Promoieties for forming prodrugs selectively cleaved by prostate-specific antigen (PSA)

Abstract

Disclosed are promoieties and compositions comprising the promoieties for use in therapeutically or diagnostically effective amounts in methods for detecting and / or treating prostate cancer in a subject, the promoieties comprising a first peptide and a second peptide linked to the first peptide, the peptide comprising a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) and a second end configured for highly selective binding to the active site of PSA, the second peptide comprising a negatively charged sequence to delay cellular uptake of the prodrug, and the second peptide being cleaved from the first peptide upon proteolysis by PSA to generate a first peptide-drug conjugate suitable for uptake by target cells.

Description

[Technical Field]

[0001] The present invention relates to promoieties for forming prodrugs that are rapidly and / or selectively cleaved by prostate-specific antigen (PSA), compositions comprising the prodrugs formed using said promoieties, and methods and uses for detecting and / or treating prostate cancer in a subject by administering a therapeutically or diagnostically effective amount of the composition to the subject.

[0002] The present invention has been developed primarily for use in the detection and / or treatment of prostate cancer and is described below with reference to this application. Those skilled in the art will appreciate that the present invention may be applied to the treatment of other conditions, particularly conditions mediated by proteolytic enzymes. [Background technology]

[0003] Prostate cancer cells express very high levels of PSA, a chymotrypsin-like serine protease also known as human kallikrein 3 (KLK3 or hK3), which can be measured in the blood and therefore can be used as a clinical test to detect prostate cancer and track response to treatment.

[0004] Prodrugs may incorporate masking moieties that are removed from the actual drug moiety under target conditions by mechanisms such as degradation or enzyme-mediated cleavage.

[0005] In this regard, prostate cancer can be targeted for drug delivery by exploiting the expression of PSA, where PSA cleaves the prodrug substrate, releasing the drug from the masking moiety. Because PSA is enzymatically active at high levels only in the extracellular fluid surrounding healthy and cancerous prostate cells, where the active form is expressed at high concentrations, exploiting PSA offers significant advantages for the diagnosis and treatment of prostate cancer.

[0006] However, for this approach to work, the bond between the drug-containing moiety and the masking moiety must be rapidly cleaved by PSA while remaining resistant to cleavage by other enzymes. These dual requirements for selectivity and timely activation are thought to have slowed the development of drugs for detecting and / or treating prostate cancer masked in this way.

[0007] Accordingly, the present invention seeks to provide promoieties for conjugation to drugs to form prodrugs that are rapidly and / or selectively cleaved by prostate-specific antigen (PSA), compositions comprising said promoieties conjugated to drugs to form prodrugs, and methods and uses for detecting and / or treating prostate cancer in a subject by administering the compositions to the subject in a therapeutically or diagnostically effective amount that overcome or substantially ameliorate, or at least provide an alternative to, at least some of the deficiencies of the prior art.

[0008] Where any prior art information is referred to herein, it should be understood that such reference does not constitute an acknowledgement that the information forms part of the common general knowledge in the art in Australia or any other country. Summary of the Invention

[0009] According to a first aspect of the invention, there is provided a promoiety comprising: a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) and at a second end for binding with high selectivity to the active site of PSA; the second peptide comprises a sequence having a negative charge to delay cellular uptake of the prodrug; The second peptide provides a promoiety that is cleaved from the first peptide upon proteolysis by PSA to generate a first peptide-drug conjugate suitable for uptake by target cells.

[0010] According to a second aspect of the invention, there is provided a composition comprising a promoiety, wherein the promoiety a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) and at a second end for binding with high selectivity to the active site of PSA; the second peptide comprises a sequence having a negative charge to delay cellular uptake of the prodrug; The second peptide provides a promoiety that is cleaved from the first peptide upon proteolysis by PSA to generate a first peptide-drug conjugate suitable for uptake by target cells.

[0011] In one embodiment, the first peptide sequence comprises HisSerSerLysLeuGln (HSSKLQ).

[0012] Preferably, the first end of the first peptide is the N-terminus.

[0013] Preferably, the second peptide comprises one or more amino acids that are negatively charged at physiological pH.

[0014] In one embodiment, the second peptide comprises the sequence D-Asp-D-Glu(de).

[0015] Preferably, the second peptide is linked to the C-terminus of the first peptide via a spacer.

[0016] More preferably, the spacer is an amino acid sequence in which the first amino acid of the spacer is compatible with the active site of PSA.

[0017] Even more preferably, the first amino acid of the spacer is a leucine residue.

[0018] In one embodiment, the spacer comprises the amino acid sequence LeuGlyGly (LGG).

[0019] According to a third aspect of the present invention there is provided a method for detecting and / or treating prostate cancer in a subject, the method comprising administering to the subject a therapeutically or diagnostically effective amount of a composition according to the second aspect.

[0020] In one embodiment, the composition is administered intratumorally and / or intraprostatically.

[0021] In one embodiment, the composition is administered intravenously, intramuscularly, and / or subcutaneously.

[0022] In one embodiment, the subject has a localized prostate tumor.

[0023] In one embodiment, the subject has a metastatic prostate tumor.

[0024] Preferably, the administration results in a reduction in prostate tumor volume.

[0025] Preferably, the administration results in a reduction in metastatic prostate tumors.

[0026] Preferably, the administration results in the treatment of metastatic prostate tumors.

[0027] According to a fourth aspect of the present invention there is provided the use of a composition according to the second aspect in the manufacture of a medicament for detecting and / or treating prostate cancer.

[0028] According to one embodiment of the present invention, there is provided a promoiety comprising: a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by a target protease and at a second end for highly selective binding to the active site of the target protease; the second peptide comprises a sequence having a negative charge to delay cellular uptake of the prodrug; The second peptide is cleaved from the first peptide upon proteolysis by the target protease to generate a first peptide-drug conjugate suitable for uptake by the target cell.

[0029] According to another embodiment of the present invention, there is provided a composition comprising a promoiety, wherein the promoiety is: a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by a target protease and at a second end for highly selective binding to the active site of the target protease; the second peptide comprises a sequence having a negative charge to delay cellular uptake of the prodrug; The second peptide is cleaved from the first peptide upon proteolysis by the target protease to generate a first peptide-drug conjugate suitable for uptake by the target cell.

[0030] In a preferred embodiment, the target protease is prostate-specific antigen (PSA).

[0031] Other aspects of the invention are also disclosed. [Brief explanation of the drawings]

[0032] Notwithstanding any other forms which may fall within the scope of the present invention, preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1]

[0023] Figure 1 shows a proof-of-concept strategy used in the development of a model prodrug containing a promoiety according to a preferred embodiment of the present invention for conjugation to a drug to form a prodrug. (a) The negatively charged sequence on the model prodrug is repelled from the cell membrane, reducing uptake prior to cleavage by prostate-specific antigen (PSA). (b) The model prodrug interacts with the active site and arginine patch of PSA and is cleaved; (c) The model active drug is taken up into the cell and the remaining negatively charged sequence is removed; (d) The model active drug is imaged in the cell using a fluorescence microscope. [Figure 2] Schematics of model prodrugs are shown in which the drug moiety of the prodrug is replaced with a fluorescent tag with a structure comparable to that of the drug. [Figure 3] Figure 1 shows the chemical structure of the promoiety HSSKLQ↓LGGde for conjugation to a drug to generate a prodrug, where ↓ represents the position of PSA cleavage (PSA cleavable bond). [Figure 4] The following chemical structures are shown: (a) a model prodrug peptide sequence, tag-HSSKLQ↓LGGde, in which the drug moiety of the prodrug is replaced with either (c) a fluorescent anthraquinone (aqueous) tag or (d) a luminescent rhenium(I) complex tag; and (b) a fragment of the tag-HSSKLQ↓LGGde peptide sequence after cleavage at the PSA-cleavable bond upon exposure to PSA (tag-HSSKLQ and LGGde), where ↓ represents the position of PSA cleavage (PSA-cleavable bond). [Figure 5] Photodiode array detection (PDA) traces (254 nm, 20–30 min) of (a) the peptide sequence of the model prodrug, AQ-HSSKLQ↓LGGde (green, RT = 27.4 min), and (b) the peptide sequence of the corresponding AQ-HSSKLQ (blue, RT = 26.2 min) fragment resulting from cleavage of the AQ-HSSKLQ↓LGGde model prodrug, are shown after 3, 6, 24, and 48 h of incubation with PSA. [Figure 6]Composite images of the fluorescent and brightfield channels of DLD-1 cells (a colorectal adenocarcinoma cell line) treated with AQ-HSSKLQ at a concentration of 50 μM for 1, 4, and 24 hours either in the absence of PSA (no PSA) or with added PSA (2 μg, 67 pmol) [7.5 × 10 cells were seeded per well on a 96-well glass-bottom plate. Scale bar represents 30 μm]. [Figure 7] Composite images of the fluorescence and bright-field channels of DLD-1 cells treated with AQ-HSSKLQ↓LGGde at a concentration of 50 μM for 1, 4, and 24 hours in the absence of PSA (no PSA) or with the addition of PSA (2 μg, 67 pmol) are shown (7.5 × 10 cells were seeded per well on a 96-well glass-bottom plate. Scale bar represents 30 μm). [Figure 8] Fluorescence, brightfield, and merged images of DLD-1 cells treated with 100 μM AQ-HSSKLQ↓LGGde for 48 hours in the absence of PSA (no PSA), 1 μg of PSA (33 pmol), or 2 μg of PSA (67 pmol) are shown. [2.5 × 10 cells were seeded per well on a 96-well plastic-bottom plate. Scale bar represents 30 μm. Fluorescence images were processed using brightness and contrast settings ranging from 20 to 1000.] DETAILED DESCRIPTION OF THE INVENTION

