Application of ENPP1 inhibitor in preparation of medicine for treating pituitary growth hormone adenoma

Abstract

The invention relates to the field of biological medicine, and discloses application of an ENPP1 inhibitor in preparation of a medicine for treating pituitary growth hormone adenoma. The ENPP1 inhibitor is a compound with a structure as shown in a formula I, or an isomer, a derivative, a solvate or a pharmaceutically acceptable salt thereof. It is found for the first time that the ENPP1 inhibitor has a remarkable treatment effect on pituitary growth hormone adenoma, not only inhibits secretion of GH, but also inhibits growth of adenoma. Specifically, the inhibitor efficiently inhibits the activity of growth hormone adenoma cells ENPP1, inhibits adenoma cell proliferation and GH secretion in an in-vitro model, reduces the GH and IGF-1 levels of tumor-bearing mice in an in-vivo model, and is suitable for GH adenoma insensitive to somatostatin analogue drugs (such as octreotide). In addition, when the ENPP1 inhibitor is combined with octreotide for use, a synergistic effect is shown, and the anti-tumor effect is further enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more particularly, it relates to the use of an ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1) inhibitor in the preparation of a drug for treating pituitary growth hormone adenoma. BACKGROUND

[0002] Pituitary growth hormone adenoma (GH adenoma) is a functional pituitary tumor, and the core pathological feature is the abnormal over-secretion of growth hormone (GH) and insulin-like growth factor 1 (IGF-1). Long-term elevated GH and IGF-1 can cause multiple system complications throughout the body, so the main treatment goal of GH adenoma is to reduce GH and IGF-1 to the normal range, i.e. biochemical remission. Epidemiological data show that patients who do not meet the biochemical criteria have a significantly increased all-cause mortality and a shortened life expectancy. Current clinical standard treatment methods include surgical resection, radiotherapy, and medical drug treatment. Despite the use of multiple modes of treatment, there is still a significant unmet clinical need: the cure rate of surgical treatment for large adenomas is only 50-60%; radiotherapy has a slow onset (about 2 to 3 years) and can cause a decrease in normal pituitary function; somatostatin analogs (SSAs) that act on somatostatin receptor 2 (SSTR2) are first-line drugs, not only are they expensive and require long-term injection, but also only 50% of patients can achieve biochemical remission after drug treatment; other drugs such as dopamine receptor agonists have limited efficacy (e.g. bromocriptine only makes 10-35% of patients biochemically remission), and GH receptor antagonists can reduce IGF-1, but cannot inhibit GH secretion and have the risk of tumor growth. Therefore, it is of great clinical significance to develop new drugs that can efficiently inhibit GH secretion and control tumor growth.

[0003] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein with enzyme activity of hydrolyzing various nucleotide substrates (such as cGAMP, ATP). In recent years, ENPP1 has attracted more and more attention in the field of tumors. Studies have shown that in solid tumors such as breast cancer and colorectal cancer, ENPP1 can inhibit the natural immune response mediated by the cGAS-STING pathway by hydrolyzing extracellular cGAMP; the adenosine catalyzed by it further inhibits T cell activity, and together promotes tumor immune escape and metastasis. In addition, it has also been reported that ENPP1 can promote epithelial-mesenchymal transition (EMT), enhance E2F1 transcription function, and maintain the proliferation of tumor stem cell-like cells (GSCs), but its specific mechanism has not been fully elucidated. Currently, there are patents approved for disease diagnosis and drug development targeting ENPP1 in rectal cancer, cervical cancer, lung cancer, and ovarian cancer (CN 111973748 B, CN 110244058 B).

[0004] Our previous multi-omics study of pituitary tumors revealed that the expression level of ENPP1 in growth hormone adenomas was significantly higher than that in normal pituitary gland tissues and pituitary tumors of other lineages, suggesting that ENPP1 may play a certain role in the pathological process of GH adenomas. However, existing studies are limited to the reporting of this phenomenon, and the specific biological function of ENPP1 in GH adenomas is still unknown.

