Application of Neurokinin-1 Receptor in Targeted Strategies for Glioma Treatment. Part I: Synthesis and Evaluation of Substance P Fragments Labeled with 99mTc and 177Lu as Potential Receptor Radiopharmaceuticals

Gliomas, particularly WHO grade IV glioblastoma multiforme, are one of the most common and aggressive primary tumors of the central nervous system. The neuropeptide, substance P (SP), is the physiological ligand of the neurokinin-1 (NK-1) receptor that is consistently overexpressed in glioblastoma cells. The aim of this work was to study physico-chemical and biological properties of different SP analogues labeled with technetium-99m and lutetium-177 radionuclides. The synthesized compounds were characterized in vitro by partition coefficients (logP) and their stability was investigated in various physiological solutions. Biological properties (Kd, Bmax) were characterized using the U373 MG cell line. The obtained lipophilicity values of the [99mTc]NS3/CN-SP and [177Lu]DOTA-SP radiobioconjugates were in the range of −0.3 to +0.6 and −2.5 to −5.0, respectively. The studied radiobioconjugates were stable in PBS buffer and CSF, as well as in 10 mM histidine and/or cysteine solutions whereas in human serum showed enzymatic biodegradation. [177Lu]DOTA-[Thi8,Met(O2)11]SP(1–11), [177Lu]DOTA-SP(4–11) and [177Lu]DOTA-[Thi8,Met(O2)11]SP(5–11) radiobioconjugates bound specifically to NK-1 receptors expressed on glioblastoma cells with affinity in the nanomolar range. To conclude, the shorter analogues of SP can be used as vectors, nevertheless they still do not fulfil all requirements for preparations in nuclear medicine.


Introduction
Gliomas are the most common primary brain tumors in adult patients. The most malignant of them is glioblastoma multiforme (GBM), also called glioblastoma or astrocytoma. Grade IV GBM is a very aggressive primary brain tumor with unknown etiology in most cases [1]. GBM appears to account for over 50% of all brain tumors and more than 20% of all intracranial tumors. The infiltrating character of these tumors (clearly defined margins are absent) and the significant morphological cell In the first series ([ 99m Tc]NS3-SP, 1), the biomolecules were labelled with diagnostic radionuclide technetium-99m (emitter , t½ = 6.01 h, Emax = 0.141 MeV) as an analog of therapeutic rhenium-188. Technetium and rhenium are congeners and owing to lanthanide contraction, they have almost identical ionic radii. This indicates that these two elements form analogous complexes (differing only in the metal center) with exactly the same chemical structure and stability. Due to the fact that these species should exhibit the same "in vivo" biological behavior [14,15]. In the second series ([ 177 Lu]DOTA-SP, 2), the biomolecules were labelled with therapeutic radionuclide lutetium-177 (emitter β, t½ = 6.71 days, Emax = 0.497 MeV). The 99m Tc radiobioconjugates contained [ 99m Tc]Tc 3+ cation coordinated by the tetradentate NS3 tripodal chelator (tris(2-mercaptoethyl)-amine; 2,2',2''nitrilotriethanethiol) and a monodentate isocyanide species CN-BFCA (bifunctional coupling agent, isocyanobutyric acid succinimidyl ester). These technetium-99m complexes are extremely stable not only in the thermodynamic sense, but also towards ligand exchange in vivo [16,17]. In the case of 177 Lu radiobioconjugates, the [ 177 Lu]Lu 3+ cation was coordinated by the macrocyclic ligand DOTA (used in the syntheses in the active ester form, mono-N-hydroxysuccinimide, DOTA-NHS). In both series, the bifunctional coupling agent (CN-BFCA or DOTA-NHS) was coupled with the selected SPanalogue beforehand. The labeled SP analogues ( Figure 2) were: SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and its modified form [Thi 8 ,Met(O2) 11 ]SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), SP (4)(5)(6)(7)(8)(9)(10)(11), and SP (5)(6)(7)(8)(9)(10)(11) and its modified form [Thi 8 ,Met(O2) 11 ]SP (5)(6)(7)(8)(9)(10)(11). All analogues contained a sequence of five amino acids at the C-terminus of the peptide, responsible for their affinity towards the NK-1 receptor [8]. The HPLC chromatograms of labeling reactions with technetium-99m (1a-e) and lutetium-177 (2a-e) are presented in Figure 3. The small peaks on the HPLC chromatograms of the SP analogue labeling reactions with technetium-99m (1a-e) recorded at RT = 12.2-12.5 min ( Figure 3A) corresponded to the