mice in each of the following
groups: no treatment, E. coli, CRISPP-Post/E. coli, and SCR-Post/E.
coli. The survival increase seen in CRISPP-Post/E. coli mice
compared to SCR-Post/E. coli mice approached statistical
significance. For all experiments in each of the three murine
models, the Log-rank (Mantel-Cox) statistical tool was used to
compare the survival curves between groups and employed the post
hoc Bonferroni correction. For all experiments in all three models,
* denotes p<0.05, ** denotes p<0.01, and *** denotes
p<0.001.
Example 13
Analyzing NET Formation in CLD Using Human Tissue and a Sheep
Model
[0191] De-identified human lung tissue from preterm infants who
died with CLD was immunohistochemically analyzed for extracellular
DNA and histone H.sub.3, two key indicators of NET formation (see
FIGS. 12A and 12B). Using similar techniques, lung tissue samples
collected from prematurely born lambs in the sheep model of CLD
were also examined. Lung tissue samples from preterm lambs
mechanically ventilated for 18-21 days demonstrated all of the
hallmarks of CLD while control tissue samples taken from lambs born
either at term or prematurely but supported with CPAP alone did
not. Lung tissue samples from the mechanically ventilated preterm
lambs demonstrated robust NET formation while that from control
lambs did not.
[0192] NET formation was assessed in paraffin-embedded human lung
tissue obtained at autopsy of neonates who died with CLD. In a
preliminary analysis, extracellular, alveolar histone H.sub.3
accumulation consistent with NET formation was found. NET formation
was also assessed in paraffin-embedded preterm lamb lung tissue in
the sheep model of CLD. Extracellular, alveolar NET formation was
detected in the preterm lamb CLD specimen but not in the control
preterm lamb. Similar deposition of NETs has been reported in a
murine model of TRALI. Other hallmarks of neonatal CLD in both
human and lamb CLD samples, including hypercellularity and alveolar
simplification, were also observed.
Example 14
Characterizing NET Formation in Sheep PMNs
[0193] Referring to FIGS. 13A and 13B, PMNs from preterm lamb cord
blood and from mature ewes were isolated. The isolated PMNs were
stimulated with LPS (100 ng/mL) for 1 hour and NET formation was
assessed qualitatively. Mature ewe PMNs were treated with CRISPP (1
nM) or a scrambled control peptide (1 nM) for 1 hour prior to
LPS-stimulation (100 ng/mL) and NET formation was assessed
qualitatively. PMNs isolated from lamb cord blood failed to form
NETs in vitro, but PMNs isolated from mature sheep robustly formed
NETs following LPS stimulation. CRISPP pre-treatment of sheep PMNs
inhibited NET formation in a dose-dependent manner while the
scrambled control peptide did not. CRISPP pre-treatment of sheep
PMNs inhibited NET formation stimulated with PMA.
Example 15
Isolating PMNs
[0194] PMNs were isolated from acid-citrate-dextrose (ACD) or
ethylenediaminetetraacetic acid (EDTA) anticoagulated venous blood
of healthy adults, healthy term infants, and prematurely born
infants. For the 11 prematurely born infants from whom cord and
peripheral blood samples were collected, cord and peripheral blood
plasma and PMN preparations were obtained at 5 separate time points
throughout the first 2 months of life. PMN suspensions (>96%
pure) were prepared by positive immunoselection using
anti-CD15-coated microbeads and an auto-MACS cell sorter (MILTENYI
BIOTEC, INC.) and were resuspended at 2.times.10.sup.6 cells/mL
concentration in serum-free M-199 media at 37.degree. C.
[0195] Table 1 (below) provides the demographics associated with
the preterm infant cohort (n=11).
TABLE-US-00003 TABLE 1 Preterm Infant Demographic Information.sup.1
Gestational ages at birth 23 6/7-29 0/7 weeks Birth weight 570-1160
g Indication for pre-term delivery Prolonged premature rupture of
membranes or 8 preterm labor Pregnancy induced hypertension 1
Placental abruption/preterm labor 0 Bacterial blood culture results
E. coli 0 Coagulase (-) Staphlococcus 2 Group B Streptococcus 0
Negative 6 Meningitis 2 Pneumonia 2 Antibiotic treatment All
treated, 2-14 d .sup.1Clinical characteristics and infectious
complications of preterm infant participants.
