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 ResearchA novel combination of Chinese medicines to treat advanced cancers and lymphomas in ratsDawn N Waterhouse Department of Advanced Therapeutics, BC Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada
Chinese Medicine 2009, 4:22doi:10.1186/1749-8546-4-22 The electronic version of this article is the complete one and can be found online at: http://www.cmjournal.org/content/4/1/22
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2009 Waterhouse; licensee BioMed Central Ltd. AbstractBackgroundChinese medicine often targets more than one system and as such comprises several compounds, often in non-purified form, with treatments therefore consisting of whole extracts of herbs rather than isolated compounds. The additive and synergistic effects of the phytochemicals in OMN54, a novel mixture of extracts from three commonly used Chinese medicine components; Ganoderma lucidum, Salvia miltiorrhiza and Scutellaria barbata, were previously demonstrated to have potent anti-cancer activity. This study aims to test whether this heterogeneous, multifunctional and multitargeted agent has an acceptable toxicity profile. MethodsWe conducted preliminary and formal preclinical tolerability determination of OMN54 in Sprague-Dawley rats. In the preliminary study rats were given OMN54 by oral feeding daily for 14 days at doses of 1000 mg/kg, 1750 mg/kg, 2500 mg/kg or 3000 mg/kg per day. A subsequent daily dosing (x 28, 60, 120 or 180) formal toxicology study was conducted in male and female Sprague-Dawley rats at a dose of single dose of 2000 mg/kg/day. ResultsSignificant body weight loss was noted in one of the rats treated at 3000 mg/kg/day, with decline beginning study day 11. This animal experienced mild GI toxicity in the form of diarrhoea. Gross observation indicated kidney damage (pale kidneys) in both this group and in one rat treated at 2500 mg/kg/day. For the later studies, no body weight loss was noted over the course of the study. Blood counts and chemistry were not substantially altered following administration of OMN54, nor were there any findings on histological assessment of organs. ConclusionOMN54 was found to be well tolerated in rat models. OMN54 did not cause any microscopic, anatomic or pathologic changes in exposed animals at the concentrations and under the conditions employed in this study. BackgroundComparative studies in cancer research have recently demonstrated some uses of Chinese medicine in the West. This is particularly true in the field of cancer research [1-4]. The present study investigates OMN54, a proprietary multifunctional and multitargeted oral formulation of extracts manufactured from the following three commonly used Chinese medicinal plants, namely Ganoderma lucidum (Lingzhi), Salvia miltiorrhiza (Danshen) and Scutellaria barbata (Banzhilian), for its effectiveness and toxicity. OMN54 is a complex mixture of phytochemicals, the additive and synergistic effects of which are likely responsible for the potent anti-cancer activity noted. Cultivated in China, Japan and Korea, Ganoderma lucidum is considered a 'superior herb' according to Shennong Bencao Jing (Shen Nung Pen Ts'ao Ching), a Chinese medicine classic dated AD 25. The cap and stem of the dried fruit body (sporophore) of Ganoderma lucidum are used in the commercial preparation of OMN54. Ganoderma lucidum inhibits nuclear factor kappa B (NFκB) and Activator Protein 1 (AP-1), thereby inhibiting the expression of urokinase-type plasminogen activator (uPA) and its receptor uPAR [5]. Ganoderma lucidum inhibits colorectal [6] and breast cancer [7] cells through down regulating oestrogen receptor and NF-κB signalling. Ganoderma lucidum also suppresses cell adhesion and cell migration of highly invasive breast [8,9] and prostate cancer [8] cells. Recent research elucidated a role for specific long chain fatty acids (nonadecanoic acid (C19:0), heptadecanoic acid(C17:0), octa-(C18:0) and hexadecanoic acids (C16:0) from the spores of Ganoderma lucidum to the observed anti-cancer activity [10] by means of in vitro HL-60 cell growth inhibition assays in addition to the previously identified polysaccharides and triterpenes components [11]. These data suggest that Ganoderma lucidum is a potential therapeutic dietary supplement to treat breast and prostate cancer. Extracts of Salvia miltiorrhiza, the second component of OMN54, are used in Chinese medicine to treat liver diseases [12]. Recent studies indicated that Salvia miltiorrhiza had anti-tumour potential and prevented Aflatoxin B1 (AFB1)-induced liver cell injury [13], suggesting that Salvia miltiorrhiza may prevent AFB1-induced hepatocarcinogenesis as evidenced by a decrease in AFB1-DNA adduct formation as well as AFB-1 induced oxidative DNA damage (8 hydroxydeoxyguanosine) in rat liver [13] following administration. Finally, Salvia miltiorrhiza treatment results in apoptosis in a number of cancer cell lines, such as hepatoma [14] and leukemia [15,16] as evidenced by cytoplasmic and nuclear shrinkage, chromatin condensation and membrane blebbing, all characteristic morphological alterations of apoptosis as well as time and dose dependent increase of apoptotic sub-G1 cells. The stems and leaves of Scutellaria barbata are the third component of OMN54. Research showed that Scutellaria barbata had anticancer activity such as apoptosis induction in lung cancer [17], hepatocellular carcinoma [18], ovarian cancer [19] and colon cancer cells [20] through down regulation of Bcl-2 [21]. Recent research identified a number of diterpenoid alkaloids that may contribute to this cytotoxic activity of Scutellaria barbata [22,23]. The present study aims to investigate in vivo safety of OMN54 and focuses on the formal repeat dose toxicology in Sprague Dawley rats conducted in preparation for Phase I clinical study. MethodsOMN54 preparationThe botanical source materials used for the pharmaceutical manufacturing of OMN54 were cultivated by Shanghai Wah Pao Chinese Herbal Medicine Company (China) and Jiangsu Tonghui Biologic Technology (China) according to Good Agricultural Practices and formulated into OMN54 by Canadian Phytopharmaceuticals Corp (batch number 54B09C60520AAXXXXX, Canada). Purity of each component was assessed for specifications and marker compounds prior to use. Briefly, herbal components were subjected to a gentle ethyl acetate extraction process to ensure that all natural components were obtained intact and that no new chemical entities were being produced. Extraction temperature was approximately 37°C, mimicking physiological temperature. Two rounds of ethyl acetate concentration by vacuum with heating were conducted to leave compounds in 50% (w/w) ethyl acetate. Following pooling of compounds, ethanol exchange was performed for removal of ethyl acetate resulting in a mixture of active components in 30% (w/w) ethyl alcohol. Oleic acid, Cremophor, Labrasol and Soya oil were then added to this material to give a homogenous mixture. The mixture was then blended under vacuum evaporation to further remove ethanol solvent residues. The result was a 50% w/w of Soya oil diluents/OMN54 in liquid excipient mixture. Botanical verification of plants was performed as required by the Natural Health Product Guidelines of Health Canada, Category II: isolate, synthetic duplicate, non-standardized and standardized extract, amino acid, essential fatty acid, vitamin, minerals. To obtain comprehensive chemical profiles on these three botanical materials, we developed and applied liquid chromatography mass spectrometry (LC/MS) and complementary gas chromatography mass spectrometry (GC/MS) methods. The LC/MS assay provided an absolute measurement of lot-to-lot consistency of selected relevant chemical markers in each qualified compound and is complimentary to a LC/MS chemical profile assay employed to survey the relative consistency of minor and major detectable chemical components, typically polar non-volatile components observable by the electrospray LC/MS process. In addition to LC/MS, a GC/MS chemical profile assay was performed in parallel for information on the relative consistency of semi-volatile chemical components. This assay also provided extra advantage of mass spectral library matching and identification of known chemical components from OMN54. Chemical markers for the three compounds were Ganoderic acid H and Apigenin (Ganoderma lucidum), Tanshinone IIA (Salviae miltiorrhizae) and Scutellarein (Scutellariae barbatae). The composition ratio of the mixture was Ganoderma lucidum (48.5%), Salviae mitiorrhizae (49.0%) and Scutellariae barbatae (2.5%) (Tables 1 and 2). Quality control certificates for each raw material are kept on file and quality assurance retains are in place for each component of each batch. Animal studiesMale and female Sprague-Dawley rats were obtained from Harlan Laboratories (USA) and housed in 12 hour light cycles with food and water supplied ad libitum. An initial tolerability study was conducted in groups of two rats per dose level. Doses tested were 1000, 1750, 2500 and 3000 mg/kg administered daily for 28 days by oral gavage. Following establishment of dose for full toxicology