Phα1β cardiovascular safety pharmacology using the telemetry system in rats

Phα1β, a peptide toxin purified from Phoneutria nigriventer spider venom, has a dual antinociceptive effect, inhibiting high-voltage-activated calcium channels and antagonising the TRPA1 receptor that differentiates it from other traditional analgesics. Safety pharmacology studies conducted during the nonclinical development of new drug candidates include the potential effects of the drug, on cardiovascular parameters. The present study, cardiovascular assessments of Phα1β performed in conscious male Sprague-Dawley rats, previously implanted with the DSI™ PhysioTel telemetry system in the abdominal aorta. Animals received either Vehicle (PBS, 1 mL/kg) or the test compound ST-164 (Phα1β) at doses of 0.2, 0.8 or 2 mg/kg, administered intravenously. Cardiovascular parameters systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR), body temperature, and electrocardiographic (ECG) readings were evaluated at baseline and at multiple time points post-dosing (0.5, 1, 1.5, 2, 3, 4, 5, and 24 h) post administration of ST- 164 (0.2, 0.8 and 2 mg/kg) resulted in minor, transient, and dose-independent changes in SBP, DBP, MAP, and HR compared to baseline values, with no statistically differences to the vehicle group. A slight, transient, and non-physiologically change in body temperature was observed only at the highest dose (2 mg/kg). Electrocardiographic analysis revealed no alterations in QRS, QT, QTc, or PR intervals at any time point with ST-164 compared to Vehicle. All values were consistent with cardiovascular parameters in Sprague-Dawley rats. The telemetry assay validation with atenolol shows that atenolol reduced systolic blood pressure and mean arterial blood pressure validating the test.

Over the past 2 decades, significant research efforts have focused on N-type calcium channel inhibitors as promising candidates for the development of novel analgesic drugs (Bowersox et al., 1996). One such compound, ω-conotoxin MVIIA—originally derived from the marine snail Conus magus —was synthesised and commercialised as ziconotide (Prialt®). Ziconotide is a selective, reversible, and potent blocker of high-voltage-activated N-type calcium channels (HVCCs) and has demonstrated efficacy in the treatment of severe chronic pain (Miljanich and Raachandran, 1995). However, its therapeutic use is limited due to a narrow therapeutic window, as maximum analgesic efficacy occurs at doses close to those causing severe adverse effects (Penn and Paice, 2000). Despite being developed as a first-in-class analgesic for neuropathic pain, ziconotide and other available treatments often fail to provide satisfactory relief of chronic pain, highlighting a critical unmet medical need in the management of chronic pain (Staats et al., 2000; van Velzen et al., 2020). Consequently, the search for new analgesics that are both effective and safe remains a priority in pain research. Phα1β, a peptide toxin derived from Phoneutria nigriventer spider venom (Cordeiro et al., 1993), inhibits high-voltage-activated calcium channels (Vieira et al., 2005) and antagonises the TRPA1 receptor (Tonello et al., 2017), which differentiates it from other traditional analgesics. Over the past 15 years, our research group has investigated the analgesic potential of both the native and recombinant forms of Phα1β in preclinical rodent models of pain, including various pain models, such as neuropathic pain. Comparative studies have demonstrated that both forms of Phα1β, when administered intrathecally, exhibit superior analgesic efficacy and a more favourable (Rosa et al., 2014). These findings have positioned Phα1β as a promising analgesic candidate, warranting further optimization and advancement into preclinical development—an essential step before initiating clinical trials (Calixto et al., 2025). To comply with regulatory requirements established by agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA), two safety studies are required. The first, an in vitro study evaluating Phα1β′s interaction with the hERG potassium channel in human cardiac cells, has already been completed in collaboration with CIEnP. This study confirmed that Phα1β does not inhibit the hERG channel (Calixto et al., 2025). The second requirement, addressed in the present study, involves a comprehensive in vivo cardiovascular safety assessment using telemetry in rats. Radiotelemetry is considered the gold standard for real-time monitoring of physiological parameters in conscious, freely moving animals, thereby minimising stress-related artefacts. This technique is widely employed in pharmacology and toxicology to predict both efficacy and safety of new drugs candidates. Telemetry has been extensively validated for assessing cardiovascular parameters in preclinical studies (Kramer and Kinter, 2003), and rats with free movement, remain one of the most commonly used species for regulatory toxicology assessment (Gad, 2012). Telemetry employs implantable sensors and wireless transmitters to continuously collect biological signals, such as arteial blood pressure, heart rate, body temperature, and electrocardiographic (ECG) parameters, which are transmitted to remote receivers. This method facilitates early “go/no-go” decisions in drug development by providing high-quality safety data. Preclinical safety studies in animal models are essential not only for evaluating the therapeutic potential of new drugs but also for identifying potential adverse effects. In particular, cardiovascular toxicity is a major cause of drug attrition during early development stages. Even minor elevations in arterial blood pressure have been associated with an increased risk of myocardial infarction and stroke, while elevated resting heart rate has been linked to higher incidence of sudden cardiac death and heart failure. Therefore, rigorous cardiovascular safety evaluations are critical in the preclinical development of new analgesic drug, such as Phα1β.