[0196] It was found that PMNs isolated from newborn infants,
whether born at term or prematurely, rapidly gained the ability to
form NETs, as demonstrated by qualitative and quantitative assays
of NET formation following in vitro stimulation with LPS (see FIGS.
14A and 14B; see also Yost et al., Blood, 2009, 113: 6419-1154; and
McInturff et al., Blood, 2012, 120: 3118-3125). Robust NET
formation was demonstrated as early as the third day of life even
for the most prematurely born infants and NET competency remained
intact in PMNs isolated serially through two months of age.
Example 16
Isolating Platelets
[0197] Human platelets were isolated as described in Weyrich et
al., J Clin Invest, 1996, 97: 1525-1534. Briefly, ACD
anticoagulated whole blood was spun at 100.times.g for 20 minutes.
The platelet rich plasma (PRP) was collected, transferred to a new
50 mL conical tube, and prostaglandin E1 (PGE; 100 nM; CAYMAN
CHEMICAL) was added. The PRP was then centrifuged at 1500 RPM for
20 minutes. After centrifugation, the supernatant was removed and
the platelet pellet was resuspended with 10 mL of 37.degree. C. PSG
media and PGE (100 nM). Platelets were resuspended in serum-free
M-199 media to 1.times.10.sup.8 cells/mL with platelet counts
acquired using a BECKMAN COULTER Particle Counter (BECKMAN
COULTER).
Example 17
Live Cell Imaging of NET Formation
[0198] It was determined whether nNIF and CRISPP inhibit NET
formation by human PMNs using qualitative and quantitative assays
of in vitro NET formation. Human PMNs isolated from healthy adult
donors were stimulated with LPS (100 ng/mL) or phorbal-12-mystirate
(PMA; 20 nM), .+-.nNIF (1 nM) or CRISPP (1 nM), pre-incubation (1
hour). Scrambled control peptide (SCR) generated using the
identical amino acid content as CRISPP but linked in a random order
was used as a control. NET formation was assessed 2 hours after
stimulation. It was found that both nNIF and CRISPP significantly
inhibited NET formation in the in vitro system as compared to the
SCR peptide control (see FIGS. 6A and 16A-16D).
[0199] NET formation was assessed by stimulated adult PMNs.+-.nNIF
or CRISPP pre-incubation, both qualitatively and quantitatively
(see FIGS. 6A and 16A-16D). nNIF or CRISPP pre-incubation (1 nM)
for 1 hour prior to stimulation (LPS, 100 ng/mL or PMA, 20 nM)
inhibited NET formation, while pretreatment with SCR (1 nM) did not
appear to inhibit NET formation. The representative images of FIG.
16A (20.times. magnification) and FIG. 6A (60.times. magnification)
show extracellular NET-associated DNA and nuclear DNA. The human
histone H.sub.3 release assay was used to quantify NET formation in
stimulated PMNs pre-incubated.+-.nNIF or CRISPP. As depicted in
FIGS. 16B and 16C, extracellular histone content (fold change over
baseline) is represented on the y-axis, and the dashed line
represents the control values, arbitrarily set at 1. The data
presented are representative of 3 separate experiments performed
using PMNs isolated from 3 different participants. * denotes
p<0.05 for CRISPP/PMA compared to PMA and SCR/PMA groups, **
denotes p<0.05 for LPS and SCR/LPS compared to control, while
.dagger. denotes p<0.05 for CRISPP/LPS and nNIF/LPS compared to
both the LPS and SCR/LPS groups. The one-way ANOVA statistical tool
with Tukey's post-hoc testing was employed.