[0033] In the following description, it should be noted that like or identical reference numbers in different embodiments indicate the same or similar features.

[0034] In a general form, the invention provides a promoiety for conjugating to a drug to form a prodrug, the promoiety comprising: (i) a first peptide conjugated to the drug; and (ii) a second peptide linked to the first peptide, wherein the first peptide comprises a sequence that binds with high selectivity to a target protease; the second peptide comprises a sequence bearing one or more negative charges to slow uptake of the prodrug by target cells and increase the selectivity of binding to PSA via association with the arginine patch; and the second peptide is cleaved from the first peptide upon proteolysis by the target protease to generate a first peptide-drug conjugate suitable for uptake by the target cell.

[0035] In a preferred embodiment, as described in more detail below, the target protease is prostate-specific antigen (PSA).

[0036] To this end, it will be understood that: (1) Prostate-specific antigen (PSA) has high substrate specificity for cleaving after glutamine (Gln) residues, a preference that is not shared by other proteases in human serum or by proteases with similar structure and function to PSA. (2) The hydrophilic HisSerSerLysLeuGln (HSSKLQ) peptide sequence is an efficiently cleaved peptide sequence specific for PSA; (3) PSA has a positively charged region near the active site (i.e., an arginine patch consisting of Arg36, Arg38, and Arg60 residues creates a high density of positive charges in the vicinity of the active site).

[0037] With this in mind, the inventors have used molecular modeling to design prodrugs that are rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) containing a promoiety for conjugation to drugs for the specific purpose of detecting and / or treating prostate cancer, where the promoiety comprises a negatively charged peptide sequence positioned to electrostatically interact with the positively charged arginine patch of PSA in order to increase the prodrug selectivity for PSA and / or cleavage rate.

[0038] composition The present invention provides compositions comprising a promoiety for conjugating to a drug to form a prodrug, wherein the drug, in a preferred embodiment, is designed to detect and / or treat prostate cancer.

[0039] Here, the prodrug comprises a peptide-based promoiety for conjugation to a drug, the peptide-based promoiety containing a negatively charged sequence that serves both to electrostatically interact with the positively charged arginine patch of prostate-specific antigen (PSA) and to slow the diffusion of the prodrug across the membrane of a cell due to charge repulsion. Only when the peptide-based promoiety is selectively cleaved in the presence of PSA to remove the negatively charged sequence can the remaining conjugate of the drug and part or all of the first peptide diffuse across the cell membrane.

[0040] The pro-moiety comprises a first peptide having a sequence that binds with high selectivity to the active site of prostate-specific antigen (PSA), and a second peptide that is linked to the first peptide sequence via a spacer sequence and includes a negatively charged sequence.

[0041] FIG. 1 shows the proof-of-concept strategy used in the development of the model prodrugs described below.

[0042] As shown in Figure 1(a), the negatively charged sequence of the promoiety associated with the model prodrug is repelled from the cell membrane, reducing uptake prior to cleavage by PSA.

[0043] Figure 1(b) shows that when PSA is present in the extracellular fluid surrounding a target cell, the model prodrug interacts with the active site and arginine patch of PSA, promoting cleavage of the second peptide from the first peptide by PSA, releasing a conjugate of the first peptide and the drug moiety.

[0044] Figure 1(c) shows that because the first peptide-drug conjugate no longer carries a net negative charge, it is no longer repelled by the cell membrane and can therefore diffuse across the cell membrane for uptake by the target cell, while the remaining negatively charged sequences are excluded.

[0045] Successful uptake of the first peptide / drug conjugate by the target cells can possibly be confirmed by imaging using a fluorescence microscope, as shown in Figure 1(d).

[0046] Computer Molecular Modeling We performed computerized molecular modeling on specific peptide sequences to identify suitable spacer residues and to identify suitable negatively charged residues (e.g., Asp or Glu) to include after the spacer to reduce any interference of the negative charge with the active site of PSA. We also used computerized molecular modeling to investigate the effect of residue chirality (D or L) on substrate-PSA interactions.

[0047] Peptide sequence Sequences with D chirality are more resistant to proteolysis than their L counterparts [see Feng et al. and Liu et al.], and therefore a comparison between the behavior of similar sequences with different chirality was used to establish whether there is an energetic penalty associated with the use of D residues.

[0048] The substrate-PSA interaction was evaluated based on the number of hydrogen bonds observed between the negatively charged sequence and PSA, and the potential energy of the system containing the substrate and PSA.

[0049] To perform the simulations, the crystal structure of enzymatically active PSA with the substrate LysGlyIleSerSerGlnTyr (KGISSQY) in the active site was downloaded from the Protein Data Bank (PDB:2ZCK) [see Menez et al.].

[0050] The KGISSQY substrate was extended at the C-terminus with the residues or sequences listed in Table 1 using the Molecule function in Visual Molecular Dynamics software, followed by molecular dynamics and energy minimization cycles using Nanoscale Molecular Dynamics software. See Theoretical and Computational Biophysics Group.

[0051] [Table 1] # - Single letter amino acid residue codes are used, with uppercase letters indicating L-chirality and lowercase letters indicating D-chirality. * -Abbreviations: L-leucine (Leu), G-glycine (Gly), D-aspartic acid (Asp), E-glutamic acid (Glu), S-serine (Ser).

[0052] After energy minimization, the potential energy of the system and the number of hydrogen bond interactions between PSA and negatively charged residues of the substrate were investigated.

[0053] Here, we found that when an extension sequence has zero or only one lipophilic spacer residue between the C-terminus of KGISSQY and the negatively charged residue, the extension sequence interacts unfavorably with the kallikrein loop of PSA. The interaction between the negative charge and the kallikrein loop that locks the substrate in position for cleavage may destabilize substrate binding and / or interfere with the enzymatic action of PSA.

[0054] Incorporation of two spacer residues resulted in a lower incidence of interactions with the kallikrein loop. There was no apparent energy penalty associated with the incorporation of residues of D-chirality compared with residues with L-chirality.

[0055] We substituted the side chain of KGISSQY in situ to generate HSSKLQ, which has Q in the S1 pocket of PSA, and subjected it to energy minimization cycles to optimize its position and geometry in the active site of PSA.

[0056] The HSSKLQ sequence was extended at the C-terminus with the sequences listed in Table 2, and the resulting geometries after further energy minimization revealed that three-residue spacers such as SerGlyGly (SGG) and LeuGlyGly (LGG) were more effective than two-residue spacers in reducing interference with the kallikrein loop.

[0057] [Table 2] # - Single letter amino acid residue codes are used, with uppercase letters indicating L-chirality and lowercase letters indicating D-chirality. * -Abbreviations: L-leucine (Leu), G-glycine (Gly), D-aspartic acid (Asp), E-glutamic acid (Glu), S-serine (Ser).

[0058] Comparing a three-residue spacer followed by two negatively charged AspGlu residues of D or L chirality, the extracted geometries did not show any interference of the negatively charged side chains with the kallikrein loop.