testing, groups of twenty male and twenty female rats for each of the 28-, 60-, 120- and 180-day studies were treated daily with 2000 mg/kg OMN54 or equivalent volume of OMN54 excipient. OMN54 was delivered by oral gavage in a volume designed to administer the prescribed dose (mg per kg of body weight) to the animals. The gavage volume was based on 2 mL per 200 g of body weight and a dose of 2000 mg/kg unless otherwise stated. Gavage was performed with a ball tipped stainless steel gavage needle attached to a syringe and was performed at approximately the same time on each dosing day. Rats were monitored daily after drug administration for general signs related morbidity. Body weights were measured three times weekly. In particular, signs of ill health were based on body weight loss, change in appetite and behavioural changes such as altered gait, lethargy and gross manifestations of stress. If signs of severe toxicity or tumour-related illness were seen, the animals were terminated (by CO2 asphyxiation) and a necropsy performed to assess other signs of toxicity. The following organs were examined: liver, spleen, lung, kidney, heart, intestine and lymph nodes. Any other unusual findings were also noted. On day 29, 61, 121 or 181 respectively, all rats were terminated by CO2 asphyxiation. Blood was collected by cardiac puncture and allocated for complete blood counts with differentials and chemistry analysis (IDEXX Laboratories, Canada). Necropsy was performed on all animals. Kidney, liver, lung, spleen, heart were harvested into formalin and fixed for a period of 24 hours. The following organs were harvested as above and fixed in formalin for a period of 7 days: brain, spinal cord (cervical, mid-thoracic and lumbar). All tissues were immediately processed for hematoxylin and eosin (H&E) staining following formalin fixation. Pathology assessmentSlides were examined by a board-certified veterinary pathologist who had no knowledge of the test article, dosage, schedule, experimental protocol or group assignment of any of the animals. Each tissue section was evaluated and either recorded as normal or a description was made of any abnormalities. Pathological findings were recorded on individual data sheets for each animal. The importance of any changes (or lack of changes) was assessed for each animal and such assessment recorded on the individual data sheets. All methodology was reviewed and approved by the Institutional Animal Care Committee (IACC) at the University of British Columbia prior to the study. And the study was conducted in compliance with the standards of the Canadian Council on Animal Care (CCAC). Statistical analysisAll experimental data was assessed for significance using a t-test analysis with SigmaPlot® v.10 (SyStat Software Inc., USA). Results were considered to be significant if the reported p-value was less than 0.05. ResultsGroups of two rats were treated by daily oral gavage over a period of 28 days at 1000, 1750, 2500 or 3500 mg/kg to assess tolerability for the full rat toxicology study. The body weights of rats treated at the high dose (3500 mg/kg) started to decline following the 10th consecutive dosing day such that by study day 13 mean body weight had decreased by 7.5% as compared to day 10 (Table 3). It was noted that the mean decrease was as a result of morbidity experienced by one rat only. This rat also demonstrated overall weakness and softened stool and was found dead following the dosing on study day 14. Although no signs of morbidity were noted in rats dosed at 2500 mg/kg, there was some degree of resistance to dosing, therefore the formal toxicology studies were designed and conducted at a dose of 2000 mg/kg. Table 3. Body weights of rats following daily oral gavage with vehicle control or OMN54 over 28 days (n = 10) Groups of 20 male and 20 female rats for each of 28-, 60-, 120- and 180-day studies were treated daily with 2000 mg/kg OMN54 or equivalent volume excipient and assessed daily for signs of morbidity, and three times weekly for body weights. At study end, rats were terminated by CO2 asphyxiation and gross and histopathological analysis performed. Body weights for all animals increased gradually over the course of the study (Table 4 males, Table 5 females). No gross signs of morbidity were noted and no resistance to dosing such as choking or gagging. Necropsies on rats from all schedules resulted in no remarkable findings. Tissues were collected in formalin and processed for H&E staining for scoring by a certified rodent pathologist. Microscopic examination of a standard set of organs collected at