Systems Chemico-Pharmacology Drugs and Methods of Use
20220409692 · 2022-12-29
Inventors
- Kirkwood Arthur Pritchard, Jr. (Elm Grove, WI, US)
- Dustin Paul Martin (Sturtevant, WI, US)
- Stephen Naylor (Elm Grove, WI, US)
Cpc classification
C12N9/0065
CHEMISTRY; METALLURGY
A61K38/12
HUMAN NECESSITIES
International classification
Abstract
As new class of drugs designated systems chemico-pharmacology drugs (SCPD) is disclosed. SCPDs work by targeting a druggable biomolecular site (the first target), resulting in a significant change in the properties of the first target. In the process, the SCPD itself is chemically modified. Subsequently, the resulting modified SCPD interacts with a second target, which is also modified in a manner that is beneficial for the patient.
Claims
1. A systems chemico-pharmacology drug (SCPD) for treating a disease or condition characterized by an increased peroxidase activity in a subject, the SCPD configured such that: i. the SCPD interacts with a first target that comprises a druggable biomolecular site; ii. the SCPD modifies the properties and functions of the first target and is itself chemically modified and activated, and ii. the chemically modified/activated SCPD interacts with one or more second targets, thus modifying each of the second targets' functions; whereby the disease or condition is treated in the subject.
2. The SCPD of claim 1, wherein the druggable biomolecular site is a protein, a peptide, a DNA segment, an RNA segment, or a metabolite.
3. The SCPD of claim 2, wherein the druggable biomolecular site is an active site of a peroxidase.
4. The SCPD of claim 3, wherein the peroxidase is a myeloperoxidase (MPO) or eosinophil peroxidase (EPO).
5. The SCPD of claim 1, wherein the SCPD is an agonist or an antagonist of the first target.
6. The SCPD of claim 1, wherein the SCPD is chemically modified after administration to a subject by being oxidized, being reduced, forming a radical, forming a salt, or undergoing energetic excitation.
7. The SCPD of claim 6, wherein undergoing energetic excitation comprises electron transfer excitation.
8. The SCPD of claim 6, wherein the chemically modified SCPD comprises a stabilized free radical.
9. The SCPD of claim 8, wherein the stabilized free radical is activated such that it is capable of reacting with each of the one or more second targets.
10. The SCPD of claim 8, wherein the chemically modified version of the SCPD is capable of auto-scavenging by forming a homodimer of chemically modified SCPDs via an oxidized linkage.
11. The SCPD of claim 8, wherein the chemically modified SCPD is capable of scavenging by forming a heterodimer with said second target via an oxidized linkage.
12. The SCPD of claim 9, wherein the at least one of the one or more second targets is a peptide or protein.
13. The SCPD of claim 12, wherein the peptide or protein is a pro-inflammatory peptide or protein, and wherein the peptide or protein's function is modified to reduce its pro-inflammatory activity.
14. The SCPD of claim 13, wherein the pro-inflammatory peptide is glutathione.
15. The SCPD of claim 13, wherein the pro-inflammatory protein is HMGB1, RAGE, TRL4, NRf2 or KEAP-1.
16. The SCPD of claim 1, wherein the SCPD has the peptide-based formula AA(n), wherein n is 2-5; said peptide comprising: i. an N-terminus amino acid that includes a basic side chain; ii. an amino acid at any one of positions 2-5 that includes a polar, non-polar, aromatic ring or hetero-atom side-chain that can stabilize a radical species and is configured such that said amino acid at position 2-5 can participate in direct radical transfer from the heme porphyrin of the MPO's active site to said amino acid's side-chain, thus yielding the chemically modified SCPD; iii. an amino acid at any one of positions 2-5 including a side chain that can interact with the MPO's active site through one or more of ionic, dipolar, pi-pi, hydrophobic or hydrophilic interactions facilitating radical transfer from the heme porphyrin of the MPO's active site to the SCPD; and iv. an amino acid at any one of positions 2-5 that includes a side chain containing a heteroatom that stabilizes the radical on the chemically modified SCPD; wherein said chemically modified SCPD is configured to: auto-scavenge by forming a homo dimer via an oxidized linkage with another chemically modified SCPD; or, optionally, to scavenge by forming a hetero dimer via an oxidized linkage with another peptide or protein.
17-36. (canceled)
37. A method of treating a disease or condition in a subject, the method comprising administering to the subject a systems chemico-pharmacology drug (SCPD), whereby the SCPD interacts with a first target that comprises a druggable biomolecular site, whereby the SCPD modifies the properties of the first target and is itself chemically modified, and further whereby the chemically modified SCPD interacts with one or more second targets, thus modifying the each of the second targets' functions; whereby the disease or condition is treated in the subject.
38-55. (canceled)
56. The method of claim 37, wherein the SCPD comprises a tripeptide KXZ having the formula AA.sub.1-AA.sub.2-AA.sub.3, wherein AA.sub.1 (K) is an amino acid comprising a basic side chain, AA.sub.2 (X) is a polar, non-polar or aromatic amino acid, and AA.sub.3 (Z) is an amino acid possessing a heteroatom that is capable of stabilizing a free radical, wherein one or more of the second targets is a protein and the protein is HMGB1, RAGE, TRL4, NRf2 or KEAP-1.
57-62. (canceled)
63. A compositional system comprising a systems chemico-pharmacology drug (SCPD) in fluid communication with (a) a first target that comprises a druggable biomolecular site, and (b) a modified first target comprising a druggable biomolecular site that has been modified by the SCPD.
64-84. (canceled)
85. The compositional system of claim 63, further comprising one or more second targets wherein at least one of the one or more second targets is a protein and the protein is HMGB1, RAGE, TRL4, NRf2 or KEAP-1.
86-92. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] The disclosure will be better understood and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description. Such detailed description makes reference to the following drawings.
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[0116] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are herein described in detail. The description of specific embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.
DETAILED DESCRIPTION
I. IN GENERAL
[0117] This invention is not limited to the particular methodology, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the language of the appended claims.
[0118] As used in this disclosure and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. The terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably. The terms “comprising”, “including”, and “having” can also be used interchangeably.
[0119] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Chemical compound names that are commonly used and recognized in the art are used interchangeably with the equivalent IUPAC name. All publications and patents specifically mentioned in this disclosure are incorporated by reference for all purposes.
[0120] Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used and all cited references are incorporated herein by reference for all purposes.
[0121] As used herein, the term “amino acid” residue or sequence refers to abbreviations used herein for designating the amino acids based on recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). Also included are the (D) and (L) stereoisomers of such amino acids when the structure of the amino acid admits of stereoisomeric forms. The term “amino acid” encompasses the 20 naturally-occurring amino acids and, as well, the “unnatural amino acids,” which include any amino acid, modified amino acid, and/or amino acid analog that is not one of the 20 common naturally occurring amino acids.
[0122] As used herein, the term “peptide” refers to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. For example, “peptide” specifically includes the non-genetically-coded amino acids that either occur naturally or are chemically synthesized including, but not limited to synthetic .alpha.- and .beta.-amino acids known to one of skill in the art.
[0123] As used herein, the phrase “protecting group” refers to a chemical group that, when attached to a functional group in an amino acid (e.g. a side chain, an alpha amino group, an alpha carboxyl group, etc.) blocks or masks the properties of that functional group. Preferred amino-terminal protecting groups include, but are not limited to acetyl, or amino groups. Other amino-terminal protecting groups include, but are not limited to alkyl chains as in fatty acids, propenol, formyl and others. Preferred carboxyl terminal protecting groups include, but are not limited to groups that form amides or esters.
[0124] As used herein, the term “retro” as applied to an amino acid sequence refers to an amino acid sequence that is in reverse order of the original reference sequence. The term “inverso” as applied to an amino acid sequence refers to an amino acid sequence composed of D-amino acids as opposed to the parent L-sequence. Because the orientation of the side-chains in a retro-inverso analog is very similar to that in a reference L-sequence, there is a high probability of functional similarity between the two sequences.
[0125] As used herein, the term “subject” includes non-human mammals and humans.
[0126] As used herein, the phrase “therapeutically effective amount” means the amount of a compound that, when administered to a subject for treating a disease or disorder, is sufficient to affect such treatment for the disease or disorder. The “therapeutically effective amount” can vary depending on the compound, the disease or disorder and its severity, and the age, weight, etc., of the subject to be treated.
[0127] As used herein, the term “treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder, or even preventing the same.
II. THE INVENTION
[0128] This disclosure is based on our discovery that certain peptide-based inhibitors of MPO are modified in the process of MPO inhibition such that they can subsequently target other pathways related to disease and/or inflammation. We have designated such agents as “systems chemico-pharmacology drugs” (SCPDs). We have demonstrated this phenomenon using a KXZ tripeptide (in KXZ amino acid #1 (AA.sub.1, K) is a native amino acid or an artificial amino acid (aa) with a basic side-chain such as an amine functional group (e.g., Lysine (K)); Amino acid #2 (AA.sub.2, X) is a native or artificial amino acid that may contain a polar, non-polar, or aromatic amino acid; and Amino acid #3 (AA.sub.3, Z) is a native or artificial amino acid that possesses a heteroatom that can stabilize a free radical. The free radical itself may act as an activated species, and is stable enough to react with proximal proteins, peptides, metabolites or small molecules to effect further change. The free radical is not limited to a single target, but may effect change multiple independent targets. In a non-limiting example, the free radical goes on to form a heteroatom-carbon bond in the form of a homo- or heterodimer of the tripeptide) in a model of bronchopulmonary dysplasia. However, SCPDs can be used to treat a wide variety of diseases and conditions.
Unique Properties of an SCPD
[0129] Both “systems pharmacology” and “polypharmacology” of therapeutic drugs are terms used as descriptors to describe efforts to overcome the one drug-one target model. Polypharmacology refers to a one drug-multi-target concept as utilized in drug repurposing. However, systems pharmacology refers to a broader concept best captured as one drug-multi targets associated with pathways and networks. Thus systems pharmacology is based on the rational design of drug therapies using information based on molecular, cellular and physiological complexity. This type of approach produces systems pharmacology drugs rooted in molecular interactions between a single drug and multi-targets present in defined pathways/networks.
[0130] A systems chemico-pharmacology drug (SCPD) has unique properties and must do the following: i) SCPD must target a druggable biomolecular site. This can be a protein/peptide, DNA, RNA, or metabolite or other small molecule. ii). The SCPD must modify the properties of the target, it can be an agonist or antagonist, that results in the target properties changing. iii) As a function of the SCPD-target interaction and change in target properties, the SCPD is itself chemically modified. iv) The modification can be oxidation/reduction, formation of a radical, salt formation, or any form of energetic excitation such as electron transfer excitation, to create a modified SCPD structure. v) this modified entity must then interact with specific new target(s) and modify the new target's function in a efficacious manner for the patient.
Systems Chemico-Pharmacology Drugs Having the Generic Formula AA.SUB.(n)
[0131] In a first aspect, the invention is directed to small end-capped peptides with a generic formula of AA.sub.(n) (where AA can represent different amino acids, and n can be 2-5) that inhibit MPO. In addition we provide a set of physicochemical properties of individual amino acid that collectively contribute to inhibition of MPO. These same criteria (for the amino acids and thus collectively for the peptide) are also applicable to any organic molecule that will inhibit MPO toxic oxidant production. The peptide studies described herein were designed to better describe and understand the optimal sequence and physicochemical properties of individual amino acids used as constituents of MPO small peptide inhibitors. These same peptides also serve as inhibitors of other members of the peroxidase enzyme family such as eosinophil peroxidase.
