RGD-(bacterio)chlorophyll conjugates for photodynamic therapy and Imaging of Necrotic tumors

• US 20110064658A1
• Filed: 03/01/2009
• Published: 03/17/2011
• Est. Priority Date: 02/27/2008
• Status: Active Grant

• Current Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: Salomon, Yoram, Scherz, Avigdor, Goldshaid, Liat
• Granted Patent: US 8,815,213 B2
• Time in Patent Office: Days
• US Class Current: 424/1.69
• CPC Class Codes: A61K 41/0057 Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• CPC Class Codes: A61K 41/0071 PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• CPC Class Codes: A61K 49/0036 Porphyrins used in photodynamic therapy A61K41/0071 or A61K41/0076; used as targeting group or modifying agent for targeting a therapeutic compound A61K47/546
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 49/085 conjugated systems
• CPC Class Codes: A61K 49/14 Peptides, e.g. proteins
• CPC Class Codes: A61K 51/044 having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins rifampin A61K51/0459
• CPC Class Codes: A61K 51/0446 having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• CPC Class Codes: A61K 51/0451 having four such rings, e.g. phorphine derivatives, bilirubin, biliverdine hemin, hematin A61K51/0472
• CPC Class Codes: A61K 51/0485 Porphyrins, texaphyrins wherein the nitrogen atoms forming the central ring system complex the radioactive metal
• CPC Class Codes: A61K 51/082 the peptide being a RGD-containing peptide
• CPC Class Codes: A61K 51/088 conjugates with carriers being peptides, polyamino acids or proteins antibodies A61K51/10
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/04 specific for metastasis
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07D 257/04 Five-membered rings

Abstract

RGD-chlorophyll and RGD-bacteriochlorophyll conjugates that home and accumulate in necrotic tumor domains much longer than in tumor non-necrotic domains are provided for use in minimally invasive tumor-targeted imaging, tumor-targeted photodynamic therapy, and/or on-line prognosis of necrotic tumors.

Claims

1. A method for minimally invasive tumor-targeted imaging, on-line prognosis and/or mapping of tumor margins before surgery, of necrotic tumors, said method comprises:

(i) administering to a subject suspected of having a tumor with necrotic domains a conjugate of an RGD-containing peptide or an RGD peptidomimetic and a photosensitizer; and

(ii) tumor-targeted imaging the subject, wherein said photosensitizer is a chlorophyll or bacteriochlorophyll of the formula I, II or III;

6. The method according to claim 1, wherein the positions 7-8 of said photosensitizer are hydrogenated and the photosensitizer is a bacteriochlorophyll of formula I or II, wherein:

(i) each R₄, independently, is acetyl, vinyl, ethyl, or 1-hydroxyethyl radical or an ether or ester of said 1-hydroxyethyl radical;

(ii) R′

₄ is methyl; and

(iii) any of the C₁-C₂₅ hydrocarbyl is a C₁-C₂₅ alkyl, alkenyl or alkenyl, optionally substituted by at least one functional group selected from the group consisting of;

(a) a negatively charged group, selected from the group consisting of COO⁻

, COS⁻

, SO₃⁻

, and PO₃²⁻

;

(b) an acidic group that is converted to a negatively charged group at the physiological pH, selected from the group consisting of COOH, COSH, SO₃H, and PO₃H₂ or a salt thereof;

(c) a positively charged group, selected from the group consisting of;

an onium group such as —

O⁺(RR′

), —

S⁺(RR′

), —

Se⁺(RR′

), —

Te⁺(RR′

), —

P⁺(RR′

R″

), —

As⁺(RR′

R″

), —

Sb⁺(RR′

R″

), and —

Bi⁺(RR′

R″

);

a cation derived from an N-containing group such as —

N⁺(RR′

R″

), —

(R)N—

N⁺(RR′

R″

), O←

N⁺(RR′

R″

)—

, >

C═

N⁺(RR′

), —

C(═

NR)—

N⁺RR′

R″

or —

(R)N—

C(═

NR)—

N⁺RR′

R″

group;

or a cation derived from a heteroaromatic compound containing one or more N atoms and optionally O or S atoms such as pyrazolium, imidazolium, oxazolium, thiazolium, pyridinium, quinolinium, isoquinolinium, pyrimidinium, 1,2,4-triazinium, 1,3,5-triazinium or purinium; and

