# Patent

## Title
Compositions and methods for the treatment of cast nephropathy and related conditions

## Abstract

Provided are novel peptides of Formula SEQ ID No. 1:

J1CysX1X2X3X4X5X6ProX7ThrCysJ2J3(J4)s(J5)t;   (SEQ ID No. 1)

pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, which are effective inhibitors of light chains to uromodulin.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 62/380,068 filed Aug. 26, 2016, the entire contents of which are incorporate herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 21, 2016, is named 119562-03502_SL.txt and is 20,665 bytes in size.
BACKGROUND
Multiple myeloma is a neoplastic disorder characterized by a clonal proliferation of malignant plasma cells, which produce monoclonal immunoglobulins (i.e., antibodies). Acute kidney injury (AKI) is a well-known complication of multiple myeloma with high tumor burden and high amounts of light chains (which are fragments of immunoglobulins) in the urine. AKI affects up to 50% of patients with multiple myeloma, and has important consequences: it exposes patients to the risk of permanent kidney damage (including end stage kidney failure), is associated with a decrease in overall survival, and impacts the treatment of the underlying myeloma.
In the vast majority of cases, AKI is due to myeloma cast nephropathy that results from the precipitation of complexes (called myeloma casts) formed by light chains and uromodulin (a protein normally present in urine) in the distal part of the nephron. See Leung et al., Adv Chronic Kidney Dis, 21: 36-47. These casts obstruct the nephrons, leading to AKI and induce an inflammatory reaction that leads to fibrosis and irreversible chronic kidney damage. The events leading to cast formation have been dissected. See Huang, et al., J Clin Invest, 92: 2975-83, 1993; Huang, et al., J Clin Invest, 99: 732-6, 1997. In particular, different light chains were found to bind to the same peptidic segment of uromodulin (later identified as D8C) and interacted with uromodulin through their CDJ3 (for complementarity-determining region 3). This finding led to the indentation of a CDJ3 sequence derived peptide that blocked the interaction between monoclonal light chains and uromodulin, and inhibited cast formation in vivo. See e.g., Ying, et al., Am J Pathol, 158: 1859-66, 2012.
Recognizing this result, peptides that inhibit the binding of light chains to uromodulin represent are an attractive targets for combating myeloma cast nephropathy and related conditions.
SUMMARY OF THE INVENTION
Provided herein are peptides and compositions thereof which are effective binding inhibitors of light chains to uromodulin. (See e.g., Table 3). Such peptides include those of Formula SEQ ID No. 1:

J1CysX1X2X3X4X5X6ProX7ThrCysJ23(J4)s(J5)t;   (SEQ ID No. 1)

or a pharmaceutically acceptable salt thereof, wherein each of X1, X2, X3, X4, X5, X6, X7, J1, J2 J3, J4, J5, s, and t are as defined and described herein.

Also provided are methods of treating myeloma cast nephropathy in e.g., patient populations with severe renal impairment, comprising administering one or more of the peptides or compositions described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the plasma pharmacokinetics following intravenous bolus injection of a peptide of Formula SEQ ID No. 1 in rat.
FIG. 2 illustrates the plasma pharmacokinetics following 2 hour intravenous infusion of a peptide of Formula SEQ ID No. 1 in rat.
FIG. 3 compares the plasma pharmacokinetics of two peptides of Formula SEQ ID No. 1 after intraperitoneal injection in rat, where a.) shows a plot of peptide vs time at various concentrations; b.) shows a plot of steady state concentration (Css) as a function of. dose; and c.) shows a plot of the Area Under the Cure (AUC) as a function of dose.
FIG. 4 illustrates the urine recovery of a peptide of Formula SEQ ID No. 1.



DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
1. General Description of Peptides
In one aspect, provided herein are peptides of Formula SEQ ID No. 1:

J1CysX1X2X3X4X5X6ProX7ThrCysJ2J3(J4)s(J5)t;   (SEQ ID No. 1)

or a pharmaceutically acceptable salt thereof, wherein

    
    
        J1 is Aha or Arg;
        X1 to X7 are each independently a natural or non-natural amino acid;
        s, and t are each independently 0 or 1;
        J2 and J3 are each independently Lys or Arg;
        J4, if present is Lys or Arg; and
        J5, if present is Lys or Arg.

