SDX-7320 was synthesized as previously described (29).

All animal experiments were carried out in accordance with the institutional guidelines and were approved by the Institutional Animal Care and Use Committee.

Male C57BL/6J mice were individually housed and fed a high-fat, high-sucrose diet (Research Diets D12492) beginning at 5 weeks of age for 12 weeks prior to study initiation. Body weight was measured every four days, and the total food consumed per week per cage was also measured. Mice were randomized to treatment groups based on body weight (N = 10/group) and treatment was conducted with subcutaneous (SC)-injected vehicle (5% w/v D-mannitol in water) Q4D or SDX-7320 (SC, Q4D) for a total of eight doses. Mice were euthanized by carbon dioxide inhalation on day 34 (two days after their last dose) and terminal blood was collected via cardiac puncture. Leptin and adiponectin levels were measured in plasma prepared from terminal blood samples using a Meso Scale Discovery mouse metabolic kit (K15124C) and adiponectin kit (K152BXC), respectively, according to the manufacturer’s instructions. Adipose tissue (epididymal, inguinal, and retroperitoneal depots) was dissected and weighed.

Male C57BL/6J mice (Jackson Laboratories) were individually housed and fed a high-fat, high-sucrose diet (Research Diets D12492) beginning at 6 weeks of age for 22 weeks before the start of the study. Body weight was measured on Day -4 and Day 0. Prior to treatment on Day -4, mice were randomized by body weight into vehicle and SDX-7320 treatment groups (N = 6/group). The mice were then administered a single dose of vehicle (5% w/v D-mannitol in water) or SDX-7320 (8 mg/kg) SC. Four days later, on Day 0, baseline blood glucose (mg/dL) was measured (from <10 µL of blood collected from a tail nick) using an EasyMax V glucometer. Insulin tolerance test (ITT) was conducted after a 4-hour fast by delivering 0.75 U insulin/kg dosed intraperitoneally (IP). Blood glucose levels were measured at the indicated times following insulin administration using a hand-held glucometer (EasyMax V).

Male, diet-sensitive (DS) Sprague-Dawley/Levin rats (30) were obtained from Taconic at 3 weeks of age. Rats were individually housed and fed a high-fat/high-sucrose diet (Harlan TD.06414, 60% of calories from fat, 21% of calories from carbohydrate rodent diet) for 9 weeks until their average body weight was approximately 600 g, and they continued to receive this diet for the remainder of the study. Treatment groups (N = 3/group) consisted of vehicle (phosphate-buffered saline, PBS), SDX-7320 (0.3, 1.0, 3.0 mg/kg; Q4D) and SDX-7539 (1.0, 3.0 mg/kg; Q4D) with all agents administered by SC injection for a total of 16 doses over 67 days. The body weight of each animal was measured every other day and food consumption was measured weekly. Blood samples for plasma SDX-7539 bioanalysis were collected on day 57; 1, 2, 4, and 24 h after dosing with SDX-7320 or SDX-7539. Adipose tissue was dissected (epidydimal, inguinal and retroperitoneal depots) and weighed at the end of the study (day 67, two days after the last dose of either test agent), following euthanasia by carbon dioxide inhalation.

Male C57BL/6J mice (Jackson Laboratories) were fed either a high-fat diet (D124592) or a low-fat diet (D12450J) beginning at 6 weeks of age for 15 weeks prior to tumor cell inoculation and were maintained on the same diet for the entire study. The B16-F10 mouse melanoma tumor cell line was obtained from ATCC. Cultures were maintained in DMEM (Gibco, Invitrogen) supplemented with 5% fetal bovine serum and housed at 37 °C in a humidified 5% CO₂ atmosphere. The study was initiated by injecting 2 × 10⁵ B16F10 melanoma cells into the flank of lean and obese mice. When the tumors reached approximately 100 mm³, treatment with the test article was commenced (N = 10/group). Animals were dosed with SDX-7320 or vehicle (5% D-mannitol in water) via SC intra-scapular injection every four days for a total of 5 doses. The study was terminated on day 17 and mice were euthanized using carbon dioxide inhalation, and adipose tissue was dissected and weighed.