(d) a basic group that is converted to a positively charged group under physiological conditions, said basic group is an end group or a group located within an alkyl chain, such as —

NRR′

, —

C(═

NR)—

NR′

R″

, —

NR—

NR′

R″

, —

(R)N—

C(═

NR)—

NR′

R″

, O←

NRR′

—

, and >

C═

NR, or the basic group is an N-containing heteroaromatic radical such as pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyrimidyl, 1,2,4-triazinyl, 1,3,5-triazinyl or purinyl,

wherein R, R′ and

R″

each independently is H, optionally substituted hydrocarbyl or heterocyclyl, or two of R, R′ and

R″

together with the N atom to which they are attached form a 3-7 membered saturated ring, optionally containing one or more heteroatoms selected from the group consisting of O, S or N, and optionally further substituted at the additional N atom, said ring is selected from the group consisting of aziridine, pyrrolidine, piperidine, morpholine, thiomorpholine, azepine or piperazine optionally substituted at the additional N atom by C₁-C₆ alkyl optionally substituted by halo, hydroxyl or amino.

8. The method according to claim 6, wherein said bacteriochlorophyll is of formula II and R₆ is —

NR₉R′

₉ wherein R₉ is H and R′

₉ is selected from the group consisting of;

(i) C₁-C₁₀ alkyl substituted by SO₃H or an alkaline salt thereof; and

(ii) C₁-C₆ alkyl substituted by a basic group —

NRR′

or —

NH—

(CH₂)_2-6—

NRR′

, wherein each of R and R′

independently is H, C₁-C₆ alkyl optionally substituted by NH₇, or R and R′

together with the N atom form a 5-6 membered saturated ring, optionally containing an O or N atom and optionally further substituted at the additional N atom by —

(CH₂)_2-6—

NH₂.

9. The method according to claim 8, wherein R₆ is (i) —

NH—

(CH₂)₂—

SO₃K or —

NH—

(CH₂)₃—

SO₃K;

or (ii) —

NH—

(CH₂)₃—

NH—

(CH₂)₃—

NH₂, —

NH—

(CH₂)₂-1-morpholino, or —

NH—

(CH₂)₃-piperazino-(CH₂)₃—

NH₂.

18. The method according to claim 1, wherein the photosensitizer is a bacteriochlorophyll of formula II and R₁ and R₆ together form a cyclic ring comprising an RGD peptide or RGD peptidomimetic.

22. The method according to claim 1, wherein the RGD-containing peptide is an all-L, all-D or an L,D-linear or cyclic peptide composed of 4-100, natural, modified natural or non-natural amino acids, wherein the natural amino acids are selected from the group consisting of Ala, Arg, Asn, Asp, Cys, His, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and the modification of said modified natural or non-natural amino acids includes D-modification, alkylation or acylation of the amino terminal group or of the free amino group of lysine, esterification or amidation of the carboxy terminal group or of the acid free carboxy group of aspartic or glutamic acid, and etherification or esterification of the free hydroxyl group of serine or tyrosine.

24. The method according to claim 22, wherein the RGD-containing peptide is a cyclic peptide, such as the pentapeptide c(RGDfK) (SEQ ID NO:

1), wherein f indicates D-Phe, c(RGDK) (SEQ ID NO;

3), c(RGDf-n(Me)K) (SEQ ID NO;

     4), c(RGDyK) (SEQ ID NO;

     5), wherein y is D-Tyr;

or a linear peptide such as CDCRGDCGC (SEQ ID NO;

     9);

the hexapeptide GRGDSP (SEQ ID NO;

     6);

the heptapeptide GRGDSPK (SEQ ID NO;

     7);

or the 25-mer (GRGDSP)₄K (SEQ ID NO;

     8).

27. The method according to claim 1, wherein the conjugate is compound 13, herein also designated c(RGDfK)-2H-MLT, or compound 24, herein also designated c(RGDfK)-Pd-MLT.

28. The method according to claim 1, wherein the tumor is a primary tumor or a metastatic tumor with necrotic domains, such as melanoma, prostate, brain, colon, ovarian, breast, colorectal, head and neck, chest wall tumor arising from breast cancer, skin, lung, esophagus and bladder cancer and tumor.

30. The method according to claim 28, wherein the tumor is localized breast cancer, particularly ductal carcinoma in situ (DCIS).

