Precursor moieties for forming prodrugs selectively cleaved by prostate-specific antigens (PSA)

Abstract

Disclosed are precursor moieties and compositions comprising the precursor moieties for use in methods of detecting and / or treating prostate cancer in a therapeutically or diagnostically effective amount. The precursor portion comprises a first peptide and a second peptide linked to the first peptide, where the peptides comprise a sequence configured for conjugation with a drug near a first terminal to form a prostate-specific antigen (PSA) rapid and / or highly selective cleavage of the prostate-specific antigen (PSA), and where the prostate-specific antigen (PSA) is selectively cleavage of the prostate-specific antigen (PSA) to the prostate-specific antigen (PSA). The first peptide is configured at a first end to bind to the active site of the PSA and at a second end to bind to the active site of the PSA with high selectivity, and the second peptide comprises a negatively charged sequence to retard uptake of the prodrug by the cell, and wherein the second peptide is cleaved from the first peptide upon hydrolysis by the PSA protein to produce a conjugate of the first peptide and the drug suitable for uptake by the target cell.

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 prodrugs formed using the 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 compositions to the subject.

[0002] The present invention has been developed primarily for use in detecting and / or treating prostate cancer and will be described below with reference to that application.It will be appreciated by those skilled in the art that the present invention may be applied to the treatment of other conditions, particularly those mediated by proteolytic enzymes. Background Art

[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 is measurable in the blood and can therefore be used as a clinical test to detect prostate cancer and track response to treatment.

[0004] Prodrugs may incorporate a masking moiety that is removed from the actual drug component by a mechanism such as degradation or enzyme-mediated cleavage under desired conditions.

[0005] In this regard, PSA expression by prostate cancer can be exploited to target drug delivery to prostate cancer, with the drug being released from the masking moiety when PSA cleaves the prodrug substrate. Utilizing PSA offers significant advantages for the diagnosis and treatment of prostate cancer because PSA has high levels of enzymatic activity only in the extracellular fluid surrounding healthy and cancerous prostate cells, where the active form is expressed in high concentrations.

[0006] However, for this approach to work, the linkage between the drug-containing moiety and the masking moiety must be rapidly cleaved by PSA while retaining resistance 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 that are masked in this way.

[0007] Therefore, the present invention seeks to provide a promoiety for conjugating to a drug to form a prodrug that is rapidly and / or selectively cleaved by prostate-specific antigen (PSA), a composition comprising the promoiety conjugated to a drug to form a prodrug, and a method and use for detecting and / or treating prostate cancer in a subject by administering a therapeutically or diagnostically effective amount of the composition to the subject, which will overcome or substantially improve at least some of the deficiencies of the prior art, or at least provide an alternative.

[0008] It should be understood that, if any prior art information is referred to herein, such reference does not constitute an admission 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 present invention, there is provided a precursor part comprising:

[0010] a first peptide; and

[0011] a second peptide, the second peptide being linked to the first peptide,

[0012] wherein the first peptide comprises a sequence configured near the first terminus for conjugation with a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA), and configured at the second terminus for binding with high selectivity to the active site of PSA, and the second peptide comprises a negatively charged sequence to slow down cellular uptake of the prodrug, and

[0013] The second peptide is cleaved from the first peptide after being hydrolyzed by PSA protein to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

[0014] According to a second aspect of the present invention, there is provided a composition comprising a precursor portion, the precursor portion comprising:

[0015] a first peptide; and

[0016] a second peptide, the second peptide being linked to the first peptide,

[0017] wherein the first peptide comprises a sequence configured near the first terminus for conjugation with a drug to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA), and configured at the second terminus for binding with high selectivity to the active site of PSA, and the second peptide comprises a negatively charged sequence to slow down cellular uptake of the prodrug, and

[0018] The second peptide is cleaved from the first peptide after being hydrolyzed by PSA protein to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

[0019] In one embodiment, the first peptide sequence comprises HisSerSerLysLeuGln (HSSKLQ).

[0020] Suitably, the first terminus of the first peptide is the N-terminus.

[0021] Preferably, the second peptide comprises one or more amino acids that are negatively charged at physiological pH.

[0022] In one embodiment, the second peptide comprises the sequence D-Asp-D-Glu(de).

[0023] Preferably, the second peptide is linked to the C-terminus of the first peptide via a spacer.

[0024] More preferably, the spacer is an amino acid sequence, wherein the first amino acid of the spacer is compatible with the active site of PSA.

[0025] Still more preferably, the first amino acid of the spacer is a leucine residue.

[0026] In one embodiment, the spacer comprises the amino acid sequence LeuGlyGly (LGG).

[0027] 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 the composition according to the second aspect.

[0028] In one embodiment, the composition is administered intratumorally and / or intraprostatically

[0029] In one embodiment, the composition is administered intravenously, intramuscularly, and / or subcutaneously.

[0030] In one embodiment, the subject has a localized prostate tumor.

[0031] In one embodiment, the subject has a metastatic prostate tumor.

[0032] Preferably, the administration results in a reduction in prostate tumor volume.

[0033] Preferably, the administration results in a reduction in metastatic prostate tumors.

[0034] Preferably, a treatment is administered that results in a metastatic prostate tumor.

[0035] According to a fourth aspect of the present invention, there is provided use of the composition according to the second aspect in the preparation of a medicament for detecting and / or treating prostate cancer.

[0036] According to one embodiment of the present invention, there is provided a precursor portion comprising:

[0037] a first peptide; and

[0038] a second peptide, the second peptide being linked to the first peptide,

[0039] wherein the first peptide comprises a sequence configured near the first terminus for conjugation with a drug to form a prodrug that is rapidly and / or highly selectively cleaved by a target protease, and configured at the second terminus for binding with high selectivity to the active site of the target protease, and the second peptide comprises a negatively charged sequence to slow down the uptake of the prodrug by cells, and

[0040] The second peptide is cleaved from the first peptide after proteolysis by a target protease to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

[0041] According to another embodiment of the present invention, there is provided a composition comprising a pro-portion, the pro-portion comprising:

[0042] a first peptide; and

[0043] a second peptide, the second peptide being linked to the first peptide,

[0044] wherein the first peptide comprises a sequence configured near the first terminus for conjugation with a drug to form a prodrug that is rapidly and / or highly selectively cleaved by a target protease, and configured at the second terminus for binding with high selectivity to the active site of the target protease, and the second peptide comprises a negatively charged sequence to slow down the uptake of the prodrug by cells, and

[0045] The second peptide is cleaved from the first peptide after proteolysis by a target protease to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

[0046] In a preferred embodiment, the target protease is prostate specific antigen (PSA).