post-mortem from 40 rats on a toxicity study at day 28, on 40 animals at day 60, 40 animals at day 90 and on a further 40 animals at 180 days did not reveal any anatomic changes that were attributed to exposure to the test article. Table 4. Body Weights of male rats following treatment with OMN54 at 2000 mg/kg by daily oral gavage for 28, 60, 120 or 180 days (n = 10) Table 5. Body Weights of female rats following treatment with OMN54 at 2000 mg/kg by daily oral gavage for 28, 60, 120 or 180 days (n = 10) Blood was collected by cardiac puncture for complete blood counts with differentials and chemistry screens at study end for each of the 28-, 60-, 90-, 120- and 180-day studies. Results are shown in Table 6 and Table 7 (complete blood counts, males and females respectively), Table 8 (differentials), Table 9 and Table 10 (males), Table 11 and Table 12 (females) (blood chemistry). Table 6. Complete blood counts from male rats treated by daily oral gavage with OMN54 at a dose of 2000 mg/kg/day for 28, 60, 120 or 180 days (n = 10) Table 7. Complete blood counts from female rats treated by daily oral gavage with OMN54 at a dose of 2000 mg/kg/day for 28, 60, 120 or 180 days (n = 10) Table 8. Blood differential analysis of rats following 28, 60, 120 or 180 consecutive days of oral gavage with OMN54 at 2000 mg/kg (× 109, absolute count; n = 10) Table 9. Blood chemistry of male rats treated daily by oral gavage with OMN54 at 2000 mg/kg/day for 28 or 60 days Table 10. Blood chemistry of male rats treated daily by oral gavage with OMN54 at 2000 mg/kg/day for 120 or 180 days Table 11. Blood chemistry of female rats treated daily by oral gavage with OMN54 at 2000 mg/kg/day for 28 or 60 days Table 12. Blood chemistry of female rats treated daily by oral gavage with OMN54 at 2000 mg/kg/day for 120 or 180 days No significant findings were noted in the blood count. While several trends were observed such as a slight increase in the red blood cell count and red cell distribution width after the first month of dosing, these observations were clinically insignificant. It should be noted that reticulocytes were not analyzed in this dataset; however this will be considered for further studies with OMN54. Glucose levels were within normal ranges compared to reference values [24]. As the rats were fed ad libitum, these results were not fasting values and therefore were likely to be irrelevant. Values of globulin showed a gradual increase over the 180 days; however values were all within normal. Female rats treated with OMN54 at 2000 demonstrated a gradual increase of gamma-glutamyl transpeptidase, with all values being greater than normal. The female rats treated with vehicle and all males had consistent low levels over the course of the study, indicating a parameter of interest in females for the clinical testing of OMN54. There were no other clinically significant findings in blood chemistry. DiscussionWhile statistically significant differences in body weights were noted in the rats treated daily with OMN54 up to 180 days (both male and female), these results were inconsistent between the sexes with females treated with OMN54 having higher mean body weights than those treated with the vehicle control while the trend in males was for higher body weights in rats treated with vehicle control. Importantly neither male nor female rats experienced any weight loss as a result of treatment, indicating the tolerability of OMN54 at the tested dose of 2000 mg/kg/day. ConclusionOMN54 was found to be well tolerated in rat models. OMN54 did not cause any microscopic, anatomic or pathologic changes in exposed animals at the concentrations and under the conditions employed in this study. AbbreviationsAFB1: aflatoxin B1; CCAC: Canadian Council on Animal Care; H&E: hematoxylin and eosin; GC/MS: gas chromatography/mass spectrometry; IACC: institutional animal care committee; LC/MS: liquid chromatography/mass spectrometry; NFκB: nuclear factor kappa B; RDW: red cell distribution width; uPA: urokinase-type plasminogen activator; uPAR: urokinase-type plasminogen activator receptor Competing interestsDNW served, in the past five years, as a consultant to Genyous Biomedical which owns OMN54, and has received compensation in this capacity. Authors' contributionsDNW had oversight for the preclinical toxicology testing, conducted the studies and drafted the manuscript. The author read and approved the final version of the manuscript. AcknowledgementsDrs Francis Law and Joseph Tai contributed to the design and conduct of the study. References
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