KIT FOR FORMULATION OF LIPOSOMAL DOXORUBICIN MODIFIED WITH BIOACTIVE PEPTIDES FOR SELECTIVE TARGETING OF RECEPTORS OVEREXPRESSED BY CANCER CELLS

Abstract

The invention relates to methods for obtaining liposomes externally modified with a targeting peptide able to selectively drive liposomal doxorubicin on membrane receptors over expressed in tumours.

The invention is based on a kit containing a first vial filled with a sterile, translucent, red dispersion of the liposomal doxorubicin drug; a second vial filled with a modified phospholipid with a reactive function such as DSPEPegmaleimide in lyophilized form; and a third vial filled with a peptide modified with an appropriate reactive function, in a 1:1 stoichiometric amount respect to modified phospholipid and in lyophilized form.

The kit could also contain an additional fourth vial with the targeting peptide modified with a chelating agent able to complex radioactive metal ions for diagnostic and imaging purposes. The patients to be treated with the peptide modified liposomal doxorubicin could be selected on the basis of the over expression of the targeting receptors.

Claims

1. A kit containing: a) a doxorubicin liposomal formulation; b) a phospholipid modified with a reactive group (compound 1), in the form of lipid or lyophilic film and in amounts ranging from 1% to 10% by weight with respect to the total lipid component, preferably from 2% to 4%; c) a peptide modified with a suitable reactive function (compound 2), in 1:1 stoichiometric amount to the compound 1 and with general formula: R-L-P in which: R is a residue containing a chemical group capable of reacting to give a stable covalent bond, with one of the chemical groups present on compound 1, L is absent or is an oxyethylene linker with molecular weight ranging from 100 to 1000 daltons, containing amine and carboxylic groups to form an amido bond with R and P, respectively; or L is a of natural o non-natural amino acid residue, in L or D conformation, or 2 or 3 amino acid residues sequentially bonded; P is a peptide with target activity to the receptors overexpressed by tumour cells belonging to the bombesin, cholecystokinin, somatostatin or neurotensin families

2. A kit according to claim 1 wherein the modified phospholipid (compound 1) is a derivative di N-(polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3phosphoethanolamine functionalized with maleimido, azido, dibenzocyclooctyl, aldehyde, carboxylic, pyridyldithiol propionate, succinyl, glutaryl groups.

3. A kit according to claim 1 further comprising a component d) consisting of a peptide P modified with a chelating agent capable of complexing radioactive metal ions.

4. A kit according to claim 1 wherein each component a), b), c) and possibly d) is contained in vials, ampoules or pre-filled syringes.

5. A kit according to claim 1 wherein the compound 1 is N-(polyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine maleimide (DSPEPeg maleimide) and the component c) (compound 2) is a modified, antagonist analogue of the bombesin peptide selected from: TABLE-US-00005 SEQID1 R-L-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH.sub.2 SEQID2 R-L-Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH.sub.2 SEQID3 R-L-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH.sub.2 SEQID4 R-L-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu- NH.sub.2 SEQID5 R-L-DPhe-Gln-Trp-Ala-Val-Gly-His-Sta- Leu-NH.sub.2 SEQID6 R-L-DPhe-Gln-Trp-Ala-Val-Gly-His-Cha- Nle-NH.sub.2 SEQID7 R-L-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta- Leu-NH.sub.2 SEQID8 R-L-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 SEQID9 R-L-DPhe-Gln-Trp-Ala-Val-NMeGly-His- Leu-Nle-NH.sub.2 in which: R is: Cys o SH(CH2)nCOOH (n ranging from 1 to 5) to react with the maleimide group; L is absent or is an oxyethylene linker with molecular weight ranging from 100 to 1000 daltons, containing amino and carboxylic group to form an amido bond with R and the peptide, respectively; or L is a natural or non-natural amino acid residue, in L or D conformation, or 2 o 3 amino acid residues sequentially bonded.

6. A kit according to claim 5 wherein the L residue present in the bombesin peptide derivative comprises Lys, Orn, Asp or Glu residues functionalized with the chelating agents DTPA, GluDTPA, DOTA, NOTA, for the subsequent labelling with metal or paramagnetic ion radioactive isotopes.

