NZ711983B2 - Product and process for mucus viscosity normalization - Google Patents
Product and process for mucus viscosity normalization Download PDFInfo
- Publication number
- NZ711983B2 NZ711983B2 NZ711983A NZ71198314A NZ711983B2 NZ 711983 B2 NZ711983 B2 NZ 711983B2 NZ 711983 A NZ711983 A NZ 711983A NZ 71198314 A NZ71198314 A NZ 71198314A NZ 711983 B2 NZ711983 B2 NZ 711983B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- mucus
- thioredoxin
- protein
- sputum
- Prior art date
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Abstract
Disclosed are compositions and methods for decreasing the viscosity and/or cohesiveness of and/or increasing the liquefaction of excessively or abnormally viscous or cohesive mucus or sputum. The composition contains a protein or peptide containing a thioredoxin monocysteinic active site in a reduced state and optionally further contains a reducing system. d state and optionally further contains a reducing system.
Description
Field of the Invention
This invention relates generally to the use of a protein or peptide containing a
thioredoxin monocysteinic active site in a reduced state to reduce viscosity and to induce,
enhance and/or increase the liquefaction of mucus or sputum.
Background of the Invention
A large unmet medical need exists for safe, well-tolerated and ive drugs for the
treatment of patients with cystic fibrosis (CF), COPD/emphysema, bronchiectasis, severe
asthma and other serious obstructive pulmonary diseases. These diseases are characterized
by oduction of thickened mucus resulting in impaired lung fianction (reviewed in
Evans, CM. and Koo, J.S., Pharmacology & Therapeutics 121: 332-348, 2009). Poor
clearance of abnormal, sticky mucus is ated with chronic infection and premature
death, especially in CF. Despite advances in antibiotic therapy and other treatments,
improved mucus clearance remains a central clinical treatment ive even while our
understanding of the mechanisms underlying mucus transportability are still limited
(Verdugo, P., Cold Spring Harb Perspect Med 2012;2:a009597).
Mucus is a continuously-secreted supramolecular polymer gel that forms a
protective barrier on epithelial surfaces and is responsible via ciliary action and cough for
transporting inhaled debris and ia out of the lung (Knowles, MR. and Boucher,
R.C., J Clin Invest 109:571-577, 2002; Cone, R.A., Adv Drug Deliv Rev 61:75-85, 2009).
Proper viscoelasticity and hydration of the mucus layer, which enables efficient
mucociliary ort, is therefore al to mucus n and the prevention of
2014/030545
infection and inflammation. Normal mucus consists of mostly water (97%) with the
remaining solids comprising mucin ns, non-mucin proteins, salts, lipids and ar
debris (Fahy, J.V. and , B.F., N Engl J Med 363:2233-47, 2010). The polymeric
mucin glycoproteins MUCSAC and MUCSB are primarily responsible for the viscoelastic
ties of the respiratory mucus gel (Matsui et al., Cell 95:1005-1015, 1998; reviewd
in Kreda, et al., Cold Spring Harb Perspect Med 2012;2:a009589). The 0-linked glycan
hydroxyl groups attached to mucins contribute water-binding, while the mucins
themselves form an entangled network (Verdugo et al., Biorheology 20:223-230, 1983)
that may also involve covalent and non-covalent interchain cross-linking as suggested
from ed studies of the digestive tract mucin MUC2 (Ambort et al., Biochem J
436:61-70, 2011). Mucins are unusually rich in Cys amino acids, with human MUCSAC
containing a able 295 Cys residues out of a total of 5030 amino acids
niprot.org/uniprot/P98088). Mucin Cys residues located near the N and C termini
are thought to be involved in formation of interchain disulf1de linkages between mucin
subunits, while the role of the internal Cys residues is less clear (Thornton et al., Annu Rev
Physiol 70:459-486, 2008). Some are located in a ‘Cys Knot’ region and might readily
form intramolecular disulfide bonds that could play a role in facilitating the non-covalent
entanglement central to the mucus gel mesh structure (Fahy, J.V. and Dickey, B.F., N Engl
JMed 33-47, 2010).
Recent work (Button et al., Science 337:937-941, 2012) has led to a new model of
the ure of the mucosal surface based on the finding that certain mucins, once thought
to be membrane bound on epithelial cells, are actually tethered to membranes on cilia
themselves. The implication of this model is that the mobile mucus layer overlays an even
denser periciliary layer, described as a “gel-on-brush”. The model explains elegantly how
liquid moves between the two layers with the mucus acting as a reservoir, and establishes
a new paradigm for understanding the role of mucus osmotic s in determining the
functionality of mucociliary transport and mucus layer hydration. The model also
provides a framework to understand how excess disulf1de bonding in the mucus protein
scaffold might cause increased mucus layer osmotic modulus, which in turn dehydrates the
underlying ciliary layer and severely constrains normal mucus transport. Such a scenario
may underpin a substantial portion of the disease mechanism of CF.
CF is an mal recessive condition. The symptoms of CF result from defects
in CFTR, the Cystic Fibrosis embrane Conductance Regulator, a key epithelial
membrane transporter for monovalent negatively d ions, primarily chloride
(Riordan, et al., Science 245: 1066-1073), but also bicarbonate and glutathione. Mutations
leading to CF, of which over 1700 are known (www.genet.sickkids.on.ca/cftr/), include
those causing complete loss of CFTR (the case for the most common CF genotype) as well
as point mutations ing in partial or full loss of anion transport activity. In addition,
as a result of the defects in CFTR, lia within the body are impermeable to chloride
ion transport (Boucher eta 1., Lung 161:1-17, 1983; Welsh, Physiol Rev 67:11443-1184,
1987). Although several organs are affected, including pancreas, intestine, and male
genital tract, complications within the lung account for 95% of the morbidity and mortality
(Means, M. Cystic Fibrosis: the first 50 years. In: Cystic Fibrosis-Current Topics Volume
1, edited by Dodge IA, Brock DJH, and Widdicombe JH. Chichester: Wiley and Sons,
1992, p. 217-250). In lung impaired by the disease, chloride transport into the airway
lumen leads to ive absorption of Na+ and fluid, reducing the volume of airway
surface liquid (Jiang et al., Science 262:424-427, 1993). Attempts have failed, however, to
restore chloride l ty to compensate for the effects caused by the non-
functioning CFTR, e. g. via ts of the P2Y2 subtype of purinergic receptor (Ratj en, F.
et al., J Cyst Fibros 11:539-49, 2012). This suggests that loride effects of CFTR
might be more significant than originally thought.
In oxidizing environments like the lung, disulfide bonds are readily formed
between adjacent oxidized Cys residues such as those present in great abundance on mucin
proteins. These bonds are highly stable, and disrupting (i.e. ng) them in order to
restore the Cys residues to their free thiol form requires the action of potent chemical or
biological reductants. In the healthy lung, excess disulfide bond formation is countered
ily by the reduced form of the ical reductant glutathione (GSH), a Cys-
ning tripeptide that is secreted in large amounts into the mucus layer (Cantin et al., J
Appl Physiol 63:152-157, 1987), and may play a key role in maintaining a normal
disulfide bond vs. free Cys thiol equilibrium in mucins. Secretion of GSH onto the airway
e is highly dependent upon CFTR, which both directly and indirectly facilitates GSH
export (reviewed in Ballatori et al., Biol Chem 390: 191-214, 2009). Consequently, levels
of pulmonary GSH in CF patients may be 30% or less than levels found in normal
individuals (Roum et al., J Appl Physiol 75:2419-24, 2003; Wetmore D.R. et al., J Biol
Chem 285:30516-22, 2010). CFTR is also responsible for secretion of bicarbonate anions,
and the ing deficiency of bicarbonate in the CF lung appears to bute to disease.
A primary chemical effect of bicarbonate is to raise pH. Since ion of disulfide bonds
by thiol-containing reductants requires the formation of an attacking deprotonated thiolate,
2014/030545
which is ted at low pH where the protonated thiol form is favored (Singh and
Whitesides, I_n: Sulphur-containing Functional Groups, 5: pp. 633-58, John Wiley & Sons,
1993), y between the ties of bicarbonate and GSH (as well as other biological
reductants known to be t in the airway surface environs) is likely. Measured pH in
CF obronchial secretions is up to 0.6 units lower vs non-diseased (Song et al., Am J
Physiol Cell Physiol 290:C741-C749), consistent with an environment in the CF lung
where reductant is both present in limiting supply as well as being less active due to an
ed ability to form disulfide-attacking thiolates. Taken together with the enormous
number of clustered Cys present in mucins, mucus in the oxidizing respiratory
environment is thus poised to be in a more highly disulf1de-bonded state if either secreted
reductant levels become limiting, or if mucin proteins are produced and secreted in excess
resulting in a superabundance of disulf1de-bondable Cys. Both ions are known to
occur in CF and certain other obstructive pulmonary diseases: mucus ns are over-
ed in response to lung stress (Rogers, Resp Care 52:1134-1149), and 70% or more
of GSH secretion can be d as a consequence of defects in CFTR (Roum et al., J
Appl Physiol 75:2419-24, 2003; Wetmore D.R. et al., JBiol Chem 285:30516-22, 2010).
This potential for excess mucus disulflde-bonding as well as general redox
imbalance to play a mechanistic role in CF has led to the clinical tion of various
thiol-containing agents as mucolytic drugs. These include N—acetylcysteine (NAC) and
Nacystelyn (NAL; N-acetylcysteine + L-lysine) (Hurst et al., Am Rev Respir Dis, 96:962-
970, 1967; Dasgupta and King, Pediatr Palmonol, 22:161-166, 1996; Nash, E.F., et al.,
Cochrane Database of Systematic Reviews, 2010(12): 1-49, 2009) as well as reduced
glutathione itself p, C., et al., CHEST Journal, 127(1): 308-317, 2005; Griese, M.,
et al., Am JResp Crit Care Med 169(7):822-828, 2004; Griese, M., et al., Am JResp Crit
Care Med 188(1):83-89, 2013; Roum, J.H., et al., JAppl Physiol, 87:438-443, 1999).
While largely safe, to date these small-molecule agents have not exhibited clear clinical
benefits in either oral or inhaled forms (reviewed in Nash, E.F., et al., Cochrane Database
of Systematic Reviews, 2010(12): 1-49, 2009). Much of this lack of efficacy may be the
result of low potency or loss of activity during delivery because of autoxidation effects, as
well as the potential for inactivation by pulmonary enzymes. GSH is subject to rapid
autoxidation to the inactive GSSG form (Curello, S. et al., Clin Chem, 33:1448-49, 1987)
and is hence pharmacologically unstable in the reduced form when aerosolized and
inhaled (Carl White M.D., pers comm.), losing a large fraction of its activity by the time
the target site in the airway is reached. In addition, y—glutamyltransferase present at high
concentrations in the pulmonary space readily degrades GSH to an inactive form (Corti et
al., Am J Resp Crit Care Med 189:233-234, 2014), the abundance of which increases
markedly upon GSH inhalation (Griese et al., Am J Resp Crit Care Med 188:83-89
mental information, 2013). Improving thiol agents by combining disulf1de-
targeting with the superior pharmacology and specificity of biologic drugs is thus a key
unmet therapeutic objective.
While the gy of CF lung disease can be attributed to the altered rheological
properties of mucus, compromised lung on is rarely evident at birth. Instead,
iectasis and airway ction progress with age of patient. This chronic lung
injury results from a persistent cycle of bacterial infection and atory response.
Airway damage results when phils recruited into the lung release matrix degrading
enzymes, such as elastase, and harmful reactive oxygen species (reviewed in Konstan and
Berger, r Pulmonol 24:137-142, 1997). Following persistent infection, interaction of
mucins with DNA (Potter et al., Am J Dis Child 100:493-495, 1960; Lethem et al., Am Rev
Respir Dis 100:493-495, 1990; Lethem et al., Eur Respir J 3:19-23, 1990) and f-actin
polymers (Sheils et al., Am J Path 148:919-927, 1996; Tomkiewicz et a1., DNA and actin
filament tructure in cystic fibrosis sputum. In: Cilia, mucus, and mucociliary
ctions, edited by Baum GL, Priel Z, Roth Y, Liron N, and Ostfeld E]. New York,
NY: Marcel , 1998) released from dying inflammatory cells may also occur, and
can be responsible for some of the dense and viscous nature of CF sputum in severe
disease. The inability to clear such mucus by cough or mucociliary clearance facilitates
further colonization of the lung with opportunistic pathogens, airway remodeling, and
eventually death.
Interventions designed to te directly the consequences of CFTR defects
ore are particularly desirable, as these may prevent or attenuate disease progression.
While direct correction of CF by gene therapy is not yet attainable, the use of potentiator
and corrector therapies to restore some degree of CFTR on to defective proteins has
been demonstrated recently (Sloane, PA and Rowe, SM, Current Opinion in Pulmonary
Medicine 16: 591—7, 2010). Such therapy is limited to a small percentage of CF patients
with a particular CFTR defect, such as the G551D mutation targeted by ivacaftor /
KalydecoT'VI (Jones, AM and Helm, JM, Drugs 69: 1903—10, 2009). However, in these
few individuals dramatic results have been observed (Accurso, FJ; Rowe, SM; Clancy, JP;
Boyle, MP; Dunitz, JM; Durie, PR; Sagel, SD; Homick, DB et al., The New England
Journal ofMedicine 363: 1991—2003, 2010) demonstrating that mechanistic intervention
in CF is capable of mitigating late-stage consequences such as those ing from
chronic infection and inflammation. Currently, however, symptomatic rather than disease-
modifying approaches including antibiotic regimens coupled with drugs that facilitate the
clearance of purulent airway secretions remain the mainstay treatments for progressive
airway disease. Inhalation of purified thNase (PulmozymeT'V'; Genentech, USA), which
digests extracellular DNA present in the CF airway, is widely used as a respiratory
estant. Such ent is clinically effective for diminishing sputum viscosity and
stabilizing the forced expiratory volume (FEV) (Fuchs et al., N Engl J Med 331:637-642,
1994). Other investigative therapies aimed at breaking down mucin or actin polymers,
including N-acetylcysteine (NAC), nacystelyn (an yl-L-cysteine derivative), and
gelsolin, can also reduce sputum viscosity experimentally, but have yet to demonstrate
al efficacy and attain approval for treatment of CF in the United States (Nash, EF et
al., Cochrane se 0fSystematic Reviews, 2010(1):CD007168, 2009).
Other approaches being utilized to improve mucus clearance e mucoactive
agents such as d onic saline and inhaled high-dose mannitol (Fahy, J.V. and
Dickey, B.F., N Engl J Med 33-47, 2010). These agents are thought to act by
pulling water osmotically into the mucus layer to increase hydration, or to improve
clearance through induction of coughing reflexes. Some evidence exists for both
mechanisms (Levin, M.H. et al., J Biol Chem 803-12, 2006; Boucher, R.C., Trends
Mol Med 13:231-240, 2007). However, mucoactives are symptomatic treatments (not
e-modifying), and efficacy is generally only te as many ts are not able
to tolerate the high doses that can have the greatest clinical effect (Aziz, I. and Kastelik,
J.A., NEnglJMed 354:1848-1851, 2006).
Findings by White and colleagues (Rancourt et al., Am J Physiol Lung Cell Mol
Physiol 286:L93l-L938, 2004; Rancourt et al., Free Radical Biol & Med 42:1441-43,
2007) have found that the use of a protein or peptide containing a thioredoxin active site in
the reduced state is useful for increasing the liquefaction of mucus or sputum in a patient
that has excessively s or cohesive mucus or sputum, including a patient having CF,
wherein the mucus or sputum is contacted with the protein or peptide (US. Patent No.
7,195,766 and US. Patent No. 7,534,438, both of which are incorporated herein by
reference in their entirety). In this system (see Figure 3), a transient mixed-disulf1de
between the N—terminal cysteine of the thioredoxin active site and a cysteine of a target
protein (found in the mucus or sputum) is formed, followed immediately by nucleophilic
attack on the intramolecular mixed disulfide e and release of oxidized thioredoxin
and the fully-reduced target (Wynn et a1,. Biochemistry 34(37):11807-11813, 1995), thus
allowing for re-formation of cysteine disulfides in the mucus or sputum but at the same
time also allowing free access of reduced or oxidized doxin to enter cells and induce
undesired off-target activities following re-reduction by the endogenous thioredoxin
reductase — NADPH system. In addition White and gues have shown reduced
thioredoxin to mitigate the abnormal viscoelasticity of human CF mucus in vitro and in ex
vivo animal implantation studies (Rancourt et a1., Free Radical Biol & Med 42:1441-43,
2007), as well as to inhibit the activity of pro-inflammatory neutrophil elastase by
disruption of active site disulfide bonds (Lee et al., Am J l Lung Cell Mol l
289:L875-L882, 2005). ed to GSH and thiol agents such as NAC, thioredoxin is a
more potent 1de bond-reducing molecule and is much less susceptible to inactivation
by autoxidation. Taken together, this creates the opportunity to restore a normal disulfide
reduction state to mucus with a pharmacologically stable le. Such a therapy may
prevent or delay the cascade of chronic infection, inflammation and lung fianction decline
that leads to early death in CF ts. However, there is also a strong motivation to
avoid the potential pro-inflammatory and other intracellular regulatory effects of
thioredoxin (Amer, ES. and A. Holmgren, Eur J Biochem 267: 6102-6109, 2000;
Rancourt et a1., Free Radical Biol & Med 1-43, 2007) as well as to increase the
y of mucus viscosity-modulation by preventing mucin Cys dation. These
improvements are the subject of the present invention.
Summary of the Invention
One ment of the present invention relates to a method to decrease viscosity
of mucus or sputum in a patient that has excessively viscous or cohesive mucus or sputum.
The method includes the step of contacting the mucus or sputum of the patient with a
composition comprising a protein or peptide containing a thioredoxin monocysteinic
active site in a reduced state effective to decrease the viscosity of the mucus or sputum as
compared to prior to the step of contacting. In one aspect of this embodiment, the t
has a lung disease in which abnormal or excessive viscosity or cohesiveness of mucus or
sputum is a symptom or cause of the disease. In one aspect, the t has a lung disease
selected from, cystic fibrosis (CF), chronic obstructive pulmonary disease, bronchiectasis
and . In a preferred aspect, the patient has CF. In another aspect of this
embodiment, the patient has a lung disease in which abnormal or excessive viscosity or
cohesiveness of mucus or sputum is associated with a deficiency of biological reductant
activity. In yet another aspect of this embodiment, the patient has a digestive tract disease
associated with thickened or abnormal mucus, including but not limited to coccidiosis.
In one aspect, the step of contacting the mucus or sputum of the patient with the
composition is performed by introducing the composition to the t by a route selected
from nasal, intratracheal, bronchial, direct installation into the lung, inhaled and oral. In
one aspect, the mucus or sputum to be contacted is located in the respiratory tract, the
digestive tract (i.e. intestinal tract) or the reproductive tract of the patient.
In another , the composition is administered to the patient in a
pharmaceutically able carrier.
In any of the foregoing aspects, the step of contacting the mucus or sputum of the
patient with the composition increases the percentage of free thiols in a sample of mucus
or sputum from the t as compared to prior contact with the ition.
In any of the foregoing aspects, after the step of contacting the mucus or sputum of
the patient with the composition the patient has at least about a 2.5% increase in forced
expiratory volume (FEV) as compared to prior to the step of contacting.
In any of the foregoing s, the thioredoxin monocysteinic active site
comprises an amino acid sequence selected from S (SEQ ID NO:24), C-X-X-X
(SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO:l9), X-C-G-P-X-X (SEQ ID , W-
C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID
NO:26), or W-C-G-P-S-K (SEQ ID NO:27) wherein the C residue is in a reduced state,
and wherein the X residues are any amino acid residue other than cysteine. In a preferred
aspect, the thioredoxin monocysteinic active site comprises the amino acid sequence C-X-
X-S (SEQ ID NO:24) as described above.
