A phase 0 study of the pharmacokinetics, biodistribution, and dosimetry of ¹⁸⁸Re-liposome in patients with metastatic tumors

An open-label, single-arm phase 0 trial was conducted at Taipei Veterans General Hospital (TVGH), Taipei, Taiwan, following the guidelines for exploratory IND study that were published by the US Food and Drug Administration (FDA) in 2006 [35] and approved by the Taiwanese FDA and the Institutional Review Board of the TVGH. All patients signed an informed consent document before participating in the study. The study was established to determine a safe starting dose for the phase I clinical trial and to obtain data for biodistribution, pharmacokinetics, radioactivity, and radiation dosimetry in patients who receive a single-intravenous bolus injection of ¹⁸⁸Re-liposome.

The major criteria for inclusion in the study were as follows: Patients had to have a pathologically confirmed diagnosis of a primary solid tumor and pathologically or radiologically documented metastases that were refractory to current standard/available therapies. Patients had to have a Karnofsky Performance Status of at least 60% (or eastern cooperative oncology group (ECOG) performance status (PS) 0 to 2) and an anticipated survival of at least 3 months. Patients had to have recovered from any adverse events due to prior treatment (grade 0 or 1 in the common terminology criteria for adverse events (CTCAE) 4.03). Patients’ laboratory data had to show: absolute neutrophil count > 1.5 × 10⁹/L; platelet count > 100 × 10⁹/L; hemoglobin > 90 g/L; AST (aspartate aminotransferase), ALT (alanine aminotransferase), and ALP (alkaline phosphatase) ≤ 2.5 × ULN (upper limit of normal) (or ≤ 5 × ULN if known hepatic metastasis); total bilirubin ≤ 1.5 × ULN; and serum creatinine ≤ 1.5 × ULN (or calculated creatinine > 50 mL/min). Patients who had been subjected to any chemotherapy, radiation therapy, immunotherapy, angiogenesis inhibitors, and/or surgical treatment within 4 weeks preceding study entry were excluded.

Between Jul 2012 and Dec 2013, 12 patients with metastatic cancers that were refractory to currently standard or available therapies received an injection of 111 MBq of ¹⁸⁸Re-liposome and then underwent imaging. Patients’ characteristics are summarized in Table 1. Fourteen patients received a ¹⁸⁸Re-liposome injection with a mean administrated activity of 111 MBq. All patients tolerated the injection well, and there was no evidence of a significant infusion reaction, anaphylaxis, or signs of distress. Two of the 14 eligible patients withdrew from the trial because of abnormal liver function or intolerable pain from a metastatic mass: patient 05 withdrew consent on day 3 after the administration of ¹⁸⁸Re-liposome due to deteriorating liver function and performance status. During a screening session, the ALT and ALP values for patient 05 were more than the ULN, which fulfilled the inclusion criteria for liver function for a patient with known hepatic metastasis (ALT and ALP ≤ 5 × ULN). However, a blood sample for patient 05 that was taken immediately before the injection of ¹⁸⁸Re-liposome on day 1 showed an increase in AST and ALT to more than 200 U/L and a further increase to more than 800 U/L was recorded on day 2. Due to the patient’s deteriorating performance status and ongoing liver failure, the patient withdrew consent and did not undergo imaging on day 3 or day 4. The patient had existing multiple liver metastases due to lung cancer and there had been a rapid progression of hepatic failure before the injection so ¹⁸⁸Re-liposome was not considered to be the cause of the deterioration in the condition of patient 05. Patient 06 withdrew consent after experiencing intolerable pain from a metastatic mass over the left axilla, extending to the upper back, while lying in a supine position during SPECT imaging. Therefore, data for dosimetry and biodistribution for these two patients is not included in the analysis. Two patients (patient 05 and 06) prematurely withdrew from the study without completing all of the imaging procedures, so 12 patients were included in the analysis of SPECT/CT imaging, pharmacokinetics, biodistribution, dosimetry, and safety of ¹⁸⁸Re-liposome. Patient characteristics are listed in Table 1.