7. A kit according to claim 1 wherein the peptide has the amino acid sequence Cys-L-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH.sub.2 wherein L is NH(CH.sub.2CH.sub.2O).sub.6-CH.sub.2CH.sub.2CO.

8. A kit according to claim 3 wherein the component d) is the DOTA-Peg-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2 derivative wherein Peg corresponds to a polyoxyethylene linker resulting from two condensed 8-amino-3,6-dioxaoctanoic acid units.

9. Method of preparing liposomal doxorubicin formulations according to claim 1, said method comprising externally modifying doxorubicin liposomes with a target peptide.

10. Method according to claim 9 wherein the target peptide is stable antagonistic bombesin analogue.

11. Method of treating ovarian tumor with the kit according to claim 1 in patients in need thereof, both first line and second line after the appearance of the resistance to platinum anticancer drugs, said method comprising administering said kit to said patients.

12. Method of diagnosing and treating patients suffering from ovarian tumor or metastatic ovarian tumor wherein overexpression of GRP receptors is present and wherein component d of the kit is used for selecting patients to be subjected to therapy with liposomal doxorubicin functionalized with a peptide of the bombesin family and monitoring the progression of the disease and the efficacy of the therapeutic treatment, said method comprising administering to said patients the kit according to claim 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0046] The invention concerns the obtainment of liposomal doxorubicin exposing a targeting peptide on the liposome external surface, according to a very efficient new method based on a kit.

[0047] The kit for the preparation contains: [0048] a) The sterile, translucent, red dispersion of the liposomal doxorubicin drug; [0049] b) A phospholipid modified with a reactive function (compound 1) in lyophilized form and in amount between 1% and 10% respect to the total lipid composition of the liposomes; better between 2% and 4%; [0050] c) A peptide modified with an appropriate reactive function (compound 2), in a 1:1 stoichiometric amount respect to compound 1 and having the general formula:

R-L-P

[0051] in which:

[0052] R is a residue containing a function able to react, giving a stable covalent bond, with one of the chemical functions present on Compound 1.

[0053] L is absent or is oxoethylene linker with a MW comprised between 100 and 1000 dalton, and containing amine and carboxylic functions to form amide bonds with R and P, respectively. or

[0054] L is an natural or non-natural amino acid residue in L or D conformation, or 2 or 3 amino acid residues sequentially linked.

[0055] P is a peptide sequence able to target with high affinity toward receptors over expressed by tumor cells belonging to the bombesin peptide family, to cholecistokinin peptide family, to the somatostatin peptide family or to the neurotensin peptide family.

[0056] The kit object of this invention could also contain an additional component (d) consisting of the targeting peptide P modified with a chelating agent capable of complexing radioactive metal ions for diagnostic and imaging in vivo applications.

[0057] The kit components a, b, c, and, possibly, d could be contained in vials, containers, little bottles or in pre-filled syringes and are here identified as (A), (B), (C) and, possibly, (D).

[0058] The modified phospholipid (compound 1) is preferably a DSPE-PEG2000 (N-(polyethylene glycol 2000)-1,2-diasteroyl-sn-glycero-3 -phosphoethanolamine) derivative modified with the introduction of reactive functions such as: maleimido, azide, dibenzocycloctyl, aldehyde, carboxy, pyridyldithiol propionate, succinyl or glutaryl groups.

[0059] Therefore, compound 1 is preferably chosen among: [0060] a) DSPEPEG(2000)Maleimide lipid; [0061] b) DSPEPEG(2000)azide lipid; [0062] c) DSPEPEG(2000)DBCO lipid (DBCO=dibenzocyclooctyl); [0063] d) DSPEPEG(2000)aldehyde lipid; [0064] e) DSPEPEG(2000)carboxilic acid lipid; [0065] f) DSPEPEG(2000)PDP lipid (PDP=pyridyldithiol propionate); [0066] g) DSPEPEG(2000)Succinyl lipid [0067] h) DSPEPEG(2000)Glutaryl lipid in which DSPE-PEG(2000) =N-(carbonyl-methoxypolyethylene glycol 2000)-1,2distearoyl-sn-glycero-3-phosphoethanolamine

[0068] In Compound 2 having the general formula R-L-P

[0069] The preferred R groups are:

[0070] Cys or SH(CH2)nCOOH (n in the range 1- 5) to react with the maleimide function present on Compound la or with PDP moiety present on compound lf;

[0071] Pra (Propargylglycine) or propyolic acid or DBCO to react with the azide function present on Compound lb, according to click chemistry procedures;

[0072] Azide to react with the DBCO moiety present on Compound lc, according to click chemistry procedures;

[0073] Amine to react with the carboxy function present on Compound 1e, 1g, or 1h.