intermediate complex [ 99m Tc](NS3) [17]. In the first series ([ 99m Tc]NS 3 -SP, 1), the biomolecules were labelled with diagnostic radionuclide technetium-99m (emitter γ, t 1 2 = 6.01 h, E max = 0.141 MeV) as an analog of therapeutic rhenium-188. Technetium and rhenium are congeners and owing to lanthanide contraction, they have almost identical ionic radii. This indicates that these two elements form analogous complexes (differing only in the metal center) with exactly the same chemical structure and stability. Due to the fact that these species should exhibit the same "in vivo" biological behavior [14,15]. In the second series ([ 177 Lu]DOTA-SP, 2), the biomolecules were labelled with therapeutic radionuclide lutetium-177 (emitter β, t 1 2 = 6.71 days, E max = 0.497 MeV). The 99m Tc radiobioconjugates contained [ 99m Tc]Tc 3+ cation coordinated by the tetradentate NS 3 tripodal chelator (tris(2-mercaptoethyl)-amine; 2,2',2"-nitrilotriethanethiol) and a monodentate isocyanide species CN-BFCA (bifunctional coupling agent, isocyanobutyric acid succinimidyl ester). These technetium-99m complexes are extremely stable not only in the thermodynamic sense, but also towards ligand exchange in vivo [16,17]. In the case of 177 Lu radiobioconjugates, the [ 177 Lu]Lu 3+ cation was coordinated by the macrocyclic ligand DOTA (used in the syntheses in the active ester form, mono-N-hydroxysuccinimide, DOTA-NHS). In both series, the bifunctional coupling agent (CN-BFCA or DOTA-NHS) was coupled with the selected SP-analogue beforehand. The labeled SP analogues ( Figure 2) were: SP(1-11) and its modified form [Thi 8 ,Met(O 2 ) 11 ]SP(1-11), SP (4)(5)(6)(7)(8)(9)(10)(11), and SP(5-11) and its modified form [Thi 8 ,Met(O 2 ) 11 ]SP(5-11). All analogues contained a sequence of five amino acids at the C-terminus of the peptide, responsible for their affinity towards the NK-1 receptor [8]. The HPLC chromatograms of labeling reactions with technetium-99m (1a-e) and lutetium-177 (2a-e) are presented in Figure 3. The small peaks on the HPLC chromatograms of the SP analogue labeling reactions with technetium-99m (1a-e) recorded at R T = 12.2-12.5 min ( Figure 3A  For all obtained [ 99m Tc]NS3/CN-SP (1a-e) and [ 177 Lu]DOTA-SP (2a-e) radiobioconjugates, studies of their physico-chemical properties (so called 'challenge experiments', which are important from the view-point of receptor radiopharmaceuticals) were performed [18]. Stability of the radiobioconjugates was investigated in various physiological solutions, namely PBS, histidine and  For all obtained [ 99m Tc]NS3/CN-SP (1a-e) and [ 177 Lu]DOTA-SP (2a-e) radiobioconjugates, studies of their physico-chemical properties (so called 'challenge experiments', which are important from the view-point of receptor radiopharmaceuticals) were performed [18]. Stability of the radiobioconjugates was investigated in various physiological solutions, namely PBS, histidine and For all obtained [ 99m Tc]NS 3 /CN-SP (1a-e) and [ 177 Lu]DOTA-SP (2a-e) radiobioconjugates, studies of their physico-chemical properties (so called 'challenge experiments', which are important from the view-point of receptor radiopharmaceuticals) were performed [18]. Stability of the radiobioconjugates was investigated in various physiological solutions, namely PBS, histidine and cysteine. The experimental results, verified by HPLC (high-performance liquid chromatography) and ITLC (instant thin layer chromatography) methods, showed that in 10 −2 M PBS solution and 10 −3 M cysteine and/or histidine solutions (containing a 1000 times excess of strongly competing natural ligands compared to the concentration of the tested radiobioconjugate) all studied compounds were stable. On the recorded HPLC chromatograms (after 24 h and 15 days of incubation in the case of 1a-e and 2a-e compounds, respectively) only single peaks were observed with R T corresponding to R T values of the tested radiobioconjugates. Thus, we consider that the studied radiobioconjugates do not undergo ligand exchange reactions with amino acids or other ligands containing strongly reactive SH or NH groups. The lipophilicity parameters (logP) for all radiobioconjugates (expressed as the logarithms of their partition coefficients, P) determined in a biphasic mixture of n-octanol and PBS (pH 7.40) are presented in Tables 1 and 2.  