Smart Drug Delivery System and Pharmaceutical Kit for Dual Nuclear Medical Cytotoxic Theranostics

Abstract

The invention generally relates to a smart drug delivery system for dual nuclear medical cytotoxic theranostics incorporating either (i) a first compound with the structure CT-L1-Chel-S1-TV or

##STR00001##

or (ii) a second compound with the structure Chel-S-TV and a third compound with the structure CT-L-TV. In the first, second and third compounds Chel is a radical of a chelating agent for complexing a radioisotope; CT is a radical of a cytotoxic compound; TV is a biological targeting vector; L1 and L are each linkers; S1, S2 and S are each spacers.

Claims

1. A compound for dual nuclear-medical/cytotoxic theranostics having the structure ##STR00145## wherein Chel is a radical of a chelator for the complexation of a radioisotope; CT is a radical of a cytotoxic compound; TV is a biological targeting vector; L1 is a linker; S1 and S2 are each a spacer.

2. A smart drug delivery system for dual nuclear-medical/cytotoxic theranostics, comprising a first compound as claimed in claim 1 having the structure ##STR00146## or a second compound having the structure Chel-S-TV and a third compound having the structure CT-L-TV; wherein, in the first, second and third compounds, Chel is a radical of a chelator for the complexation of a radioisotope; CT is a radical of a cytotoxic compound; TV is a biological targeting vector; L1 and L are each a linker; S1, S2 and S are each a spacer.

3. A pharmaceutical kit for dual nuclear-medical/cytotoxic theranostics as claimed in claim 1, consisting of a first vessel containing a first compound or a first carrier substance containing the first compound; or a second vessel containing a second compound or a second carrier substance containing the second compound, and a third vessel containing a third compound or a third carrier substance containing the third compound; wherein the first compound has the structure ##STR00147## the second compound has the structure Chel-S-TV; and the third compound has the structure CT-L-TV, wherein Chel is a radical of a chelator for the complexation of a radioisotope; CT is a radical of a cytotoxic compound; TV is a targeting vector selected from one of the structures [1] to [18] ##STR00148## ##STR00149## where the structures [1] to [8] and [18] denote amino acid sequences; L and L1 independently have a structure selected from ##STR00150## in which M1, M2, M3, M4, M5, M6, M7, M8 and M9 are independently selected from the group comprising amide, carboxamide, phosphinate, alkyl, triazole, thiourea, ethylene, maleimide radicals, —(CH.sub.2)—, —(CH.sub.2CH.sub.2O)—, —CH.sub.2—CH(COOH)—NH— and —(CH.sub.2).sub.mNH— with m=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n1, n2, n3, n4, n5, n6, n7, n8 and n9 are independently selected from the set of {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}; Clv is a cleavable group; QS is a squaric acid radical ##STR00151## S is the same as L (S=L); and/or S, S1 and S2 independently have a structure selected from ##STR00152## in which O1, O2 and O3 are independently selected from the group comprising amide, carboxamide, phosphinate, alkyl, triazole, thiourea, ethylene, maleimide radicals, —(CH.sub.2)—, —(CH.sub.2CH.sub.2O)—, —CH.sub.2—CH(COOH)—NH— and —(CH.sub.2).sub.qNH— with q=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and p1, p2 and p3 are independently selected from the set of {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}.

4. The radiopharmaceutical kit as claimed in claim 3, wherein CT is a radical of a cytotoxic compound selected from adozelesin, alrestatin, anastrozole, anthramycin, bicalutamide, bizelesin, bortezomib, busulfan, camptothecin, capecitabine, carboplatin, carzelesin, CC-1065, chlorambucil, cisplatin, cyclophosphamide, cytarabine (ara-C), dacarbazine (DTIC), dactinomycin, daunorubicin, dexamethasone, disulfiram, docetaxel, doxorubicin, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, erismodegib, etoposide (VP-16), fludarabine, fluorouracil (5-FU), flutamide, fulvestrant, gemcitabine, goserelin, idarubicin, ifosfamide, L-asparaginase, leuprolide, lomustine (CCNU), mechlorethamine (nitrogen mustard), megestrol acetatr, melphalan (BCNU), menadione, mertansine, metformin, methotrexate, milataxel, mitoxantrone, monomethylauristatin E (MMAE), motesanib, maytansinoid, napabucasin, NSC668394, NSC95397, paclitaxel, prednisone, pyrrolobenzodiazepine, pyrvinium pamoate, resveratrol, rucaparib, S2, S5, salinomycin, saridegib, shikonin, tamoxifen, temozolomide, tesetaxel, tetrazole, tretinoin, verteporfin, vinblastine, vincristine, vinorelbine, vismodegib, α-chaconine, α-solamargine, α-solanine, or α-tomatine.