[0200] NET formation was assessed in the peritoneum using live cell
imaging. A cell impermeable DNA dye stained all extracellular DNA,
and a cell permeable DNA dye served as a nuclear counterstain. A
qualitative decrease in NET formation in the peritoneal fluid of
nNIF or CRISPP pretreated mice was found compared to the SCR
control and a significant quantitative decrease in NET formation as
assayed by histone H.sub.3 release assay of the peritoneal fluid
(see FIGS. 17D and 17E). NET formation was also examined on freshly
resected peritoneal membrane tissue using live cell imaging. A
qualitative and quantitative decrease in peritoneal
membrane-associated NET formation was seen (see FIGS. 17F and 17G).
These results demonstrate that nNIF and the NRP CRISPP can inhibit
NET formation stimulated in in vivo as well as in vitro model
systems.
[0201] The ability of CRISPP to inhibit NET formation induced by
bacterial and viral pathogens was also tested. CRISPP inhibited NET
formation induced by incubation with S. aureus bacteria (MOI 100)
and Dengue viral infection (MOI 0.05) (see FIGS. 7A, 8A, and 8B;
see also Example 18) demonstrating that nNIF can inhibit NET
formation in reductionist experimental models of bacterial and
viral infection.
[0202] Qualitative assessment of NET formation was performed as
previously referenced in Yost et al., Blood, 2009, 113: 6419-6427
and McInturff et al., Blood, 2012, 120: 3118-3125. Briefly, primary
PMNs isolated from preterm infants, healthy term infants, and
healthy adults (2.times.10.sup.6 cells/mL) were incubated with
control buffer or stimulated with LPS (100 ng/mL), PMA (20 nM), or
S. aureus bacteria (MOI 100) for 1 hour at 37.degree. C. in 5%
CO.sub.2/95% air on glass coverslips coated with poly-L-lysine. For
selected experiments, primary PMNs were pre-incubated with
autologous plasma, cord blood plasma, nNIF (1 nM), CRISPP (1 nM),
or SCR (1 nM) for 1 hour prior to imaging. After pre-incubation
and/or stimulation, PMNs were gently washed with PBS and incubated
with a mixture of cell permeable (SYTO Green, MOLECULAR PROBES) and
impermeable (SYTOX Orange, MOLECULAR PROBES) DNA fluorescent dyes.
Confocal microscopy was accomplished using a FV300 1.times.81
Microscope and FLUOVIEW software (OLYMPUS). Both 20.times. and
60.times. objectives were used. Z-series images were obtained at a
step size 1 .mu.m over a range of 20 .mu.m for each field. OLYMPUS
FLUOVIEW and ADOBE PHOTOSHOP CS2 software were used for image
processing.
Example 18
Imaging of Dengue Virus-Induced NET Formation
[0203] Primary PMNs isolated from healthy adults (2.times.10.sup.6
cells/mL) were incubated with mock infection buffer or live dengue
virus (MOI 0.05), as for the live cell imaging discussed above.
After a 1 hour incubation, the infected PMNs were fixed with 2%
p-FA for 10 minutes prior to incubation with fluorescently-labeled,
cell permeable and cell impermeable DNA dyes, and imaged as for
live cell imaging using confocal microscopy.
Example 19
Quantitating NET Formation and Supernatant Histone H.sub.3
Content
[0204] Supernatant total histone H.sub.3 content was determined as
previously referenced in McInturff et al., Blood, 2012, 120:
3118-3125. After live cell imaging of control and stimulated
primary PMNs (2.times.10.sup.6/mL; various agonists), the cells
were incubated with PBS containing DNase (40 U/mL) for 15 minutes
at room temperature to break down and release NETs formed in
response to stimulation. The supernatant was gently removed and
centrifuged at 420.times.g for 5 minutes. The cell-free supernatant
was then mixed 1:3 with 4.times. Laemmli buffer prior to western
blotting. A polyclonal primary antibody against human histone
H.sub.3 protein (CELL SIGNALING TECHNOLOGY) and infrared secondary
antibodies (LI-COR BIOSCIENCES) were used. Imaging and densitometry
were performed on the ODYSSEY.TM. infrared imaging system (LI-COR
BIOSCIENCES).
