Phytopharmaceutical Research Summary
Sherman Zhao, MD, PhD
ABSTRCT
An integrative approach for treating cancer should target the multiple intracellular pathways that support tumour development and should minimize normal-tissue toxicity. Many traditional medicine manifests anticancer activities. The present article focuses on Dammarane Sapogenins, a group of compounds with anticancer activity generated from panax ginseng. Preclinical studies conducted by Dr. William Jia’s team at University of British Columbia indicated Dammarane Sapogenins have pleiotropic anti-cancer pharmacological effects: inducing tumor cell differentiation and apoptosis through various signalling pathways, reversing multidrug resistance by inhibiting p-gp and synergistic effects with some chemotherapy agents. Moreover, these benefits were also demonstrated in large amount of cancer patients through a number of pilot clinical studies world-wide. Both preclinical and clinical data confirmed the high safety of Dammarane Sapogenins as medicinal preparations.
KEY WORDS Dammarane Sapogenins; cancer apoptosis; multidrug resistance
Panax ginseng background
Ginseng (Panax ginseng) has been used for centuries in Asian medicine as an adaptogen to promote longevity, a tonic for well-being, and a curative, especially in China, Korea, and Japan [1]. The root, legendary for the uncanny resemblance it sometimes bears to the human body, grows slowly and should be several years old before harvesting. Ginseng is referred to as the lord or king of herbs in ancient China [2]. Its medicinal efficacy was first documented in Shennong Ben Cao Jing early in the 3rd century and it was later summarized by Li Shizhen in Ben Cao Gang Mu and Zhong Yao Zhi (Chinese Materia Medica by People’s Health Publishing House, Beijing, published in 1596 and 1959 respectively) [2, 3]. In the 18th century, the effectiveness of ginseng was recognized in the West, and subsequently, a large number of investigations were conducted on its botany, chemistry, pharmacology and therapeutic applications [4-7]. Ginseng is now one of the most popular herbal medicines used nutraceutically worldwide. The pharmacological and therapeutic effects of ginseng have been demonstrated to affect the central nervous system (CNS), cardiovascular system, endocrine regulation, immune function, metabolism, biomodulation action, anti-stress, and anti-aging [5]. The most prominent constituent of ginseng is saponin glycoside compounds known as ginsenosides. Recent research indicates that most of the pharmacological effects of ginseng are attributed to ginsenosides [8]. In general, the contents of ginsenosides vary widely ranging from 2 to 20% depending on the species, age, and part of ginseng, and even vary with the preservation or extraction method [9-11]. More than 30 ginsenosides have been isolated, and characterized from various Panax species [12, 13]. In terms of their chemical structural characteristics, ginsenosides can be classified into three major categories, namely protopanaxadiols (PPD) (e.g., Rb1, Rb2, Rc, Rd, Rg3, Rh2), protopanaxatriols (PPT) (e.g., Re, Rf, Rg1, Rg2, Rh1) and the oleanolic acid derivatives. Ginsenosides are metabolized into Dammarane Sapogenins by β-glucosidase bacteria in the gastrointestinal tract. All Dammarane Sapogenins share a pharmacologically active tetracyclic triterpene backbone. Figure 1 shows the structure of protopanaxadiol. Experimental studies from the past decades have shown that both the anti-tumor activities and therapeutic effects of ginseng are mainly attributed to Dammarane Sapogenins.
Almost all the studies were concentrated on pre-clinical stages [14-28], few clinical results were reported regarding ginseng products. This situation ascribes to several factors, but cardinally, on one hand, the technology barrier to acquire high concentration of Dammarane Sapogenins limited its medicinal extendibility; on the other hand, very low bioavailability is the most difficult part for this potential drug. With recent advances in technology, the isolation and standardization of these active ingredients for therapeutic use is now possible [29]. Under this advantaged circumstance, Dr. William Jia in collaboration with Pegasus Pharmaceuticals not only developed preparations (in form of soft gel capsule and injection) from ginseng but also conducted in vitro, in vivo, and clinical studies systematically and extensively on the therapeutic effects in cancer. In this review, we focus on the recent advances in the studies of Dammarane Sapogenins on the conquering of cancer which completed by Dr. Jia’s group.