¹⁸⁸Re was obtained from an alumina-based ¹⁸⁸W/¹⁸⁸Re generator. Elution of the ¹⁸⁸W/¹⁸⁸Re generator with normal saline yielded solutions of carrier-free ¹⁸⁸Re as sodium perrhenate (NaReO₄). A slightly modified form of the labeling procedure for ¹⁸⁸Re-liposome that is described in [25 and 28] was used. The lipid compositions of pegylated liposomes contained hydrogen soybean phosphatidylchloine (HSPC), cholesterol, mPEG₂₀₀₀-DSPE (molar ratio 3:2:0.3), and ammonium sulfate solution with 250 mM (NH₄)₂SO₄ (pH 5.0) in the inner water phase. Pegylated nanoliposomes have an average particle size of about 82.59 nm and contain 13.16 μmol/mL phospholipids. ¹⁸⁸Re-liposome was manufactured in compliance with PIC/S GMP. BMEDA (ABX, Germany). Stannous chloride was used as the reductant, and sodium gluconate was used as an intermediate ligand to produce ¹⁸⁸Re-SNS/S complexes. BMEDA (3.08 mg) was pipetted into a fresh glass vial, and sodium gluconate dissolved in 10% acetate solution was then added (0.34 mol/L). Stannous chloride was then added. When the solution had been flushed with N₂ gas, ¹⁸⁸Re-sodium perrhenate (0.9 mL) with a high specific activity (5.2~9.4 GBq/mL) was added. The vial was sealed and heated in an 80 °C dry bath for 1 h. The ¹⁸⁸Re-BMEDA solution was adjusted to a pH of 6.0, prior to liposome (5 mL) labeling. The liposomes encapsulating (NH₄)₂SO₄ were mixed and after-loaded with ¹⁸⁸Re-BMEDA solution and incubated in a 60 °C dry bath for 30 min. A PD-10 column (G-25, GE Healthcare) chromatography with normal saline was used to separate radiolabeled liposomes from free ¹⁸⁸Re-BMEDA, and a 0.22-μm filter was used to filter the solution. The ¹⁸⁸Re-liposome injection was formulated with normal saline. The specification for the concentration of phospholipid was 3~6 μmol/mL, for particle size was 80~100 nm, for zeta potential was − 3~2 mV, and for radiochemical purity was more than 90%.

The dose of ¹⁸⁸Re-liposome that was injected for this exploratory clinical study was determined based on previous animal studies [33] and the US FDA guidelines. In a study of ¹⁸⁸Re-liposome toxicity in rats, the no adverse effect level (NOAEL) was 185 MBq per 200-g rat. The recommended starting dose for exploratory IND is 1/100 of NOAEL, so following the guidelines for exploratory IND study published by the US Food and Drug Administration (FDA) in 2006 [35], 111 MBq was selected for the low radioactivity phase 0 study. On day one, patients received an intravenous (i.v.) bolus injection of 111 MBq of ¹⁸⁸Re-liposome and the time at which ¹⁸⁸Re-liposome was administered was established as 0 h for the study. Information on biodistribution and dosimetry was derived from SPECT/CT data that was collected at 1, 8, 24, 48, and 72 h after injection with ¹⁸⁸Re-liposome. Whole-body SPECT images were acquired over 60 min (1- and 8-h time points) or 90 min (24-, 48-, and 72-h time points) using a dual-headed SPECT/CT (Discovery™ NM/CT 670, GE Healthcare, Milwaukee, WS, USA) that was equipped with a medium-energy, general purpose collimator, for which the energy window was 155 + 10% KeV (139–170). Three bed positions (40 cm each) from head to thigh of SPECT/CT were performed for each time point set with a matrix size of 128 × 128 per image. The SPECT images were collected in step-and-shoot mode at 6° intervals over 180°. Sixteen-second projection views were obtained from each camera head. The images were reconstructed and analyzed using filtered back projection methods. Whole-body planar images were acquired at 8 cm/min from head to feet using the same machine. To identify the radioactivity level in tumors, major body organs, and muscle, volumes of interest (VOIs) were drawn based on CT images that were obtained within 24 h and merged with SPECT images using Velocity™ v2.8.1 (Velocity Medical Solutions, Atlanta, Georgia). VOIs for the muscle were placed over the left front thigh of each patient (Volume: 17.99 ± 2.66 ml). The VOI counts for normal and tumor areas at each time point were converted to a radioactivity level by multiplying factor 1 [36]. Factor 1 was calculated using the formula:

Factor 1 = A₁/whole-body VOI counts

A₀ and A₁, respectively, refer to the radioactivity of the ¹⁸⁸Re-liposome at injection time and the radioactivity at 1 h after injection. The results for biodistribution are presented as a percentage of the injected activity per kilogram (%IA/kg) for organs or tumors.

On the basis of the VOI counts for the whole body, major organs, and tumor obtained using Velocity™, the percentage of injected activity (%IA) for organs was calculated at various time points (1, 8, 24, 48, and 72 h after the injection of ¹⁸⁸Re-liposome) and the %IA value for major organs was entered into the OLINDA/EXM [37, 38] software (Vanderbilt University, Nashville, TN, USA) to determine the dose that is absorbed by the brain, skin, bone, spleen, kidney, heart, liver, lung, intestine (large/small), bladder, muscle, stomach, testes (male only), ovaries (female only), and pancreas.