2. Peptides and Definitions

    
    


“Amino acid” refers to an organic compound containing an amine (—NH2) and a carboxylic acid (—COOH) functional group, usually along with a side-chain specific to each amino acid. Amino acids can be classified according to the core structural functional groups' locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to polarity, pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur, etc.). In one embodiment, amino acid includes
“Natural amino acid” is used interchangeably with proteinogenic amino acid and refers to the 20 standard amino acids encoded by the universal genetic code along with selenocysteine and pyrolysine. The 20 standard amino acids encoded by the universal genetic code include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, and histidine. Unless otherwise specified, when a natural amino acid is defined without indicating the stereochemistry it will be understood that the defined amino acid is present as the L-enantiomer. Thus, when used as part of a formula, the letter A or Ala means L-alanine.
The full name of a natural amino acid and its corresponding three-letter or one-letter code are used interchangeably. For example, glycine is interchangeable with three letter abbreviation Gly or the one letter abbreviation G; alanine is interchangeable with three letter abbreviation Ala or the one letter abbreviation A, valine is interchangeable with three letter abbreviation Val or the one letter abbreviation V, leucine is interchangeable with three letter abbreviation Leu or the one letter abbreviation L, isoleucine is interchangeable with three letter abbreviation Ile or the one letter abbreviation I, proline is interchangeable with three letter abbreviation Pro or the one letter abbreviation P, phenylalanine is interchangeable with three letter abbreviation Phe or the one letter abbreviation F, tyrosine is interchangeable with three letter abbreviation Tyr or the one letter abbreviation Y, tryptophan is interchangeable with three letter abbreviation Trp or the one letter abbreviation W, serine is interchangeable with three letter abbreviation Ser or the one letter abbreviation S, threonine is interchangeable with three letter abbreviation Thr or the one letter abbreviation T, cysteine is interchangeable with three letter abbreviation Cys or the one letter abbreviation C, methionine is interchangeable with three letter abbreviation Met or the one letter abbreviation M, asparagine is interchangeable with three letter abbreviation Asn or the one letter abbreviation N, glutamine is interchangeable with three letter abbreviation Gln or the one letter abbreviation Q, aspartate is interchangeable with three letter abbreviation Asp or the one letter abbreviation D, glutamate is interchangeable with three letter abbreviation Glu or the one letter abbreviation E, lysine is interchangeable with three letter abbreviation Lys or the one letter abbreviation K, arginine is interchangeable with three letter abbreviation Arg or the one letter abbreviation R, histidine is interchangeable with three letter abbreviation His or the one letter abbreviation H, selenocysteine is interchangeable with three letter abbreviation Sec or the one letter abbreviation U, and pyrolysine is interchangeable with three letter abbreviation Pyl or the one letter abbreviation O. In one embodiment, the term “natural amino acid” or proteinogenic amino acid refers only to the 20 standard amino acids encoded by the universal genetic code, i.e., G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, and H.
“Non-natural amino acid” refers to a non-proteinogenic amino acid that is not found in proteins (e.g., carnitine, gamma-aminobutyric acid, and D-forms of natural amino acids except glycine) or not produced directly and in isolation by standard cellular machinery (e.g., hydroxyproline and selenomethionine). Other examples include, but are not limited to, β-amino acids (β3 and β2), homo-amino acids, alanine derivatives, alicyclic amino acids, arginine derivatives, asparagine derivatives, aspartic acid derivatives, cysteine derivatives, 2,4-diaminobutyric acid, glycine derivatives, isoleucine derivatives, leucine derivatives, lysine derivatives (such as 6-aminohexanoic acid abbreviated herein as Aha), methionine derivatives, norleucine (nL) and norleucine derivatives, phenylalanine derivatives, phenylglycine derivatives, proline and pyruvic acid derivatives, pyroglutamine derivatives, serine derivatives, threonine derivatives, tryptophan derivatives, norvaline derivatives, 2,3-diaminopropionic acid, ornithine derivatives, valine derivatives, linear core amino acids, and N-methyl amino acids. In one embodiment, the term “non-natural amino acid” refers only to D-forms of the 20 standard amino acids encoded by the universal genetic code. These forms include D-Ala, D-Val, D-Leu, D-Ile, D-Pro, D-Phe, D-Tyr, D-Trp, D-Ser, D-Thr, D-Cys, D-Met, D-Asn, D-Gln, D-Asp, D-Glu, D-Lys, D-Arg, and D-His.
The abbreviation “oxC” means a compound of the structure