Pharmacological actions of Dammarane Sapogenins In Vitro Studies:
Arresting Cancer cell growth and inducing differentiation
At low concentrations, Dammarane Sapogenins can arrest the cell division at the G1-S transition phase in wild type p53 cancer cells, and at G2 phase in p53 mutated cancer cells.
Furthermore, Dammarane Sapogenins can induce malignant cells to differentiate into cells resembling benign cell morphology. Following a 48-hour treatment with Dammarane Sapogenins, the differentiated, spindle-shaped phenotype (Fig. 2b), resemble benign melanocyte morphology (Fig. 2a) was observed in original malignant melanoma cells. Continuously treatment with low concentrations of Dammarane Saponins caused the cells to go apoptosis.
Dammarane Sapogenins induce apoptosis through various signalling cascades [30-32].
Activation of multiple Caspase pathways: Caspase pathway is one of the most important and well documented apoptotic pathways. Dammarane Sapogenins directly activate multiple caspase pathways, including caspases 8 and 9 (the initiators) and caspase 3, 6 and 7 (the executors). At high concentrations, Dammarane Sapogenins directly activate the “executor” caspases to cause apoptosis without the mediation of their up-stream initiators. In this way, Dammarane Sapogenins short cut apoptotic mechanisms, leading to the rapid programmed cell death of cancer cells. Dammarane Sapogenins can also induce apoptosis in certain cancer cells in which the caspase pathways were disabled. This indicates that Dammarane Sapogenins have apoptotic mechanisms that are both caspase dependent and independent.
Inhibition of Akt phosphorylation: Akt is a kinase that inhibits cancer cell apoptosis by opening multiple survival pathways. Akt is activated through the process of phosphorylation. Once activated, not only does Akt inhibits apoptosis, but it also stimulates cell growth. Therefore, one way to inhibit the survival pathway is to down regulate Akt activity. Genetic malfunction in cancer cells, such as mutation in the PTEN gene, results in constitutively activation of the survival pathway. Dr. William Jia and his team at the University of British Columbia discovered that Dammarane Sapogenins effectively inhibit the survival pathway in cancer cells lacking PTEN expression. Western bolting confirmed that the level of Akt phosphorylation is significantly reduced in cancer cells treated with Dammarane Sapogenins. Furthermore, Dammarane Sapogenins arrest Akt biosynthesis in certain cancer cells (MIAPaCa, MCF7, PC3, H460).
Amplification of free radical production in cancer cells, which also trigger apoptosis in cancercells. Recent studies in Dr. Jia’s lab have discoveredanother mechanism for Dammarane Sapogeninsmediated apoptosis. They found that freeradicals within cancer cells are significantly increasedfollowing treatment with DammaraneSapogenins. Most cancer cells have elevatedconcentrations of free radicals following 1 hour ofDammarane Sapogenins administration. Freeradicals can activate various apoptotic pathwaysthrough caspases dependent and caspase independent mechanisms.
Effectively Inhibiting Cancer Cells with Various Mutations
In addition, Dammarane Sapogenins induce apoptosis in cancer cells with different genetic backgrounds. The normal expression of p53 arrests cellular division, which eventually results in apoptosis. However, p53 is prone to mutation. This mutation can cause normal cells to become cancerous. Furthermore, some chemotherapeutic drugs require the normal function of p53 in order to be effective. Therefore, a mutation of p53 may lead to multidrug resistance. PTEN mutations are also common in cancer cells. As previously mentioned, PTEN regulates the survival pathway through the activation of Akt. Once the PTEN gene is lost, cancer cells can become drug resistant through the activation of Akt. Dr. Jia’s studies found that Dammarane Sapogenins exhibit anticancer effects regardless of whether the normal functions of the p53 or PTEN genes have been compromised, this efficacy demonstrated by the induction of apoptosis in SF188 (lacks the p53 gene) cell line and U87 (lost the PTEN gene) cell line.