[0047] Other aspects of the invention are also disclosed. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Although any other forms 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:

[0049] Figure 1 The proof-of-concept strategy used in developing a model prodrug comprising a promoiety according to a preferred embodiment of the present invention, which is used to conjugate with a drug to form a prodrug. (a) The negatively charged sequence on the model prodrug is repelled by the cell membrane, thereby reducing the uptake before being cleaved 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 by the cell and the residual negatively charged sequence is eliminated; (d) The model active drug in the cell is imaged using a fluorescence microscope;

[0050] Figure 2 Schematic representation of a model prodrug in which the drug component of the prodrug is replaced by a fluorescent tag having a comparable structure to the drug is shown;

[0051] Figure 3The chemical structure of the promoiety HSSKLQ↓LGGde used for conjugation with a drug to generate a prodrug is shown, where ↓ represents the position of PSA cleavage (PSA cleavable bond);

[0052] Figure 4 The following chemical structures are shown: (a) the peptide sequence Tag-HSSKLQ↓LGGde of a model prodrug, in which the drug component of the prodrug is replaced by (c) a fluorescent anthraquinone (AQ) tag or (d) a luminescent rhenium (I) complex tag; and (b) a fragment of the Tag-HSSKLQ↓LGGde peptide sequence (Tag-HSSKLQ and LGGde) after cleavage at the PSA cleavable bond when exposed to PSA, wherein ↓ represents the position of PSA cleavage (PSA cleavable bond); and

[0053] Figure 5 The photodiode array detection (PDA) traces (254 nm, 20–30 min) of (a) and (b) are shown: (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 generated by cleavage of the AQ-HSSKLQ↓LGGde model prodrug incubated with PSA for 3, 6, 24, and 48 h;

[0054] Figure 6 Composite images of fluorescence and bright field 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 in the absence of PSA (No PSA) or the addition of PSA (2 μg, 67 pmol) are shown [7.5 × 10 cells / well in a 96-well glass bottom plate]. 3 cells. Scale bar represents 30 μm];

[0055] Figure 7 Composite images of fluorescence and bright field channels of DLD-1 cells [7.5 × 10 cells / well in a 96-well glass bottom plate] treated with AQ-HSSKLQ↓LGGde at a concentration of 50 μM for 1, 4, and 24 h in the absence of PSA (No PSA) or with the addition of PSA (2 μg, 67 pmol) are shown. 3 cells. Scale bar represents 30 μm]; and

[0056] Figure 8Fluorescence channel, bright field channel, and composite images of DLD-1 cells treated with AQ-HSSKLQ↓LGGde at a concentration of 100 μM for 48 h in the absence of PSA (No PSA), the presence of 1 μg PSA (33 pmol), or the presence of 2 μg PSA (67 pmol) [2.5 × 10 cells / well of a 96-well plastic plate] are shown. 3 cells. Scale bar represents 30 μm. Fluorescence images were processed using a range of 20–1000 in brightness and contrast settings]. DETAILED DESCRIPTION

[0057] In the following description, it is noted that similar or identical reference numerals in different embodiments represent the same or similar features.

[0058] Generally, the present invention provides a prodrug moiety for conjugation with a drug to form a prodrug, wherein the prodrug moiety comprises (i) a first peptide, which is conjugated to the drug, and (ii) a second peptide, which is linked to the first peptide, wherein the first peptide comprises a sequence that binds to a target protease with high selectivity, and the second peptide comprises a sequence with one or more negative charges to slow down the uptake of the prodrug by the target cell and to increase the selectivity of binding to PSA by associating with an arginine patch, and wherein the second peptide is cleaved from the first peptide after proteolysis by the target protease to produce a conjugate of the first peptide and the drug suitable for uptake by the target cell.

[0059] In a preferred embodiment, and as will be described in more detail below, the target protease is prostate specific antigen (PSA).

[0060] For this purpose, it should be understood that:

[0061] (1) Prostate-specific antigen (PSA) has high substrate specificity for cleavage after glutamine (Gln) residues, which is a preference not possessed by other proteases in human serum or those with similar structures and functions to PSA.

[0062] (2) the hydrophilic HisSerSerLysLeuGln (HSSKLQ) peptide sequence is an effective cleavage peptide sequence specific for PSA, and

[0063] (3) PSA has a positively charged region near the active site (i.e., an arginine patch consisting of Arg36, Arg38, and Arg60 residues, which creates a high density of positive charges near the active site).

[0064] 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), comprising a promoiety for conjugation to a drug for the specific purpose of detecting and / or treating prostate cancer, wherein the promoiety comprises a negatively charged peptide sequence that is positioned for electrostatic interaction with the positively charged arginine patch of PSA, with the aim of increasing the selectivity and / or cleavage rate of the prodrug towards PSA.

[0065] Composition

[0066] The present invention provides compositions comprising a promoiety for conjugation with a drug to form a prodrug, wherein in a preferred embodiment, the drug is designed for the detection and / or treatment of prostate cancer.

[0067] Herein, the prodrug comprises a peptide-based promoiety for conjugation with a drug, wherein the peptide-based promoiety includes a negatively charged sequence, the purpose of which is to electrostatically interact with the positively charged arginine patch of prostate-specific antigen (PSA) and slow down the diffusion of the prodrug across the cell membrane due to charge repulsion. Only when the peptide-based promoiety is selectively cleaved in the presence of PSA to remove the negatively charged sequence, the conjugate of the remaining drug with part or all of the first peptide can diffuse across the cell membrane.

[0068] The promotor portion comprises a first peptide having a sequence that binds to the active site of prostate specific antigen (PSA) with high selectivity, and a second peptide linked to the first peptide sequence via a spacer sequence, the second peptide comprising a negatively charged sequence.

[0069] Figure 1 The proof-of-concept strategy used in the development of the model prodrug described below is shown.

[0070] like Figure 1 As shown in (a), the negatively charged sequence of the promoiety associated with the model prodrug is repelled by the cell membrane, thereby reducing uptake prior to cleavage by PSA.

[0071] Figure 1 (b) shows that when PSA is present in the extracellular fluid surrounding the target cells, the model prodrug interacts with the active site and arginine patch of PSA, promoting PSA to cleave the second peptide from the first peptide to release the conjugate of the first peptide and the drug component.

[0072] Figure 1 (c) shows that since the conjugate of the first peptide and the drug no longer has a net negative charge, it is no longer rejected by the cell membrane and is therefore able to diffuse across the cell membrane to be taken up by the target cell, while the residual negatively charged sequence is eliminated.

[0073] Successful uptake of the first peptide / drug conjugate by target cells can be confirmed by fluorescence microscopy imaging, e.g. Figure 1 (d) may be shown.

[0074] Computational molecular modeling

[0075] The inventors performed computational molecular modeling on certain peptide sequences to identify appropriate spacer residues and to identify negatively charged residues (e.g., Asp or Glu) suitable for inclusion behind the spacer to reduce any interference of the negative charge with the PSA active site. The inventors also used computational molecular modeling to study the effect of residue chirality (D or L) on substrate-PSA interactions.

[0076] Peptide sequence

[0077] Sequences with D-chirality are more resistant to proteolysis than their L-chirality counterparts [see Feng et al. and Liu et al.], therefore, comparisons between the behavior of similar sequences with different chirality were used to determine if there was an energetic penalty associated with the use of D-residues.

[0078] 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.

[0079] For simulations, the crystal structure of the enzymatically active PSA with the substrate LysGlyIleSerSerGlnTyr(KGISSQY) in the active site was downloaded from the Protein Data Bank (PDB: 2ZCK) [see Ménez et al.

[0080] The KGISSQY substrate was extended at the C-terminus with the residues or sequences listed in Table 1 using the Molefacture function in the Visual Molecular Dynamics software, followed by cycles of molecular dynamics and energy minimization using the Nanoscale Molecular Dynamics software. [See Theoretical and Computational Biophysics Group]

[0081] Table 1 #,* - Amino acid residue sequence

[0082]

[0083] # -Use the single letter amino acid residue code, upper case indicates L chirality, lower case indicates D chirality.

[0084] *-Abbreviations: L-leucine (Leu), G-glycine (Gly), D-aspartic acid (Asp), E-glutamic acid (Glu), S-serine (Ser).

[0085] After energy minimization, the potential energy of the system and the amount of hydrogen bonding interactions between PSA and the negatively charged residues of the substrate were examined.