Example 20
Isolating and Identifying nNIF in Umbilical Cord Blood Plasma
[0205] Two plasma samples from a single preterm infant, one from
the umbilical cord blood, and one from a peripheral blood sample
taken on day of life 28, underwent abundant plasma protein removal
(PROTEOSPIN, NORGEN) prior to 2D-electrophoresis using separation
first by isoelectric focusing (pH range 3-8) and then by size (TGX
PRECAST GEL, BIORAD). The resulting gels were compared for
differential protein content. Six differentially expressed protein
clusters were analyzed. Following trypsin digestion and tandem mass
spectrometry using an LTQ-FT ion-trap/FTMS hybrid mass spectrometer
(THERMOELECTRON), candidate proteins/peptides were identified as
potential NET-inhibitory substances.
Example 21
Affinity Purifying nNIF
[0206] To determine whether the 29 amino acid peptide identified in
Example 5 were the substances circulating in umbilical cord blood
responsible for the inhibition of NET formation, cord blood plasma
was depleted of nNIF via affinity purification using the
carboxy-terminus AAT antibody. nNIF was then eluted from the column
and used in experiments to assess the NET-inhibitory activity of
unaltered cord blood plasma, nNIF-depleted cord blood plasma, and
affinity purified nNIF from cord blood plasma. As a control,
recombinant AAT in nNIF-depleted cord blood was also assessed (see
FIGS. 5A-5F). PMNs isolated from healthy adult donors responded
with robust NET formation following 2 hour incubation with LPS (100
ng/mL). Pre-incubation with unaltered cord blood plasma inhibited
in vitro NET formation, consistent with earlier results (see FIGS.
3B and 14C). Pre-incubation with nNIF-depleted cord blood
plasma.+-.rAAT (1 nM) did not. Pre-incubation with affinity
purified nNIF, however, inhibited NET formation by LPS-stimulated
PMNs to a similar degree as the unaltered umbilical cord blood.
Further experiments also showed that full-length, enzymatically
active human AAT failed to inhibit NET formation by PMNs isolated
from healthy adult donors while nNIF did (see FIG. 15C). These
results suggested that the 29 amino acid peptides nNIF and CRISPP
detected in umbilical cord blood have NET-inhibitory activity.
Synthesis of nNIF and CRISPP for in vitro studies of their NET
inhibitory capacities was then performed.
[0207] As discussed above, following abundant plasma protein
removal (PROTEOSPIN, NORGEN), a polyclonal antibody raised against
the last 18 amino acids of the carboxy terminus of full length AAT
(LIFESPAN BIOSCIENCES) was used to affinity purify nNIF from cord
blood plasma using an immunoprecipitation kit (THERMO SCIENTIFIC).
Cord blood plasma depleted of both nNIF and full length AAT was
collected as were the peptides captured by affinity purification
for use in in vitro assays of NET formation. Full length,
enzymatically active AAT (SIGMA) was also resuspended in elution
buffer and tested in parallel.
Example 22
Analyzing Peritoneal Fluid and Membranes
[0208] Animals were treated with CRISPP (10 mg/kg), nNIF (10
mg/kg), or SCR (10 mg/kg) by i.p. injection 1 hour prior to
infection (E. coli, 4.5.times.10.sup.7 cfu/mouse, i.p. injection)
or stimulation (LPS, 25 mg/kg, i.p. injection (SIGMA)). Control
mice were injected with saline alone. The mice were sacrificed in a
CO.sub.2 chamber 3 hours post-infection/stimulation with the
peritoneal contents harvested and analyzed. Briefly, the skin of
the abdomen was cut open in the midline after thorough disinfection
and without injuring the muscle. The peritoneal cavity was lavaged
with sterile saline solution (100 mL) and analyzed for in vivo NET
formation, bacteriology, and leukocyte accumulation. NETs in the
peritoneal fluid were qualitatively and quantitatively analyzed
using live cell imaging with confocal microscopy and histone
release assays. NETs on the inner surface of the peritoneal
membrane were assessed quantitatively using live cell imaging
followed by standardized grid analysis of 5 randomly selected high
power visual fields per tissue sample (IMAGE-J software, NIH).