To determine the dose that is absorbed for different sizes of tumor, the %IA/kg value for the tumor, obtained at various time points (1, 8, 24, 48, and 72 h after injection with ¹⁸⁸Re-liposome), was directly fitted with exponential models to calculate the number of disintegrations. This value was then input to the OLINDA/EXM computer program using the unit density sphere model [38].

From days 1 to 4 and during follow-up visits on days 9 to 16 and 28 to 30 post-injection, the safety of ¹⁸⁸Re-liposome was assessed by monitoring vital signs and checking for any indication of adverse events. Particular attention was given to infusion reaction, anaphylaxis, myelosuppression, or changes in hepatic or renal function. Blood samples were taken just before each SPECT scan at 1-, 8-, 24-, 48-, 72-h time points and at one extra time point (4 h) and during two follow-up visits for blood and plasma radioactivity analyses and hematological and biochemical studies. Urine samples were collected every 24 h to determine the daily and cumulative urinary excretion of ¹⁸⁸Re-liposome. The severity of adverse events was graded using CTCAE v4.03. Laboratory tests (hematology, biochemistry, and urinalysis) were conducted during the screening visit, which occurred less than 10 days before ¹⁸⁸Re-liposome was administered on day 1. These results were used as baseline data. Any adverse events (graded using CTCAE v4.03) or concomitant medications/therapies were recorded throughout the study using the case report forms (CRFs).

Urine samples were collected every 24 h to determine the daily urinary excretion of ¹⁸⁸Re-liposome. Radioactivity in urine was measured using a gamma counter at 24, 48, and 72 h after injection and the results are expressed as %IA/mL. In terms of pharmacokinetics, blood samples with anticoagulants were collected at 1, 4, 8, 24, 48, and 72 h and these were measured for radioactivity using the same gamma counter. The results are expressed as the percentage of injected activity per milliliter (%IA/ml). The pharmacokinetic parameters for ¹⁸⁸Re-liposome in the blood were determined using WinNonlin software version 5.3 (Pharsight Corp., Mountain View, CA, USA). Non-compartmental analysis (NCA) was used to determine the pharmacokinetic parameters, including the terminal elimination half-life (T_1/2z), the maximum concentration (C_max), the total body clearance (Cl), and the area under the curve (AUC).

The characteristics of patients are listed in Table 1. The specification includes the concentrations of phospholipid as previously described. The radiochemical purity of ¹⁸⁸Re-liposome was determined by running samples through PD-10 desalting columns containing Sephadex™ G-25 resin. The purity is 98.5 ± 1.3%, the mean specific activity is 63.2 ± 11.7 MBq/μmol (n = 12), the nanoparticle ¹⁸⁸Re-liposome size range is 83.7 + 11.4, and the zeta potential of the nanoliposome is − 1.1 ± 0.7 mV.

The clearance of ¹⁸⁸Re-liposome from the blood, plasma, and urine over the 72-h period of the study is shown in Fig. 1. Mean values for radioactivity in the blood and plasma of all patients decrease with time. At 1-h post-injection, the mean value for radioactivity is 0.012 ± 0.012%IA/ml in the blood and 0.017 ± 0.016%IA/ml in the plasma and this decreases to 0.002 ± 0.002%IA/ml in the blood and 0.002 ± 0.002%IA/ml in the plasma at day 4, post-injection. Urine samples were examined on days 2, 3, and 4, post-injection. The mean respective radioactivity values for the three examination times are 0.008 ± 0.005%IA/ml, 0.002 ± 0.001%IA/ml, and 0.002 ± 0.001%IA/ml, so the radioactivity level in urine also decreases over time, after injection.

The pharmacokinetic parameters are listed in Table 2. The maximum respective concentrations (C_max) of ¹⁸⁸Re-liposome in blood and plasma are 0.01 ± 0.01%IA/mL and 0.02 ± 0.02%IA/mL. The respective clearance rates (Cl) for ¹⁸⁸Re-liposome from blood and plasma are 749.40 ± 663.19 mL/h and 494.26 ± 447.01 mL/h. The respective area under the time curve (AUC_{0 → ∞}) for ¹⁸⁸Re-liposome in blood and plasma is 0.33 ± 0.32% IA/g*h and 0.50 ± 0.46% IA/g*h. The respective values for T_1/2z (terminal elimination half-life) for ¹⁸⁸Re-liposome in blood and plasma are 36.73 ± 14.00 h and 52.02 ± 45.21 h.

The urinalysis showed no clinically significant abnormalities that are related to the drug during the study period, with the exception of Patient 12, who showed a significant abnormality in the urine parameters, with significant proteinuria, hematuria, and leukocytes between day 2 post-injection and the last observation visit. In this case (Pt. 12), hematuria was only discovered at the screening visit and became clinically significant on day 2, post-injection. Although hematuria was identified as a SAE (serious adverse event), this adverse event appears to be unrelated to the study drug. No clinically significant abnormalities that are related to the ¹⁸⁸Re-liposome injection are noted.