The abbreviation “Pen” means a compound of the structure




A “D-amino acid” or D-form of an amino acid means that the indicated amino acid is present as the D-enantiomer. Shorthand notation for the D-enantiomer of an amino acid can be represented by an asterisk (*). For example, I* or I*le, wherein * represents a D-amino acid, refers to the D-enantiomer of Ile (isoleucine).
The peptides described herein may be present in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts are art-recognized and include e.g., relatively non-toxic inorganic and organic acid addition salts, or inorganic or organic base addition salts that are suitable for human consumption. Examples of such salts include, but are not limited to, sodium, potassium, calcium, magnesium, acetate, benzoate, bicarbonate, carbonate, citrate, dihydrochloride, gluconate, glutamate, hydrochloride, and tartrate.
The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not adversely affect the pharmacological activity of the peptide with which it is formulated, and which is also safe for human use. Pharmaceutically acceptable carriers that may be used include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose Phthalate), starch, lactose monohydrate, mannitol, trehalose sodium lauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The terms “subject” and “patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.
The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
An “effective amount” or “therapeutically effective amount” is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of or a decrease in the symptoms associated with a condition that is being treated, e.g., the conditions described herein.
3. Description of Exemplary Peptides
In a first embodiment, provided herein are peptides of the Formula SEQ ID No. 1:

J1CysX1X2X3X4X5X6ProX7ThrCysJ2J3(J4)s(J5)t;   (SEQ ID No. 1)

or a pharmaceutically acceptable salt thereof, wherein the variables are as described above.