[0132] Small peptides of generic formula AA(n) (where AA represents different amino acids, and n can be 2-5) are both substrates and inhibitors of MPO (alternatively, inhibitors of EPO). These peptides are reduced (gain an electron in the form of a radical) at the active site of MPO, which subsequently decreases the production of toxic oxidants that includes but not restricted to hypochlorous acid. All MPO activities (i.e., hypochlorous acid production) described herein were quantified by the 3,3′,5,5′-tetramethylbenzidine (TMB) assay. In order to inhibit the production of toxic oxidants such as hypochlorous acid by MPO each peptide must contain amino acids that possess: i) an N-terminus amino acid #1 (AA1), that is a native amino acid or an artificial amino acid with a basic side-chain such as an amine functional group (e.g. Lysine (K)). ii) Amino acid #2-5 (AA2-5), is a native or artificial amino acid that may contain a polar, non-polar, aromatic ring or hetero-atom containing side-chain that can stabilize a radical species and has specific proximity to the iron/heme of theMPO active site, and can participate in direct radical transfer to the amino acid side-chain. iii) Amino acid #2-5 (AA2-5), is a native or artificial amino acid that also possesses functional groups that interact with any functionality of the overall MPO active site (iron/heme or side-chains of other active site amino acids) through ionic, dipolar, pi-pi, hydrophobic or hydrophilic interactions. These interactions “lock” the peptide into the MPO active site and facilitate radical transfer from MPO to the peptide. iv) Amino acid #2-5 (AA2-5,) is a native or artificial amino acid that also possesses a heteroatom that can further stabilize a free radical. The free radical itself may act as an activated species, and is stable enough to react with proximal proteins, peptides, metabolites or small molecules to effect further change. The free radical is not limited to a single target, but may effect change multiple independent targets. In a non-limiting example, the free radical goes on to form a hetero- or homo dimer via an oxidized —S—S— disulfide (or di-Selenium) linkage. Finally, the more interactions between the peptide and active site of MPO that occurs the lower numerical IC50 value of the peptide inhibitor.
[0133] Operationally, an AA(n) (where AA represents different amino acids, and n can be 2-5) peptide is a substrate that is oxidized by MPO to ultimately generate a peptide radical that autoscavenges when it forms a disulfide homodimer. The AA(n) peptide radical may also be scavenged when it forms a disulfide with the thiol of cysteine, glutathione or the thiol of cysteine in another peptide or protein. The free radical itself may act as an activated species, and is stable enough to react with proximal proteins, peptides, metabolites or small molecules to effect further change. The affinity of the AA(n) peptide for the active site of MPO should be higher than the affinity of endogenous MPO substrates (such as chloride and nitrite) in order to prevent MPO from binding chloride or nitrite and oxidizing these ions to hypochlorous acid and nitronium ion, respectively.
[0134] AA.sub.(n) peptides compete with MPO's native substrates, such as chloride, and nitrite, etc., and prevents them from entering the active site of MPO. AA.sub.(n) competitive inhibition of the binding of these native substrates prevents MPO from generating toxic oxidants such as hypochlorous acid and nitronium ion (
The KXZ Tripeptide: An Exemplary SCPD
[0135] We have demonstrated this phenomenon using an exemplary KXZ tripeptide (in KXZ amino acid #1 (AA.sub.1, K) is a native amino acid or an artificial amino acid (aa) with a basic side-chain such as an amine functional group (e.g., Lysine (K)); Amino acid #2 (AA.sub.2, X) is a native or artificial amino acid that may contain a polar, non-polar, or aromatic amino acid; and Amino acid #3 (AA.sub.3, Z) is a native or artificial amino acid that possesses a heteroatom that can stabilize a free radical that then goes on to form a heteroatom-carbon bond in the form of a homo- or heterodimer of the tripeptide) in a model of bronchopulmonary dysplasia. However, SCPDs can be used to treat a wide variety of diseases and conditions.
[0136] An example of a SCPD is any one of the KXZ peptides in the library of peptides as it pertains to MOA in Bronchopulmonary Dysplasia (see example 1 below). KXZ is a tripeptide substrate and inhibitor of the peroxidase enzyme MPO. This enzyme is present in myeloid cells such as neutrophils, monocytes and macrophages. Humans employ MPO primarily as an anti-infective agent that produces the toxic oxidant HOCl.
[0137] When KXZ interacts with MPO the tripeptide is reduced (gains an electron in the form of a radical) to form KXZ* (where * represents a free electron radical species) as well as inhibits the production of HOCl. The KXZ* can go on to thiolate proximal proteins of MPO, such as HMGB1, resulting in the loss of pro-inflammatory activity of this protein. Additional proteins such as RAGE, TRL4, NRf2, and KEAP-1 also appear to be regulated by the presence of KXZ/KXZ*.
[0138] Such results indicate that KXZ is a systems pharmacology drug. However, the novel mechanism of action requires a distinction from all known current systems pharmacology drugs. In this specific case, KXZ is chemically modified by MPO, whilst effecting MPO activity and function, to subsequently produce KXZ*, and it is the latter that then produces further regulatory effect by thiolation of proximal proteins such as HMGB1.
[0139] In order to differentiate the mechanism of action of KXZ from other systems pharmacology drugs, we have labeled this class of compounds as systems chemico-pharmacology drugs. The definition of such a drug is that it predicated on molecular interactions between a single drug and multi-targets present in defined pathways/networks. In addition the parent systems pharmacological agent must react chemically with at least one target to produce a new chemical entity (in this case a radical species (KXZ.fwdarw.KXZ*) that then continues to modulate additional new pathway/network associated targets.
[0140] In sum, KXZ peptides in the presence of MPO are a clear example of a SCPD. KXZ peptides are multimodal in function and appear to regulate a number of different proteins, especially when modified by the initial target. Examples of such proteins include, but are not limited to, MPO, HMGB1, RAGE, TRL4, Nrf2, and KEAP1.
Benefits of SCPDs
[0141] A systems chemico-pharmacology drug exemplified by KXZ is designed to both inhibit MPO generation of toxic oxidants and subsequently inhibit other pro-inflammatory peptides/proteins associated with neutrophil mediated inflammation. In addition it is designed to provide the following non-limiting advantageous properties.
[0142] First, a single systems chemico-pharmacology drug with multi-target activity should have a more predictable, therefore superior pharmacokinetic (PK) and pharmacodynamic (PD) profile compared to a number of individual drugs administered either as a single drug or in combination.
[0143] Second, acute toxicity may be enhanced in more non-selective single/combination therapies.
[0144] Third, adverse synergistic effects may be more pronounced in single/combination therapies.
[0145] Fourth, the probability of developing target-based resistance to multi-target drugs is statistically lower than is the probability of developing resistance against single-target drugs.
[0146] Fifth, the administration of a single systems pharmacology drug results in a more consistent and predictable ADME profile.
[0147] Sixth, drug-drug interactions do not exist in a systems chemico-pharmacology regime.
[0148] Finally, a single agent binding to multiple targets might be easier to develop, given that the regulatory requirements showing safety/efficacy of a drug combination.
KXZ Structure
[0149] As discussed above, the inventors have discovered that peptide-based inhibitors of peroxidase activity designated as “KXZ” may act as SCPDs in the presence of MPO. Such peptide inhibitors are particularly useful for improving vascular function, decreasing pulmonary inflammation and increasing cardioprotection in a subject.
[0150] In view of the inventors' discovery, certain peptide-based SCPDs of the invention (KXZ) have the formula AA.sub.1-AA.sub.2-AA.sub.3. Amino acid #1 (AA.sub.1, K) is native amino acid or an artificial amino acid (aa) with a basic side-chain such as an amine functional group (e.g., Lysine (K)). Amino acid #2 (AA.sub.2, X) is a native or artificial amino acid that may contain a polar, non-polar, or aromatic amino acid. Amino acid #3 (AA.sub.3, Z) is a native or artificial amino acid that possesses a heteroatom that can stabilize a free radical. The free radical itself may act as an activated species, and is stable enough to react with proximal proteins, peptides, metabolites or small molecules to effect further change. The free radical is not limited to a single target, but may effect change multiple independent targets. In a non-limiting example, the free radical goes on to form a heteroatom-carbon bond in the form of a homo- or heterodimer of the tripeptide. A non-limiting example of KXZ is the tripeptide KYC.
[0151] In some embodiments, the amino termini may be protected by an acetyl group, and/or the carboxyl termini may be protected by an amide. The disclosure also encompasses peptides comprising retro and retro-inverso analogs of each of the sequences. In retro forms, the direction of the amino acid sequence is reversed. In inverso forms, the chirality of the constituent amino acids is reversed (i.e., L form amino acids become D form amino acids and D form amino acids become L form amino acids). In the retro-inverso form, both the order and the chirality of the amino acids are reversed. A given amino acid reference amino acid sequence and its retro-inverso form are mirror images of each other, and typically have similar functions.
[0152] In certain embodiments the peptide can be a cyclic peptide.
[0153] In certain embodiments the SCPD is a deuterated (either partially or per-deuterated) compound/peptide.
[0154] In certain embodiments, peptides of the invention further contain a protecting group coupled to the amino or carboxyl terminus of the peptide, or a first protecting group coupled to the amino terminus of the peptide and a second protecting group coupled to the carboxyl terminus of the peptide. Possible protecting groups for use in this embodiment include without limitation amide, 3 to 20 carbon alkyl groups, Fmoc, Tboc, 9-fluorene acetyl group, 1-fluorene carboxylic group, 9-florene carboxylic group, 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bz1), 2-chlorobenzyloxycarbonyl (2-Cl—Z), 2-bromobenzyloxycarbonyl (2-Br—Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), acetyl (Ac), and Trifluoroacetyl (TFA).
[0155] In certain other embodiments, the peptide contains a protecting group coupled to the amino terminal and the amino terminal protecting group is acetyl. In other embodiments, the peptide contains a protecting group coupled to the carboxyl terminal and the carboxyl terminal protecting group is an amide. In still other embodiments, the peptide contains a first protecting group coupled to the amino terminus that is an acetyl, and a second protecting group coupled to the carboxyl terminal that is an amide.
[0156] The disclosure also includes pharmaceutical compositions for simultaneously inhibiting peroxidase activity and targeting other disease-related pathways, containing one or more of the peptides described above and a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. It is also envisioned that the peptides of the present invention may be incorporated into transdermal articles designed to deliver the appropriate amount of peptide in a continuous fashion.
[0157] It is not critical whether an inhibitor according to the invention is administered directly to a peroxidase, to a tissue comprising the peroxidase, a body fluid that contacts the peroxidase, or a body location from which the inhibitor can diffuse or be transported to the peroxidase. It is sufficient that the inhibitor is administered to the subject in an amount and by a route whereby an amount of the inhibitor sufficient to inhibit the peroxidase arrives, directly or indirectly at the peroxidase.
[0158] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutically acceptable carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid pre-formulation composition containing a homogeneous mixture for a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these pre-formulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer, which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[0159] The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium caboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.
[0160] In certain embodiments, the peptides of the invention will be provided as pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts, which may, for example, be formed by mixing a solution of the peptide according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g. sodium or potassium salts, alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.
[0161] Suitable dosage levels for the inhibition of peroxidase activity in a human subject (i.e, an effective therapeutic amount to inhibit peroxidase activity) is about 0.01-5000 mg/kg, per day, preferably about 0.1-500 mg/kg per day, and especially about 0.1-50 mg/kg per day.