[0059] The peptide with a three-residue spacer and a D-Asp-D-Glu sequence positioned the negatively charged residues of AspGlu sufficiently to form hydrogen bonds and short-range electrostatic interactions. The Asp and Glu residues with D-chirality formed hydrogen-bonding interactions with Arg36, Arg38, and Arg60 of the arginine patch of PSA, resulting in no significant energy penalty compared with the corresponding sequences with L-chirality.

[0060] Because the AspAsp sequence can be difficult to synthesize, the LGGde sequence, consisting of an LGG lipophilic spacer and a de(D-Asp-D-Glu) sequence contributing three negative charges from the side chain and C-terminus, was selected to yield HSSKLQ↓LGGde as the prodrug sequence to be investigated.

[0061] Based on this computer modeling, a promoiety in which the first peptide contained the sequence HisSerSerLysLeuGln (HSSKLQ) and the second peptide contained the negatively charged sequence D-Asp-D-Glu(de) was selected for further investigation.

[0062] Spacer The drug is conjugated to the N-terminus of the HisSerSerLysLeuGln(HSSKLQ) sequence or to an amino acid sequence added to the N-terminal side of this sequence, while the negatively charged D-Asp-D-Glu(de) sequence is linked to the C-terminus of the HisSerSerLysLeuGln(HSSKLQ) sequence via a spacer.

[0063] Preferably, the spacer is an amino acid sequence in which the first amino acid of the spacer is compatible with the active site of PSA, such as leucine or serine.

[0064] In one embodiment, the spacer comprises a leucine residue.

[0065] In one embodiment, the spacer comprises the amino acid sequence LeuGlyGly (LGG).

[0066] The spacer sequence LeuGlyGly (LGG) was introduced to reduce interference between the negatively charged sequence (de) and the kallikrein loop, allowing the negative charge to be placed near the positively charged arginine patch of PSA. The electrostatic interaction between the negative charge of the substrate and the arginine patch may increase the selectivity of the prodrug for PSA and increase the cleavage rate.

[0067] FIG. 3 shows the chemical structure of the HSSKLQ↓LGGde promoiety for conjugation to a drug to generate a model prodrug for use in understanding the effect PSA may have on a prodrug in vitro, where ↓ represents the position of PSA cleavage (PSA cleavable bond).

[0068] Model Prodrugs As described in more detail below, the inventors synthesized two exemplary prodrug models and studied the effectiveness of PSA to selectively cleave one of these model prodrugs in vitro and in the presence of cancer cells.

[0069] For the purposes of this study, the example prodrugs contain fluorescent tags as a substitute for the drug moiety. Anthraquinone (aqueous) fluorescent and rhenium(I) luminescent tags, structurally similar to drugs and / or imaging agents, were selected for conjugation to peptides to enable tracking by fluorescence microscopy in vitro.

[0070] Figure 4(a) shows the chemical structure of a model prodrug peptide sequence tag-HSSKLQ↓LGGde according to a preferred embodiment of the present invention in which the drug moiety is replaced with a fluorescent tag, and Figure 4(b) shows the chemical structures of the fragments (tag-HSSKLQ and LGGde) generated after cleavage of the HSSKLQ↓LGGde peptide sequence at the PSA-cleavable bond upon exposure to PSA, where ↓ represents the position of PSA cleavage (PSA-cleavable bond).

[0071] Fluorescent anthraquinone tag In one embodiment, the fluorescent tag selected for this purpose is structurally similar to a component of an established drug and was selected for conjugation to the peptide to allow for imaging of the distribution of the conjugate in vitro using fluorescence imaging.

[0072] Figure 4(c) shows the chemical structure of the fluorescent anthraquinone (aqueous) tag, which shares structural similarity with antitumor anthracycline drugs such as doxorubicin and mitoxantrone. See Kizek et al. and Hortobagyi. The AQ tag was synthesized with a carboxylic acid group to allow facile coupling to the N-terminus of the HSSKLQLGGde sequence under standard solid-phase peptide synthesis (SPPS) conditions.

[0073] Luminescent rhenium(I) complex tags Figure 4(d) shows the chemical structure of a luminescent rhenium(I) complex tag with a quinolyl-based ligand. Such Re(I) complexes exhibit their long-lived triplet metal-ligand charge transfer (T) potential, which allows in vitro monitoring using fluorescence microscopy. 3 It has attracted significant attention due to its MLCT condition.

[0074] In the second example, the luminescent tag is fac-[Re(CO)3bisquinolylamine] + The complex is a fluorophore (see Figure 4(d)). The stability of luminescent polypyridyl Re(I) complexes based on the facial-{M(CO)3} core makes them particularly suitable for imaging purposes. A fluorescent Re(I) complex tag was conjugated to an HSSKLQ-based vector, allowing for comparison of PSA-mediated activation and in vitro distribution with those of an AQ model prodrug. The Re(I) complex tag was also synthesized with a carboxylic acid linker to enable easy coupling to the N-terminus of the HSSKLQLGGde sequence under standard SPPS conditions.

[0075] Cleavage assay Figure 5 shows a set of PDA traces (254 nm, 20–30 min) of (a) AQ-HSSKLQ↓LGGde (green, retention time (RT) = 27.4 min) and (b) the peptide fragment, AQ-HSSKLQ (blue, RT = 26.2 min), resulting from cleavage of the AQ-HSSKLQ↓LGGde model prodrug after incubation with PSA for 3, 6, 24, and 48 h.

[0076] Incubation of AQ-HSSKLQ↓LGGde with PSA resulted in cleavage after Q to generate a fragment corresponding to AQ-HSSKLQ and a negatively charged sequence of LGGde, which coeluted with persistent solvent contaminants (15.7 min, not shown). Here, the intensity (a.u.) of the uncleaved AQ-HSSKLQ↓LGGde sequence (green, RT = 27.4 min) was observed to decrease over time, whereas the intensity of the cleaved AQ-HSSKLQ fragment (blue, RT = 26.2 min) increased.

[0077] The normalized integrals of the peaks in the UV trace (254 nm) corresponding to the fragments corresponding to AQ-HSSKLQ↓LGGde and AQ-HSSKLQ in Table 3 show that AQ-HSSKLQ↓LGGde, which is in 200-fold excess over PSA, continued to be cleaved by PSA up to 48 h, although cleavage was still incomplete at that time point.

[0078] [Table 3]

[0079] As evidenced by the data in Table 3, cleavage of AQ-HSSKLQ↓LGGde occurred at a higher rate than previously reported for a related substrate, where 23% remained uncleaved after 24 hours of incubation (Di Marco et al.). We believe that the negatively charged LGGde sequence may assist in positioning the substrate for cleavage, allowing for a greater cleavage rate. We also believe that this same sequence (LGGde) may continue to interact with the arginine patch of PSA after cleavage, thereby slowing the rate of subsequent substrate binding and cleavage.

[0080] Fluorescence microscopy Cell imaging studies were designed to compare the effect of the absence or presence of PSA on the distribution of tag-peptide conjugates in colorectal cancer cells, DLD-1 cells. The DLD-1 cell line was chosen explicitly because the cells do not express PSA, thereby allowing a controlled comparison between cells administered substrate in the absence of PSA (PSA-) and cells administered substrate in the presence of exogenous PSA of known concentration and uniform activity (PSA+).

[0081] For these studies, the use of human-derived prostate cancer cell lines did not allow for a comparative model based on PSA expression, as expression of PSA by prostate cancer cell lines varies as a function of culture conditions and the PSA that is expressed tends to have low levels of enzymatic activity.

[0082] We predicted that in the presence of PSA, the AQ-HSSKLQ↓LGGde model prodrug would be cleaved to generate AQ-HSSKLQ for cellular uptake. However, in the absence of PSA, the model prodrug would remain uncleaved, and thus, administered cells would exhibit background levels of fluorescence.

[0083] It was also predicted that cells would exhibit fluorescence upon administration of AQ-HSSKLQ regardless of the presence of PSA.

[0084] Cells were incubated with AQ-HSSKLQ↓LGGde or AQ-HSSKLQ at a concentration of 50 μM for 1, 4, or 24 h in the absence or presence of exogenous PSA (20 μg / mL), unless otherwise stated.

