Preclinical and phase I studies of an antisense oligonucleotide drug targeting IGF-1R in liver cancer

Primary liver cancer is a common malignant tumor with high morbidity and mortality rates globally. Liver cancer is the sixth most common cancer and the third leading cause of cancer deaths in the world [1]. In 2020, the annual number of new cases of liver cancer reached 906,000, and the number of deaths reached 830,000 worldwide. The number of new cases of liver cancer is predicted to reach 1 million per year by 2025 [2].

For patients with intermediate and advanced liver cancer who are not eligible for surgical intervention or are not suitable for locoregional therapies, the survival benefits of other treatments are relatively limited. Thus, other treatment strategies need to be integrated, including transcatheter arterial chemoembolization, radiation therapy, chemotherapy and targeted therapy [3].

Given the limited efficacy of classical radiation therapy and chemotherapy, molecular targeted therapy is a promising new area of liver cancer treatment, especially for advanced liver cancer. With the development of biomedical technology, targeted drug therapy has become a hot topic in tumor treatment in recent years. Multitarget kinase inhibitors, immune checkpoint inhibitors [4,5], antiangiogenic molecular targeting drugs [2,6], mTOR signaling pathway-specific inhibitors [7] and other drugs with different molecular targeting [8,9] have been discovered and applied clinically. These targeted drugs have brought new hope to patients. For the treatment of liver cancer, sorafenib [10], atezolizumab [11], bevacizumab [12] and sindilizumab [13] have been approved for the treatment of liver cancer, but some of them have serious adverse effects which seriously affect their clinical applications [14,15]. With many diagnostic and prognostic biomarkers found in liver cancer [16], new targeted drugs are expected for the treatment of liver cancer.

Recently, IGF/IGF-1R signaling has become a potential target for hepatocellular carcinoma (HCC) treatment. It is reported that IGF/IGF-1R signaling mediates cell proliferation, cell survival, migration and protein synthesis and can block apoptosis by expressing Myc and Akt1 in the liver, thereby causing invasion and metastasis of tumor cells [17]. An IGF-1 binding to IGF-1R signaling pathway could activate the Ras-MAPK and PI3K pathways [18] to inhibit tumor proliferation in various types of cancer [19]. IGF-2 also binds to IGF-1R and may lead to cancer cell proliferation in HCC patients [20]. Thus, as the receptor for both IGF-1 and IGF-2, IGF-1R is a potential target for HCC therapies. In addition, IGF-1R is reported to be overexpressed in liver cancer cells where the IGF signaling pathway plays a critical role in cancer development and progression [21,22]. The expression level of IGF-1R is significantly higher in human liver cancer tissues than in paraneoplastic tissues [23], indicating that altered IGF-1R expression is closely related to the occurrence and development of primary liver cancer. In nude mice bearing orthotopic xenografts of human liver tumor cells, tumor growth in the liver was decreased by shRNA-IGF1R, resulting in prolonged overall survival of treated mice [24]. Therefore, the inhibition of IGF-1R and its downstream signaling pathways in liver cancer cells is considered an ideal strategy for liver cancer treatment.

To expand the range of interference with the target gene expression, RNA-based therapies, including small interfering RNAs (siRNA or shRNA), antisense oligonucleotides (ASOs), microRNAs, aptamers, circular RNAs, messenger RNAs and guide RNAs (CRISPR/Cas-based gene editing) offer unique opportunities for treatments [25]. Among them, ASOs are the senior antisense technology, with current rapid development in the investigation and approval of drugs [26]. CT102 is an ASO of 20 nucleotides in length that targets IGF-1R mRNA. Preclinical studies have demonstrated that CT102 inhibited tumor growth in HepG2 hepatoma cells and xenograft nude mice in a sequence-specific and dose-dependent manner [27]. Subsequently, CT102 was further verified to have an antitumor effect by inhibiting IGF-1R expression specifically in an orthotopic transplant model of HCC xenografts in nude mice [28]. Another in vivo study has shown that CT102 could also inhibit recurrence and metastasis of liver cancer after hepatectomy [28]. These studies have shown the antitumor potential of CT102 in liver cancer treatment, which provides the basis for the new drug investigation and development of CT102.