In any of the above aspects, the protein haVing a thioredoxin monocysteinic active
site comprises thioredoxin ed from the group consisting of prokaryotic thioredoxin,
fungal thioredoxin, plant thioredoxin, and mammalian thioredoxin. In a preferred aspect,
the protein comprises human thioredoxin.
In any of the above aspects of the invention, the composition r comprises a
reducing agent for reducing the thioredoxin monocysteinic active site of the protein. In a
further aspect, the composition ses thioredoxin reductase and NADH or NADPH.
Another ment of the invention relates to a composition for use in
sing viscosity of mucus or sputum, sing a protein or peptide containing a
thioredoxin monocysteinic active site in a reduced state and at least one additional agent
for treatment of excessively viscous or cohesive mucus or sputum. In one aspect of this
W0 2014/145735
embodiment, the thioredoxin steinic active site comprises an amino acid sequence
selected from C-X-X-S (SEQ ID N024), C-X-X-X (SEQ ID NO:l7), X-C-X-X-X-X
(SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID N021), W-C-G-P-X-K (SEQ ID N023), X-C-
X-X-S-X (SEQ ID NO25), X-C-G-P-S-X (SEQ ID N026), or W-C-G-P-S-K (SEQ ID
N027) wherein the C residue is in a reduced state, and wherein the X residues are any
amino acid residue other than ne. In a preferred aspect, the thioredoxin
monocysteinic active site comprises the amino acid ce C-X-X-S (SEQ ID N024)
as described above. In any of the foregoing aspects for this embodiment, the protein
having a thioredoxin monocysteinic active site comprises thioredoxin selected from a
group ting of prokaryotic thioredoxin, fugal thioredoxin, plant thioredoxin, and
mammalian thioredoxin. In one aspect, the protein comprises human thioredoxin.
Still fiarther, in any one of the foregoing aspects of this embodiment, the
ition comprises a reducing agent. In still a fiarther aspect, the composition further
comprises thioredoxin ase and NADH or NADPH.
Yet another embodiment of the present invention relates to a pharmaceutical
composition sing a protein or peptide ning a thioredoxin steinic
active site in a reduced state. In one aspect, the composition is formulated for aerosol
administration to the lung. In still another aspect, the composition is ated for oral
administration. In any of the foregoing aspects for this embodiment, the thioredoxin
monocysteinic active site comprises an amino acid ce selected from C-X-X-S (SEQ
ID NO: 24), C-X-X-X (SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X
(SEQ ID N021), W-C-G-P-X-K (SEQ ID N023), X-C-X-X-S-X (SEQ ID N025), X-C-
G-P-S-X (SEQ ID N026), and W-C-G-P-S-K (SEQ ID N027), wherein the C residue is
in a d state, and wherein the X residues are any amino acid residue other than
cysteine. In still another aspect, the pharmaceutical composition is formulated for aerosol
administration to the lung by a nebulizer device. In one aspect, the nebulizer device is a
vibrating-mesh nebulizer. In another aspect, the pharmaceutical composition fiarther
comprises a reducing agent. In any of the foregoing aspects of this invention, the
pharmaceutical composition fiarther comprises doxin reductase and NADH or
NADPH.
Yet another ment of the present invention relates to a composition
comprising a protein or peptide containing a thioredoxin monocysteinic active site,
wherein the cysteine in the monocysteinic active site is covalently bound to a cysteine
residue in a mucus protein. In one , the doxin monocysteinic active site
W0 2014/145735
comprises an amino acid sequence selected from C-X-X-S (SEQ ID NO: 24), C-X-X-X
(SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:2l), W-
C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID
NO:26), and W-C-G-P-S-K (SEQ ID NO:27), wherein the C residue is in a reduced state,
and wherein the X residues are any amino acid residue other than cysteine. In any one of
the above aspects, the mucus protein is a atory mucus n or a digestive tract
mucus protein. In r aspect, the mucus protein is a mucin.
Another embodiment of the present invention relates to a method to decrease
viscosity of mucus or sputum in a patient that has excessively viscous or cohesive mucus
or sputum. The method es the step of contacting the mucus or sputum of the patient
with a composition comprising a de bond reducing agent and a cysteine-blocking
agent. In one aspect, the de bond reducing agent and a cysteine-blocking agent are
the same molecule. In a further aspect, the same molecule is a n or peptide
containing a thioredoxin monocysteinic active site. In yet another , the de
bond reducing agent and the cysteine-blocking agent are different molecules. In still
another aspect, the disulfide bond ng agent can be dithiothreitol (DTT),
ethylenediaminetetraacetic acid (EDTA), glutathione, dithioglycolic acid, 2-
mercaptoethanol, N—acetyl cysteine or Tris-(2-carboxyethyl)phosphene. In yet a further
aspect, the ne-blocking agent can be iodoacetamide, iodoacetic acid, or other
alkylation agents.
Yet another embodiment of the t invention relates to a method to treat a
patient having excessively viscous or cohesive mucus. The method includes the step of
administering to the patient a composition comprising a compound having a thioredoxin
active site that is incapable of cellular uptake. In one aspect, the compound can be a
protein or peptide comprising a doxin monocysteinic active site, a fusion protein
comprising a thioredoxin portion and a cell surface or ligand portion, or a
combination of a protein or peptide comprising a thioredoxin active site and a blocking
compound for the ne corresponding to the cysteine at position 35 of SEQ ID NO: 12.
Another embodiment of the present invention relates to a method of preventing
systemic exposure to a drug nce in a patient. The method includes the step of
administering the drug to the patient by a delivery route including but not limited to a
pulmonary, oral or topical delivery route. In yet another aspect, the drug forms a covalent
bond to its target site once administered. In still r aspect, the drug substance is a
thiol-containing drug and can be, for example, a protein or peptide containing a
W0 2014/145735
thioredoxin monocysteinic active site in a d state. In still another aspect the drug
substance is an antibiotic or nfective agent and is fiased or attached by linker to a
thioredoxin monocysteinic active site in a reduced state. In still another aspect the drug
substance is an anti-inflammatory agent and is fiased or attached by linker to a thioredoxin
steinic active site in a reduced state. In still another aspect the drug substance is a
nucleic acid-hydrolyzing agent and is fused or attached by linker to a thioredoxin
monocysteinic active site in a d state. In still another aspect the drug substance is a
herapeutic agent and is fused or ed by linker to a thioredoxin monocysteinic
active site in a reduced state.
Still another embodiment of the t invention relates to a pharmaceutical
composition comprising a protein or peptide containing a doxin monocysteinic
active site in a reduced state and further comprising at least one saccharide or saccharide
derivative capable of stabilizing the reduced redox-active thiol group. In one aspect, the
saccharide or saccharide derivative can be sucrose, sucralose, lactose, trehalose, maltose,
galactose, raffinose, mannose or mannitol. In one aspect, the thioredoxin monocysteinic
active site comprises an amino acid sequence selected from C-X-X-S (SEQ ID NO: 24),
C-X-X-X (SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID
NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X
(SEQ ID N026), and W-C-G-P-S-K (SEQ ID NO:27), wherein the C residue is in a
reduced state, and wherein the X residues are any amino acid residue other than cysteine.
Another embodiment of the present invention relates to an animal feed
composition comprising a protein or peptide containing a thioredoxin monocysteinic
active site in a reduced state. In one aspect, the thioredoxin monocysteinic active site
ses an amino acid sequence selected from C-X-X-S (SEQ ID NO: 24), X
(SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:21), W-
C-G-P-X-K (SEQ ID , X-C-X-X-S-X (SEQ ID NO:25), P-S-X (SEQ ID
N026), and W-C-G-P-S-K (SEQ ID NO:27), wherein the C residue is in a reduced state,
and wherein the X residues are any amino acid residue other than cysteine.
In one aspect of any of the ments of the present invention, the patient is a
vertebrate, including but not limited to mammals and birds. In still another aspect, the
patient is a human. In yet another aspect, the patient is a chicken or a turkey.
Yet r embodiment of the present invention relates to a use of a composition
sing a protein or peptide containing a thioredoxin steinic active site in a
reduced state to decrease viscosity of mucus or sputum in a patient that has excessively
s or cohesive mucus or sputum, wherein contacting the mucus or sputum of the
patient with the composition decreases the viscosity of the mucus or sputum as compared
to prior to the step of contacting. In one , the thioredoxin monocysteinic active site
comprises an amino acid sequence selected from C-X-X-S (SEQ ID NO: 24), C-X-X-X
(SEQ ID NO:l7), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:2l), W-
X-K (SEQ ID NO:23), X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID
NO:26), and W-C-G-P-S-K (SEQ ID NO:27) n the C residue is in a reduced state,
and wherein the X residues are any amino acid residue other than cysteine.
Brief Description of the gs
Figs la-lb shows the enzymatic activity of a protein or peptide ning a
thioredoxin monocysteinic active site (referred to as r(Cys)hTrX) ed with a protein
or peptide containing a wild-type thioredoxin active site (referred to as X). Figure
la shows non-specific 5.5'—dithiobis—(2~nitrobenzoic acid) (DTNB or Ellman’s reagent)
reduction reflects loss of l reducible cysteine in thioredoxin monocysteinic active site vs.
wild-type. Figure lb shows that a n or peptide containing a thioredoxin
monocysteinic active site (r(Cys)hTrx) had similar or greater potency vs. a protein or
peptide containing a wild-type thioredoxin active site (WTrhTrX) in a human sputum
compaction assay at concentration amounts of 12.5 uM, 25 uM and 50 uM.
Fig 2 shows the effect of wildtype rhTrx, r(Cys)hTrx and various controls
including dithiothreitol (DTT) at two concentrations (0.58 mM and 1.5 mM), N—acetyl
cysteine (NAC) and recombinant human DNase e) at equimolar concentrations on
normalization of patient sputum s (n=6 per treatment) in a sputum-compaction
assay.
Fig 3 illustrates the mechanism of disulfide bond formation with a protein or
peptide having a thioredoxin monocysteinic active site at position 35 of SEQ ID NO:l2.
The mechanism of native Trx ide-reduction involves a two-step reaction. As shown
in figure 3, steps I and II show the formation of a transient mixed-disulfide between the N-
terminal Cys (located at position 32 in the human TRX-l amino acid sequence) of the Trx
active site and one Cys of the target protein disulf1de, followed by step 111. Step III shows
the nucleophilic attack on the intermolecular mixed disulfide bond by the C-terminal Cys
of the Trx active site, located at position 35 in the human TRX-l amino acid sequence.
This second reduction resolves the mixed-disulfide linkage and releases oxidized Trx and
the reduced target. By mutating the C-terminal active site Cys residue at position 35
of Trx to a non-cysteine amino acid, such as a serine residue (a monocysteinic variant), or
otherwise modifying the protein sequence such as to interfere with the nucleophilic attack
by the C-terminal active site Cys at position 35, thioredoxin is still able to function as a
reducing agent, but unlike the wild-type enzyme such monocysteinic active site variants
remain covalently ed to the reduced target protein via the unresolved intermolecular
mixed-disulf1de linkage.
Detailed Description of the Invention
The present invention generally relates to the use of a protein or peptide containing
a thioredoxin monocysteinic active site in a reduced state to induce, enhance and/or
increase the liquefaction of mucus or sputum. More specifically, the present inventor has
discovered that proteins or peptides with a monocysteinic thioredoxin active site se
the viscosity and/or cohesiveness of sputum or mucus and thereby are effective agents for
enhancing or increasing the liquefaction of sputum or mucus. Accordingly, ns or
peptides containing a monocysteinic thioredoxin active site in reduced state, or nucleic
acid molecules encoding such proteins, can be used alone or in a composition to treat a
variety of conditions or diseases associated with undesirable mucus or tenacious and
viscous sputum. For example, respiratory diseases such as cystic fibrosis, chronic
obstructive pulmonary disease, bronchiectasis and asthma are particularly amenable to
treatment using the product and process of the invention. Also, ive tract diseases
associated with thickened or adherent mucus such as coccidiosis are also particularly
amenable to treatment using the product and process of the ion. Therefore, the
t invention relates to the use of proteins containing a monocysteinic active site of
thioredoxin in a reduced state for decreasing the viscosity of mucus or sputum, particularly
mucus or sputum that is abnormally or ively viscous and/or cohesive. The proteins
are stered to a patient that is suffering from or affected by such abnormal or
excessive mucus or sputum in a manner and amount ive to decrease the viscosity of
the mucus or sputum and preferably, to provide a therapeutic benefit to the patient.
Thioredoxin and proteins containing either the ype (or native) thioredoxin
active site (also referred to herein as “rthr”) or containing the thioredoxin monocysteinic
active site (also referred to herein as “r(Cys)hTrx”) have advantages over other reducing
agents for use in the treatment of ions such as cystic fibrosis. For example, unlike
other reducing agents such as N—acetylcysteine (NAC), elyn (NAL), dithiothreitol
(DTT), or reducted glutathione (GSH), thioredoxin is less susceptible to inactivation by
tic or auto-oxidative mechanisms, including ons to produce superoxide,
hydrogen peroxide, hydroxyl l and other toxic oxygen metabolites. Furthermore,
native or wildtype thioredoxin is a naturally-occurring compound which is normally
secreted extracellularly onto the airway surface, and ore, introduction of thioredoxin
into the airway should be non-irritating and unlikely to induce an immune response.
Thioredoxin is also not glycosylated, and as such, it is more easily manufactured, and
administration of the protein in natural or recombinant form should not induce an innate
immune response. Perhaps even more significantly, d thioredoxin, in contrast to
other reducing agents, more rapidly and potently es the treated mucus or sputum to a
normal viscosity level, and this normalization lasts for a longer on. NAC, NAL,
DTT, and GSH, for example, become "spen " or oxidized over time and at this stage,
normalized sputum or mucus can revert back to an abnormal viscosity state. In contrast,
the decrease in viscosity produced by doxin appears to endure , most likely
due to its cyclic re-reduction by its reducing . Further, by remaining covalently
bound to mucin Cys residues r(Cys)hTrx creates an even more potent and longer-duration
reduction in viscosity vs native rhTrx. Finally, thioredoxin is both more potent and more
specific for disulfide bond-reduction than other reducing agents and therefore, it can be
used at significantly lower doses than other agents to achieve a beneficial effect.
In addition to the above-described advantages, thioredoxin has other benefits
which increase its usefulness in disease ions. For e, it is known that
thioredoxin induces MnSOD (e.g., see US. Patent No. 5,985,261 to White et al.,
incorporated herein by reference in its entirety) which is predicted to decrease the toxicity
of certain bacterial toxins (including, but not limited to, endotoxin from ial cell walls
of gram-negative bacteria, nin from Pseudomonas aeruginosa, and others) in
disease sputum (e.g., cystic fibrosis sputum). In addition, thioredoxin has extracellular
anti-inflammatory properties (Lee, R.L., et al., Am J l Lung Cell M01 Physio],
289(5):L875-82, 2005) that can enhance the l treatment of a respiratory condition.
Thioredoxin (Trx) is a protein disulfide reductase that catalyzes numerous thiol-
dependent cellular reductive processes. Native thioredoxin contains two redox-active
cysteines that are highly conserved across species. In their oxidized form, these cysteines
form a disulfide bridge that protrudes from the three dimensional structure of the protein
(Holmgren, Annu Rev Biochem 54:237-271, 1985). ion of this active center by the
NADPH-dependent thioredoxin reductase (TR) enzyme allows Trx to filnction as an
electron carrier with dithiol/disulfide exchange capability (Oblong et al., Biochemistry
32:7271-7277, 1993). Protein des are a preferred substrate for Trx-mediated
reducing action. Modification of the Trx C-terminal active site cysteine produces a
monocysteinic active site, which as discussed below, has substantial advantages over Trx
having the native or wildtype active site. The persistent and viscous nature of airway
secretions in cystic fibrosis disease leads to airway obstruction, opportunistic infection,
and deterioration of lung function. Recognizing that respiratory mucins contain multiple
cysteine domains that are believed to play an essential role in polymerization (Bell et al.,
Biochem J 357:203-209, 2001; Asker et al., Biochem J 333:381-387, 1998) as well as
increasing the entanglement of mucins via numerous intramolecular disulfide bonds, the
present inventor sought to determine whether Trx containing a monocysteinic active site
could serve as an effective mucus viscosity modulator by reduction of mucin des.
There are several advantages and benefits of thioredoxin containing a
monocysteinic active site versus thioredoxin containing the native or wildtype active site.
The monocysteinic modification is designed to minimize potential side effects of
thioredoxin associated with intracellular signaling or systemic exposure such as those
described by Rancourt et al. (Free Radical Biol & Med 42:1441-43, 2007). This
modification prevents nucleophilic attack on the mixed disulf1de formed between
thioredoxin and a target protein disulf1de that is catalyzed by the N—terminal thioredoxin
active site cysteine (for example located at position 32 in human thioredoxin, SEQ ID
NO: 14). Surprisingly, the present inventor has determined that the thioredoxin ning
a steinic active site has greater potency than wild-type doxin decreasing
ing toward liquefying) and normalizing the ity of diseased human mucus.
Even though thioredoxin ning the monocysteinic active site logically would be
expected to have less reducing potential due to the gross perturbation caused by loss of an
active site cysteine and therefore following an initial catalytic reaction at the inal
thioredoxin active site cysteine, be bound covalently to mucus proteins (such as the
heavily disulf1de-bonded mucins MUCSAC and MUCSB) and would be unable to be
reduced and m repeated catalysis, the present inventor has found that not only does
the thioredoxin containing the monocysteinic active site not show impaired activity
compared to ype doxin, it exhibits r quantitative ability to reduce human
CF mucus viscosity in a rheological assay. Based on this cted result, the or
has ded that the enhanced potency of thioredoxin containing the monocysteinic
active site must be a consequence of the covalently-linked thioredoxin and mucus
functioning to block re-formation of cysteine disulfides in mucins, thus providing an
exceedingly e and long-lived change in mucin oligomer gel structure and pore size.
At the same time, covalent linkage of a thioredoxin containing the monocysteinic active
site to its mucin target sequesters and hence prevents cellular uptake and internalization of
inhaled thioredoxin, which has the duel s of preventing off-target effects due to
undesired thioredoxin activity within cells, while at the same time facilitating clearance of
mucus-linked spent drug from the body. As the only therapeutic use envisioned
previously in the art for Cys-modif1ed monocysteinic thioredoxins is to facilitate uptake of
non-reduced forms via lipid rafts in endothelial cells following injection into the systemic
circulation (Hara et al., Antiox Redox Sig 9:1427-37, 2007; Kondo et al., Antiox Redox Sig
9:1439-48, 2007; US Patent Applications 20080119398, 20090075871, and
20100184215), and thus teaching away from the present invention which is focused on
ting systemic exposure and intracellular uptake, the extracellular ism for
enhanced potency and safety of monocysteinic active site doxin in a reduced form is
cipated and highly novel.
Mucus obstruction of the airways can cause cant morbidity and mortality in
patients with CF. The present inventor has demonstrated that the viscoelastic properties
facilitating the persistence of these secretions within airways are markedly diminished by
Trx containing a monocysteinic active site. This conclusion is supported by two lines of
experimental evidence. First, compaction assay results indicate that large amounts of
liquid are ed from the gel matrix of CF sputum during incubation with
monocysteinic Trx. Occurring simultaneously with this release are ses in the
volume of solid matter, indicating that the gel forming constituents of sputum were being
solubilized. This normalization of CF sputum viscosity could often be observed y in
CF sputum samples during the incubation period, and ore, is not an artifact of
centrifugal disruption. The liberation of liquid by monocysteinic Trx is expected to have
important therapeutic ations since restoration of water volume at airway es
can restore the mucociliary transport ability of CF epithelium (Jiang et al., Science
4-427, 1993) and relief of excess viscosity will, based on the Button et al. (Science,
2012) gel-on-brush model, allow hydration of the underlying periciliary layer and restore
mucociliary transport, the loss of which is the primary cause of pathology in CF. Second,
magnetic microrheometry measurements provide direct evidence that sputum
viscoelasticity es as a result of reduction of sputum components by monocysteinic
Trx.