[0074] L is absent or is a linker such as oxoethylene linker with a MW comprised between 100 and 1000 dalton, and containing amine and carboxylic functions to form amide bonds with R and P, respectively; or

[0075] L is an natural amino acid residue or a Beta-alanine residue, or 2 or 3 amino acid residues sequentially linked. If Lys, Orn, Glu, or Asp are present in the linker, they could also contain, on their side chain, a chelating agent such as DTPA (2,2,2,2-((((carboxymethyl)azanediyl) bis(ethane-2,1-diyl))bis(azanetriyl))tetraacetic acid), GluDTPA (N,N-Bis[2- [bis [2-(1,1-dimethylethoxy)-2-oxoethyl]-amino]ethyl]-L-glutamic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1, 4,7-triacetic acid) for labeling with a radio-active isotope or with a paramagnetic ion such as Gd(III).

[0076] P is a peptide sequence able to interact with nanomolar affinity toward target receptors over expressed by tumor cells. P belongs to one of the following peptide families.

[0077] 1) Peptide P Belongs to the Bombesin Family and is Chosen Among: [0078] the wild-type sequence of bombesin; [0079] the C-terminal fragments of peptide bombesin containing 6-9 amino acidic residues; [0080] analogues (agonist or antagonist) of the C-terminal fragment of the bombesin peptide with a general formula:

AA1-Gln-Trp-Ala-Val-AA2-His-AA3-AA4-NH.sub.2

[0081] wherein: [0082] AA1 is DPhe, D-Cpa (3-cyclopropyl-D-alanine), D-Tyr, D-Trp or is absent, [0083] AA2 is NMeGly, Gly or -Ala [0084] AA3 is Leu, Cha (cycloesil-alanine), Sta (statine), Met or Nle (Norleucine). [0085] AA4 is Met, Leu or Nle (Norleucine).

[0086] Peptides are bound to L (or in absence of L, directly to R) through their amino function on the N-terminus with formation of an amide bond.

[0087] The preferred peptide sequences, containing natural or unnatural amino acid residues are reported in Table 1.

TABLE-US-00001 TABLE1 Preferredpeptidesequences(P)inthe Bombesinfamily SEQID1 -Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH.sub.2 SEQID2 -Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH.sub.2 SEQID3 -Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH.sub.2 SEQID4 -Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH.sub.2 SEQID5 -DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu- NH.sub.2 SEQID6 -DPhe-Gln-Trp-Ala-Val-Gly-His-Cha-Nle- NH.sub.2 SEQID7 -DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta- Leu-NH.sub.2 SEQID8 -Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 SEQID9 -DPhe-Gln-Trp-Ala-Val-NMeGly-His-Leu- Nle-NH.sub.2

[0088] 2) Peptide P Belongs to the Cholecystokinin Family and is Chosen Among: [0089] the wild-type sequence of CCK8 peptide; [0090] the wild-type sequence of CCK8 peptide having Tyr in the sulphated form; [0091] the wild-type sequence of CCK8 peptide having Tyr in the sulphated form or in non-sulphated form and having Norleucine residues in substitution of the Met residues; [0092] the C-terminal fragments of peptide cholecystokinin containing between 5 and 9 amino acidic residues; [0093] the sequence of the peptide gastrin containing 1 or more D or L Glu residues at the N-terminus; [0094] analogues (agonist or antagonist) of the C-terminal fragment of the cholecystokinin peptide.

[0095] Peptides are bound to L (or in absence of L, directly to R) through their amino function on the N-terminus with formation of an amide bond.

[0096] The preferred peptide sequences, containing natural or unnatural amino acid residues are reported in Table 2.

