Synthesis, identification and in vivo studies of tumor-targeting agent peptide doxorubicin (PDOX) to treat peritoneal carcinomatosis of gastric cancer with similar efficacy but reduced toxicity

PDOX was successfully synthesized according to the previously reported 7-step chemical process (Figure 1) [16‐18].

PDOX (Ac-Phe-Lys-PABC-Dox · HCl) was a red solid powder with molecular weight of 1046.51 (MS: m/z calculated for C₅₂H₆₀ClN₅O₁₆: 1046.52, found: 1046.50), chemical purity of 99.1% (by HPLC), structure of C₅₂H₆₀ClN₅O₁₆ (by both of H¹-NMR and C¹³-NMR) (Figure 2) and melting point of 180°C (decomposition), which is stable at -5°C to 0°C for 36-54 months, ambient temperature for 24 months, solvable in water, partially solvable in methanol and ethanol. PDOX used in this study was stored in the dark, dry area at 4°C.

Rabbit model of gastric PC was established in all animals (100%, 40/40). On d 8 after tumor cells inoculation, small, hard and transparent tumor nodules developed on the greater omentum, and typical ulcerative cancer about 0.5-1.0 cm in diameter formed on the antrum of the stomach. No ascites was observed. No obvious PC was found in other regions. There were no differences in the PC severity among all the rabbits. This could be equivalent to clinical stage I PC by Gilly criteria [22] (Figure 3A).

All investigated tumor specimens showed extensive invasive growth and tissue destruction. The tumors, on the greater curvature of the gastric antrum, penetrated the mucosal layer to form ulcers. Histopathological study showed tumor nests penetrating the entire stomach wall, with typical invasion into the muscle layer and the gastric glands (Figure 3B). The tumor cells are round, oval or atypical morphology with many pathological mitotic figures. There were also conspicuous infiltration of lymphocytes, plasma cells and other inflammatory cells (Figure 3C). Apoptotic and necrotic tumor cells were observed in the central region of the tumor nodules (Figure 3D). Typical PC presented as tumor nodules on the surface of the omentum (Figure 3E) and intraperitoneal lymph node metastases were also observed (Figure 3F).

Typical ulcerative cancer with PC was observed in post mortem pathological examinations of rabbits in the Control. The stomach wall was totally invaded by the tumor to create cancer ulcer encased by confluent nodules on the greater omentum, forming a big tumor block. The abdominal wall and diaphragm were totally invaded by the tumor. Many tumor nodules formed on the intestinal wall, the mesentery and the retroperitoneum. Bloody ascites could be more than 100 mL. All the features are similar to the clinicopathologic characteristics of gastric PC in patients (Figure 3G).

Animal was observed to record OS. Two long surviving rabbits (one in the PDOX and another in the DOX, living more than 100 d) were euthanized on the 100th day by an overdose injection of 2% pentobarbital sodium. The median (95% confidence interval, CI) OS was 23.0 d (19.9 - 26.1 d) in the Control, 41.0 d (36.9 - 45.1 d) in the HIPEC, 65.0 d (44.1 - 71.9 d) in the PDOX and 58.0 d (39.6-54.4 d) in the DOX. Compared with the Control, the OS was extended by at least 70% in the HIPEC (p < 0.001, log rank test). Compared with the HIPEC, the OS was further extended by 40% in the DOX (p = 0.029, log rank test) and by 58% in the PDOX (p = 0.021, log rank test) (Figure 4A).

At the study endpoint, the tumor burden in the Control was heaviest, with tumor weight of (137.51 ± 16.09) g, tumor-bearing ratio (tumor weight to body weight) of (7.88 ± 0.85)%, bloody ascites of (65.50 ± 33.45) mL, and ePCI score of (9.50 ± 2.17). Compared with the Control, the HIPEC had significantly reduced tumor weight (76.50 ± 11.41) g (p = 0.007) and tumor-bearing ratio (3.94 ± 0.54)% (p = 0.001), but not ePCI score (9.50 ± 2.17)% (p = 0.420) and bloody ascites [(29.16 ± 15.30) mL, p = 0.085]. Compared with the HIPEC, PC severity was further decreased in both the PDOX and the DOX, with ePCI score (6.40 ± 2.07, p = 0.020 in both PDOX and DOX vs. HIPEC) and bloody ascites (0.00 mL, p < 0.001 in PDOX and DOX vs. HIPEC); while there were no significant differences in tumor weight [(65.30 ± 14.55) g, p = 0.552, PDOX vs. HIPEC; (48.74 ± 16.31) g, p = 0.180, DOX vs. HIPEC)] and tumor-bearing ratio[(2.91 ± 0.69)%, p = 0.552, PDOX vs. HIPEC; (2.13 ± 0.78)%, p = 0.180, DOX vs. HIPEC]. There were no significant differences in tumor burden between the PDOX and the DOX (Table 1).