[0086] Herein, the inventors have found that when the extension sequence has zero or only one lipophilic spacer residue between the C-terminus and the negatively charged residue of KGISSQY, the extension sequence interacts unfavorably with the kallikrein loop of PSA. The interaction between the negative charge and the kallikrein loop (which locks the substrate in the cleavage position) may destabilize substrate binding and / or interfere with the enzymatic action of PSA.

[0087] Incorporation of two spacer residues resulted in a lower incidence of interactions with the kallikrein loop. There was no significant energy penalty associated with incorporation of residues of D chirality compared to residues of L chirality.

[0088] The inventors replaced the side chain of KGISSQY in situ to generate HSSKLQ with Q in the S1 pocket of PSA, which was subjected to energy minimization cycles to optimize its position and geometry in the active site of PSA.

[0089] The HSSKLQ sequence was extended at the C-terminus with the sequences listed in Table 2 and the geometries obtained after further energy minimization indicated 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.

[0090] Table 2 #,* - Amino acid residue sequence

[0091] LGddd SGddd LGeee SGeee LGG SGD LGGDE SGGDE

[0092] # - Single-letter amino acid residue codes are used, with uppercase letters representing L chirality and lowercase letters representing D chirality.

[0093] * -Abbreviations: L-leucine (Leu), G-glycine (Gly), D-aspartic acid (Asp), E-glutamic acid (Glu), S-serine (Ser).

[0094] The three residue spacer followed by two negatively charged AspGlu residues of either D or L chirality was compared and the extracted geometries did not show any interference of the kallikrein loops by the negatively charged side chains.

[0095] The peptide with a three-residue spacer and the D-Asp-D-Glu sequence well positioned the negatively charged residues of AspGlu for hydrogen bonding 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 without incurring a significant energy penalty compared to the corresponding L chiral sequence.

[0096] Since the synthesis of AspAsp sequences can be challenging, the LGGde sequence consisting of the LGG lipophilic spacer and the de(D-Asp-D-Glu) sequence, which contributes three negative charges from the side chain and the C-terminus, was selected to provide HSSKLQ↓LGGde as the prodrug sequence to be studied.

[0097] Based on this computational modeling, the precursor portion 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 study.

[0098] Spacer

[0099] The drug is conjugated to the N-terminus of the HisSerSerLysLeuGln(HSSKLQ) sequence or to an amino acid added to the N-terminal side of the 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.

[0100] Preferably, the spacer is an amino acid sequence, wherein the first amino acid of the spacer is compatible with the active site of PSA (eg, leucine or serine).

[0101] In one embodiment, the spacer comprises a leucine residue.

[0102] In one embodiment, the spacer comprises the amino acid sequence LeuGlyGly (LGG).

[0103] The spacer sequence LeuGlyGly (LGG) was introduced to reduce the interference between the negatively charged sequence (de) and the kallikrein loop and allow the negative charge to be positioned close to the positively charged arginine patch of PSA. The electrostatic interaction between the negative charge of the substrate and the arginine patch can increase the selectivity of the prodrug for PSA and increase the cleavage rate.

[0104] Figure 3The chemical structure of the HSSKLQ↓LGGde promoiety used for conjugation with drugs to generate a model prodrug for understanding the effect of PSA on the prodrug in vitro is shown, where ↓ represents the position of PSA cleavage (PSA cleavable bond).

[0105] Model prodrug

[0106] As will be described in more detail below, the inventors have synthesized two exemplary prodrug models and investigated the efficacy of PSA selective cleavage of one of these model prodrugs in vitro in the presence of cancer cells.

[0107] For the purpose of this study, the exemplary prodrugs contained fluorescent tags as a surrogate for the drug component. Anthraquinone (AQ) fluorescent and rhenium (I) luminescent tags and / or imaging agents that are structurally similar to the drug were selected for conjugation to the peptides for tracking by fluorescence microscopy in vitro.

[0108] Figure 4 (a) shows the chemical structure of the peptide sequence tag-HSSKLQ↓LGGde of the model prodrug according to the preferred embodiment of the present invention, in which the drug component is replaced by a fluorescent tag, and Figure 4 (b) shows the chemical structure of the fragments (Tag-HSSKLQ and LGGde) produced 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).

[0109] Fluorescent Anthraquinone Label

[0110] In one embodiment, the fluorescent tag chosen for this purpose is structurally similar to a component of a validated drug that is selected for conjugation to the peptide, thereby enabling the distribution of the conjugate to be imaged in vitro using fluorescence imaging.

[0111] Figure 4 (c) shows the chemical structure of a fluorescent anthraquinone (AQ) tag that has structural similarity to anti-tumor anthracyclines such as doxorubicin and mitoxantrone. (See Kizek et al. and Hortobagyi) The AQ tag was synthesized with a carboxylic acid group to allow easy coupling to the N-terminus of the HSSKLQLGGde sequence under standard solid phase peptide synthesis (SPPS) conditions.

[0112] Luminescent Re(I) Complex Label

[0113] Figure 4 (d) shows the chemical structure of a luminescent Re(I) complex tag with a quinoline-based ligand. The long-lived triplet metal-ligand charge transfer ( 3This Re(I) complex has attracted considerable interest due to its MLCT state, which allows for in vitro monitoring using fluorescence microscopy.

[0114] In the case of the second example, the luminescent label is fac-[Re(CO)3 bisquinolinylamine] + Metal complexes (see Figure 4 (d)). The stability of the luminescent polypyridyl Re(I) complexes based on the fac-{M(CO)3} core makes them particularly suitable for imaging purposes. Fluorescent Re(I) complex tags have been conjugated to HSSKLQ-based carriers to allow comparison of PSA-mediated activation and in vitro distribution with those of the AQ model prodrug. The Re(I) complex tags were also synthesized with a carboxylic acid linker for easy coupling to the N-terminus of the HSSKLQLGGde sequence under standard SPPS conditions.

[0115] Cleavage assay

[0116] Figure 5 The following set of PDA traces (254 nm, 20-30 min) are shown: (a) AQ-HSSKLQ↓LGGde (green, retention time (RT) = 27.4 min) and (b) the peptide fragment AQ-HSSKLQ↓LGGde (blue, RT = 26.2 min) generated by cleavage after the AQ-HSSKLQ↓LGGde model prodrug was incubated with PSA for 3, 6, 24 and 48 hours.

[0117] Incubation of AQ-HSSKLQ↓LGGde with PSA resulted in cleavage after Q and the generation of fragments corresponding to AQ-HSSKLQ and negatively charged LGGde sequences that co-eluted with persistent solvent contaminants (15.7 min, not shown). Here, the intensity (au) of the uncut AQ-HSSKLQ↓LGGde sequence (green, RT = 27.4 min) was observed to decrease over time, while the intensity of the cleaved AQ-HSSKLQ fragment (blue, RT = 26.2 min) increased.

[0118] Normalized integration of the UV trace (254 nm) peaks corresponding to AQ-HSSKLQ↓LGGde and the equivalent fragment to AQ-HSSKLQ in Table 3 shows that AQ-HSSKLQ↓LGGde outcompetes PSA 200-fold and continues to be cleaved by PSA for up to 48 hours, but cleavage is still incomplete at this time.

[0119] Table 3: Normalized integral of AQ-HSSKLQ fragment cleaved from AQ-HSSKLQ↓LGGde by PSA over time. Standard errors are given for experiments performed in triplicate.

[0120]

[0121] It is evident from the data in Table 3 that the cleavage rate of AQ-HSSKLQ↓LGGde is higher than that of a previously reported related substrate (Di Marco et al.), where 23% remained uncleaved after 24 hours of incubation. The inventors believe that the negatively charged LGGde sequence may help position the substrate for cleavage, thereby achieving a higher cleavage rate. It is also believed that this same sequence (LGGde) may continue to interact with the arginine patch of PSA after cleavage, resulting in a slower binding and cleavage rate for subsequent substrates.