In this study, preclinical pharmacodynamic and pharmacokinetic studies of CT102 were performed to provide a basis for the clinical trials, and a phase I clinical trial was conducted to evaluate the safety and tolerability of CT102 following administration of a single intravenous dose in patients with primary liver cancer.

Materials & methods

CT102 was chemically synthesized by Kelaiying Life Science Technology Co., Ltd (Tianjin, China) as a freeze-dried powder and stored at 2–8°C. The freeze-dried powder was first dissolved in 0.9% saline to prepare a working solution, and then the dosage was determined according to the body weight of animals. HepG-2 orthotopic xenograft mice would receive 3.75, 7.5 and 15 mg/kg of CT102 for treatment; rhesus monkeys would receive 1.5, 5 and 15 mg/kg of CT102; and SD rats would receive 3, 10 and 30 mg/kg of CT102 for pharmacokinetic analysis. All experiments were approved by the ethics committee of the Fifth Medical Center of Chinese PLA General Hospital.

HepG-2 orthotopic mouse xenograft model & in vivo treatment

The HepG-2 hepatoma cell line was cultured in Dulbecco’s modified Eagle medium (Hyclone, Beijing, China) supplemented with 10% fetal bovine serum (Solarbio, Beijing, China) at 37°C in a humidified incubator with 5% CO₂ for proliferation. BALB/c nude mice (female, body weight 20–22 g) were purchased from Weitonglihua Co., Ltd (Beijing, China). Mice were held five or six per cage (individually ventilated) with sterilized food and pure water at room temperature under a constant 12 h light/dark cycle.

Next, the cultured HepG-2 cells were digested into a suspension for implantation and 2 × 10⁶ HepG-2 cells were subcutaneously inoculated in the axilla of the right forelimb. Once the tumors had grown to 1–2 cm (>1000 mm³), they were isolated, homogenized to 1.0 mm³ and transplanted into the livers of mice. The mice were anesthetized by 2% isoflurane and secured for the surgery. After disinfection of the abdominal skin, a 1 cm incision was made in the upper right abdomen to expose the liver. The 1.0 mm³ tumor was then carefully inserted into a specialized inoculation cannula needle and implanted into the liver. Disinfectant gauze was used for bleeding management. Following the surgery, the abdominal muscles and skin were sutured sequentially with a 4/0 surgical suture needle.

After 3 weeks, 40 mice with similar tumor sizes were selected based on abdominal ultrasonography and randomly divided into five groups (eight mice for each group). The control group was untreated, while the other three groups were injected with CT102 through the tail vein at doses of 3.75, 7.5 and 15 mg/kg on days 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. The sorafenib group was intragastrically administered a dose of 20 mg/kg once daily for 20 days. After the last administration, the mice were sacrificed and the tumors were removed and weighed.

For assessing the tissue distribution of CT102, a single dose of 15 mg/kg was administered via the tail vein to the orthotopic xenograft mice. A total of 36 mice were divided into six groups, which were euthanized at 15 min and 1, 6, 24, 48 and 72 h to harvest the samples of tissues. Urine and excrement samples were collected until 72 h after administration. Plasma, heart, liver, spleen, lung, brain, muscle, fat, marrow, tumor and gonads were collected. The blood sample was collected into an anticoagulant tube on ice and centrifuged at 4°C, 1500 × g for 10 min. The resulting supernatant was collected and preserved as a plasma sample, stored at -70°C for further analysis. The dissected tissues were immediately placed into cryovials and frozen in liquid nitrogen, then stored at -70°C. Before analysis, the frozen tissues were thawed in pre-chilled PBS for homogenization, and subsequently centrifuged at 4°C, 10,000 × g for 10–20 min. The resulting supernatant was collected for further analysis.