[0005] In summary, ENPP1 is highly expressed in GH adenomas, suggesting that ENPP1 may serve as a therapeutic target for GH adenomas, and ENPP1 inhibitors have clinical translational value for treating GH adenomas. SUMMARY

[0006] The present application aims to overcome the limitations of target heterogeneity in drug treatment, address the limited response rate and drug resistance of existing drug treatment, and provide a targeted drug that can simultaneously inhibit tumor growth and hormone secretion.

[0007] To achieve the above-mentioned application purposes, the following technical solutions are adopted in the present application: In a first aspect, the present application provides the use of an ENPP1 inhibitor in the preparation of a drug for treating pituitary growth hormone adenoma.

[0008] Further, the ENPP1 inhibitor is preferably a compound capable of specifically inhibiting the enzyme activity of ENPP1 and having the structure shown in Formula I below, or known isomers and derivatives, solvates, or pharmaceutically acceptable salts thereof;

[0009] Formula I.

[0010] The use of an ENPP1 inhibitor in the preparation of a drug for treating pituitary growth hormone adenoma. Further, the pituitary growth hormone adenoma is one that is not sensitive or resistant to somatostatin analogs.

[0011] In a second aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of an ENPP1 inhibitor.

[0012] Further, the ENPP1 inhibitor is a compound capable of specifically inhibiting the enzyme activity of ENPP1 and having the structure shown in Formula I below, or known isomers and derivatives, solvates, or pharmaceutically acceptable salts thereof;

[0013] Formula I.

[0014] Further, the pharmaceutical composition further comprises a somatostatin analog.

[0015] In a third aspect, the application provides use of the pharmaceutical composition in the preparation of a drug for treating pituitary growth hormone adenoma.

[0016] Further, the pituitary growth hormone adenoma is an adenoma that is not sensitive or resistant to somatostatin analogs.

[0017] Further, the somatostatin analog is octreotide.

[0018] In a fourth aspect, the application provides a method for constructing a pituitary growth hormone adenoma organoid model, comprising the following steps: (a) obtaining a fresh human GH adenoma sample; (b) using collagenase IV for digestion treatment to obtain a cell suspension; (c) mixing the cell suspension with Matrigel at a volume ratio of about 1:5, and inoculating into a culture plate for solidification; and (d) culturing in complete culture medium.

[0019] In a fifth aspect, the application provides use of a pituitary growth hormone adenoma organoid model prepared by the construction method in the pathogenesis research, drug screening and efficacy evaluation of GH adenoma.

[0020] In summary, the application has the following remarkable beneficial effects relative to the prior art, which are directly brought about by the technical features or are inevitable technical effects resulting therefrom: 1. Efficiently inhibiting ENPP1 activity. The ENPP1 inhibitor of the application significantly inhibits the enzyme activity of ENPP1 by specifically binding to the active site of ENPP1, thereby effectively blocking the function of ENPP1.

[0021] 2. Significantly inhibiting the growth of GH adenoma. Experimental data show that the ENPP1 inhibitor of the application can significantly inhibit the proliferation of tumor cells and induce tumor cell apoptosis in GH3 cell lines and primary cells, organoid models of human GH adenoma. This effect solves the problem of limited tumor treatment effect in the prior art, and provides a more effective means for tumor treatment.

[0022] 3. Suitable for GH adenoma that is not sensitive to somatostatin analogs. Experimental results show that the ENPP1 inhibitor of the application can still effectively inhibit GH secretion and tumor growth in GH adenoma that is SSTR2 negative or has low inhibition rate of octreotide. This effect solves the problem of poor treatment effect on GH adenoma that is not sensitive to somatostatin analogs in the prior art, and expands the therapeutic indications.