The body weight of each animal was recorded every 4 d. No significant differences were found in initial body weight of 4 groups before the treatment. Perioperative body weight decreased in all groups because of the overnight fasting. In the Control, the body weight recovered once food intake was resumed but again decreased progressively till the study endpoint. In the 3 treatment groups, postoperative body weight decreased considerably after model construction and to the lowest 4 d after CRS + HIPEC, and then stayed at a low level during the post-operative phase until chemotherapy had been completed. The body weight began to increase at d 44 in the HIPEC and d 56 in both the PDOX and the DOX, and gradually increased to a higher level, which was related to both tumor growth of the living tumor-bearing rabbits and death of the failure rabbits with lesser body weight. Thereafter, body weight decreased progressively again until the study endpoint in both the PDOX and the DOX (Figure 4B).

By the time of animal death, detailed information on postmortem pathological examinations was listed in Table 2.

In addition to systematic examinations of all anatomic sites for possible cancer metastases, particular attention was paid to lung and liver metastases. As shown in Figure 5, in the Control, extensive liver metastases were observed in 8 animals, but no pulmonary metastases were evident in all animals, because of the very aggressive growth behaviors of the tumor led to short OS in this group of animals (Figure 5 A2, B2). In the HIPEC, as animals lived much longer because of CRS + HIPEC, fewer liver metastases were observed in 4 animals, but considerably much more pulmonary metastases were observed in 7 animals (Figure 5 A3, B3). In the PDOX and DOX, animals developed much fewer metastases in both the livers and the lungs, although the OS was further improved remarkably, because the animals received not only CRS + HIPEC to control local regional metastases but also systemic chemotherapy to control hematogenous metastases and lymphatic metastases (Figure 5 A4, B4, A5, B5).

There were no differences in peripheral blood cell counts on d 2, d 6 and d 14 among the 4 groups, but the white blood cells and platelets were significantly decreased on d 36 after systemic chemotherapy in the DOX (p < 0.05). There were no differences in the hepatic and renal functions throughout the experiment among the 4 groups. In terms of cardiac functions parameters, there were no differences in creatine kinase and creatine kinase mb isoenzyme on d 2, d 6 and d 14 among the 4 groups, but creatine kinase mb isoenzyme was significantly increased on d 36 after systemic chemotherapy in the DOX (p < 0.05). Of particular attention, 2 animals in the DOX with short OS of 47 d and 53 d had exceptionally higher serum creatine kinase mb isoenzyme levels, 467.5 U/L and 656.4 U/L, respectively (p < 0.05) (Table 3).

Histopathological studies under both light microscope and transmission electron microscope found no myocardium toxicities in the Control, the HIPEC and the PDOX, but significant histopathological changes of myocardium were found in all animals (100%, 10/10) in the DOX, including coagulation necrosis of the myocardium with deep red stained cytoplasm, and the karyopyknosis (Figure 6 C1, H1, P1, D1 black arrows). Electron transmission micrographs showed that normal myocardium with abundant mitochondria packed around normal nucleus in the Control and HIPEC, rich normal but with slightly dilated cristae mitochondria scattered around normal nucleus in the PDOX, nearly absent mitochondria around the morphologically senile and degenerative nucleus in the DOX. In addition, partial myocardium-lytic necrosis (yellow arrow) and mitochondria-pyknosis away from the nucleus (red arrow) were also evident in the DOX. Complete, clear and continuous lines of normal intercalated discs in the Control, the HIPEC and the PDOX, but obscure, loosing and discontinuous intercalated discs were found in the DOX (white arrows).

All reactions were carried out in dried flasks under an atmosphere of dry nitrogen unless otherwise specified. The reactions monitored either on HPLC or TLC for completion. All reagents and solvents used were used as received without further purification unless otherwise stated. Chemical purity of the intermediates and final compound was determined on Agilent 1100 series instrument. LC-MS was performed on Agilent 1100 instrument with Zorbax column (4.6 × 150 mm) eluted with 0.1% trifluoroacetic acid water/0.1% trifluoroacetic acid acetonitrile at 1 mL/min flow rate. Column chromatography was performed on silica gel 60 (40-63 μm). Nuclear magnetic resonance (NMR) spectra were recorded on a Varian 300 instrument operating at 300 MHz (¹H) or Bruker AC 500 instrument operating at 500 MHz (¹H), 125 MHz (¹³C). Chemical shifts δ were reported in parts per million (ppm) from tetramethylsilane (TMS) as internal standards (0.00 ppm). Abbreviations for signal coupling are as follows: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, m = multiplet. High resolution mass spectrum (ES, positive) was determined on a Thermo LTQ FT Ultra Mass Spectrometer.

PDOX was synthesized according to the previously reported chemical process [16‐18], with the following 7 major steps.