[0162] In another aspect, the invention provides a method of treating a disease or condition in a subject that is associated with excess peroxidase activity. The method includes the step of administering to a subject in need of such therapy one or more of the peptides as described above. In certain preferred embodiments, the subject is a human or a non-human mammal. Preferably, the method includes the additional step of mixing the peptide with a pharmaceutically acceptable carrier before the peptide is administered. In preferred embodiments of the invention, the method is carried out to improve vascular function, decrease pulmonary inflammation, and/or increase cardioprotection in the subject. However, it can be appreciated that the inventive peptides act on a molecular process common to a plethora of medical diseases and conditions. Therefore, the present peptides are envisioned to be useful in treating a wide range of diseases and conditions attributable to aberrant peroxidase activity, including but not limited to, wound inflammation, hypersensitivity, digestive disease, cardiovascular disease, neuronal disease, lung disease, autoimmune disease, degenerative neurological disease, degenerative muscle disease, infectious disease, disease associated with graft transplantation, allergic disease, musculo-skeletal inflammation, and sepsis.
[0163] Methods of the invention are further envisioned to be useful in treating hypertension, peripheral vascular disease, pulmonary inflammation, asthma, atherosclerosis, diabetes, persistent pulmonary hypertension, sickle cell disease, neurodegenerative disease, multiple sclerosis, Alzheimer's disease, lung cancer, lupus, ischemic heart disease, Parkinson's disease, Crohn's disease, inflammatory bowel disease, necrotizing enterocolitis, arthritis, polymyocytis, cardiomyopathy, psoriasis, amyotrophic lateral sclerosis, muscular dystrophy, cystic fibrosis, attention deficiency hyperactive disorder, acute lung injury, acute respiratory distress syndrome, flu (including H1N1), heart failure, chemotherapy-induced heart failure, arthritis, rheumatoid arthritis, acute myocardial infarction, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), ischemic or hemorrhagic stroke, or bronchopulmonary dysplasia.
[0164] Further, the disclosed methods will find use in the promotion of angiogenesis in tissues of a subject, or the promotion of angiogenesis impaired by persistent pulmonary hypertension, peripheral vascular disease or vascular disease in the myocardium in the subject, or the treatment of a disease or condition associated with abnormal, excessive blood vessel development in the subject. The disclosed methods of are additionally useful in treating subjects for the reduction and/or prevention of ischemic injury to a subject's heart following an ischemic event or insult.
[0165] The disclosure also encompasses the use of a peptide as described herein for the manufacture of a medicament for inhibiting peroxidase activity and subsequently targeting a second pathway in a subject. Such methods include the steps of (a) providing a peptide as described herein, and (b) mixing the peptide with a pharmaceutically acceptable carrier. As well, the invention encompasses the manufacture and use of medicaments specifically-purposed for treatment of one or more of the diseases/conditions described above.
[0166] The following example is offered for illustrative purposes only and is not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the following examples and fall within the scope of the appended claims.
III. EXAMPLES
Example 1
Demonstrating Systems Chemico-Pharmacology Properties of Exemplary KXZ Tripeptide in Bronchopulmonary Dysplasia Model
[0167] This example provides “proof of principle” by demonstrating the use of an exemplary KXZ tripeptide to target multiple pathways in a bronchopulmonary dysplasia disease model.
[0168] Summary.
[0169] Although many consider myeloid cells to play important roles in hyperoxia-induced bronchopulmonary dysplasia (BPD), the role of myeloperoxidase (MPO) is unclear. We hypothesize that MPO increases BPD by inducing an oxidative and inflammatory destructive cycle.
[0170] To test this hypothesis Sprague-Dawley neonatal pups were raised in normoxic or hyperoxic (>90% oxygen) environments from day one (P1) until day ten (P10) of life. Hyperoxic neonatal pups were treated each day with either N-acetyl lysyltyrosylcysteine amide (KYC) to inhibit MPO, or phosphate buffered saline from P1 through P10 and then euthanized. Lungs were examined for changes in lung morphometrics, oxidative stress, inflammation, and myeloid cell counts.
[0171] Chronic hyperoxia impaired lung development, decreased microvascular and alveolar complexity, and increased lung pathology based on marked increases in MPO+ myeloid cell counts; MPO; 3-chlorotyrosine (Cl-Tyr); 3-nitrotyrosine (NO.sub.2-Tyr); high mobility group box 1(HMGB1); receptor for advanced-glycosylated end-products (RAGE), and, toll-like receptor 4(TLR4). In contrast, KYC treatment reduced MPO+ myeloid cell counts, increased microvascular and alveolar complexity, and decreased MPO, Cl-Tyr, NO.sub.2-Tyr, HMGB1, TLR4, and RAGE levels, and lung pathology.
[0172] Taken together, these data indicate that MPO establishes a destructive cycle predominantly mediated by excess oxygen (O.sub.2), myeloid cells, MPO, HOCl, and pulmonary release of HMGB1. Clearly, MPO oxidants play a causal role in propagating this cycle to increase lung pathology since inhibiting MPO toxic oxidant production reduces lung pathology and improves lung development in hyperoxic neonatal rat pups.
[0173] Introduction.
[0174] Bronchopulmonary dysplasia (BPD) is caused by complications from respiratory distress syndrome of the newborn. BPD affects more than 10,000 infants annually (32), making it the most common pulmonary morbidity of premature infants in the United States (14). Premature neonates have immature lungs that do not function well enough to support life and therefore must be provided respiratory support, such as supplemental oxygen and mechanical ventilation. Both interventions are known to increase oxidative lung injury and BPD (14).
[0175] Experimental strategies for treating BPD range from treating with antioxidants to scavenge oxidants (24, 47), supplementing the antioxidant defense system (40), as well as blocking myeloid cell recruitment (2, 3, 15). Since myeloid cell recruitment always precedes BPD (13), inhibiting recruitment should be an effective therapeutic strategy for reducing BPD. Although anti-cytokine-induced neutrophil chemoattractant-1 (CINC-1) antibodies and CXC chemokine receptor-2 (CXCR2) antagonist have both been used to reduce neutrophil infiltration and myeloperoxidase (MPO) release in the lungs of established neonatal rat models of BPD (3, 15) neither approach has progressed towards clinical implementation.
[0176] MPO is considered by many to play a causal role in inducing oxidative injury and inflammatory lung disease (4, 10, 17, 28). However, to our knowledge, few if any studies have examined the mechanistic role of MPO in BPD. Recently, we reported that MPO generation of toxic oxidants increases oxidative damage in secondary brain injury in a murine model of stroke (56), neuroinflammation in experimental autoimmune encephalomyelitis (EAE) mice (60), and impaired endothelial- and eNOS-dependent vasodilatation in a murine model of sickle cell disease (61). MPO is a robust peroxidase that has evolved to generate large quantities of free radicals and oxidants to kill invasive bacteria. MPO oxidation of chloride anions (Cl−) produces hypochlorous acid (HOCl), while MPO oxidation of nitrite (NO.sub.2.sup.−) generates predominantly nitrogen dioxide (NO.sub.2*) (11). As HOCl oxidizes protein tyrosine to form 3-chlorotyrosine (Cl-Tyr) (54), many consider tissue levels of Cl-Tyr to be an index of MPO-dependent oxidative damage (45). In contrast, peroxynitrite and NO.sub.2* are both capable of oxidizing tyrosine to 3-nitrotyrosine (NO.sub.2-Tyr) (19, 42). Accordingly, NO.sub.2-Tyr is considered a footprint of both forms of nitrosative stress. Data supporting the idea that MPO oxidants are involved in disease pathology are often indirect, based on relative differences in the levels of Cl-Tyr and/or NO.sub.2-Tyr in lung tissues before and after treatment or from comparison of biomarker levels in wild-type and MPO-knockout mice (27, 28, 60). Selective attenuation of MPO activity by either transgenic knockout or selective inhibition with non-toxic small molecules are both logical strategies for determining the extent of MPO's contribution to BPD pathology.
[0177] MPO knockout rats are not currently available, hence we investigated the role of MPO in hyperoxia-induced BPD in neonatal rat pups by inhibiting MPO oxidant production with N-acetyl lysyltyrosylcysteine amide (KYC). KYC is a novel tripeptide inhibitor of MPO toxic oxidant production that reduces MPO-dependent secondary brain injury in a murine model of stroke (56, 57), neuroinflammation in a murine model of multiple sclerosis (58, 60), dose-dependently protects endothelial cell cultures from MPO-mediated cell death (59), and improves endothelial- and eNOS-dependent vasodilatation in an established murine model of sickle cell disease (61). Mechanistically, KYC is unique in its ability to reduce MPO toxic oxidant production because it is a substrate that reduces the iron heme of MPO to ground state and in so doing shuttles MPO oxidants directly into the glutathione (GSH) system (
[0178] Here, we present data showing KYC inhibition of MPO breaks the proposed destructive cycle between MPO, HMGB1, and myeloid cell recruitment to reduce oxidative damage and inflammation in an established neonatal rat pup model of chronic hyperoxia-induced BPD model.
[0179] Materials and Methods.
[0180] Peptide Synthesis: KYC was synthesized using Fmoc [N-(9-fluorenyl)methoxy-carbonyl] chemistry, prepared and purified as an acetate salt by Biomatik USA, LLC (Wilmington, Del.), as previously described (56, 60). Trifluoroacetic acid (TFA) in tripeptide preparations was reduced by the Peptide Core (Blood Research Institute, Medical College of Wisconsin) by dissolving KYC in distilled water containing 6 mM HCl followed by lyophilization (2-3×). TFA content in peptide preparations was quantified by NMR (30). TFA in KYC preparations was reduced to <0.01% prior to being used for experiments. All other chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.).
[0181] Antibodies: Rabbit antibodies for myeloperoxidase heavy chain (MPO, sc-33596) and for TLR4 (sc-10741) were from Santa Cruz Biotechnology (Dallas, Tex.). Rabbit antibody for Cl-Tyr (HP5002) was from Hycult Biotech (Plymouth Meeting, Pa.). Rabbit antibody for NO.sub.2-Tyr (N0409) was from Millipore-Sigma (St. Louis, Mo). Chicken antibody for HMGB1 (326052233) was from SHINO-TEST Corporation (Kanagawa, Japan). Rabbit antibody for RAGE (GTX23611) was from GeneTex (Irvine, Calif.). Mouse antibodies for PECAM-1/CD31 (ab24590) and for rat endothelial cell antigen-1 (RECA-1, ab9774) were from Abcam (Cambridge, Mass.).
[0182] Rats and Experimental Protocols: Sprague-Dawley rats were obtained from Harlan Laboratories (Madison, Wis.) and pregnancy was achieved naturally in our animal facility. Animal protocols were submitted to, and approved by, the Medical College of Wisconsin's Institutional Animal Care and Use Committee and conformed to NIH Guide for the Care and Use of Laboratory Animals. The animals were housed in barrier cages with a 12-h dark-light cycle and were given free access to chow and water. The dam and pups were placed in a cage in either room air (normoxia) or >90% oxygen chamber (hyperoxia) from postnatal day 1 to day 10 (P1-P10) to generate BPD as previously reported (25, 51). Oxygen concentrations were continuously monitored with an oxygen sensor (Reming Bioinstruments Co., Redfield, N.Y.).
