Method of treating radiation induced toxicity in human lens epithelial cells

Abstract

A method for reducing radiation damage in a lens epithelial cell from a subject in which the subject has a proliferative disease and is undergoing radiation therapy to treat the disease. The method includes administering to the lens epithelial cell a radio¬ modulating polypeptide in an amount sufficient to reduce or repair DNA double strand breaks. The polypeptide is administered to the cell in an amount sufficient to reduce radiation damage to the lens epithelial cell.

Description

AFFIRMED-00120 METHOD OF TREATING RADIATION INDUCED TOXICITY IN HUMAN LENS EPITHELIAL CELLS RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 63 / 657,832, field June 8, 2024, the entire teachings of which are incorporated herein by reference. GOVERNMENT FUNDING This invention was made with government support under Grant No. 1R41EY033583-01, awarded by the National Institute of Health. The United States government has certain rights to the invention. TECHNICAL FIELD

[0001] The present generally concerns radiation-induced toxicity in human lens epithelial (HLE) cells. BACKGROUND

[0002] Proton (PRT) and photon (XRT) radiotherapy-induced cataract formation (RICF) is a common adverse complication for patients with head and neck cancers including cataracts, which can lead to impaired vision or blindness. Currently, there are no available preventative treatments other than surgery to remove the cataracts.

[0003] Thrombin peptide derivatives from amino acids 508-530 of human pro- thrombin have been described for promoting thrombin receptor mediated cell stimulation and for their use in the treatment of wounds, bone fractures and cardiovascular ischemia. Because of their biological activity, these polypeptides show great potential as pharmaceuticals. Rusalatide acetate is one such example.

[0004] DNA double strand breaks (DSBs) of human lens epithelial cells are a possible cause of RICF. We studied the cytotoxic and molecular effects of PRT versus XRTAFFIRMED-00120 in HLE cells and the impact of a radio-modulating peptide acetate, specifically rusalatide acetate, on these effects. BRIEF SUMMARY

[0005] We have significantly reduced, or essentially eliminated, the problems associated with cytotoxic and molecular effects of PRT and XRT in HLE cells and investigated the impact of a radio-modulating peptide, known as rusalatide acetate, and its effect thereon. Based on our data, rusalatide acetate formulations may be used to prevent proton and photon radiotherapy-induced cataract formation in human subjects. Moreover, this discovery was made using an aqueous preparation of rusalatide acetate, which was added to cultured HLE cells in petri dishes or plates prior to proton or photon (X-ray) radiation.

[0006] Accordingly, there is provided a method for reducing radiation damage in a lens epithelial cell from a subject, the subject having a proliferative disease and undergoing radiation therapy to treat same, the method comprising:

[0007] administering to the lens epithelial cell a radio-modulating polypeptide in an amount sufficient to reduce or repair DNA double-strand breaks, the radio-modulating polypeptide being administered to the cell in an amount sufficient to reduce radiation damage to the human lens epithelial cell.

[0008] In one example, the proliferative disease is head and / or neck cancer,

[0009] In one example, the radiation therapy is proton (PRT) radiotherapy.

[0010] In one example, the radiation therapy is photon (X-ray or XRT) radiotherapy.

[0011] The radio-modulating polypeptide used in the disclosed methods has the amino acid sequence: Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu- Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 1). Alternatively, the polypeptide is H- Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-AFFIRMED-00120 Pro-Phe-Val-NH2 SEQ ID NO: 2). In one example, the polypeptide is the acidic acid salt of the polypeptide represented by SEQ ID NO: 2 and is referred to as “rusalatide acetate”.

[0012] In one example, the subject is a human subject. BRIEF DESCRIPTION OF THE FIGURES

[0013] These and other features of that described herein will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0014] FIG. 1 is a graphical representation of an MTT toxicity assay using primary human lens epithelial cells (HLEpiC or HLE-P), used to select optimum rusalatide acetate concentration. Based on the data, we selected 100 ug / ml as the rusalatide concentration for all subsequent assays;

[0015] FIG. 2 is a graphical representation showing clonogenic survival assay (CSA) and assessment of proton relative biological effectiveness (RBE) by clonogenic survival assays with rusalatide acetate treatments of primary (HLEpiC or HLE-P) using 100 ug / ml;