CF sputum is a non-Newtonian fluid exhibiting both liquid and solid
characteristics. Polymers when present in solutions at low concentration are able to rotate
. When polymers become concentrated or cross-linked to such a degree that their
rotation is hindered, a solution has reached a transition phase called the percolation
threshold (Forgacs, J Cell Sci 108:2131-2143, 1995). At the ation threshold the
solution begins to acquire characteristics of a solid, and the elastic moduli continue to
increase as more cross-polymer interactions are added, until each filament in the sample is
incorporated into the matrix. Biochemical es have revealed that mucins MUCSAC
and MUCSB, secreted by cells lining the respiratory tract, are the major gel forming
polymers components of airway mucus (Hovenberg et al., Glycoconj J 13:839-847, 1996;
Thornton et al., Biochem J 316:967-975, 1996; Thornton et al., JBi0l Chem 272:9561-
9566, 1997). Cysteine domains present on these mucins contribute to r formation,
and possibly interaction with neighboring mucin chains, by intramolecular disulfide bond
formation (Bell et al., Biochem J 357:203-209, 2001; Asker et al., Biochem J 333381-
387, 1998) which is a likely contributor to gel mesh entanglement. Since disulfide bonds
on proteins are the preferred substrates for Trx enzymatic actiVity, mucin polymers are
s for reduction during the liquefaction of sputum by Trx. This is supported by PAS
staining which reveals s in the solubility of high molecular weight glycoforms in
Trx-treated sputum. Detection of r concentrations of glycoproteins in the liquid
phase of posed sputum was further indicated by a more intense yellow color and
had greater opacity than liquid phase derived from diluent-treated s. The enhanced
electrophoretic mobility of PAS-detectable glycoproteins in Trx-exposed sputum also
suggests that these macromolecules may decrease in size during enzymatic reduction.
Findings from this electrophoretic analysis are in agreement with compaction assay
measurements by demonstrating that glycoprotein release into liquid phase coincides with
the decrease in mass of the gel matrix during exposure to Trx, as well as the observation of
increased labeling of free thiols in sputum following Trx treatment (Rancourt, R. et al.,
Free Radic Biol Med, 42(9): 1441-1453, 2007).
Once chronic effects of inflammation and ion have established in a CF
patient, phil lysis within the airways of ed CF lungs results in the deposition
of ellular DNA into airway secretions (Lethem et al., Eur Respir J 3:19-23, 1990).
By valent interactions, this DNA becomes entangled within mucin glycoproteins,
increasing mucus gel Viscoelasticity (SachdeV et al., Chest 81:41S-43S, 1982). DNA
present in sputum becomes increasingly soluble following Trx treatment. A logical
explanation is that Trx actiVity causes structural changes within the gel matrix which are
sufficient to relieve entanglement interactions between DNA and the ed
macromolecules. It is uncertain what the relative contribution of this increased DNA
WO 45735
lity has toward viscoelastic changes observed during exposure of CF sputum to Trx.
eless, from a clinical standpoint, ing or removal of DNA from the insoluble
gel phase of sputum could render it more susceptible to DNase activity during such
treatment in CF. In addition, the action of monocysteinic r(Cys)hTrx to reduce mucus
viscosity and prevent rapid re-formation of disulfide bonds on mucin cysteines will
function to create a more permeable, accessible mucus layer as well as king
accumulated mucus plugs. These actions are expected to tate access of other
therapeutics to the deep lung and the pulmonary lial surface. Therefore, the
mechanistic method of the invention has a strong potential for synergy with existing
symptomatic therapies for CF and other obstructive pulmonary diseases such as delivery
of inhaled antibiotics, mucoactive substances or mucolytic DNA-hydrolyzing agents.
Trx containing a monocysteinic active site has both higher activity and greater
oxidative stability than reduced glutathione and acts extracellularly in the airway mucus
and does not enter lung cells. Trx comprising a monocysteinic active site decreases
viscosity, increases the liquid fraction and diminishes the viscoelasticity of sputum, for
example in CF sputum. The development of mucus-reducing systems that stimulate
release of liquid, and reduce the ity of airway secretions, is expected to have
therapeutic potential for diseases such as CF, as well as for the treatment of excessive or
abnormal mucus viscosity and/or cohesiveness that may be associated with other
respiratory conditions (e.g., chronic or acute bronchitis; bronchiectasis;
COPD/emphysema; asthma; acute tracheitis; acute or chronic sinusitis; tasis
resulting from acute or chronic mucus plugging of the airways; bronchiolitis) or with
various ive disorders (i.e., gastrointestinal), such as coccidiosis or reproductive
disorders associated with or exacerbated by excessive or abnormal mucus viscosity and/or
veness (e.g., acute, te or chronic bowel obstruction due to mucus
inspissation; infertility due to obstruction of vital reproductive structures). Since Trx
containing a monocysteinic active site in a d state becomes covalently linked to
mucin once it reacts with mucin disulfide bonds, such a mechanism of action will also
facilitate clearance of spent (oxidized) drug along with mucus, may prevent or ate
cellular update and thioredoxin-mediated redox signaling, and may prevent or attenuate
presentation to immune cells.
Accordingly, one embodiment of the present invention relates to a method to
normalize and decrease the ity of mucus or sputum in a patient that has excessively
viscous or cohesive mucus or sputum. The method es the step of contacting the
2014/030545
mucus or sputum of the patient with a composition comprising a protein or peptide
containing a thioredoxin monocysteinic active site in a reduced state. The n is
effective to decrease the viscosity of the mucus or sputum as compared to prior to the step
of contacting.
According to the present invention, the term "mucus" generally refers to a usually
clear viscid fluid that is secreted by mucous membranes in various tissues of the body,
including by the respiratory, gastrointestinal, and reproductive tracts. Mucus moistens,
lubricates and protects the tissues from which it is secreted. It ses mucin
macromolecules (including mucus ns, nucleic acids and carbohydrates), which are
the gel-forming constituents of mucus. Mucus proteins include but are not limited to
respiratory mucus ns and digestive tract mucus proteins. The viscoelastic properties
of normal mucus are dependent on the concentration, molecular weight, and degree of
entanglement between mucin polymers. The term m" generally refers to a mixture
of saliva and discharge from the respiratory passages, including mucus. Sputum is
typically an expectorated mixture of saliva and mucus (and other discharge from the
respiratory tissues). Therefore, mucus is a primary ent of sputum, and as such, the
presence of excessively viscous mucus results in a sputum which is itself excessively
viscous. The present ion s to decreasing the viscosity of the mucus or sputum.
The term "liquefaction" refers to the act of becoming more liquid. Therefore, an increase
in the liquefaction of mucus or sputum refers to the increase in liquid phase or liquid state
of mucus or sputum, as ed to a more solid or viscous phase. In the case of
abnormally viscous or excessive mucus associated with disease, the objective is to restore
a normal level of mucus viscosity. Hence, liquefaction may also be considered as a
reduction in mucus ity.
It is iated that normal mucus function is achieved by haVing the appropriate
ratio of biological reductants to oxidizable cysteines. Hence, a deficiency of biological
reductant actiVity is ore caused by either an excess of able cysteines or a lack
of biological reductants.
The general functions of mucus and sputum in the body require that the mucus
(and thus the mucus component of the sputum) have lastic properties. In an
indiVidual with normal mucus and sputum (i.e., a healthy indiVidual, or more particularly,
an individual who does not suffer from symptoms or a condition caused or exacerbated by
the viscosity or cohesiveness of mucus or sputum), the viscoelasticity is dependent on the
concentration, molecular weight, and entanglements between mucin polymers (Verdugo et
al., Biorheology 20:223-230, 1983). ally in CF, when mucins in the mucus interact
with DNA (Potter et al., Am JDis Child 100:493-495, 1960; Lethem et al., Am Rev Respir
Dis 100:493-495, 1990; Lethem et a1., Eur Respir J 3:19-23, 1990) and f-actin polymers
(Sheils et al., Am J Path 148:919-927, 1996; wicz et a1., DNA and actin filament
ultrastructure in cystic fibrosis sputum. In: Cilia, mucus, and mucociliary interactions,
edited by Baum GL, Priel Z, Roth Y, Liron N, and Ostfeld E]. New York, NY: Marcel
Dekker, 1998) released from dying inflammatory cells, the mucus (and thus sputum) can
additionally become even more dense and viscous. The inability to clear abnormal,
thickened mucus by cough or mucociliary clearance facilitates colonization of the lung
with opportunistic pathogens. Therefore, abnormally or excessively viscous and/or
ve mucus is characterized as mucus that is measurably or detectably more viscous or
cohesive than mucus from a normal or healthy t (preferably an age and sex-matched
patient), and/or as mucus which, by virtue of its level of viscosity and/or cohesiveness,
causes or butes to at least one symptom in a patient that causes discomfort or pain to
the patient, or that causes or exacerbates a condition or disease. In other words,
abnormally or excessively viscous and/or cohesive sputum is a deviation from normal
mucus or sputum wherein it is desirable to treat the patient to provide some relief from the
condition or other therapeutic benefit.
The method and composition of the present invention can be used to treat any
patient in whom it is desirable to se the ity of mucus or sputum. In particular,
patients that have certain lung, sinus, nasal, digestive or gastrointestinal, or reproductive
diseases or conditions can benefit from treatment using the method of the present
invention. The present invention is most useful for ameliorating or reducing at least one
symptom of a condition or disease that is caused by or exacerbated by abnormal or
excessive viscosity and/or cohesiveness of the mucus or sputum, which of course can
include lung-associated diseases such as cystic fibrosis, as well as digestive diseases, such
as coccidiosis. Other es may, at least some of the time, be associated with abnormal
or excessive viscosity and/or cohesiveness of the mucus or sputum, and when such a
symptom , the method of the present invention can be used to decrease viscosity of
the mucus or sputum and provide at least some relief or therapeutic benefit to the patient.
Examples of such es include, but are not d to: cystic fibrosis; chronic or acute
bronchitis; bronchiectasis (non-CF and CF bronchiectasis); COPD/emphysema; acute
tracheitis (bacterial, viral, mycoplasmal or caused by other organisms); acute or chronic
sinusitis; atelectasis (lung or lobar collapse) resulting from acute or chronic mucus
plugging of the s (sometimes seen in a variety of diseases such as asthma);
bronchiolitis (viral or other); acute, subacute or c bowel obstruction due to mucus
inspissation including, but not limited to meconium ileus or meconium ileus equivalent in
CF or similar disorders; other ive diseases and infertility due to obstruction of (but
not d to) the , seminal ducts or other vital reproductive structures. In addition,
as improved liary clearance is ated with clearance of bacteria and other
pathogens from the lung, the composition and method of the present invention may be
useful for reducing symptoms associated with excessive viscosity and/or cohesiveness of
the mucus or sputum in patients with a variety of atory infections, ing both
viral and bacterial ions.
As such, a therapeutic benefit is not necessarily a cure for a particular disease or
condition, but rather, preferably encompasses a result which most typically includes
ation of the disease or condition, elimination of the disease or condition, reduction or
elimination of a m associated with the disease or condition, tion or
alleviation of a secondary disease or condition resulting from the occurrence of a primary
e or condition (e.g., infectious disease caused by opportunistic pathogenic
microorganisms that take advantage of the excessively viscous mucus in the respiratory
tract), and/or prevention of the underlying disease or condition, or a symptom associated
with the disease or condition. As used herein, the phrase "protected from a disease" refers
to reducing the symptoms of the disease; palliative therapy (relieving or soothing a
symptom of the disease without ing a cure); reducing the occurrence of the disease,
and/or reducing the severity of the disease or to alleviate disease at least one symptom,
sign or cause of the disease or condition. Preventing refers to the y of a composition
of the present invention, when administered to a patient, to prevent a disease from
occurring. Curing (or disease-modifying) refers to the ability of a composition of the
present invention, when administered to a patient to cure the disease. To protect a patient
from a disease includes treating a patient that has a disease (therapeutic treatment).
Preventing a disease/condition includes preventing disease occurrence (prophylactic
treatment). In particular, protecting a patient from a disease (or preventing disease) is
accomplished by increasing (normalizing) the action of an abnormally viscous
mucus or sputum in the patient by contacting the mucus or sputum with a protein or
peptide comprising a doxin monocysteinic active site in a reduced state such that a
beneficial effect is obtained. A beneficial effect can easily be assessed by one of ordinary
skill in the art and/or by a trained clinician who is treating the patient. The term "disease"
refers to any deviation from the normal health of a patient and includes a state when
disease symptoms are t, as well as conditions in which a deviation (e.g., infection,
gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested.
Contact of the mucus and/or sputum of a patient with the protein or peptide
comprising a thioredoxin monocysteinic active site in a reduced state (or compositions
comprising such a protein) is intended to result in decreased viscosity / sed
liquefaction of the mucus or sputum as compared to prior to contact with the composition.
According to the present invention, an increase in liquefaction of mucus or sputum can be
any measurable or detectable increase in the level of liquefaction of mucus or sputum as
compared to a prior level of liquefaction, and is preferably a statistically significant
increase (i.e., differences in measured level of action between the patient sample and
a baseline control are statistically significant with a degree of nce of at least
p<0.05). Typically, the "baseline l" is a patient sample prior to the administration of
the treatment, since normal, y individuals generally cannot produce a quantity of
sputum sufficient to serve as a control, gh sputum from a normal, healthy individual
is not excluded as a baseline control. Additionally, a decrease is viscosity results in an
ement of lung fianction. This improvement can be ined by various means
including patient reported outcomes, mean time of exacerbation to al admission
and/or an increase in forced expiratory volume (FEV). In one aspect of the invention, an
increase in FEV is described as an increase of at least about 2.5%, about 3.0%, about 3.5
%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about
7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, and 9.5% and about 10% as
compared to a sample from the patient prior to contact with a composition or protein of the
present invention. Preferably, contact of a protein or composition of the present invention
with the mucus or sputum of a patient sample results in an increase of about 2.5% as
compared to a sample from the patient prior to contact with a composition or protein of the
present invention. Liquefaction of mucus or sputum and / or decrease in viscosity can be
measured using any suitable technique known in the art, including, but not d to,
compaction assays as described in the Examples n. In such an assay, the amount of
mucus or sputum in a solid phase (gel) versus s phase (liquid) is measured. In
other s of the invention, the relative viscosity or cohesiveness of mucus or sputum
can be measured using other parameters or indicators including, but not d to,
viscoelasticity (measured, for example, by magnetic microrheometry), glycoprotein
content, or DNA content. In another aspect of the ion the change in mucus protein
disulf1de bonding can be ted by the use of ts such as NEM (NEthylmaleimide
) that preferentially react with unbound (free) Cys residue thiol groups that
are created by the disruption of disulfide bonds (Rancourt, R. et a1., Free Radic Biol Med,
42(9):1441-1453, 2007). In one aspect of the invention, the level of liquefaction is
described as the amount of a given mucus or sputum sample that is in an aqueous (liquid)
phase as a tage of the total volume of the mucus or sputum sample. In a patient
with cystic s, for example, the level of liquefaction of mucus or sputum can be as
low as less than 10% or even less than 5% of the total volume. Preferably, contact of a
protein or composition of the invention with the mucus or sputum results in a change in
the liquefaction of the mucus or sputum of at least about such that at least about 15% of
the total volume is in liquid phase, and more preferably, at least about 20% of the total
volume is in liquid phase, and more ably, at least about 25% of the total volume is in
liquid phase, and more preferably, at least about 30% of the total volume is in liquid
phase, and more preferably, at least about 35% of the total volume is in liquid phase, and
more preferably, at least about 40% of the total volume is in liquid phase, and more
preferably, at least about 45% of the total volume is in liquid phase, and more preferably,
at least about 50% of the total volume is in liquid phase or until the ge or inhibition
of function caused by the mucus has cleared (e.g., until the patient airways are cleared
sufficiently to begin expectorating the fluid). In l, it is preferred that the
liquefaction of the sputum or mucus in increased in small, gradual increments until the
airway or other blocked passage (e.g., in the gastrointestinal or reproductive tract) is
cleared, but without excessively liquefying the sputum. Excessive liquefaction of the
mucus or sputum is not desired, as it can be detrimental to the patient (e.g., liquefied
sputum could flow backward and flood the small airways with a thin liquid, that may also
be infected, before the sputum can be cleared by the patient). Preferably, the contact of a
protein, peptide or composition of the invention with mucus or sputum es at least
about a 1% increase in the action of the mucus or sputum by volume as compared to
prior to the treatment, more preferably, at least about a 2% increase, and so on, in
increments of 1%, until the t s or other clogged passages are cleared. Once
such clearing is attained, e.g. by removal of so-called “mucus plugs” to improve access of
drug to the small airways and alveoli, then a lower-dose maintenance therapy may be
undertaken in order to keep newly-secreted mucin proteins at a normal state of disulf1de
bonding.
In one aspect, the therapy is conducted in conjunction with methods to clear the
d material from the ed tissue (respiratory tract, digestive tract, reproductive
tract) of the patient. For example, in the case of the respiratory system, one can use the
method of the present invention in conjunction with postural drainage, huff coughing and
other respiratory exercises, or any other le method for expectorating the liquefied
mucus or sputum.
According to the t invention, the mucus or sputum in the patient to be treated
is contacted with a protein (or composition sing the protein) that contains a
thioredoxin monocysteinic active site in a reduced state. The protein is effective to reduce
the ity and cohesiveness of sputum or mucus and/or to increase the liquefaction of
sputum or mucus as compared to prior to the step of contacting. As described usly,
thioredoxin is a protein disulfide reductase found in most organisms that participates in
many thiol-dependent cellular reductive processes. In humans, thioredoxin is also referred
to as adult T cell leukemia-derived factor (ADF). Intracellularly, most of this ubiquitous
low molecular weight (11,700) protein remains reduced. Reduced or ed thioredoxin
may be able to enter intact cells or absorb to the cell membrane, where a small amount is
gradually internalized over time. Native thioredoxin has two vicinal cysteine es at
the active site that in the oxidized protein form a disulfide bridge located in a protrusion
from the protein's three-dimensional structure. The flavoprotein thioredoxin reductase
catalyzes the NADPH-dependent reduction of this disulf1de. In addition, engineered
ns of thioredoxin reductase modified for altered or specificity may utilize
NADH instead or in on to NADPH as described in United States patent 7,071,307,
hereby incorporated by reference. Small increases in thioredoxin can cause nd
changes in sulfhydryl-disulfide redox status in proteins.
In addition to its ability to effect the reduction of cellular proteins, it is ized
that thioredoxin can act directly as an antioxidant (e.g. by preventing oxidation of an
oxidizable substrate by scavenging reactive oxygen species) although, unlike other thiols,
thioredoxin does not generally contribute to the oxidative stress in a cell by autooxidizing
(e.g. generating superoxide radicals through autooxidation). US. Patent No. 261 to
White et al., supra, showed that thioredoxin directly induces the production of MnSOD
and that such induction is ed by doxin in a reduced state.