TABLE-US-00002 TABLE2 Preferredpeptidesequences(P)inthe cholecystokininfamily SEQID1 Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2 SEQID2 Asp-Tyr(SO4)-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2 SEQID3 Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH.sub.2 SEQID4 Asp-Tyr(SO4)-Nle-Gly-Trp-Nle-Asp-Phe-NH.sub.2 SEQID5 DGlu-Glu.sub.n-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH.sub.2 (nfrom0to10) SEQID6 Asp-Tyr-Met-Gly-DTrp-NMeLeu-Asp-Phe-NH.sub.2

[0097] 3) Peptide P Belongs to the Somatostatin Family and is Octreotide or its Analogues with Amino Acidic Sequences Reported in Table 3:

TABLE-US-00003 TABLE3 Preferredpeptidesequences(P)inthesomatostatinfamily SEQID1 Octreotide:D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol)Cys-Cys cyclization SEQID2 Lanreotide:Beta-D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH.sub.2Cys-Cys cyclization SEQID3 Tyr3-Octrotide:D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr(ol)Cys-Cys cyclization SEQID4 Tyr3-Octrotate:D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-NH.sub.2Cys-Cys cyclization SEQID5 RC-160:D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH.sub.2Cys-Cys cyclization

[0098] 4) Peptide P belongs to the neurotensin family and is selected among the following peptides [0099] the peptide neurotensin with 13 residues and having the following sequence:

TABLE-US-00004 QLYENKPRRPYIL [0100] the peptide neurotensin with 6 residues and having the following sequence:

[0101] RRPYIL [0102] peptide QLYENKPRRPYIL in a tetrameric form linked to a Lysine scaffold having three lysine residues; [0103] peptide RRPYIL in a tetrameric form linked to a Lysine scaffold having three lysine residues; [0104] peptide QLYENKPRRPYIL in a dimeric form with the two peptides linked to the alpha and epsilon amine functions of a lysine residue; [0105] peptide RRPYIL in a dimeric form with the two peptides linked to the alpha and epsilon amine functions of a lysine residue;

[0106] The following examples clearly describe the new procedure and the use of the kit for the efficient preparation of the liposomal doxorubicin externally modified with a targeting peptide.

[0107] In particular, examples 1, 2 , 3 describes procedures and analytical controls for the preparation, by using the kit object of this invention, of liposomal Doxorubicin having liposomes derivatized on their external surface with an analogue and antagonist of the bombesin peptide to target tumors overexpressing GRP receptors. The final product is usable for the treatment of ovarian and prostate cancers.

EXAMPLES

Example 1

Post-Insertion of Compound I in Doxil, Lipodox (or Other Approved Analogues) Liposomal Drugs

[0108] In this example, Compound 1, contained in Vial (B), is DSPEPeg2000maleimide. It is inserted in the amount of 2% w/w of the total phospholipids, by using a post-insertion method in preformed liposomes loaded with doxorubicin such as the Doxil/Caelix drugs or the approved generic Lipodox drug.

[0109] In detail, Vial (A) contains a sterile, translucent, red liposomal dispersion of the doxorubicin drug (Doxil or Lipodox, Volume 25.0 mLDoxorubicin quantity 2.0 mg/mL corresponding to 50.0 mg). Vial (B) contains 32.3 mg of a GMP preparation of DSPEPeg2000maleimide, in lyophilized form (compound 1).

[0110] The procedure consists in the transferring of the entire content of Vial (A) into Vial (B). The final vial is placed on a shaker at room temperature and leaved to shake for 60 minutes. A complete dissolution of the lyophilized powder previously present into the vial is immediately observed. After shaking for 60 minutes, the complete insertion of DSPEPeg2000maleimide in preformed doxorubic containing liposomes could be confirmed by DLS experiments.

[0111] DLS measurements performed on a small aliquot of the solution should evidence the presence of liposomes with diameter around 100 nm and the absence of DSPEPeg2000maleimide pure micelles with a smaller diameter (below 50 nm).