[0183] Since the number of neonatal rat pups per pregnant dam limits the number of experimental conditions that can be compared, we modified our standard experimental design protocol. The first experiment was designed to determine if hyperoxia increased BPD, while the second was designed to determine if inhibiting MPO oxidant production reduced BPD. At least three sets of neonatal pups from three different dams were used for each experiment. Pups were fed ad libitum from nursing dams. Pups were weighed and inspected daily in room air for less than ten minutes. Nursing dams were switched daily to avoid the impact of nutritional status. Experience has taught us that severity of hyperoxia-induced BPD is litter dependent. To minimize litter differences, we determined the effects of hyperoxia vs. normoxia on BPD in neonatal pups by mixing pups from the litters and randomly reallocating pups with nursing dams.
[0184] To determine the effects of KYC on BPD in hyperoxic neonatal pups we randomly assigned pups to the phosphate buffer solution (PBS) control group or the KYC treatment group. The dose of KYC (5 mg/kg twice per day) was chosen based on experience treating chronic inflammation in other disease states (58, 60, 61). KYC was injected i.p., starting on P2 to half of the randomly assigned pups in each litter using a sterile insulin syringe fitted with a 30G needle (Beckon Dickinson, New York, N.Y.) until P10. Equal volumes of PBS were injected i.p. into the remaining half of the pups as an injection control. Pups were euthanized on P10 with carbon dioxide and lungs removed en bloc. A small cut was created on the left atrium and ice-cold normal saline was gently infused through the right ventricle to flush blood from the lungs before inflation for histology or snap-frozen in liquid nitrogen for protein studies. Pups from at least three litters were used in each experiment. Survival rates were determined by plotting the deaths of hyperoxic neonatal rat pups treated with either PBS or KYC on the postnatal day of discovery. Kaplan-Meier survival curves were plotted and analyzed from Kaplan-Meier tables constructed in GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, Calif.).
[0185] Immunohistochemistry and Immunocytochemistry: After cannulating the trachea with an Instech Solomon (20G) stainless steel feeding tube (Plymouth Meeting, Pa.), the lungs were inflated with 10% neutral buffered formalin at 25 cm-H2O (2.4 kPa) for 1 h. Lungs were removed after the trachea were securely tied with surgical silk to maintain the pressure, and perfusion fixed with an additional aliquot of 10% neutral buffered formalin for 24 h before paraffin embedding. Lung sections (5 μm) were mounted on SuperFrost plus-coated slides (Denville Scientific, Metuchen, N.J.). Slides were deparaffinized and sections stained with hematoxylin and eosin (H&E). Histology images were captured with a mounted digital camera using an Olympus IX 51 microscope and a 10× objective. Inflammatory cells, myeloid cells including neutrophils, monocytes, and macrophages, were stained with MPO antibody (1:200) overnight at 4° C. and HPR-conjugated anti-rabbit antibody (1:1000) at room temperature for one hour then visualized by diaminobenzidine to generate a dark-brown color. Blood vessels were stained with RECA-1 and visualized with horseradish-conjugated secondary antibody and diaminobenzidine. The average of three sections per pup, and five counts per section (15 counts/pup) was used for statistical analysis. Quantified data were obtained and entered into the record using predetermined codes by one of the coauthors who was not involved in taking the images.
[0186] Morphometric Analysis: The mean linear intercept (MLI), or chord length, was used as a method to estimate the volume-to-surface ratio of acinar airspaces whereas radial alveolar count (RAC) and secondary septa were investigated to study the complexity of lung structure (21). Ten equally spaced horizontal lines were drawn on each picture, and the number of intercepts through the alveolar wall was counted. MLI was obtained by the number of times the traverses are placed on the lung times the length of the traverse then divided by the sum of all the intercepts. For the RAC, a line from the center of the respiratory tract perpendicular to the nearest connective tissue septum was drawn, and alveoli intercepting with the line were counted. For measurement of secondary septa, elastin was stained with resorcin fuchsin and Van Gieson's solution.
[0187] Immunoblot Analysis: Whole lung lysates were obtained by homogenizing in MOPS buffer (20 mM 3-N-morpholino-propanesulfonic acid, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 10 mM β-glycerophosphate, 10 mM Na pyrophosphate, 2 mM Na orthovanadate, 1 mM PMSF, 0.5% NP-40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktails 2 and 3, pH 7.0) by Bullet Blender (Next Advance, Inc., Averill Park, N.Y.). For protein immunoblots, 30 μg of protein lysate was separated by SDS-PAGE, transferred to nitrocellulose membranes (0.22 μm), and then probed with the appropriate primary antibodies overnight at 4° C. on a continuously rocking platform. Signals were generated after incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:8,000) using Western Bright ECL Chemiluminescent HRP Substrate (Advansta Inc., Menlo Park, Calif.). Integrated optical density (IOD) was calculated using ImageJ software and normalized to β-actin, a loading control.
[0188] Glutathione Reductase Reduction of KYC Homo- and Hetero-disulfides: Disulfide formation was induced by incubating either KYC alone (2.2 mM) or KYC with equal concentration glutathione (GSH) in PBS (pH 7.4) with shaking overnight at room temperature. The reducing conditions used to assess glutathione reductase (GR) activity on KYC-KYC and KYC-GSH disulfides were carried out using previously established methods (12, 53). Mixed KYC-KYC and KYC-GSH disulfides were incubated with 3.8 μM GR (Thermo-Fisher, Waltham, Mass.) in a 100 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA, and 1 mM NADPH for 1 hr. at room temperature with shaking. The reaction was halted by snap freezing at −80° C. Negative controls omitted either GR or NADPH. Samples were analyzed by HPLC (Beckman, Brea, Calif.) at the Blood Research Institute of Wisconsin Protein Core Lab. Samples were diluted 1:1 in 0.1% TFA, loaded on a Jupiter Proteo column (C-12 surface, dimensions: 4.6×50 mm, 4 μm particle size, Phenomenex, Torrence, Calif.) and run with a gradient (solvent B into solvent A) of 2-40% over 10 min with a flow rate of 1 ml/min and read using a UV detector at 220 nm. Solvent A was 0.08% TFA in HPLC grade H2O and solvent B was 0.1% TFA in HPLC grade acetonitrile. Unless otherwise indicated chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.).
[0189] Statistics: Representative images of lung sections and immunoblots are presented in the figures. Data from lung sections and immunoblots from the samples were analyzed for statistical differences using GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla, Calif.). Quantitative morphometric and cell count data are presented as mean±SD scatter-plots adjacent to the representative images. Significant differences between groups were determined by either student's t-test or unpaired Mann-Whitney U test, depending on distribution. A p-value<0.05 was considered statistically significant.
[0190] Results.
[0191] Effects of Hyperoxia: Chronic hyperoxia increased the number of MPO (+) myeloid cells in the lungs from neonatal pups compared with the number in lungs from normoxic pups (brown stained cells,
[0192] Effects of Hyperoxia on Lung Morphometrics: Hyperoxic lungs had decreased RAC, secondary septa and blood vessel counts by approximately 27%, 52%, and 52%, respectively, and increased MLI values by approximately 40% compared with normoxic lungs (
[0193] Effects of KYC on Hyperoxia-induced Myeloid Cell Recruitment, MPO, and Oxidative Stress: KYC reduced the number of MPO+ myeloid cells in hyperoxic lungs (brown cells in
[0194] Effects of KYC on Hyperoxia-induced BPD: If MPO plays a causal role in the mechanisms by which hyperoxia induces BPD, then KYC, which inhibits MPO toxic oxidant production but not peroxidase activity (59), should reduce hyperoxia-induced changes in lung architecture. Morphometric analysis shows that KYC treatment of hyperoxic pups increased RAC, secondary septa and blood vessel counts by approximately 35%, 55%, and 92%, respectively, while decreasing MLI (approximately 22%) in the lungs of the hyperoxic pups compared with the stunted development of lung architecture in PBS-treated hyperoxic pups (
[0195] Effects of Hyperoxia and KYC on Lung HMGBJ: Immunoblots show that chronic hyperoxia increased HMGB1 levels in lung homogenates by 170% compared with HMGB1 levels in lung homogenates from normoxic pups (
[0196] Effects of Hyperoxia and KYC Treatment on RAGE and TLR4 Expression in Lungs of Hyperoxic Neonatal Rat Pups: Oxidative stress and inflammation are reported to increase pulmonary expression of RAGE and TLR4 (16, 29). Lung homogenates were examined for changes in RAGE and TLR4 expression to determine if MPO-dependent oxidative stress modulates.
[0197] Effects of KYC on Survival of Neonatal Rat Pups Exposed to Hyperoxia. A total of 66 neonatal rat pups were used in this study. The survival rate for PBS-treated neonatal rat pups exposed to hyperoxia was 82.8% at P10 (
[0198] Effects of Glutathione Reductase on Reduction of KYC-KYC Homo-disulfide and KYC-GSH Hetero-disulfide to KYC Monomer: One of the mechanisms by which KYC is hypothesized to reduce MPO-dependent oxidant production is by forming KYC homodimers (KYC-KYC) that can be reduced to an active KYC monomeric inhibitor by GSH (59). GSH homodimer (GS-SG) can be reduced to monomeric GSH by glutathione reductase (GR) (43). GSH homodimers can also undergo thiol exchange to form mixed heterodimers (GS-SR). To determine if KYC homo-disulfide and KYC-GSH hetero-disulfide are reduced by GR, KYC-KYC homodimer and KYC-GSH heterodimer were incubated with a GR\NADPH reaction mixture and KYC monomer formation determined by HPLC as before (59).
[0199] Discussion.
[0200] Cycle of Destruction: Our findings demonstrate that hyperoxia increases BPD in neonatal rat pups by a destructive cycle containing at least five basic components, namely; myeloid cells, excess oxygen (O.sub.2), MPO, toxic oxidants (i.e., HOCl), and HMGB1 (
[0201] The destructive cycle induced by hyperoxia is characterized by increased myeloid cell recruitment and MPO release, as seen in the comparative data of
[0202] We previously reported that KYC is a highly selective inhibitor of MPO (59), and as such, can serve as a unique probe for determining the extent to which MPO is involved in disease, including BPD. It is noteworthy that KYC treatment reduces all the biomarkers for oxidation, inflammation, and improves lung structure (
[0203] Hyperoxia increases pulmonary inflammation by a variety of mechanisms. HMGB1 (18, 41), RAGE (44), as well as TLR4 (7, 33) have all been reported to increase pulmonary inflammation in hyperoxic animals. As mentioned above immunoblots show that HMGB1, RAGE and TLR4 levels are all increased in the lungs of hyperoxic neonatal rat pups (
[0204] Potential Use of KYC in BPD: One of the potential benefits in thinking that the cycle of destruction is a causal mediator in BPD onset and progression is it provides focus for treatment strategies. Since KYC effectively inhibits MPO-dependent HOCl production and HMGB1 expression and promotes H.sub.2O.sub.2, consumption it may be useful for treating BPD in neonates. Support for this idea comes from data showing that not only does MPO and HOCl play causal roles in the oxidative mechanisms mediating BPD but that inhibiting MPO reduces lung pathology, improves lung architecture, and increases alveologenesis, and vasculogenesis in hyperoxic neonatal rat pups. Inhibiting MPO toxic oxidant generation should be more effective for reducing oxidative damage to the lung because inhibiting oxidant production at its enzymatic source is more effective than trying to scavenge oxidants after they have been formed. The importance of this fundamental concept is underscored by the fact that after HOCl is generated, it is fully capable of oxidizing biomolecules such as membrane phospholipids and lipids to secondary chlorinated lipids that possess extremely cytotoxic properties (23). Our findings illustrate the archetypal principle that inhibiting oxidant generation at its biological source is the most efficient method for preventing oxidative damage.