A "thioredoxin monocysteinic active site" of the present invention comprises the
amino acid sequence C-X-X-X (SEQ ID NO:17) (native or wild-type sequence comprises
the amino acid sequence C-X-X-C having SEQ ID NO:16). As used herein, amino acid
residues denoted "C" are cysteine residues and amino acid es denoted "X" can be
any amino acid e other than a cysteine residue, and in particular, any of the
remaining standard 20 amino acid residues. Such a thioredoxin monocysteinic active site
of the present invention preferably comprises the amino acid ce C-G-P-X (SEQ ID
NO:18), wherein the native or ype sequence comprises the amino acid sequence C-
G-P-C (SEQ ID NO: 1). A thioredoxin monocysteinic active site can further comprise the
amino acid sequence X-C-X-X-X-X (SEQ ID NO:l9), wherein the native or ype
sequence comprises the amino acid sequence X-C-X-X-C-X (SEQ ID NO:20). Preferably,
a thioredoxin monocysteinic active site of the present invention comprises the amino acid
sequence X-C-G-P-X-X (SEQ ID NO:2l), wherein such amino acid residue denoted "G"
is a glycine residue, and wherein such amino acid residue d "P" is a proline residue,
wherein the native or wild-type sequence comprises the amino acid ce P-C-
X (SEQ ID NO:22). More preferably, a thioredoxin monocysteinic active site of the
t invention comprises the amino acid sequence W-C-G-P-X-K (SEQ ID NO:23),
wherein such amino acid residue denoted "W" is a tryptophan residue, and wherein such
amino acid residue denoted "K" is a lysine residue and wherein the native sequence
comprises the amino acid sequence W-C-G-P-C-K (SEQ ID NO:3). Preferably, a
thioredoxin monocysteinic active site can comprise the amino acid sequence C-X-X-S
(SEQ ID . Such a thioredoxin monocysteinic active site of the present invention
preferably comprises the amino acid sequence C-G-P-S (SEQ ID NO:1). A thioredoxin
monocysteinic active site can further comprise the amino acid sequence X-C-X-X-S-X
(SEQ ID NO:25), X-C-G-P-S-X (SEQ ID NO: 26) or W-C-G-P-S-K (SEQ ID NO:27),
wherein amino acid residues denoted "X" can be any amino acid residue other than a
cysteine residue. Reference to “thioredoxin active site” es thioredoxin
monocysteinic active sites and native or pe thioredoxin active sites.
In one aspect of the invention, the protein containing a thioredoxin monocysteinic
active site is a ength thioredoxin protein or any fragment thereof containing a
thioredoxin monocysteinic active site as described urally and fianctionally above.
Preferred thioredoxin proteins having monocysteinic active sites include prokaryotic
thioredoxin, yeast doxin, plant thioredoxin, and mammalian thioredoxin, with human
doxin being particularly preferred. The nucleic acid and amino acid sequences of
thioredoxins from a variety of organisms are well known in the art and are intended to be
encompassed by the present invention. For example, SEQ ID NOs:4-15 represent the
amino acid sequences for thioredoxin from Pseudomonas syringae (SEQ ID NO:4),
Porphyromonas gingivalis (SEQ ID NO:5), Listeria togenes (SEQ ID NO:6),
Saccharomyces cerevisiae (SEQ ID NO:7), Gallas gallas (SEQ ID NO:8), Mas mascalus
(SEQ ID NO:9), Rattas norvegz'cas (SEQ ID , Bos taaras (SEQ ID NOill), Homo
sapiens (SEQ ID NOilZ), Arabidopsis thaliana (SEQ ID NOil3), Zea mays (SEQ ID
NO:l4), and Oryza sativa (SEQ ID NO:15). Referring to each of these sequences, the X-
C-G-P-C-X (SEQ ID NO:22) motif (which includes the CGPC motif of SEQ ID NO:1)
can be found as follows: SEQ ID NO:4 (positions 33-38), SEQ ID NO:5 (positions 28-
33), SEQ ID NO:6 (positions 27-32), SEQ ID NO:7 (positions , SEQ ID NO:8
(positions 31-36), SEQ ID NO:9 (positions 31-36), SEQ ID NO:lO (positions 31-36), SEQ
ID NO:11 (positions 31-36), SEQ ID NO:12 (positions 31-36), SEQ ID NO:l3 (positions
59-64), SEQ ID NO:l4 (positions 88-93) and SEQ ID NO:15 (positions 94-99).
Moreover, the three-dimensional structure of several thioredoxin proteins has been
resolved, including human and bacterial thioredoxins. Therefore, the structure and active
site of thioredoxins from multiple organisms is well known in the art and one of skill in
the art would be able to readily identify and produce fragments or homologues of full-
length thioredoxins, ing thioredoxins having monocysteinic active sites that can be
used in the present invention.
The phrase "in a reduced state" specifically describes the state of the cysteine
residues in the active site of a protein or peptide of the present invention. In a reduced
state, adjacent cysteine residues form a dithiol (i.e. two free sulfhydryl , -SH). In
contrast, in oxidized form, such cysteine residues form an intramolecular disulfide bridge;
such a molecule can be referred to as cystine. In a reduced state, a steinic
thioredoxin active site is e of participating in redox reactions through the reversible
ion of its active site thiol to a disulfide, and catalyzes thiol-disulfide exchange
reactions that result in covalent linkage to one of the target ide Cys. For proteins or
peptides of the t invention containing a doxin monocysteinic active site, the
N—terminal cysteine in the active site is in a reduced state as a monothiol and is therefore
able to form a stable mixed-disulfide with a cysteine on the target protein.
As used herein, a protein of the present invention containing a thioredoxin
monocysteinic active site can be a thioredoxin monocysteinic active site per se or a
thioredoxin monocysteinic active site joined to other amino acids by glycosidic linkages.
Thus, the minimal size of a n or e of the present invention is from about 4 to
about 6 amino acids in length, with preferred sizes depending on r a full-length,
fusion, multivalent, or merely onal portions of such a protein is d. Preferably,
the length of a protein or peptide of the present invention extends from about 4 to about
100 amino acid residues or more, with peptides of any interim length, in whole integers
(i.e., 4, 5, 6, 7...99, 100, 101...), being specifically oned. It may also be a short
thioredoxin mimetic peptide d at the N and C termini as described by Bachnoff et
al., Free Radical Biol Med 50:1355-67, 2011. In a fiarther preferred embodiment, a
protein of the present invention can be a ength n or any homologue of such a
protein. As used herein, the term "homologue" is used to refer to a protein or e
which differs from a lly occurring protein or e (i.e., the "prototype" or
"wildtype" protein) by modifications to the naturally-occurring protein or peptide, but
which maintains the basic protein and side chain structure of the naturally-occurring form,
and/or which maintains a basic three-dimensional structure of at least a biologically active
portion (e.g., the thioredoxin active site) of the native protein. Such changes include, but
are not limited to: changes in one or a few amino acid side chains; changes in one or a few
amino acids, including deletions (e.g., a truncated version of the protein or e
(fragment)), insertions and/or substitutions; changes in stereochemistry of one or a few
atoms; and/or minor derivatizations, including but not limited to: methylation,
glycosylation, phosphorylation, acetylation, oylation, prenylation, oylation,
amidation and/or addition of glycosylphosphatidyl ol. According to the present
invention, any protein or peptide useful in the present ion, including homologues of
natural thioredoxin proteins, have a thioredoxin monocysteinic active site such that, in a
reduced state, the protein or peptide is capable of participating in redox reactions through
the oxidation of its active site thiol to a disulfide and/or of decreasing the viscosity or
cohesiveness of mucus or sputum or increasing the liquefaction of mucus or sputum. As
used herein, a protein or peptide containing a thioredoxin steinic active site can
have characteristics similar to thioredoxin, and preferably, is a thioredoxin selected from
the group of prokaryotic thioredoxin, fiangal thioredoxin (including yeast), plant
thioredoxin, or mammalian thioredoxin. In a particularly preferred embodiment, the
protein is human thioredoxin.
Homologues can be the result of natural allelic variation or natural mutation. A
lly occurring allelic variant of a nucleic acid encoding a protein is a gene that occurs
at essentially the same locus (or loci) in the genome as the gene which encodes such
protein, but which, due to l variations caused by, for example, mutation or
recombination, has a similar but not identical ce. Allelic variants typically encode
ns haVing similar actiVity to that of the protein encoded by the gene to which they
are being compared. One class of allelic variants can encode the same protein but have
different nucleic acid sequences due to the degeneracy of the genetic code. Allelic
variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e. g.,
in regulatory control regions). Allelic variants are well known to those skilled in the art.
Homologues can be produced using techniques known in the art for the production
of proteins including, but not limited to, direct modifications to the ed, naturally
occurring protein, direct n synthesis, or modifications to the nucleic acid ce
encoding the protein using, for example, classic or recombinant DNA techniques to effect
random or targeted mutagenesis.
Modifications in homologues, as compared to the wild-type protein, either e,
antagonize, or do not substantially change, the basic ical activity of the homologue
as compared to the naturally occurring protein. In general, the ical activity or
biological action of a protein refers to any filnction(s) exhibited or performed by the
protein that is ascribed to the naturally occurring form of the protein as measured or
observed in viva (i.e., in the natural physiological nment of the n) or in vitro
(i.e., under laboratory conditions). Modifications of a n, such as in a homologue or
mimetic (discussed below), may result in proteins having the same biological activity as
the naturally-occurring protein, or in proteins having decreased or increased biological
activity as compared to the naturally occurring protein. Modifications which result in a
se in protein expression or a decrease in the ty of the protein, can be referred
to as inactivation (complete or partial), down-regulation, or decreased action of a protein.
Similarly, modifications which result in an se in protein expression or an increase in
the activity of the protein, can be referred to as amplification, overproduction, activation,
enhancement, up-regulation or increased action of a protein.
In one embodiment, proteins or peptides ning a thioredoxin monocysteinic
active site can be products of drug design or selection and can be produced using various
methods known in the art. Such ns or peptides can be referred to as cs. A
mimetic refers to any peptide or non-peptide compound that is able to mimic the
ical action of a naturally-occurring peptide, often because the mimetic has a basic
structure that mimics the basic structure of the naturally-occurring peptide and/or has the
salient biological properties of the naturally occurring peptide. Mimetics can include, but
are not limited to: peptides that have substantial modifications from the prototype such as
no side chain similarity with the naturally occurring e (such modifications, for
example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic
2014/030545
antibodies, or fragments thereof; non-proteinaceous portions of an isolated protein (e.g.,
carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids
and drugs identified through combinatorial chemistry, for example. Such mimetics can be
designed, selected and/or otherwise fied using a y of methods known in the art.
Various methods of drug design, useful to design or select mimetics or other therapeutic
compounds useful in the present ion are disclosed in Maulik et al., 1997, Molecular
Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is
orated herein by reference in its entirety. Thioredoxin mimetic peptides e of
potent and selective redox activity are described by Bachnoff et al., Free Radical Biol Med
50: 1355-67 (2011) and incorporated herein by reference in its entirety.
A mimetic can be obtained, for example, from molecular diversity strategies (a
combination of d strategies allowing the rapid construction of large, chemically
e molecule libraries), libraries of natural or synthetic compounds, in particular from
chemical or combinatorial libraries (i.e., ies of compounds that differ in sequence or
size but that have the similar ng blocks) or by rational, directed or random drug
. See for example, Maulik et al., supra.
In a molecular diversity strategy, large compound libraries are synthesized, for
example, from peptides, ucleotides, carbohydrates and/or synthetic organic
molecules, using biological, enzymatic and/or chemical approaches. The al
parameters in developing a molecular diversity gy include subunit diversity,
molecular size, and library ity. The general goal of screening such libraries is to
utilize sequential application of combinatorial selection to obtain high-affinity ligands for
a desired target, and then to optimize the lead molecules by either random or directed
design strategies. Methods of molecular ity are described in detail in Maulik, et al.,
ibid.
Maulik et al. also disclose, for example, methods of directed design, in which the
user directs the process of creating novel molecules from a fragment library of
appropriately selected fragments; random design, in which the user uses a genetic or other
algorithm to randomly mutate fragments and their combinations while simultaneously
applying a selection criterion to evaluate the fitness of ate ligands; and a grid-based
approach in which the user calculates the interaction energy between three dimensional
or structures and small fragment probes, followed by linking together of favorable
probe sites.
Diversity-creation methods such as the foregoing can be ed with other
techniques designed to improve fianction or pharmacology, especially for reduced-size
les like -site mimetics. For example, one ch that has shown promise in
early-stage studies is hydrocarbon-stapled (x-helical peptides, a novel class of synthetic
miniproteins locked into their bioactive (x-helical fold through the site-specific
introduction of a chemical brace, an all-hydrocarbon staple. Stapling can greatly improve
the pharmacologic performance of peptides, increasing their target affinity and proteolytic
resistance, while creating r peptide versions of larger proteins/enzymes that are
suitable for chemical synthesis (Verdine, G. L. and Hilinsky, G. J., Methods Enzymol,
5033-33, 2012).
In one embodiment of the present invention, a protein suitable for use in the
present invention has an amino acid ce that comprises, consists essentially of, or
consists of a full length sequence of a thioredoxin protein or any fragment thereof that has
a thioredoxin steinic active site as described herein. For e, any one of the
native ces of SEQ ID NOs 4-15 or a fragment or other homologue thereof that
contains a thioredoxin monocysteinic active site as described herein is encompassed by the
invention. Such homologues can include proteins having an amino acid sequence that is at
least about 10% identical to the amino acid sequence of a full-length thioredoxin n,
or at least 20% identical, or at least 30% identical, or at least 40% identical, or at least
50% identical, or at least 60% identical, or at least 70% identical, or at least 80% identical,
or at least 90% identical, or greater than 95% identical to the amino acid sequence of a
full-length thioredoxin protein, including any percentage between 10% and 100%, in
whole integers (10%, 11%, 12%,...98%, 99%, 100%).
As used herein, unless otherwise ed, reference to a t (%) identity
refers to an tion of homology which is performed using: (1) a BLAST 2.0 Basic
BLAST homology search using blastp for amino acid searches and blastn for nucleic acid
searches with standard default parameters, wherein the query sequence is filtered for low
complexity regions by default (described in Altschul, S.F., Madden, T.L., Schaaffer, A.A.,
Zhang, J., Zhang, Z., Miller, W. & Lipman, DJ. (1997) "Gapped BLAST and PSI-
BLAST: a new generation of n database search programs." Nucleic Acids Res.
:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment
(using the parameters described ; (3) and/or PSI-BLAST with the standard default
parameters ion-Specific Iterated BLAST. It is noted that due to some differences in
the rd parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific
sequences might be recognized as having significant homology using the BLAST 2
program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the
sequences as the query sequence may not identify the second sequence in the top matches.
In addition, PSI-BLAST provides an automated, easy-to-use version of a "profile" search,
which is a ive way to look for ce homologues. The program first performs a
gapped BLAST database search. The PSI-BLAST program uses the information from any
significant alignments returned to construct a on-specific score , which
replaces the query sequence for the next round of database searching. Therefore, it is to be
understood that percent identity can be determined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST 2 sequence as
described in Tatusova and Madden, (1999), "Blast 2 sequences - a new tool for comparing
protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250, incorporated
herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or
blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0)
between the two ces allowing for the introduction of gaps (deletions and insertions)
in the resulting alignment. For purposes of y herein, a BLAST 2 ce alignment
is med using the standard t parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match = 1
Penalty for mismatch = -2
Open gap (5) and extension gap (2) penalties
gap x_dropoff (50)w (10) word size (11) filter (on)
For blastp, using 0 BLOSUM62 matrix:
Open gap (11) and extension gap (1) penalties
gap x_dropoff (50) e_xILct (10) word size (3) filter (on).
A n useful in the present invention can also include proteins having an amino
acid sequence comprising at least 10 contiguous amino acid residues of any fiJll-length
thioredoxin protein containing a monocysteinic active site (native sequences represented
by SEQ ID NOs:4-15, i.e., 10 contiguous amino acid residues having 100% identity with
contiguous amino acids of a reference sequence). In other embodiments, a homologue
of a doxin protein includes amino acid ces comprising at least 15, or at least
, or at least 25, or at least 30, or at least 35, or at least 40, or at least 45, or at least 50, or
at least 55, or at least 60, or at least 65, or at least 70, or at least 75, or at least 80
contiguous amino acid residues of the amino acid sequence of a naturally occurring
thioredoxin protein, and so on, up to the ength of the protein, including any
intervening length in whole integers (10, ll, 12,..) and which comprises a steinic
active site.
According to the present invention, the term “contiguous” or “consecutive”, with
regard to sequences described herein, means to be connected in an unbroken sequence.
For example, for a first sequence to comprise 30 contiguous (or consecutive) amino acids
of a second sequence, means that the first sequence includes an unbroken ce of 30
amino acid residues that is 100% identical to an unbroken sequence of 30 amino acid
residues in the second sequence. Similarly, for a first sequence to have “100% identity”
with a second sequence means that the first sequence exactly matches the second sequence
with no gaps between tides or amino acids.
In another embodiment, a protein useful in the present invention includes a protein
having an amino acid sequence that is sufficiently r to a natural thioredoxin amino
acid sequence that a nucleic acid sequence encoding the homologue is capable of
izing under moderate, high or very high stringency conditions (described below) to
(i.e., with) a nucleic acid molecule encoding the natural thioredoxin protein (i.e., to the
complement of the nucleic acid strand encoding the natural thioredoxin amino acid
ce). Such hybridization conditions are described in detail below.
A nucleic acid sequence complement of nucleic acid sequence encoding a
thioredoxin protein of the t invention refers to the nucleic acid sequence of the
c acid strand that is complementary to the strand that s thioredoxin. It will be
appreciated that a double-stranded DNA which encodes a given amino acid sequence
comprises a single strand DNA and its complementary strand having a sequence that is a
complement to the single strand DNA. As such, nucleic acid molecules of the present
invention can be either double-stranded or -stranded, and include those nucleic acid
molecules that form stable hybrids under stringent hybridization conditions with a nucleic
acid sequence that encodes an amino acid sequence of a thioredoxin protein, and/or with
the complement of the nucleic acid ce that encodes such amino acid sequence.
Methods to deduce a complementary sequence are known to those skilled in the art.
As used herein, reference to hybridization ions refers to standard
hybridization conditions under which nucleic acid molecules are used to identify similar
nucleic acid molecules. Such rd conditions are sed, for example, in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989.
Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically,
pages 9.31-9.62). In addition, ae to calculate the appropriate hybridization and
wash conditions to e hybridization permitting varying degrees of mismatch of
nucleotides are sed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-
284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.
More ularly, moderate stringency hybridization and washing conditions, as
referred to , refer to conditions which permit isolation of nucleic acid molecules
having at least about 70% nucleic acid sequence identity with the nucleic acid molecule
being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or
less mismatch of nucleotides). High stringency hybridization and washing conditions, as
referred to , refer to conditions which permit isolation of nucleic acid molecules
having at least about 80% nucleic acid sequence identity with the nucleic acid molecule
being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or
less mismatch of nucleotides). Very high stringency hybridization and g
conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid
molecules having at least about 90% nucleic acid sequence ty with the nucleic acid
molecule being used to probe in the hybridization reaction (i.e., conditions permitting
about 10% or less ch of nucleotides). As discussed above, one of skill in the art
can use the formulae in Meinkoth et al., ibid. to calculate the appropriate hybridization and
wash conditions to achieve these particular levels of nucleotide mismatch. Such
conditions will vary, depending on whether A or DNA:DNA hybrids are being
formed. Calculated melting temperatures for DNA:DNA hybrids are 10°C less than for
DNA:RNA hybrids. In particular embodiments, ent hybridization conditions for
DNA:DNA hybrids include hybridization at an ionic th of 6X SSC (0.9 M Na+) at a
temperature of between about 20°C and about 35°C (lower ency), more preferably,
between about 28°C and about 40°C (more stringent), and even more preferably, between
about 35°C and about 45°C (even more stringent), with appropriate wash conditions. In
particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include
hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between
about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and
even more preferably, between about 45°C and about 55°C, with similarly stringent wash
ions. These values are based on ations of a g temperature for molecules
larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%.