Test item–ST-164 (Phα1β)

The synthesis of the recombinant form of ST-164 (Phα1β) by Giotto Biotech® https://www.giottobiotech.com) uses Escherichia coli expression systems. Purification was achieved through a proprietary process combining ion exchange and size exclusion chromatography. The final yield of the purified product was 0.5 mg/mL. Previous studies have demonstrated that both the native and recombinant forms of Phα1β exhibit equivalent analgesic efficacy in preclinical models of pain (Rigo et al., 2020). The recombinant peptide has a molecular weight (Mw) of 6.045 kDa and consists of 55 amino acids, with the following sequence.

ACIPRGEICTDDCECCGCDNQCYCPPGSSLGIFKCSCAHANKYFCNRKKEKCKKA

This sequence is identical to that of the native Phα1β, confirming structural and functional equivalence between the two forms.

Study design and regulatory compliance

The safety pharmacology evaluation of the test item ST-164 (Phα1β) on the cardiovascular system was conducted at CIEnP (Centro de Inovação e Ensaios Pré-Clínicos, Florianópolis, Santa Catarina, Brazil, following international standards for nonclinical safety studies. The intravenous administration of 0.2 mg/kg recombinant Phα1β caused 100% inhibition in both acute and chronic hyperalgesia (Rigo et al., 2020). The study adhered to the guidelines proposed by (Accardi et al., 2016), as well as the ICH S7A guideline on Safety Pharmacology Studies for Human Pharmaceuticals issued by the International Council for Harmonisation (ICH).

Animals

Male Sprague Dawley rats with free of moviment (n = 6 per group), sourced from Charles River Laboratories (United States), were used in the study. At the time of the experiments, the animals had a mean body weight of 449.58 g, ranging from 359.67 g to 539.50 g. This species and strain were selected based on their widespread use and suitability in cardiovascular safety assessments, as recommended by international regulatory guidelines (Huetteman and Bogie, 2009; Sugai et al., 2020).

Telemetry implantation surgery

Cardiovascular monitoring was performed using radiotelemetry in two stages. In the first stage, surgical implantation of the DSI™ PhysioTel telemetry system (Data Sciences International, St. Paul, MN, United States) was conducted under aseptic conditions, following CIEnP’s Standard Operating Procedure (SOP E.40). Pre-anesthesia was induced via intraperitoneal (i.p.) injection of diazepam (5 mg/kg), followed by a combination of ketamine (42 mg/kg) and xylazine (8 mg/kg, i.p.) for anesthetic induction. Anesthesia was maintained using inhaled isoflurane (1%–5%) delivered with a continuous oxygen flow of 3–4 L/min Supportive medications administered preoperatively included tramadol (10 mg/kg, subcutaneous (s.c.), hydrocortisone (10 mg/kg, s.c.), and benzathine penicillin G (30,000 IU, s.c.). The abdominal region was shaved and disinfected with 70% ethanol. Lidocaine (5 mg/kg, s.c.) was administered locally prior to the incision. A midline laparotomy was performed to expose the abdominal aorta. The aorta was briefly occluded to allow for insertion of the telemetry catheter, which was secured with biological adhesive. The telemetry transmitter was anchored to the peritoneal wall, and ECG electrodes (positive and negative) were fixed to the base of the thorax. These electrodes recorded electrical cardiac activity, enabling real-time electrocardiographic monitoring. Postoperative care included subcutaneous fluid therapy with sterile saline. Animals were allowed a recovery period of 6–10 days before proceeding to data collection.