The lipophilicity value of a radiobioconjugate very strongly depends on the hydrophilichydrophobic properties of the radionuclide complex and the nature of the biomolecule used as a vector. In the series of [ 99m Tc]NS 3 /CN-SP radiobioconjugates, the lipophilicity values were several orders of magnitude higher than those in the series of [ 177 Lu]DOTA-SP radiobioconjugates. This was due to the high hydrophobic character of the NS 3 ligand and the hydrophilic character of the macrocyclic ligand DOTA, respectively. Further, analysis of determined values showed that radiobioconjugates contain shorter SP fragments. Therefore, they are of lower molecular weight, and characterized by higher lipophilicity parameters. In contrast, replacement of amino acids in positions 8 and 11 (Phe and Met by Thi and Met(O 2 ), respectively) to increase the half-life of the peptide resulted in a decrease in lipophilicity. In both series, despite the very different ranges of logP values from about −0.3 to +0.6 and −2.5 to −5 for 1a-e and 2a-e radiobioconjugates, respectively, we observed the same trend in changes of radiobioconjugate lipophilicity values. This remained in good accordance with the direction of the logP changes of the SP fragments (theoretically calculated by the program, MarvinSketch) used for radiobioconjugate synthesis. Bearing in mind the potential application of new radiobioconjugates based on SP fragments in GBM treatment (containing diagnostic or therapeutic radionuclides, e.g., 68 Ga, 213 Bi or 225 Ac, chelated by the macrocyclic ligand, DOTA), stability studies for [ 177 Lu]DOTA-SP radiobioconjugates in cerebrospinal fluid (CSF) and human serum (HS) were also performed. Stability studies in CSF showed that 2a-e radiobioconjugates were stable in this medium with no enzymatic biodegradation of [ 177 Lu]DOTA-SP radiobioconjugates. More specifically, there was no enzymatic biodegradation of biomolecules (vectors) observed. The percentage of the 2a-e radiobioconjugate bound by CSF components was in the range of 1-4%, while about 96% of the studied compounds remained in the liquid phase in unchanged form. Similar to previous 'challenge experiment' studies, the HPLC chromatograms recorded after different time intervals of incubation (up to 15 days) showed the existence of one main radioactive species in the solution, with the retention time characteristic for the studied radiobioconjugate. However, stability studies of the 2a-e radiobioconjugates in HS showed an unacceptable compound stability in these conditions ( Figure 4). from about −0.3 to +0.6 and −2.5 to −5 for 1a-e and 2a-e radiobioconjugates, respectively, we observed the same trend in changes of radiobioconjugate lipophilicity values. This remained in good accordance with the direction of the logP changes of the SP fragments (theoretically calculated by the program, MarvinSketch) used for radiobioconjugate synthesis. Bearing in mind the potential application of new radiobioconjugates based on SP fragments in GBM treatment (containing diagnostic or therapeutic radionuclides, e.g., 68   HPLC chromatograms of the liquid phases (after precipitation and separation of the radiobioconjugate bound by protein components present in HS) showed new peaks while the peak corresponding to the studied radiobioconjugate disappeared ( Figure 5). Similarly, we observed more than one stain on the ITLC strips ( Figure 6). HPLC chromatograms of the liquid phases (after precipitation and separation of the radiobioconjugate bound by protein components present in HS) showed new peaks while the peak corresponding to the studied radiobioconjugate disappeared ( Figure 5). Similarly, we observed more than one stain on the ITLC strips ( Figure 6).  The appearance of new peaks on HPLC chromatograms indicated enzymatic biodegradation of SP, as well as all its analogues (protein content in HS is about 200-fold higher than in CSF) [19]. The results also showed that enzymatic biodegradation of radiobioconjugates containing shorter SP fragments occurred faster than with radiobioconjugates containing SP molecules with the full amino acid sequence (Table 2, Figure 5). The enzyme most specific for degrading SP is neutral endopeptidase (NEP) [20]. NEP cleaves SP into shorter fragments [SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), SP(4-11), SP(5-11), SP(6-11) and SP (8)(9)(10)(11)], which generally have a higher formation rate. The lower stability of radiobioconjugates