Alternatively, TIn can be calculated empirically as set forth in Sambrook et al., supra,
pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and
should be appropriate for the chosen hybridization conditions. For example, ization
conditions can include a combination of salt and temperature conditions that are
approximately 20-25°C below the ated Tm of a particular hybrid, and wash
ions typically include a combination of salt and temperature conditions that are
approximately l2-20°C below the calculated TIn of the particular . One example of
hybridization ions suitable for use with DNA:DNA hybrids includes a 2-24 hour
hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that
include one or more washes at room temperature in about 2X SSC, followed by additional
washes at higher temperatures and lower ionic strength (e.g., at least one wash as about
37°C in about 0. lX-0.5X SSC, followed by at least one wash at about 68°C in about 0.lX-
0.5X SSC).
A protein of the present invention can also be a fusion protein that includes a
segment containing a thioredoxin monocysteinic active site and a fusion segment that can
have a variety of functions. For example, such a fusion t can on as a tool to
simplify ation of a protein of the present invention, such as to enable purification of
the ant fusion protein using affinity chromatography. A suitable qulOI‘l t can
be a domain of any size that has the desired function (e.g., imparts increased stability to a
protein, imparts increased immunogenicity to a protein, and/or simplifies purification of a
protein). It is within the scope of the present invention to use one or more fusion
segments. Fusion segments can be joined to amino and/or carboxyl termini of the segment
containing a doxin steinic active site. Linkages n fusion segments
and thioredoxin active ontaining domains of fusion proteins can be susceptible to
cleavage in order to enable straightforward recovery of the thioredoxin monocysteinic
active site-containing domains of such proteins. Fusion proteins are preferably produced
by culturing a recombinant cell transformed with a fusion nucleic acid le that
encodes a n including the fusion segment attached to either the carboxyl and/or
amino terminal end of a thioredoxin monocysteinic active site-containing domain.
In one embodiment, a protein or peptide containing a thioredoxin monocysteinic
active site suitable for use with the method of the present invention comprises a protein or
peptide containing a thioredoxin monocysteinic active site derived from a substantially
similar species of animal as that to which the protein is to be administered. In another
embodiment, any protein or peptide containing a thioredoxin monocysteinic active site,
including from diverse sources such as microbial, plant and fiangus can be used in a given
patient.
In one embodiment of the present invention, any of the amino acid sequences
described herein, such as the amino acid sequence of a naturally occurring doxin
protein or thioredoxin containing a monocysteinic active site, can be produced with from
at least one, and up to about 20, additional heterologous amino acids flanking each of the
C- and/or N—terminal ends of the specified amino acid sequence. The resulting protein or
ptide can be referred to as "consisting ially of‘ the specified amino acid
sequence. According to the present invention, the heterologous amino acids are a
sequence of amino acids that are not naturally found (i.e., not found in , in viva)
flanking the specified amino acid sequence, or that are not related to the filnction of the
specified amino acid sequence, or that would not be encoded by the nucleotides that flank
the naturally-occurring nucleic acid sequence ng the specified amino acid sequence
as it occurs in the gene, if such nucleotides in the naturally occurring sequence were
translated using rd codon usage for the organism from which the given amino acid
sequence is d. Similarly, the phrase "consisting essentially of‘, when used with
reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a
specified amino acid sequence that can be flanked by from at least one, and up to as many
as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the
nucleic acid sequence encoding the specified amino acid sequence. The heterologous
nucleotides are not lly found (i.e., not found in , in viva) flanking the nucleic
acid ce encoding the specified amino acid sequence as it occurs in the natural gene
or do not encode a protein that imparts any additional function to the protein or changes
the fianction of the protein having the specified amino acid sequence.
In another embodiment, a protein or peptide ning a thioredoxin
monocysteinic active site suitable for use with the method of the present invention
comprises an isolated, or biologically pure, protein. As such, "isolated" and "biologically
pure" do not necessarily reflect the extent to which the protein has been purified. An
isolated protein of the present ion can, for example, be obtained from its natural
source, be produced using inant DNA technology (e. g., polymerase chain reaction
(PCR) amplification, cloning), or be sized chemically.
In yet another embodiment, a chemically-synthetic protein or peptide ning a
doxin monocysteinic active site of the present invention may also refer to a
stabilized version, such as one containing an active site constrained structurally by stapled
peptide technology, by cyclization, or by constraint at the N or C i. Preferably, the
protein containing a thioredoxin monocysteinic active site to be used in methods of the
invention have a half-life in viva that is sufficient to cause a measurable or detectable
increase in liquefaction (or decrease in the viscosity or veness) of mucus or sputum
in a patient, and or to cause a measurable, detectable or perceived therapeutic benefit to
the patient that is associated with the mucus and sputum in the patient. Such ife can
be effected by the method of delivery of such a protein. A protein of the present invention
preferably has a half-life of greater than about 5 minutes in an animal, and more preferably
r than about 4 hours in an animal, and even more preferably greater than about 16
hours in an animal. In a preferred embodiment, a n of the present invention has a
half-life of between about 5 minutes and about 24 hours in an animal, and preferably
between about 2 hours and about 16 hours in an animal, and more preferably between
about 4 hours and about 12 hours in an animal.
Further embodiments of the present invention include nucleic acid molecules that
encode a n or peptide containing a thioredoxin monocysteinic active site. Such
nucleic acid molecules can be used to produce a protein that is useful in the method of the
present invention in vitro or in vivo. A nucleic acid molecule of the present invention
includes a nucleic acid molecule comprising, consisting essentially of, or consisting of, a
nucleic acid sequence encoding any of the ns described previously herein. In
ance with the present invention, an isolated nucleic acid molecule is a nucleic acid
le (polynucleotide) that has been removed from its natural milieu (i.e., that has
been subject to human manipulation) and can e DNA, RNA, or derivatives of either
DNA or RNA, including cDNA. As such, "isolated" does not reflect the extent to which
the nucleic acid molecule has been purified. Although the phrase "nucleic acid molecule"
primarily refers to the physical nucleic acid le and the phrase "nucleic acid
sequence" primarily refers to the sequence of tides on the nucleic acid molecule, the
two phrases can be used interchangeably, especially with respect to a nucleic acid
molecule, or a nucleic acid sequence, being capable of encoding a protein. An ed
nucleic acid molecule of the present invention can be isolated from its natural source or
produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR)
cation, cloning) or chemical synthesis. Isolated c acid molecules can e,
for example, genes, l allelic variants of genes, coding regions or portions thereof,
and coding and/or tory regions modified by nucleotide insertions, deletions,
substitutions, and/or inversions in a manner such that the modifications do not
substantially interfere with the nucleic acid le's ability to encode the desired protein
of the present invention or to form stable hybrids under stringent conditions with natural
gene isolates. An ed nucleic acid molecule can include degeneracies. As used
herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be
encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid
molecule that s a given protein useful in the present ion can vary due to
racies.
According to the present invention, reference to a gene includes all nucleic acid
sequences related to a natural (i.e. wildtype) gene as well as those d to the
thioredoxin steinic active site, such as tory regions that control production
of the protein encoded by that gene (such as, but not limited to, transcription, translation or
post-translation control regions) as well as the coding region itself In another
embodiment, a gene can be a naturally occurring allelic variant that includes a similar but
not cal sequence to the nucleic acid sequence encoding a given protein. Allelic
ts have been usly described above. The phrases “nucleic acid molecule” and
“gene” can be used interchangeably when the nucleic acid molecule comprises a gene as
described above.
Preferably, an isolated nucleic acid molecule of the present invention is produced
using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification,
cloning) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic
acid molecules and homologues thereof, ing, but not limited to, natural allelic
variants and modified nucleic acid molecules in which nucleotides have been inserted,
deleted, substituted, and/or inverted in such a manner that such modifications e the
desired effect on protein biological activity. Allelic variants and protein homologues (e.g.,
ns encoded by nucleic acid homologues) have been sed in detail above.
A nucleic acid molecule homologue can be produced using a number of methods
known to those skilled in the art (e. g., as described in Sambrook et al., ibid). For example,
nucleic acid molecules can be modified using a variety of techniques including, but not
d to, by classical mutagenesis and recombinant DNA techniques (including without
limitation site-directed mutagenesis, chemical treatment, restriction enzyme cleavage,
ligation of nucleic acid nts and/or PCR amplification), or synthesis of
oligonucleotide mixtures and chemical ligation, or in vitro or in viva recombination, of
mixtures of molecular groups to " a re-assorted library of nucleic acid molecules
comprising a multiplicity of combinations thereof by the process of gene shuffling (i.e.,
lar breeding; see, for example, US. Patent No. 793 to Stemmer; Minshull
and Stemmer, Curr. Opin. Chem. Biol. 3:284-290, 1999; Stemmer, P.N.A.S. USA
91:10747-10751, 1994, all of which are incorporated herein by reference in their entirety).
These and other similar techniques known to those skilled in the art can be used to
efficiently introduce multiple simultaneous changes in the protein. Nucleic acid le
homologues can subsequently be selected by hybridization with a given gene, or be
ed by expression directly for function and biological activity of proteins encoded by
such c acid les.
One embodiment of the present invention relates to a recombinant nucleic acid
molecule that comprises the isolated nucleic acid molecule described above which is
operatively linked to at least one transcription control sequence. More ularly,
according to the present invention, a recombinant nucleic acid le typically
ses a recombinant vector and the isolated nucleic acid molecule as described .
According to the present invention, a recombinant vector is an engineered (i.e., artificially
ed) nucleic acid molecule that is used as a tool for manipulating a nucleic acid
sequence of choice and/or for introducing such a nucleic acid sequence into a host cell.
The recombinant vector is therefore suitable for use in cloning, sequencing, and/or
otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or
delivering the c acid sequence of choice into a host cell to form a recombinant cell.
Such a vector typically contains heterologous nucleic acid sequences, that is, nucleic acid
sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or
delivered, although the vector can also contain tory nucleic acid sequences (e.g.,
promoters, untranslated regions) which are naturally found adjacent to nucleic acid
sequences of the present invention or which are useful for expression of the nucleic acid
molecules of the t invention (discussed in detail below). The vector can be either
RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can
be maintained as an extrachromosomal element (e.g., a replicating plasmid) or it can be
integrated into the chromosome of a recombinant host cell, although it is preferred if the
vector remain separate from the genome for most applications of the ion. The entire
vector can remain in place within a host cell, or under certain conditions, the plasmid
DNA can be deleted, leaving behind the nucleic acid molecule of the t invention.
An ated nucleic acid molecule can be under chromosomal promoter control, under
native or plasmid promoter control, or under a combination of several promoter controls.
Single or multiple copies of the nucleic acid molecule can be integrated into the
chromosome. A recombinant vector of the present invention can contain at least one
selectable marker.
In one ment, a recombinant vector used in a recombinant nucleic acid
molecule of the present invention is an expression vector. As used herein, the phrase
ssion vector" is used to refer to a vector that is suitable for production of an encoded
product (e. g., a protein of st). In this embodiment, a nucleic acid sequence encoding
the product to be produced (e.g., the protein containing a thioredoxin monocysteinic active
site) is ed into the recombinant vector to e a recombinant nucleic acid
molecule. The nucleic acid sequence encoding the protein to be produced is inserted into
the vector in a manner that operatively links the nucleic acid sequence to regulatory
sequences in the vector that enable the transcription and ation of the nucleic acid
sequence within the recombinant host cell.
In another embodiment of the invention, the recombinant nucleic acid molecule
comprises a viral vector. A viral vector includes an isolated c acid le of the
present invention integrated into a viral genome or portion thereof, in which the c
acid molecule is packaged in a viral coat that allows entrance of DNA into a cell. A
number of viral vectors can be used, including, but not d to, those based on
alphaviruses, poxviruses, adenoviruses, herpesviruses, lentiviruses, adeno-associated
viruses and retroviruses.
Typically, a inant nucleic acid molecule includes at least one nucleic acid
molecule of the present ion operatively linked to one or more expression control
sequences. As used herein, the phrase "recombinant molecule" or “recombinant nucleic
acid molecule” refers primarily to a nucleic acid molecule or nucleic acid sequence
operatively linked to an expression control sequence, but can be used interchangeably with
the phrase "nucleic acid molecule”, when such nucleic acid molecule is a recombinant
molecule as discussed herein. According to the present invention, the phrase "operatively
linked" refers to linking a nucleic acid molecule to an expression control sequence in a
manner such that the molecule is able to be expressed when transfected (i.e., ormed,
transduced, transfected, conjugated or ed) into a host cell. Transcription control
sequences are expression l sequences that l the initiation, elongation, or
ation of transcription. Particularly important transcription control sequences are
those that control transcription initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control sequences include any transcription
l sequence that can function in a host cell or organism into which the recombinant
c acid molecule is to be introduced. Recombinant nucleic acid les of the
present ion can also contain additional tory sequences, such as ation
regulatory sequences, origins of replication, and other regulatory sequences that are
compatible with the recombinant cell. In one embodiment, a recombinant molecule of the
present invention, including those that are integrated into the host cell chromosome, also
contains secretory signals (i.e., signal-segment or signal-sequence nucleic acid sequences)
to enable an expressed protein to be secreted from the cell that produces the protein.
Suitable signal segments include a signal segment that is naturally associated with the
protein to be expressed or any heterologous signal segment capable of directing the
secretion of the protein according to the t invention. In r embodiment, a
recombinant molecule of the t invention comprises a leader sequence to enable an
expressed protein to be delivered to and inserted into the ne of a host cell. Other
signal sequences include those capable of directing periplasmic or extracellular secretion,
or retention within desired compartments. Suitable leader sequences include a leader
sequence that is naturally associated with the protein, or any heterologous leader sequence
capable of directing the delivery and insertion of the n to the membrane of a cell.
According to the present invention, the term “transfection” is used to refer to any
method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid
molecule) can be inserted into a cell. The term "transformation" can be used
interchangeably with the term "transfection" when such term is used to refer to the
introduction of c acid molecules into microbial cells or plants. In microbial systems,
the term "transformation" is used to describe an ted change due to the acquisition of
exogenous c acids by the microorganism and is essentially synonymous with the
term "transfection." However, in animal cells, transformation has acquired a second
meaning which can refer to changes in the growth properties of cells in culture (described
above) after they become cancerous, for example. Therefore, to avoid confiJsion, the term
fection" is preferably used with regard to the introduction of exogenous c acids
into animal cells, and is used herein to generally encompass ection of animal cells
and transformation of plant cells and microbial cells, to the extent that the terms pertain to
the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques
include, but are not limited to, transformation, particle bombardment, electroporation,
nj ection, lipofection, adsorption, infection and last fusion.
In one embodiment, a composition comprising a protein or peptide containing a
thioredoxin monocysteinic active site in a reduced state is used for decreasing the viscosity
of excessively viscous mucus or sputum. The ition comprises the protein
containing a thioredoxin monocysteinic active site, and may include one or more
additional agents or compounds, such as other agents or compounds that can be used to
reduce/decrease excessively viscous or cohesive mucus or sputum or increase the
liquefaction of such mucus or sputum. Examples of such as other agents or compounds
are known in the art and include, but are not limited to, purified , N-
acetylcysteine, nacystelyn (an N—acetyl-L-cysteine derivative), GSH, and gelsolin. In
addition, mucoactive agents like mannitol or hypertonic saline may be used in
combination with monocysteinic active site thioredoxin.
In one embodiment, a composition, including a pharmaceutical composition can be
used to deliver a nucleic acid molecule encoding a protein or peptide containing a
thioredoxin monocysteinic active site to a cell in the patient to be treated (e.g., an
lial cell in the lung or airways), such that the cell can become transfected with and
express the protein, and so that the protein can contact mucus or sputum in the
microenvironment of the cell.
A composition, including a pharmaceutical composition, can also include, for
example, a pharmaceutically acceptable carrier, which includes ceutically
able ents and/or delivery vehicles, for delivering a protein or nucleic acid
molecule or other regulatory compound to a patient. Additionally, a composition,
including a pharmaceutical composition of the present invention can be administered to a
patient in a pharmaceutically acceptable carrier. As used herein, a pharmaceutically
acceptable carrier refers to any substance suitable for delivering a therapeutic protein,
nucleic acid or other nd useful in the method of the t invention to a suitable
in vivo or ex vivo site. Preferred pharmaceutically acceptable carriers are capable of
maintaining a protein, nucleic acid molecule or compound in a form that, upon arrival of
the protein, nucleic acid molecule or compound at the desired site (e. g., the site where the
mucus or sputum to be treated is ed or drains), is capable of ting the mucus or
sputum (in the case of a protein or compound) or of entering the cell and being expressed
by the cell and secreted (in the case of a nucleic acid molecule) so that the expressed
protein in a reduced state can t the mucus or sputum. Suitable excipients of the
present invention include excipients or formularies that transport or help ort, but do
not specifically target a eutic agent (protein, nucleic acid or compound) to a cell,
tissue or fluid (mucus or sputum) (also referred to herein as non-targeting carriers).
Examples of pharmaceutically acceptable excipients e, but are not limited to water,
phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing
solutions, Hank's solution, other aqueous physiologically balanced solutions, oils, esters
and glycols. Aqueous carriers can n suitable auxiliary substances required to
approximate the physiological conditions of the ent, for example, by enhancing
chemical stability and icity. ations for inhalation of therapeutic agents may
also include surfactant molecules.
le auxiliary substances include, for example, sodium acetate, sodium
de, sodium lactate, ium chloride, calcium chloride, and other substances used
to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can
also include preservatives, such as thimerosal, m- or o-cresol, formalin and benzol alcohol.
Compositions of the present invention can be sterilized by conventional methods and/or
lyophilized.
One type of pharmaceutically acceptable carrier includes a controlled-release
formulation that is capable of slowly releasing a composition of the present invention into
a patient. As used herein, a controlled-release formulation ses one or more
therapeutic agents of the present invention in a controlled-release vehicle. Suitable
controlled-release vehicles include, but are not limited to, biocompatible polymers, other
polymeric matrices, capsules, apsules, microparticles, bolus preparations, osmotic
pumps, diffiJsion devices, liposomes, lipospheres, and transdermal delivery systems. Such
lled-release vehicles may also incorporate reducing agents to maintain a thioredoxin
monocysteinic active site in a reduced state during storage and delivery. Suitable delivery
vehicles for nucleic acids include, but are not limited to liposomes, viral vectors or other
delivery vehicles, including ribozymes.
A suitable, or effective, amount of a protein or peptide ning a thioredoxin
monocysteinic active site to administer to a patient is an amount that is capable of:
ipating in redox reactions h the reversible oxidation of its active site thiol to a
disulfide, catalyzing thiol-disulfide exchange reactions, and particularly, decreasing the
viscosity or cohesiveness of mucus or sputum and/or increasing the action of mucus
or sputum in a patient, sufficient to provide a therapeutic benefit to the patient. Decreases
in the ity or cohesiveness or increases in the liquefaction of mucus or sputum can be
measured, detected or determined as described usly herein or by any suitable
method known to those of skill in the art. As discussed above, such ements include
determining and comparing the percentage of free thiols in a sample of mucus or sputum
from the patient prior to after contact with a suitable or effective amount of a protein or
e containing a thioredoxin monocysteinic active site, as well as determining and
comparing the FEV level of the patient prior to after t with a suitable or effective
amount of a n or peptide containing a thioredoxin steinic active site in a
reduced state.