5. The radiopharmaceutical kit as claimed in claim 3, wherein the cleavable group Clv is selected from the group comprising ##STR00153##

6. The radiopharmaceutical kit as claimed in claim 3, wherein the chelator Chel is selected from the group comprising H.sub.4pypa, EDTA (ethylenediaminetetraacetate), EDTMP (diethylenetriaminepenta(methylenephosphonic acid)), DTPA (diethylenetriaminepentaacetate) and derivatives thereof, DOTA (dodeca-1,4,7,10-tetraaminetetraacetate), DOTAGA (2-(1,4,7,10-tetraazacyclododecane-4,7,10)-pentanedioic acid) and other DOTA derivatives, TRITA (trideca-1,4,7,10-tetraaminetetraacetate), TETA (tetradeca-1,4,8,11-tetraaminetetraacetate) and derivatives thereof, NOTA (nona-1,4,7-triaminetriacetate) and derivatives thereof, TRAP (triazacyclononanephosphinic acid), NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)-phosphinic acid]-7-[methylene(2-carboxyethyl)-phosphinic acid]), PEPA (pentadeca-1,4,7,10,13-pentaamine pentaacetate), HEHA (hexadeca-1,4,7,10,13,16-hexaamine hexaacetate) and derivatives thereof, HBED (hydroxybenzylethylene-diamine) and derivatives thereof, DEDPA and derivatives thereof, DFO (deferoxamine) and derivatives thereof, trishydroxypyridinone (THP) and derivatives thereof, TEAP (tetraazacyclodecanephosphinic acid) and derivatives thereof, AAZTA (6-amino-6-methylperhydro-1,4-diazepine-N,N,N′,N′-tetraacetate) and derivatives; SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) and salts thereof, (NH.sub.2).sub.2SAR (1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane) and salts and derivatives thereof, or aminothiols and derivatives thereof.

7. The radiopharmaceutical kit as claimed in claim 3, wherein the spacer S is the same as L (S=L).

8. The radiopharmaceutical kit as claimed in claim 3, wherein the first, second and third carrier substances are independently selected from the group comprising water, 0.45% aqueous NaCl solution, 0.9% aqueous NaCl solution, Ringer's solution (Ringer's lactate), 5% aqueous dextrose solution and aqueous alcohol solutions.

9. The radiopharmaceutical kit as claimed in claim 3, wherein the NOTA derivative is NOTAGA (1,4,7-triazacyclononane, 1-glutaric acid, 4,7-acetate), the DEDPA derivative is H.sub.2DEDPA (1,2-[[6-(carboxylate-)pyridin-2-yl]methylamino]ethane), the trishydroxypyridinone derivative is YM103, and the AAZTA derivative is DATA ((6-pentanoic acid)-6-(amino)methyl-1,4-diazepine triacetate).

Description

EXAMPLES

Example 1: Dual Active Ingredient Conjugates

[0155] Schemes 13 to 22 show examples of inventive dual active ingredient conjugates according to FIG. 1a, comprising a targeting vector, a chelator for labeling with a radioisotope and a cytotoxic active ingredient.

##STR00114##

##STR00115##

##STR00116##

##STR00117##

##STR00118##

##STR00119##

##STR00120##

##STR00121##

##STR00122##

##STR00123##

Example 2: Dual Active Ingredient Conjugates According to FIG. 1b

[0156] Schemes 23, 24, 25 and 26 show examples of inventive dual active ingredient conjugates according to FIG. 1b, comprising a targeting vector, a chelator for labeling with a radioisotope, a cleavable linker and a cytotoxic active ingredient.

##STR00124##

##STR00125##

##STR00126##

##STR00127##

Example 3: Active Ingredient Conjugates According to FIG. 1d

[0157] Schemes 27, 28, 29 and 30 show examples of inventive active ingredient conjugates according to FIG. 1d, comprising a targeting vector, a cleavable linker and a cytotoxic active ingredient.