Effects of Dammarane Sapogenins on multidrug resistance
The development of multi-drug resistance (MDR) is a major problem in cancer chemotherapy. Agents that can enhance the accumulation of chemotherapeutic drugs in tumor cells by targeting the MDR proteins is a novel approach to overcome this problem. Dr. Jia’s works revealed that Dammarane Sapogenins could be the one. Several in vitro studies suggest that Dammarane Sapogenin completely reversed P-gp induced drug resistance to Taxol or Doxorubicin on MCF-7adr cells. Dammarane Sapogenins are effective P-gp blocker with a mechanism different from that of verapamil. Given extremely low toxicity of the compound, Dammarane Sapogenin is a potential candidate of chemosensitizer for treatment of multidrug resistant tumors [33-35].
Many other literatures supported that Dammarane Sapogenins could exert chemosensitizing agents [36-39].
Dammarane Sapogenins is a competitive inhibitor of Estrogen Receptors
Some Dammarane Sapogenins can bind to estrogen receptors, but they have very weak estrogen like actions. In this way, these Dammarane Sapogenins competitively inhibit the growth-stimulating effects of estrogen, as illustrated in Figure 4. This mechanism has clinical significance for breast cancer prevention, treatment, and lasting remission [40]. This effect has been found by other researchers.
Dammarane Sapogenins are Effective in Advanced and Multi-drug Resistant Cancers
The treatment of advanced cancers has yet to be adequately developed. Despite the most innovative standard medical treatment, cancers relapse in the majority cases. Most recurrent cancers are drug resistant. Advanced, multidrug resistant cancers are difficult to control. However, Dammarane Sapogenins have demonstrated efficacy in the treatment of advanced and multidrug resistant cancers. Dr. Jia’s team conducted studies on wild type cell line P388wt and its P-gp over expression pair P388adr. Results indicated that Doxorubicin is effective in killing P388wt, IC50 at 0.064μM. Meanwhile, Doxorubicin is far less effective in killing MDR cancer P388adr, with an IC50 at 20μM, which is over three-hundred times greater than the amount required for P388wt. However, when Dammarane Sapogenins is incubated with P388adr, it turned out to be more sensitive cytotoxic agent than doxorubicin. Thus, it seems that Dammarane Sapogenins is equally effective in causing cancer cell cytotoxicity on drug resistant cancer cells. These results have been confirmed by the clinical application of Dammarane Sapogenins for advanced and MDR cancer patients.
Anti-angiogenic effects of Dammarane Sapogenins
Accumulating evidence demonstrated antiangiogenic activity of Dammarane Sapogenins in different tumor models. In those animal models which have been orthotopically implanted with different tumor cells such as human breast infiltrating duct carcinoma, ovarian carcinoma SKOV3 cells, B16-BL6melanoma cells and colon 26-M3.1 carcinoma, Rg3 or Rb2, was found to inhibit angiogenesis in vivo. A significant reduction of intra- tumoral microvessels density (MVD), VEGF (vascular endothelial growth factor) mRNA and VEGF protein levels in tumor tissues and sera of the tumor-bearing animals was observed [42, 43]. Though these two components of ginseng are ginsenoside, it’s believed they are metabolized to Dammarane Sapogenins by enzymatic reactions, and the form of aglycone is their active type. Similar results were also reported when Dammarane Sapogenins was used in combination with a low dose of cyclophosphamide in bearing mice Lewis lung carcinoma [44]. These data indicate that one of the mechanisms of anticancer effect of Dammarane Sapogenins is probably related to suppression of tumor induced angiogenesis [45]. All these mechanisms in combination with findings by Dr. Jia indicated Dammarane Sapogenins are multi-targeting.
Pharmacological Actions of Dammarane Sapogenins In Vivo:
Dammarane Sapogenins Are Effective in Treating Brain Cancers
Dammarane Sapogenins were studied in brain cancer animal models. Subcutaneous implantation of glioma was allowed to develop to a given size before the administration of Dammarane Sapogenins treatment or placebo. Results are expressed in a Kaplan-Meier survival curve. The entire control group died within 24 days. Meanwhile, 40% of animals in the 25 mg/kg group survived for 40 days, the longest period observed. The survival was dose dependent. Dammarane Sapogenins administration causes a significant reduction in the size of glioma in a subcutaneous xenograft model. Prostate and pancreatic cancer models also demonstrated similar results.