Cardiovascular assessment protocol

Following recovery, animals were randomized based on body weight and assigned to one of four treatment groups: vehicle (PBS, 1 mL/kg, i.v.), ST-164 at 0.2, 0.8 or 2.0 mg/kg (i.v.). Each animal was placed individually in a recording chamber with a telemetry receiver plate. Animals had free access to water throughout the experiment. A 30-min acclimation period was observed. During the final 10 min (20–30 min), the DSI™ telemetry implant was activated using a magnet to record baseline cardiovascular parameters. Following baseline acquisition, animals received their respective treatments via intravenous administration. Physiological parameters—including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR), body temperature, and electrocardiogram (ECG)—were recorded at basal and after 0.5, 1, 1.5, 2, 3, 4, 5, and 24 h post-treatment. Telemetry recordings were conducted for 10 min at each time point, after which the implants were deactivated. After the 24-h post-treatment, a washout interval of 7 days was adopted, also used before each subsequent experimental bloco (2, three and 4) in which the same animals were employed. This period allowed for the reuse of the animals, maximising ethical and economic benefits, ensuring data integrity and complying with the 3Rs principles (replacement, reduction and refinement) (Deshmuk et al., 2008; Bod et al., 2010) This approach maximized data integrity while ensuring ethical and economic efficiency.

Assay validation with reference compound

For assay validation, the reference compound atenolol (POP D27), a known β1-adrenergic antagonist with hypotensive effects in rats, was administered at a dose of 100 mg/kg (n = 3–6 animals), in line with previously published data (Accardi et al., 2016; Pereira de Souza Neto et al., 2000; Carmichael et al., 2016). At the end of experiment animals were humanely euthanized using a CO 2 chamber supplemented with 20%–30% oxygen to minimize distress. A laparotomy was performed for the complete removal of the DSI™ PhysioTel telemetry device to conclude the study.

Analysis of cardiovascular parameters and body temperature

Baseline values for all parameters were recorded during the 30-min acclimation period before to treatment administration. Specifically, baseline measurements were calculated as the average of data collected during the final 5 min of this period (i.e., from 25 to 30 min). Following baseline acquisition, animals were treated intravenously with either vehicle (PBS, 1 mL/kg) or the test item ST-164 (Phα1β) at doses of 0.2, 0.8 or 2.0 mg/kg. Post-treatment evaluations of cardiovascular parameters—including systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), heart rate (HR)—as well as body temperature were performed at multiple time points. B (basal) and 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, and 24.0 h post-dosing. At each time point, the values for each parameter were derived from the average of the final 5 min of the corresponding 10-min recording session (e.g., minutes 25–30, 55–60, 85–90, 115–120, etc.). This standardised approach ensured consistency and minimized transient variability in the measurements.

Statistical analysis

For each cardiovascular parameter and body temperature, results were expressed as mean ± standard error of the mean (SEM) of the absolute values. Statistical analyses were conducted using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, United States). To evaluate the effects of the test item ST-164 (Phα1β) at doses of 0.2, 0.8 and 2.0 mg/kg compared to the vehicle group or to their respective baseline values, a two-way analysis of variance (ANOVA) was employed. Depending on the data structure, either a repeated measures two-way ANOVA or a mixed-effects model was applied, followed by Dunnett’s multiple comparisons test to identify specific differences between groups and time points.

Study validation

For the validation of the telemetry system, the effects of the reference compound atenolol (100 mg/kg) were statistically compared to the vehicle group using the Student’s t-test, based on values recorded before and after treatment. A significance level of p < 0.05 was considered statistically significant for all analyses.