##STR00128##

##STR00129##

##STR00130##

##STR00131##

Example 4: Synthesis Strategy for PSMA Labeling Precursors

[0158] In the synthesis of the active ingredient conjugates of the invention, preference is given to using squaric diesters. In this way, it is possible to prepare a multitude of in some cases very complex active ingredient conjugates by means of simple reactions. Squaric diesters are notable for their selective reaction with amines, such that protecting groups are not required for the coupling of chelators, linkers, spacers and targeting vectors. Moreover, the coupling reaction is controllable via the pH.

[0159] First, a targeting vector for PSMA is synthesized (see scheme 31a) and, after purification, in aqueous medium at pH=7, reacted with squaric diester to give a precursor for coupling with a chelator (see scheme 32). Alternatively, the coupling can also be conducted in an organic medium with triethylamine as base.

##STR00132##

[0160] The target vector synthesized for PSMA by means of a known method is, for example, the PSMA inhibitor L-lysine-urea-L-glutamate (KuE) (cf. scheme 31b). This involves reacting a polymer resin-bound and tert-butyloxycarbonyl-protected (tert-butyl-protected) lysine with di-tert-butyl-protected glutamic acid. After the protected glutamic acid has been activated by triphosgene and coupled to the solid-phase-bound lysine, L-lysine-urea-L-glutamate (KuE) is eliminated by means of TFA and at the same time fully deprotected. The product can subsequently be separated from free lysine by means of semipreparative HPLC with a yield of 71%.

##STR00133##

[0161] The PSMA inhibitor KuE (1) can then be coupled by means of diethyl squarate as coupling reagent to a labeling precursor (cf. scheme 32). The coupling of KuE (1) to squaric diester is effected in 0.5 M phosphate buffer at a pH of pH 7. After the two reactants have been added, the pH has to be readjusted with sodium hydroxide solution (1 M) since the buffer capacity of the phosphate buffer is insufficient. At pH 7, the single amidation of the acid proceeds at room temperature with a short reaction time. KuE-QS (2) is obtained after HPLC purification with an overall yield of 16%.

##STR00134##

[0162] The KuE squaric acid monoester thus obtained is storable and can be used as a building block for further syntheses.

Example 5: Solid-Phase-Based Synthesis of the KuE Unit and of the PSMA-617 Linker

[0163] The conjugation of the glutamate-urea-lysine binding motif KuE to an aromatic linker unit was effected by a solid-phase peptide synthesis described by Benesova et al. (Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors; J Med Chem, 2016, 59, 1761-1775). The synthesis reported by Benesova et al. was modified slightly (cf. scheme 33).

##STR00135##

##STR00136##

##STR00137##

##STR00138##

Example 6: Synthesis of the Coupling-Capable DOTAGA Chelator and Coupling Thereof to the PSMA-617 Target Vector-Linker Unit

[0164] The synthesis proceeds from commercially available DO2A(tBu)-GABz, which is functionalized on the secondary amine with a Boc-protected amino group (cf. scheme 34).

[0165] This enables the late introduction of the cytostatic-linker unit.

##STR00139##

[0166] The benzyl protecting group of the glutaric acid side chain of the DOTAGA(COOtBu)3(NHBoc)-GABz 4 is reductively removed in order to enable coupling to the PSMA target vector via a linker.

[0167] Then the linker-PSMA conjugate is coupled to the chelator 6 by means of amide coupling.

##STR00140##

[0168] The coupling of the chelator 6 to the KuE-bound linker is described in scheme 35. The protected PSMA617 derivative 7 obtained by the amide coupling is deprotected with the aid of trifluoroacetic acid (TFA) and separated from the solid phase. The overall yield of the two-stage synthesis after HPLC purification is 6%.

Example 7: Synthesis of the Inventive Compound MMAE.ValCit.QS.617.KuE

[0169] ##STR00141##

[0170] The synthesis of the compound MMAE.ValCit.QS.617.KuE proceeds from commercially available MMAE.ValCit, which is coupled to diethyl squarate at a pH of 7 in phosphate buffer (0.5 M) with addition of DMSO (cf. scheme 36). This is followed by solid-phase-based coupling of the MMAE.ValCit.QS unit and the 617.KuE-linker-target vector unit in ethanol with addition of 2% triethylamine. After HPLC purification, the yield of the synthesis was 43%.

Example 8: Radiolabeling

[0171] For the radiolabeling of the PSMA labeling precursors, .sup.68Ga was eluted from an ITG Ge/Ga generator with 0.05 M HCl and process by means of aqueous ethanol elution through a cation exchange column. According to the chelator, radiolabeling is effected at pH values between 3.5 and 5.5 and temperatures between 25° C. and 95° C. The progress of the reaction was recorded by means of HPLC and IPTC in order to ascertain the kinetic parameters of the reaction.