[0085] The administered cells were washed and then imaged using a confocal fluorescence microscope to assess the distribution of the conjugate in the cells. Emission from the conjugated AQ tag was monitored over a range of 570-670 nm. Cell images were processed using brightness and contrast settings ranging from 20-300 unless otherwise noted.

[0086] Figure 6 shows merged images of the fluorescence and bright-field channels of DLD-1 cells treated with AQ-HSSKLQ at a concentration of 50 μM for 1, 4, and 24 h either in the absence of PSA (no PSA) or with the addition of PSA (2 μg, 67 pmol) [7.5 × 10 cells per well in a 96-well glass-bottom plate]. 3 cells were seeded. Scale bar represents 30 μm].

[0087] Significant levels of fluorescent precipitate were observed in cells treated with AQ-HSSKLQ, and the amount of precipitate increased over time regardless of the presence of PSA, with little or no evidence of cellular uptake even after 24 hours of incubation (Figure 6). When cells were treated with AQ-HSSKLQ for 1 and 4 hours in the presence of PSA, significantly more precipitate appeared to form compared to cells treated in the absence of PSA. However, similar levels of fluorescent precipitate were formed for the PSA+ and PSA conditions after 24 hours of incubation.

[0088] The observed increase in precipitate formation over time may reflect the time interval required for precipitates to form in vitro. AQ-HSSKLQ may have been taken up by cells on a timescale too short to be measured by these studies, and the AQ tag or variants may have been exocytotically excreted to form precipitates. The formation of AQ-HSSKLQ precipitates or their degradation products likely inhibited the entry of fluorescent compounds into cells by passive diffusion and active uptake, contributing to the low levels of intracellular fluorescence.

[0089] Figure 7 shows merged images of the fluorescence and bright-field channels of DLD-1 cells treated with AQ-HSSKLQ↓LGGde at a concentration of 50 μM for 1, 4, and 24 h either in the absence of PSA (no PSA) or with PSA (2 μg, 67 pmol).

[0090] These images of cells treated with AQ-HSSKLQ↓LGGde for 1, 4, and 24 hours in the absence of PSA showed that a smaller amount of fluorescent precipitate formed, and the particles were smaller, compared to the images of cells treated with AQ-HSSKLQ (Figure 7). The extent of precipitation in cells treated with AQ-HSSKLQ↓LGGde in the absence of PSA increased over time but remained at a lower level than that observed in cells treated with AQ-HSSKLQ, demonstrating the utility of the LGGde sequence for improving solubility.

[0091] Background levels of intracellular fluorescence indicated that AQ-HSSKLQ↓LGGde was not internalized in cells in the absence of PSA, supporting the incorporation of amino acids with D chirality to increase the resistance of AQ-HSSKLQ↓LGGde to sequence clipping by other proteases.

[0092] Images of cells administered AQ-HSSKLQ↓LGGde in the presence of exogenous PSA showed that significantly greater amounts of precipitate formed at earlier incubation time points (1 and 4 h) than seen in images collected in the absence of PSA, consistent with in vitro cleavage of AQ-HSSKLQ↓LGGde to give AQ-HSSKLQ and subsequent formation of a fluorescent precipitate.

[0093] In cells treated with AQ-HSSKLQ↓LGGde and exogenous PSA, the amount of precipitate formed increased over time, consistent with the progression of AQ-HSSKLQ production when AQ-HSSKLQ↓LGGde was cleaved by PSA.

[0094] The extent of precipitation observed in cells incubated with AQ-HSSKLQ↓LGGde for 24 hours in the presence or absence of PSA cannot be easily distinguished based on these data alone.

[0095] Because negligible levels of intracellular fluorescence were observed after 24 h of incubation of AQ-HSSKLQ↓LGGde in the presence of exogenous PSA, cells were incubated with AQ-HSSKLQ↓LGGde at a concentration of 100 μM and 0, 1 (10 μg / mL), or 2 μg (20 μg / mL) PSA per well for 48 h (Figure 8).

[0096] Figure 8 shows the fluorescent channel, bright field channel, and composite images of DLD-1 cells treated with AQ-HSSKLQ↓LGGde at a concentration of 100 μM for 48 hours in either the absence of PSA (No PSA), 1 μg PSA (33 pmol), or 2 μg PSA (67 pmol).

[0097] Brightness and contrast settings were adjusted during imaging to avoid washout of the luminescence signal due to background noise. Cells incubated with AQ-HSSKLQ↓LGGde for 48 h without the addition of PSA showed background intracellular fluorescence, indicating that the prodrug was not internalized into the cells, even after extended incubation times.

[0098] Co-incubation of cells with AQ-HSSKLQ↓LGGde and 1 or 2 μg of PSA resulted in the formation of a fluorescent precipitate, indicating that PSA cleaves AQ-HSSKLQ↓LGGde in vitro to form AQ-HSSKLQ, followed by precipitate formation, with the amount of precipitate formed increasing with increasing concentrations of PSA. We observed some signs of intracellular fluorescence after 48 hours in cells treated with AQ-HSSKLQ↓LGGde and 2 μg of PSA, suggesting that some of the fluorescent compound may have entered the cells over the extended incubation period.

[0099] Proteolysis of AQ-HSSKLQ, the fragment generated upon cleavage of AQ-HSSKLQ↓LGGde, may result in the generation of an insoluble sequence that readily precipitates. The poor solubility of the AQ tag likely significantly affected the precipitate formation observed in vitro. The extended planar structure of the AQ tag readily allows for intermolecular π-π stacking, which promotes precipitation in aqueous environments. This is consistent with the observation of some precipitate after 48 hours of incubation of AQ-HSSKLQ with Tris buffer in the PSA cleavage assay.

[0100] The charge present on the AQ-HSSKLQ fragment may also have contributed to the poor cellular uptake.

[0101] At physiological pH, the AQ-HSSKLQ fragment has a negatively charged C-terminus and a positively charged side chain on the Lys residue, resulting in no net charge. The L-chirality of the HSSKLQ sequence, which is necessary for the correct interaction with PSA to enable cleavage, makes the HSSKLQ sequence in the AQ tag vulnerable to proteolysis in vitro and in vivo.

[0102] Proteases present in the medium or expressed extracellularly by DLD-1 cells could cleave the AQ tag at any of the present amide bonds, generating less soluble sequences such as AQ-HS or AQ-HSS, which have a net charge of −1 and are electrostatically repelled from the cell membrane.

[0103] Regardless of the presence of PSA, smaller and fewer precipitated particles were observed in images of cells treated with AQ-HSSKLQ↓LGGde compared with those observed in images of cells treated with AQ-HSSKLQ.

[0104] The negative charge of the D-Asp-D-Glu sequence in AQ-HSSKLQ↓LGGde may confer greater hydrophilicity, thereby increasing solubility, and may also hinder π-π stacking interactions that may promote precipitation.

[0105] In the absence of PSA, AQ-HSSKLQ↓LGGde appears to be subject to little or no degradation by proteases that may be expressed by DLD-1 cells or present in supplemented media. Proteolysis involving removal of the AspGlu sequence would generate less soluble sequences, such as AQ-HSSKLQ↓LGG or shorter sequences similar to AQ-HSSKLQ, which are expected to form significant amounts of precipitate. This in vitro resistance to proteolysis is likely enhanced by the D-chirality of the C-terminal AspGlu sequence of AQ-HSSKLQ↓LGGde.

[0106] Although the formation of an in vitro fluorescent precipitate in these studies is disappointing and complicates interpretation, these results suggest that AQ-HSSKLQ↓LGGde was successfully cleaved by PSA in the presence of cancer cells, presumably to generate AQ-HSSKLQ, followed by precipitate formation. The increase in the amount of precipitate formed over time in cells administered AQ-HSSKLQ↓LGGde in the presence of PSA indicates that PSA retained its enzymatic activity and continued to cleave the substrate over time.

[0107] Treatment method The present invention also provides methods and uses of the above compositions in the manufacture of a medicament for detecting and / or treating prostate cancer in a subject.

[0108] The compositions include prodrugs formed using a drug moiety (i.e., an active pharmaceutical ingredient (API)) in place of the AQ fluorescent tag described above, which can be administered to any subject (including a human or non-human animal) in an amount effective to treat a disorder, in a therapeutically effective amount, or in a diagnostically effective amount.

[0109] These prodrugs can be administered parenterally by injection or by gradual infusion over time. The prodrugs can be administered intratumorally, intraprostatically, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0110] Other methods of administration are known to those skilled in the art.