[0023] 4. Synergistic effect of combination therapy. When the ENPP1 inhibitor of the application is used in combination with octreotide, it shows a significant synergistic effect, further enhancing the effect of inhibiting GH secretion and tumor growth. This effect provides a scientific basis for the combination of ENPP1 inhibitors and somatostatin analogs. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Changes in cell viability of GH3 cells 24 hours after intervention with different concentration gradients of ENPP1-IN-1. Figure 1 It can be seen that ENPP1-IN-1 reduces GH3 cell viability, and the reduction in cell viability is more pronounced with increasing dosage.

[0025] Figure 2 Changes in apoptosis in GH3 cells 24 hours after intervention with different concentration gradients of ENPP1-IN-1 were detected by Annexin V-FITC / PI double staining. Figure 2 It can be seen that ENPP1-IN-1 increased early and late apoptosis in GH3 cells, and the apoptosis was more pronounced with increasing dosage.

[0026] Figure 3 Changes in the cell cycle were detected by PI staining 24 hours after GH3 cells were treated with ENPP1-IN-1 (20 μM). Figure 3 It can be seen that ENPP1-IN-1 reduces the proportion of cells in S phase and G2 / M phase, thereby slowing down cell replication and division.

[0027] Figure 4 This study investigated the viability and growth hormone secretion of 32 primary GH adenoma cells after drug intervention using CCK-8 cells. Drug treatment was administered on days 0, 1, and 2. Drug groups included ENPP1-IN (20 μM), Octreotide (1 μM), and a combination of ENPP1-IN and Octreotide. The control group received DMSO. Results showed that compared to the DMSO group, both the ENPP1-IN and Octreotide groups significantly inhibited GH secretion (*DMSO vs ENPP1-IN, P<0.05; #DMSO vs Octreotide, P<0.05; $Octreotide vs ENPP1-IN+Octreotide, P<0.05) and cell viability (*P<0.05). Furthermore, the combination of Octreotide and ENPP1-IN further inhibited GH secretion compared to Octreotide alone ($P<0.05). These results indicate that both ENPP1-IN and Octreotide can effectively inhibit the viability and GH secretion of primary cells, and that the combined use of these drugs has a synergistic effect.

[0028] Figure 5The results of growth hormone (GH) secretion in primary GH adenomas were analyzed, and the cells were divided into two groups based on SSTR2 expression levels (pathologically positive and negative) and octreotide inhibition rates ≥83% and <83%. The results showed that the ENPP1 inhibitor (ENPP1-IN) significantly inhibited GH secretion in GH pituitary adenomas (GHPA) that were SSTR2 negative or had low octreotide inhibition rates, indicating that ENPP1-IN also has a good therapeutic effect on GH adenomas that are insensitive to octreotide.

[0029] Figure 6 Results of growth hormone secretion and organoid diameter changes in a GH tumor organoid model after drug intervention. Experimental drugs included ENPP1-IN (20 μM) and Octreotide (1 μM), with DMSO as the control group. Results showed that, compared with the DMSO group, both the ENPP1-IN and Octreotide groups significantly inhibited GH secretion and organoid growth.

[0030] Figure 7 Evaluation of the in vivo efficacy and safety of ENPP1 inhibitors in a GH3 cell tumor-bearing mouse model. (A) Photographs of tumors taken from each group at the end of the experiment. Compared with the solvent control group, the tumor volume of the ENPP1 inhibitor (20 mg / kg, 40 mg / kg) treatment groups was significantly reduced, and the 40 mg / kg dose group was more effective than the octreotide group. (B) Changes in tumor volume of mice in each group over time (n=8 / group). ENPP1 inhibitors showed a dose-dependent tumor-suppressing effect, and the 40 mg / kg dose group was superior to the octreotide positive control group in tumor inhibition rate. (C) Changes in body weight of mice in each group during the experiment. There was no significant difference in body weight among all groups, indicating that ENPP1 inhibitors did not show significant systemic toxicity at effective doses. (D) Measurement of serum IGF-1 levels in mice at the end of the experiment. All doses of ENPP1 inhibitors and the octreotide group significantly reduced serum IGF-1 levels. (E) Random blood glucose and fructosamine levels in mice at the end of the experiment. Compared with the control group and the ENPP1 inhibitor group, octreotide treatment led to an increase in random blood glucose and fructosamine, while ENPP1 inhibitors had no significant effect on glucose metabolism, demonstrating their potential advantage in terms of metabolic safety.