[0122] Fluorescence microscopy

[0123] Cell imaging studies were designed to compare the effects of the presence or absence of PSA on the distribution of the tag-peptide conjugate in DLD-1 cells, which are colorectal cancer cells. The DLD-1 cell line was specifically chosen because it does not express PSA, allowing for a controlled comparison between cells given substrate in the absence of PSA (PSA-) and cells given substrate in the presence of exogenous PSA of known concentration and uniform activity (PSA+).

[0124] For these studies, the use of human-derived prostate cancer cell lines would not allow for a comparative model based on PSA expression, as PSA expression by prostate cancer cell lines varies with culture conditions and the PSA expressed tends to have lower levels of enzymatic activity.

[0125] The inventors predict that in the presence of PSA, the AQ-HSSKLQ↓LGGde model prodrug will be cleaved to generate AQ-HSSKLQ for cellular uptake; however, in the absence of PSA, the model prodrug will remain uncleaved and therefore the dosed cells will exhibit background levels of fluorescence.

[0126] It is also predicted that cells will exhibit fluorescence when AQ-HSSKLQ is administered regardless of the presence or absence of PSA.

[0127] Unless otherwise stated, cells were incubated with AQ-HSSKLQ↓LGGde or AQ-HSSKLQ at a concentration of 50 μM in the absence or presence of exogenous PSA (20 μg / mL) for 1, 4, or 24 h.

[0128] The dosed cells were washed and then imaged using confocal fluorescence microscopy to assess the conjugate distribution in the cells. The emission of the conjugated AQ tags was monitored in the range of 570-670 nm. Unless otherwise stated, cell images were processed using brightness and contrast settings in the range of 20-300.

[0129] Figure 6 Composite images of fluorescence and bright field channels of DLD-1 cells [7.5 × 10 cells / well in a 96-well glass bottom plate] treated with AQ-HSSKLQ 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. 3 cells. The scale bar represents 30 μm].

[0130] In cells given AQ-HSSKLQ, significant levels of fluorescent precipitate were observed, and the amount of precipitate increased over time regardless of the presence or absence of PSA, with little or no evidence of cellular uptake even after 24 h of incubation ( Figure 6 ). Significantly more precipitate appeared to form when cells were treated with AQ-HSSKLQ in the presence of PSA for 1 and 4 hours compared to cells treated in the absence of PSA; however, after 24 hours of incubation, similar levels of fluorescent precipitate formed in PSA+ and PSA- conditions.

[0131] The observed increase in precipitate formation over time could reflect the time interval required for precipitates to form in vitro. AQ-HSSKLQ may have been taken up by cells, and the AQ tag or variants may have been expelled by exocytosis to form precipitates, a process that has a time scale too short to be measured by these studies. The formation of precipitates of AQ-HSSKLQ or its degradation products may have inhibited the entry of fluorescent compounds into cells by passive diffusion and active uptake, resulting in low levels of intracellular fluorescence.

[0132] Figure 7 Composite images of 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.

[0133] These images of cells given AQ-HSSKLQ↓LGGde for 1, 4, and 24 hours in the absence of PSA show that a smaller amount of fluorescent precipitate was formed and the particles were smaller ( Figure 7 ). In the absence of PSA, the extent of precipitation in cells given AQ-HSSKLQ↓LGGde increased over time but remained below the levels observed in cells given AQ-HSSKLQ, demonstrating the utility of the LGGde sequence in improving solubility.

[0134] The background level of intracellular fluorescence indicated that AQ-HSSKLQ↓LGGde was not taken up into cells in the absence of PSA. This supports the incorporation of amino acids with D chirality to enhance the resistance of AQ-HSSKLQ↓LGGde to other protease cleavage sequences.

[0135] Images of cells administered AQ-HSSKLQ↓LGGde in the presence of exogenous PSA showed that the amount of precipitate formed at earlier incubation time points (1 and 4 h) was significantly greater than that shown in images collected in the absence of PSA, consistent with in vitro cleavage of AQ-HSSKLQ↓LGGde to produce AQ-HSSKLQ, followed by formation of a fluorescent precipitate.

[0136] In cells given AQ-HSSKLQ↓LGGde and exogenous PSA, the amount of precipitate formed increased over time, consistent with the continued generation of AQ-HSSKLQ when AQ-HSSKLQ↓LGGde is cleaved by PSA.

[0137] The extent of precipitation observed in cells incubated with AQ-HSSKLQ↓LGGde for 24 h in the presence or absence of PSA cannot be easily distinguished based on these data alone.

[0138] Since the intracellular fluorescence level observed after 24 h of incubation of AQ-HSSKLQ↓LGGde in the presence of exogenous PSA was negligible, 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) of PSA per well for 48 h ( Figure 8 ).

[0139] Figure 8 Shown are fluorescence channels, bright field channels, and composite images of DLD-1 cells administered AQ-HSSKLQ↓LGGde at a concentration of 100 μM for 48 hours in the absence of PSA (No PSA), the presence of 1 μg PSA (33 pmol), or the presence of 2 μg PSA (67 pmol).

[0140] Brightness and contrast settings were adjusted during image processing to avoid background noise from diluting the emission signal. Cells incubated with AQ-HSSKLQ↓LGGde for 48 h without added PSA showed background intracellular fluorescence, indicating that the prodrug was not taken up into the cells even after extended incubation times.

[0141] Co-incubation of cells with AQ-HSSKLQ↓LGGde and 1 or 2 μg of PSA resulted in the formation of fluorescent precipitates, indicating that PSA cleaved AQ-HSSKLQ↓LGGde in vitro to generate AQ-HSSKLQ, which subsequently formed precipitates, and the amount of precipitates formed increased with increasing PSA concentrations. After 48 h, we observed some signs of intracellular fluorescence in cells given AQ-HSSKLQ↓LGGde and 2 μg of PSA, suggesting that some fluorescent compounds may have entered the cells during the extended incubation time.

[0142] Proteolytic degradation of AQ-HSSKLQ, the fragment generated by AQ-HSSKLQ↓LGGde cleavage, can lead to the generation of insoluble sequences that are prone to precipitation. The poor solubility of the AQ tag may have a significant impact on the precipitate formation observed in vitro. The extended planar structure of the AQ tag easily enables intermolecular π-π stacking, which will promote precipitation in an aqueous environment. This is consistent with some precipitation observed after incubation of AQ-HSSKLQ with Tris buffer for 48 hours in the PSA cleavage assay.

[0143] The charge present on the AQ-HSSKLQ fragment may also contribute to the poor cellular uptake.

[0144] 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 is essential for proper interaction with PSA to achieve cleavage, which makes the HSSKLQ sequence in the AQ tag susceptible to proteolytic degradation in vivo and in vitro.

[0145] Proteases present in the culture medium or expressed extracellularly by DLD-1 cells can cleave the AQ tag at any amide bonds present to generate less soluble sequences, such as AQ-HS or AQ-HSS, with a net charge of -1, which will be electrostatically repelled by the cell membrane.

[0146] Regardless of the presence or absence of PSA, the precipitate particles observed in the images of cells administered with AQ-HSSKLQ↓LGGde were smaller and fewer in number than those observed in the images of cells administered with AQ-HSSKLQ.

[0147] The negative charge of the D-Asp-D-Glu sequence in AQ-HSSKLQ↓LGGde may impart greater hydrophilicity, thereby increasing solubility, and may also prevent π-π stacking interactions that may promote precipitation.