Figure 2 shows the transverse SPECT/CT images for patient 3, who exhibited hepatocellular carcinoma and multiple tumor masses over the liver, lung, and mediastinum. The patient had previously received transarterial chemoembolization (TACE), which left a region with hyperdense lipoid and a central necrotic area. Several necrotic lesions show as hypodense in the CT image and are less radioactive. Some viable tumor masses form a rim-like structure that surrounds the necrotic regions. Patient 14, who had a nasopharyngeal tumor (NPC) over the left nasal cavity, shows a high uptake and targeting (3.99%IA/kg) area at 24 h after injection with ¹⁸⁸Re-liposome, which corresponds with the soft tissue lesion (blue arrow) that is seen on the corresponding MRI (Fig. 3a, b). Anterior planar images of patient 14 were obtained at different time points after injection with ¹⁸⁸Re-liposome and show a discernible uptake of radioactivity in the left nasopharyngeal region (Fig. 3c).

The uptake of ¹⁸⁸Re-liposome and tumor targeting measured using Velocity™ imaging software was used to calculate biodistribution of ¹⁸⁸Re-liposome (%IA/kg) in normal organs for 12 patients from 1 h to 72 h. The results are listed in Table 3 for the spleen, kidney, heart, liver, lung, and muscle. The most prominent activity is noted in the spleen and liver at 1 h post-injection, but this decreases with time. The biodistribution of ¹⁸⁸Re-liposome for the highest values for tumor targeting and uptake at each time point for 12 patients are summarized in Additional file 1: Table S1.

Normal organ assessment for the 12 subjects shows that the liver has the greatest biodistribution at 1 h post-injection (14.7 ± 5.4%IA/kg), followed by the spleen (13.2 ± 8.0%IA/kg) and lung (9.4 ± 2.3%IA/kg). Radioactivity in normal organs peaks at 1 h post-injection and then decreases slowly. The differences in tumor targeting and the uptake of radioactivity for the bone, pelvis, lung, liver, bladder, and NPC tumors are summarized in Additional file 1: Table S1.

Using the biodistribution results for normal organs and tumors in Table 3 and Additional file 1: Table S1, the dosimetry of the absorbed radiation dose after intravenous injection of ¹⁸⁸Re-liposome (111 MBq) was calculated. The results in Table 4 show that normal organs that absorb high doses include the spleen (1.38 ± 1.81 mGy/MBq), liver (0.92 ± 0.35 mGy/MBq), lungs (0.58 ± 0.28 mGy/MBq), red marrow (0.06 ± 0.01 mGy/MBq), and kidneys (0.32 ± 0.09 mGy/MBq). It is seen that the doses that are absorbed by these normal organs were much lower than the vital organ maximum tolerance doses.

The dose that is absorbed by tumor lesions because of the intravenous injection of ¹⁸⁸Re-liposome (111 MBq) was then calculated. The highest doses of ¹⁸⁸Re-liposome that are absorbed by tumor lesions for 12 patients are listed in Table 5. The highest dose that is absorbed in a liver tumor lesion is 0.86~1.70 mGy/MBq. Lung tumors (0.21~1.04 mGy/MBq) and bone tumors (0.12~0.28 mGy/MBq) also absorb higher doses than other tumor lesions.

The results for complete blood count (CBC) and the biochemical data for blood are shown in Additional file 1: Figure S1 and Figure S2. The mean hemoglobin level, RBC and lymphocyte counts for the 12 subjects are within a normal or near-normal range throughout the study. No clinically significant abnormalities are seen. No serious adverse reaction is noted for any of the participants in the study.

The exploratory study determines the effectiveness with which tumors are targeted and the pharmacokinetics, biodistribution, and dosimetry of ¹⁸⁸Re-liposome but a study of patients’ subsequent imaging results and survival times (Table 1) shows that at least two patients exhibit a therapeutic response (Pt. 11 and Pt. 14). Patient 11 had esophageal cancer with progressive multiple lung metastases, as shown in Fig. 4. Within 4 months after injection with ¹⁸⁸Re-liposome, during which time, the patient received no other therapy, and the follow-up CT sows cavitation of the metastatic lung lesions. Patient 14 experienced NPC with multiple lung metastases. The results of the nasopharyngoscopic examination show a NPC image with engorged blood vessels and irregular surface prior to the injection of ¹⁸⁸Re-liposome, but significant ¹⁸⁸Re-liposome uptake in the corresponding site (Fig. 3a, b).