[0031] Figure 8: Expression of ENPP1 in GH pituitary adenoma tissues and cells and its relationship with GH. (A) Western blot results of 3 GH pituitary adenoma tissues showed the protein expression level of ENPP1 in different tumor samples, indicating that ENPP1 is generally highly expressed in GH pituitary adenomas. (B) Western blot results of GH3 cells verified the expression of ENPP1 in GH3 cells. (C) Immunohistochemical staining results of ENPP1 in GH pituitary adenoma tissues showed that ENPP1 was significantly expressed in tumor tissues. (D) Immunofluorescence staining of primary GH pituitary adenoma cells showed co-localization of GH (green fluorescence) and ENPP1 (red fluorescence).

[0032] Figure 9 Immunohistochemical staining results of ENPP1 in GH pituitary adenoma tissue and adjacent tissue. As shown in the figure, ENPP1 expression in tumor tissue was significantly higher than in adjacent tissue (n=25, p<0.0001), with significantly enhanced staining intensity, and it was mainly localized in the cytoplasm and cell membrane of tumor cells. ENPP1 expression in adjacent tissue was weak or almost non-existent. This result further confirms the specific high expression of ENPP1 in GH pituitary adenomas, suggesting that ENPP1 may play an important role in tumor occurrence and development, providing a basis for ENPP1 as a therapeutic target. Detailed Implementation

[0033] The technical solutions and effects of this application will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, the accompanying drawings show only the parts relevant to the invention, not the entire structure.

[0034] Example 1: In vitro experimental verification of anti-tumor effect This embodiment conducted a series of in vitro experiments to verify the direct antitumor effect of ENPP1-IN-1. CCK-8 and EdU assays showed that ENPP1-IN-1 significantly inhibited the viability and proliferation of GH3 cells and primary cells in a dose-dependent manner. Annexin V-FITC / PI double staining assays confirmed that ENPP1-IN-1 significantly increased the early and late apoptosis rates of GH3 cells. PI staining flow cytometry showed that ENPP1-IN-1 arrested GH3 cells in the G0 / G1 phase. GH assays demonstrated that ENPP1-IN-1 significantly reduced GH levels in GH3 cells, primary cells, and organoid model culture supernatants. In primary cells, ENPP1-IN-1 effectively inhibited GH secretion in SSTR2-negative GH adenomas or those with low octreotide inhibition rates. The specific implementation scheme is as follows: (1) Inhibits tumor cell viability and proliferation The effect of ENPP1-IN-1 on tumor cell viability was evaluated using the CCK-8 assay. Specifically, rat GH3 cell lines or human GH adenoma primary cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 10 μM / well in 96-well plates. After cell attachment, the medium was replaced with fresh medium containing different concentrations of ENPP1-IN-1 (1, 10, 20, and 50 μM), 1 μM octreotide (positive control), 20 μM ENPP1-IN-1 combined with 1 μM octreotide, or an equal volume of DMSO (solvent control). For primary cells, drug intervention was performed on days 0, 1, and 2. All cells were cultured at 37°C and 5% CO2 for 24 hours (GH3 cells) or until the specified detection time point. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 2 hours. The absorbance (OD value) was then measured at 450 nm using a microplate reader, and the relative cell viability was calculated. The results showed that in GH3 cells, treatment with 10 μM, 20 μM, and 50 μM ENPP1-IN-1 for 24 hours significantly reduced cell viability in a dose-dependent manner (P<0.05). In primary cells, compared with the DMSO control group, the use of 20 μM ENPP1-IN-1 or 1 μM octreotide alone significantly inhibited cell viability (*P<0.05).