Example 9: Squaric Acid as Complexing Aid

[0172] For clinical use, it is very important that complexation proceeds efficiently at low temperature. Squaric acid complexes free metals and can thus protect the chelator site from non-specific coordination. This effect has been observed in the case of radiolabeling of TRAP.QS at different temperatures. TRAP complexes quantitatively at room temperature. By contrast, under the same conditions, in the case of TRAP.QS, an RCY value of only 50% was measured. If the temperature is increased, there is a rise in the labeling yield of TRAP.QS to quantitative values. This shows the influence that squaric acid has on complexation. This effect, illustrated in scheme 37, enables the stable complexation of metals having a high coordination number, for example zirconium, with the aid of the chelator AAZTA.QS.

##STR00142##

[0173] In appropriate embodiments of the pharmaceutical kit of the invention, the first, second and/or third compound contains one or more squaric acid radicals QS. The use of squaric diesters allows coupling reactions to be simplified considerably.

Example 10a: Squaric Acid as Affinity Promoter

[0174] Moreover, the inventors have found that, surprisingly, the incorporation of squaric acid groups QS improves pharmacological properties and increases the binding affinity of PSMA-specific targeting vectors. The inventors suspect that the binding affinity is increased by ionic interaction of the squaric acid group QS with ARG463. To verify this hypothesis, docking studies were conducted. FIGS. 3 and 4 show the arrangements favored on the basis of the docking studies. ARG463 is located in what is called the arginine patch of PSMA. A further putative mechanism of action is based on hydrogen bonds to Trp541, which increase affinity for the arene binding pocket of PSMA.

[0175] The squaric acid group interacts with Arg463 in the arginine-rich region (dark region) and with Trp541 in the arene binding pocket. The dotted light-colored lines represent the distance in Å. The zinc ions present in the active binding pocket are shown as spheres. The structure data are based on the structure, determined by means of x-ray diffraction, of PSMA in complex with PSMA 1007 (PDB 5O5T).

[0176] FIG. 5 shows the putative binding mode of AAZTA.QS.KuE in the binding pocket of PSMA. The AAZTA chelator project out of the PSMA pocket. The QS linker interacts with the hydrophobic portion of the binding pocket. The binding motif is in the pharmacophore portion of the pocket and is complexed by the two zinc ions. FIG. 6 shows the putative binding mode of DATA.QS.EuE. The EuE binding motif causes an extension of the linker and associated spatial shift of the QS linker, which impairs electrostatic interaction with the amino acids in the binding pocket. Subsequent in vitro assays confirmed the results of the docking analyses.

Example 10b: Squaric Acid as Modulator of Excretion

[0177] Scheme 38 shows an example of an active ingredient conjugate or labeling precursor with a targeting vector for PSMA and a squaric acid group conjugated to the targeting vector.

##STR00143##

[0178] The conjugation of squaric acid (QS) to the PSMA Tracer reduces accumulation in the kidneys and the associated masking or disturbance of the PET signal from the adjacent prostate, which crucially improves sensitivity and reliability in the imaging diagnosis of prostate carcinoma by means of PET. FIGS. 7a and 7b show μPET images (60 min p.i.) Of [.sup.68Ga]Ga.DOTA.QS.PSMA (A), [.sup.68Ga]Ga-PSMA-11 (B) and [.sup.68Ga]Ga-PSMA-617 (C) and a diagram with SUV values (standard uptake value: SUV) for tumor tissue, kidney and liver.

[0179] Scheme 39 shows a further QS derivative that has been tested in vivo in tumor-carrying animals.

##STR00144##

[0180] DATA.QS.KuE was labeled with .sup.68Ga and tested in vivo on LNCaP tumor-carrying Balb/c mice. FIG. 8 shows the accumulation of [.sup.68Ga]-DATA.QS.KuE in the organs (biodistribution). The selectivity of binding was determined by means of competitive co-injection of the PSMA inhibitor PMPA. By way of comparison, FIG. 9 shows the biodistribution of [.sup.68Ga]-PSMA-11.

[0181] FIGS. 10a and 10b show the maximum-intensity projections from μPET studies with [.sup.68Ga]-PSMA-11 and, respectively, [.sup.68Ga]-DATA.QS.KuE in LNCaP tumor-carrying Balb/c.