[0205] Another potential advantage in inhibiting oxidant generation compared to intervening downstream (e.g. HMGB1) is highlighted by the following consideration. Yu and colleagues (55) reported on the effectiveness of a polyclonal antibody against HMGB1 in BPD. They reported that survival rates of mouse pups exposed to 85% O.sub.2 increased from ˜75% to ˜91% (P10) after treatment with anti-HMGB1 antibody. In contrast, KYC increased survival of hyperoxic (>90%) neonatal rat pups from ˜83% to ˜98% at P10. While anti-HMGB1 antibodies are effective at reducing myeloid cell recruitment, existing myeloid cells in the hyperoxic lung are still be able to release MPO, produce HOCl, generate H.sub.2O.sub.2, and therefore cause lung injury. In contrast, KYC not only inhibits MPO HOCl production but is a substrate that causes MPO to consume H.sub.2O.sub.2 without generating HOCl. Reduced HOCl production, results in less oxidative damage and as shown here lower levels of HMGB1. While both agents are likely reducing oxidative stress and myeloid cell recruitment, physiologically it appears KYC improves chances for survival more than anti-HMGB1 antibodies.
[0206] Previously we reported that when MPO oxidizes KYC, the resultant oxidized product is a KYC radical that auto-scavenges by forming a KYC homo-disulfide (59). However, additional studies suggest that the formation of a disulfide may not be the end of KYC's role as an inhibitor of MPO. As a simple homo-disulfide, KYC disulfide is capable of undergoing thiol exchange reactions and/or direct reductions to a KYC monomer by physiological concentrations of GSH (59). To better understand how KYC modifies thiol cell biology, we determined if glutathione reductase (GR) reduces KYC homo- and hetero-disulfides. Our studies showed that while GR cannot reduce KYC homodimers in the presence of NADPH alone (data not shown), it can reduce KYC homo- and hetero-disulfides in the presence of GSH and NADPH (
[0207] Although the association of neutrophils with neonatal BPD was reported over 35 years ago in 1983 (34), the role that MPO played in BPD has largely been ignored. Consequently, major gaps in our knowledge exist concerning MPO's role in BPD. Support for this point comes the BPD literature itself, which although excellent reviews for BPD exist, their discussion of MPO's role in BPD is sparse to nonexistent (1, 26, 39, 49). When it comes to standard journal reports, most studies use MPO as a biomarker of neutrophil and myeloid cell recruitment, not as a major mechanism of acute or chronic lung injury in BPD. As a result, investigators have focused on inhibiting myeloid cell recruitment and infiltration rather than targeting MPO. Relegating MPO to biomarker status also influences how one develops therapeutic strategies to inhibit BPD. For example, melatonin and vitamin E are broad-spectrum antioxidants that are capable of scavenging oxidants and free radicals although both have been shown to inhibit BPD in animal models and improve outcomes in clinical studies with mixed results (20, 38, 40, 48). Although melatonin has been used to inhibit MPO, since it is classified as a general antioxidant, its ability to reduce BPD cannot be attributed solely to inhibiting MPO.
[0208] Conclusion: Chronic hyperoxia increases BPD in neonatal rat pups by a 5-component destructive cycle: 1) excess O.sub.2, 2) adherent and recruited myeloid cells, 3) MPO, 4) toxic oxidants (i.e., HOCl), and 5) HMGB1 release. As MPO peroxidase activity plays a central role in this cycle, inhibiting MPO should be an effective strategy for reducing the oxidative damage associated with BPD as well as improving lung development in at-risk premature neonates receiving supplemental oxygen.
[0209] In 2014, Berkelhamer and Farrow suggested that progress in treating BPD would require the development of novel antioxidant agents that target multiple systems of oxidants (8). Such a statement implies that a systems biology approach should be used to treat BPD, which to our knowledge does not exist. From the studies here, we learned that KYC effectively reduced inflammation and restored development in hyperoxic neonatal lungs. The breadth and depth of KYC's antioxidant and anti-inflammatory effects on hyperoxic neonatal lungs underscores the importance of HOCl-dependent oxidative damage to BPD pathology. Accordingly, inhibiting HOCl production may restore homeostatic balance to multiple cells and systems in BPD simply by preventing the formation of secondary chlorinated phospholipids and lipids that others have shown are toxic and have severe pathological effects (23, 36, 37).
Example 2
Demonstrating Systems Chemico-pharmacology Properties of Exemplary AA.SUB.(n) .Peptides
[0210] Background and reference data for our new discoveries involving AA.sub.(n) peptides are presented herein. Note that N-acetylated, C-amidated AA.sub.(n) inhibition of MPO is amino acid sequence dependent and the MOA can change as a function of pH.
[0211] i). KYC Peptide
[0212] The peptide KYC inhibits MPO employing three of the four attributes of individual amino acids described above. It contains i) an N-terminus amino acid #1 (AA.sub.1), with an amine functional group (e.g. Lysine (K)). ii) Amino acid #2 (AA.sub.2), contains an, aromatic ring side chain (Tyr) that can stabilize a radical species. iii) Amino acid #3 (AA.sub.3,) contains a heteroatom (the sulfhydryl sulfur of Cys) that can further stabilize a free radical. The free radical itself may act as an activated species, and is stable enough to react with proximal proteins, peptides, metabolites or small molecules to effect further change. The free radical is not limited to a single target, but may effect change in multiple independent targets. In a non-limiting example, the free radical goes on to form a hetero- or homo dimer via an oxidized —S—S— disulfide linkage. The changing of pH has limited effect on the IC.sub.50 values, as shown in the MPO v KYC (
[0213] ii) KWC Peptide
[0214] A model peptide such as KWC, does indeed inhibit MPO toxic oxidant production, as shown in
[0215] iii) KFC
[0216] The significant higher IC.sub.50 values for KFC reinforce the importance of a direct interaction with the iron/heme of MPO (where complex I, II and III all reside). The structure difference between KYC and KFC is simply the hydroxyl group of the aromatic ring (KYC) is removed in KFC, otherwise the peptide structures are identical. This removal results in a ˜four-fold drop of inhibition by KFC versus KYC, as seen in
[0217] iv) Different MOA-KLC Peptide
[0218] KLC peptide data (see
[0219] v) Different MOA-KVC and KVVC Peptides
[0220] In this case the addition of an amino acid, KVC versus KVVC results in an improvement of inhibitory properties (i.e. IC.sub.50 numerical values decrease—see
[0221] Taken together, the data disclosed herein suggests that the structural features of the AA.sub.(n) peptides (where n can be 2-5) are important for determining the effectiveness of a library of model peptides that can inhibit MPO toxic oxidant production. Data in this disclosure describe the structural and biochemical features of peptide inhibitors of MPO toxic oxidant production. Findings presented in the current disclosure show that peptides must possess a number of distinct physicochemical properties to inhibit MPO toxic oxidant production. In addition depending on the sequence and amino acid composition inhibition may occur through different MOA pathways. Also these rules are applicable in the design of a library of organic molecules. [0222] Data shown here for the first time demonstrate that the AA.sub.(n) peptide model of MPO inhibitors identifies new mechanisms not previously described. Accordingly, the AA.sub.(n) peptide model KXZ expands the number of amino acids that can be used to make peptide inhibitors of MPO toxic oxidant production beyond any previous teachings.
Example 3
Demonstrating Systems Chemico-pharmacology Properties for Exemplary KXZ Tripeptide in Inhibiting Pro-inflammatory Peptides and Proteins
[0223] KYZ is reduced (gains an electron in the form of a radical) in the presence of MPO, which subsequently decreases the production of hypochlorous acid. All MPO activities (i.e., hypochlorous acid production) were quantified by the 3,3′,5,5′-tetramethylbenzidine (TMB) assay. The tripeptide KXZ, requires amino acid #1 (AA.sub.1, K) to be a native amino acid or an artificial amino acid (aa) with a basic side-chain such as an amine functional group (e.g., Lysine (K)). Amino acid #2 (AA.sub.2, X) is a native or artificial amino acid that may contain a polar, non-polar, or aromatic amino acid. Amino acid #3 (AA.sub.3, Z) is a native or artificial amino acid that possesses a heteroatom that can stabilize a free radical that then goes on to form a heteroatom-carbon bond in the form of a homo- or heterodimer of the tripeptide.
[0224] Although no one mode of operation is adopted herein, it is postulated that when KXZ enters the active site of MPO (and presumably any other Peroxidase), it is “activated” through a series of redox chemistry processes and KXZ* is created (where Z* is a radical located on the heteroatom (HA) [e.g. S, or Se or other HAs] of the Z amino acid side-chain). The radical of KXZ* must be located on a HA that can form a new “lower energy” chemical bond such as —S—R. In addition, the KXZ* may go on to form either a neutral homodimer (KXZ).sub.2, radical homodimer (KXZ).sub.2 *, or a radical heterodimer (KXZ-GSH)*. This process can play a role in facilitating the regeneration of neutral KXZ via the GSH pathway.
[0225] However, a second process occurs, and is postulated to occur as follows although no one mode of operation is adopted herein: (1) KXZ* is formed in the active site of MPO; (2) KXZ* exits the active site and interacts with GSH to form (KXZ-GSH)* (where R is a molecule of neutral GSH), which is relatively more stable than KXZ* alone; and (3) (KXZ-GSH)* then can react with a PROXIMAL peptide/protein containing an accessible, free —SH group (assuming, but not restricted to, from Cys or homocysteine), which results in the thiolation of the peptide/protein to form P-KXZ (where P represents the peptide or protein).
[0226] In support of this activity,
[0227] The incubation of Endothelial cells in the presence of MPO and H.sub.2O.sub.2 with KYC results in the production of KYC* (* represents radical). The resultant activated peptide KYC* can then go on further to modify specific proximal proteins such as HMGB1. The HMGB1-thiolated protein with KYC no longer functions in a pro-inflammatory manner. As a consequence downstream proteins, such as Nrf2 are also significantly ameliorated in their pro-inflammatory actions (see
Example 4
N-Acetyl-Lysyltyrosylcysteine Amide, a Novel Systems Pharmacology Agent, Reduces Bronchopulmonary Dysplasia in Hyperoxic Neonatal Rat Pups
[0228] Abstract
[0229] Bronchopulmonary dysplasia (BPD) is caused primarily by oxidative stress and inflammation. To induce BPD, neonatal rat pups were raised in hyperoxic (>90% O2) environments from day one (P1) until day ten (P10) and treated with N-acetyl-lysyltyrosylcysteine amide (KYC). In vivo studies showed that KYC improved lung complexity, reduced myeloperoxidase (MPO) positive (+) myeloid cell counts, MPO protein, chlorotyrosine formation, increased endothelial cell CD31 expression, decreased 8-OH-dG and COX1/COX2, HMGB1, RAGE, TLR4, had little effect on weight gain but improved survival in hyperoxic pups. EPR studies showed that MPO reaction mixtures oxidize KYC to a KYC thiyl radical. Adding recombinant HMGB1 to an MPO reaction mixture containing KYC resulted in KYC thiylation of HMGB1. In rat lung microvascular endothelial cell (RLMVEC) cultures, KYC thiylation of RLMVEC proteins was increased the most in RLMVEC cultures treated with MPO+H2O2, followed by H2O2, and then KYC alone. KYC treatment of hyperoxic pups decreased total HMGB1 in lung lysates, increased KYC thiylation of HMGB1, terminal HMGB1 thiol oxidation, decreased HMGB1 association with TLR4 and RAGE, and shifted HMGB1 in lung lysates from a non-acetylated to a lysyl-acetylated isoform, suggesting that KYC reduced lung cell death and that recruited immune cells had become the primary cellular sources of HMGB1 released in the lung. MPO-dependent and independent KYC-thiylation of Keap1 were both increased in RLMVEC cultures. Treating hyperoxic pups with KYC increased KYC thiylation and S-glutathionylation of Keap1, Nrf2 activation. These data, taken together, suggest that KYC is a novel system pharmacological agent that exploits MPO to inhibit toxic oxidant production and to be oxidized into a thiyl radical that inactivates HMGB1, activates Nrf2, and increases antioxidant enzyme expression to improve lung complexity and reduce BPD in the lungs of hyperoxic rat pups.