Apart from PDOX described above, other agents were obtained from commercial sources, including DOX hydrochloride for Injection (Pharmacia, Milan, Italy), Docetaxel (Wanle Pharmaceuticals Co., Ltd. Shenzhen, China) and Carboplatin (Qilu Pharmaceuticals Co., Ltd. Shandong, China).

The animal study protocol was approved by the Animal Welfare Committee of Wuhan University, complied with the Helsinki Declaration. The animal study was completed in the Animal Biosafety Level 3 Laboratory at the Animal Experimental Center of Wuhan University (Animal Study Certificate ID: No.00022310).

Forty male New Zealand white rabbits, body weight between 2.5-3.0 kg, were obtained from Animal Biosafety Level 3 Laboratory at the Animal Experimental Center of Wuhan University (Approval ID: SCXK 2008-0004). The animals were individually housed and allowed free access to standard laboratory food and water as well as 12 h of light and dark cycle per day.

Rabbit VX2 carcinoma was used to establish GC with PC in this study. The VX2 tumor is a transplantable rabbit squamous cell carcinoma, characterized by rapid tumor growth and early metastasis, established from a virus-induced papilloma by Rous and coworkers [43]. The tumor was maintained by successive in vivo transplantation into the hind leg of a carrier rabbit used for every passage.

When the VX2 tumor grew to about 2 cm in diameter on the carrier rabbit, the animal was anesthetized by ear vein injection of 2% pentobarbital sodium (30 mg/kg). After skin preparation and disinfection, the tumor was excised from the carrier rabbit and placed in icy cold 0.9% sodium chloride solution. Tumor tissue was minced into approximately 1.0-2.0 mm³ fragments and suspended in 2 mL of normal saline, then drawn into a 2 mL injector. Other tumor tissues about 3.0-5.0 mm³ were placed into the homogenizer embedded in ice bath, to which 3 mL of icy cold normal saline was added, and the tumor cells suspension was made, with the tumor cells concentration adjusted to 5 × 10¹⁰ vial cells/L.

Rabbit VX2 carcinoma was used to establish gastric PC in this study. All rabbits had overnight fasting before experiment, but water was given ad libitum. The animals were anesthetized by ear vein injection of 2% pentobarbital sodium (30 mg/kg). The abdominal skin was cleaned and disinfected. Tumor cells were injected into the stomach submucosa layer to construct rabbit model of gastric PC as described previously [24, 25]. Briefly, a midline incision of 3 cm long was made beginning 2 cm below the xyphoid and the upper abdomen was open. The stomach was exposed, 0.1 mL of tumor cells (5 × 10¹⁰ vial cells/L) was injected into the submucosal layer of the stomach, through the serosal layer and the muscle layer, the injection site was pressed for 1 min to keep the injected tumor cells in place, and the abdomen was closed with a double layer 3-0 vicryl interrupted suture. After tumor inoculation, Penicillin G at the dose of 100,000 IU/d was intramuscularly injected to each animal for 3 d.

When animal model construction has been confirmed successful on d 8 after operation, these rabbits were randomly divided into 4 groups according to a computer generated randomize number, 10 animals in each group: the Control (n = 10) was observed for natural course of disease progression without any intervention; the HIPEC (n = 10) receiving CRS plus HIPEC (docetaxel 10.0 mg and carboplatin 50.0 mg in 250 mL normal saline, at 42.5 ± 0.5°C for 30 min); the PDOX (n = 10) and the DOX (n = 10) receiving systemic chemotherapy with PDOX 50.0 mg/kg (10.0 mg/kg every 4 d for 5 cycles) or DOX 5.0 mg/kg (1.0 mg/kg every 4 d for 5 cycles) after CRS + HIPEC, respectively; The CRS + HIPEC was performed on d 8 while the systemic chemotherapy was initiated on d 16 after model construction.