Peritoneal bacterial colony forming units (cfu) counts were
quantified by permeabilizing all recovered leukocytes with 0.1%
Triton-X 100 for 10 minutes and performing serial dilutions and
bacterial cultures on 5% sheep blood agar plates (HARDY
DIAGNOSTICS). After a 24-hour incubation, bacterial counts were
determined. Total leukocyte counts in the peritoneal lavage were
determined in Neubauer chambers using an optical microscope after
dilution in Turk's solution (2% acetic acid). Differential
leukocyte analysis was performed using a 60.times. oil immersion
objective to assess morphology of cyto-centrifuged cells stained
with May-Grunwald-Giemsa dye.
Example 23
Assaying Chemotaxis
[0209] Potential CRISPP-mediated effects on key neutrophil
activities besides NET formation were assessed: chemotaxis,
phagocytosis, reactive oxygen species generation, and bacterial
killing. It was found that nNIF and CRISPP did not significantly
inhibit PMN chemotaxis, phagocytosis, or reactive oxygen species
generation (see FIGS. 18A-18C). Total, phagocytotic, and
NET-mediated bacterial killing of a pathogenic strain of E. coli
was also assessed. It was found that while CRISPP pre-incubation of
LPS-stimulated PMNs significantly decreased total and NET-mediated
extracellular bacterial killing in comparison to control PMNs, the
phagocytotic component of bacterial killing was not different
between the three groups (see FIG. 18D).
[0210] Chemotaxis by PMNs isolated from healthy adult donors was
assessed using a modified Boyden Chamber assay.+-.a one hour
pre-incubation with nNIF (1 nM), CRISPP (1 nM), or SCR (1 nM).
Recombinant human IL-8 (2 ng/mL) was used as the chemoattractant.
Chemotaxis through a 5 micron filter was determined by counting
PMNs in 10 randomly selected high-power fields as previously
described in Hill et al., Lancet, 1974, 2: 617-619. In separate
experiments, nNIF, CRISPP, or SCR (all at 1 nM) were evaluated for
chemoattractant activity using the same system.
Example 24
Assaying Phagocytosis
[0211] As discussed above, it was found that nNIF and CRISPP did
not significantly inhibit phagocytosis activity in neutrophils.
PMNs were isolated from blood of healthy adult donors and
resuspended in M-199 at a concentration of 2.times.10.sup.6
cells/mL. Leukocytes were pre-incubated for 60 minutes under
standard conditions with cytochalasin B and D (10 .mu.M), nNIF (1
nM), CRISPP (1 nM), or SCR (1 nM). Following pre-incubation, PMNs
were incubated with 6.times.10.sup.6 E. coli BIOPARTICLES (E-13231,
MOLECULAR PROBES) on a rotator for 4 hours under standard
conditions. The PMNs were then washed and resuspended in the
starting volume in M-199 before being spun down onto glass
coverslips and fixed with 2% paraformaldehyde for 10 minutes and
permeabilized with 0.1% Triton-X-100 for 10 minutes. Leukocytes
were stained with WGA 555 (INVITROGEN) and TOPRO-3 (MOLECULAR
PROBES) and randomly selected high power visual field images were
captured using confocal microscopy. IMAGE-J software (NIH) was used
to determine the percentage of PMNs that were positive for
fluorescently labeled E. coli BIOPARTICLES detected at 488 nm.
Example 25
Assaying Reactive Oxygen Species Generation
[0212] Also, as discussed above, it was found that nNIF and CRISPP
did not significantly inhibit ROS generation activity in
neutrophils. Human PMNs isolated from healthy adult whole blood
were resuspended to a concentration of 2.times.10.sup.6 cells/mL in
M-199 media and pre-incubated.+-.CRISPP (1 nM) or SCR (1 nM)
peptide for 1 hour under standard conditions. The PMNs were then
stimulated with LPS (100 ng/mL) for 1 hour, washed, and resuspended
with a dihydrorhodamine (7.25 mM; D-632, MOLECULAR PROBES) and
catalase (1000 Units/mL; C-40, SIGMA) mixture and incubated at
37.degree. C. for 10 minutes. After incubation, samples were placed
at 4.degree. C. and analyzed for ROS-dependent fluorescence using a
BECTON-DICKINSON FACSCAN device equipped with CELLQUEST
software.