Female C57BL/6J mice that had undergone surgical ovariectomy at 4 weeks of age to mimic the postmenopausal state were obtained from the Jackson Laboratory at 6 weeks of age. The mice were then maintained for approximately 20 weeks on either a high-fat/high-sucrose diet (D12451) or a low-fat/high-sucrose diet (D12450B) until the start of the study. EO771 tumor cells (a model of TNBC, obtained from CH3 Biosystems) were implanted into the fourth mammary fat pad (1 × 10⁵/mouse; implantation was conducted after mice were anesthetized with 4% isoflurane and 1.2% oxygen) at approximately 26 weeks of age. Treatment with vehicle (5% w/v D-mannitol in water, SC, Q4D) or SDX-7320 (8 mg/kg, SC, Q4D) was initiated when the tumors were approximately 60 mm³ (N = 9/group). All mice were euthanized on day 15 (after receiving a total of 4 doses) using carbon dioxide inhalation, after which the tumors and adipose tissues were excised.

Male DIO and lean age-matched C57BL/6J mice (Jackson Laboratories) were fed a 60% kcal high-fat/high-sucrose diet (D12492) or a 10% kcal low-fat/high-sucrose diet (D12450B) beginning at 6 weeks of age for 13 weeks prior to study initiation. Mice were housed to 1–3 per cage for the duration of the study. Body weight was recorded twice weekly and changes in body weight were calculated relative to the baseline body weight at the start of the study. MC38 tumor cells (Kerafast, RRID: CVCLB288) were suspended in 50% PBS and 50% Matrigel before being implanted into the SC space of the right rear flank (2x10⁵/mouse). Tumor volume was measured twice weekly using wireless Mitutoyo UWAVE-T digital calipers in conjunction with UWAVE-R to digitally record the measurements. Tumor volume was calculated ((l × w²) × π/6) using Microsoft Office Excel software. Once the average tumor volume reached approximately 100 mm³, mice were randomized to treatment groups based on tumor volume. Treatment with vehicle (5% w/v D-mannitol in water, SC, Q4D) or SDX-7320 (6 mg/kg, SC, Q4D) was initiated for a total of five doses (N = 10/group). Upon termination of the study (on day 17, one day following the last dose), the mice were euthanized by carbon dioxide inhalation, blood was collected via cardiac puncture, and serum and plasma were prepared. Tumor tissues were dissected, weighed, and placed in RNAlater tubes. The adipose tissues (epididymal, retroperitoneal, and inguinal) were dissected and weighed.

An additional study was conducted in which SC MC38 tumors (MC38 cells (Kerafast), 1 × 10⁶ cells/mouse) were established in DIO mice (Taconic, male C57Bl/6TANac mice that had been fed a high-fat/high-sucrose diet (D12492) beginning at 6 weeks of age for 12 weeks). When the tumors reached approximately 60 mm³, treatment with vehicle, SDX-7320 (6 mg/kg, SC, Q4D), or tirzepatide (30 nmol/kg, SC, QD) was initiated (N = 8/group). Body weight was measured every other day, and tumor volume was assessed twice per week. Upon termination of the study (eighteen days after initiation and two days after the last dose of SDX-7320 and one day after the last dose of tirzepatide; a total of 5 doses of SDX-7320 and 18 doses of tirzepatide were delivered), the mice were euthanized by carbon dioxide inhalation and adipose tissue depots were dissected and weighed.

Time-dependent data were analyzed using repeated measures two-way ANOVA (Geisser-Greenhouse correction with Sidak’s multiple comparisons test). For the analysis of endpoint data (i.e., adipose tissue mass, plasma biomarkers) non-parametric Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparisons test was used. All data were analyzed as described using GraphPad v10.0.

Total RNA from MC38 tumors was extracted using the Qiagen RNeasy Plus Universal Mini Kit following the manufacturer’s instructions (Qiagen). RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies), and RNA integrity was checked using a TapeStation (Agilent Technologies). The RNA sequencing library was prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs), validated on TapeStation (Agilent Technologies), and quantified using a Qubit 2.0 Fluorometer (Invitrogen) and quantitative PCR (KAPA Biosystems). The sequencing library was clustered in one lane of a flow cell and loaded on an Illumina HiSeq instrument (4000 or equivalent), according to the manufacturer’s instructions. The sample was sequenced using a 2x150bp Paired End (PE) configuration at a depth of 3 x 10⁷ reads/sample. Image analysis and base calling were performed using HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated were converted into fastq files and de-multiplexed using Illumina’s bcl2fastq 2.17 software. One mismatch was allowed for index sequence identification. Analysis was performed in the Watershed^® Cloud Data Lab (CDL). Raw RNA sequence reads were aligned to the mouse reference genome (GRCm38/mm10) with Gencode vM10 annotations using STAR v2.7.5c. RNA-Seq data were uploaded to the GEO database (GSE295063). Principal component analysis (PCA) was performed using the PCA function in the Python package scikit-learn. Normalized gene expression was used as an input to identify the first 10 components of global gene expression. Differential gene expression analysis was performed using the R DESeq2 package version 1.40.2 (31). Gene set enrichment analyses (GSEA) were performed using the R clusterProfiler package, version 4.12.0 (32) and the mouse hallmark gene set collection from MsigDB v2024.1 (33). For this analysis, the Wald statistic calculated by DESeq2 analysis was used to rank genes for GSEA analysis.