Example 2

Reaction of Compound 2 With The Product Obtained in Example 1

[0112] In this example the preparation of the final compound starting from the product obtained in Example 1 is described. In this example is used a peptide derivative (compound 2) belonging to the bombesin family, in particular a stable antagonist analogue of the 1-9 C-terminal sequence of Bombesin. In this way the final product, liposomal doxorubicin modified with the bombesin analogue peptide, will be able to target in a selective way GRP receptors over expressed in several human tumor and could be used for therapy of such tumors such as ovaric cancer. The content of Vial B, obtained as described above, is transferred to Vial C containing compound 2. This vial contains 17.2 mg of a GMP preparation of a Bombesin analogue modified at N-terminus with a Cys residue, in lyophilized form. The preferred peptide sequence, used according to this example, is: Cys-L-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH.sub.2 in which L is NH(CH.sub.2CH.sub.2O).sub.6CH.sub.2CH.sub.2CO. The quantity of the peptide derivative (compound 2) (17.2 mg) is in 1:1 molar ratio with DSPEPeg2000maleimide (compound 1) inserted in liposomes according to the previously reported step (example 1). The vial is placed on a shaker at room temperature and leaved to shake for 30 minutes.

[0113] During the reaction time of 30 minutes a complete quantitative reaction is performed between the maleimide function present on the liposome external surface and sulphydryl function present on the peptide derivative. This guarantees on the absence of free peptide derivative in the final product.

Example 3

Characterization of the Final Product

[0114] Characterization of the final product aims to determine: i) if all the peptide derivative has completely reacted with the maleimido function and, thus the absence of the free peptide in solution; ii) if dimensions and polydispersity index of the final liposomes are the same of the non modified product initially contained in Vial (A); iii) if more than 90% of the total Doxorubicin is still contained in liposomes as in the starting product.

[0115] To verify point i) the procedure consists in the following steps: [0116] Transfer 250 microliters from final solution present in vial (C) to a clean eppendorf and add 150 microliters of distilled water. [0117] Transfer the solution to a Sephadex G50 column (i.e.: a GE HealthcareIllustra Nick Column) and elute with 800 microliters of water to obtain the liposomal fraction; [0118] Elute with additional 2.0 mL of water to obtain the free peptide fraction; [0119] Analysis by UV-Vis spectrometry: place in a 1.0 cm path length cell the free peptide fraction and read UV absorbance at 280 nm. The absence of free peptide is confirmed if Abs.sub.280<0.016. [0120] Analysis by HPLC chromatography: perform HPLC analysis on the free peptide fraction by using the following conditions: Column C18 (i.e.: JupiterPhenomenoex 15025); eluents: water/0.1% TFA (A) and Acetonitrile/0.1% TFA (B); method from 5% (B) to 70% (B) in 10 min; UV revelation at 210 nm. The peptide derivative should eluate at 8.0 min. The absence of free peptide is confirmed in absence of the peak at 8.0 min or with a peak around 8.0 min having an area <500. [0121] Analysis by fluoresce spectroscopy: analyze by fluorescence spectroscopy the liposomal fraction by exciting at 280 nm (excitation for the tryptophan residue) measuring fluorescence emission between 300 and 400 nm; an emission peak around 350 nm confirm the presence of the peptide on external surface of the liposomal drug.

[0122] To verify point ii) the procedure consists in the following steps: [0123] Transfer 250 microliters from final solution present in vial (C) to a clean eppendorf; [0124] Measure liposome dimensions by DLS; positive results are obtained if liposome diameter is 9815 nm with a polydispersity index of 0.200.05.

[0125] To verify point iii) the procedure consists in the following steps: [0126] Transfer 100 microliters from the starting liposomal doxorubicin product present in vial (A) to a clean eppendorf and add 300 microliters of distilled water; [0127] Transfer the solution to a Sephadex G50 column (i.e.: a GE HealthcareIllustra Nick Column) and elute with 800 microliters of water to obtain the liposomal fraction; [0128] Elute with additional 2.0 mL of water to obtain the free Doxorubicin fraction; [0129] Analysis by HPLC chromatography: perform HPLC analysis on the free doxorubicin fraction by using the following conditions: Column C18 (i.e.: JupiterPhenomenoex 15025); eluents: water/0.1% TFA (A) and Acetonitrile/0.1% TFA (B); method from 5% (B) to 70% (B) in 10 min; UV revelation at 210 nm. The free doxorubicin should eluate at 10.7 min. Measure the area of the peak. [0130] Perform the same operations starting from the final product present in Vial (C). The area of the peaks corresponding to free doxorubicin in the analysis of sample in the starting product (Vial A) and in the analysis of sample in final product (Vial C) should be comparable with an error of 20%.