For the three treatment groups, CRS + HIPEC was performed on 8 d after tumor cells inoculation. Rabbits were given 2% pentobarbital sodium (30 mg/kg) intravenously (i.v) for anesthesia. The abdominal skin was cleaned and disinfected. The abdominal exploration was performed through a midline incision of 8 cm long beginning 1 cm below the xyphoid. Once the abdomen was open, detailed evaluation of the PC was conducted in different regions including the parietal peritoneum, visceral peritoneum, the omentum, stomach, liver, spleen, intestinal wall, bladder and other pelvic organs. An ePCI score system [44] was developed to record the experimental peritoneal carcinomatosis index of the animals in each group, which took into consideration of tumor nodule sizes, distributions and the characteristics of ascites. In this system, the abdominal cavity of rabbit was divided into 4 regions: region I, sub-diaphragm; region II, the liver, spleen, stomach and affiliated ligaments; region III, small intestine, colon, mesenterium and abdominal wall; and region IV, pelvic cavity urogenital system and rectum. The detailed scoring criteria were modified from similar reporting systems on rat and mice PC models and set as the following: score 0, no tumor nodules throughtout the region; score 1, nodule size less than 5 mm in greastest diameter; score 2, nodule size greater than 5 mm and up to 20 mm; score 3, nodule size greater than 20 mm. If bloody ascites occurred, it was set as score 1. The sum of all the scores was the ePCI of the animal (ranging from 0 to 13). Thereafter, maximal CRS was performed including a routine omentectomy, and optimal removal of tumor nodules. Unresectable tumors were cauterized. The gastric tumor itself, however, was not removed but treated by injection of absolute alcohol. HIPEC was performed just before the closure of abdominal cavity after completion of CRS, as this open technique was believed to provide optimal thermal homogeneity and spatial diffusion [25], with 250 mL of heated saline containing 10 mg of docetaxel and 40 mg of carboplatin for each animal. The abdominal cavity was rinsed twice with 250 mL of normal saline preheated to 42.5°C and perfusion tube was placed in pelvic cavity just before HIPEC. The perfusion equipment consisted of a miniature heat exchanger and a roller pump, allowing perfusion with a variable dynamic flow of 6-12 mL/min. An inflow catheter was inserted into the upper abdomen between the hepatic and diaphragmatic surface and an outflow catheter was placed at the pelvic floor. The perfusion solution was heated to 42.5 ± 0.5°C and infused into the peritoneal cavity at a rate of 10 mL/min through the inflow tube introduced from the automatic perfusion pump. The perfusion in the peritoneal cavity was stirred manually to make equal spatial distribution.

The temperature of the perfusion solution in peritoneal space was kept at 42.5 ± 0.5°C and monitored using a thermometer on real time. The total HIPEC time was 30 min, after which the perfusion solution in the abdominal cavity was removed. Twenty min before surgery, 100 mL of 0.9% sodium chloride solution with 1 g of ceftriaxone powder, 2 mL of 10% potassium chloride solution and 20 mL of 50% glucose solution was infused intravenously for rehydration, nutrition support and infection control in all the four groups of animals. Such treatment was continued for 3 d.

After CRS + HIPEC, the PDOX (n = 10) received systemic chemotherapy with PDOX 50.0 mg/kg (10.0 mg/kg and 3.0 mL/kg every 4 d for 5 cycles) on d 16, d 20, d 24, d 28, and d 32 after tumor cells inoculation respectively; the DOX (n = 10) received systemic chemotherapy with DOX 5.0 mg/kg (1.0 mg/kg and 3.0 mL/kg every 4 d for 5 cycles) at the same time; the Control and the HIPEC received 3.0 mL/kg 5% dextrose instead of chemotherapy drugs at the same time. All drugs were slowly injected i.v through the ear vein of rabbits. For each animal, 2.5 mg of dexamethasone was injected i.v 20 min before chemotherapy, and an additional 2 mL of 5% dextrose was injected i.v after PDOX or DOX injection to reduce local vascular damage.

The general status of the animals was daily recorded in a standard form. For pathological studies, euthanasia was performed on the rabbits by overdose injection of 2% pentobarbital sodium through the ear vein. Post mortem pathological examinations included gross pathology such as tumor size and distributions; local tumor features of GC such as ulcer formation, intestinal obstruction and perforation; special features of PC such as bloody ascites, discrete or confluent tumor nodules on the peritoneum and omentum cake; metastases to major organs such as the livers and the lungs.

For laboratory studies, 3 mL of blood was harvested from ear vein on 2 d before tumor cells inoculation as the baseline (D-2), 2 d before CRS + HIPEC (D6), 2 d before chemotherapy (D14), and 4 d after completion of chemotherapy (D36). The samples were used for routine peripheral blood test, liver and kidney functions tests and detection of creatine kinase and creatine kinase mb isoenzyme.

During the procedure of CRS, the resected omentum, tumor nodules, enlarged mesenteric lymph nodes were harvested for HE staining and observation under optical microscope. After post mortem pathological examinations, myocardial tissues of all the rabbits were harvested at the first time for the electronic microscope, and all the suspected organ metastasis tissues were sampled for routine histopathology study with sections stained by hematoxylin and eosin (HE stain).

All data were integrated into a central database. The numerical data were directly recorded, and the category data were recorded into different categories. The primary endpoint was OS, and secondary endpoint was toxicity profile. Survival was analyzed on Kaplan-Meier survival plots and statistical significance was analyzed with SPSS software version 15.0 (SPSS Inc., IL, USA), using the log-rank test. The differences in blood routine and biochemistry among different groups were tested using nonparametric test at each time point. Categorized variables were compared by chi square test (χ²) or Fisher’s exact test. Two-sided p < 0.05 was considered to be statistically significant.