Rat plasma samples were subjected to protein precipitation followed by separation on a Waters Acquity UPLC system with a Waters Acquity BEH C18 reverse-phase column (50 mm × 2.1 mm) using an acetonitrile-water solvent gradient containing 0.1% formic acid. Detection was achieved using an AB Sciex API 5500 mass spectrometer with positive electrospray ionization in MRM mode, providing an acceptable concentration range for SDX-7539 of up to 500 ng/mL (lower limit of quantitation was 0.1 ng/ml).

Murine plasma (50 µL) was transferred onto an Oasis solid-phase extraction (SPE) system (Waters). Quality control (QC) samples were generated by pooling 10 µL plasma from each sample. Next, 200 µL of 1:1 methanol:acetonitrile (MeOH: ACN) was added to each well and shaken for 1 min at room temperature at 360 rpm, followed by 10 min of incubation. Next, 150 µL 2:2:1 MeOH: ACN:water was added to each well and incubated again for 10 min. Polar metabolites were eluted into a 96-well collection plate using a positive pressure manifold. This procedure was repeated using 100 µL of 2:2:1 MeOH: ACN:H₂O in the same collection plate. The polar eluates were then covered and stored at -80 °C until Liquid Chromatography-Mass Spectrometry (LC/MS) analysis. The SPE plates from the polar metabolite extraction were then washed twice with 500 µL of 1:1 methyl tert-butylether:methanol to elute non-polar metabolites into a new collection plate using a positive pressure manifold. The combined eluates were dried under a stream of nitrogen at room temperature, reconstituted with 200 µL of 1:1 isopropanol:methanol, and stored at -80 °C prior to LC/MS analysis.

All untargeted metabolomics data were acquired using an Agilent Q-TOF 6546 mass spectrometer. The mobile phases for polar metabolite analysis were as follows: A) 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide, 5% ACN, 2.5 mM medronic acid in water, and B) 95% ACN. Chromatography was performed using a HILICON iHILIC (P)-Classic (2.1 x 100 mm). A 4 µL injection volume was used for both positive and negative ionization modes. HILIC/MS was performed using the following linear gradient at a flow rate of 250 µL/min: 0–1 min: 90% B, 1–12 min: 90-35% B, 12-12.5 min: 35-25% B, 12.5-14.5 min: 25% B. The column was re-equilibrated with 20 column volumes of 90% B. Mass spectrometry analysis was performed with a mass range of 100–1700 Da and 1 scan/s. MS/MS data were acquired in a data-dependent iterative fashion with a 1.3 m/z isolation window.

The mobile phases for non-polar metabolite analysis were: A) 10 mM ammonium formate and 5 µM InfinityLab Deactivator Additive (Agilent) in 5:3:2 water:ACN: IPA, and B) 10 mM ammonium formate in 1:9:90 water:ACN: IPA. Reverse phase liquid chromatography (RPLC) was performed using a Waters Acquity HSS T3 (2.1 x 100 mm). A 2 µL injection volume was used when data were acquired in positive ion mode, and a 4 µL injection volume was used when data were acquired in negative ion mode. RPLC/MS was performed using the following linear gradient at a flow rate of 400 µL/min: 0-2.5 min: 15-50% B, 2.5-2.6 min: 50-57% B, 2.6–9 min: 57-70% B, 9-9.1 min: 70-93% B, 9.1–11 min: 93-96% B, 11-11.1 min: 96-100% B, 11.1–12 min: 100% B, 12-12.2 min: 100-15% B. The column was re-equilibrated for 3.8 min. Mass spectrometry analysis was performed with a mass range of 100–1700 m/z and 1 scan/s. MS/MS data were acquired in a data-dependent iterative fashion with a 1.3 m/z isolation window.