[0148] In the absence of PSA, AQ-HSSKLQ↓LGGde appears to undergo little or no degradation by proteases, which may be proteases expressed by DLD-1 cells or may be present in the supplemented culture medium. Proteolytic degradation 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 large precipitates. The D-chirality of the C-terminal AspGlu sequence of AQ-HSSKLQ↓LGGde is likely to enhance this in vitro resistance to proteolysis.

[0149] Although the formation of fluorescent precipitates in vitro in these studies was disappointing and complicated the interpretation, these results do indicate that in the presence of cancer cells, AQ-HSSKLQ↓LGGde is successfully cleaved by PSA, presumably to generate AQ-HSSKLQ, which then forms a precipitate. The increase in the amount of precipitate formed over time in cells given AQ-HSSKLQ↓LGGde in the presence of PSA suggests that PSA retains its enzymatic activity and continues to cleave substrate over time.

[0150] Treatment

[0151] The present invention also provides a method and use of the above composition in producing a drug for detecting and / or treating prostate cancer in a subject.

[0152] The composition comprises a prodrug in an amount effective for treating a disease condition, wherein the prodrug is formed by replacing the AQ fluorescent label with a drug component (i.e., an active pharmaceutical ingredient (API)), and can be administered to any subject, including human or non-human animals, in a therapeutically or diagnostically effective amount.

[0153] These prodrugs can be administered parenterally by injection or gradual infusion over time. These prodrugs can be administered intratumorally, intraprostatically, intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitary or transdermally.

[0154] Other methods of administration will be known to those skilled in the art.

[0155] In one embodiment, a therapeutically or diagnostically effective amount of a prodrug composition is administered to a subject with a localized or metastatic prostate tumor for treatment thereof, wherein the treatment is to reduce the volume or size of the tumor.

[0156] Preparations for parenteral administration of the prodrug 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 (e.g. olive oil) and injectable organic esters (e.g. ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (e.g. supplements based on Ringer's dextrose) etc. Preservatives and other additives may also be present, for example, antibacterial agents, antioxidants, chelating agents and inert gases.

[0157] definition

[0158] As used herein, the term "prodrug" means a compound that is a drug precursor, which, after administration to a subject, releases the drug in vivo by some chemical or physiological process (e.g., the prodrug is converted into the desired active drug form when the physiological pH is reached or by enzyme action). The prodrug can be converted into a product that is toxic to tumor cells.

[0159] Those skilled in the relevant art will appreciate that the terms "drug" and "prodrug" as used herein may include diagnostic agents and pro-diagnostic agents, respectively.

[0160] 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.

[0161] As used herein, the term "prostate specific antigen" (PSA) means prostate specific antigen, also known as human kallikrein 3 (KLK3 or hK3), and all other proteases that have the same or substantially the same proteolytic cleavage specificity as prostate specific antigen.

[0162] As described herein, amino acid sequences are presented according to the standard convention with the amino terminus of the peptide on the left and the carboxyl terminus on the right.

[0163] 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 within the skill of the art, for example, as described in Remington's Pharmaceutical Science, 18th edition, Mack Publishing Company, Easton, Pa. (1990), and Remington: the Science and Practice of Pharmacy, 20th edition, Lippincott Williams & Wilkins, (2003).

[0164] As used herein, the term "administering" or "administration" as used herein means the introduction of a foreign molecule into a cell or a host. The term is intended to be synonymous with the term "delivery".

[0165] As used herein, the term "treating" or the phrase "to treat" refers to any type of treatment that benefits a subject suffering from a disease, including improving the subject's condition (e.g., one or more symptoms), slowing the progression of the condition.

[0166] As used herein, when referring to a measurable value (e.g., a parameter, an amount, a duration, etc.), the term "about" is intended to encompass variations of ±10% or less, preferably ±-5% or less, more preferably ±1% or less, and still more preferably ±0.1% or less of the specified value, as long as such variations are suitable for implementation in the disclosed invention. It should be understood that the value to which the modifier "about" refers is itself also specifically and preferably disclosed.

[0167] As used herein, the singular forms "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0168] As used herein, the terms "amino acid" and "peptide" are understood to include groups that can be incorporated into the peptide sequence such that it retains the ability to bind to the active site of a protease and be cleaved by that protease.

[0169] As used herein, residues of a peptide amino acid sequence having L chirality are written in upper case, while residues having D chirality are written in lower case (eg, LGGde).

[0170] Materials and Methods

[0171] Synthesis of AQ tags

[0172] This method was adapted from Di Marco et al. (2019).

[0173] During the synthesis and work-up process, the reaction mixture and the product were protected from light. 1-Chloroanthraquinone (2.64 g, 10.9 mmol, 1.1 eq.) was combined with anhydrous DMA (60 mL) and TEA (3.0 mL, 21.4 mmol, 2.2 eq.). 4-Aminobutyric acid (1.04 g, 10.1 mmol, 1.0 eq.) was added and the reaction mixture was heated to 160 ° C for 5 hours. The reaction mixture was cooled to 55 ° C and concentrated overnight under a nitrogen stream. 0.1 M HCl was used. (aq) The mixture was acidified with a solution (60 mL) and extracted into DCM (2 x 80 mL), then washed with water (2 x 60 mL) and quenched with 0.1 M NaOH (aq) The solution (2 x 60 mL) was extracted into the aqueous layer. The aqueous layer was washed with DCM until the organic layer was clear, and then washed with 1 M HCl (aq) The solution acidified the aqueous layer to precipitate the product. The product was collected by vacuum filtration as a dark red solid (0.19 g, 0.601 mmol, 6% yield).

[0174] 1 H NMR (400 MHz, MeOD-d 4 , ppm): δ8.27 (m, 2H), 7.85 (ddd, J = 7.5 Hz, J = 1.5 Hz, 2H), 7.64 (dd, J = 7.4 Hz, J = 8.6 Hz, 1H), 7.29 (dd, J = 0.7 Hz, J = 8.6 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 7.2 Hz, 2H), 2.04 (quintet, J = 7.2 Hz, 2H).

[0175] 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.

[0176] LRMS-ESI(-)m / z:C 18 H 14 NO4 - [M–H] - The measured value is 308.25 and the calculated value is 308.09.

[0177] Method for synthesizing Re(I) complex tags

[0178] Unless otherwise stated, the following synthetic methods were adapted from Green (2013).

[0179] Synthesis of Methyl Quinoline Ligand

[0180] TEA was dried over anhydrous sodium sulfate for 48 hours. 4-(Aminomethyl)benzoic acid methyl ester hydrochloride (0.61g, 3.03mmol, 1.0eq.) was combined with anhydrous MeCN (10mL) and anhydrous TEA (0.42mL, 3.03mmol 1.0eq.) to neutralize the mixture. 2-(Chloromethyl)quinoline hydrochloride (1.43g, 6.65mmol, 2.2eq.) was combined with anhydrous MeCN (10mL) and anhydrous TEA (0.93mL, 6.67mmol, 2.2eq.) to neutralize the mixture. Both mixtures were ultrasonically treated to aid the formation of fine particles and combined under inert conditions. 2.2eq. of anhydrous TEA (0.93mL, 6.67mmol) was further added, and the reaction mixture was refluxed under an inert atmosphere for 24 hours. The mixture was cooled to room temperature and concentrated under a nitrogen stream, then heated to room temperature with saturated NaHCO 3(aq) The product was added into 4% ethyl acetate (0.71 g, 1.60 mmol, 53% yield).Then the mixture ... product was added into 4% ethyl acetate (0.71 g, 1.60 mmol, 53% yield).Then the solution (20 mL) was diluted. The product was extracted into chloroform (4 × 20 mL) and washed with brine (3 × 15 mL), then dried over anhydrous sodium sulfate. The mixture was filtered, and the solvent was removed under reduced pressure from the crude product. 10-55% EtOAc in hexane was used as eluent, and the product was purified by column chromatography without flashing on a silica gel stationary phase. The solvent was removed under reduced pressure from the column to obtain a golden brown solid (0.71 g, 1.60 mmol, 53% yield).