containing shorter SP fragments is probably caused by easier matching of the smaller peptide to the enzyme cellular matrix (more accurate adjustment of the substrate to the active sites of the enzyme). The stability study results for the 2a-e radiobioconjugates in HS assessed by HPLC method corresponded very well with the results obtained using the ITLC method ( Figure 6). For each of the 2a-e radiobioconjugates, two TLC strips were presented, showing the location of spots   The appearance of new peaks on HPLC chromatograms indicated enzymatic biodegradation of SP, as well as all its analogues (protein content in HS is about 200-fold higher than in CSF) [19]. The results also showed that enzymatic biodegradation of radiobioconjugates containing shorter SP fragments occurred faster than with radiobioconjugates containing SP molecules with the full amino acid sequence (Table 2, Figure 5). The enzyme most specific for degrading SP is neutral endopeptidase (NEP) [20]. NEP cleaves SP into shorter fragments [SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), SP(4-11), SP(5-11), SP(6-11) and SP(8-11)], which generally have a higher formation rate. The lower stability of radiobioconjugates containing shorter SP fragments is probably caused by easier matching of the smaller peptide to the enzyme cellular matrix (more accurate adjustment of the substrate to the active sites of the enzyme). The stability study results for the 2a-e radiobioconjugates in HS assessed by HPLC method corresponded very well with the results obtained using the ITLC method ( Figure 6). For each of the 2a-e radiobioconjugates, two TLC strips were presented, showing the location of spots The appearance of new peaks on HPLC chromatograms indicated enzymatic biodegradation of SP, as well as all its analogues (protein content in HS is about 200-fold higher than in CSF) [19]. The results also showed that enzymatic biodegradation of radiobioconjugates containing shorter SP fragments occurred faster than with radiobioconjugates containing SP molecules with the full amino acid sequence (Table 2, Figure 5). The enzyme most specific for degrading SP is neutral endopeptidase (NEP) [20]. NEP cleaves SP into shorter fragments [SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), SP(4-11), SP(5-11), SP(6-11) and SP(8-11)], which generally have a higher formation rate. The lower stability of radiobioconjugates containing shorter SP fragments is probably caused by easier matching of the smaller peptide to the enzyme cellular matrix (more accurate adjustment of the substrate to the active sites of the enzyme). The stability study results for the 2a-e radiobioconjugates in HS assessed by HPLC method corresponded very well with the results obtained using the ITLC method ( Figure 6). For each of the 2a-e radiobioconjugates, two TLC strips were presented, showing the location of spots after incubation (left-hand strip) and, as a blank, without incubation in HS (right-hand strip). In the case of 2a, 2b and 2c radiobioconjugates (within 24 h of incubation), the enzymatic biomolecule biodegradation was only partial. However, for the 2d and 2e compounds (within the same incubation time), virtually all of the radiobiomolecule that was deposited onto the strip was degraded enzymatically (there were no stains at the origin on the strip). During incubation in HS, the proportion of the 2a-e compounds bound by serum protein components was in the range of 8-11%. The stability data presented in Table 2, and Figures 5 and 6, shows that modification of the SP(5-11) fragment in positions 8 and 11 (replacement of Phe and Met by Thi and Met(O 2 ), respectively) caused a significant improvement in radiobioconjugate stability in HS (stability of 2e was significantly higher than that of 2d). The same modification of the entire peptide, SP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11), had no effect on radiobioconjugate stability (the stability of 2b and 2a radiobioconjugates in HS were comparable). This led us to conclude that the radiobioconjugate based on the fragment SP(4-11), with prior modification in an analogous way (i.e., [ 177 Lu]DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP(4-11) compound), would also be characterized by better stability in HS than the compound 2c. However, simultaneously the lipophilicity parameter of this compound would become slightly lower.