[0035] (2) Inducing tumor cell apoptosis The role of Annexin V-FITC / PI double staining in inducing apoptosis was investigated by flow cytometry. GH3 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and treated with 1 μM, 10 μM, and 20 μM ENPP1-IN-1 or DMSO for 24 hours. Cells were then digested and collected, washed with PBS, resuspended in 1× Binding Buffer, and 5 μL Annexin V-FITC and 5 μL PI staining solution were added. Cells were incubated at room temperature in the dark for 15 minutes, followed by flow cytometry analysis. The proportion of apoptotic cells was analyzed using FlowJo software. Flow cytometry scatter plots and statistical analysis clearly showed that, compared with the DMSO control group, the proportions of early apoptotic (Annexin V-FITC positive / PI negative) and late apoptotic (Annexin V-FITC positive / PI positive) cells in the ENPP1-IN-1 treatment group showed a significant dose-dependent increase (P<0.05), demonstrating that ENPP1-IN-1 can effectively initiate and promote the apoptosis process of GH3 cells.

[0036] (3) Inhibit tumor cell cycle progression To further elucidate the mechanism by which ENPP1-IN-1 inhibits cell proliferation, we further analyzed its effect on cell cycle distribution using PI staining flow cytometry. GH3 cells were treated with 20 μM ENPP1-IN-1 for 24 hours, then digested and collected, and fixed overnight at 4°C with pre-chilled 70% ice-cold ethanol. After fixation, the cells were washed with PBS, resuspended in staining solution containing 50 μg / mL PI and 100 μg / mL RNase A, and incubated at 37°C in the dark for 30 minutes. Cell DNA content was then detected by flow cytometry, and the cell cycle proportions at each phase were analyzed using the cycle fitting function of FlowJo software. The results showed that compared with the DMSO control group, the proportion of cells in the G0 / G1 phase was significantly increased in the ENPP1-IN-1 treatment group, while the proportions of cells in the S and G2 / M phases were correspondingly significantly decreased. This result indicates that ENPP1-IN-1 can arrest the cell cycle of GH3 cells at the G0 / G1 phase, thereby effectively inhibiting cells from entering the DNA synthesis and mitosis stages.

[0037] (4) Inhibit growth hormone secretion To analyze the effect of ENPP1 inhibitors on GH secretion function, we quantitatively detected GH levels in the GH3 cell line, primary human GH adenoma cells, and organoid models. For the GH3 cell line, we used enzyme-linked immunosorbent assay (ELISA). The specific method is as follows: GH3 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 24-well plates. After cell attachment, the culture medium was replaced with different treatments: DMSO solvent control, 20 μM ENPP1-IN-1, 1 μM octreotide, and a combination of both. After culturing for another 24 hours, the cell culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes at 4°C to remove cell debris. The supernatant was aliquoted and stored at -80°C for later analysis. Following the instructions of the rat GH ELISA kit, samples and standards were added to pre-coated antibody-impregnated plates. After incubation, washing, and the addition of biotinylated detection antibody, horseradish peroxidase-labeled streptavidin, and substrate TMB, the absorbance was measured at 450 nm using a microplate reader. The GH concentration in each sample was calculated based on the standard curve.