Injection of CT102 in rhesus monkeys & rats

18 rhesus monkeys (nine male and nine female, body weight 4.2–5.3 kg) were purchased from the Institute of Xieerxin Biology Resource (Beijing, China). They were divided into three groups (six animals in each group; three male and three female) which were intravenously administered with CT102 as a single dose at 1.5, 5 and 15 mg/kg, respectively. Blood samples were collected before treatment (0 min), at 10, 20, 30, 35 and 40 min and at 1, 1.5, 2, 3, 4, 6, 8, 10, 24 and 48 h after injection. In the low-dose group, another four doses were administered every other day, and blood samples were collected after the fifth dose as above. Blood samples were collected from the saphenous vein of the rhesus monkeys (0.5–1.0 ml each time) into an anticoagulant tube on ice. After centrifuging at 4°C, 1500 × g for 10 min, the plasma was collected and stored at -70°C until further analysis.

18 Sprague Dawley rats (nine males and nine females, body weight ~0.3 kg) were purchased from Weitonglihua Co., Ltd and divided into three groups (six animals in each group; three male and three female). CT102 was administered as a single dose via the tail vein at doses of 3, 10 and 30 mg/kg. Blood samples were collected at 0 min (before treatment), at 5, 10, 20, 30, 35 and 40 min and 1, 1.5, 2, 3, 4, 6, 8 and 24 h after injection from the retro-orbital sinus of the rats (0.15–0.25 ml each time). The processing of blood samples was the same as described above.

Measurement of CT102 & pharmacokinetic analysis

The concentration of CT102 in the collected samples was measured using an enzyme-linked bridging assay. In brief, the CT102 present in biological samples was immobilized using a capture probe that complemented the CT102 sequence at the 3′ end. A detection probe was then connected to the hybridization complex, with the 5′ end complementing the capture probe and the 3′ end coupled to an enzyme-labeling color system, which was achieved through ligation to establish a bridging system. S1 is an endonuclease that specifically targets single-stranded nucleic acids and can either degrade a single strand in a double-stranded nucleic acid or cut double-stranded DNA at the gap [29]. In the aforementioned bridging system, the enzyme can remove the differential CT102 sequence, which cannot be connected to the detection probe to form a complete double-stranded sequence (i.e., the degradation product of CT102 has more than one base notch between the 3′ end and the 5′ end of the detection probe), to ensure the sequence length specificity of the bridging system. Finally, CT102 was quantified by using the 3′ end-coupled enzyme color development system.

The original data were processed using Origin v.7.5 software ( www.originlab.com/ftp/dist/origin75sr7/Origin7.5SR7.exe), and a standard curve was generated using a four-parameter logistic regression model (weighting method: statistical). The concentration of each sample was then calculated using the parameters obtained from the standard curve. During the calculation, the response value of each sample needs to be subtracted from the blank.

Any individual blood drug concentration below the lower limit of quantification (15.625 ng/ml) was marked as being below the quantification limit and assigned a value of 0 for calculation.

The pharmacokinetic parameters were calculated using a noncompartmental model with WinNonLin v 5.2.1 software (Pharsight Corp., Inc., CA, USA). Individuals with plasma drug concentrations below the lower limit of quantification were excluded from the pharmacokinetic calculation.

Clinical trial participants

Signed informed consent was obtained from all participants. This human clinical trial was registered at www.chictr.org.cn (ChiCTR2100044235). Patients with advanced liver cancer were screened according to the inclusion and exclusion criteria.

The ten inclusion criteria for the participants in this study were as follows: subjects aged ≥18 years with advanced liver cancer; Barcelona Clinic Liver Cancer stage C or B; subjects who were refractory to standard treatment; at least one measurable target lesion according to Response Evaluation Criteria In Solid Tumors v.1.1; appropriate organ and hematopoietic function is available; routine blood counts showing absolute neutrophil count ≥1.5 × 10⁹/l, platelets ≥75 × 10⁹/l and hemoglobin ≥90 g/l; biochemical tests showing serum total bilirubin ≤two-times the upper limit of normal (ULN), serum creatinine ≤1.5-times the ULN, alanine transaminase (ALT) and aspartate transaminase (AST) ≤2.5-times the ULN in the absence of liver metastases or ALT and AST ≤five-times the ULN in the presence of liver metastases; coagulation results showing prothrombin time, thrombin time and activated partial thromboplastin time within the normal range (±15%); an Eastern Cooperative Oncology Group (ECOG) physical performance status score of 0–1; and an expected survival time of at least 3 months.