In one embodiment, a suitable, or ive, amount of a protein or peptide
containing a thioredoxin monocysteinic active site to be administered to a patient
comprises between about 10 umoles/kg, 15 umoles/kg, 20 umoles/kg, 25 umoles/kg, 30
umoles/kg, 35 umoles/kg, 40 /kg, 45 umoles/kg, 50 umoles/kg, 55 umoles/kg, 60
umoles/kg, 65 umoles/kg, 70 umoles/kg, 75 umoles/kg, 80 umoles/kg, 85 umoles/kg, 90
umoles/kg, 95 umoles/kg, 100 umoles/kg, 105 umoles/kg, 110 umoles/kg, 115
umoles/kg, 120 /kg, 125 umoles/kg, 130 umoles/kg, 135 umoles/kg, 140
umoles/kg, 145 umoles/kg, 150 umoles/kg, 175 umoles/kg, 200 /kg, 225
umoles/kg, 250 umoles/kg, 275 umoles/kg, 300 umoles/kg, 325 umoles/kg, 350
umoles/kg, 375 umoles/kg, 400 umoles/kg, 425 umoles/kg, 450 umoles/kg, 475
umoles/kg, 500 umoles/kg, 525 umoles/kg, 550 umoles/kg, 575 umoles/kg, 600
umoles/kg, 625 umoles/kg, 650 umoles/kg, 675 umoles/kg, 700 umoles/kg, 725
umoles/kg, 750 umoles/kg, 775 umoles/kg, 800 /kg, 825 umoles/kg, 850
umoles/kg, 875 umoles/kg, 900 umoles/kg, 925 umoles/kg, 950 umoles/kg, 975
umoles/kg, 1000 umoles/kg, 1100 umoles/kg, 1200 umoles/kg, 1300 umoles/kg, 1400
umoles/kg, 1500 umoles/kg, 1600 umoles/kg, 1700 umoles/kg, 1800 umoles/kg, 1900
umoles/kg, 2000 umoles/kg, 2100 umoles/kg, 2200 umoles/kg, 2300 umoles/kg, 2400
umoles/kg or about 2500 umoles/kg body weight of a patient.
In r embodiment, if the route of delivery is aerosol delivery to the lung or a
similar route, an amount of a n or peptide containing a thioredoxin monocysteinic
active site to be administered to a patient comprises between about 0.25 mg per dosing
unit (e.g., a dosing unit for a human is typically about 2-3 ml) to about 100 mg per dosing
unit. Preferably, an amount of a n or peptide containing a thioredoxin monocysteinic
active site to be administered to a patient comprises about 0.25 mg, 0.50 mg, 1.0 mg, 5.0
mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65
mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg or about 100 mg per dosing unit.
ing on the device used for aerosol delivery, some aerosol delivery devices only
allow for about 10% of the volume in the aerosol to actually be delivered to the lung.
r, when the delivery device is a vibrating mesh nebulizer, about 90% of the
volume in the aerosol can be delivered. Electronic vibrating-mesh nebulizers, are capable
of delivering drugs far more rapidly and are smaller, more portable devices that are greatly
preferred by CF patients (Geller, D.E., Pediatric Pulmonology, 43(S9):SS-Sl7, 2008).
Vibrating-mesh nebulizers also are more efficient at delivering drugs with less residual
dose vs. air-jet nebulizers. This is particularly significant for reducing ent costs as
smaller doses are required to achieve therapeutic . Devices such as these also do
not result in reduced biological activity of proteins (Kesser, K.C., et al. Resp Care,
54(6):754-768, 2009; Scherer, T., et al. JPharm Sci, 100(1):98-109, 2011). Therefore, for
other routes of administration when the volume of the composition that will be red
to the site is greater, it will readily be seen that lower doses of the protein or peptide
comprising a thioredoxin active site may be used.
The optimum amount of a protein of the present invention to be administered to an
animal will vary depending on the route of administration. For instance, if the protein is
administered by an inhaled (aerosol) route, the optimum amount to be administered may
be different from the optimum amount to be administered by intratracheal microspray. It
is within the y of one d in the art to vary the amount depending on such route of
administration. It is important to note that a suitable amount of a protein of the present
invention is an amount that has the desired function without being toxic to an animal.
Other routes of administration e but are not limited to oral administration, especially
for the treatment of digestive mucus, or topical for the treatment of reproductive mucus.
In a one embodiment of the present ion, a composition, ing a
pharmaceutical composition, of the present invention that contains a protein comprising a
thioredoxin monocysteinic reactive site is filrther formulated with one or more agents that
maintains the thioredoxin active site in a reduced state following initial reduction using
reducing agents. Such reducing agents used in the present invention include, but are not
d to, dithithreitol (DTT), lipioc acid, NADH or dependent thioredoxin
reductase, ethylenediaminetetraacetic acid (EDTA), reduced glutathione, dithioglycolic
acid, 2-mercaptoehtanol, 2-carboxyethyl)phoshene, N—acetyl ne, NADPH,
NADH and other biological or chemical reductants.
As discussed above, a composition, including a pharmaceutical composition, of the
present invention is stered to a t in a manner effective to deliver the
composition, and particularly the protein comprising a thioredoxin monocysteinic active
site and/or any other nds in the composition, to a target site (e.g., mucus or sputum
to be treated for proteins and nds, a target host cell that will be or is in the
environment of the mucus or sputum to be treated for recombinant nucleic acid
molecules). Suitable administration protocols e any in vivo or ex vivo administration
protocol.
ing to the present invention, an effective administration protocol (i.e.,
administering a composition of the present invention in an effective manner) comprises
suitable dose parameters and modes of administration that result in contact of the protein
containing a thioredoxin monocysteinic active site and/or other compound in the
composition with the mucus or sputum to be treated, preferably so that the t obtains
some measurable, observable or ved benefit from such administration. In some
situations, by sampling the mucus or sputum from the patient, ive dose parameters
can be determined using methods as described herein for assessment of mucus or sputum
viscosity or liquefaction. Alternatively, effective dose parameters can be determined by
experimentation using in vitro samples, in vivo animal models, and eventually, clinical
trials if the patient is human. Effective dose parameters can be determined using s
standard in the art for a particular disease or condition. Such methods include, for
example, ination of al rates, side effects (i.e., ty) and progression or
regression of disease, as well as relevant logical parameters such as forced
expiratory volume in one second (FEVl).
According to the present invention, suitable s of administering a
composition of the present invention to a patient include any route of in vivo
administration that is suitable for delivering the composition to the desired site into a
patient. The preferred routes of administration will be apparent to those of skill in the art,
depending on whether the compound is a protein or other compound (e.g., a drug), to what
part of the body the composition is to be administered, and the disease or ion
experienced by the patient. In general, suitable methods of in vivo administration of a
monocysteinic active site thioredoxin include, but are not limited to, dermal delivery,
intratracheal administration, inhalation (e.g., aerosol), nasal, oral, pulmonary
administration, and impregnation of a catheter. Aural delivery can include ear drops,
intranasal delivery can include nose drops or asal ion, and intraocular delivery
can include eye drops or the use of suitable devices for passage of the drug across the
sclera. Aerosol (inhalation) delivery can also be performed using methods standard in the
art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992,
which is incorporated herein by reference in its entirety). Oral delivery can include solids
and liquids that can be taken through the mouth, for example, as tablets or capsules, as
well as being formulated into food and ge ts or animal feed or feed pellets.
Other routes of administration that are useful for mucosal tissues e bronchial,
intranasal, other inhalatory, , topical, transdermal, vaginal, transcervical, pericervical
and urethral routes. In addition, administration protocols can include atment
devices, such as application of the protein, e or composition in a diaphragm (e.g., to
the cervix) for use in applications such as infertility. In a preferred embodiment of the
t invention, when the protein or composition of the ion is administered to treat
excessively or abnormally viscous or cohesive sputum or mucus in the respiratory tract
(airways), a n or peptide (or composition) containing a thioredoxin monocysteinic
active site or other compound is administered by a route ing, but not limited to,
inhalation (i.e. by inhaling an aerosol, e. g., in or with tants); direct installation into
the lung via a oscope, endotracheal tube and/or via any artificial ventilation device;
nasal administration (intranasal or transnasal), bronchial, or intratracheally (i.e. by
injection directly into the trachea or tracheostomy), either directly or via lipidencapsulation
or surfactant. Any vable method of introducing the composition or
protein into the airways so that it can contact the mucus or sputum therein is encompassed
by the invention.
In the methods of the present invention, compositions, including pharmaceutical
compositions can be administered to any member of the Vertebrate class, including,
without limitation, primates, rodents, livestock, chickens, turkeys and domestic pets.
Preferred patients to t are humans.
Another embodiment of the present invention relates to a composition comprising
a protein or peptide containing a thioredoxin monocysteinic active site, wherein the
cysteine in the monocysteinic active site is covalently bound to a cysteine residue in a
mucus protein. Mucus proteins include but are not limited to atory mucus ns
and digestive tract mucus proteins. Mucus proteins include mucins, such as the heavily
disulf1de-bonded mucins MUCSAC and MUCSB. For example, following an initial
catalytic reaction at the N—terminal thioredoxin active site ne in a exchange
reaction with a disulfide bond of a mucus n, the protein or peptide containing a
thioredoxin monocysteinic acid can be bound covalently via the N—terminal cysteine to a
mucus protein and thus be refractory to repeated reduction and catalysis. Covalent linkage
of a thioredoxin containing the monocysteinic active site to its mucin target prevents
cellular uptake and alization of thioredoxin by sequestration on the mucus, which
has the dual benefits of preventing epithelial uptake and off-target effects due to undesired
2014/030545
thioredoxin activity within cells, while at the same time facilitating clearance of mucus-
linked spent drug from the body.
A further embodiment of the present invention s to a method to decrease
viscosity of mucus or sputum in a patient that has excessively viscous or cohesive mucus
or sputum by administering a composition comprising a protein or e containing a
thioredoxin monocysteinic active site, wherein the protein covalently binds to a ne
residue in a mucus protein. In one aspect, the mucus protein is a mucin. In yet another
aspect, the thioredoxin monocysteinic active site is in a reduced state. In yet another
aspect, the mucus n can be a respiratory mucus protein or a ive tract mucus
protein, or a reproductive tract mucus protein.
Another embodiment of the present invention relates to a method to se
viscosity of mucus or sputum in a t that has excessively viscous or cohesive mucus
or sputum by contacting the mucus or sputum of the patient with a composition
comprising a disulfide bond reducing agent and a cysteine-blocking agent. The disulfide
bond reducing agent and a cysteine-blocking agent can be the same molecule, including
but not limited to a protein or peptide containing a thioredoxin steinic active site,
or they can be different les. The disulfide bond reducing agent can be dithithreitol
(DTT), ethylenediaminetetraacetic acid (EDTA), GSH, dithioglycolic acid, 2-
mercaptoethanol, N—acetyl cysteine, Tris-(2-carboxyethyl)phosphine, or other
pharmaceutically-compatible reducing agents known to the art. The cysteine-blocking
agent can be etamide, iodoacetic acid, or other alkylation agents, or a cysteine-
specific dy or other affinity-encoding protein or peptide composition or antibody
mimetic. The cysteine-blocking agent binds to a cysteine that was in a disulfide bond in
the mucus protein prior to the bond being d. The cysteine-blocking agent prevents
the thiol group of the cysteine in the mucus from reforming a disulfide bond.
Yet another ment of the present invention relates to a method to treat a
patient having excessively viscous or cohesive mucus by administering to the patient a
composition comprising at least one compound haVing a thioredoxin active site that is
incapable of ar uptake. The compound can be a protein or peptide comprising a
thioredoxin monocysteinic active site. The compound can also be a fusion protein
comprising a thioredoxin portion and a cell surface receptor ligand portion. In this
embodiment, the cell surface receptor ligand portion binds to a cell surface or
thereby preventing cellular uptake of the fusion protein. The compound can be a
combination of a protein or peptide comprising a thioredoxin active site and a blocking
compound for the cysteine corresponding to the cysteine at position 35 of SEQ ID NO: 12.
In a preferred ment, the blocking nd can be an antibody or antibody
mimetic that binds to the thioredoxin molecule and thus blocks the cysteine at position 35
of SEQ ID NO: 12. In this regard, the term “blocks” refers to interfering with the ability of
the cysteine at position 35 of SEQ ID NO:l2 for example to form an intramolecular
disulfide bond with the cysteine at position 32 of SEQ ID NO:12.
A further embodiment of the present ion relates to a method of preventing
systemic exposure to a drug substance in a patient. The method includes the step of
administering the drug to the patient by a delivery route including but not limited to a
pulmonary, oral or topical delivery route. The drug can form a covalent bond to its target
site once administered. This mechanism of action is distinct from known drug
mechanisms of action as many drugs act by molecular interactions wherein ligands bind to
receptors with reversible binding of the molecules to the receptors. In a preferred
embodiment, the drug substance is a thiol-containing drug wherein the thiol group forms a
covalent bond with another thiol group at its target site. For example, the drug can
comprise a protein or peptide containing a thioredoxin monocysteinic active site in a
reduced state. In yet another preferred embodiment the target site is extracellular and the
drug is administered by an extracellular delivery route.
Still r embodiment of the present invention relates to a pharmaceutical
composition comprising a protein or peptide containing a thioredoxin monocysteinic
active site in a reduced state and r comprising at least one ride or saccharide
derivative capable of stabilizing the redox-active thiol group. The saccharide or
saccharide derivative can be sucrose, sucralose, lactose, trehalose, maltose, galactose,
raff1nose, mannose or mannitol. By redox-active thiol group it is meant a thiol group that
may exist in either a reduced state (-SH) or an oxidized state (-S-S-). The term
"stabilizing" includes, for example, reducing the rate of oxidation of the redox-active thiol
group in a reduced state when the ptide is present in a pharmaceutical composition
with the saccharide or saccharide derivative ve to a composition in which the
saccharide or saccharide derivative is omitted. By "saccharide" it is meant any mono-, di-
or of saccharides are
, oligo- poly-saccharide. Examples glucose, fructose, sucrose,
e, maltose, galactose, ose, inulin, dextran trehalose, sucralose, mannose and
mannitol. By saccharide derivative it is meant a compound that structurally resembles the
saccharide from which it is derived. For example, sucralose, which is a nated
sucrose, would be ered a ride derivative of sucrose. r derivatives
include, for example, alditol tives for example mannitol and xylitol. red
compositions of the present invention comprise non-reducing saccharides, for example
raffinose, trehalose, stachyose and particularly sucrose.
Another embodiment of the present invention relates to an animal feed
composition comprising a protein or peptide containing a thioredoxin monocysteinic
active site in a reduced state. Examples of animal feed include but are not d to hay,
straw, silage, ssed and ed feeds, oils and mixed rations, sprouted grains,
legumes, crop residue, grain, cereal crop, and corn.
The following es are provided for the purpose of illustration and are not
intended to limit the scope of the present invention.
Examples
Example 1
This e demonstrates the expression and cation wild-type Trx (also
referred to herein as “rthr”) and monocysteinic active site rhTrx (also referred to herein
as “r(Cys)hTrx”) proteins. n expression levels and post-transcriptional expression
fidelity of rhTrx and r(Cys)hTrx were maximized by codon optimization of the human
Trx-l DNA ce as described by Harris et al., Biotechnol Biogen 109:1987-97 (2012),
herein orated by reference in its entirety. g of synthetic constructs into
suitable vectors for production in E. coli was performed allowing both wildtype and
monocysteinic active site r(Cys)hTrx mutant proteins to be produced at the ug scales.
Several expression tag and purification strategies were evaluated at laboratory scale, as
well as affinity cleavage to facilitate purification away from endotoxin and endogenous
host thioredoxins.
Preparation of rhTrx and r(Cys)hTrx in E. coli. The rhTrx gene encoding the
105 amino acid mature rhTrx protein including the initiator nine was optimized for
E. coli expression and synthesized (DNA2.0, Inc.). The gene was subcloned into the pEV
vector, which contained an inducible T7 promoter, kanamycin resistance marker, a 6-
histidine tag for Ni-affmity purification and a tobacco etch virus protease cleavage site
(TEV). To s the rhTrx, the rhTrx pEV plasmid (verified by sequencing) was
transformed into C43 E. coli (Lucigen) grown to an optical density of ~0.6 (600nm) and
induced with isopropyl B-D-l-thiogalactopyranoside (IPTG). The protein was extracted
from the E. 0011' cells using homogenization and detergent lysis followed by centrifilgation.
The rhTrx was purified from the E. 6012' lysate using Ni-affinity chromatography. The Ni-
affinity purified rhTrx was protease d (TEV) to remove the nickel affinity tag and
further purified using ion exchange chromatography and e phase high pressure
liquid chromatography . The purity of the protein was assessed by SDS-PAGE
and ion spray mass spectroscopy (LC/MS/MS) and the sequence of the protein was
verified by trypsin cleavage of the rhTrx followed by ion trap mass spectroscopy. The
protein concentration and endotoxin concentration was determined by the bicinchoninic
acid assay (Pierce) and s Amoebacyte Assay (Charles River Lab.), respectively.
The r(Cys)hTrx protein was prepared following similar s, but with a hTrx
gene optimized and synthesized by DNA2.0. These rhTrx and r(Cys)hTrx proteins were
reduced in vitro using DTT which was subsequently d by dialysis and desalting
column treatment.
Example 2
A preferred expression, purification and reduction (activation) strategy for native
rhTrx and monocysteinic active site rhTrx (r(Cys)hTrx) proteins utilizing direct expression
of mature n rather than an affinity binding approach is described in this example.
r(Cys)hTrx and native Trx control proteins were produced in shake-flask batch cultures
and tch fermentations of E. 6012' BLZl, and KlZ-dervived hosts using standard
techniques of bacterial fermentation with a range of common constitutive or inducible
promoter systems, however inducible or IPTG-inducible systems were preferred, as
these resulted in the highest-expressing strains. Following cell disruption by
microfluidizer, supematants containing soluble Trx were collected by centrifugation and
treated with ammonium sulfate (AS) to entially precipitate host cell proteins. The
resulting Trx-enriched supematants were filtered using ultraf1ltration/diafiltration (UF/DF)
for buffer ge to 15 mM Hepes, 10 mM beta mercaptoethanol (b-ME), 250 mM
NaCl. In this buffer, Trx does not bind to a Q-Sepharose HP anion-exchange
chromatography (AEC) resin whereas DNA and other impurities bind with high affinity.
Following high-salt AEC the Trx-enriched flow-through was ged into a lt
buffer for a second AEC step on a Hi-Trap Q-HP column (GE Healthcare) under reducing
conditions (10 mM DTT) followed by buffer exchange to formulation buffer ibed
below). Alternatively, after AS precipitation the e fraction was filtered at 30 kD
cutoff and loaded on a hydrophobic interaction chromatography (HIC) column. The
eluted material was concentrated and buffer exchanged using a UF/DF cassette of lKD
and loaded onto a Source Q (anion exchange) column. Thioredoxin proteins were eluted
from the ion exchange column using a salt nt and then an additional UF/DF step to
exchange into storage buffer.
Endotoxin was quantified using a Limulus Amoebacyte Lysate kit (LAL, Pierce)
and any residual endotoxin was removed using a 0.2 micron Mustang E-filter (Pall).
Protein ty and yield were determined by SDS-PAGE and Western blot/ELISA at
appropriate steps, and total mass and final sequence identity was verified by MALDI and
electrospray mass spectrometry. The reducing activity of the Trx formulations were
quantified using a DTNB (Ellman’s Reagent) reduction assay. Fifty microliters of 2.5 mM
rhTrx or r(Cys)hTrx was added to a 96-well plate, ed by 175 microliters of sample
buffer and 25 microliters of 6 mM DTNB (5,5'-dithiobis-(2-nitrobenzoic acid). After
reactions were initiated by the addition of DTNB, the change in absorbance at 412 nm due
to DTNB reduction was measured ophotometrically at 300 C after 15 minutes.
A range of storage buffer conditions, such as those discussed below, are used for
compatibility with lyophilization and their y to stabilize the reduced state of
r(Cys)hTrx. The buffer conditions include low pH with reducing sugars, ammonium
e pH 5.5 with 10 mM b-ME, as well as a buffer formulation previously identified as
enhancing e stability of reduced native Trx (40mM Na acetate pH 5.5, 0.05%
EDTA, 9.25% sucrose). Each buffer condition with Trx is lyophilized and stored for
varying times in a dried form, and redox stability of stored protein ed using DTNB
and protein activity assays as described above. For aerosol stability assessment, a Penn-
Century aerosolizer or nebulizer may be used. Aerosol compounds are collected into
0.1M Tris buffer pH 8.0, lmM EDTA containing DTNB at different time points of
incubation to determine aerosol stability of the r(Cys)hTrx and native rhTrx. ates
are not expected to react with DTNB and hence only the reduced form will be detected.
