# US 2023/0055441 A1 · Plasmin-resistant peptides for treating stroke and related conditions

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- Number: US 2023/0055441 A1
- Document ID: US20230055441A1
- Status: Published application
- Kind: A1
- Application number: 17/791,711
- Filed: 2021-01-08
- Published: 2023-02-23
- Earliest claimed: 2020-01-09
- Assignees: NoNO Inc.
- Applicants: Nono Inc.
- Inventors: Michael Tymianksi; Jonathan David Garman; Diana Mayor
- Web page: https://patentgrep.com/patent/US20230055441A1

## CPC

- C07K 14/70571: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C07K 7/06: having 5 to 11 amino acids
- C07K 14/47: from mammals
- C12N 9/0075: {Nitric-oxide synthase (1.14.13.39)}
- C12Y 114/13039: Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The invention provides variants of a previously described active agent for treating stroke, Tat-NR2B9c, in which target binding characteristics are retained by inclusion of L-amino acids at the C-terminus and plasmin-resistance is conferred by inclusion of D-amino acids elsewhere. An exemplary agent has the sequence ygrkkrrqrrrklssIETDV (SEQ ID NO:62). The resulting active agents have several advantages including administration at the same time as thrombolytic agents without significant loss of activity due to plasmin digestion. The resulting agents are also more suitable for administration by alternative routes to intravenous infusion, such as subcutaneous, intranasal and intramuscular, and for multi-dosing regimes for treatment of chronic conditions.

## Claims

### Claim 1

An active agent comprising an internalization peptide linked to an inhibitor peptide, which inhibits PSD-95 binding to NOS and/or NMDAR2B, wherein the internalization peptide has an amino acid sequence comprising YGRKKRRQRRR (SEQ ID NO:1) and the inhibitor peptide has a sequence comprising KLSSIESDV (SEQ ID NO:2), or a variant thereof with up to five substitutions or deletions total in the internalization peptide and inhibitor peptide, wherein at least the four C-terminal amino acids of the inhibitor peptide are L-amino acids, and a contiguous segment of amino acids including all of the R and K residues are D-amino acids.

### Claim 2 (depends on 1)

The active agent of claim 1, wherein the residue immediately C-terminal to the most C-terminal R or K residue is also a D-residue.

### Claim 3 (depends on 1, 2)

The active agent of claim 1 or 2, wherein the C-terminal of the internalization peptide is linked to the N-terminus of the inhibitor peptide as a fusion peptide.

### Claim 4

The active agent of any preceding claim, wherein the inhibitor peptide comprises [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:3) at the C-terminus.

### Claim 5

The active agent of any preceding claim, wherein the inhibitor peptide comprises I-E-[S/T]-D-V (SEQ ID NO:4) at the C-terminus.

### Claim 6 (depends on 1)

The active agent of claim 1, wherein the inhibitor peptide comprises IESDV (SEQ ID NO:5) at the C-terminus.

### Claim 7

The active agent of any preceding claim, wherein each of the five C-terminal amino acids of the inhibitor peptide are L-amino acids.

### Claim 8 (depends on 7)

The active agent of claim 7, wherein every other residue of the active agent is a D-amino acid.

### Claim 9 (depends on 1)

The active agent of claim 1 having the amino acid sequence ygrkkrrqrrrklsslESDV (SEQ ID NO:6), ygrkkrrqrrrksslESDV (SEQ ID NO:7), ygrkkrrqrrrkslESDV (SEQ ID NO:8), or ygrkkrrqrrrklESDV (SEQ ID NO:9).

### Claim 10 (depends on 1)

The active agent of claim 1, having the amino acid sequences ygrkkrrqrrrklsslESDV (SEQ ID NO:6), wherein the lower case letter are D-amino acids and the upper case letters are L-amino acids.

### Claim 11

The active agent of any preceding claim having enhanced stability in plasma compared with Tat-NR2B9c.

### Claim 12

The active agent of any preceding claim having enhanced plasmin resistance compared with Tat-NR2B9c.

### Claim 13

The active agent of any preceding claim having a binding affinity for PSD-95 within 2- fold of Tat-NR2B9c.

### Claim 14

The active agent of any preceding claim having an IC50 for inhibiting PSD-95 binding to NMDAR2B within 2-fold of Tat-NR2B9c.

### Claim 15

The active agent of any preceding claim, which competes with Tat-NR2B9c for binding to PSD-95.

### Claim 16

The active agent of any preceding claim as a chloride salt.

### Claim 17

A formulation of an active agent of any preceding claim, further comprising histidine and trehalose.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

A formulation of an active agent of any of claims 1-16, further comprising a phosphate buffer.