Example 26
Assaying Platelet Activation
[0213] Platelets reportedly play a role in regulating NET formation
by human PMNs (see Clark et al., Nat Med, 2007, 13: 463-469). It
was determined whether CRISPP alters platelet activation and
platelet-PMN interactions. P-selectin expression was determined by
platelets pre-incubated with CRISPP and SCR peptides followed by
stimulation with thrombin. It was found that CRISPP and SCR
peptides did not alter platelet p-selectin expression when given
alone or following thrombin stimulation (see FIG. 18E).
Furthermore, in experiments where LPS-stimulated PMNs were mixed
with thrombin-stimulated platelets isolated from the same donor,
CRISPP and SCR did not alter platelet/PMN association as assessed
via flow cytometry (see FIG. 18F). To further assess whether
platelets take up CRISPP from the inflammatory milieu, CRISPP and
SCR peptides were synthesized with a FLAG tag motif added to the
carboxy terminus of each peptide (CRISPP-F, SCR-F). It was then
demonstrated that the CRISPP-F maintains NET-inhibitory activity
similar to that of un-tagged CRISPP (see FIGS. 21A and 21B), and
immunocytochemical experiments were performed with freshly isolated
human platelets to determine whether platelets take up CRISPP-F
from the inflammatory milieu. It was found that platelets do not
take CRISPP-F or SCR-F into their cytoplasm (see FIG. 18G) while
PMNs co-incubated with platelets do. This may suggest that the NET
inhibitory effects of nNIF and CRISPP do not result from
alterations in platelet activation or signaling to trigger NET
formation by PMNs.
[0214] Using protocols modified from van Velzen, et al., Thromb
Res, 2012, 130: 92-98, human platelets (1.times.10.sup.8 cells/mL)
were incubated with CRISPP or SCR (0.1-10 ng/ml) for 1 hour
followed by stimulation with thrombin (0.1 IU/mL) or control M-199
media for 15 minutes. The cells were prepared for FACS analysis
with the following primary, monoclonal antibodies for 20 minutes:
PE-conjugated CD41a (platelet-specific marker) and FITC-conjugated
CD62P (P-selectin). Isotype and fluorochrome matched control
antibodies were used. After incubation at room temperature in the
dark, the cells were lysed with FACS lysis buffer and analyzed by
flow cytometry (BECTON-DICKINSON FACSCAN, CELLQUEST software).
Example 27
Assaying Platelet/PMN Aggregation
[0215] Using protocols modified from Saboor et al., Malays J Med
Sci, 2013, 20: 62-66, human PMNs (2.times.10.sup.6 cells/mL) were
isolated and incubated with CRISPP or SCR (0.1-10 ng/ml) for 1 hour
prior to addition of freshly isolated human platelet at a ratio of
100:1 for another 15 minute incubation. After this period, the
cells (PMNs and platelets) were stimulated with thrombin (0.1
IU/mL) or control M-199 media for 1 hour. The cells were stained
for 20 minutes with the following primary monoclonal antibodies:
PE-conjugated CD41 and FITC-conjugated CD16. Isotype and
fluorochrome matched control antibodies were used. Flow cytometry
was performed using BECTON-DICKINSON FACSCAN and CELLQUEST
software.
Example 28
Assaying Nuclear Decondensation
[0216] A possible mechanism by which nNIF and CRISPP inhibit NET
formation was ascertained. Using techniques modified from
Papayannopoulos et al., J Cell Biol, 2010, 191: 677-691, nuclear
area assays were used for PMA-stimulated PMNs as a surrogate for
NET formation. PMNs were stimulated with PMA (20 nM) for 2 hours
and then visualized via live cell imaging with a cell permeable DNA
dye. Images from 5 randomly selected high power fields were
captured and the nuclear area of all PMNs on each visual field was
quantitated using IMAGEJ software. Nuclear areas were compared for
PMNs stimulated with PMA with PMNs pre-incubated with CRISPP (1
nM), SCR (1 nM), or the NE inhibitor Sivelestat (200 nM).