In a second embodiment, X6 in the peptide of Formula SEQ ID No. 1 is not Glu, Thr, Asn, or Asp; and X7 is not Glu or Asp, wherein the remaining variables in SEQ ID No. 1 are as described above.
In a third embodiment, X1 in the peptide of Formula SEQ ID No. 1 is selected from Arg, Lys, His, Pro, Cys, Thr, Ser, Gln, Glu, Leu, Ile, Met, Ala, Val, Gly, n-Leu, Met, Asp, and Ile, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment. Alternatively, X1 in the peptide of Formula SEQ ID No. 1 is selected from Arg, Gln, Glu, Leu, n-Leu, Met, Asp, and Ile, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment. In another alternative, X1 in the peptide of Formula SEQ ID No. 1 is selected from Gln, Leu, and n-Leu, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment.
In a forth embodiment, X2 in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. Alternatively, X2 in the peptide of Formula SEQ ID No. 1 is selected from Ser, Thr, Cys, Met, Asn, Lys, Gln, His, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. In another alternative, X2 in the peptide of Formula SEQ ID No. 1 is selected from Ser, Gln, His, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. In yet another alternative, X2 in the peptide of Formula SEQ ID No. 1 is selected from Gln and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment.
In a fifth embodiment, X3 in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. Alternatively, X3 in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Trp, Phe, Ser, Thr, Cys, Met, Asn, Ala, Lys, His, Glu, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. In another alternative, X3 in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Ser, Ala, Glu, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. In yet another alternative, X3 is selected from Tyr, Ser, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment.
In a sixth embodiment, X4 in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. Alternatively, X4 in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Asp, Glu, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. In another alternative, X4 in the peptide of Formula SEQ ID No. 1 is selected from Leu, Asp, Gly, Tyr, Glu, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. In yet another alternative X4 in the peptide of Formula SEQ ID No. 1 is selected from Leu, Asp, Tyr, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment.
In a seventh embodiment, X5 in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment. Alternatively, X5 in the peptide of Formula SEQ ID No. 1 is selected from Ser, Thr, Cys, Met, Asn, Gln, Asp, Glu, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment. In another alternative, X5 in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Ser, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment.
In an eighth embodiment, X6 in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Gln, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. Alternatively, X6 in the peptide of Formula SEQ ID No. 1 is selected from Ile, Leu, Pro, Val, Ala, Gly, His, Lys, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. In another alternative, X6 in the peptide of Formula SEQ ID No. 1 is selected from Ile, Leu, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. In yet another alternative, X6 in the peptide of Formula SEQ ID No. 1 is selected from Ile and Leu, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment.
In a ninth embodiment, X7 in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. Alternatively, X7 in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Phe, His, Lys, Arg, and Trp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. In another alternative, X7 in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Arg, and Trp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. In yet another alternative, X7 in the peptide of Formula SEQ ID No. 1 is Tyr, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment.
In a tenth embodiment, s and t are each 0 in the peptide of Formula SEQ ID No. 1, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment.
In an eleventh embodiment, s and t are each 1 in the peptide of Formula SEQ ID No. 1, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth embodiment.
In a thirteenth embodiment, J1 in the peptide of Formula SEQ ID No. 1 is Aha, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh embodiment.
Specific examples of the disclosed peptides are provided in Table 1 and Table 2 below as well as in the EXEMPLIFICATION sections. Pharmaceutically acceptable salts as well as the neutral forms of these peptides are included in the present disclosure.