[0016] FIG. 3 is a graphical representation showing 53BP1 Foci after 4-Gy of XRT or PRT of primary (HLEpiC or HLE-P) with and without rusalatide acetate treatment 100 ug / ml. As assayed by 53BP1 foci, rusalatide acetate mitigated RT-induced persistent DSBs (at 1- hour and 4-hours for XRT and PRT; at 24 hours for PRT) (all P ≤0.03); and

[0017] FIG. 4 is a graphical representation showing primary (HLEpiC or HLE-P) senescence by β-galactosidase activity at 6 days following 4-Gy of XRT or PRT with and without rusalatide acetate treatment 100 ug / ml. Treatments appear to reduce HLEpiCs senescence at 6 days.AFFIRMED-00120 DETAILED DESCRIPTION

[0018] Definitions

[0019] Unless otherwise specified, the following definitions apply:

[0020] The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

[0021] As used herein, the term "comprising" is intended to mean that the list of elements following the word "comprising" are required or mandatory but that other elements are optional and may or may not be present.

[0022] As used herein, the term "consisting of” is intended to mean including and limited to whatever follows the phrase "consisting of”. Thus, the phrase "consisting of” indicates that the listed elements are required or mandatory and that no other elements may be present.

[0023] Abbreviations for amino acids used herein are as follows: Alanine (Ala), Arginine (Arg), Aspartic acid (Asp), Cysteine (Cys), Glutamic acid (Glu), Glycine (Gly), Lysine (Lys), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Tyrosine (Tyr), and Valine (Val).

[0024] As used herein, the term "residue" when referring to α-amino acids is intended to mean a radical derived from the corresponding α-amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the α-amino group. For example, the terms Glu, Ala, Gly, Arg, Asp, Phe, Ser, Cys, and Tyr represent the residues of L- glutamic acid, L-alanine, L-glycine, L-arginine, L-aspartic acid, L-phenylalanine, L- serine, L-cysteine, L-tyrosine, and L-valine respectively.

[0025] As used herein, the term "subject" is intended to mean humans and non- human mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses, rabbits, rats, mice and the like.AFFIRMED-00120

[0026] As used herein, the term “treatment” with a formulation, pharmaceutical composition or dosage form of a disclosed polypeptide such as rusalatide acetate, in accordance with the present invention, include subjects with diseases or conditions that can be treated with a pharmaceutical composition or dosage form of a disclosed polypeptide such as rusalatide acetate to achieve a beneficial therapeutic or prophylactic result. I: Compositions

[0027] In one embodiment, the polypeptide used in the disclosed methods has the 23 amino acid sequence herein identified as SEQ ID NO: 1. The 23 amino acid sequence for this polypeptide can also be depicted based on the one-letter amino acid code: AGYKPDEGKRGDACEGDSGGPFV (SEQ ID NO: 1).

[0028] Alternatively, the polypeptide used in the disclosed methods is: H-Ala-Gly- Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe- Val-NH2 SEQ ID NO: 2. In one example, the polypeptide is the acetic acid salt of the polypeptide represented by SEQ ID NO: 2 and is referred to herein as rusalatide acetate.

[0029] For the sake of clarity, the one-letter amino acid code designations and the three-letter amino acid code designations are provided below\

[0030] A represents Alanine (ALA)

[0031] R represents Arginine (ARG)

[0032] D represents Aspartic Acid (ASP)

[0033] C represents Cysteine (CYS)

[0034] E represents Glutamic Acid (GLU)

[0035] G represents Glycine (GLY)

[0036] K represents Lysine (LYS)AFFIRMED-00120

[0037] F represents Phenylalanine (PHE)

[0038] P represents Proline (PRO)

[0039] S represents Serine (SER)

[0040] V represents Valine (VAL)

[0041] The disclosed polypeptides, e.g., rusalatide acetate, or other pharmaceutically acceptable salts of the polypeptide represented by SEQ ID NO: 1 and 2 may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. In preferred embodiments of the present invention, a disclosed polypeptide such as rusalatide acetate is present in the natural (L) form. The present invention is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. The racemic mixtures may be prepared and thereafter separated into individual optical isomers or these optical isomers may be prepared by chiral synthesis. The enantiomers may be resolved by other methods, for example by formation of diastereoisomeric salts which may then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. It will also be appreciated that where the desired enantiomer is converted into another chemical entity by a separation technique, an additional step is then required to form the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents or by converting one enantiomer to another by asymmetric transformation.