Data files were converted to the mzML format using MSConvert. Features were detected using proprietary peak detection and curation software (Panome Bio, Inc). Features were aligned across samples, and those with intensities greater than 1/3 of the corresponding intensity in the QC sample were classified as contaminants and were removed from future analysis. Feature degeneracies (isotopes, adducts, fragments, etc.) were identified using clustering and ion assignment. Metabolites were structurally identified by comparing isotope patterns and MS/MS fragmentation data (when available) against a database composed of known metabolites found in RefMet, LipidMaps, and HMDB, in addition to an in-house standard library from Panome Bio (34). Metabolite signals were discarded if the coefficient of variation among the QC samples was greater than 25%. Missing values were imputed using half the minimum intensity detected for each metabolite.

Principal component analysis was performed using the PCA function within the Python package scikit-learn. Prior to hypothesis testing, data were log-transformed to ensure normality. Metabolites showing significant differences between the control, tirzepatide, and SDX-7320 treated mice were identified using one-way ANOVA.

The data for this study were generated by multiple contract research organizations. Reports containing raw and derived data supporting the findings of this study are available from the corresponding author upon reasonable request. Gene expression data are available in the GEO database under accession number GSE295063.

Small molecule METAP2 inhibitors exhibit anti-obesity and anti-diabetes activities, both in non-clinical models and in humans (20, 35–37). Therefore, the activity of SDX-7320, the polymer-conjugated METAP2 inhibitor, was evaluated in standard rodent models of DIO and insulin resistance. Treatment with SDX-7320 SC led to a dose-dependent reduction in body weight in DIO mice (Figure 1A). Consistent with the initial weight loss mediated by SDX-7320, food intake (normalized to body weight) was significantly reduced during the first week of the study, but then increased, returning to vehicle group levels after the second week despite continued weight loss (Figure 1B). Analysis of adipose tissue weight at the end of the study demonstrated a significant reduction in response to SDX-7320 in each of the three adipose depots (Figures 1C–E). Mice treated with SDX-7320 had significantly reduced leptin levels (Figure 1F) and increased adiponectin levels (Figure 1G), which resulted in a significant reduction in the leptin-to-adiponectin ratio (Figure 1H).

In a second study in DIO mice, SDX-7320 elicited effects on body weight, food intake, and fat mass similar to those shown in Figure 1 (Supplementary Figure S1). To assess the impact of weight loss in response to SDX-7320 on glycemic control, an intraperitoneal glucose tolerance test (IPGTT) was conducted after four weeks of SDX-7320 treatment. IPGTT results showed a significant dose-dependent reduction in glucose excursion, indicating enhanced glucose disposal in mice treated with SDX-7320 (Supplementary Figures S1D, E). Consistent with the reductions in body weight and fat mass in response to SDX-7320, decreases in plasma levels of ALT, cholesterol, triglycerides, as well as in liver weight, were observed (Supplementary Figures S1F–I).

Because DIO mice treated with SDX-7320 exhibited improved glucose responses in an IPGTT, a separate study was conducted to directly assess whether SDX-7320 improved peripheral insulin sensitivity. An ITT was conducted in DIO mice that received vehicle or a single dose of SDX-7320 four days before the ITT. The acute response to insulin (i.e., a decline in blood glucose) in mice pretreated with SDX-7320 was significantly greater than that observed in mice pretreated with the vehicle (Supplementary Figure S2). These results demonstrated that SDX-7320 enhances peripheral insulin sensitivity.

SDX-7320 was designed to minimize CNS toxicity previously observed with small-molecule METAP2 inhibitors, such as TNP-470, by attaching a novel small-molecule METAP2 inhibitor (SDX-7539) to a high-molecular-weight, water-soluble, and biocompatible polymer. Pharmacokinetic studies of unconjugated SDX-7539 versus SDX-7539 derived from the PDC SDX-7320 showed that the half-life of SDX-7539 in plasma was approximately 40-fold longer following administration of the PDC (29). To investigate whether the unique plasma PK profile observed for SDX-7539 delivered via the polymer conjugate had any effect on its pharmacodynamics, we compared the anti-obesity activity of SDX-7320 with that of unconjugated SDX-7539 in obese male Levin rats. Subcutaneous administration of SDX-7320 elicited a dose-dependent attenuation in weight gain over time (Figure 2A), accompanied by a reduction in adipose tissue (Supplementary Figure S3), and improved insulin sensitivity (Supplementary Figure S3). Significant differences in SDX-7539 C_max and AUC were observed; dosing with SDX-7320 yielded far lower systemic plasma levels of SDX-7539 than dosing with unconjugated SDX-7539 (Figure 2B; Supplementary Table S1). PK-PD analysis of SDX-7539 pharmacokinetics (C_max, AUC) against the efficacy data (i.e., % increase from baseline in body weight) showed greater anti-obesity efficacy in response to SDX-7320, despite much lower plasma exposure to polymer-released SDX-7539 (Figures 2C, D).