TABLE 1





SEQ ID



No.
Sequence
















11
AhaCQQSYSIPWTCKK


12
AhaCQQSYSIPYTCKK


13
AhaCQQYDSLPLTCKK


14
AhaCQQYSYLPITCK*K*


15
AhaCQQYSTAPWTCKK


16
AhaCQQYYSAPPTCKK


17
AhaCQQYKNYPWTCKK


18
AhaCQHYDYLPITCK*K*


20
AhaCQQSYSIPWTCK*K*


21
AhaCQQSYSIPYTCK*K*


22
AhaCQQ*SYSIPWTCK*K*


23
AhaCQQS*YSIPWTCK*K*


24
AhaCQQSY*SIPWTCK*K*


25
AhaCQQ*SYSIPYTCK*K*


26
AhaCQQS*YSIPYTCK*K*


27
AhaCQQSY*SIPYTCK*K*


28
AhaCEQSYSIPYTCKK


29
AhaCQESYSIPYTCKK


30
AhaCQQEYSIPYTCKK


31
AhaCQQSESIPYTCKK


32
AhaCQQSYEIPYTCKK


33
AhaCQQSYSEPYTCKK


34
AhaCQQSYSIEYTCKK


35
AhaCQQSYSIPETCKK


36
AhaCQQSYSIPYECKK


37
AhaCRQSYSIPYTCKK


38
AhaCQRSYSIPYTCKK


39
AhaCQQRYSIPYTCKK


40
AhaCQQSRSIPYTCKK


41
AhaCQQSYRIPYTCKK


42
AhaCQQSYSRPYTCKK


43
AhaCQQSYSIRYTCKK


44
AhaCQQSYSIPRTCKK


45
AhaCQQSYSIPYRCKK


46
AhaCQQSY*SIPYTCK*K*K*K*


50
AhaCQQYSYLPITCK*K*


51
AhaCQQYSYLPITCR*R*


52
AhaCQQSYLPITCK*K*


53
AhaCQQYSLPITCK*K*


54
AhaCQQYDLPITCK*K*


55
AhaCQQSYSIPYTCR*R*

















TABLE 2





SEQ ID



No.
Sequence
















2
AhaCAAWDDSLNGPVCKK


3
AhaCnLQALRTPLYTCKK


4
AhaCLSADSSGSYLYVCKK


5
AhaCQVWDNSVGVCKK


6
AhaCQSYDNTLSGSYVCKK


7
AhaCQSYDNTLSGSLCKK


8
AhaCQSYDARNV


9
AhaCQSYDHNNQ


10
AhaCQSYDSTNEGVCKK


19
AhaCQHYGSSALTCKK


47
K*K*CQQSY*SIPYTCK*K*


48
AhaPenQQSY*SIPYTPenK*K*


49
oxCCQQSY*SIPYTCK*K*









4. Uses, Formulation, and Administration

In certain embodiments, the present disclosure provides a method of treating a subject (e.g., a human) suffering from myeloma cast nephropathy, comprising administering to the subject a therapeutically effective amount of a peptide of Formula SEQ ID No. 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the amount of peptide of SEQ ID No. 1 is such that it is an effective binding inhibitor of light chains to uromodulin. In certain embodiments, a provided composition is formulated for administration to a subject in need of such composition.
Pharmaceutically acceptable compositions are included and can be administered to humans and other animals by other methods such as e.g., orally, rectally, parenterally, intracisternally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, as an oral or nasal spray, or the like. In certain embodiments, the disclosed peptides may be administered parenterally (e.g., intravenous).
In certain embodiments, the present disclosure also provides a method of treating a subject (e.g., a human) suffering from myeloma cast nephropathy, comprising administering to the subject a therapeutically effective amount of composition comprising a peptide of Formula SEQ ID No. 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. adjuvant, or vehicle. In certain embodiments, the amount of peptide of SEQ ID No. 1 in a provided composition is such that it is an effective binding inhibitor of light chains to uromodulin.
It will be understood that a specific dosage for any particular subject will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided peptide in the composition will also depend upon the particular peptide in the composition.
The amount of provided peptides that may be combined with carrier materials to produce a composition in a single dosage form will vary depending upon the patient to be treated and the particular mode of administration.
EXEMPLIFICATION