[0042] The disclosed polypeptides, such as rusalatide acetate, may exist in Zwitterionic form and the present invention includes Zwitterionic forms of these isomers and mixtures thereof.AFFIRMED-00120

[0043] It is also contemplated that the disclosed polypeptides, e.g., rusalatide acetate, can be manufactured or prepared by any suitable technique, including but not limited to any suitable, reliable and effective method for peptide synthesis. In certain embodiments, the disclosed polypeptides, such as rusalatide acetate, can be synthesized by a solid phase peptide synthesis method (for example, but not limited to, BOC or FMOC), or in other embodiments by solution phase synthesis. The present invention also contemplates that the disclosed polypeptides, e.g., rusalatide acetate can be synthesized by other suitable techniques including, e.g., combinations of other methods of peptide synthesis described herein, or using recombinant peptide synthesis. The disclosed polypeptides, e.g., rusalatide acetate can also be synthesized and purified according to Good Manufacturing Practice (GMP) requirements.

[0044] It is also contemplated that the disclosed polypeptides, can exist in different salt forms, i.e. as one or more peptide salts, and desired salt forms can be screened, selected and used in the preparation of pharmaceutical compositions, formulations or dosage forms.

[0045] It is to be understood that the present invention contemplates the use of any suitable and pharmaceutically acceptable excipient in the manufacture and production of formulations, compositions and dosage forms comprising the disclosed polypeptides, e.g., rusalatide acetate. For example, the present invention contemplates the use of any suitable and pharmaceutically acceptable excipient, for example, as disclosed in the “Handbook of Pharmaceutical Excipients” (Sixth Edition, Pharmaceutical Press and American Pharmacists Association 2009), which is incorporated by reference herein in its entirety.

[0046] The present invention contemplates that one or more stabilizing agents may be utilized to enhance the stability of (i) a disclosed polypeptide such as rusalatide acetate or (ii) a composition, formulation or dosage form of a disclosed polypeptide such as rusalatide acetate.AFFIRMED-00120

[0047] The present invention also contemplates that one or more other types of excipients may also be utilized in a composition, formulation or dosage form of a disclosed polypeptide such as rusalatide acetate to enhance the stability of (i) a disclosed polypeptide or (ii) a composition, formulation or dosage form of a disclosed polypeptide. Pharmaceutical Compositions. Formulations and Dosage Forms to treat proton and photon radiotherapy-induced cataract formation

[0048] Generally speaking, the mode of ocular delivery of the pharmaceutical composition will use one or more FDA- approved ointments, liquid or gel known to those skilled in the art. Alternatively the mode of delivery of the pharmaceutical composition will be by an ocular or a systemic injection.

[0049] It is understood that the description provided herein provides non-limiting examples, and the examples are for illustration purposes only. The examples do not limit the scope of the invention in any way. It is understood that any other type of pharmaceutical composition, formulation, dosage form or mode of delivery can also be used in accordance with the present invention.

[0050] Methods of Treatment

[0051] According to a preferred embodiment, the present invention contemplates methods useful for treating a subject in need of treatment with an “effective amount” of a composition, formulation or dosage form of a disclosed polypeptide such as rusalatide acetate.

[0052] An “effective amount” refers to an amount, concentration, dose or quantity of a formulation, pharmaceutical composition or dosage form of a disclosed polypeptide such as rusalatide acetate that results in an improved clinical outcome of a condition in a subject being treated with the formulation, pharmaceutical composition or dosage form, compared with the absence of treatment. The amount, concentration, dose or quantity of the formulation, pharmaceutical composition or dosage form administered will depend on the degree, severity, and type of the disease or condition, the amount of therapyAFFIRMED-00120 desired, and the release characteristics of the formulation, pharmaceutical composition or dosage form. It will also typically depend on the subject's health, size, weight, age, sex and tolerance to drugs. Typically, the formulation, pharmaceutical composition or dosage form is administered for a sufficient period of time to achieve the desired therapeutic effect.

[0053] The polypeptides used in the disclosed invention, e.g., rusalatide acetate, can be administered concurrently with radiotherapy, before radiotherapy or after radiotherapy. For example, the polypeptide can be administered up to 5 hours before radiotherapy and up to 5 hours afterwards. Alternatively, the polypeptide can be administered up to 3 hours before radiotherapy and up to 3 hours afterwards. In another alternative, the polypeptide can be administered up to one hour before radiotherapy and up to one hour afterwards.