[0111] In one embodiment, the prodrug composition is administered in a therapeutically or diagnostically effective amount to a subject having a localized or metastatic prostate tumor for the treatment thereof, said treatment being for the purpose of reducing the volume or size of the tumor.

[0112] Preparations for parenteral administration of the prodrugs of the present invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives (e.g., antimicrobials, antioxidants, chelating agents, and inert gases) may also be present.

[0113] definition As used herein, the term "prodrug" refers to a compound that is a drug precursor that, after administration to a subject, releases the drug in vivo through some chemical or physiological process (e.g., the prodrug is brought to physiological pH or converted to the desired active drug form through enzymatic action). Prodrugs can be converted into products that are toxic to tumor cells.

[0114] Those skilled in the art will appreciate that the terms "drug" and "prodrug" as used herein can include diagnostic agents and prodiagnostic agents, respectively.

[0115] As used herein, the term "promoiety" refers to a functional portion of a prodrug that is used to modify the structure of a drug to improve the physicochemical, biopharmaceutical and / or pharmacokinetic properties of the drug.

[0116] As used herein, the term "prostate-specific antigen" (PSA) refers to prostate-specific antigen, also known as human kallikrein 3 (KLK3 or hK3), as well as all other proteases that have the same or substantially the same proteolytic cleavage specificity as prostate-specific antigen.

[0117] As described herein, amino acid sequences are presented according to standard convention, ie, with the amino terminus of the peptide on the left and the carboxy terminus on the right.

[0118] As used herein, the term "therapeutically or diagnostically effective amount" refers to the amount of a drug or pharmaceutically active ingredient delivered to a subject to provide a desired physiological response. Methods for preparing pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Science, 18 th ed., Mack Publishing Company, Easton, Pa. (1990) and Remington: the Science and Practice of Pharmacy, 20. th ed., Lippincott Williams & Wilkins, (2003), are within the skill of the art.

[0119] As used herein, the term "administering" or "administration" as used herein means the introduction of a foreign molecule into a cell or host. This term is intended to be synonymous with the term "delivery."

[0120] As used herein, the term "treating" or phrase "treat" refers to any type of treatment that benefits a subject afflicted with a disease, including improving the subject's condition (e.g., one or more symptoms) or slowing the progression of the condition.

[0121] As used herein, the term "about," when referring to a measurable value such as a parameter, amount, duration, etc., is meant to encompass a variation of no more than ±10%, preferably no more than ±5%, more preferably no more than ±1%, and even more preferably no more than ±0.1% of the specified value, insofar as such variations are appropriate for practicing the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, preferably disclosed.

[0122] As used herein, the singular forms "a," "an," and "said" include both singular and plural referents unless the context clearly dictates otherwise.

[0123] As used herein, the terms "amino acid" and "peptide" are understood to include groups that can be incorporated into a peptide sequence so as to retain the ability to bind to and be cleaved by the active site of a protease.

[0124] As used herein, residues of a peptide amino acid sequence that have L-chirality are written in upper case, while residues that have D-chirality are written in lower case (eg, LGGde).

[0125] Materials and Methods Synthesis of AQ tags This method was adapted from DiMarco et al. (2019).

[0126] The reaction mixture and product were protected from light during synthesis and workup. 1-Chloroanthraquinone (2.64 g, 10.9 mmol, 1.1 equiv.) was combined with dry DMA (60 mL) and TEA (3.0 mL, 21.4 mmol, 2.2 equiv.). 4-Aminobutyric acid (1.04 g, 10.1 mmol, 1.0 equiv.) was added and the reaction mixture was heated to 160° C. for 5 h. The reaction mixture was cooled to 55° C. and concentrated under a stream of nitrogen gas overnight. The mixture was diluted with 0.1 M HCl. (水溶液) (60 mL), extracted into DCM (2×80 mL), then washed with water (2×60 mL) and 0.1 M NaOH (水溶液) The aqueous layer was extracted with a solution of 1 M HCl (2×60 mL). The aqueous layer was washed with DCM until the organic layer was clear. (水溶液) The product was precipitated by acidifying with a solution of 100 ml of HCl and collected by vacuum filtration as a dark red solid (0.19 g, 0.601 mmol, 6% yield).

[0127] 1 H NMR (400 MHz, MeOD-d 4 ,ppm):δ8.27(m,2H),7.85(ddd,J=7.5Hz,J=1.5Hz,2H),7.64(dd,J=7.4Hz,J=8.6Hz,1H),7.29(d d,J=0.7Hz,J=8.6Hz,1H),3.47(t,J=7.2Hz,2H),2.51(t,J=7.2Hz,2H),2.04(quintet,J=7.2Hz,2H).

[0128] 13 C NMR (75 MHz, DMSO-d 6 ,ppm)δ183.96,182.79,173.97,151.28,135.57,134.41,134.30,133.92,13 3.38,132.31,126.36,126.19,118.49,115.00,112.00,41.45,30.99,24.06.

[0129] LRMS-ESI(-)m / z:[MH] - Measured value 308.25, calculated value 308.09:C 18 H 14No. 4 - .

[0130] Synthesis of Re(I) complex tags The following synthetic methods are adapted from Green (2013) unless otherwise stated.

[0131] Synthesis of methyl ester quinolyl ligands The TEA was dried over anhydrous sodium sulfate for 48 hours. Methyl 4-(aminomethyl)benzoate hydrochloride (0.61 g, 3.03 mmol, 1.0 equiv.) was combined with dry MeCN (10 mL) and dry TEA (0.42 mL, 3.03 mmol, 1.0 equiv.) to neutralize the mixture. 2-(Chloromethyl)quinoline hydrochloride (1.43 g, 6.65 mmol, 2.2 equiv.) was combined with dry MeCN (10 mL) and dry TEA (0.93 mL, 6.67 mmol, 2.2 equiv.) to neutralize the mixture. Both mixtures were sonicated to aid in the formation of microparticles and combined under inert conditions. An additional 2.2 equiv. of dry TEA (0.93 mL, 6.67 mmol) was added, and the reaction mixture was refluxed under an inert atmosphere for 24 hours. The mixture was cooled to room temperature, concentrated under a stream of nitrogen gas, and then diluted with saturated NaHCO 3 (水溶液) The mixture was diluted with 20 mL of chloroform (4 × 20 mL), washed with brine (3 × 15 mL), and then dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed from the crude product under reduced pressure. The product was purified by column chromatography without flash on a silica stationary phase using 10–55% EtOAc in hexanes as the eluent. The solvent from the column was removed under reduced pressure to give a golden-brown solid (0.71 g, 1.60 mmol, 53% yield).

[0132] 1 H NMR (400 MHz, CDCl3-d 1 ,ppm):δ8.14(d,J=8.5Hz,2H),8.05(d,J=8.5Hz,2H),7.99(d,J=8.1Hz,2H),7.78( d,J=8.1Hz,2H),7.69(m,4H),7.51(m,4H),4.01(s,4H),3.89(s,3H),3.81(s,2H).

[0133] 13 C NMR (75 MHz, DMSO-d 6 ,ppm)δ166.95,159.92,147.50,144.49,136.48,129.66,129.46,128.99,128.96,127.51,127.35,126.25,120.95,60.93,58.46,52.02.

[0134] LRMS-ESI(+)m / z:[M+H] + Measured value 448.17, calculated value 448.20:C 29 H 26 N3O2 + .R f =0.23, in a 50% mixture of EtOAc in hexanes, on a silica plate.

[0135] Synthesis of quinolyl ligands Methyl-4-((bis(quinolin-2-ylmethyl)amino)methyl)benzoate (0.47 g, 1.05 mmol) was dissolved in MeCN and 1 M KOH (水溶液) and heated with stirring at 95°C for 4 hours. After cooling to room temperature, the reaction mixture was concentrated under a stream of nitrogen gas and then diluted with water (10 mL). (水溶液) The acidity of the mixture was adjusted to pH 4 with HCl to precipitate the product, which was extracted into chloroform (6 × 15 mL) while maintaining the acidity of the aqueous layer at pH 4. The chloroform was removed under reduced pressure to give the product as a golden brown solid (0.39 g, 0.91 mmol, 85%).