Standard solid phase techniques were employed to synthesize the disclosed peptides. Fmoc (fluorenylmethyloxycarbonyl) chemistry occurred on PEG-base Rink Amide resin, 0.45 mmol/g loading. Side-chain protection of Fmoc amino acid included t-Bu (t-butyl) on Asp, Ser, Tyr and D-Tyr; Trt (trityl) on Asn and Cys; Boc (tert-butyloxycarbonyl) on Lys; Pbf (2,2,4,6,7-pentamethyldihydrobenzofurane) on Arg, Fmoc on K* (i.e., Fmoc-Lys(Fmoc)-OH was used to coupled to the resin).
General Procedure
1. Fmoc Deprotection
Fmoc deprotection was performed using 20% piperidine in DMF (dimethylformamide) or 20% piperidine/0.1% 6-Cl HOBt (Hydroxybenzotriazole) in DMF, 5 min+20 min for each deprotection.
2. Coupling of Fmoc Amino Acids
Acylations were carried out using 3-fold Fmoc amino acids activated with DIC (3 eq.) (N,N′-Diisopropylcarbodiimide) in the presence of 6-Cl-HOBt (3 eq.). Each coupling reaction took on average 1-3 h for completion. Kaiser test was used throughout for the in-process control of the coupling completion and the Fmoc deprotection. In the case that the Kaiser test was not satisfactory, re-couplings were done using DIC/6-Cl HOBt or HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate)/OxymaPure.
3. Resin Cleavage and Peptide Recovery
Peptidyl resin was extensively washed with DCM (dichloromethane) and methanol to remove the trace of DMF and dried at RT overnight. Peptidyl resin was cleavaged with the mixture of TFA/TIS/EDT/H2O (trifluoracetic acid/triethylsilane/1,2-ethanedithiol/water) (92.5/2.5/2.5/2.5, v/v/v/v) for 2.5-3 h under nitrogen bubbling. Peptides were collected by precipitation in cold diethyl ether/Hexane (1/1, v/v).
4. Purification of Linear Peptide
Crude liner peptide was purified using Phenomenex Gemini C-18 prep column. 21.5 mm×250 mm. The peptide was then dried by lyophilization.
5. Disulfide Bridge Formation
Purified linear peptide was dissolved in 0.1M ammonium bicarbonate (3-5 mg/ml), stand open to air for 2 days. Frequent HPLC testings were done to monitor the cyclization process. After cyclization, use TFA to adjust to acidic. isolated the product by lyophilization.
6. Cyclic Peptide Purification
Peptides were purified using the following conditions. Column: Phenomenex Gemini, 5 u, C18, 110 A, 250×21.2 mm Mobile phase A: 0.1% TFA in Acetonitrile/H2O (50/50). Mobile phase B: 0.1% TFA/H2O. Gradient: 0-100% A in 120 min Wavelength: 229 nm. Flow rate: 16-20 ml/min.
7. Isolation of Human Uromodulin
Uromodulin was precipitated from fresh urine from healthy donors using 0.58M NaCl. The precipitate was then washed with 0.58M NaCl and resuspended in water. The uromodulin was dialyzed extensively against water to remove salt and other contaminants (MWCo=50 KD). Samples were then stored at −20° C. without lyophilization. Good overall purity was seen by gel electrophoresis.
8. General Binding Assays
Enzyme-linked immunosorbant assays (ELISA) were performed on the disclosed peptides. Plates were coated with the following light chain clones overnight at 4° C.: 0.05 uM BER (subtype lambda, isolated from SP2/0 cells), 0.2 uM ROC (subtype kappa, isolated from CHO cells), and 0.3 uM BEN (subtype lambda, isolated from CHO cells). The plates were washed and treated with bovine serum albumin (BSA) overnight at 4° C. For peptide plates, 10 nM biotin-uromodulin was mixed with peptide overnight at 4° C. Plates were then washed and results were revealed using streptaviding-HRP (horseradish peroxidase) and 3,3′,5,5′-tetramethylbenzidine (TMB). Results are shown in Table 3.