[0181] 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).

[0182] 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.

[0183] LRMS-ESI(+)m / z:C 29 H 26 N3O2 + [M+H] + Found 448.17, Calculated 448.20. On silica gel plates, in a 50% mixture of EtOAc in hexanes, R f =0.23.

[0184] Synthesis of Quinoline Ligands

[0185] Methyl 4-((bis(quinolin-2-ylmethyl)amino)methyl)benzoate (0.47 g, 1.05 mmol) was dissolved in MeCN and 1 M KOH (aq) 1:1 mixture of 50% HCl and heated at 95°C for 4 hours with stirring. After cooling to room temperature, the reaction mixture was concentrated under a nitrogen stream and then diluted with water (10 mL). 1 M HCl was used (aq) The acidity of the solution mixture was adjusted to pH 4 to precipitate the product, and the product was extracted into chloroform (6 x 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%).

[0186] 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).

[0187] 13 C 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.

[0188] LRMS-ESI(-)m / z:C 28 H 22 N3O2 + [MH]- Found 432.18, calculated 432.17. On silica gel plates, in a 50% mixture of ethyl acetate in hexane, R f =0.00.

[0189] Synthesis of triaquatricarbonylrhenium(I)bromide

[0190] This procedure was adapted from Lazarova et al. (2004).

[0191] Water (25mL) was added to bromopentacarbonylrhenium (I) (3.33g, 8.19mmol) and heated to reflux. During the reaction, the condenser was regularly rinsed with water. After 24 hours, the reaction mixture was cooled to room temperature and filtered to remove the remaining starting material. Water was removed from the product under reduced pressure to obtain a white green solid, and trihydrate tricarbonylrhenium (I) bromide (1.44g, 3.55mmol, 43% yield) was identified. The product was used for subsequent composite reactions without further purification.

[0192] Synthesis of Re(I) complex tags

[0193] Combine 4-((Bis(quinolin-2-yl)amino)-methyl)benzoic acid (0.33 g, 0.763 mmol, 1.0 eq.) and tricarbonylrhenium(I) bromide trihydrate (0.45 g, 1.11 mmol, 1.5 eq.) in MeOH. Use 0.5 M NaOH (aq) The acidity of the mixture is adjusted to pH 8, and the mixture is refluxed for 4 hours. The reaction mixture is concentrated under a stream of nitrogen and extracted into DCM (15mL), then washed twice with water (2 × 10mL). DCM is removed under reduced pressure from the reaction mixture, and the crude product is dissolved in 3: 1 MeOH: MeCN, and the unbuffered MeCN with a gradient of 30-60% in 0.1% w / v NH4OAc-buffered milliQ water is purified by preparative HPLC in 30 minutes (RT = 21.3min). The fractions containing the pure product are merged, and the residual buffer is removed by continuous lyophilization. The purity of the product is determined by LCMS, and LCMS (RT = 21.0min) is carried out in 30 minutes with a gradient of 0-50% buffered MeCN. The purified product is concentrated under nitrogen, then lyophilized twice to remove residual NH4OAc, and collected as a light brown solid (0.30g, 0.382mmol, 50%).

[0194] 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).

[0195] 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.

[0196] LRMS-ESI(+)m / z:C 31 H 23 N3O5Re + [M] + The measured value is 703.96 and the calculated value is 704.12.

[0197] HRMS(+)m / z:C 31 H 23 N3O5Re + [M] + The measured value is 404.1194 and the calculated value is 704.1190.

[0198] IR:ν max (cm -1 )=2025,1916(fac-[Re(CO)3] + ); on silica gel plates, in a 50% mixture of EtOAc in hexanes, R f =0.00.

[0199] Peptide synthesis

[0200] Peptides were synthesized manually in a sintered syringe using a loaded solid phase resin support and the Fmoc protecting group strategy. 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. Considering the resin loading, the amounts of reagents and solvents were specified to be equivalent to 0.1 g of loaded resin.

[0201] Between each step, the resin was washed with 5×DMF, 5×DCM, and then 5×DMF, with a volume of approximately 1.5 mL per wash. HSSKLQ was prepared using commercially available Fmoc-Gln(Trt)-Wang resin (loading: 0.52 mmol g -1 ) was synthesized, while HSSKLQL was synthesized using commercially available Fmoc-Leu-Wang resin (load: 0.66 mmol g). Ctc resin was manually loaded with Fmoc-D-Glu(OtBu)-OH to synthesize HSSKLQLGGde.

[0202] In order to load ctc resin, 0.1g resin is expanded in DCM, and 30 μ L thionyl chloride (thionylchloride) is slowly added, and 0.5mL DMF is added subsequently. Cover container and vibrate 4 hours, then discharge DMF solution, and use DCM (5 × 3mL) washing resin. Add freshly prepared Fmoc-D-Glu (OtBu) -OH (28mg, 66 μ mol, 1.1eq.) and DIPEA (17 μ L, 0.18mmol, 3.0eq.) solution in DCM (1.0mL) immediately, then cover container and vibrate 45min. Solution is poured out, and fresh same solution is added, then container is vibrated 16 hours.

[0203] The loaded resin was washed and used for peptide synthesis. Based on the absorbance of the dibenzofulvene-piperidine adduct at 301 nm, this procedure produced 1.2 mmol g -1 Loading, which was chosen to compensate for the short Fmoc removal time of Fmoc-e-ctc and Fmoc-de-ctc, which resulted in low yields when synthesizing HSSKLQ↓LGGde. Fmoc removal of all peptides without Asp residues was performed by exposing the resin to 20% piperidine in DMF solution (3×3 min, 9 mL total), except for Fmoc-e-ctc, where shorter interval times were used to avoid cleavage from the resin (2×2 min, 1×1 min, 9 mL total). Fmoc was removed from Fmoc-de-ctc using 20% ​​piperidine in DMF containing 0.1 M Oxyma Pure with short interval times to reduce the formation of diketopiperazine and asparagine (1×2 min, 1×1 min, 6 mL total). Fmoc was removed from peptide sequences containing Asp residues in 20% piperidine in DMF containing 0.1 M Oxyma Pure.

[0204] For all steps, the extent of Fmoc removal was monitored using the Kaiser test, and except for the short time intervals specified above, the Fmoc removal step was repeated if the Kaiser test indicated that removal was incomplete. After Fmoc removal was complete, the resin was washed as detailed above.

[0205] To couple the residues, the amino acid (4 eq.), PyBOP (4 eq.) and NMM (12 eq.) were mixed in DMF to give a 0.5 M solution relative to the amino acid. All couplings were performed for 45 min and repeated. The coupling solution was drained and the resin washed. The extent of coupling was monitored using the Kaiser test, where a negative result for the free amine indicated that the coupling was complete.