[0136] 1 H NMR (400 MHz, DMSO-d 6 ,ppm): δ8.35(d,J=8.5Hz,2H),7.94(m,6H),7.74(m,4H),7.57(m,4H),3.93(s,4H),3.77(s,2H).

[0137] 13C NMR(75MHz,MeOD,ppm)δ169.77,161.09,148.09,145.09,138.56,131.00,130.83,130.20,128.90,128.77,127.72,122.43,79.44,61.80,59.99.

[0138] LRMS-ESI(-)m / z:[MH] - Measured value 432.18, calculated value 432.17:C 28 H 22 N3O2 + R in a 50% mixture of ethyl acetate in hexane on silica plates. f =0.00.

[0139] Synthesis of triaquatricarbonylrhenium(I) bromide. This procedure was adapted from Lazarova et al. (2004).

[0140] Bromopentacarbonylrhenium(I) (3.33 g, 8.19 mmol) was added to water (25 mL) and heated to reflux. The condenser was periodically flushed with water during the reaction. After 24 h, the reaction mixture was cooled to room temperature and filtered to remove any remaining starting material. Water was removed from the product under reduced pressure to give a whitish-green solid identified as triaquatricarbonylrhenium(I) bromide (1.44 g, 3.55 mmol, 43% yield). The product was used in the subsequent complexation reaction without further purification.

[0141] Synthesis of Re(I) complex tag 4-((bis(quinolin-2-yl)amino)-methyl)benzoic acid (0.33 g, 0.763 mmol, 1.0 equiv.) and triaquatricarbonylrhenium(I) bromide (0.45 g, 1.11 mmol, 1.5 equiv.) were combined in MeOH. The acidity of the mixture was adjusted to 0.5 M NaOH. (水溶液)The pH was adjusted to 8 with HCl, and the mixture was refluxed for 4 h. The reaction mixture was concentrated under a stream of nitrogen gas, extracted into DCM (15 mL), and then washed twice with water (2 × 10 mL). DCM was removed from the reaction mixture under reduced pressure, and the crude product was dissolved in 3:1 MeOH:MeCN and purified by preparative HPLC using a gradient of 30–60% unbuffered MeCN in 0.1% w / v NHOAc-buffered milliQ water over 30 min (RT = 21.3 min). Fractions containing pure product were combined, and residual buffer was removed by sequential lyophilization. The purity of the product was confirmed by LCMS performed with a gradient of 0–50% buffered MeCN over 30 min (RT = 21.0 min). The purified product was concentrated under nitrogen gas and then lyophilized twice to remove residual NHOAc and recovered as a light brown solid (0.30 g, 0.382 mmol, 50%).

[0142] 1 H NMR (400 MHz, MeOD-d 4 ,ppm)δ8.55(d,J=9.2Hz,2H),8.47(d,J=8.4Hz,2H),8.14(d,J=8.2Hz,2H),7.97(d,J=8.1Hz,2H), 7.86(t,J=8.0Hz,2H),7.78(d,J=8.2Hz,2H),7.68(t,J=7.2Hz,2H),7.55(d,J=8.5Hz,2H),5.46(d, 2 J=17.4Hz,2h),5.17(s,2H),4.73(d, 2 J = 17.2 Hz, 2H).

[0143] 13 C NMR(100MHz,MeOD,ppm)δ166.04,163.04,148.16,142.82,140.24,137.16,134.73 ,133.98,132.96,131.07,130.76,129.71,129.53,129.40,121.07,71.93,68.77.

[0144] LRMS-ESI(+)m / z:[M] + Measured value 703.96, calculated value 704.12:C 31 H 23N3O5Re + .

[0145] HRMS(+)m / z:[M] + Measured value 404.1194, calculated value 704.1190:C 31 H 23 N3O5Re + .

[0146] IR:ν max (cm -1 ) = 2025, 1916(fac-[Re(CO)3] + ). R f =0.00, in a 50% mixture of EtOAc in hexanes, on a silica plate.

[0147] Peptide synthesis Peptides were synthesized manually using a packed solid-phase resin support and an Fmoc protecting group strategy in fritted syringes. The side chains of His, Ser, Lys, Gln, d-Asp, and d-Glu were protected with Trt, tBu, Boc, Trt, OtBu, and OtBu protecting groups, respectively. The amounts of reagents and solvents are specified as equivalents per 0.1 g of packed resin, taking into account the resin loading.

[0148] Between each step, the resin was washed with 5x DMF, 5x DCM, and then 5x DMF, where each wash was approximately 1.5 mL in volume. HSSKLQ was obtained from commercially available Fmoc-Gln(Trt)-Wang resin (loading: 0.52 mmol g). 1 ) and HSSKLQL was synthesized using commercially available Fmoc-Leu-Wang resin (loading: 0.66 mmol g). For the synthesis of HSSKLQLGGde, Ctc resin was manually loaded with Fmoc-D-Glu(OtBu)-OH.

[0149] To load the ctc resin, 0.1 g of resin was swelled in DCM and 30 μL of thionyl chloride was slowly added, followed by 0.5 mL of DMF. The vessel was capped and shaken for 4 h. The DMF solution was then drained and the resin was washed with DCM (5 × 3 mL). A freshly prepared solution of Fmoc-D-Glu(OtBu)-OH (28 mg, 66 μmol, 1.1 equiv.) and DIPEA (17 μL, 0.18 mmol, 3.0 equiv.) in DCM (1.0 mL) was immediately added, and the vessel was capped and shaken for 45 min. The solution was drained, a fresh identical solution was added, and the vessel was shaken for 16 h.

[0150] The loaded resin was washed and then used for peptide synthesis. This procedure yielded 1.2 mmol g of dibenzofulvene-piperidine adduct, calculated from the absorbance of the adduct at 301 nm. -1 The short Fmoc removal times for Fmoc-e-ctc and Fmoc-de-ctc were chosen to offset the short loading and low yields that resulted when synthesizing HSSKLQ↓LGGde. Fmoc removal for all peptides without Asp residues was performed by exposing the resin to 20% piperidine in DMF (3 × 3 min, 9 mL total), except for Fmoc-e-ctc, where shorter intervals (2 × 2 min, 1 × 1 min, 9 mL total) were used to avoid cleavage from the resin. Fmoc removal from Fmoc-de-ctc was performed using 20% ​​piperidine in DMF with 0.1 M Oxyma Pure for short intervals to reduce the formation of diketopiperidine and aspartimide (1 × 2 min, 1 × 1 min, 6 mL total). Fmoc removal from peptide sequences containing Asp residues was performed in 20% piperidine in DMF with 0.1 M Oxyma Pure.

[0151] For all steps, the extent of Fmoc removal was monitored using the Kaiser test, and the Fmoc removal step was repeated if the Kaiser test indicated incomplete removal, except for the short time intervals specified above. After Fmoc removal was complete, the resin was washed as detailed above.

[0152] To couple the residues, amino acid (4 equivalents), PyBOP (4 equivalents), and NMM (12 equivalents) were combined in DMF to give a 0.5 M solution with respect to the amino acid. All couplings proceeded for 45 minutes and were performed in duplicate. The coupling solution was drained and the resin was washed. The extent of coupling was monitored using a Kaiser test, where a negative result for the free amine indicated that the coupling had proceeded completely.

[0153] Coupling of fluorescent tags When the sequences (HSSKLQ, HSSKLQL, and HSSKLQLGGde) were synthesized on the resin, the N-terminal Fmoc group was removed, followed by washing and adding the coupling solution for the tag. Coupling of the AQ tag proceeded similarly to the amino acid coupling, but at a lower level and over a longer interval to attach the Re(I) complex tag (3 eq. Re(I) tag, 3 eq. PyBOP, 9 eq. NMM in DMF, 0.5 M solution, 24 h).

[0154] Cutting from resin The tag-peptide conjugates were cleaved from the resin and globally deprotected by shaking in 95% TFA, 2.5% TIS, and 2.5% water (2 mL) for 2.5 hours. The cleavage solution was drained, and the resin was exposed to pure TFA for 1 hour. The cleavage solutions were combined and concentrated under a stream of nitrogen gas. Analysis by MALDI-TOF confirmed the presence of each conjugate and global deprotection of the AQ-peptide, but indicated that some tBu groups remained on the side chains of the Re-peptide conjugates. The Re-peptide was exposed to 90% TFA, 5% TIS, and 5% water for an additional 1.5 hours to completely remove the tBu groups, as confirmed by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF).