The extinction coefficient of DTNB (14150 at 412nm) is used to calculate free thiol (SH)
groups as a function of recovered protein concentration. Previous studies of intratracheal
(IT) delivery of native rhTrx (Rancourt, R. et al., Free Radic Biol Med, 1441-1453,
2007) used normal saline. Addition of redox-stabilizing agents known to be compatible
with inhalation ry, such as methionine, may also be included.
This example demonstrates construction and E. coli s for expressing
r(Cys)hTrx and native rhTrx. r(Cys)hTrx and native rhTrx l are readily sible
as mature, soluble proteins with s vector systems and laboratory BL21 or K12
strains of E. 0012'. To produce higher-yielding (> 1 g/L) Trx-expressing strains of E. coli,
codon-optimized r(Cys)hTrx and native rhTrx DNA sequences were synthesized and
cloned into a range of expression vectors using different intracellular promoters systems
including nutrient-depletion based inducible systems, sugar-inducible systems (e. g.,
se, xylose, ose or other inducible promoters known in the art, e.g. US patent
7871815), or IPTG-inducible systems based on promoters from bacteriophage T3, T5 or
T7. Following DNA sequence confirmation expression constructs were transformed into
different commonly used BL21 / K12 E. coli host strains, including strains engineered to
lack utilization of molecules used for induction, or which also may have included over-
expressed methionine eptidase to e fidelity of expression at high titers (Liu,
M. et al., Protein Expr Purif84(1):130-139, 2012). The resulting production host / vector
ations were grown in medium to high-throughput format deep-well microtiter
plates, with shaking at 30-37°C in minimal medium and screened for expression using
methods commonly used in the art, such as gel or capillary electrophoresis. Positive
clones were confirmed via SDS-PAGE and Western blot and/or ELISA/ELISPOT. Fed-
batch fermentation of the strain was conducted and the yields of protein (r(Cys)hTrx) were
ed by SDS-PAGE and Western blot and/or ELISA/ELISPOT. Proteins were
purified and d using the methods described in Examples 1 and 2 above, and
ion state of the activated protein was assessed by DTNB assay and specific disulfide
bond reduction activity by insulin reduction assay. ce identity was analyzed by
mass spectrometry (MALDI or electrospray) in order to confirm translational fidelity and
proper molecular weight.
Example 4
This example trates the in vitro activity and function of wildtype and
steinic active site rhTrx. The activity of rhTrx was assessed by measuring its
reducing activity in a standard colorimetric DTNB assay, and by determining its y to
inhibit neutrophil elastase activity. In addition, the ability of r(Cys)hTrx to liquefy human
CF mucus as compared to rhTrx was determined, by using a sputum compaction assay,
and optionally, by examining the reduction of the viscoelasticity of CF sputum using a
specialized rheometer. The rhTrx and r(Cys)hTrx was compared to the standard of care
CF mucolytic Pulmozyme (thNase I; domase alfa).
Initial characterization of rhTrx: Reducing activity. The general reducing
activity of pre-reduced Trx formulations was quantified using the DTNB reduction assay
as previously reported (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-1453, 2007).
For enzymatic ion of rhTrx and r(Cys)hTrx, 50 microliters of assay buffer (100 mM
potassium ate, pH 7.0, 10 mM EDTA, and 0.05 mg/ml bovine serum albumin)
containing 5 or 50 mM rhTrx and 0.5 mM purified Ter were added to a 96-well plate.
Next 175 microliters of sample buffer containing 720 mM NADPH were added, followed
by 25 microliters of 6 mM DTNB in sample . To measure the reducing activity of
ally pre-reduced rhTrx, 50 microliters of 2.5 mM rhTrx was added to a 96-well
plate, followed by 175 microliters of sample buffer and 25 microliters of 6 mM DTNB.
After reactions were initiated by the addition of DTNB, the change in kinetic absorbance
at 412 nm due to DTNB reduction was monitored spectrophotometrically at 30 s C.
ing the same protocol, r(Cys)hTrx was subjected to this assay as well as those
described below. The total reducing activity of r(Cys)hTrx was less than rhTrx reflecting
the one fewer reduced Cys present in r(Cys)hTrx. However, both proteins were reduced to
over 90-95% of their potential reduction state, reflecting full activation.
Inhibition of neutrophil elastase (NE) activity. An tion system for
determining the effect of rhTrx on NE ty has been described previously (Lee, R., et
al. (2005) Am J. Physiol. Lung Cell. Mol. Physiol. 289(5):L875-882). , Trx was
diluted to the desired concentration in phosphate-buffered saline (PBS, pH 7.2). The
mixture was added to purified human NE (100 micrograms per ml in PBS, pH 7.2, 0.01%
Triton X-100) and ted at 37 s C for 1 h. The final volume during incubation
was 210 microliters, and the concentration of NE was 1.6 micrograms per ml. After
incubation, 60 microliter aliquots were tested in triplicate for elastolytic activity. Elastase
activity was ined by adding 120 microliters of 0.8 mM N-methoxysuccinyl-Ala-
Ala-Pro-Val 4-nitroanilide in PBS (pH 7.2) to the experimental sample and kinetic
absorbance was monitored at 405 nm at 37°C for 4 min. The concentration of elastase
used in these experiments was determined to be within the linear range of the assay. The
percent NE activity inhibition was determined as: percent inhibition = (1 — absorbance
change for experimental group / absorbance change for PBS l group). The elastase
activity of r(Cys)hTrx was found to be least comparable to rhTrx.
Compaction assay to assess liquefaction of human CF sputum. For all
manipulations, CF sputum was handled under a microbiologic hood. rhTrx, r(Cys)hTrx or
diluent alone (25 microliters), in relevant concentrations, was added to CF sputum (275
microliters) in a 1.5 ml Eppendorf conical tube and mixed by very brief vortexing. After
incubation at 37 s C for 30 min, samples were again briefly mixed and loaded into
hematocrit tubes and sealed at both ends. After a 5 minute centrifilgation in a hematocrit
fuge, the percent solids (gel) and percent liquid was determined by direct linear
measurement and expressed as percent liquid as s: 100 x liquid / (liquid+solids). For
each condition, sputum from at least five different CF patients was measured in triplicate.
The liquefaction ability of r(Cys)hTrx was at least comparable to, if not much greater than,
rhTrx reflecting the unexpected potency of the r(Cys)hTrx mechanism of action.
Sputum viscoelasticity determination. These measurements are made using a
cone-and-plate rheometer as well as qualitatively via direct ation of sputum flow
following ent with thioredoxin. Incubations are conducted using the same sputum
handling protocol as described above. Viscoelasticity is assessed using an AR—lOOO
rheometer (TA Instruments, New Castle, DE) in oscillation mode with an angular velocity
of l rad/second at 37°C. For each preparation of Trx, ability to se sputum
viscoelasticity is assessed in triplicate in sputum obtained from at least 5 different CF
patients. The ability to decrease sputum visoelasticity of r(Cys)hTrx is expected to be at
least comparable to, if not much greater than, rhTrx reflecting the unexpected potency of
the r(Cys)hTrx mechanism of action. Measurement of sputum viscoelasticity by
observing the rate of flow of sputum mixed with rhTrx or r(Cys)hTrx in inverted
Eppendorf tubes was performed, and in this assay the flow of r(Cys)hTrx was found to be
more ive than rhTrx, DTT and Pulmozyme, and much greater than that of negative
(vehicle) controls which exhibited essentially no sputum flow.
Comparison of efficacy with standard of care mucolytic agent e). For
these studies, relevant concentrations of rhTrx, r(Cys)hTrx and thNase mpared by
incubation as described above. After 30 minutes, s were assessed for changes in
liquefaction of sputum (compaction assay) and alteration of viscoelasticity. In addition,
synergy studies were performed. In these, sputum samples were exposed to rhTrx for 30
min and then thNase for 30 min, and, alternatively, thNase for 30 min ed by
rhTrx for 30 min. These results were compared to the effects of either agent alone. For
each condition, sputum from at least five different CF patients were measured in cate.
ent sputum from individual donors on a given day were required in order to test
each of 12 samples (rhTrx x 3, thNase x 3, rhTrx + thNase x3, thNase + rhTrx x3)
for wildtype and mutant rhTrx variants. In practice, this is, lly, 4.5 - 5.0 ml of well-
mixed sputum from a single donor/day. The liquefaction ability of hTrx was at least
comparable to, if not much r than, thNase. thNase, rhTrx, or DTT positive
controls.
Human CF sputum collection. Sputum was obtained from adult and pediatric
patients with CF as ined by a health care provider. Patients were diagnosed with CF
if they demonstrated clinical symptoms and had a sweat chloride value in excess of 60
millimolar in two separate pilocarpine iontophoresis sweat tests and exhibited two allelic
ducing mutations in subsequent genetic is. All samples were donated by
either spontaneous expectoration or hypertonic saline induction. Sputum samples
ning visibly detectable saliva were discarded. Sputum was kept on ice until
delivered to the laboratory, then held at -80 degrees C until use in O-ring-sealed vials to
prevent ation.
Example 5
This example trates the enzymatic (Figure lb) and non-enzymatic (Figure
la) activity of a protein or peptide containing a doxin monocysteinic active site as
compared with a protein or peptide containing a wild-type doxin active site. As
rated in Figure la a non-specific 5,5'—dithiolfsis—(IZ—niti‘obenzoic acid) (DTNB or
Ellman’s reagent) reduction s the loss of one reducible cysteine in the thioredoxin
monocysteinic active site as compared to wildtype. As illustrated in Figure lb and Figure
2, a protein or peptide containing a thioredoxin monocysteinic active site surprisingly
exhibited greater potency as compared to a protein or peptide containing a wild-type
doxin active site in a human sputum compaction assay as well as greater potency on
an equimolar basis versus DNase or NAC (Figure 2). This result is unexpected, given the
substantial modification at the active site due to mutation of Cys35, and the observation
that the total reducing power was 4/5 that of native thioredoxin by virtue of the loss of one
reducible Cys residue in the prior DTNB assay. The surprising increase in potency of the
thioredoxin monocysteinic active site was realized to be due to the covalent linkage to
mucin protein Cys residues, which had the unanticipated consequence of preventing these
Cys from re-forming new disulfide bonds.
Example 6
This example trates the decreased propensity of r(Cys)hTrX (in a reduced,
active state) to stimulate release of pro-inflammatory cytokines by cultured primary
human bronchial epithelial cells (HBE) as compared to native rhTrx. Donor tissues and
cells are provided under the es of approved protocols for the protection of the rights
of human subjects. HBE cells from normal (i.e. non-diseased) lungs are harvested by
tic digestion as previously described (Fulcher, M.L., Methods M01 Med 107:183-
206, 2005). Disaggregated HBE cells are seeded on 12 mm diameter Transwell Clear
supports (Corning) at a density of 2.5 x 105/cm2 in a well-defined airway cell media
(Fulcher, M.L., 2005 ibid). Cultures are maintained at an air-liquid interface until fully
differentiated (about 4-6 weeks) before use.
Thioredoxin delivery protocol: For this study, a device capable of delivering
nanoliter volumes of test agent (or control) to the surface of HBE cultures is utilized. This
system is designed to be an in vitro model system to mimic the in viva delivery of an
ultrafine mist of nebulized medications to the surface of airway epithelial cells, and
represents the ideal way to study the effect of adding eutics, such as r(Cys)hTrx.
Based on the results of multiple-path particle dosimetry modeling (Anjilvel, S., et al.
Fundam App] Toxicol 28(2):4l-50, 1995) demonstrating the average deposition rates of
Pari LC Star nebulizer over the first 20 generations of airways, the average deposition over
the first 6 generations of airways, where the majority of les are expected to be
delivered, is estimated to be ~50 nl/min/cmz, over the course of 15 minutes. In these
studies, nebulization on a total volume of 750 nl (over 15 minutes) to a total of five
different experimental groups is performed. These include: 1) e control (isotonic
saline), 2) Native recombinant Trx (500 um), and 3) three doses of r(Cys)hTrX (10 um,
250 um, and 1000 um). These concentrations represent the “final” airway surface
concentration. Following nebulization of the each test reagent (or vehicle control), cultures
are returned to the tissue culture incubator and ted for 24 hours before ne
Cytokine Immunoassay: In this study, the effect of native and r(Cys)hTrx) on
stimulation of the four major cytokines which are released by HBE cells, including IL-6,
IL-8, TNF-u, and IL-lB is assessed. Cytokines in cell-free, un-concentrated culture
supematants are measured using commercially available enzyme-linked immunosorbent
assay (ELISA) kits (R&D systems). Samples of teral ALI media (Fulcher, M.L.,
2005, ibid) are obtained at 24 hours post-nebulization of all five groups, and frozen before
analysis. As a ound control, clean (unused) ALI media is cted from all
values. For each ne analysis, positive controls over three decades of concentrations
are used to generate appropriate standard curves. Additionally, each “unknown” sample is
analyzed in duplicate. Cytokines measured are: TNF-u (lower detection limit of 0.3
pg/ml), IL-6 (lower detection limit of 0.35 pg/ml), IL-8 (lower detection limit of 2.4
pg/ml), and IL-1 [3 (lower detection limit of 1.5 . Cross-reactivity of these assays
with each other and with other recombinant human cytokines (IL-la, IL-2, IL-3, IL-4, IL-
7, tumor necrosis factor-S, granulocyte colony-stimulating , and transforming
growth factor-Ill) has been previously shown to all below limits of detection.
Sample size and statistical is: Each of the five conditions are evaluated on
a total of nine individual HBE cultures. This represents three cultures (n=3) from three
different patients (n=3). Such an approach is sufficiently powered statistically to provide
an understanding of the inter- and intra-sample ion of each condition. For data
analysis, comparison between individual samples/groups is made using iled
Student’s t-test. For multiple group comparisons, a one-way analysis of variance
) is used. Significance for all analyses is set at p<0.05.
ammatory evaluation of r(Cys)hTrx in HBE cultures from CF patient
donors. Human CF primary airway epithelial cell culture: Recent techniques to
proliferate and e human primary airway epithelial cells at an air-liquid interface with
mucociliary differentiation, is employed based on methods ed by Schlegel and
colleagues (Liu, M. et al., Protein Expr Purz’f. 84(1):130-139, 2012; owicz, F.A., et
al. Proc. Natl. Acad. Sci. U.S.A.. 109(49):20035-20040, 2012). These methods allow a
virtually unlimited supply of primary airway epithelial cells that are still capable of
terminal differentiation at ALI, without genetic manipulation. 30 wells are cultured to
differentiation at ALI over 30 days from each of three unique CF donors, homozygous for
F508del. The cultures are exposed at the apical surface to three concentrations (10 uM,
250 uM and 1000 uM) of rhTrx (wild type) and r(Cys)hTrx followed by collection of
both apical and basolateral media samples at 4 and 24 hours after re challenge (see
Table 1). The media used is serum free, centrifuged to remove debris and stored at -80°C
until use. ELISA assays for human atory cytokines are performed on all samples
taken from both the apical and basolateral media. Apical collection occurs by placing 200
uL of sterile PBS on the apical e and recovering this after 15 min incubation.
ELISA for IL-8 and IL-6 are performed for each sample in duplicate (Becker, M.N. et al.,
Am J Resp Crit Care Med 169(5):645-653, 2004). The plan above involving 30 ALI
cultures is repeated for each of the three CF donors. ison of the results of native
rhTrx and r(Cys)hTrx demonstrates that the propensity of thioredoxin to induce
proinflammatory cytokine release from differentiated airway epithelial cells of CF ts
is attenuated in the monocysteinic Trx vs. native at the concentrations tested.
Table 1
Time Diluent
after
Control
exposure
This example demonstrates the biophysical and biochemical characterization of the
effect of r(Cys)hTrx versus native rhTrx on uniform mucus ted from in vitro HBE
cell cultures.
ation of cell culture mucus: Human ial epithelial cells are grown
and maintained as bed (Matsui, H., et al., J Clin Invest 102(6):ll25-l 131, 1998). In
brief, mucus harvested from cultures is pooled and stored at 4°C. Samples are loaded into
dialysis tubes (MWCO=3,500) and concentrated with a polymer absorbent (Spectra/Gel)
for l — 5 days at 4°C. The concentrated mucus is then dialyzed against PBS containing
500 uM MgClz and 800 uM CaClz at 4°C to establish a proper salt balance (Matsui, H. et
al., Proc NatlAcad Sci USA 103(48):l8l3 1-18136, 2006).
Macroscopic Rheology: Concentration and time course assays using a Bohlin
Gemini Rheometer in both cone and plate and parallel geometries are performed to assess
the bulk, copic biophysical effects of d r(Cys)hTrx and native thioredoxin on
in vitro HBE mucus properties. Creep ry experiments are performed in which a
known stress (between 0.05 and ~ 100Pa) is applied to treated or control mucus for 10
seconds, and the rheological recovery of the fluid is recorded for an additional 50 seconds.
In successive runs, the applied stress is sed in a logarithmic fashion until the yield
stress of the fluid is reached (i.e. the stress at which the viscosity of the fluid suddenly and
dramatically decreases). From the measured parameters the viscosity and elasticity of the
fluid is determined as a on of applied stress. Frequency sweeps are performed at both
constant stress and strains and used to determine the baseline physical properties of mucus
(G’ and G” respectively), as well as the viscosity and shear thinning behavior of the fluid.
All experiments are med at 23°C.
High Pressure Liquid Chromatography: The concentration and molecular mass
of mucins in treated and control mucus is assessed by differential refractometry to
determine the effect of native thioredoxin and r(Cys)hTrx on mucin structure. Samples
(500 ul) are loaded onto a Sepharose S1000 column (Amersham Pharmacia), and eluted
with 200 mM sodium chloride/10 mM EDTA at a flow rate 0.5 ml/min. An in—line Dawn
EOS laser photometer coupled to a Wyatt/Optilab DSP inferometric refractometer is used
to measure light scattering and sample concentration, respectively (Wyatt Technology
ation). The tration of the mucins is calculated by integrating the refractive
index peak associated with the material eluted in the void volume of the column and
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employing a value for the refractive-index increment (dn/dc) of 0.165 ml/g, which has
been measured previously at 650 nm and found to be reproducible within 5%. The total
protein content (mucins and small proteins) of a given mucus sample is determined by
similar methods, but with a G-25 column ((1de = 0.170). The non-mucin (or small
protein) content of the sample is determined by subtracting the mass of the mucin content
(determined by elution the sample through the S-1000 column) from the total mass of the
mucin and protein content (detected by the eluting the sample through the G-25 column).
In comparison with gel-based methods, e. g. reducing or non-reducing PAGE, differential
refractometry is advantageous in that exact molecular weights and molecular weight
distributions are ible rather than qualitative comparisons. The quantitative
age of these techniques are particularly important for the study of large
glycoproteins such as mucins where no size standards exist and lar s often
exceed illis, R.B., et al., ydr Polym 93(1):178-183, 2013).