According to the results presented in Figure 1, the single ST-164 treatment (0.2, 0.8 or 2.0 mg/kg i.v.) did not significantly alter systolic blood pressure (SBP) compared to the Vehicle group (PBS, 1 mL/kg) at any of the evaluated time points. Compared to the respective baseline values, there was a significant reduction in systolic blood pressure (SBP) after treatment with the vehicle (PBS, 1 mL/kg, i.v. (120.00 mmHg versus 125.93 mmHg)), 5 and 24 h after treatment with St-164 (0.2 mg/kg i.v.) 114.13 mmHg versus 127.45, 117.16 mmHg versus 127.45 mmHg and 117.16 mmHg versus 127.45 mmHg, respectively. 1.5 h after treatment with ST-164 (0.8 mg/kg, i.v.) (120.34 mmHg versus 128.28 mmHg and 5 h after treatment with St-164 (2 mg/kg, i.v) 120.09 mm/Hg versus 130.93 mm/Hg. Analysis of diastolic blood pressure (DBP), showed that single treatment with ST-164 (0.2, 0.8 or 2 mg/kg, i.v.) did not promote significant changes in this parameter compared to the Vehicle group (PBS, 1 mL/kg, i.v.) at any of the evaluated time points (Figure 2). However, when compared to their respective baseline values, significant reductions in diastolic blood pressure were observed 5 h after treatment with ST-164 (0.2 mg/kg, i.v.) 83.73 mmHg vs. 93.67 mmHg, 1 and 1.5 h after treatment with ST-164 (0.8 mg/kg, i.v.) 86.22 mmHg vs. 92.56 mmHg and 87.71 mmHg vs. 92.56 mmHg, respectively) and 24 h after treatment with ST-164 (2 mg/kg, i.v.) 90.70 mmHg vs. 96.25 mmHg. Figure 3 shows that a single treatment with ST-164 (0.2; 0.8 or 2 mg/kg, i.v.) did not significantly alter mean arterial pressure (MAP) compared with the Vehicle group (PBS, 1 mL/kg, i.v.) at any of the time points evaluated (Figure 3). Regarding baseline values, significant reductions in MAP were observed 1 h after treatment with the Vehicle group (PBS, 1 mL/kg, i.v.) 102.26 mmHg vs. 107.36 mmHg, 5 and 24 h after treatment with ST-164 (0.2 mg/kg, i.v.) 97.68 mmHg vs. 109.32 mmHg and 99.38 mmHg vs. 109.32 mmHg, respectively), and 1.5 h after treatment with ST-164 (0.8 mg/kg, i.v.) 102.59 mmHg vs. 109.26 mmHg. Treatment with ST-164 (2 mg/kg, i.v.) did not result in significant changes in MAP at any of the evaluated time points. Regarding heart rate (HR), a single treatment with ST-164 (0.2; 0.8 or 2 mg/kg, i.v.) did not cause significant changes compared to the Vehicle group (PBS, 1 mL/kg, i.v.) at any of the evaluated time points (Figure 4). However, when compared to baseline values, significant reductions in HR were observed at 2 h 312.62 bpm vs. 355.96 bpm, 3 h 308.65 bpm vs. 355.96 bpm, 4 h (313.54 bpm vs. 355.96 bpm, 5 h (308.97 bpm vs. 355.96 bpm and 24 h (318.69 bpm vs. 355.96 bpm) after treatment with ST-164 (0.2 mg/kg, i.v.), as well as 2 h 307.16 bpm vs. 338.18 bpm and 4 h 310.43 bpm vs. 338.18 bpm after ST-164 treatment (0.8 mg/kg, i.v.). It is important to mention that the changes observed for SBP, DBP, MAP, and HR were sporadic and discreet, remaining within the expected physiological range for adult Sprague-Dawley rats (SBP: 115–130 mmHg; DBP: 73–107 mmHg; MAP: 86–112 mmHg; HR: 250–400 bpm)as reported in the literature (Carmichael et al., 2016; Kapsdorferová et al., 2024; Detweiller et al., 2004). Figures 5A,B present the results for absolute body temperature and its variation relative to baseline (ΔT), respectively. Regarding absolute body temperature (Figure 5A), no significant differences were observed between groups treated with ST-164 at doses of 0.2, 0.8, and 2.0 mg/kg compared to the vehicle group (PBS, 1 mL/kg) at any time point. Similarly, for the variation in body temperature (ΔT) shown in Figure 5B, no significant differences were detected between ST-164-treated groups and the vehicle group throughout the observation period. The magnitude of temperature changes was minimal and without physiological relevance, as variations remained close to baseline values across all groups. Figure 6 shows the electrocardiogram (ECG) evaluation in conscious, free movement rats after a single intravenous administration of the Vehicle or test item ST-164. In this study, the QRS (Figure 6A), QT (Figure 6B), QTc (Figure 6C), and PR (Figure 6D) intervals were recorded using electrocardiography. The QT interval represents the duration of ventricular electrical systole, encompassing the total time for depolarization and repolarization of the ventricles. Because the QT interval varies with heart rate, the corrected QT interval (QTc) is considered a more reliable measure (Chugh et al., 2009). Prolongation of the QTc interval has been associated with various cardiovascular and non-cardiovascular diseases and is recognized as an independent predictor of ventricular arrhythmias, increased mortality, and sudden cardiac death (Elming et al., 2003; Guyton and Hall, 2011; Konopelski and Ufnal, 2016). The QRS interval comprises the Q (initial downward deflection), R (upward deflection), and S (subsequent downward deflection) waves, which collectively represent ventricular depolarization (contraction). The PR interval measures the time from the onset of atrial depolarisation (P wave) to the beginning of ventricular depolarisation (QRS complex), reflecting conduction through the atria, the atrioventricular node, and the His–Purkinje system (Guyton and Hall, 2011). The results shown in Figure 6 correspond to electrocardiographic recordings obtained after a single intravenous administration of vehicle (PBS, 1 mL/kg) or test item ST-164 at doses of 0.2, 0.8 or 2.0 mg/kg. No significant changes in QRS, QT, QTc, or PR intervals were observed in animals treated with ST-164 compared to the vehicle group at any time point. When comparing values to their respective baselines, ST-164 at 0.2 mg/kg and 2.0 mg/kg did not cause significant alterations in these parameters. However, a modest but statistically significant increase in the QRS interval was noted 0.5 h after administration of ST-164 at 0.8 mg/kg (19.39 ms vs. 18.18 ms baseline). Similarly, significant increases in the QT 62.75 ms vs. 55.77 ms) and QTc 108.26 ms vs. 98.90 ms intervals were detected 2 h post-treatment at the same dose (Detweiler et al., 2004). Despite statistical significance, these increases—1.2 ms in QRS, 6.98 ms in QT, and 9.39 ms in QTc—were minimal and considered physiologically irrelevant for male Sprague-Dawley rats (Konopelski and Ufnal, 2016). Moreover, these values fall within the historical range for vehicle-treated animals in multiple studies performed by CIEnP, reinforcing the absence of any adverse effects on ventricular conduction or repolarization associated with ST-164 treatment. For the Telemetry assay validation with Atenolol), three animals were orally treated with the reference item Atenolol, a β1-adrenergic receptor antagonist, at a dose of 100 mg/kg (Accardi et al., 2016; Munos, 2009). The results shown in Figure 7 indicate that atenolol reduced systolic and mean arterial blood pressure compared to the vehicle (PBS, 1 mL/kg), with findings consistent with historical data from CIEnP’s internal database (PL-400) validating this preclinical study.