From the 2a-e compound series, the radiobioconjugates 2c and 2e were selected for biological studies due to their relatively low molecular weight and high lipophilicity. The stability of both radiobioconjugates in HS was found to be unsatisfactory. However, for GBM treatment these preparations are administered locally into the solid cancer or into the cavity after surgical tumor removal (where the content of serum components is rather low). Therefore, relatively low radiobioconjugate stability in HS does not appear to be a crucial parameter for its potential application. Biological studies were also performed for the 2b compound-this radiobioconjugate contains, as a vector, the same biomolecule [ 11 ]-SP currently used in GBM therapy. As values obtained experimentally in biological studies are determined in different conditions and laboratories, they cannot be considered 'absolute' and their comparison can only be qualitative. Therefore, the biological properties of the new 2c and 2e radiobioconjugates were compared with the properties of compound 2b used in our studies as a reference. The biological properties of 2b, 2c and 2e radiobioconjugates were characterized in vitro by investigation of their affinity to the NK-1 receptor using the U373 MG cell line (Uppsala, human glioblastoma astrocytoma derived from brain malignant tumor). Biodistribution studies in vivo, usually performed in biological tests for novel preparations, were not planned for 2b, 2c and 2e radiobioconjugates (containing as a vector SP peptide or its analogues) for two reasons. First, in GBM targeted therapy, the radiobioconjugates are administered directly into the solid cancer or into the postsurgical cavity. Second, SP is a potent vasodilator, which results in a reduction in blood pressure. To characterize the receptor-binding properties of SP radiobioconjugates, saturation binding experiments on the U373 MG cells were performed (Figure 7), with determination of K d and B max values (Table 3). These results demonstrated that both newly synthesized 2c and 2e radiobioconjugates bind specifically to NK-1 receptors expressed on GBM cells with high affinity in the nanomolar range. Nevertheless, the receptor affinity of the 2c radiobioconjugate (K d = 13.3 ± 1.6 nM) to NK-1 receptor was higher (a lower K d value corresponds to higher affinity) than compound 2e (K d = 48.7 ± 5.1 nM) and very close to the K d value of 2b (K d = 11.1 ± 2.7 nM). Our results showed that new radiobioconjugates, based on the modified SP(1-11) analogues, e.g., SP (4)(5)(6)(7)(8)(9)(10)(11)

Scheme 2.
Coupling reaction of DOTA-NHS with SP fragments.  A wide peak located at R T 6.7-9 min visible in each chromatogram corresponds to the DMF solvent used in the coupling reactions. This peak covers small peak corresponding to the reaction by product NHS (R T = 6.8 min), while the small peak at R T = 6.6 min corresponds to the reaction substrate DOTA-NHS.  according to the following procedure: to the vial containing 15 nmol of lyophilized DOTA-SP dissolved in 300 µL of 0.4 M acetate buffer (pH 5.0) the solution of [ 177 Lu]Cl3 (in 0.04 M HCl, 5-10 MBq) was added (Scheme 4). The reaction mixture was heated for 30 min at 95C and the reaction progress was checked by ITLC using iTLC-SG strips (Agilent, Santa Clara, CA, USA) and 0.05 M sodium citrate as a developing solvent. Additionally, product efficiency was confirmed by HPLC (system 2).

Physico-Chemical Studies of Radiobioconjugates
All radioconjugates were purified on a semi-preparative column (HPLC method) before using them for physico-chemical and biological tests. Stability tests (so called "challenge experiments") of 1a-e and 2a-e radiobioconjugates were carried out in solutions imitating physiological body fluids, such as 10 −2 M phosphate buffered saline (PBS), 10 -3 M cysteine and 10 -3 M histidine. The radiobioconjugates isolated from the reaction mixture (semi-preparative HPLC method, system 2) were incubated in the solutions at 37C for 24 h. At selected time intervals (1, 2, 4 and 24 h), the samples of the incubated solutions were taken and analyzed by TLC and/or HPLC method (system 2). The presence of only one stain on the TLC strip or one peak on an HPLC chromatogram was proof of radiobioconjugate stability.