[0155] Peptide purification Peptides were purified to >95% purity by reverse phase using milliQ water and MeCN buffered with 0.1% v / v TFA. Semi-preparative HPLC was performed using a Waters 2695 controller and pump and a Waters Sunfire C18 column (OBD 5 μm, 10 mm × 250 mm, 4 mL / min).

[0156] Preparative HPLC was performed using a Waters 2535 Quaternary Gradient module and a Waters Sunfire C18 column (OBD 5 μm, 19 mm × 150 mm, 7 mL / min). The UV trace was monitored at 254 nm, and peaks were collected using an automated fraction collection system.

[0157] AQ-HSSKLQ↓LGGde: Preparative, 0–100% MeCN over 30 min, RT = 17.4 min; AQ-HSSKLQ: Semi-preparative, 0–50% MeCN over 30 min, RT = 22.1 min; AQ-HSSKLQ↓L: Preparative, 20–80% MeCN over 30 min, RT = 12.8 min; Re-HSSKLQ↓LGGde: Preparative, 30–40% MeCN over 30 min, RT = 8.1 min; Re-HSSKLQ: Preparative, 20–80% over 30 min, RT = 15.3 min; Re-HSSKLQ↓L: Preparative, 30–40% MeCN over 30 min, RT = 8.3 min.

[0158] Peptide characterization The peptides were characterized by MALDI-TOF mass spectrometry and analytical HPLC. MALDI-TOF analysis was performed using a Bruker autoflex speed TOF in reflectron positive mode, with samples co-crystallized with an α-cyano-4-hydroxycinnamic acid matrix on a steel plate. Reverse-phase analytical HPLC was performed using a Waters 2695 controller and pump and a Waters Sunfire C18 column (OBD 5 μm, 2.1 mm × 150 mm, 0.2 mL / min) using milliQ and MeCN buffered with 0.1% v / v TFA. Samples were dissolved in 1:1 milliQ:MeCN containing 0.1% TFA, with a 1 M concentration of guanidine hydrochloride to prevent peptide folding. Analytical HPLC traces were acquired with a gradient of 0–100% MeCN over 30 min and monitored at 254 nm.

[0159] AQ-HSSKLQ↓LGGde: Yield 7.9 mg, 5.41 μmol, 4%, LRMS m / z 729 [M] 2 , RT=16.5 min; AQ-HSSKLQ: Yield 44.3 mg, 44.8 μmol, 68%, LRMS m / z 990.41 [M] + , RT = 13.9 min; AQ-HSSKLQ↓L: Yield 43.6 mg, 39.5 μmol, 54%, MS m / z 1103.52 [M] + , RT=17.4 minutes.

[0160] PSA cleavage study The methods and conditions used for PSA cleavage studies were based on those previously reported. Enzymatically active PSA purified from human semen was obtained from GenWay Biotech (0.59 mg / mL solution in 10 mM PBS, >95% purity by SDS-PAGE, pH 7.4, 30 kDa, no preservatives). AQ-HSSKLQ↓LGGde, AQ-HSSKLQ, and AQ-HSSKLQ↓L were dissolved in milliQ water to make 5 mM stock solutions. Reverse-phase liquid chromatography-mass spectrometry (LCMS) traces were obtained using a Shimadzu UFLC LCMS system equipped with a CBM-20A controller, a DGU-20A3 degasser, two LC-20AD pumps, a CTO-20A column oven, an SPD-M20A photodiode array detector, and an LCMS-2020 mass spectrometer. Separation was achieved using a Waters Xbridge BEH130 C18 analytical column (OBD 5 μm, 4.6 mm × 150 mm, 0.2 mL / min) using milliQ water and MeCN containing 0.1% v / v formic acid buffer, respectively. PSA (200 pmol, 10 μL of a 20 μM solution in PBS) was incubated with each substrate (40,000 pmol, 8 μL of a 5 mM stock solution) in Tris-buffered saline (TBS) at 37 °C to a total volume of 200 μL. For compounds AQ-HSSKLQ↓LGGde, AQ-HSSKLQ, and AQ-HSSKLQ↓L, 20 μL aliquots of the incubated solution were collected once at 3 and 6 h of incubation and once at 24 and 48 h of incubation, respectively.

[0161] Each aliquot was made up to 100 μL with 50% MeCN in milliQ water, centrifuged, and then syringe filtered. A 20 μL sample was injected into the LCMS and analyzed over a 60-minute gradient of 0 to 100% MeCN in milliQ water over the m / z range of 200–2000. Component compounds eluted over the course of the gradient were monitored at 254 nm. Control incubations containing only substrate in buffer or only PSA in buffer were also performed and analyzed in the same manner to confirm the stability of the conjugate under the experimental conditions and that cleavage of the Gln-Leu bond in AQ-HSSKLQ↓LGGde was PSA-mediated.

[0162] Cell lines and maintenance Human-derived colorectal cancer DLD-1 cells (a colorectal adenocarcinoma cell line) were obtained from the American Type Culture Collection and used from passages 6–20 within 3 months of resuscitation. Cells were maintained in exponential growth in Adv DMEM supplemented with 2 mM glutamine and 2% FBS in a humidified atmosphere containing 5% CO2. Cells were seeded into 96-well plates containing 100 μL of Adv DMEM supplemented with 2% FBS and 2 mM glutamine. The number of cells seeded per well and the plate material (glass or plastic) are listed in the cell image figure caption. Cells were allowed to adhere overnight under standard incubation conditions. Each experimental condition was performed in triplicate. Prior to treatment, cells were gently washed with Adv DMEM supplemented with 2 mM glutamine (3 x 100 μL). Cells were treated in Adv DMEM supplemented with 2 mM glutamine at a final substrate concentration of 50 μM unless otherwise noted. Cells were treated without PSA or with added PSA (2 μg, 67 pmol, resulting in a final concentration of 20 μg / mL unless otherwise stated). Plates were incubated under standard conditions.

[0163] Prior to imaging, the medium was removed from each well and the cells were gently washed with PSA (3 x 100 μL). Fluorobrite DMEM supplemented with 2 mM glutamine (100 μL) was added to each well before imaging at the specified time points.

[0164] Confocal fluorescence microscopy Confocal fluorescence microscopy was performed using an Olympus FV3000 inverted fluorescence microscope equipped with an UPLSAPO 10X dry objective (NA = 4.0) and a thermally controlled stage maintained at 37°C and 5% CO2. The AQ peptide conjugate was excited using a 561 nm LED laser, and emission was monitored over a range of 570–670 nm. All images were acquired using line averaging (3 times per line), a scan speed of 2 μs per pixel, and a digital zoom of 5.0. Three different positions within each well were selected for imaging. Micrographs were imaged with cells in best focus in bright field. Images were acquired using Olympus FV31S-SW-v2.4.1.198 software. The 561 nm laser was used at 50% power, 650 V, 1× gain, and 3% offset. The scale bar represents 30 μm. Images were processed using Fiji ImageJ v1.53c. All fluorescent images were processed using brightness and contrast settings ranging from 20 to 300 unless otherwise stated.