[0038] For primary human GH adenoma cells, we used electrochemiluminescence immunoassay for detection. Primary cells were treated with DMSO, 20 μM ENPP1-IN-1, 1 μM octreotide, or a combination of drugs (interventions were performed on days 0, 1, and 2, respectively), and the culture supernatant was collected. Before detection, considering the high concentration of GH secreted, the samples were appropriately diluted using diluents determined in preliminary experiments (typically 1:50 to 1:200). Subsequently, automated detection was performed using a Roche electrochemiluminescence immunoassay analyzer and a human GH detection kit, following the manufacturer's standard operating procedures. This method is based on the double-antibody sandwich principle, where a ruthenium-labeled antibody binds to GH to form a complex, which undergoes an electrochemiluminescence reaction in an electric field. The intensity of the generated light signal is proportional to the GH content in the sample. Experimental results showed that ENPP1-IN-1 significantly inhibited GH secretion. Figure 4 In the three-drug-treatment experiments of primary cells shown, both the ENPP1-IN-1 group and the octreotide group consistently and significantly reduced GH levels in the culture supernatant compared to the DMSO control group (*DMSO VS ENPP1-IN, P<0.05; #DMSO VS Octreotide, P<0.05). More importantly, the combined use of ENPP1-IN-1 and octreotide showed a significantly stronger inhibitory effect on GH secretion than octreotide alone ($ Octreotide VS ENPP1-IN+Octreotide, P<0.05), indicating a synergistic effect between the two in inhibiting hormone secretion.

[0039] In three-dimensional organoid models that better simulate the in vivo tumor microenvironment, we also observed the effectiveness of ENPP1 inhibitors. The constructed human GH adenoma organoids were cultured in media containing DMSO, 20 μM ENPP1-IN-1, or 1 μM octreotide, with regular medium changes, drug administration, and collection of supernatant for photographic recording. Figure 6 As shown, the growth of organoids was assessed by measuring the GH concentration in the culture supernatant (using the same detection method as primary cells) and simultaneously measuring the organoid diameter under a microscope. The results showed that, compared with the DMSO control group, both the ENPP1-IN-1 treatment group and the octreotide treatment group significantly inhibited GH secretion and slowed the diameter growth of the organoids. This confirms, from both functional and morphological perspectives, that ENPP1 inhibitors can effectively inhibit tumor hormone secretion and proliferation in an in vitro model that more closely resembles the true state of human GH adenomas.

[0040] (5) Overcoming octreotide resistance To further validate the efficacy of ENPP1 inhibitors against octreotide-insensitive tumors, we stratified the samples based on SSTR2 immunohistochemical expression (positive / negative) in primary cell-derived tumor tissues and in vitro octreotide inhibition rates (with 83% as the cutoff). Figure 5 As shown, ENPP1-IN-1 (20 μM) could still significantly inhibit GH secretion in primary GH adenoma cells that were SSTR2 negative or poorly responsive to octreotide inhibition (inhibition rate <83%). This result suggests that the efficacy of ENPP1 inhibitors is independent of somatostatin receptors, providing a new alternative for overcoming resistance to existing targeted drugs.

[0041] Example 2: In vivo experiments to verify anti-tumor effects This embodiment established a GH3 cell tumor-bearing mouse model to verify the in vivo efficacy and safety of the ENPP1 inhibitor. Compared with the control group, treatment with the ENPP1 inhibitor (20 mg / kg, 40 mg / kg) significantly reduced tumor volume in a dose-dependent manner, with the 40 mg / kg group showing better efficacy than the octreotide group. The specific implementation method is as follows: Five-week-old male BALB / c nude mice were selected, and GH3 cells (5 × 10⁻⁶) in the logarithmic growth phase were subcutaneously inoculated under the right axilla. 6 (One tumor per 200 μL PBS). Allow the tumor to grow to approximately 100 mm in volume. 3 Mice were randomly divided into four groups (n=8 / group): a solvent control group (containing 5% DMSO + 5% Cremophor EL + 90% saline), a low-dose ENPP1-IN-1 group (20 mg / kg), a high-dose ENPP1-IN-1 group (40 mg / kg), and an octreotide positive control group (1 mg / kg). All drugs were administered via intraperitoneal injection once daily for a total of 14 doses. During the experiment, the long diameter (a) and short diameter (b) of the tumor were measured every 3 days using calipers and calculated according to the formula V = ab. 2 / 2 The tumor volume was calculated, and the weight changes of mice in each group were recorded simultaneously. The experimental results are as follows: Figure 7 As shown. Figure 7 A shows photographs of tumors isolated from mice in each group at the end of the experiment. It is clearly shown that the tumor volume in each dose group of ENPP1 inhibitor was significantly smaller than that in the solvent control group, and the tumor volume in the 40 mg / kg dose group was smaller than that in the octreotide monotherapy group. Figure 7Quantitative analysis of tumor growth curves in group B confirmed that ENPP1 inhibitors significantly inhibited tumor growth in a dose-dependent manner. Throughout the experimental period, the tumor volume in both the high- and low-dose ENPP1-IN-1 groups was significantly smaller than that in the solvent control group (P<0.05). The high-dose ENPP1-IN-1 group (40 mg / kg) showed significantly better tumor-suppressive effects than the octreotide positive control group (P<0.05), demonstrating its stronger in vivo antitumor activity. Regarding safety evaluation, Figure 7 The body weight change curves for C mice showed that all experimental groups experienced steady weight gain throughout the administration period, with no statistically significant differences between groups. This indicates that ENPP1-IN-1 did not cause significant systemic toxicity under the administration regimen used in this experiment and was well-tolerated.