Having established the anti-obesity activity of SDX-7320 including its effects on host metabolic factors (insulin sensitivity, leptin and adiponectin levels), we next determined its ability to suppress obesity-accelerated tumor growth. Three different mouse tumor models were selected because obesity is known to accelerate the growth of each of these tumors (38–40). Initially, the B16F10 melanoma model was used to evaluate the effects of SDX-7320 on obesity-accelerated tumor growth. Subcutaneous B16F10 tumors grew significantly faster and achieved a larger size in obese animals than in lean age-matched animals (Figure 3A). SDX-7320 significantly inhibited tumor growth in both obese and lean animals in a dose-dependent manner (Figures 3B, D). SDX-7320 significantly reduced absolute body weight in both obese and lean mice compared to their respective vehicle-treated controls (Figures 3C, E). In obese mice, weight loss induced by SDX-7320 resulted in part from significant reduction in adipose tissue mass (Figures 3F–H). SDX-7320 did not significantly reduce adipose tissue mass in lean mice bearing B16F10 tumors (Figures 3F–H).

To further investigate the efficacy of SDX-7320 in obesity-accelerated tumor growth, a study was conducted in obese vs. lean ovariectomized female C57BL/6 mice (intended to mimic the post-menopausal state) harboring syngeneic, orthotopic EO771 mammary gland tumors. The mice were fed either a high-fat diet (DIO mice) or a low-fat diet (lean mice). As shown in Supplementary Figure S4A, tumor growth was significantly accelerated in obese compared to lean mice. Obese and lean mice bearing EO771 mammary gland tumors were randomized to treatment with vehicle or SDX-7320 (8 mg/kg, SC, Q4D) once the mean tumor volume had reached approximately 60 mm³. Treatment with SDX-7320 significantly reduced the rate of tumor growth in obese mice (Supplementary Figure S4B), whereas only a trend was observed in lean mice (Supplementary Figure S4D). There was also a trend toward reduced body weight with SDX-7320 in obese mice with EO771 tumors, and no effect on body weight was observed in lean mice with EO771 tumors (Supplementary Figures S4C, E). SDX-7320 significantly reduced the mass of retroperitoneal, inguinal and parametrial adipose tissue as well as parametrial adipose tissue in lean mice (Supplementary Figures S4F–H).

To extend the findings described above in models of obesity-accelerated melanoma and breast cancer, the effect of SDX-7320 on MC38 tumor growth, a model of colorectal cancer, was tested. Male C57BL/6 mice were initially fed either a high-fat/high-sucrose diet or a low-fat diet for 12 weeks to generate obese and lean mice, respectively. Subsequently, obese and lean mice were injected SC with MC38 tumor cells and tumor growth was monitored. Consistent with the models mentioned above, tumor growth in obese mice was significantly greater than that in lean mice (Figure 4A). Treatment with SDX-7320 was initiated when the average tumor volume reached approximately 100 mm³.

SDX-7320 treatment significantly inhibited tumor growth in obese mice whereas a trend toward reduction in tumor growth was observed in lean mice (Figures 4B, D). SDX-7320 elicited modest but significant reduction in body weight in cohorts of obese and lean mice (Figures 4C, E).