The 19 exclusion criteria for the participants in this study were as follows: serious coagulation or hemorrhagic diseases in the past or present; anticoagulation or thrombolytic therapy within 4 weeks before the start of treatment; cachexia or multiorgan failure; nephrotic syndrome or moderate-to-severe proteinuria; severe cardiac disease; uncontrolled hypertension; resting dyspnea or requiring oxygen therapy support due to progressive malignancy or other disease; had received first-line or second-line treatment for liver cancer within 4 weeks; malignancies other than liver cancer; HIV infection; major surgery or trauma within the last 4 weeks without full recovery; toxic reactions after anticancer therapy that had not returned to grade 0 or 1 levels (except for alopecia); received a liver transplant; participated in other clinical studies within 4 weeks before the start of the trial; allergic individuals with known hypersensitivity to the components of the test drug or a history of substance abuse or alcohol addiction; pregnant or lactating people; those with childbearing potential who were unable or unwilling to use effective contraception during and 6 weeks after the end of the trial; any other comorbidities that might interfere with the study or evaluation, or may result in an individual being at increased risk during the study in the opinion of the investigator; and those who were believed by the investigator to be unable to comply with the requirements of the clinical trial scheme and have poor compliance to complete the trial.

The exclusion criteria including subjects with severe cardiac disease and uncontrolled hypertension were combined and modified as subjects with uncontrolled or significant cardiovascular disease, including: New York Heart Association class II or higher congestive heart failure, unstable angina, myocardial infarction within 6 months, and arrhythmia requiring treatment with left ventricular ejection fraction <50% at screening; primary cardiomyopathy; history of clinically significant QTc interval prolongation or screening QTc interval >470 ms in women and >450 ms in men; symptomatic coronary heart disease requiring pharmacological treatment and hypertension (systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg) not well controlled with antihypertensive medication.

Phase I trial design

This open-label, nonrandomized phase I study followed a two-stage dose-escalation scheme (single subject and traditional 3 + 3 design) to determine the dose-limiting toxicity (DLT) in subjects with liver cancer. Dose escalation was determined using a modified Fibonacci method. The initial cohort was at a dose level of 0.33 mg/kg; there were dose escalations of 100% for the second cohort (0.66 mg/kg), 100% for the third cohort (1.32 mg/kg), 100% for the fourth cohort (2.64 mg/kg), 67% for the fifth cohort (4.40 mg/kg), 50% for the sixth cohort (6.60 mg/kg) and 33% for the seventh cohort (8.80 mg/kg). For the dosages of 3.52 and 3.96 mg/kg, a 33 and 50% dose escalation from 2.64 mg/kg could be attempted if DLT occurred. Among them, the 0.33 and 0.66 mg/kg dose groups were in the ‘accelerated titration’ phase. One subject was enrolled in each dose group; if one DLT occurred, the test was stopped, otherwise the test was escalated to the next dose group. Starting with the 1.32 mg/kg dose cohort, we conducted a ‘3 + 3’ dose-escalation study. At this stage, if one subject experienced DLT during the DLT assessment window, that cohort would be expanded to six subjects; if two or more subjects experienced DLT, further enrollment at that dose level and dose escalation would be ceased, and the previous dose level was considered the maximum tolerated dose (MTD); otherwise, the trial continued.

Two or more subjects were prohibited from enrolling in each dose group simultaneously. The new subject would not be enrolled until there was a determination that the prior subject had no serious adverse reactions and the subject’s DLT observation was completed.

Method of administration & visit procedure

CT102 was administered as a single dose via intravenous drip. The final concentration of CT102 should not exceed 1.0 mg/ml and should be administered over 120 min by intravenous intermittent infusion.

Safety was evaluated before administration, up to 120 h after dosing and 1 week after discharge (day 13 ± 2). Safety – the primary objective – was assessed using vital signs, physical examination, ECOG performance status, laboratory tests, electrocardiography, echocardiography and the incidence of adverse events (AEs). AEs were graded using the Common Terminology Criteria for Adverse Events, version 5.0 [30].