Dammarane Sapogenins Inhibit Breast Cancer in Animal Models, and are synergistic with Tamoxifen
According to the studies conducted by Dr. William Jia at UBC, oral administration of Dammarane Sapogenins is effective in the treatment and prevention of breast cancer both estrogen-dependent and independent. The studies demonstrated that Dammarane Sapogenins have competitive inhibitory effects on estrogen receptor sites, decreasing the estrogen-to-estrogen receptor binding. Decreased estrogen receptor binding leads to a reduction in the growth of estrogen-dependent breast cancer. In a breast cancer animal model, Dammarane Sapogenins completely arrested the growth of estrogen-dependent breast cancer in high estrogen exposure. Furthermore, when used in combination, Dammarane Sapogenins can enhance the effects of Tamoxifen independent of the estrogen dependency. Dammarane Sapogenins have also demonstrated the ability to inhibit the stimulatory effects of estrogen on early-stage breast cancers. Tamoxifen does not possess such an effect. Based on these studies, Dammarane Sapogenins therapy has demonstrated the potential to outperform Tamoxifen in the prevention of breast cancer recurrence and metastasis.
The Pharmacological Actions of Dammarane Sapogenins Intravenous Treatment
Animal studies demonstrated that intravenous administration of Dammarane Sapogenins inhibit the growth murine sarcoma S180, murine melanoma B16, murine Lewis lung cancer, and murine colon cancer colon-26 in a dose dependent manner. The inhibition rate of murine sarcoma S180 and murine melanoma was 36-55% and 32-48% respectively, following 7 consecutive daily intra- venous injections of Dammarane Sapogenins at dosages of 15-60 mg/kg. The inhibition rate of murine Lewis lung cancer and murine colon cancer colon-26 was 36-54% and 31-47% respectively, following 10 consecutive daily intravenous injections of Dammarane Sapogenins at dosages ranging from 15-60 mg/kg. In the model of intestine cancer LOVO implanted in nude mice, high dose (60 mg/kg) caused a 60% inhibition rate following a course of 10 consecutive daily intravenous injections.
A low dose of 15 mg/kg causes a 30% inhibition. A dose of 10 mg/kg of intravenous administration of Dammarane Sapogenins was used in combination with Adriamycin, cisplatin, and paclitaxel in the treatment of murine sarcoma S180. The results revealed that Dammarane Sapogenins enhancing the effects of these drugs.
Intravenous administration of Dammarane Sapogenins has demonstrated an affect on the immune systems in mice bearing Lewis lung cancer. Although low dose did not show obvious evidence of immunomodulation, higher dosages can enhance the growth of the spleen lymph cells and increase the activation of Natural Killer cells.
In summary, pre-clinical studies demonstrate that intravenous Dammarane Sapogenins therapy has strong anticancer activities in many types of xenograft animal models. Furthermore, the results indicate a dose dependence mode. In addition, Dammarane Sapogenins enhance the immune function of the tumor-bearing animals.
Toxicology Studies of Dammarane Sapogenins
1. Toxicology of Orally Administered Dammarane Sapogenins
Acute toxicology studies demonstrated that Dammarane Sapogenins, at dosages (4,000 mg/kg) equivalent to 550 times higher than the human dose do not cause any adverse reactions. Long-term toxicology studies (8 weeks) demonstrated that the long-term use of Dammarane Sapogenins at dosages equivalent to 50 times higher than the human dose do not cause any abnormality in the heart, liver, kidney, and blood counts; nor did these dosages cause damage to the nervous system.
2. Toxicology of Dammarane Sapogenins in Injectable Dosage Form
Hemolysis and pyrogen tests, blood vessel irritation test, allergy tests in Cavia porcellus, all these examinations necessary for injection preparations demonstrated Dammarane Sapogenins’ safety.
Acute toxicity studies obtained LD50’s for injectable Dammarane Sapogenins, LD50 for female mice is 511.28 mg/kg body weight; for male mice it is 494.70 mg/kg. These dosages are 15 times higher than effective clinical doses.