To investigate whether there were distinct alterations in gene expression across the different cohorts and to elucidate the mechanisms underlying SDX-7320-mediated inhibition of MC38 tumor growth in both obese and lean mice, RNA sequencing was conducted on tumor tissue. A total of 463 up-regulated and 61 down-regulated differentially expressed genes (DEGs; p.adj<0.05, fold change>1.5) were found in tumors from obese mice treated with SDX-7320 vs. vehicle (Figures 5A, B; Supplementary Table S2; Supplementary Figure S5) using DESeq2. In tumors from lean mice, SDX-7320 treatment was associated with only 14 up-regulated and 11 down-regulated DEGs (Figures 5A, B; Supplementary Table S2; Supplementary Figure S5). To better understand the significance of these global transcriptome differences, pathway enrichment analysis was performed. The results of ranked gene set enrichment analysis (GSEA) showed that the expression of numerous pathways was enriched in tumors from SDX-7320 vs. vehicle-treated obese mice (Figure 5C), including the immune response elements interferon gamma response, interferon alpha response, inflammatory response, IL6-JAK-Stat3 signaling, and TNF-α signaling via NFKappaB. The negatively enriched pathways included G2M checkpoint and E2F target pathways. In lean mice, fewer pathways were positively enriched by SDX-7320 treatment, although two of the same immune response elements were enriched (i.e., interferon alpha response and interferon gamma response; Figure 5D), but not IL6-JAK-Stat3 or TNF-α signaling. Negatively enriched pathways in tumors from lean mice treated with SDX-7320 also included G2M checkpoint and E2F target pathways, as was observed in obese mice. The responses in lean mice generally showed lower enrichment scores and less robust false discovery rates than those observed in obese mice. As multiple immune response pathways were enriched in SDX-7320-treated tumors from both obese and lean mice, we next determined whether there were any overlapping effects of SDX-7320 on specific DEGs. Venn diagrams for SDX-7320-treated groups vs. vehicle treatment indicated that there were 10 shared up-regulated and 3 shared down-regulated DEGs in tumors from obese and lean mice treated groups (p.adj<0.05, fold change >1.5; Figures 5A, B). The overlapping up- and down-regulated genes suggest a consistent effect of METAP2 inhibition on tumor gene expression regardless of body weight (highlighted in orange and purple, respectively, in Supplementary Table S2). For example, the expression of numerous granzymes and perforin1 was increased in tumors from both obese and lean mice treated with SDX-7320 (Figures 5E–G), suggesting that inhibiting METAP2 enhanced the host anti-tumor immune response. Furthermore, the expression of CD-206/MRC1, a marker of immune-suppressive macrophages, was significantly decreased in tumors from both lean and obese mice, suggesting that SDX-7320 favorably alters the tumor immune microenvironment (Figure 5H).

With an increasing focus on the link between obesity and cancer progression and the clinical success of incretin-based therapies for weight loss and improvement of obesity-related conditions, we compared the anti-tumor efficacy of SDX-7320 with tirzepatide in the MC38 syngeneic model of obesity-accelerated colon cancer. DIO mice with established MC38 tumors were treated with vehicle, SDX-7320 or tirzepatide beginning when the tumors were approximately 60 mm³. Interestingly, tirzepatide modestly but significantly inhibited MC38 tumor growth, whereas SDX-7320 exerted a significantly greater inhibitory effect on MC38 tumor growth compared to tirzepatide (Figure 6A). In contrast, the effect of tirzepatide on body weight loss was significantly greater than that of SDX-7320 (Figure 6B). Unsurprisingly, when adipose tissue was measured at the end of the study, the effect of tirzepatide on fat mass was greater than that of SDX-7320 (Figures 6C–E). Analysis of circulating adipokines leptin and adiponectin showed that tirzepatide elicited a greater reduction in plasma leptin levels than SDX-7320 (Figure 6F), whereas only SDX-7320 significantly increased levels of plasma adiponectin (Figure 6G). The leptin:adiponectin ratio (LAR) was determined and only tirzepatide significantly decreased LAR (Figure 6H).

To explore potential mechanistic similarities and differences between SDX-7320 and tirzepatide, global untargeted metabolomics was performed on plasma samples collected at the end of the study. The overall chemical profiles of each sample were investigated using PCA, which revealed a clear separation between the treatment groups (Figure 6I), suggesting non-overlapping mechanisms of action. Treatment with tirzepatide resulted in increased levels of six metabolites and decreased levels of 22 metabolites compared to vehicle controls (Supplementary Table S3). Treatment with SDX-7320 resulted in 15 metabolites showing significant alterations, of which four were significantly up-regulated and 11 were significantly down-regulated, whereas only five of the metabolites were significantly altered by both treatments (Supplementary Table S3; Supplementary Figure S6). One steroid, ST 24:1;O5, was up-regulated by both compounds, whereas dihydrouracil, ureidopropionic acid, and diacylglycerophosphocholines PC 44:10 and PC 40:6 were down-regulated by both agents (Figure 6J). Changes in the remaining 10 compounds exclusively altered by SDX-7320 included increases in artemin and glycerophosphoglycerols PG O-36:5 and PG 34:1, in tandem with the depletion of several additional lipids (Supplementary Table S3). Interestingly, the depleted lipid profile included ceramides and an additional phosphatidylcholine (PC O-38:4). Lastly, investigating the significantly altered metabolites exclusive to tirzepatide showed that most down-regulated metabolites were phospholipids and di/triglycerides (Supplementary Table S3).