TABLE 3









Binding


SEQ ID

Inhibition (IC50 uM)











No.
Sequence
BER
BEN
ROC














2
AhaCAAWDDSLNGPVCKK
500
500
500


3
AhaCnLQALRTPLYTCKK
16.0
12.0
14.9


4
AhaCLSADSSGSYLYVCKK
500
66
500


5
AhaCQVWDNSVGVCKK
500
>120
18.5


6
AhaCQSYDNTLSGSYVCKK
>120
19
>120


7
AhaCQSYDNTLSGSLCKK
500
500
500


8
AhaCQSYDARNV
500
58.2
500


9
AhaCQSYDHNNQ
500
26.7
500


10
AhaCQSYDSTNEGVCKK
500
500
500


11
AhaCQQSYSIPWTCKK
34.4
3.2
20.8


12
AhaCQQSYSIPYTCKK
22.9
2.5
27.3


13
AhaCQQYDSLPLTCKK
21.3
3.3
31.9


14
AhaCQQYSYLPITCK*K*
17.3
2.8
20.9


15
AhaCQQYSTAPWTCKK
500
16.7
500


16
AhaCQQYYSAPPTCKK
500
14.3
>120


17
AhaCQQYKNYPWTCKK
500
7.3
>120


18
AhaCQHYDYLPITCK*K*
14.8
0.9
13.7


19
AhaCQHYGSSALTCKK
52
3.3
16.4


20
AhaCQQSYSIPWTCK*K*
60.1
2.6
nt


21
AhaCQQSYSIPYTCK*K*
49.8
nt
75.5


22
AhaCQQ*SYSIPWTCK*K*
96.9
nt
nt


23
AhaCQQS*YSIPWTCK*K*
54.1
nt
nt


24
AhaCQQSY*SIPWTCK*K*
67.8
nt
nt


25
AhaCQQ*SYSIPYTCK*K*
35.9
nt
22.0


26
AhaCQQS*YSIPYTCK*K*
30.1
nt
27.5


27
AhaCQQSY*SIPYTCK*K*
27.1
2.8
26.5


28
AhaCEQSYSIPYTCKK
24.9
nt
25.2


29
AhaCQESYSIPYTCKK
26.3
nt
25.5


30
AhaCQQEYSIPYTCKK
26.5
nt
25.9


31
AhaCQQSESIPYTCKK
25.9
nt
24.1


32
AhaCQQSYEIPYTCKK
Nt
nt
28


33
AhaCQQSYSEPYTCKK
>120
nt
>120


34
AhaCQQSYSIEYTCKK
>500
nt
>120


35
AhaCQQSYSIPETCKK
>120
nt
>120


36
AhaCQQSYSIPYECKK
>120
nt
>500


37
AhaCRQSYSIPYTCKK
49.8
nt
27.9


38
AhaCQRSYSIPYTCKK
55
nt
26.4


39
AhaCQQRYSIPYTCKK
20.7
nt
22.1


40
AhaCQQSRSIPYTCKK
23.5
nt
26.2


41
AhaCQQSYRIPYTCKK
20.9
nt
21.6


42
AhaCQQSYSRPYTCKK
27.8
nt
29.5


43
AhaCQQSYSIRYTCKK
>500
nt
>120


44
AhaCQQSYSIPRTCKK
24.9
nt
25


45
AhaCQQSYSIPYRCKK
>500
nt
>500


46
AhaCQQSY*SIPYTCK*K*K*K*
23.4
nt
35.0


47
K*K*CQQSY*SIPYTCK*K*
32.3
nt
33.5


48
AhaPenQQSY*SIPYTPenK*K*
120.0
nt
120.0


49
oxCCQQSY*SIPYTCK*K*
43.3
nt
41.2


50
AhaCQQYSYLPITCK*K*
17.3
nt
nt


51
AhaCQQYSYLPITCR*R*
13.6
nt
nt


52
AhaCQQSYLPITCK*K*
69.0
nt
nt


53
AhaCQQYSLPITCK*K*
120.0
nt
nt


54
AhaCQQYDLPITCK*K*
120.0
nt
nt


56
AhaCnLQALRTPLYTCR*R*
10.6
2.0
18.1





nt = not tested






8. Ex Vivo Conditions

Binding inhibition, stability, and solubility data was obtained for peptides of the Formula SEQ ID No. 1. These results are shown in Table 4. MLC90 represents the lowest IC50 at which uromodulin binding to 90% of the light chains tested was inhibited. KBB is kidney brush border enzyme.












TABLE 4








Binding














SEQ ID
Inhibition
Stability
Solubility












No.
MLC90
Plasma50%
KBB50%
Urine80%
(saline, mg/mL)





11
nt
 <5 min
 5 min
>2 hrs
≧100


27
27.1
>120 min
>60 min
>2 hrs
≧100









9. Pharmacokinetic Evaluations

The plasma pharmacokinetics of SEQ ID No. 27 was evaluated following intravenous (IV) bolus injection (FIG. 1) and 2 hour IV infusion (FIG. 2) in rats. Similarly, FIG. 3 plasma pharmacokinetics of SEQ ID No. 27 following intraperitoneal injection of SEQ ID No. 27 when compared with SEQ ID No. 4 in rat. This data establishes that peptides having SEQ ID No. 1, in particular SEQ ID No. 27, has high efficacy and very good dose-exposure proportionally over a broad range of doses.
Surprisingly, and perhaps most notably, the clearance rate of SEQ ID No. 27 was essentially equal to the glomerular filtration rate, e.g., after infusion of 10 mg/kg of SEQ ID No. 27 for 2 hours to 6 rats, the percentage of intact peptide recovered in the urine was 100%±22% (mean±SD). See FIG. 4.
It will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

## Claims

1. A peptide of the Formula:


AhaCQQSY*SIPYTCK*K*;


or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the peptide of claim 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

3. A method of treating cast nephropathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the peptide of claim 1 peptide, or a pharmaceutically acceptable salt thereof.