Qualitative and quantitative results of these assays demonstrated a
significant increase in nuclear area for PMA-stimulated PMNs as
compared to control PMNs or PMNs pre-incubated with each of the
three reagents but without PMA (see FIG. 19A). Pre-incubation of
PMA-stimulated PMNs with CRISPP and Sivelestat, however, resulted
in a significant decrease in nuclear area compared to PMA alone
while no change was seen in the SCR group (see FIGS. 19A and 19B).
Next, immunocytochemical experiments designed to see whether CRISPP
is taken up by human PMNs were performed. The CRISPP-F and SCR-F
peptides and FLAG-tag specific primary antibodies were used to
assess cellular location. It was found that CRISPP-F and SCR-F are
taken up into the cytoplasm by PMNs during a one hour incubation
(see FIGS. 18G and 19C). No evidence was found of nuclear
translocation of CRISPP-F or SCR-F (see FIGS. 18G, 19C, and 19D).
These results may suggest that NRPs rapidly enter PMNs but not
platelets (see FIGS. 18G, 19C, and 19D), and that uptake of NRPs by
PMNs is a function of size and may not be dependent on amino acid
sequence or specific receptor-mediated transport. Furthermore,
protein co-localization assays using the DUOLINK protein proximity
assay (see Carlo et al., FASEB J, 2013, 27: 2733-2741) and primary
antibodies targeting F-CRISPP and NE, demonstrate that F-CRISPP and
NE can co-localize within 40 nm of each other following CRISPP
incubation for 1 hour (see FIG. 19D). CRISPP can inhibit nuclear
chromatin decondensation, which is consistent with the selective
inhibition of NET formation by CRISPP and nNIF. This process may
involve inhibition of NE activity.
[0217] PMNs were isolated and resuspended to 2.times.10.sup.6
cells/mL in M-199 media. They were pre-incubated with CRISPP (1
nM), SCR (1 nM), or the neutrophil elastase inhibitor Sivelestat
(100 .mu.M) under standard conditions. Under the same conditions,
cells were treated.+-.PMA (20 nM) on poly-L-Lysine coated glass
coverslips for 2 hours. 5 randomly selected high power visual
fields per sample were captured via confocal microscopy and
analyzed for nuclear area using the cell-permeable, fluorescent DNA
dye SYTO Green. The nuclear pixel areas of >100 individual cells
per high power field were determined using IMAGE-J software
(NIH).
Example 29
Determining CRISPP Peptide Cellular Localization
[0218] The cellular locations of FLAG-tagged CRISPP (F-CRISPP) and
FLAG-tagged SCR (F-SCR) peptides were determined using
immunocytochemistry. Adult neutrophils were pre-incubated with
either F-CRISPP (1 nM) or F-SCR (1 nM) for 1 hour under standard
conditions followed by stimulation with LPS (100 ng/mL) for 2
hours. The PMNs were then spun down onto glass coverslips with 2%
p-FA fixation and 0.1% Triton-X-100 permeabilization. FLAG-tagged
peptide was detected using a monoclonal anti-FLAG antibody (F1804,
SIGMA) with TOPRO-3 as a nuclear counterstain.
Example 30
Using a DUOLINK Protein Proximity Assay
[0219] The DUOLINK Protein Proximity Assay (SIGMA) was used to
determine whether CRISPP and NE co-localize within PMNs isolated
from healthy adults. PMNs were isolated and pre-incubated for 1
hour with F-CRISPP (10 nM) or F-SCR (10 nM) under standard
conditions prior to stimulation with LPS (100 ng/mL) on
poly-L-lysine coated glass coverslips. The DUOLINK Protein
Proximity Assay was performed using a rabbit anti-neutrophil
elastase antibody (ab131260, ABCAM) and a mouse anti-FLAG antibody
(F1804, SIGMA). Fluorescence detected via confocal microscopy at
546 nm indicated FLAG-tagged peptide and NE co-localization.
Example 31
Assaying Bacterial Killing
[0220] As discussed above, it was found that while CRISPP
pre-incubation of LPS-stimulated PMNs significantly decreased total
and NET-mediated extracellular bacterial killing in comparison to
control PMNs, the phagocytotic component of bacterial killing was
not different between total, phagocytotic, and NET-mediated
bacterial killing of a pathogenic strain of E. coli (see FIG.