[0042] To assess the effect of the drug on IGF-1 levels in vivo, we collected mouse serum at the end of the experiment and used a mouse IGF-1 ELISA kit to detect serum IGF-1 levels. Figure 7 As shown in Figure D, compared with the solvent control group, all doses of the ENPP1 inhibitor and the octreotide group significantly reduced the serum IGF-1 concentration (P<0.05), demonstrating that the ENPP1 inhibitor can effectively block IGF-1 secretion in vivo. Furthermore, we investigated the drug's effect on glucose metabolism. Random blood glucose levels were measured in mice after the last administration, and serum fructosamine concentrations (reflecting the average blood glucose level over the past 2-3 weeks) were measured at the end of the experiment using a mouse fructosamine ELISA kit. Results ( Figure 7 E) showed that, compared with the solvent control group and each dose group of ENPP1 inhibitor, the random blood glucose and fructosamine levels in the octreotide treatment group were significantly increased (P<0.05). However, the blood glucose and fructosamine levels in each dose group of ENPP1 inhibitor were not significantly different from those in the solvent control group. Clinically, some somatostatin analogs may cause fluctuations in patients' blood glucose levels, while ENPP1 inhibitors, while exerting tumor-suppressive effects, do not cause glucose metabolism disorders, demonstrating their potential safety advantage in clinical applications.

[0043] Example 3: Validation of ENPP1 distribution in tumor samples This embodiment elucidates the expression characteristics of ENPP1 in pituitary growth hormone-secreting adenomas and its relationship with GH at the tissue, cell line, and primary cell levels. It clarifies that ENPP1 is expressed in pituitary growth hormone-secreting adenomas and co-localizes with GH-secreting cells. The specific implementation scheme is as follows: Fresh tissue samples from three surgically resected GH pituitary adenomas were collected, flash-frozen in liquid nitrogen, and then lysed on ice for 20 minutes using RIPA lysis buffer (containing 1% PMSF and protease inhibitors). After centrifugation at 12000 g for 15 minutes at 4°C, the supernatant was collected, and the total protein concentration was determined using the BCA method. 20 μg of total protein was separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After blocking, the membrane was incubated overnight at 4°C with mouse anti-human ENPP1 primary antibody (1:500 dilution) and mouse anti-GAPDH monoclonal antibody (1:10000 dilution, internal control). The next day, after washing, the membrane was incubated at room temperature for 1 hour with HRP-labeled goat anti-mouse secondary antibody (1:10000 dilution). Finally, the membrane was developed using ECL chemiluminescence reagent, and images were acquired using a chemiluminescence imaging system. Results ( Figure 8 The first image shows that ENPP1 protein bands were detected in all three independent GH pituitary adenoma tissue samples, indicating that ENPP1 is widely expressed in GH pituitary adenomas.