[0230] Introduction
[0231] Bronchopulmonary dysplasia (BPD) affects more than 10,000 infants annually [1], making it the most common pulmonary morbidity of premature infants in the United States [2]. BPD is caused by complications from respiratory distress syndrome (RDS) of the newborn. Currently, treatments for RDS include surfactant, caffeine, supplemental oxygen, and gentle mechanical ventilation [2]. Although supplemental oxygen is essential for preventing hypoxemic organ injury in premature neonates, supplemental oxygen always increases oxidative stress and inflammation. One way hyperoxia increases inflammation is by activating resident myeloid cells and vascular endothelial cell NADPH oxidase (NOX) dependent superoxide anion production [3-5]. Activated resident myeloid cells release MPO and also generate superoxide anion, which dismutates into hydrogen peroxide (H2O2) to provide the substrate for MPO to oxidize chloride or nitrite anions into hypochlorous acid (HOCl) [6] or nitrogen dioxide radical (NO2*) [7], respectively.
[0232] A consequence of chronic increases in oxidative stress and inflammation in the lung is pulmonary cell injury and cell death. Dead and dying cells release high mobility group box1 (HMGB1). Under normal conditions, HMGB1 serves as a nuclear DNA binding protein that performs many vital roles in maintaining DNA structure and regulating gene expression [8]. However, after cells die or immune cells and platelets become activated, HMGB1 is released into extracellular spaces, where it turns into a danger-associated molecular pattern (DAMP) molecule that possesses potent cytokine- and chemokine-like properties [9, 10]. HMGB1's ability to recruit myeloid cells to the lung begins to explain how hyperoxia and excessive mechanical ventilation increase myeloid cell recruitment, and MPO-dependent oxidative damage to the premature neonate's lung to induce a form of lung injury [11] that is far greater than that which is initially induced by hyperoxia. Support for HMGB1 playing a causal role in inflammation in BPD comes from clinical studies showing that the concentration of HMGB1 in tracheal aspirates directly correlates with BPD severity and death in ventilated premature neonates [12].
[0233] One of the reasons premature neonates are at increased risk of BPD is that they often lack the antioxidant enzymes that protect against the oxidative stress induced by supplemental oxygen [13, 14]. Exactly why premature neonates lack antioxidant enzymes is unclear. A potential explanation however is that hypoxic in utero environments do not require a robust antioxidant system and some premature neonates do not respond to hyperoxia appropriately, possibly because of genetic predispositions [14]. However, reports by Cho and Kleeberger [15-17] have defined the relationship between Keap1 and Nrf2, linked hyperoxic lung injury to Nrf2 [18], and confirmed that Nrf2 is the primary regulatory pathway for modulating antioxidant enzyme expression in the lung [19]. Although developing antioxidant agents that scavenge multiple oxidants is attractive [14], effective targeting of oxidative stress to reduce lung injury in neonates has not been achieved and optimizing scavengers to target specific oxidants and free radicals is extremely difficult. As hyperoxia has been linked to Nrf2 [18], and premature neonates who are at risk of hyperoxic lung injury lack the antioxidant enzymes required for protection [13, 14], it might be possible to exploit the Keap1/Nrf2 signaling pathway to protect premature neonates against supplemental oxygen.
[0234] The above reports, taken together, suggest that BPD is a complex, multifactorial disease process that is induced primarily by oxidative stress and inflammation. Such a statement implies that a systems biology approach would be useful for treating BPD. However, to our knowledge, no such agent exists. If a therapeutic agent could be developed to inhibit MPO, inactivate HMGB1, or active Nrf2, then such an agent might be useful for decreasing oxidative stress and inflammation in the lungs of neonates that lung development will proceed even in high-risk premature neonates treated with supplemental oxygen.
[0235] In the present study, we report on the effects of KYC on BPD in hyperoxic neonatal rat pups. KYC is a tripeptide that was initially designed to inhibit MPO toxic oxidant (HOCl) production and increase MPO-dependent catalase activity [20]. Our studies suggest that KYC exploits MPO peroxidase activity to be oxidized into a KYC thiyl radical that inactivates HMGB1 and activates Nrf2. When hyperoxic neonatal rat pups are treated with KYC, oxidative stress and inflammation decrease, and antioxidant enzymes increase. Studies here show that KYC prevents oxidative lung injury and improves lung development in neonatal rat pups raised in hyperoxic environments.
[0236] Material and Methods
[0237] Peptide Synthesis: KYC and biotin-aminohexanoyl-N-[Ahx]-KYC-amide or biotinylated-KYC (B-KYC) were synthesized using Fmoc [N-(9-fluorenyl)methoxy-carbonyl] chemistry, prepared and purified as an acetate salt by Biomatik USA, LLC (Wilmington, Del.) as previously described [21-23]. Trifluoroacetic acid (TFA) in the tripeptide preparations was removed by dissolving KYC in distilled water containing 6 mM HCl followed by lyophilization (2-3×). TFA in KYC and B-KYC was quantified by NMR in the Biomolecular NMR Facility at MCW [24]. TFA was reduced to <0.01% before being used for experiments.
[0238] Rats and Experimental Protocols: Time-dated pregnant Sprague-Dawley rats were obtained from Envigo (Madison, Wis.) and acclimated in the animal facility for one week. Animal protocols were approved by MCW's Institutional Animal Care and Use Committee and conformed to NIH Guide for the Care and Use of Laboratory Animals. Animals were housed in barrier cages with a 12-h dark-light cycle and were given free access to chow and water. Pups from two dams were mixed and randomly distributed to each nursing dam by balancing sex and size of the pups. The sex of the pups was determined from the relative distance between the genitalia and anus. The dam and pups were placed in cages in either room air (normoxia) or in >90% oxygen (hyperoxia) in an enclosed chamber from postnatal day 1 to day 10 (P1-P10) to induce BPD as previously reported [25, 26]. Oxygen concentrations were continuously monitored with an oxygen sensor (Reming Bioinstruments Co., Redfield, N.Y.).
[0239] Since the number of neonatal rat pups per pregnant dam limits the number of experimental conditions that can be compared, we modified our standard experimental protocol. The first study was designed to determine if hyperoxia increased BPD and caused a change in proteins of interest. Data from these studies can be found in on-line supplemental data. The second study was designed to determine if inhibiting MPO toxic oxidant production reduced BPD. At least three sets of neonatal pups from three different dams were used for each study. Pups were fed ad libitum from nursing dams. Pups were inspected daily starting on P1 and weighed starting on P3 in room air for less than ten minutes. Nursing dams were switched daily to avoid the impact of oxygen toxicity. Experience has taught us that severity of hyperoxia-induced BPD is litter dependent. To minimize litter differences in both types of experiments pups from different litters were mixed and randomly reallocated to the nursing dams.
[0240] Previously we reported that KYC was highly effective at reducing MPO-dependent oxidative injury to cultured endothelial cells and that KYC had little to no effect on endothelial cell viability, apoptosis, necrosis, or mitochondrial function when added to culture media up to 4000 μM [27]. In other studies, we observed that injection of increasing single doses (IP) of KYC into control mice did not induce observable adverse effects until the dose reached 800 mg/kg (unpublished, personal observations). KYC has been used in a variety of animal models and has been shown to reduce chemically-induced tumor formation [28], vasculopathy in sickle cell mice [21], secondary brain injury in middle cerebral artery occlusion mice [22, 29], neurological disease scores in EAE [23, 30], and improve vasculogenesis and reduce neutrophil infiltration in hindlimb ischemia in diabetic mice [31]. With such an abundance of murine models of vascular disease and injury showing that KYC effectively reduced myeloid cell recruitment and oxidative damage, with little to no evidence of toxicity, we focused our efforts on determining if KYC reduced BPD in hyperoxic neonatal rat pups.
[0241] No KYC dose-response studies were performed in neonatal rat pups in preparation for the current study. Instead, we decided to treat neonatal rat pups empirically with KYC at 5 mg/kg twice per day based on published and unpublished data in adult mice. A review of doses in previous studies in adult mice suggests that the therapeutic window for KYC is from 0.3 mg/kg (once per day) [23] and up to 15 mg/kg (twice per day) [30]. The higher dose is slightly more than 26 times less than the concentration of KYC that was observed to induce adverse effects. The dose of KYC used in the current study is based on our experience in treating chronic inflammation in other disease states [21, 23, 30] and is within the range that we consider appropriate for neonatal rat pups and adult rats.
[0242] To determine the effects of KYC on BPD in hyperoxic neonatal rat pups we randomly assigned pups to the PBS injection control group, or the KYC treatment group. KYC was injected (intraperitoneally, IP), starting at P2 to half of the randomly assigned pups in each litter using a sterile insulin syringe fitted with a 30G needle (Beckon Dickinson, New York, N.Y.) until P10. To determine the effects of KYC on KYC thiylation of HMGB1 and Keap-1, pups were injected IP twice daily with KYC at P2-P9, and then once with B-KYC (5 mg/kg), or an equal volume of phosphate buffer solution (PBS) . Pups were euthanized at P10 with carbon dioxide and lungs removed en bloc. A small cut was created on the left atrium and ice-cold normal saline was gently infused through the right ventricle to flush blood from the lungs before inflation for histology or snap-frozen in liquid nitrogen for protein studies. For weight-gain and survival studies, comparisons were made between the effects of normoxia and hyperoxia and between the effects of KYC and PBS on hyperoxic pups. Weight was recorded starting on P3 and death was recorded each day from P1 to P10.
[0243] Cells, Materials, and Antibodies:
[0244] Rat lung microvascular endothelial cells (RLMVEC) from neonatal Sprague-Dawley rats (RN-6011) and rat endothelial growth medium (EGM, M1266SF) were from Cell Biologics (Chicago, Ill.). Amicon®Ultra-4 centrifugal concentrators with 3K cut-off (UFC800324) were from Merck Millipore Ltd (Carrigtwohill, Ireland). PierceTM Streptavidin (#88816), Protein G (#88848), Protein A Magnetic Beads (#88845), PierceTM ECL Western Blotting Substrate (#32106), and SuperSignalTM West Femto Maximum Sensitivity Substrate (#34095) were from Thermo Fisher Scientific (Waltham, MA). Rabbit antibodies for myeloperoxidase heavy chain (MPO, sc-33596) and TLR4 (sc-10741) were from Santa Cruz Biotechnology (Dallas, Texas). Rabbit antibody for Cl-Tyr (HP5002) was from Hycult Biotech (Plymouth Meeting, PA). The chicken antibody for HMGB1 (326052233) was from SHINO-TEST Corporation (Kanagawa, Japan). Rabbit antibody for RAGE (GTX23611) was from GeneTex (Irvine, CA). Mouse antibodies for PECAM-1/CD31 (ab24590), for 8-OH-dG (ab48508), and for rat endothelial cell antigen-1 (RECA-1, ab9774) were from Abcam (Cambridge, MA). Rabbit antibody for Cox1 (sc-7950) and Cox2 (sc-7951) were from Santa Cruz Biotechnology. Mouse (ab190377) and rabbit (ab77302b) antibodies for HMGB1, and streptavidin-HRP (ab7403) were from Abcam (Cambridge, Mass.). Rabbit antibody for acetylated-HMGB1 (OASG03545) was from Aviva System Biology (San Diego, Calif.). Rabbit antibodies for Nrf2 (sc-722) and TLR4 (sc-30002) were from Santa Cruz Biotechnology (Dallas, Tex.). Rabbit antibody against Keap1 (ABS97) and mouse antibody for β-actin (A2228) were from Millipore-Sigma (St. Louis, Mo.). Rabbit antibody for cysteine sulfonyl (ADI-OSA-820) was from Enzo Life Sciences (Farmingdale, N.Y.). Mouse antibody for glutathionylated proteins (D8) and all other chemicals were from Sigma-Aldrich (St. Louis, Mo.). All antibodies were certified either by the manufacturer or were used previously and certified by the authors, as was the case for the anti-HMGB1 antibody [32].