Pharmacokinetics of Dammarane Sapogenins was also investigated. It is found that hard shell capsules with Dammarane Sapogenins powder have the poorest oral absorption. Oral solution has increased absorption. However, the soft gel capsules resulted in the best overall oral absorption. The blood level peaked between 30 and 40 Dammarane Sapogenins minutes. The organ distribution of Dammarane Sapogenins is positively correlated with the dose in animals and showed that Dammarane Sapogenins may be able to cross the blood-brain barrier. These findings demonstrate that Dammarane Sapogenins may be useful in the treatment of malignant brain tumors, whether primary or metastatic.
A New Class of drug: Clinical Studies of Dammarane Sapogenins
Two formulations of Dammarane Sapogenins, soft gel and intravenous injection have been developed. Dammarane Sapogenins preparations are indicated as adjuvant treatment in combination with chemotherapy agents. To date, thousands of advanced cancer patients have benefited from Dammarane Sapogenins therapy, generally these benefits are effectively reduced tumor size, stabilize disease condition, improve life quality and physical conditions, prolong survival time, and prevent recurrence and metastases. With prostate and colorectal cancer patients, the tumor marker CEA and PSA had lowered or stabilized during treatment. In 2005 and 2006, another clinical study was conducted. High doses Dammarane Sapogenins I.V (5000mg ~ 7000mg per day) were used in the treatment of advanced cancers. The overall response rate (including complete and partial responses, and stable disease) for primary lung tumor lesions was over 80- percent. Furthermore, 90-percent of patients saw an increase in their WBC count, which demonstrates bone marrow function restoration from damage because of conventional chemotherapy. When these preparations were in combination with other cytotoxic agents such as Paclitaxel et al, less adverse events and improved outcomes were observed. The study also demonstrated that Dammarane Sapogenins was well tolerated by patients, with very high clinical safety. Most commonly, occurred side effects concerning Dammarane Sapogenins include gastrointestinal discomfort, mild allergic reactions such as rush, redness et al [46, 47].
It is especially noteworthy that Dammarane Sapogenins cross the Blood-Brain Barrier and have been used to treat primary and metastatic brain cancers. Numerous cases showed, as single agent, Dammarane Sapogenins can be administered to patients with metastasis brain cancer, and promising outcomes had been observed (significant shrinkage even disappearance of tumor).
Additional from above mentioned effects, Dammarane Sapogenins have demonstrated Pleiotropic Effects for Cancer Patients:
Analgesic Effect
An analgesic effect lasting between 12 and 57- hours following administration in all 13 patients evaluated in a clinical trial for middle to late-stage primary and metastatic NSCLC. All 13 patients (100%) responded with NRS below 3. The pain relief after administration was obvious, accompanied with improvement of sleep, appetite, and other behavioral benefits. The relief lasted for 2 to 5 days after administration.
Impact on bone marrow functions
Dammarane Sapogenins showed a significant effect on improving the bone marrow function after chemotherapy and/or radiotherapy, especially neutropenia. Some patients who had above normal WBC due to infection saw the count fall back.
Effect on immune system
Dammarane Sapogenins demonstrated impact on immune system. The proportion of TH1/Th2 was reduced by 20, and the average values of IgE (Allergy) and IL-6 were decreased. Suggesting that Dammarane Sapogenins preparations enhanced humoral immune function.
Conclusion:
At present, ginseng is not only used as therapeutics by traditional medical practitioners but is also as medications available in the commercial market. The diversity of highly desirable pharmacological effects of ginseng has intrigued scientists for years. Two Dammarane Sapogenins based ginseng products have been commercially available and large-scale phase II studies are undergoing right now. Although the modulatory and therapeutic effects of ginseng components have been extensively investigated, the actual molecular mechanisms remain largely unknown. Recently, it has been found that ginsenosides can act as functional ligands to activate different cellular receptors including some steroid receptors [48-51]. The interaction between ginsenosides and various nuclear steroid hormone receptors may explain the diverse activities of ginseng, which may eventually lead to further development of ginseng-derived therapeutics for a variety of diseases.
Acknowledgement:
Dr. William Jia’s team at University of British Columbia.
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Sherman Zhao, MD, PhD
Correspondence: Sherman_zhao@hotmail.com