Stability tests of 2a-e compounds in CSF and HS were performed similarly to the previous one. The solution (0.1 mL) of isolated 2a-e radiobioconjugate in 0.1 M PBS buffer (pH 7.4), was added to 0.9 mL of CSF or HS and incubated at 37°C. After specific time intervals (1, 2, 4 and 24 h), a small sample (0.1-0.2 mL) of the mixture was taken, mixed with ethanol (0.3-0.5 mL) and shaken vigorously to precipitate protein components as well as part of the tested radiobioconjugate bound to protein components. The sample was then centrifuged (14,000 rpm, 5 min) and the supernatant separated. The radioactivities of both supernatant and precipitate were measured using the well-type NaI(Tl) detector (Institute of Nuclear Chemistry and Technology, Warsaw, Poland). Finally, the HPLC chromatograms (system 2) of the reaction mixtures of the SP analogue labeling reactions with lutetium-177 (for 2a-e radiobioconjugates) were presented earlier in Figure 3B. The R f values of 2a-e radiobioconjugates obtained in the ITLC method were about 0.1. The spots did not migrate with the mobile phase and stayed at the origin. All [ 99m Tc]NS 3 /CN-SP and [ 177 Lu]DOTA-SP radiobioconjugates were formed with a yield in the range 96-98% and purity higher than 98%.

Physico-Chemical Studies of Radiobioconjugates
All radioconjugates were purified on a semi-preparative column (HPLC method) before using them for physico-chemical and biological tests. Stability tests (so called "challenge experiments") of 1a-e and 2a-e radiobioconjugates were carried out in solutions imitating physiological body fluids, such as 10 −2 M phosphate buffered saline (PBS), 10 -3 M cysteine and 10 -3 M histidine. The radiobioconjugates isolated from the reaction mixture (semi-preparative HPLC method, system 2) were incubated in the solutions at 37 • C for 24 h. At selected time intervals (1, 2, 4 and 24 h), the samples of the incubated solutions were taken and analyzed by TLC and/or HPLC method (system 2). The presence of only one stain on the TLC strip or one peak on an HPLC chromatogram was proof of radiobioconjugate stability.
Stability tests of 2a-e compounds in CSF and HS were performed similarly to the previous one. The solution (0.1 mL) of isolated 2a-e radiobioconjugate in 0.1 M PBS buffer (pH 7.4), was added to 0.9 mL of CSF or HS and incubated at 37 • C. After specific time intervals (1, 2, 4 and 24 h), a small sample (0.1-0.2 mL) of the mixture was taken, mixed with ethanol (0.3-0.5 mL) and shaken vigorously to precipitate protein components as well as part of the tested radiobioconjugate bound to protein components. The sample was then centrifuged (14,000 rpm, 5 min) and the supernatant separated. The radioactivities of both supernatant and precipitate were measured using the well-type NaI(Tl) detector (Institute of Nuclear Chemistry and Technology, Warsaw, Poland). Finally, the supernatant fraction was analyzed by HPLC to verify that the studied radiobioconjugate still existed in its intact form.
The lipophilicity (logP) of 1a-e and 2a-e radiobioconjugates was characterized as the logarithm of their partition coefficients (P) determined according to the standard procedure used in studies of biological compounds. The partition coefficient was measured for each compound in a biphasic system of n-octanol/PBS (pH 7.4). The biphasic mixture was shaken (vortex, 1 min) and then centrifuged (14,000 rpm, 5 min) before the organic (n-octanol) and aqueous (PBS, pH 7.4) phases were separated. The activity of each phase (corresponding to the content of radiobioconjugate) was determined by measuring γ-radiation, using a well-type NaI(Tl) detector. P was calculated as the ratio of activity in the organic versus aqueous phases (as an average value from at least three independent measurements). Immediately after the γ-radiation measurements, the aqueous phase was analyzed by HPLC (system 2) to determine whether the tested radiobioconjugate existed in the liquid phase in an unchanged form.  11 ]SP (5)(6)(7)(8)(9)(10)(11), 2e, to the NK-1 receptors expressed on glioblastoma cells (U373 MG), saturation binding studies were performed. One day before the experiment, 8 × 10 5 cells/well were seeded in six-well plates. To determine total binding, different concentrations of peptides (15.3 pM to 100 nM) were added to the cells and incubated at 37 • C for 1.5 h. Cellular uptake was stopped by aspirating the reaction supernatant, followed by washing twice with 1 mL of cold PBS. Finally, the cells were lysed using 1 M NaOH to detach them from the well plates. To determine nonspecific binding, the cells were incubated with 1000-fold excess of unlabelled peptides. The radioactivities of collected samples with cell pellets and supernatants were measured using Perkin Elmer automatic γ-counter. The specific binding was obtained by subtracting nonspecific binding from total binding. For estimating the receptor density (B max ) and the dissociation constant (K d ) of the receptor for the studied radiobioconjugates, the Scatchard analysis method was used. Results are means ±SD of three individual experiments.