[0245] Immunohistochemistry and Immunocytochemistry:
[0246] After euthanasia, the trachea was cannulated with an Instech Solomon (20G) stainless steel feeding tube (Plymouth Meeting, Pa.) and the lungs were inflated with 10% neutral buffered formalin at 25 cm-H2O (2.4 kPa) for one hour. After the trachea was securely tied to the stainless feeding tube to maintain pressure with surgical silk, the lungs were perfusion fixed with an additional aliquot of 10% neutral buffered formalin, surgically removed and then stored in 10% buffered formalin for 24 h before paraffin embedding. Lung sections (5 μm) were mounted on SuperFrost plus-coated slides (Denville Scientific, Metuchen, N.J.). Slides were deparaffinized, and sections stained with hematoxylin and eosin (H&E). Histology images were captured with a mounted digital camera using an Olympus IX 51 microscope and a 10× objective. Inflammatory cells, myeloid cells, including neutrophils, monocytes, and macrophages, were stained with MPO antibody (1:200) overnight at 4° C. and HPR-conjugated anti-rabbit antibody (1:1000) at room temperature for one hour then visualized by diaminobenzidine to generate a dark-brown color. Blood vessels were stained with RECA-1 and visualized with horseradish-conjugated secondary antibody and diaminobenzidine. Immunofluorescence of 8-OH-dG was used as a biomarker for oxidative DNA damage. Lung sections were stained with the 8-OH-dG antibody (1:100) for overnight at 4° C. then treated with AlexaFluor488-conjugated secondary antibody for one hour in room temperature and counterstained with DAPI before imaging with a fluorescent microscope. The average of three sections per pup, and five counts per section (15 counts/pup) was used for statistical analysis. Quantified data were obtained and entered into the record using predetermined codes by one of the coauthors who was not involved in taking the images.
[0247] Morphometric Analysis:
[0248] The mean linear intercept (MLI), or chord length, was used as a method to estimate the volume-to-surface ratio of acinar airspaces whereas radial alveolar count (RAC) and secondary septa were investigated to study the complexity of lung structure [33]. Ten equally spaced horizontal lines were drawn on each picture, and the number of intercepts through the alveolar wall was counted. MLI was obtained by multiplying the number of times the traverses are placed on the lung times the length of the traverse and dividing the result by the sum of all the intercepts. For RAC, a line from the center of the respiratory tract perpendicular to the nearest connective tissue septum was drawn, and alveoli intercepting with the line were counted. For measurement of secondary septa, elastin was stained with resorcin fuchsin and Van Gieson's solution.
[0249] Immunoblot Analysis: Whole lung lysates were obtained by homogenizing in MOPS buffer (20 mM 3-N-morpholino-propane sulfonic acid, 2 mM EGTA, 5 mM EDTA, 30 mM NaF, 10 mM β-glycerophosphate, 10 mM Na pyrophosphate, 2 mM Na orthovanadate, 1 mM PMSF, 0.5% NP-40, 1% protease inhibitor cocktail, and 1% phosphatase inhibitor cocktails 2 and 3, pH 7.0) by Bullet Blender (Next Advance, Inc., Averill Park, N.Y.). For protein immunoblots, 30 μg of protein lysate was separated by SDS-PAGE, transferred to nitrocellulose membranes (0.22 μm), and then probed with the appropriate primary antibodies overnight at 4° C. on a continuously rocking platform. Signals were generated after incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (1:8,000) using Pierce™ ECL Western Blotting Substrate or SuperSignal™ West Femto Maximum Sensitivity Substrate. Integrated optical density (IOD) was calculated using ImageJ software and normalized to β-actin, a loading control.
[0250] Statistical Analysis:
[0251] Representative images of lung sections and immunoblots are presented in the figures. Data from lung sections and immunoblots from the samples were analyzed for statistical differences using GraphPad Prism version 8.4.3, for Windows (GraphPad Software, La Jolla, Calif.). Quantitative morphometric and cell count data are expressed as mean±SD scatter-plots adjacent to the representative images. Student t-test, or Mann-Whitney U test, was used for comparing two groups wherever appropriate. One-way analysis of variance with post-hoc Student-Newman-Keuls test was used when more than two groups of data were analyzed. Significant differences between groups were determined by either unpaired student's t-test or Mann-Whitney U test, depending on the distribution. Weight gain and survival data were analyzed using MedCalc Statistical Software version 15.2.1 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc.org). Kaplan-Meier survival curves were plotted using Kaplan-Meier tables constructed in GraphPad Prism. A p-value<0.05 was considered statistically significant.
[0252] Results
[0253] Effects of Hyperoxia on BPD in Neonatal Rat Pups: Chronic hyperoxia increased BPD in neonatal rat pups on P10 based on differences in lung morphometrics and various mechanism-based biomarkers of oxidative stress and inflammation (see supplemental
[0254] Effects of KYC on Hyperoxia-induced Myeloid Cell Recruitment, MPO, and Oxidative Stress: KYC reduced the number of MPO+ myeloid cells in hyperoxic lungs by approximately 50% (brown cells in
[0255] Effects of KYC on Hyperoxia-induced BPD: If MPO plays a causal role in the mechanisms by which hyperoxia induces BPD, then KYC, which inhibits MPO toxic oxidant production but not peroxidase activity [27], should reduce hyperoxia-induced changes in lung architecture. KYC treatment of hyperoxic pups increased RAC, secondary septa, and blood vessel counts by approximately 35%, 55%, and 92%, respectively, while decreasing MLI by approximately 22% (
[0256] Effects of KYC on 8-OH-dG in Hyperoxic Lungs: Hyperoxia increased 8-OH-dG, a biomarker of DNA oxidative damage in the lungs of neonatal rat pups (see supplemental
[0257] Effects of KYC on Cox1/Cox2 in Hyperoxic Lungs: Hyperoxia increased Cox1 slightly and Cox2 markedly in the lungs of neonatal rat pups (see supplemental
[0258] Effects of KYC on HMGB1 in Hyperoxic Lungs: Hyperoxia increased HMGB1 levels in lung lysates of neonatal rat pups (see supplemental
[0259] Effects of KYC on RAGE and TLR4 in Hyperoxic Lungs: Hyperoxia increased RAGE and TLR4 in lungs of neonatal rat pups (see supplemental
[0260] Effects of Oxidative Stress on KYC Thiylation on Endothelial Cell Proteins in RLMVEC Cultures: To determine the effects of oxidative stress on KYC thiylation, RLMVEC cultures were treated with B-KYC to follow KYC thiylation with streptavidin-affinity blotting as outlined in Methods. Three different levels of oxidative stress were used to determine the effects of oxidative stress on KYC thiylation of endothelial cell proteins: media alone (baseline); media containing MPO+H.sub.2O.sub.2 (MPO-dependent); and media containing H.sub.2O.sub.2 alone (H.sub.2O.sub.2-dependent) (
[0261] Immunoblot band densities for Nrf2 (29B), Keap1 (29C), and HMGB1 (29D) relative to β-actin (29E) reveal that protein expression for the three proteins is differentially modulated by oxidative stress. Nrf2 expression is increased in RLMVEC cultures subjected to MPO-dependent KYC thiylation more than in RLMVEC cultures subjected to KYC thiylation under baseline or H.sub.2O.sub.2-dependent oxidation (
[0262] MPO Oxidizes KYC to a Thiyl Radical that Thiylates HMGB1:
[0263] MPO Oxidation of KYC Results in KYC Thiylation of HMGB1 Released from RLMVEC Cultures: To determine if MPO oxidizes KYC to a thiyl radical that thiylates HMGB1 released from RLMVEC cultures, proteins in the media and the cell lysates from RLMVEC cultures at baseline with no KYC, with KYC and with MPO+H.sub.2O.sub.2+KYC were affinity blotted for KYC-thiylated proteins and immunoblotted for HMGB1. HMGB1 immunoblots showed that all RLMVEC cultures released low amounts of HMGB1 (
[0264] Effects of KYC Treatment on HMGB1 Association with TLR4 and RAGE in Hyperoxic Lungs: Although KYC treatment reduced total HMGB1 in lung lysates of hyperoxic neonatal rat pups (
[0265] Effects of KYC Thiylation on HMGB1, Terminal HMGB1 Thiol Oxidation, and Shifts in Cell Sources for HMGB1 in Hyperoxic Lungs: Immunoblots of normoxic and hypoxic lung lysates revealed that lungs from normoxic pups had higher levels of sulfonyl HMGB1 than lungs from hyperoxic pups (
[0266] Effects of KYC on Keap1 KYC Thiylation and S-Glutathionylation and Modulation of Nrf2 in Normoxic and Hyperoxic Lungs: Band density analysis of streptavidin affinity and immunoblots revealed that KYC treatment of hyperoxic pups increased B-KYC thiylation of Keap1 and S-glutathionylation of Keap1 in lung lysates from hyperoxic pups (
[0267] Effects of KYC Treatment on HO-1, GST, and Trx Expression in Hyperoxic Lungs. The immunoblots show that treating hyperoxic pups with KYC increased the expression of HO-1, GST, and Trx, which are all members of the antioxidant enzyme system known to be upregulated by Nrf2 (
[0268] Effects of KYC on Weight Gain and Survival of Hyperoxic Pups: KYC-treated normoxic pups gained more weight than untreated normoxic pups based on curve analysis (
[0269] Discussion
[0270] Our findings suggest that the MPO, HMGB1, and Nrf2 play essential roles in BPD. MPO, released from resident myeloid cells, plays an initiating role by amplifying the oxidative stress induced by hyperoxia. HMGB1, released from dead and dying lung cells, plays a propagating role by recruiting the myeloid cells that enter the interstitium of the lung and release MPO and increasing inflammation. The arrival and activation of the recruited myeloid cells induces a second wave of oxidative injury and cell death that is always greater than that which is induced initially by hyperoxia. Reduced Nrf2 activation plays a permissive role in BPD by preventing premature neonates who lack antioxidant enzymes from defending themselves against the oxidative stress mediated by chronic hyperoxia. In this way, blunted Nrf2 responses to the oxidative stress induced by hyperoxia allows hyperoxia to induce greater pulmonary cell injury and death [14], which result in proportionately greater release of HMGB1 [12]. Viewed in this fashion, failure to target all three mediators adequately permits the cycle to continue, resulting in varying degrees of BPD severity. Our findings suggest that BPD is caused by a destructive cycle that is mediated by oxidative stress and inflammation, where activation of one mediator results in the activation of a second mediator to worsen BPD. As myeloid cells are capable of releasing various mediators of oxidative stress and inflammation, and HMGB1 also increases vascular inflammation, it is likely that additional mediators of inflammation and oxidative stress be added to this list over time.