Previous datasets have shown good concordance of drug-induced changes in heart rate and blood pressure between animals and humans (sensitivity: 78%; specificity: 79%). Although minor fluctuations were observed in SBP, DBP, MAP, and HR following treatment, these changes were transient, discrete, and dose-independent, remaining within the established physiological range for adult Sprague-Dawley rats, as reported in the literature: SBP: 115–130 mmHg; DBP: 73–107 mmHg; MAP: 86–112 mmHg; and HR: 250–400 beats per minute (b.p.m.) (Carmichael et al., 2016; Kapsdorferová et al., 2024; Detweiller et al., 2004). These findings indicate that ST-164 did not induce any cardiovascular alterations of toxicological or pharmacological concern at the tested doses. The results of this study indicate that Test Item 164 (Phα1β), at the evaluated doses and under the experimental conditions applied, induced specific and discrete changes, regardless of the dose used. These findings may warrant further investigation in clinical studies since all cardiovascular parameters remained within the physiological ranges expected for adult Sprague-Dawley rats, indicating the absence of clinically relevant effects.

Raw data recording

All data generated during the study were recorded directly, promptly, accurately, and legibly by the individual responsible for generating the data (NIT-DICLA-035). All entries were documented in appropriate spreadsheets and/or forms, signed and dated, in accordance with SOP B.03.

Archiving

An original copy of the Study Plan, Raw Data,raw data, and Final Reportraw data, final report was archived at CIEnP (Florianópolis, Brazil) for a minimum of 5 years, in accordance with SOP B.07. The ST-164 test item (Phα1β) was stored in the CIEnP archive until its expiration date or, if undetermined, for at least 5 years, as per SOP F.12.