[0044] Total protein was collected from rat GH3 cells. Sample preparation and Western blotting procedures were the same as before, with β-actin used as the internal control. Results ( Figure 8 The second image confirms that ENPP1 protein is also clearly expressed in GH3 cells.

[0045] Paraffin sections of GH pituitary adenoma tissue were routinely dewaxed to water and subjected to heat-induced antigen retrieval using sodium citrate antigen retrieval solution (pH 6.0). Endogenous peroxidase was then blocked with 3% hydrogen peroxide solution. After serum blocking, ENPP1 primary antibody (1:500 dilution) was added, and the sections were incubated overnight at 4°C in a humidified chamber. The following day, HRP-labeled secondary antibody was added, and the sections were incubated at room temperature for 30 minutes. DAB staining was performed, and the nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated, cleared, and mounted with neutral resin. Microscopic observation revealed (…). Figure 8 The third image shows that the ENPP1 positive signal (brownish-yellow) is mainly located in the cytoplasm and cell membrane of tumor cells, further confirming the expression of ENPP1 in GH adenoma cells at the histomorphological level.

[0046] Cultured primary human GH adenoma cells were seeded in culture dishes, fixed and permeabilized, and then blocked with 5% BSA. Rabbit anti-ENPP1 antibody (1:500 dilution) and mouse anti-GH monoclonal antibody (1:200 dilution) were then added simultaneously, and incubated overnight at 4°C. The next day, the cells were incubated for 1 hour at room temperature in the dark with Cy3-labeled goat anti-rabbit IgG (red fluorescence) and Alexa Fluor 488-labeled goat anti-mouse IgG (green fluorescence), respectively. Finally, the cells were stained with DAPI for 10 minutes and observed under a microscope. Results ( Figure 8The fourth figure shows the co-localization of ENPP1 (red) and GH (green) in tumor cells, suggesting that ENPP1 is expressed on GH-secreting cells and that the two may be functionally related.

[0047] To further confirm the specificity of ENPP1 expression, we selected paired GH adenoma tissues and adjacent pituitary tissues (pathologically confirmed as non-tumor tissues) for immunohistochemical staining comparison, following the same experimental procedures as before. Results ( Figure 9 The study clearly showed that, compared with the adjacent tissue, the staining intensity of ENPP1 in GH adenoma tissue was significantly increased, and the proportion of positive cells was significantly higher. In the adjacent normal pituitary tissue, only weak background staining or a few positive cells were observed. This contrast provides direct histological evidence for ENPP1 as a specific therapeutic target for GH pituitary adenomas.

[0048] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Use of ENPP1 inhibitors in the preparation of drugs for treating pituitary growth hormone adenomas.

2. The use according to claim 1, characterized in that, The ENPP1 inhibitor is a compound that can specifically inhibit ENPP1 enzyme activity and has the structure shown in Formula I below, or a known isomer and derivative, solvate, or pharmaceutically acceptable salt thereof; Formula I.

3. The use according to claim 1, characterized in that, The pituitary growth hormone adenoma is an adenoma that is insensitive to or resistant to somatostatin analogues.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of an ENPP1 inhibitor.

5. The pharmaceutical composition according to claim 4, characterized in that, The ENPP1 inhibitor is a compound that can specifically inhibit ENPP1 enzyme activity and has the structure shown in Formula I below, or a known isomer and derivative, solvate, or pharmaceutically acceptable salt thereof; Formula I.

6. The pharmaceutical composition according to claim 4, characterized in that, It also includes somatostatin analogues.

7. Use of the pharmaceutical composition according to any one of claims 4-6 in the preparation of a medicament for treating pituitary growth hormone adenoma.

8. The use according to claim 7, characterized in that, The pituitary growth hormone adenoma is an adenoma that is insensitive to or resistant to somatostatin analogues.

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