[0271] The mechanisms by which hyperoxia impairs lung development and increases BPD are complex. To begin to understand the cellular mechanisms mediating BPD, we first determined the effects of hyperoxia on the lungs of neonatal rat pups. After establishing baseline differences, we determined KYC's effects on preventing oxidative lung damage in the hyperoxic pups. Our studies showed that chronic hyperoxia decreased lung complexity and CD31 expression, a cellular biomarker for endothelial cell content. These data, taken together, suggest hyperoxia impaired angiogenesis. Hyperoxia increased lung MPO+ myeloid cell counts, MPO protein, and Cl-Tyr formation (a biomarker for HOCl-dependent oxidative damage). These data suggest that hyperoxia increased oxidative stress. In this context, it is significant that the increase in Cl-Tyr was paralleled by a second biomarker, 8-OH-dG, a biomarker of DNA oxidative damage, and that both biomarkers increased as lung development decreased and cell death increased [37]. Inflammation was assessed by immunoblotting, which showed that hyperoxia increased HMGB1, Cox1/Cox2, RAGE and TLR4. HMGB1 is a biomarker for lung cell death and injury, myeloid cell recruitment and vascular inflammation. Cox1/Cox2, RAGE and TLR4 are all biomarkers of inflammation. Finally, other immunoblots revealed that hyperoxia decreased Nrf2 activation. The inability of the lungs of neonatal rat pups to activate Nrf2 makes the pups more susceptible to oxidative stress (see supplemental).
[0272] Treating hyperoxic pups with KYC improved lung complexity, reduced oxidative stress and inflammation, and reversed the biomarkers for oxidative stress, DNA damage and inflammation in the hyperoxic pups' lungs. Thus, KYC treatment effectively reduced oxidative stress and inflammation, and restored lung development in the hyperoxic pups and, in so doing, likely increased survival.
[0273] The fact that KYC inhibits multiple mechanisms suggests that KYC is a systems pharmacology agent. In 2013, we reported that KYC not only inhibited MPO-dependent HOCl and nitrogen dioxide (NO.sub.2°) production, but also increased MPO-dependent H.sub.2O.sub.2 consumption [27], which, all things being equal, should reduce oxidative stress. Our idea that KYC increases MPO-dependent H.sub.2O.sub.2 consumption is consistent with the report by Kettle and Winterbourn [20], who showed that tyrosine increases MPO-dependent catalase activity. In 2013, we also reported that MPO oxidized KYC to a KYC thiyl radical that prevented further oxidative damage by autoscavenging to form either a homodimer with a second KYC or a heterodimer with GSH [27]. As all thiols require activation before they can thiylate a thiol group or a protein cysteine, we reasoned that KYC should reduce oxidative stress in the very places that myeloid cells released MPO. Such a model implies that the tissues benefiting the most from KYC would be proximal to MPO. Although our intent in developing this model was to address non-specificity, having such a model demanded that us to take a closer look at KYC thiylation.
[0274] To better understand thiylation biotin-labeled KYC was added to RLMVEC cultures under baseline, H.sub.2O.sub.2-dependent, and MPO-dependent conditions of oxidative stress. At baseline KYC thiylation of RLMVEC proteins was low. However, under H.sub.2O.sub.2-dependent oxidative stress conditions, KYC thiylation of RLMVEC proteins was only slightly increased, the majority of RLMVEC proteins that were KYC thiylated were not heavily thiylated and were scattered throughout the full range of molecular weights. Under MPO-dependent oxidative stress conditions, the level of KYC thiylation of RLMVEC proteins was markedly increased throughout a wide range of molecular weights. These data support our model that KYC thiylation occurs predominantly in cells proximal to MPO. Although oxidative stress may increase KYC thiylation in a few cell proteins, the KYC thiylation that occurs is diffuse and not much more than that which occurs at baseline. Protein expression data show that Keap1 and Nrf2 were increased in RLMVEC cultures treated MPO+H.sub.2O.sub.2+B-KYC, while HMGB1, a likely injury mechanism, was increased in the RLMVEC cultures treated with H.sub.2O.sub.2-dependent oxidative stress+B-KYC but not in RLMVEC cultures treated with MPO+H.sub.2O.sub.2+B-KYC. These data support our idea that B-KYC is an MPO substrate that increases MPO-dependent catalase activity [20] and reduces oxidative injury and damage to RLMVEC cultures.
[0275] Previously we reported that KYC reduced MPO-dependent oxidative damage because KYC competes with native substrates, such as chloride and nitrite, that are oxidized to “toxic” oxidants. In 2013, we argued that KYC reduced MPO-dependent oxidative damage because the resultant KYC thiyl radical would autoscavenge to prevent radical propagation [27]. HMGB1 expression data in
[0276] Although the exact mechanisms by which KYC thiylation of HMGB1 promotes terminal HMGB1 thiol oxidation remain unclear, some insight may be gained into the reactions promoting terminal thiol oxidation from in vitro studies. In vitro incubations show that GST reduces KYC disulfide to KYC monomers only in the presence of excess GSH (see supplemental data,
[0277] In animal studies relative rates of weight gain in neonatal rat pups provide an independent measure of animal development. Slower rates of weight gain suggest that the pups are under-developed or suffering from excessive stress. Survival curves reveal which experimental test groups are under stress or lacking the nutrition required to survive. In this context, it is important to note that KYC treatment of normoxic rat pups significantly increased weight (
[0278] In conclusion, BPD is caused by a destructive cycle mediated by oxidative stress and inflammation. The major mediators are the recruited myeloid cells that release MPO and generate H.sub.2O.sub.2 that activates MPO to generate toxic oxidants, HMGB1 released from dead and dying pulmonary cells, and inadequate Nrf2 activation. KYC is a novel systems pharmacology agent that we previously reported inhibits MPO toxic oxidant production and enhances MPO-dependent catalase activity [27]; and, that we now show here exploits MPO peroxidase activity to be oxidized into a KYC thiyl radical that thiylates and inactivates HMGB1, thiylates Keap-1 and increases Nrf2 activation. As Nrf2 mediates antioxidant enzyme expression, KYC reduces oxidative stress and inflammation and increases the lung's ability to protect itself against oxidative stress induced by hyperoxia to improve lung development even under conditions of chronic hyperoxia.
[0279] Supplemental Data
[0280] These supplemental data describe the effects of hyperoxia on oxidative stress, inflammation and bronchopulmonary dysplasia in the lungs of neonatal rat pups, and the effects of glutathione and glutathione metabolizing enzymes on reduction of KYC homo- and heterodisulfides.
[0281] Material and methods not provided here can be found in the manuscript.
[0282] Rats and Experimental Protocols: Sprague-Dawley rats were obtained from Envigo Bioproducts, Inc. (Madison, Wis.), and pregnancy was achieved naturally in our animal facility. Animal protocols were submitted to and approved by MCW's Institutional Animal Care and Use Committee and conformed to NIH Guide for the Care and Use of Laboratory Animals. The animals were housed in barrier cages with a 12-h dark-light cycle and were given free access to chow and water. The pups from two dams were mixed and randomly distributed to each nursing dam by balancing sex and size of the pups. The sex was determined by the relative distance between the genitalia and anus. The dam and pups were placed in cages in either room air (normoxia) or in >90% oxygen chamber (hyperoxia) from postnatal day 1 to day 10 (P1-P10) to induce BPD as previously reported [25, 26]. Oxygen concentrations were continuously monitored with an oxygen sensor (Reming Bioinstruments Co., Redfield, N.Y.).
[0283] Glutathione Reductase Reduction of KYC Homo- and Hetero-disulfide: Disulfide formation was induced by incubating either KYC alone (2.2 mM) or KYC with equal concentration glutathione (GSH) in PBS (pH 7.4) with shaking overnight at room temperature. The conditions used to assess the effects of glutathione reductase (GR) on KYC-KYC homodisulfide and KYC-GSH heterodisulfides were in the presence and absence of GSH as before [22, 31, 32]. Mixtures of KYC-KYC homodisulfide and KYC monomer and KYC-GSH heterodisulfide and GSH were incubated with 3.8 μM GR (Thermo-Fisher, Waltham, Mass.) in a 100 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA, and 1 mM NADPH for 1 hr at room temperature with shaking. The reaction was halted by snap freezing at −80° C. Negative controls omitted either GR or NADPH. Samples were analyzed by HPLC (Beckman, Brea, Calif.) at the Blood Research Institute of Wisconsin Protein Core Lab. Samples were diluted 1:1 in 0.1% TFA, loaded on a Jupiter Proteo column (C-12 surface, dimensions: 4.6×50 mm, 4 μm particle size, Phenomenex, Torrence, Calif.) and run with a gradient (solvent B into solvent A) of 2-40% over 10 min with a flow rate of 1 ml/min and read using a UV detector at 220 nm. Solvent A was 0.08% TFA in HPLC grade H2O, and solvent B was 0.1% TFA in HPLC grade acetonitrile. Unless otherwise indicated, chemicals were obtained from Sigma-Aldrich (St. Louis, Mo.).
[0284] Results
[0285] Effects of Hyperoxia on BPD: Chronic hyperoxia increased the infiltration of MPO (+) myeloid cells in the lungs from neonatal pups compared with the number in lungs from normoxic pups (brown stained cells,
[0286] Effects of Hyperoxia on Lung Morphometrics: Hyperoxic lungs had decreased RAC, secondary septa, and blood vessel counts by approximately 27%, 52%, and 52%, respectively, and increased MLI values by approximately 40% compared with normoxic lungs (
[0287] Effects of Hyperoxia and KYC on Lung 8-OH-dG: Immunofluorescence staining shows that chronic hyperoxia increased the fluorescent density of 8-OH-dG in the stained cells by 1.8-fold (
[0288] Effects of Hyperoxia and KYC on Lung Cox1/Cox2: Immunoblots for Cox1 and Cox2 show that chronic hyperoxia increased Cox1 slightly and Cox2 markedly in the lungs of neonatal rat pups (
[0289] Effects of Hyperoxia on Lung HMGB1: Immunoblots show that chronic hyperoxia increased HMGB1 levels in lung homogenates by 170% compared with HMGB1 levels in lung homogenates from normoxic pups (
[0290] Effects of Hyperoxia on RAGE and TLR4 Expression in Lungs of Hyperoxic Neonatal Rat Pups: Hyperoxia increased RAGE and TLR4 in the lungs of neonatal rat pups. Hyperoxia increased the expression of RAGE by approximately 130% (
[0291] Effects of Hyperoxia on Nrf2 Activation in Lungs of Neonatal Rat Pups: Hyperoxia Decreased Nrf2 Activation in the Lungs of Neonatal Rat Pups. Hyperoxia decreased Nrf2 activation by approximately 70% (
[0292] Effects of Glutathione Reductase on Reduction of KYC-KYC Homo-disulfide and KYC-GSH Hetero-disulfide to KYC Monomer: One of the mechanisms by which KYC is hypothesized to reduce MPO-dependent oxidant production is by forming KYC homodimers (KYC-KYC) that can be reduced to an active KYC monomeric inhibitor by GSH [22]. GSH homodimer (GS-SG) can be reduced to monomeric GSH by glutathione reductase (GR) [35]. GSH homodimers can also undergo thiol exchange to form mixed heterodimers (GS-SR). The KYC-KYC and KYC-GSH mixtures were incubated with a GR\NADPH reaction mixture as described in methods and KYC monomers determined by HPLC as before [22]. The HPLC tracings in
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