Phα1β is a peptide toxin purified from the venom of the spider Phoneutria nigriventer with a well-documented antinociceptive profile (Rosa et al., 2014; Rigo et al., 2020). Based on its efficacy and tolerability in preclinical pain models, Phα1β (test item ST-164) has progressed to the optimization final report optimisation and nonclinical development stages, which are essential steps before human studies. Given the high attrition rate during drug development despite substantial investment in research and development (Brockway et al., 1993), early identification of cardiovascular liabilities remains critical for informed go/no-go decisions. Radiotelemetry in conscious, freely moving animals is widely considered the gold standard for cardiovascular safety pharmacology because it enables real-time monitoring while minimising stress-related artefacts (Kilkenny et al., 2010; Kramer and Kinter, 2023; Lindgreen et al., 2008). This approach improves the physiological relevance of measurements and reduces variability associated with restraint or anesthesia, thereby increasing the precision of blood pressure, heart rate, body temperature, and electrocardiographic (ECG) endpoints {30,31]. Rats remain one of the most frequently used species in regulatory cardiovascular safety pharmacology packages, and radiotelemetry has been broadly validated for detecting drug-induced effects on arterial pressure, heart rate, and ECG intervals (Kramer and Kinter, 2023). Under the experimental conditions applied, a single intravenous administration of ST-164 (0.2, 0.8, 2.0 mg/kg) did not produce statistically significant differences versus vehicle for systolic, diastolic, or mean arterial pressure, nor for heart rate, across the evaluated time points (Figures 1–4). When baseline comparisons reached statistical significance at isolated time points, the direction and magnitude of changes were small, transient, and not dose-dependent. Importantly, the absolute values remained within physiological ranges reported for adult Sprague–Dawley rats (Carmichael et al., 2016; Kapsdorferová et al., 2024; Detweiller et al., 2004), supporting the interpretation that these findings are not clinically relevant. Consistent with the hemodynamic, profile, ST-164 did not cause meaningful effects on body temperature (Figure 5). Any observed variations were minimal and remained close to baseline, indicating no thermoregulatory liability under the tested conditions. Together, the blood pressure, heart rate, and temperature data suggest that ST-164 does not elicit pharmacologically or toxicologically concerning cardiovascular responses following single-dose intravenous exposure in rats. ECG interval analysis further supports a favourable haemodynamic and cardiac safety profile. ST-164 did not significantly alter QRS, QT, QTc, or PR intervals versus vehicle at any time point (Figure 6). Evaluation of ventricular depolarisation and conduction (QRS) is important because QRS prolongation has been associated with clinically relevant adverse events across multiple drugs depolarisation (Cros et al., 2012; Laverty et al., 2011). Likewise, preclinical assessment of QT/QTc is recommended because QT prolongation links to increased proarrhythmic risk, including torsades de pointes, and remains a key regulatory concern (Vlaminckx et al., 2008; Redfern et al., 2009). In the present study, occasional statistically significant baseline-related changes observed at isolated time points were small in magnitude and within the expected physiological range for rats (Konopelski and Ufnal, 2016), supporting the conclusion that they are unlikely to reflect an adverse effect on ventricular conduction or repolarization. (Vlaminckx et al., 2008; Redfern et al., 2009). These in vivo telemetry findings complement the previously reported in vitro assessment, which showed that ST-164 does not inhibit the human Kv11.1 (hERG) channel (Calixto et al., 2025). In line with the intent of ICH S7B, integrating ion-channel information with in vivo ECG monitoring strengthens confidence that the compound does not present an early signal for delayed ventricular repolarisation. Taken together, the absence of meaningful QT/QTc effects in conscious rats and the lack of hERG inhibition support the interpretation that ST-164 has a low risk of QT-related liabilities under the conditions tested.

Assay sensitivity was confirmed using atenolol, which produced the expected reductions in systolic and mean arterial blood pressure (Figure 7), consistent with literature reports (Accardi et al., 2016; Pereira de Souza Neto et al., 2000; Carmichael et al., 2016). This positive-control response supports the ability of the telemetry platform and study design to detect pharmacologically relevant cardiovascular effects and increases confidence in the negative findings obtained with ST-164.

This study has limitations inherent to single-dose assessments in one species and sex, and the telemetry recordings were performed at discrete post-dose intervals rather than continuously over the full 24-h period. Although these conditions are appropriate for a core cardiovascular safety pharmacology evaluation, additional studies may be warranted as development advances (e.g., alternative dosing regimens or expanded nonclinical packages), particularly to further characterise exposure–response relationships.

The results of this study indicate that test item ST-164 (Phα1β), at the evaluated doses and under the experimental conditions applied, induced minor and transient changes, regardless of the dose used. These findings may warrant further investigation in clinical studies since all cardiovascular parameters remained within the physiological ranges expected for adult Sprague-Dawley rats, indicating the absence of clinically relevant effects.