[0165] References 1. Di Marco, L. et al. "Modulating the Cellular Uptake of Fluorescently Tagged Substrates of Prostate-Specific Antigen before and after Enzymatic Activation", Bioconjugate Chemistry 30, 124-133, doi:10.1021 / acs.bioconjchem.8b00792(2019). 2.Eissler, S. et al. “Substitution determination of Fmoc-substituted resins at different wavelengths”, Journal of Peptide Science 23, 757-762 (2017). 3.Feng,Z.& Xu,B.「Inspiration from the mirror:D-amino acid containing peptides in biomedical approaches」,Biomol Concepts 7,179-187,(2016). 4.Frenette,G.,Gervais,Y.,Tremblay,R.R.& Dube,J.Y.「Contamination of purified prostate-specific antigen preparations by kallikrein hK2」,The Journal of Urology 159,1375-137,(1998). 5.Green,B.P.「Matrix metalloproteinase binding agents for luminescence and radioimaging of metastatic tumours」,Doctor of Philosophy thesis,The University of Sydney,(2013). 6.Greene,R.F.&Pace,C.N.「Urea and guanidine hydrochloride denaturation of ribonuclease,lysozyme,α-chymotrypsin,andβlactoglobulin」,Journal of Biological Chemistry 249,5388-5393(1974). 7.Hortobagyi,G.「Anthracyclines in the treatment of cancer」,Drugs 54,1-7(1997). 8.Kaiser,E.,Colescott,R.,Bossinger,C.& Cook,P.「Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides」,Analytical Biochemistry 34,595-598(1970). 9.Kizek,R.et al.「Anthracyclines and ellipticines as{DNA}-damaging anticancer drugs:Recent advances.Pharmacology & Therapeutics」,133,26-39(2012). 10.Lazarova,N.,James,S.,Babich,J.& Zubieta,J.「A convenient synthesis,chemical characterization and reactivity of[Re(CO)3(H2O)3]Br:the crystal and molecular structure of[Re(CO)3(H2O)3]Br」,Inorganic Chemistry Communications 7,1023-1026(2004). 11.Liu,M.et al.「D-Peptides as Recognition Molecules and Therapeutic Agents」,The Chemical Record 16,1772-1786,(2016). 12.Menez,R.et al.「Crystal Structure of a Ternary Complex between Human Prostate-specific Antigen,Its Substrate Acyl Intermediate and an Activating Antibody」,Journal of Molecular Biology 376,1021-1033(2008). 13.Mergler,M.,Dick,F.,Sax,B.,Weiler,P.& Vorherr,T.「The aspartimide problem in Fmoc-based SPPS」Part I.Journal of Peptide Science 9,36-46,doi:doi:10.1002 / psc.430(2003). 14. Remington's Pharmaceutical Science, 18th ed., Mack Publishing Company, Easton, Pa. (1990). 15. Remington: the Science and Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, (2003). 16. Theoretical and Computational Biophysics Group, www.ks.uiuc.edu, www.ks.uiuc.edu (March 2006, Accessed 2018).

[0166] conclusion This work involved the design and synthesis of a model prodrug targeted to prostate cancer that incorporates a promoiety with a peptide sequence that is cleaved with high selectivity by prostate-specific antigen (PSA), a negatively charged sequence for both electrostatic interaction with the arginine patch of PSA and delayed cellular uptake, and a fluorescent tag that allows imaging of the in vitro distribution of the model prodrug.

[0167] The results described herein demonstrate that negatively charged peptide sequences containing lipophilic spacers can be easily modeled and designed in the active site of PSA using computerized molecular simulations. The modeled interaction between HSSKLQ↓LGGde and PSA demonstrated the desired electrostatic interaction between the negatively charged sequence and the arginine patch of PSA.

[0168] Furthermore, these studies showed that amino acids with D chirality can be easily incorporated into the model and that they do not incur an energy penalty compared to corresponding sequences with L chirality.

[0169] Despite the limitations of the AQ-peptide conjugates in these studies, these results demonstrate their resistance to cellular uptake in the absence of PSA. The model prodrugs appear not to be vulnerable to proteolysis in this in vitro model, likely due to the incorporation of a C-terminal D-Asp-D-Glu negatively charged sequence. Furthermore, the prodrugs are activated by PSA in vitro, even at PSA concentrations 1000-fold lower than those found in the extracellular fluid of prostate cells.

[0170] advantage As described in embodiments of the invention herein, promoieties and compositions comprising the promoieties are used in forming prodrugs for detecting and / or treating prostate cancer, which provide many advantages, including but not limited to: the pro-moiety of the prodrug has a peptide sequence that is selectively cleavable in the presence of prostate-specific antigen (PSA) to yield a cleaved peptide-drug conjugate; The cleaved peptide-drug conjugate is expected to be taken up by cells in the immediate environment; · The prodrugs generated from the promoieties described herein are selective for prostate cancer.

[0171] Embodiments: References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0172] Similarly, in the foregoing description of exemplary embodiments of the invention, it should be understood that various features of the invention may be grouped together in a single embodiment or description thereof for the purpose of streamlining the disclosure and facilitating understanding of one or more of the various inventive aspects. This method of disclosure, however, should not be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description of a particular embodiment are expressly incorporated into that detailed description of the particular embodiment, with each claim standing on its own as a separate embodiment of the invention.

[0173] Furthermore, although some embodiments described herein include some features and not other features included in other embodiments, as will be understood by those skilled in the art, combinations of features from different embodiments are intended to be within the scope of the present invention and form different embodiments. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0174] Other embodiments It will be understood by those skilled in the art that the model prodrugs and compositions thereof described in the above embodiments of the present invention are not limited solely to conjugates formed between the peptide sequences and the above anthraquinone (aqueous solution) type drugs, but may be adapted to be conjugated to other drugs for detecting and / or treating prostate cancer, including, for example, drugs approved for prostate cancer by the NIH National Cancer Institute (see https: / / www.cancer.gov / about-cancer / treatment / drugs / prostate).

[0175] Equipped with and including In the following claims and the foregoing description of the invention, unless the context requires otherwise by express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of stated features but do not exclude the presence or addition of further features in various embodiments of the invention.

[0176] Any one of the following terms: "including" or "which includes" or "that includes" as used herein is also an open term meaning to include at least the elements / features that follow that term, but not to exclude others. Thus, including is synonymous with and means comprising.

[0177] Problems that the invention aims to solve Thus, while there has been described what are believed to be preferred embodiments of the present invention, those skilled in the art will recognize that other and further modifications can be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications that fall within the scope of this invention. For example, any formulas given above are merely representative of procedures that may be used.

[0178] Although the present invention has been described with reference to particular examples, those skilled in the art will appreciate that the present invention can be embodied in many other forms.

[0179] Industrial Applicability From the above, it is clear that the described arrangements are applicable to the medical and healthcare industry.

Claims

1. The pro part is a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) and at a second end for binding with high selectivity to the active site of PSA; and the second peptide comprises a negatively charged sequence for delaying cellular uptake of the prodrug; A promoiety wherein the second peptide is cleaved from the first peptide upon proteolysis by PSA to generate a conjugate of the first peptide and the drug suitable for uptake by target cells.

2. 1. A composition comprising a promoiety, said promoiety comprising: a first peptide; a second peptide linked to the first peptide, the first peptide comprises a sequence configured near a first end for conjugation to a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA) and at a second end for binding with high selectivity to the active site of PSA; and the second peptide comprises a negatively charged sequence for delaying cellular uptake of the prodrug; A composition wherein the second peptide is cleaved from the first peptide upon proteolysis by PSA to generate a conjugate of the first peptide and the drug suitable for uptake by target cells.

3. 3. The promoiety of claim 1 or the composition of claim 2, wherein the sequence of the first peptide comprises HisSerSerLysLeuGln (HSSKLQ).

4. 3. The promoiety of claim 1 or the composition of claim 2, wherein the first end of the first peptide is an N-terminus.

5. 3. The promoiety of claim 1 or the composition of claim 2, wherein the second peptide comprises one or more amino acids that are negatively charged at physiological pH.

6. 3. The promoiety of claim 1 or the composition of claim 2, wherein the second peptide comprises the sequence D-Asp-D-Glu(de).

7. 3. The promoiety of claim 1 or the composition of claim 2, wherein the second peptide is linked to the C-terminus of the first peptide via a spacer.

8. 8. The promoiety or composition of claim 7, wherein the spacer is an amino acid sequence, the first amino acid of the spacer being compatible with the active site of PSA.

9. 9. The promoiety or composition of claim 8, wherein the first amino acid of the spacer is a leucine residue.

10. 10. The promoiety or composition of claim 9, wherein the spacer comprises the amino acid sequence LeuGlyGly (LGG).

11. 10. A method for detecting and / or treating prostate cancer in a subject, the method comprising administering to the subject a therapeutically or diagnostically effective amount of the composition of claim 2.

12. The method of claim 11 , wherein the composition is administered intratumorally and / or intraprostatically.

13. 12. The method of claim 11, wherein the composition is administered intravenously, intramuscularly, or subcutaneously.

14. 12. The method of claim 11, wherein the subject has a localized prostate tumor.

15. 12. The method of claim 11, wherein the subject has a metastatic prostate tumor.

16. 12. The method of claim 11, wherein the administration results in a reduction in prostate tumor volume.

17. 12. The method of claim 11, wherein the administration results in a reduction of metastatic prostate tumors.

18. 18. The method of claim 17, wherein administering results in treatment of the metastatic prostate tumor.

19. 10. Use of the composition of claim 2 in the manufacture of a medicament for detecting and / or treating prostate cancer.

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