[0054] According to another preferred embodiment, the present invention also contemplates the use of a composition, formulation or dosage form of a disclosed polypeptide such as rusalatide acetate for treating one or more conditions in a human or non-human subject, specifically treating or preventing DNA double strand breaks in human lens epithelial cells caused by radiation therapy EXAMPLES The following, non-limiting examples illustrate certain aspects of the invention. These examples shall not be construed as limiting the scope of the invention in any way. EXAMPLE 1 We analyzed primary human lens epithelial cells (HLEpiCs, ScienCell, Carlsbad, CA) with or without rusalatide acetate pretreatment 1 hour prior to radiotherapy (RT) as follows: Optimal dose by MTT assay; proton relative biological effectiveness (RBE) by clonogenic survival assays (CSA); radiation-induced DSBs by 53BP1 foci; and cell senescence by CellEventTMSenescence Green Probe stain (Invitrogen). The effects induced by protonAFFIRMED-00120 (PRT) versus Photon (XRT) with or without rusalatide acetate were compared using Student’s t-tests. As illustrated in Figures 1 through 4: Fig 1: With the MTT assay, rusalatide acetate improved HLEpiC cell viability 4-fold at concentrations of 50 or 100µg / ml. Fig 2: With CSA, PRT killed more HLEpiC (HLE-P) cells than XRT at all tested doses (all RBEs ≥ 1.16); Rusalatide acetate protected HLEpiCs from RT killing (survival protection factor [SPF]; 1.14 [XRT]; 1.44 [PRT] at 4-Gy) while slightly promoting the RBE (all RBEs ≥ 1.16), SBF and RBE were RT dose dependent. Fig 3: With or without rusalatide acetate, PRT caused more unrepaired DSBs (at 1-, 4- , and 24-hours, P ≤0.001). As assayed by 53 BP1 foci, rusalatide acetate mitigated RT- induced persistent DSBs (at 1- hour and 4-hours for XRT and PRT; at 24 hours for PRT) (all P ≤0.03) Fig 4: Rusalatide acetate appears to reduce HLEpiC cell senescence following XRT and PRT at 6 days, (P ≤0.001). Conclusions: Advantageously and importantly, our observations demonstrated that human lens epithelial cells were more sensitive to PRT than XRT. Rusalatide acetate appeared to protect human lens epithelial cells from RT killing, especially at higher doses (4-Gy). PRT increased DSBs and cell senescence compared to XRT. Rusalatide acetate mitigated these effects, especially those caused by PRT.

Claims

AFFIRMED-00120 CLAIMS What Is Claimed Is:

1. A method for reducing radiation damage in a lens epithelial cell from a subject, the subject having a proliferative disease and undergoing radiation therapy to treat same, the method comprising: administering to the lens epithelial cell a radio-modulating polypeptide in an amount sufficient to reduce or repair DNA double-strand breaks, the radio-modulating polypeptide being administered to the cell in an amount sufficient to reduce radiation damage to the lens epithelial cell.

2. The method, according to claim 1, in which the proliferative disease is head and / or neck cancer, 3. The method, according to claim 1, in which the radiation therapy is proton radiotherapy.

4. The method, according to claim 1, in which the radiation therapy is photon radiotherapy 5. The method, according to claim 1, in which the radio-modulating polypeptide is H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly- Gly-Pro-Phe-Val-NH2 (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

6. The method, according to claim 1, in which the subject is a human subject.

7. A method of reducing or reducing the likelihood of developing proton or photon radiotherapy-induced cataract formation in a human subject undergoing proton or photon radiotherapy, comprising administering to the subject an effective amount of the polypeptide H-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-AFFIRMED-00120 Asp-Ser-Gly-Gly-Pro-Phe-Val-NH2 (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof.

8. The method of claim 7, wherein the subject is undergoing proton radiotherapy or photon radiotherapy for the treatment of a proliferative disease.

9. The method of claim 8, wherein the proliferative disease is head or neck cancer.

10. The method of any one of clams 7-9, wherein the subject is undergoing proton radiotherapy.

11. The method of any one of claims 7-9, wherein the subject is undergoing photon radiotherapy.

12. The method of any one of claims 7-11, wherein the polypeptide is administered between an hour prior to or an hour after undergoing radiation or radiotherapy.

13. The method of any one of claim 1-12, wherein the polypeptide is rusalatide acetate.

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