Data analysis

Measurement data are generally described as means, medians, standard deviations, coefficients of variation, maxima and minima. Count data or grade data are expressed as frequencies. SAS version 9.4 (SAS Institute, Inc., NC, USA) was used for all statistical analyses.

Results

In vivo activity & tissue distribution of CT102 in orthotopic mouse xenograft model

The HepG2 orthotopic xenograft mice were administered with CT102 every other day for 10 doses; none of the mice died during the 20-day treatment period. CT102 suppressed tumor growth at all three doses and was comparable to sorafenib in inhibitory activity at low doses (71.9 vs 72.2%, respectively; Figure 1 & Table 1). However, the medium- and high-dose groups showed an improvement in inhibition rates (77.8 and 83.9%, respectively). These results verified the antitumor effect of CT102 in a dose-dependent manner. The tissue distribution of CT102 was then assayed as follows.

After a single intravenous administration of CT102, the area under the curve (AUC) for each tissue was measured (Table 2).The concentration of CT102 was initially high in the plasma, but rapidly decreased and was distributed to other tissues through circulation. The AUC of each tissue was observed in descending order: liver, kidney, spleen, muscle, heart, fat, bone marrow, tumor, gonads, plasma, lungs and brain. At 24 h, the serum CT102 level decreased to below the detection limit (Figure 2).

CT102 was most abundant in the liver and kidney. The highest level was detected in the liver at 1 h and was maintained at a high level thereafter until 72 h, when the level was similar to that at 15 min. The concentration of CT102 in the orthotopic xenograft tumors was much lower than that in the liver (5–10%). This suggests that the blood supply and drug uptake in hepatoma cells differ from those in hepatocytes. Although the CT102 level in the tumor was low, it remained stable. At 48 h, the concentration of CT102 (1359.30 ng/g) in the tumor was more than half the highest level observed at 1 h (2464.55 ng/g). The stable level of CT102 in the tumor can be attributed to the hepatoma being submerged in a high concentration of CT102 in the liver, where drug access is easier than in other tissues. The lowest concentration of CT102 among all the observed tissues was in the brain, which may indicate that the drug has difficulty crossing the blood–brain barrier. In the prototype form, a small amount of the drug was eliminated through stool and urine.

Pharmacokinetics of CT102

After a single intravenous injection, CT102 was measured in the serum of rats and rhesus monkeys for up to 48 h. The plasma drug concentration, as indicated by the area under the plasma concentration–time curve (AUC_0-∞) and maximum concentration (C_max), was positively linked with the administration dose after a single dose of CT102 with doses of 1.5, 5 and 15 mg/kg in rhesus monkeys. The terminal half-life period (t_1/2) of CT102 was 4.2 ± 1.9 h for the low dose, and the terminal t_1/2 values for the medium- and high-dose groups were approximately 13 h (13.5 ± 1.5 and 13.5 ± 1.7 h, respectively). Because the low dose had a much lower t_1/2 than the medium and high doses, we assumed that the former dose had a better clearance rate than the latter (Figure 3A). Clearance values were 98.1, 63.3 (p < 0.05) and 76.7 ml/h/kg for the dosages of 1.5, 5 and 15 mg/kg, respectively. The medium dose had the lowest clearance.

Another four intravenous doses were administered to the 1.5 mg/kg CT102 group on alternate days. CT102 was measured after the fifth dose and compared with that after the first dose. The mean peak concentration variation was comparable between the last and the first administration. The AUC_0-∞, C_max and clearance values were also similar between the two groups. There was no significant change in the kinetic behavior, suggesting that the drug exposure in the body was relatively stable (Figure 3B). However, the t_1/2 and mean residence time of CT102 were significantly longer after the fifth dose (p < 0.001), at nearly twice as long as in the first dose, indicating a stable concentration in the tumor.

In rats, the pharmacokinetics of CT102 were similar to those in rhesus monkeys. According to AUC_0-∞ and C_max measurements, the plasma exposure to CT102 was dose dependent. Clearance was also significantly lower (p < 0.05) in rats administered a dose of 10 mg/kg than in those administered a dose of 3 or 30 mg/kg. By contrast, t_1/2 was significantly longer (p < 0.05) in rats receiving the medium dose of 10 mg/kg than in those receiving 3 or 30 mg/kg (Figure 3C). In rats and rhesus monkeys, the lowest clearance was attained at the medium dose, which led to the highest t_1/2.