[0206] Fluorescent label conjugation

[0207] After the sequences (HSSKLQ, HSSKLQL, HSSKLQLGGde) had been synthesized on the resin, the N-terminal Fmoc group was removed, the resin was then washed, and the tag coupling solution was added. The AQ tag was coupled as for the amino acids, while the Re(I) complex tag was coupled using lower levels and longer intervals (3 eq. Re(I) tag, 3 eq. PyBOP, 9 eq. NMM in DMF to make a 0.5 M solution; 24 hours).

[0208] Cutting from resin

[0209] The tag-peptide conjugate was cleaved from the resin and overall 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. MALDI-TOF analysis confirmed the presence of each conjugate and the overall deprotection of the AQ-peptide, but showed that some tBu groups remained on the side chains of the Re-peptide conjugate. The Re-peptide was exposed to 90% TFA, 5% TIS and 5% water for another 1.5 hours to completely remove the tBu group and was confirmed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF).

[0210] Peptide purification

[0211] The peptide was purified in reverse phase to >95% purity 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 x 250 mm at 4 mL / min).

[0212] Preparative HPLC was performed using a Waters 2535 quaternary gradient module and a Waters Sunfire C18 column (OBD 5 μm, 19 mm×150 mm, at 7 mL / min). The UV trace was monitored at 254 nm and the peaks were collected using an automated fraction collection system.

[0213] AQ-HSSKLQ↓LGGde: preparative type, 0-100% MeCN within 30 min, RT=17.4 min; AQ-HSSKLQ: semi-preparative type, 0-50% MeCN within 30 min, RT=22.1 min; AQ-HSSKLQ↓L: preparative type, 20-80% MeCN within 30 min, RT=12.8 min; Re-HSSKLQ↓LGGde: preparative type, 30-40% MeCN within 30 min, RT=8.1 min; Re-HSSKLQ: preparative type, 20-80% within 30 min, RT=15.3 min; Re-HSSKLQ↓L: preparative type, 30-40% MeCN within 30 min, RT=8.3 min.

[0214] Peptide characterization

[0215] 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, and the samples were 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 5μm, 2.1mm×150mm, at 0.2mL / min) and milliQ and MeCN buffered with 0.1% v / v TFA. The sample was dissolved in 1:1 milliQ:MeCN containing 0.1% TFA and guanidine hydrochloride at a concentration of 1M to prevent peptide folding. The analytical HPLC trace was obtained by a gradient of 0-100% MeCN in 30min and monitored at 254nm.

[0216] AQ-HSSKLQ↓LGGde: yield 7.9 mg, 5.41 μmol, 4%, LRMS m / z 729[M] 2 ,RT=16.5min;AQ-HSSKLQ:yield 44.3mg, 44.8μmol, 68%,LRMS m / z990.41[M] + ,RT=13.9min;AQ-HSSKLQ↓L:yield 43.6mg, 39.5μmol, 54%,MS m / z 1103.52[M]+ ,RT=17.4min.

[0217] PSA cleavage study

[0218] The methods and conditions used in the PSA cleavage study are based on the methods and conditions reported previously. PSA with enzymatic activity purified from human semen was obtained from GenWay Biotech (0.59 mg / mL solution in 10 mM PBS, measured by SDS-PAGE> 95% purity, pH 7.4m 30 kDa, no preservatives). AQ-HSSKLQ↓LGGde, AQ-HSSKLQ and AQ-HSSKLQ↓L were dissolved in milliQ water to prepare a 5 mM stock solution. Liquid chromatography mass spectrometry (LCMS) traces in reverse phase were obtained using Shimadzu UFLC LCMS, and Bruker autoflex speed TOF included CBM-20A controller, DGU-20A3 degasser, two LC-20AD pumps, CTO-20A column oven, SPD-M20A photodiode array detector and LCMS-2020 mass spectrometer. Separation was achieved using a Waters Xbridge BEH130C18 analytical column (OBD 5 μm, 4.6 mm×150 mm, at 0.2 mL / min) using milliQ water and MeCN (each containing 0.1% v / v formic acid buffer). 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, a 20 μL aliquot of the incubation solution was collected once at 3 and 6 hours of incubation, and three times at 24 and 48 hours of incubation.

[0219] Each aliquot was prepared to 100 μL with 50% MeCN in milliQ solution and centrifuged and then syringe filtered. 20 μL of the sample was loaded into the LCMS and analyzed over the m / z 200-2000 range with a gradient of 0-100% MeCN in milliQ water over 60 min. The component compounds eluting during 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 the cleavage of the Gln-Leu bond in AQ-HSSKLQ↓LGGde was mediated by PSA.

[0220] Cell lines and maintenance

[0221] Human colorectal cancer DLD-1 cells (colorectal adenocarcinoma cell line) were obtained from the American Type Culture Collection and used from passage 6-20 within 3 months of recovery. Cells were maintained in exponential growth in Adv DMEM supplemented with 2mM glutamine and 2% FBS in a humidified environment of 5% CO2. Cells were seeded into 96-well plates containing 100 μL of Adv DMEM supplemented with 2% FBS and 2mM glutamine. The number of cells seeded in each well and the plate material (glass or plastic) are listed in the captions of the cell images. The cells were allowed to adhere overnight under standard incubation conditions. Each experimental condition was repeated three times. Before dosing, cells were gently washed with Adv DMEM supplemented with 2mM glutamine (3×100 μL). Cells were dosed in Adv DMEM supplemented with 2mM glutamine, and the final substrate concentration was 50 μM, unless otherwise stated. Cells were dosed in the absence or addition of PSA (2 μg, 67 pmol, yielding a final concentration of 20 μg / mL unless otherwise stated). Plates were incubated under standard conditions.

[0222] Prior to imaging, media was removed from each well and cells were gently washed with PSA (3 x 100 μL). Prior to imaging at specific time points, Fluorobrite DMEM supplemented with 2 mM glutamine (100 μL) was added to each well.

[0223] Confocal fluorescence microscopy

[0224] Confocal fluorescence microscopy was performed using an Olympus FV3000 inverted fluorescence microscope equipped with a UPLSAPO 10X dry objective (NA=4.0) and a thermally controlled stage maintained at 37°C with a supply of 5% CO2. A 561nm LED laser was used to excite the AQ peptide conjugate and the emission was monitored in the range of 570-670nm. All images were acquired using line averaging (3 times per line), a scan rate of 2μs per pixel, and a digital zoom of 5.0. Three different positions were selected in each well for imaging. Micrographs of cells with the best focus in bright field were imaged. Images were acquired using Olympus FV31S-SW-v2.4.1.198 software. The 561nm laser was used at 50% power, 650V, 1× gain, and 3% offset. The scale bar represents 30μm. Images were processed using Fiji ImageJ v1.53c. Unless otherwise stated, all fluorescence images were processed using a brightness and contrast setting range of 20–300.

[0225] References

[0226] 1.Di Marco,L.et al.“Modulating the Cellular Uptake of FluorescentlyTagged Substrates of Prostate-Specific Antigen before and after EnzymaticActivation”,Bioconjugate Chemistry 30,124-133,doi:10.1021 / acs.bioconjchem.8b00792(2019).

[0227] 2.Eissler,S.et al.“Substitution determination of Fmoc-substitutedresins at different wavelengths”,Journal of Peptide Science 23,757-762(2017).

[0228] 3.Feng,Z.&Xu,B.“Inspiration from the mirror:D-amino acid containingpeptides in biomedical approaches”,Biomol Concepts 7,179-187,(2016).

[0229] 4.Frenette,G.,Gervais,Y.,Tremblay,R.R.&Dube,J.Y.“Contamination ofpurified prostate-specific antigen preparations by kallikrein hK2”,TheJournal of Urology 159,1375-137,(1998).