Conclusions
The obtained results showed that the lipophilicity of the radiobioconjugates strongly depended on the hydrophilic-hydrophobic properties of the radionuclide complex and the structure of the SP fragment. Radiobioconjugates containing shorter SP fragments were characterized by a lower molecular weight and higher lipophilicity, which allows more effective migration into GBM tissue or into the post-surgery cavity walls. Further, replacement of Phe by Thi in position 8 and Met by its oxidized form, MetO 2 , in position 11 (to increase the half-life of the peptide) resulted in a lipophilicity decrease.
Stability studies of [ 99m Tc]NS 3 /CN-SP and [ 177 Lu]DOTA-SP radiobioconjugates showed that all preparations were stable in different physiological solutions (PBS, histidine and cysteine). The [ 177 Lu]DOTA-SP radiobioconjugate was also stable in CSF, but not sufficiently stable in HS. Radiobioconjugates containing shorter SP fragments were characterized by lower stability in HS due to the faster enzymatic biodegradation of biomolecules. We performed biological studies on the two newly synthesized and tested radiobioconjugates, [ 177 Lu]DOTA-SP(4-11) and [ 177 Lu]DOTA-[Thi 8 ,Met(O 2 ) 11 ]SP (5)(6)(7)(8)(9)(10)(11), and showed that they specifically bound with high affinity (in the nanomolar range) to NK-1 receptors expressed on the U373 MG cell line (their K d values were close to the K d values of preparations currently used in medical experiments for GBM treatment). At the same time, both formulations had a lower molecular weight and higher lipophilicity compared to preparations used currently.
To summarize, the SP analogues tested in this work can be used (taking into account the lipophilicity and receptor affinity of [ 177 Lu]DOTA-SP radiobioconjugates) as biologically active molecules (potential vectors) able to lead the diagnostic or therapeutic radionuclide to NK-1 receptors overexpressing on glioblastoma cells. The unsatisfactory stability of [ 177 Lu]DOTA-SP radiobioconjugates tested in HS may be an additional reason (apart from the low lipophilicity and high molecular weight of the preparations) for inefficient migration of the receptor radiopharmaceuticals into the walls of post-surgery cavity. After enzymatic vector biodegradation, the therapeutic radionuclides remain at the administration site, so they do not reach and destroy single glioblastoma cancer cells (including stem cells) placed in the tissues around the post-surgery cavity. On the other hand, the unsatisfactory stability of [ 177 Lu]DOTA-SP radiobioconjugates in HS does not seem to be a disqualifying parameter because the content of serum components in the post-surgery cavity is rather low. However, it is possible that a hematoma will form in the post-surgical cavity increasing local concentrations of serum components. Application of receptor radiopharmaceuticals, both diagnostic and therapeutic, in accordance with the rules of the use of preparations in nuclear medicine, requires individual consideration (personalized medicine) for each patient [26]. Imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) performed before application of radiobioconjugates allows assessment of the advisability and safety of selected treatment strategies. Our overall conclusion is that potential receptor radiopharmaceuticals based on SP analogues do not fulfil all requirements for preparations used in nuclear medicine. As a result, our current studies are focused on the search for other vectors including peptidic and nonpeptidic antagonists of the NK-1 receptor. This work is in progress and obtained results will be the subject of the new work "Application of neurokinin-1 receptor in therapeutic targeted strategies for glioma treatment, Part II."