Participants in phase I clinical trial

Based on preclinical studies, a phase I clinical trial was conducted with participants with advanced liver cancer. A total of 132 subjects signed an informed consent form for this trial, of whom 115 were excluded based on the aforementioned inclusion and exclusion criteria. Finally, 17 subjects were sequentially entered into the corresponding dose groups in order of increasing doses and received a single intravenous dose of CT102. One subject was enrolled in each of the 0.33 and 0.66 mg/kg dose groups, while three subjects were enrolled in each of the 1.32, 2.64, 4.4, 3.52 and 3.96 mg/kg dose groups. All 17 subjects completed the dosing and corresponding visits as planned, and no subjects withdrew from the trial (Figure 4).

Regarding the 17 subjects who participated in this trial, the male:female ratio was 16:1, the median age was 57 years (range: 35–79) and the median BMI was 24.4 kg/m² (range: 14.9–28.5). The ECOG score was 0 for six subjects (35.3%) and one for the other 11 subjects (64.7%). The Barcelona Clinic Liver Cancer stage analysis showed two subjects (11.8%) at stage B and 15 subjects (88.2%) at stage C. Among these subjects, the numbers of subjects with one, two and three lesions were 14 (82.3%), two (11.8%) and one (5.9%), respectively (Supplementary Table 1). The mean size of the target lesions was 85.9 mm. In addition, there were eight subjects with hepatitis B and two subjects with hepatitis C, among whom one had received antihepatitis treatment. Two subjects had undergone antitumor therapy, including immunotherapy (Table 3).

In total, 118 AEs were reported in 17 subjects (100%) during the trial (Supplementary Table 2). AEs with an incidence of >10% were reported as follows: abnormal laboratory tests (elevated AST, elevated C-reactive protein, elevated γ-glutamyl transferase, elevated interleukin levels, decreased white blood cell count, elevated ALT, increased procalcitonin, elevated conjugated bilirubin, decreased percentage of lymphocytes, decreased lymphocyte count, positive occult blood, increased blood bilirubin and increased neutrophil percentage), fever and hypertension. Most of these AEs are commonly observed adverse reactions in clinical studies of liver cancer or ASO drugs [31,32]. All subjects returned to their baseline levels with or without interventions.

The AE with the highest incidence was fever (7/17 subjects, 41.2%; Table 4). One of these subjects had a fever on day 6 after dosing, and the cause of the fever was a cold, which the investigator deemed was unrelated to CT102. The other six subjects (two in the 4.4 mg/kg dose group, three in the 3.52 mg/kg dose group and one in the 3.96 mg/kg dose group) were regarded by the investigator as having fever possibly related to CT102. All fevers started within 3 h of dosing and reached their highest level 3–6 h after the end of drug administration. The highest body temperature did not exceed 40°C. Because this clinical trial is the first of CT102 in humans and very limited reference information is available, based on the investigator’s judgment some subjects were administered interventions, including physical cooling, compound amobarbital injection, or Xinhuang tablets, for subject safety. Within 10–15 h after drug administration, all six subjects, with or without intervention, had normal body temperatures, and the fever did not recur.

In the highest dose (4.4 mg/kg) group, two subjects who were reported as having DLT had intermittent grade 3 hypertension (Table 4). Hypertension started after administration and was highest 2–6 h after dosing. The highest blood pressure measured was 166/108 mm Hg. Blood pressure recovered to baseline (within 10–20 h after dosing) after intervention with blood pressure medication (80 mg oral valsartan capsules or 30 mg oral nifedipine controlled-release tablets). Thus, two additional dose groups (3.52 and 3.96 mg/kg), were used to further investigate the MTD of single-dose CT102. No DLT occurred in the dose of 3.96 mg/kg, and so 3.96 mg/kg was regarded as the MTD for CT102.

