Preclinical studies of the Chinese Herbal Medicine formulation PHY906 (KD018) as a potential adjunct to radiation therapy

Abstract

Objective: Abdominal and pelvic radiotherapy is limited by the radiosensitivity of the small and large intestine. PHY906 (KD018), a state-of-the-art, well defined adaptation of a traditional Chinese medicine, decreased intestinal injury from chemotherapy in preclinical studies and is in clinical trials with chemotherapy. This project assessed whether PHY906 would also reduce intestinal injury from abdominal irradiation in mice.

Materials and methods: BALB/c mice received whole-abdomen irradiation (2 Gy/day) ± PHY906 by oral gavage twice daily for 4 days. Intestinal injury was assayed by physiological observations and histological studies. Effects of PHY906 on EMT6 mouse mammary tumors were assayed in tumor growth studies.

Results: PHY906 decreased toxicity from fractionated abdominal irradiation. Radiation alone produced marked blunting and loss of villi, crypt hyperplasia and irregular crypt morphology, which were reduced by PHY906. The radiation-induced reduction in viable crypt numbers was also mitigated by PHY906. PHY906 did not alter radiation-induced weight loss, but resulted in more rapid recovery. PHY906 did not alter tumor growth, local invasion or metastatic spread and did not protect tumors from growth delays produced by single-dose or fractionated irradiation.

Conclusion: In this mouse model, PHY906 (KD018) decreased the toxicity of abdominal irradiation without protecting tumors and thereby increased the therapeutic ratio.

Figures

Figure 1

PHY906 does not alter the radiosensitivity of EMT6 tumor cells in cell culture. Survival curves for EMT6 mouse mammary tumor cells in exponential growth in cell culture treated with radiation alone or in combination with 2 mg/ml of PHY906 present for 4 h before, during and 20 h after irradiation. The surviving fractions for cells treated with PHY906 plus radiation was normalized to the survivals for cells with PHY906 alone. Points are means ± SEM of data from 3 independent experiments.

Figure 2

Effect of PHY906 on the weight of mice receiving fractionated whole-abdomen irradiation. Mice were randomized to receive whole abdomen irradiation (4 daily 2 Gy fractions, days 1-4) alone or in combination with PHY906 treatment (~8:30 AM and ~4PM daily by oral gavage; 500 mg/kg/dose) for 4 days. Mice received one 4-day course of PHY906 or two 4-day courses of PHY906 separated by a 3-day interval. Crosshatched box: radiation and PHY906. Single hatched box: second course of PHY906. The weight for each mouse was normalized to the pretreatment weight of that animal. Data are means ± SEM from three replicate experiments, each with 6 mice per group.

Figure 3

Histopathology reveals decreased intestinal damage from radiation in mice treated with PHY906. Representative HE-stained sections of the jejunum, collected 1 day after the final irradiation from control mice, mice treated with PHY906 alone, radiation alone, and radiation plus PHY906. The overall morphology of villi and crypts in control and PHY906 treated mice were similar, with numerous long villus tips (V) and small regular crypts (C) within the lamina propria of the intestinal mucosa. Mice receiving radiation alone had marked blunting and loss of villi (arrow), crypt loss, and crypt hyperplasia with irregular crypt morphology (arrow heads). Overall, mice receiving radiation and PHY906 were histologically similar to control mice and mice treated with PHY906 alone in crypt number and morphology. However, there were sections with minor loss of villi and with edema (*) within the villus lamina. Upper panels: scale bar = 500 microns, lower panels: scale bar = 200 microns.

Figure 4

Representative HE sections of small intestine from mice receiving radiation alone (left panels), or radiation plus PHY906 (right panels) 2 weeks earlier. At this time, there was still mild to marked villus blunting and villus loss in irradiated mice, which was variable in different sections. Crypt numbers also remained lower than normal. In mice treated with PHY906 along with radiation, there was preservation of villus length and distribution, as well as preservation of crypt numbers. Scale Bars = 200 microns.

Figure 5

Number of Crypts per circumference in the mid jejunum of mice given fractionated whole abdomen irradiation (4 × 2 Gy) alone or in combination with PHY906 (500 mg/kg, twice daily). Hatched box shows days of treatment. Open gray box shows SEM of crypt counts in control mice. Points are means ± SEM.

Figure 6

Effect of PHY906 on the tumor growth delay induced by fractionated irradiation. Tumors were stratified by volume on day 10 into groups (6 mice/group) which were randomly assigned to receive no treatment, radiation alone, or radiation + PHY906. PHY906 was given twice daily by oral gavage at a dose of 500 mg/kg/treatment. Tumors were irradiated locally with 4 daily fractions of 2.5 Gy per fraction, given 30 min after the morning PHY906 treatment. Points are means ± SEM. This graph shows tumor growth curves from a representative experiment. Analyses of the growth delays from this experiment are shown in row 3 of the Table. Similar results were found in a replicate experiment using this same regimen, and also in two experiments examining tumor growth after a single dose of 10 Gy. Growth delay data from all 4 tumor growth experiments are summarized on the Table.