### Claim 19

A coformulation comprising the active agent of any preceding claim and an anti-inflammatory agent.

### Claim 20 (depends on 19)

The co-formulation of claim 19, wherein the anti-inflammatory is a mast cell degranulation inhibitor or antihistamine.

### Claim 21

A co-formulation comprising the active agent of any preceding claim and a thrombolytic agent.

### Claim 22

A method of treating a subject having or at risk of a condition selected from stroke, cerebral ischemia, traumatic injury to the CNS, subarachnoid hemorrhage, pain, anxiety, epilepsy, comprising administering an effective regime of the active agent of any preceding claim to the subject.

### Claim 23

A method of treating ischemic stroke in a subject having or at risk of stroke, comprising administering an effective regime of an active agent to the subject, wherein the subject is co-administered a thrombolytic agent, wherein the active agent comprises an internalization peptide linked to an inhibitor peptide, which inhibits PSD-95 binding to NOS and/or NMDAR2B, wherein at least the four C-terminal amino acids of the inhibitor peptide are L-amino acids, and at least one of the remaining amino acids of the active agent is a D-amino acid, wherein the active agent and thrombolytic agent are administered sufficiently proximate in time that cleavage of the active agent induced by the thrombolytic agent is reduced by the inclusion of the at least one D-amino acid.

### Claim 24 (depends on 23)

The method of claim 23, wherein the internalization peptide is linked at its N-terminus to the C-terminus of the inhibitor peptide as a fusion protein.

### Claim 25 (depends on 23)

The method of claim 23, wherein the inhibitor peptide comprises [E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L](SEQ ID NO:3) as the last four residues.

### Claim 26 (depends on 23)

The method of claim 23, wherein the inhibitor peptide comprises [I]-[E/D/N/Q]-[S/T]-[D/E/Q/N]-[V/L] (SEQ ID NO:10) as the last five residues, each of which is an L amino acid.

### Claim 27 (depends on 23)

The method of claim 23, wherein the internalization peptide is a tat peptide.

### Claim 28 (depends on 27)

The method of claim 27, wherein at least 8 residues of the tat peptide are D-amino acids.

### Claim 29 (depends on 27)

The method of claim 27, wherein each residue of the tat peptide is a D-amino acid.

### Claim 30 (depends on 23)

The method of any preceding claim 23, comprising YGRKKRRQRRR (SEQ ID NO:1) as the internalization peptide linked at its N-terminus to KLSSIESDV (SEQ ID NO:2) or KLSSIETDV (SEQ ID NO:12) as the inhibitor peptide forming a fusion protein.

### Claim 31 (depends on 30)

The method of claim 30, wherein the active agent comprises a contiguous segment of D-residues including each of the K and R residues.

### Claim 32 (depends on 23)

The method of claim 23, wherein the active agent comprises ygrkkrrqrrrklsslESDV (SEQ ID NO:6), wherein lower case letters represent D-amino acids and upper case letters are L-amino acids.

### Claim 33

The method of any preceding claim, wherein the thrombolytic agent is administered within a window of 60, 30 or 15 minutes before the active agent.

### Claim 34

The method of any preceding claim, wherein the active agent and thrombolytic agent are administered at the same time.

### Claim 35

A method of delivering an active agent to a subject in need thereof, comprising administering the active agent as defined in any preceding claim by a nonintravenous route, wherein the active agent is delivered to the plasma at a therapeutic level.

### Claim 36 (depends on 35)

The method of claim 35, wherein the active agent is administered subcutaneously.

### Claim 37 (depends on 35)

The method of claim 35, wherein the active agent is administered intramuscularly.

### Claim 38 (depends on 35)

The method of claim 35, wherein the active agent is administered intranasally or intrapulmonarily.

### Claim 39 (depends on 35)

The method of claim 35, wherein the dose is greater than 3 mg/kg.

### Claim 40 (depends on 35)

The method of claim 35, wherein the dose is greater than 10 mg/kg.

### Claim 41 (depends on 35)

The method of claim 35, wherein the dose is greater than 20 mg/kg.

### Claim 42 (depends on 35)

The method of claim 35, wherein the dose is below 10 mg/kg and the active agent is administered without co-administration of a mast cell degranulating inhibitor or anti-histamine.

### Claim 43 (depends on 35)

The method of claim 35, wherein the dose is above 10 mg/kg and the active agent is administered

### Claim 45 (depends on 35)

The method of claim 35, wherein the subject has or is at risk of a condition selected from stroke, cerebral ischemia, traumatic injury to the CNS, pain, anxiety, epilepsy, subarachnoid hemorrhage, Alzheimer's disease or Parkinson's disease.