Carbohydrate and n Blotting: The ial effects of thioredoxin
treatments on mucin fine structure is assessed by ming protein blotting is on
intact HBE mucus. 50-200 uL aliquots of samples are loaded onto nitrocellulose
membranes and a vacuum applied for five min to pull the entire loaded sample into the
membrane. Samples are washed 2x in distilled water. For periodic acid Schist (PAS)
assays of carbohydrate content, loaded and washed samples are incubated for 30 minutes
in 0.25% periodic acid + 3% acetic acid in water. Following two distilled water washes,
samples are incubated in NaMBS on (0.1% sodium metabisultate, 1% HCl) twice for
five minutes. Next, samples are incubated with SchifPs reagent for 5-15 min and washed
twice in NaMBS, once in distilled water, and quickly vacuum dried. Protein-specific
antibody blots are blocked with 1% milk in TBST buffer (1.21% Tris HCl, 8.76% NaCl,
and 0.5% Tween, pH 8) following loading and the first distilled water washes. After two
five-min TBST washes, samples are incubated in primary antibody (MAN-5ACI for
MUC5AC, MUC5BIII and K5B for MUC5B) for 30 min. Following two additional five-
min washes in TBST, samples are incubated in secondary antibody for two hours.
ne blots are developed using a Li-Cor Odyssey infrared detector. Additionally,
anti-thioredoxin dies may be used to detect monocysteinic vs. native thioredoxin
bound to mucins. Compared to standard n immunoblotting following separation on
PAGE gels this direct blotting approach allows for more rapid and precise determination
ofmucin content and to visualize and quantify covalent interaction between monocysteinic
active site thioredoxin (r(Cys)hTrx) and mucin disulfide bonds.
Example 8
This example demonstrates the ability of steinic active site Trx
(r(Cys)hTrx) to attenuate the propensity of native rhTrx to induce proinflammatory and
pathophysiological effects in the lung following intratracheal delivery to rats and mice.
Evaluation of pro-inflammatory cytokine release and cell migration in normal
rats dosed intratracheally with r(Cys)hTrx and native rhTrx. To determine
r(Cys)hTrx’s ability to induce pro-inflammatory signaling as compared to native rhTrx, a
comparative in vivo study in rats utilizing intratracheal (IT) delivery of increasing
concentrations of thioredoxin protein compared to vehicle control is performed. The two
study components are 1) an initial study designed to replicate previous findings (Rancourt,
R. et al., Free Radic Biol Med, 42(9):l44l-l453, 2007) ed with purified native
rhTrx, and 2) a Main Study to compare the pro-inflammatory effects of r(Cys)hTrx to
native rhTrx. All studies utilize purified, endotoxin-free protein that has been treated with
DTT or other suitable reductant to reduce (activate) the Trx active site Cys residues.
Following reduction, DTT or reductant is removed via size-exclusion chromatography.
Full reduction of Trx Cys residues is verified by in vitro assay (DTNB reduction) and
catalytic disulfide-bond reduction activity of r(Cys)hTrx is assayed using an insulin-
ion or HPLC target-binding assay.
Initial Study: Twenty-four rats are randomized into four experimental groups with
six animals per group dosed as follows: Group 1 vehicle control; Group 2 oxidized
ive) rhTrx; Group 3 d e) rhTrx; Group 4 Human Serum Albumin (HSA;
negative control). All test es are delivered once by the IT route. The end points of
this study include 1) cytokine analysis of TNF, ne induced neutrophil chemo-
tant-2 (CINC2) and macrophage inflammatory protein-3 (MIP3) by enzyme-linked
immunosorbent assays ); and 2) cell counts from bronchoalveolar lavage (BAL)
with Wright’s staining to elucidate the percentage of inflammatory cells ophil vs
macrophage). Group 2 (treated with oxidized rhTrx) has a similar level of cytokine
activities and cell counts in BAL ed to Group 1, the e control and Group 4,
HSA. Group 3, treated with a reduced rhTrx has an increased level of cytokine activities
and cell counts compared to Group 1 and Group 2.
Main Study: Comparison of cytokine release in rats when stered native
thioredoxin rhTRX vs. r(Cys)hTRX. In this comparative study, the relative degree of in
vivo cytokine e and cell migration following IT administration for rhTrx vs.
r(Cys)hTrx is determined. Three dose levels of reduced rhTrx and reduced r(Cys)hTrx are
tested (50 uM, 200 uM and 1000 uM). End points are the same as for the Pilot study with
the addition of immunohistochemistry (IHC) is of lung tissues. In r(Cys)hTrx
mutation of the C- terminal active site cysteine motif (change CXXC to CXXX) has been
shown to eliminate the second thiol- disulfide exchange capability of thioredoxin and
results in covalent linkage to disulfide-bond targets. The most prominent extracellular
disulfide-bond targets in the lung, which IT-delivered TRX encounters first, are located in
the mucus layer and on epithelial cell-surface proteins. uently, as compared to
native rhTrx, an increased binding of r(Cys)hTrx to lung epithelial cells and associated
mucus occurs and this binding may be detected by IHC using an anti-human thioredoxin
antibody. Lung tissues are collected from two animals from each group for IHC is
without BAL and ed to two post-BAL animals from each of the same groups.
Hence, for each group eight animals are lavaged, and two animals are treated but not
subject to BAL prior to lung collection and IHC. r(Cys)hTrx exhibits an attenuated dosedependent
ability to increase cytokine activities and cell counts compared to the same dose
level of rhTrx.
tion of toxicity and lung pathology in normal rats dosed intratracheally
with r(Cys)hTrx. To assess pulmonary effects of r(Cys)hTrx under exaggerated dosing
conditions a range of single doses of r(Cys)hTrx from 0.5 to 20 mg/kg is stered by
the IT route, and evaluation of whether pathological changes have occurred at 2 and 14
days post-dosing is med. Male and female Sprague-Dawley rats are randomized into
three experimental groups with 20 animals per group x). All animals are given a pre-
study physical examination. e (saline formulation) is used as a negative control.
The end points of this study include 1) clinical and histopathological examination to look
for gross adverse changes; and 2) terization of serum to detect the presence of test
article and dies to the test article. Monocysteinic r(Cys)hTrx results in less severe
effects at comparable dosages vs. native rhTrx.
Evaluation of pro-inflammatory cytokine release and cell migration in normal
and bENaC mice dosed intratracheally with r(Cys)hTrx and native rhTrx. Mice are
briefly anesthetized with isoflurane and placed on a tilting rodent workstation. Using a
rodent oscope fitted with a magnifying loupe, the larynx is directly Visualized and a
microsprayer (PennCentury) is passed into the distal trachea. A fixed volume (25-50 uL)
of test article solution is administered in the airway and the mice d to recover.
Weight at time of airway instillation and euthanasia is recorded. Following Institutional
Animal Care and Use tee (IACUC) approved protocols, five mice per condition
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are euthanized at six hours and another five mice at 24 hours after IT instillation. The
s are lavaged with a total of 1.5 mL of cold, sterile saline with protease inhibitor
(Pierce). Whole lungs are collected and all specimens are stored on ice until processed
further. oalveolar lavage fluid (BALF) is centrifuged to pellet leukocytes and other
cell debris. Cell-free BALF is frozen at -80C until ELISA g is performed. The BAL
cell pellet is resuspended in 1 mL of sterile saline and total leukocyte counts with
differentials are performed using a Coulter counter and manual counts of 300 cells from
stained cytospin preparations. When all samples have been collected, BAL fluid is thawed
on ice and ELISAs are performed for KC (analog of IL-8), TNF alpha, IL-6, and IL-lbeta
(ElisaTech, Denver, CO). BALF arity is characterized by both % and total
ytes (neutrophils, macrophages, lymphocytes).
Both wild-type and mutant TRX are tested at two concentrations and two time
points, along with control animals, to determine the acute airway inflammatory response
to both mutant and wildtype TRX, relative to t control (Table 2). The response is
characterized by: change in body weight, total leukocyte count in BALF, relative and total
neutrophil, macrophage, and lymphocyte counts, and the inflammatory cytokines listed
above. r(Cys)hTrx exhibits an attenuated dose-dependent ability to increase cytokine
activities and cell counts, as well as adverse effects, compared to the same dose level of
rhTrx.
Table 2
Diluent rhTrx r(Cys)hTrx r(Cys)hTrx r(Cys)hTrx
Control (500 pM) (10 pM) (250 HM) (1000 HM)
Evaluation of pathophysiological and inflammatory s in normal and
BENaC mice dosed intratracheally with r(Cys)hTrx and native rhTrx. Previous
studies by Rancourt and colleagues (Rancourt, R. et al., Free Radic Biol Med, 42(9): 1441-
1453, 2007) demonstrated that the presence of additional nous) mucus on the
airways could significantly reduce the stimulation of ne release by native Trx. While
this might suggest that CF patients with a higher mucus burden might exhibit a reduced
inflammatory response due to extracellular reduction of the Trx, there is currently no
information regarding the effect of thioredoxin in an airway model with 1) increased
endogenous mucus production and 2) a preexisting inflammatory response (as in CF). The
goal of these studies is to use a mouse model of chronic mucus obstruction/inflammation
to investigate the effect of native Trx and r(Cys)hTrx. For these studies, the BENaC mouse
model which presses the Beta subunit of the ENaC channel and exhibits water
hyperasborption in the lungs is used (Mall, M., et al., Nat Med. 10(5):487-493, 2004). The
BENaC mice produce excess mucus and p a CF/COPD-like lung phenotype with
mucus airway ng and inflammation. These mice have previously been used as a
model of CF lung disease in a number of preclinical studies (e.g., Graeber, S.Y., et al., Am
'r Cell Mol Biol 49(3):410-4l7, 2013). In the present study 1) the effect of native
Trx vs r(Cys)hTrx on airway/lung histopathology and inflammation ; and 2) the
effect of these agents on mucus burden in BENaC mice (see below) is determined to
investigate the relative effect of native and monocysteinic Trx (r(Cys)hTrx) over a range
of delivery doses and understand how chronic mucus overproduction and pre-existing
inflammation modulates the potential pulmonary toxicities of these compounds.
The effects of three concentrations (100 uM, 500 uM and 1000 uM) of native
thioredoxin in viva using WT and BENaC mice and comparison of toxicity and drug
efficacy with the r(Cys)hTrx compound at the same concentrations is determined. A
minimum of seven mice per ion are tested. All nds are administered via
intratracheal instillation (10-25 ul) at a given concentration. A single dose treatment is
used and toxicity and drug efficacy is monitored at four time points (4h, 24h, 72h and 7
days post-dosing). Early time points (4h-24h) are carefully monitored. Later time points
(72h-Day 7) are monitored if drug toxicity is maintained at 24h. The effects of the lower
concentrations (100 uM, 500 uM) are expected to not be sustained past 24h or Day 3,
respectively. The protocol used allows instillation of volumes as small as 10 ul reliably
into mouse lungs. Briefly, the animals are anesthetized with isoflurane, placed onto a
mouse intubation rm wherein the harynx is visualized using a small
laryngoscope. Drugs are instilled either directly into the trachea (intratracheal instillation,
IT) or, for a more homogenous and deeper deposition, 25 ul are red by microspray
using the Penn Century device. After treatment, animals are euthanized at the appropriate
time point. Intact lungs are collected for histology and oalveolar lavage fluids for
various measurements, i.e., cell count, mucin content and reduction status and cytokines.
In more details, the left lung is tied up via a ligature around the em bronchus and
surgically isolated for histology. A bronchoalveolar lavage of the opposite lung (or right
lobes) is performed via a tracheotomy and cannula using 500 ul of sterile PBS. After
on of the whole lung, longitudinal sections are analyzed by H&E and AB-PAS
WO 45735 2014/030545
staining to assess inflammation and retained mucus. BALs are analyzed for cell
counts/differentials, mucin content and reduction status (agarose gel separation method
and Western blotting), and cytokines.
Sample size and tical analysis: Each of the compounds are evaluated at
various concentrations (100 umol, 500 umol, 1000 umol) for a total of 7 mice per
condition. Two groups are tested, wildtype (WT) and BENaC mice (Table 3). As indicated
by “X’s” in the following table a total of 36 conditions x 7 mice (252 mice total) are
treated and analyzed to e a sufficient understanding of the inter- and intra-sample
variation of each condition. For data analysis, comparison n indiVidual
samples/groups is made using two-tailed Student’s t-test. For multiple group comparisons,
a one-way analysis of variance (ANOVA) is used. Significance for all analyses will be set
at p<0.05. Monocysteinic r(Cys)hTrx s in less severe effects at comparable dosages
vs. native rhTrx, and this effect is further attenuated by the ce of endogenous mucus
in the BENaC mice.
Table3
Model Concn 4h " 72h Day7
umol
WT X X
r(Cys)hTrx X
X X
BENaC X X
r(Cys)hTrx X
X X
“X” represents times x concentrations tested
Example 9
This example demonstrates that r(Cys)hTrx improves mucus-normalizing ty
versus wildtype rhTrx. The effect of wildtype rhTrx, r(Cys)hTrx and various controls
including dithiothreitol (DTT) at two concentrations, N—acetyl cysteine (NAC) and
recombinant human DNase (thNase) at lar concentrations on normalization of
patient sputum samples (n=6 per treatment) in a sputum-compaction assay was
determined. Sputum studies were conducted using four to six spontaneously-expectorated
(non-induced) samples each from up to three indiVidual CF patients for each experiment.
The “favorable” part of the sputum (i.e. the mucus) was collected and combined from each
2014/030545
sample and the total sample is gently homogenized by stirring or mild vortexing before
separating into tubes and exposing to either: diluent (i.e. Tris buffer), DTT in diluent (at
0.58 mM and 1.5 mM), NAC in diluent (0.58 mM), thNase in diluent (0.58 mM), rhTrx
in t (0.58 mM) or r(Cys)hTrx in diluent (0.58 mM). DTT was made fresh for each
experiment as a positive control. Figure 2 shows that r(Cys)hTrx was able to increase
ly the liquefaction of t sputum as compared to wildtype thioredoxin (referred
to as “rhTrx” in Figure 2), thus showing an improved mucus-normalizing activity versus
both native Trx and the standard of care recombinant human DNase, PulmozymeTM
(“thNase”). Both thioredoxin and DNase were in turn more potent than equimolar
amounts of NAC, commonly used as a mucolytic outside of the US. These results using
human CF patient sputum demonstrate that monocysteinic r(Cys)hTrx s fully
competent for CF sputum Viscoelasticity normalization (and is even more potent than
unmodified native rhTrx), despite the expected covalent binding to mucin Cys. However,
the potential for the small 12 kD r(Cys)hTrx to cross the lung epithelium should be greatly
minimized by binding to mucin. The ability of active r(Cys)hTrx to then subsequently
enter the cell nucleus and interact with redox regulatory target proteins such as NFkB is
even further attenuated, as is any signaling due to ction with extracellular
transmembrane domains on immune cells as has been theorized for Trx-mediated
tion of the CD30 TNF receptor (Schwertassek, U., et al., EMBO J 26(13):3086-
3097, 2007). The increased y observed for r(Cys)hTrx es la, lb and 2) is
consistent with mucin Cys covalent binding by the molecule, as bound Cys would be
blocked from re-forming disulfide bonds unlike treatment with native rhTrx (or ,
any small-molecule thiol agent).
Each of the publications and other references discussed or cited herein is incorporated
herein by reference in its entirety.
While various embodiments of the present invention have been described in detail,
it is apparent that modifications and adaptations of those ments will occur to those
skilled in the art. It is to be expressly understood, however, that such modifications and
tions are within the scope of the present invention, as set forth in the following
claims.
Claims (13)
1. Use of a composition comprising a protein or peptide containing a thioredoxin monocysteinic active site in a reduced state for the manufacture of a medicament for use in sing viscosity of mucus or sputum, in a human patient that has excessively viscous or cohesive mucus or sputum, wherein the thioredoxin monocysteinic active site comprises an amino acid sequence selected from the group consisting of C-X-X-S (SEQ ID NO: 24), C-X-X-X (SEQ ID NO:17), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID NO:26), and W-C-GP-S-K (SEQ ID NO:27) wherein the C residue is in a reduced state, and wherein the X residues are any amino acid residue other than cysteine.
2. The use according to Claim 1, wherein the human patient has a disease selected from the group consisting of cystic fibrosis, c obstructive pulmonary disease, iectasis, asthma, ocular disease and a digestive tract disease.
3. The use according to Claim 1, n the composition is formulated for administration by a route selected from the group consisting of nasal, intratracheal, bronchial, direct installation into the lung or eye, inhaled and oral.
4. The use according to Claim 1, n the ition includes a pharmaceutically acceptable carrier.
5. The use according to any one of Claims 1-4, wherein the use of the ition increases the percentage of free thiols in a sample of mucus or sputum from the human patient as ed to prior to the use of the composition.
6. The use according to any one of Claims 1-4, wherein the use of the composition increases the forced expiratory volume (FEV) of the human patient by at least about 2.5% as compared to prior to the use of the composition.
7. The use according to any one of Claims 1-4 wherein the protein comprises thioredoxin selected from the group consisting of prokaryotic thioredoxin, fungal thioredoxin, plant thioredoxin, mammalian doxin and human thioredoxin.
8. The use according to Claim 1, wherein the composition is formulated for oral administration, or aerosol administration to the lung, and wherein the medicament is for use in the treatment of a human having a e selected from the group consisting of cystic fibrosis, chronic ctive pulmonary disease, bronchiectasis, asthma and a digestive tract disease.
9. The use of Claim 8, wherein the aerosol administration to the lung is to be administered by a nebulizer device.
10. The use of Claim 9, wherein the nebulizer device is a vibrating-mesh nebulizer.
11. A method of treating a digestive tract disease in a man animal, comprising administering an animal feed composition sing a protein or peptide containing a thioredoxin monocysteinic active site in a reduced state, wherein the thioredoxin monocysteinic active site ses an amino acid sequence selected from the group consisting of C-X-X-S (SEQ ID NO: 24), C-X-X-X (SEQ ID NO:17), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID NO:26), and W-C-G-P-S-K (SEQ ID NO:27) and wherein the X residues are any amino acid residue other than cysteine.
12. A method for decreasing viscosity of mucus or sputum, in a non-human patient that has excessively s or cohesive mucus or sputum, comprising ting the mucus or sputum of the man patient with a composition comprising a protein or peptide containing a thioredoxin monocysteinic active site in a reduced state wherein the step of contacting decreases the viscosity of the mucus or sputum as compared to prior to the step of contacting, wherein the thioredoxin monocysteinic active site comprises an amino acid sequence selected from the group consisting of C-X-X-S (SEQ ID NO: 24), C-X-X-X (SEQ ID NO:17), X-C-X-X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), P-S-X (SEQ ID NO:26), and W-C-G-P-S-K (SEQ ID NO:27) wherein the C residue is in a reduced state, and wherein the X residues are any amino acid e other than
13. A method of treating a non-human patient having a disease selected from the group consisting of cystic is, chronic obstructive pulmonary disease, bronchiectasis, asthma and a digestive tract disease, sing administering to the non-human patient a pharmaceutical composition comprising a protein or peptide containing a thioredoxin monocysteinic active site in a reduced state, n the composition is formulated for oral stration, or aerosol administration to the lung, wherein the thioredoxin monocysteinic active site comprises an amino acid sequence selected from the group ting of C-X-X-S (SEQ ID NO: 24), C-X-X-X (SEQ ID NO:17), X-X-X (SEQ ID NO: 19), X-C-G-P-X-X (SEQ ID NO:21), W-C-G-P-X-K (SEQ ID NO:23), X-C-X-X-S-X (SEQ ID NO:25), X-C-G-P-S-X (SEQ ID NO:26), and W-C-G- P-S-K (SEQ ID NO:27), wherein the C residue is in a reduced state, and wherein the X residues are any amino acid e other than cysteine.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361792198P | 2013-03-15 | 2013-03-15 | |
US61/792,198 | 2013-03-15 | ||
PCT/US2014/030545 WO2014145735A2 (en) | 2013-03-15 | 2014-03-17 | Product and process for mucus viscosity normalization |
Publications (2)
Publication Number | Publication Date |
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NZ711983A NZ711983A (en) | 2021-09-24 |
NZ711983B2 true NZ711983B2 (en) | 2022-01-06 |
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