[0182] FIGS. 11a and 11b Showtime-activity curves of [.sup.68Ga]-PSMA-11 and, respectively, [.sup.68Ga]-DATA.QS.KuE. With approximately the same tumor enrichment, DATA.QS.KuE, by comparison with PSMA-11, shows considerably lower kidney exposure/dose. In the case of treatment with highly ionizing radionuclides, for example .sup.177Lu, rather than .sup.68Ga, DATA.QS.KuE enables a crucial reduction in nephrotoxicity.

Example 11a: Evaluation of the In Vitro PSMA Binding Affinity of Selected Compounds and Compound Constituents

[0183] By means of a cell-based assay, the affinity of the target vector-linker units QS.KuE, QS.K.EuE and KuE with a lipophilic linker—analogously to PSMA-617—and the affinity of the substructures NH.sub.2.DOTAGA.617.KuE and NH.sub.2-DOTAGA.QS.KuE was determined. In addition, the PSMA affinity of the structure MMAE.ValCit.QS.617.KuE which is preferred in accordance with the invention (see scheme 30) was determined.

[0184] For the essay, LNCaP cells were pipetted into multiwell plates (Merck Millipore Multiscreen™). The compounds to be analyzed were each admixed with a defined amount or concentration of the reference compound .sup.68Ga[Ga]PSMA-10 with a known K.sub.d value and incubated in the wells with the LNCaP cells for 45 min. After repeated washing, the cell-bound activity was determined. The inhibition curves obtained were used to calculate the IC.sub.50 values and K.sub.i values reported in table 1.

TABLE-US-00003 TABLE 3 PSMA binding affinities Compound IC.sub.50 (nM) K.sub.i (nM) PSMA-617 15.1 ± 3.8 12.3 ± 3.1 QS.KuE-TV linker unit 35.9 ± 2.6 29.3 ± 2.1 QS.EuE-TV linker unit 17.2 ± 5.2 14.0 ± 4.2 617.KuE-TV linker unit 21.5 ± 1.9 17.5 ± 1.5 NH2.DOTAGA.617.KuE 20.2 ± 3.6 16.5 ± 3.0 [natGa]Ga-NH2.DOTAGA.617.KuE 20.4 ± 9.4 16.8 ± 7.7 [natLu]Lu-NH2.DOTAGA.617.KuE 26.0 ± 4.7 21.4 ± 3.9 NH2.DOTAGA.QS.KuE 20.2 ± 3.5 18.1 ± 2.9 DATA.QS.EuE 386.0 ± 81.0 315.4 ± 66.2 MMAE.ValCit.QS.617.KuE 198.1 ± 1.9  161.9 ± 3.3 

[0185] In order to determine non-specific binding, all compounds were additionally admixed with an excess of the PSMA inhibitor 2-PMPA (2-(phosphonomethyl)-pentanoic acid) and subjected to the same LNCaP assay—as described above.

[0186] Both the TV linker units and the chelator-TV linker units have similar affinity for PSMA to the reference compound PSMA-617. Accordingly, the use of QS as linker unit leads to an affinity comparable to the use of the peptidic PSMA-617. Neither coupling to the DOTAGA chelator nor labeling thereof with the radionuclides gallium-68 and lutetium-177 leads to any decrease in affinity.

[0187] The use of the binding unit EuE rather than KuE leads to a considerable deterioration in PSMA affinity. The results confirm the findings of the docking studies with regard to the unfavorable orientation of the EuE derivative in the PSMA binding pocket.

[0188] The coupling of the sterically demanding cytostatic MMAE and the ValCit linker and the TV linker unit QS.617.KuE leads to a distinct lowering of affinity.

Example 7b: Determination of the Cytotoxic Action of the Dimeric Compound MMAE.ValCit.QS.617.KuE In Vitro

[0189] In a CellTiter Blue assay, LNCaP cells were incubated with the substance to be studied for 72 hours, and then the IC.sub.50 of the compound was determined. Table 4 shows the IC.sub.50 values of the compound MMAE.ValCit.QS.617.KuE which is preferred in accordance with the invention (scheme 30) compared to the pure active ingredient MMAE.

TABLE-US-00004 TABLE 4 Cytotoxic action in vitro Compound IC.sub.50 (nM) MMAE 0.29 ± 0.12 MMAE.ValCit.QS.617.KuE 32.2 ± 5.7 

[0190] Although the inventive compound MMAE.ValCitQS.617.KuE shows somewhat lower cell cytotoxicity in vitro than the pure active ingredient MMAE, it is nevertheless in the lower nanomolar range.