18D).
[0221] Bacterial killing efficiency of primary human PMNs was
determined as previously described in Yost et al., Blood, 2009,
113: 6419-6427. PMNs were incubated for 30 minutes at 37.degree. C.
in 5% CO.sub.2/95% air alone or with the phagocytosis inhibitors
cytochalasin B and D (10 .mu.M). The leukocytes were then
stimulated with LPS (100 ng/mL), placed in poly-L-lysine-coated
wells of a 24-well tissue culture plate, and incubated at
37.degree. C. for 1 hour to induce cellular activation and
formation of NETs. To inhibit NET-mediated bacterial killing, we
incubated selected wells with DNase (40 U/mL) for 15 minutes prior
to addition of bacteria. E. coli (1 cfu/100 PMN) were added to the
PMNs, followed by continued incubation for 2 hours. The PMNs were
then permeabilized with 0.1% Triton-X 100 for 10 minutes and each
well was scraped to free all cells. Serial dilutions were performed
and bacterial cultures were grown on 5% sheep blood agar plates
(HARDY DIAGNOSTICS). After a 24-hour incubation, bacterial counts
were determined. Total, phagocytotic, and fractional NET-mediated
bacterial killing were determined as described above.
Example 32
Studying Survival in Murine Models of Sepsis
[0222] Animals were treated with CRISPP (10 mg/kg), nNIF (10
mg/kg), or SCR (10 mg/kg) by i.p. injection 1 hour prior to and 6
hours after infection (E. coli, 4.5.times.10.sup.7 cfu/mouse, i.p.
injection) or stimulation (LPS, 25 mg/kg, i.p. injection). Other
mice were subjected to CL/P as a model of polymicrobial sepsis (see
Araujo et al., Microvasc Res, 2012, 84: 218-221). Still other mice
were injected with nNIF, CRISPP, or SCR 4 hours after
injection/stimulation to evaluate post-infection impact of
nNIF/CRISPP on survival. Control mice were injected with saline
alone and control mice for the CL/P mice were subjected to sham
surgery. The mice in these studies were not treated with fluid
resuscitation or antibiotic treatment, but were provided with ample
food and water during the experiments. Survival was assessed over 6
days at 6 hour to 12 hour intervals.
Example 33
Performing Statistical Analysis
[0223] GRAPHPAD PRISM statistical software (version 4, GRAPHPAD
SOFTWARE) was used to analyze results. Reported values are the
mean.+-.SEM. To assess differences between more than two sets of
normally distributed data the one-way ANOVA test with Tukey's or
Bonferroni's post-hoc testing was employed. For comparison of two
groups of continuously distributed data, the unpaired, single or
two tailed Student's t-test was used where appropriate. For
comparison of murine survival curves, the Log-rank (Mantel-Cox)
test was used. All P values of less than 0.05 were considered
statistically significant.
[0224] It will be apparent to those having skill in the art that
many changes may be made to the details of the above-described
embodiments without departing from the underlying principles of the
invention. The scope of the present invention should, therefore, be
determined only by the following claims.
Sequence CWU 1
1
2160PRTHomo sapiens 1Lys Ala Val Leu Thr Ile Asp Glu Lys Gly Thr
Glu Ala Ala Gly Ala 1 5 10 15 Met Phe Leu Glu Ala Ile Pro Met Ser
Ile Pro Pro Glu Val Lys Phe 20 25 30 Asn Lys Pro Phe Val Phe Leu
Met Ile Glu Gln Asn Thr Lys Ser Pro 35 40 45 Leu Phe Met Gly Lys
Val Val Asn Pro Thr Gln Lys 50 55 60 230PRTHomo
sapiensmisc_feature(2)..(2)Xaa can be any naturally occurring amino
acid 2Met Xaa Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe
Leu 1 5 10 15 Met Ile Asp Gln Asn Thr Lys Val Pro Leu Phe Met Gly
Lys 20 25 30
* * * * *