In summary, a single dose of CT102 was escalated up to 4.4 mg/kg with a DLT of intermittent grade 3 hypertension, and a dose of 3.96 mg/kg was found to be tolerable. No grade 4 or higher AEs occurred during the two dosage treatments. None of the subjects discontinued the drug or withdrew due to AEs, and no severe AEs were reported. The most common AE was transient fever, and the body temperature would recover in a short time with or without intervention. These results indicate that CT102 has low toxicity and was proved safe for further investigation.

Discussion

In this study, the antitumor effect of CT102 was verified in orthotopic xenograft mice with hepatoma cells; the drug was distributed to tissues immediately after intravenous administration, with the liver having the greatest abundance of the drug. However, the CT102 level was lower in the orthotopic xenograft than in the liver. The organ uptake of ASOs is reported to be generally heterogeneous and cell type-specific [33]. This finding might be attributed to differences in blood supply, drug intake and affinity for CT102 between hepatoma cells and hepatocytes. In addition, the study showed that the t_1/2 of CT102 in the orthotopic hepatoma was longer than 48 h. This phenomenon can be explained by an equilibrium that is established following distribution between tissues [33]. In rats and rhesus monkeys, AUC and C_max increased dose-proportionally, whereas clearance was the lowest at the medium dose for both animals, leading to delayed t_1/2 and plasma concentration. Based on these results, the first human clinical trial of CT102 was conducted in patients with liver cancer and demonstrated the safety of the drug when administered intravenously, with acceptable tolerability.

In the clinical trial on liver cancer patients, fever was the most common AE, possibly related to CT102, with an incidence of 41.2% (7/17 patients). After excluding the risk factors for fever of unknown origins such as infection, malignancy, autoimmune and autoinflammatory conditions, stroke, endocrine disorders and conditions associated with substance use (e.g., alcohol withdrawal), we analyzed the related mechanism of CT102. CT102 is a thiol-modified oligonucleotide drug, and the literature has reported that some thiol modifications and unmethylated CpG sequences may cause immune reactions when thiol oligonucleotide drugs are used continuously or at excessive doses [34]; or the mammalian immune system recognizes unmethylated CpG as foreign DNA (viral or bacterial) and the Toll-like receptor signaling in immune cells is triggered, which may lead to the production of proinflammatory cytokines [35]. ASO use has been reported to result in nonspecific proinflammatory effects owing to enhanced binding to immune receptors [26], and fever has also been reported as an AE associated with ASOs [36,37]. We also monitored transient elevations in IL-6, C-reactive protein and procalcitonin levels in subjects with liver cancer. These subjects developed a fever; however, their corresponding pathogenic tests were negative. Therefore, this topic requires further attention in subsequent clinical trials.

In this study, two DLT events were reported in the highest-dose group (4.4 mg/kg), both of which were grade 3 hypertension. Among the marketed ASO drugs, hypertension has not been reported as a common AE or adverse reaction; however, other anti-IGF-1R drugs have been associated with the AE of hypertension in clinical trials [38,39]. Previous studies have demonstrated that IGF-1R mediates the biological effects of IGF-1, subsequently activating the downstream PI3K signaling pathway involved in cell proliferation [40], and disruption of the PI3K/Akt pathway is reported to be a contributor to hypertension [41]. Thus, the intravenous infusion of CT102 might affect the PI3K signaling pathway by the IGF-1/IGF-1R axis, increasing human blood pressure. The cause of hypertension requires further observation.

The observed fever and hypertension events were transient and were rapidly restored to baseline levels with pharmacological intervention. Therefore, from a risk/benefit perspective, we suggest that fever and hypertension should not be used as DLTs in subsequent studies of CT102.

This study has three main limitations: an insufficient number of subjects was assessed, the ratio of male to female participants was unbalanced and the observation period was too short. Thus, further research should aim to overcome those limitations.

Conclusion

The preclinical trials provided the basis for the phase I clinical trial, which indicated that CT102 has good tolerability and safety up to a dose of 3.96 mg/kg for liver cancer treatment. We plan to gradually perform safety, tolerability and human pharmacokinetics and pharmacodynamics studies in multiple-dose clinical trials of CT102 in the future.