[0230] 5.Green,B.P.“Matrix metalloproteinase binding agents for luminescenceand radioimaging of metastatic tumours”,Doctor of Philosophy thesis,TheUniversity of Sydney,(2013).

[0231] 6.Greene,R.F.&Pace,C.N.“Urea and guanidine hydrochloride denaturationof ribonuclease,lysozyme,α-chymotrypsin,andβlactoglobulin”,Journal ofBiological Chemistry 249,5388-5393(1974).

[0232] 7.Hortobagyi,G.“Anthracyclines in the treatment of cancer”,Drugs 54,1-7(1997).

[0233] 8.Kaiser,E.,Colescott,R.,Bossinger,C.&Cook,P.“Color test fordetection of free terminal amino groups in the solid-phase synthesis ofpeptides”,Analytical Biochemistry 34,595-598(1970).

[0234] 9.Kizek,R.et al.“Anthracyclines and ellipticines as{DNA}-damaginganticancer drugs:Recent advances.Pharmacology&Therapeutics”,133,26-39(2012).

[0235] 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 andmolecular structure of[Re(CO)3(H2O)3]Br”,Inorganic Chemistry Communications 7,1023-1026(2004).

[0236] 11. Liu, M., et al. “D-Peptides as Recognition Molecules and Therapeutic Agents”, The Chemical Record 16, 1772 - 1786, (2016).

[0237] 12. Ménez, 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).

[0238] 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).

[0239] 14. Remington's Pharmaceutical Science, 18th ed., Mack Publishing Company, Easton, Pa. (1990).

[0240] 15. Remington: the Science and Practice of Pharmacy, 20th ed., Lippincott Williams & Wilkins, (2003).

[0241] 16. Theoretical and Computational Biophysics Group, www.ks.uiuc.edu, www.ks.uiuc.edu (March 2006, Accessed 2018).

[0242] Conclusion

[0243] This work involves the design and synthesis of a model prodrug targeting prostate cancer that incorporates a promotor portion with a peptide sequence that is highly selectively cleaved by prostate-specific antigen (PSA), a negatively charged sequence for electrostatic interaction with the arginine patch of PSA and slowing down cellular uptake, and a fluorescent tag that allows imaging of the distribution of the model prodrug in vitro.

[0244] The results described herein provide that negatively charged peptide sequences (including lipophilic spacers) can be easily modeled and designed in the active site of PSA using computational molecular simulations. Modeled interactions between HSSKLQ↓LGGde and PSA showed that the desired electrostatic interactions occur between the negatively charged sequence and the arginine patch of PSA.

[0245] Furthermore, these studies revealed that amino acids with D-chirality can be easily incorporated into the models and that they do not incur an energetic penalty compared to the corresponding sequences with L-chirality.

[0246] Despite the limitations of the AQ-peptide conjugates in these studies, these results suggest resistance to cellular uptake in the absence of PSA. In this in vitro model, the model prodrug appears to be less susceptible to proteolytic degradation, likely due to the incorporation of a negatively charged C-terminal D-Asp-D-Glu sequence. Furthermore, the prodrug was activated in vitro by PSA, even at PSA concentrations 1000-fold lower than those found in prostate extracellular fluid.

[0247] advantage

[0248] As described herein in embodiments of the invention, promoieties and compositions comprising the same for forming prodrugs for detecting and / or treating prostate cancer provide numerous advantages, including, but not limited to:

[0249] The prodrug's proportion has a peptide sequence that is selectively cleavable in the presence of prostate-specific antigen (PSA) to produce a cleaved peptide-drug conjugate;

[0250] The cleaved peptide-drug conjugate is expected to be taken up by cells in the surrounding environment;

[0251] - The resulting prodrugs produced from the promoieties described herein are selective for prostate cancer.

[0252] Implementation plan:

[0253] References in this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, it will be apparent to one of ordinary skill in the art from this disclosure that the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0254] Similarly, it should be understood that in the above description of exemplary embodiments of the present invention, various features of the present invention are sometimes grouped together in a single embodiment or description thereof in order to simplify the disclosure and facilitate understanding of one or more of the various inventive aspects. However, this disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than those explicitly recited in each claim. On the contrary, as reflected in the following claims, inventive aspects lie in less than all features of the aforementioned disclosed individual embodiments. Therefore, the claims following the detailed description are hereby expressly incorporated into the detailed description, with each claim itself serving as a separate embodiment of the present invention.

[0255] In addition, although some embodiments described herein include some features included in other embodiments but not other features, the combination of features of different embodiments is intended to be within the scope of the present invention and form different embodiments, as will be understood by those skilled in the art. For example, in the following claims, any claimed embodiment can be used in any combination.

[0256] Other Implementations

[0257] It should be understood by those skilled in the relevant art that the model prodrug and its composition described above in the embodiments of the present invention are not simply limited to the conjugate formed between the above peptide sequence and the anthraquinone (AQ) type drug, but can be suitable for conjugation with other drugs for detecting and / or treating prostate cancer, including, for example, drugs approved by the National Cancer Institute of the National Institutes of Health (NIH) for treating prostate cancer (see https: / / www.cancer.gov / about-cancer / treatment / drugs / prostate ).

[0258] Contains and includes

[0259] In the following claims and foregoing description of the invention, unless the context requires otherwise by explicit language or necessary implication, the word "comprise" or variations (such as "comprises" or "comprising") are used in an inclusive sense, i.e. specifying the presence of stated features, but not excluding the presence or addition of further additional features in various embodiments of the invention.

[0260] As used herein, any term: including or which includes or that includes is also an open term, which also means at least including the elements / features following the term, but not excluding other elements / features. Therefore, including is synonymous with including or means including.

[0261] Scope of the Invention

[0262] Therefore, although there have been described what are considered to be preferred embodiments of the present invention, those skilled in the art will recognize that other and further modifications may be made thereto without departing from the spirit of the present invention, and it is intended to claim all such changes and modifications that fall within the scope of the present invention. For example, any recipe given above represents only procedures that may be used.

[0263] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

[0264] Industrial Applicability

[0265] As is evident from the above, the described setup is suitable for the medical and healthcare industries.

Claims

1. A precursor part, comprising: a first peptide; and a second peptide, the second peptide being linked to the first peptide, wherein the first peptide comprises a sequence configured to be conjugated to a drug near a first terminus to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA), and configured to bind to an active site of PSA with high selectivity at a second terminus, and the second peptide comprises a negatively charged sequence to slow cellular uptake of the prodrug, and The second peptide is cleaved from the first peptide after being hydrolyzed by PSA protein to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

2. A composition comprising a precursor portion, the precursor portion comprising: a first peptide; and a second peptide, the second peptide being linked to the first peptide, wherein the first peptide comprises a sequence configured to be conjugated to a drug near a first terminus to form a prodrug that is rapidly and / or highly selectively cleaved by prostate-specific antigen (PSA), and configured to bind to an active site of PSA with high selectivity at a second terminus, and the second peptide comprises a negatively charged sequence to slow cellular uptake of the prodrug, and The second peptide is cleaved from the first peptide after being hydrolyzed by PSA protein to produce a conjugate of the first peptide and the drug suitable for uptake by target cells.

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

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

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

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

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

10. A promotor or composition according to claim 8 or 9, wherein the spacer comprises the amino acid sequence LeuGlyGly (LGG).

11. 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 according to claim 2.

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

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

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

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

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

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

18. The method of claim 17, wherein administration is of a therapy that results in said metastatic prostate tumor.

19. Use of the composition according to claim 2 in the preparation of a medicament for detecting and / or treating prostate cancer.

Images