31. The method according to claim 1 for imaging of a tumor comprising necrotic domains, or mapping the margins of said tumor before surgery, by dynamic fluorescence imaging, which comprises:

(a) administering to a subject suspected of having a tumor with necrotic domains a conjugate as defined in claim 1, wherein M is 2H or a metal selected from Pd and Zn;

(b) illuminating the subject and measuring the fluorescence of the suspected areas during at least 24-48 hours after administration of the conjugate at time intervals of 1-8 hours, wherein the areas that exhibit fluorescence after 24-48 hours or longer indicate the presence of said tumor necrotic domains.

32. The method according to claim 31, wherein the conjugate is compound 13 or compound 24, the tumor is mammary or ovarian tumor, and the necrotic domains are visualized 3 to 8 days post drug injection.

33. The method according to claim 1, for diagnosis of a tumor comprising necrotic domains by radiodiagnostic technique, which comprises:

(a) administering to said subject suspected of having a tumor a conjugate as defined in claim 1, wherein M is a radioisotope selected from the group consisting of ⁶⁴Cu, ⁶⁷Cu, ^99mTc, ⁶⁷Ga, ²⁰¹Tl, ¹⁹⁵Pt, ⁶⁰Co, ¹¹¹In or ⁵¹Cr.

(b) scanning the subject in an imaging scanner during at least 24-48 hours after administration of the conjugate at time intervals of 1-8 hours, and measuring the radiation level of the suspected areas, wherein the areas that exhibit radiation after 24-48 hours or longer indicate the presence of said tumor necrotic domains.

34. The method according to claim 33, wherein said radiodiagnostic technique is positron emission tomography (PET) and M is ⁶⁴Cu or ⁶⁷Cu, or single photon emission tomography (SPET) and M is a radioisotope selected from the group consisting of ^99mTc, ⁶⁷Ga, ¹⁹⁵Pt, ¹¹¹In, ⁵¹Cr and ⁶⁰Co.

36. The method according to claim 1, for diagnosis of a tumor comprising necrotic domains by magnetic resonance imaging (MRI), which comprises the steps of:

(a) administering to a subject suspected of having a tumor a conjugate as defined in claim 1, wherein M is a paramagnetic metal selected from Mn³⁺, Cu²⁺, Fe³⁺, Eu³⁺, Gd³⁺ or Dy³⁺; and

(b) subjecting the patient to magnetic resonance imaging by generating at least one MR image of the target region of interest within the patient'"'"'s body prior to said administration (zero time) and one or more MR images at a second or more time points at least 24-48, preferably 96, hours after said administration;

(c) processing and analyzing the data to diagnose the presence or absence of said tumor necrotic domains.

37. The method according to claim 1 for mapping of tumor margins before surgery comprising administering to a subject in need a conjugate as defined in claim 1, and imaging the tumor, e.g. breast tumor, margins, at the first 2-24 h after administration of the conjugate.

38. A minimally invasive treatment, detection and prognosis strategy for localized breast cancer, in particular ductal carcinoma in situ (DCIS), comprising (i) administering to a subject a conjugate as defined in claim 1 that specifically homes and accumulates in the tumor necrotic domains, (ii) tumor-targeted imaging the subject treated with said conjugate by the method defined in claim 1 for tumor detection and tumor margin definition at high precision as well as prognosis by MRI, fluorescence, and PET SCAN approaches;

and (iii) tumor-targeted photodynamic therapy (PDT) of the localized necrotic areas allowing breast conservation and remodeling.

39. A method for tumor-targeted photodynamic therapy (PDT) of necrotic tumors, comprising:

(i) administering to a subject suspected of having a tumor with necrotic domains a conjugate of an RGD-containing peptide or an RGD peptidomimetic and a photosensitizer as defined in claim 1;

(ii) tumor-targeted imaging the subject treated with said conjugate according to the method of claim 1 for minimally invasive tumor-targeted imaging, on-line prognosis and/or mapping of tumor margins; and

(iii) irradiating the local of the tumor and its necrotic domains after determining the presence of necrotic domains in (ii) at least 24 hours after injection of the conjugate.

40. The method according to claim 39, wherein the conjugate is compound 13 or compound 24, the tumor is mammary or ovarian tumor, and the necrotic domains are treated 2 to 8 days post drug injection.

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