A Randomized, Open-Label, Safety and Exploratory Efficacy Study of Kanglaite Injection (KLTi) plus Gemcitabine versus Gemcitabine in Patients with Advanced Pancreatic Cancer.

Background: This study was designed to assess the safety and preliminary efficacy of KLTi plus gemcitabine in patients with locally advanced or metastatic pancreatic cancer. Methods: In a randomized, open-label study, patients with locally advanced or metastatic pancreatic cancer were randomized 2:1 to receive KLTi plus gemcitabine or gemcitabine monotherapy. Three sequential cohorts were tested at 30 g/day, 50 g/day, and 30 g/day. Gemcitabine was administered at 1000 mg/m2 on days 1, 8 and 15 of each 28 day cycle. KLTi was administered on days 1-5, 8-12, and 15-19 of each 28 day cycle. Patients received study treatment until disease progression. The primary endpoint was progression-free survival in the ITT population. Safety evaluation was based on patients who received any study treatment. ClinicalTrials.gov identifier NCT00733850. Results: Eighty-five patients were randomized including 41 (28:13) in Cohort 1, 18 (12:6) in Cohort 2, and 26 (17:9) in Cohort 3. Due to a different dose and/or shift in patient populations in Cohort 2 and 3, efficacy data for the 30 gm dose are presented in this manuscript for Cohort 1 alone, and for the combination of Cohort 1+3. The 30 gm KLTi + gemcitabine group had a statistically significant improvement in progression-free survival (PFS) as assessed by blinded independent radiology review in the ITT population, with a median of 112 days, versus 58 days in the gemcitabine group (HR 0.50; 95% CI: 0.27, 0.92), p = 0.0240. The incidence rates of TEAEs, CTCAE Grade 3 or higher TEAEs, and SAEs were similar between the two arms. There were no deaths related to KLTi + gemcitabine treatment. Conclusion: Kanglaite Injection (30 g/day) plus a standard regimen of gemcitabine demonstrated encouraging clinical evidence of anti-neoplastic activity and a well-tolerated safety profile.

A Selective Estrogen Receptor Beta Agonist for the Treatment of Hot Flushes: Phase 2 Clinical Trial.

OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, and efficacy of a selective estrogen receptor beta (ERß) agonist, Dr. Tagliaferri's Menopause Formula (MF102), to treat the symptoms of menopause. METHODS: An open-label trial of MF102 taken for 12 weeks by 30 postmenopausal women aged 40-65 years, who experienced a minimum of five moderate to severe hot flushes per day. The primary efficacy outcome was a change in the frequency of moderate to severe hot flushes from baseline to week 12. A change in the frequency of hot flushes that woke participants from their sleep from baseline to 12 weeks was a secondary endpoint. Lipid profile and endometrial thickness were also evaluated. RESULTS: Thirty postmenopausal women with an average of nine moderate to severe hot flushes per day were treated with MF102 4 g/day; 27 participants completed the study. The median percent reduction in moderate to severe hot flushes was 71% (p < 0.001). The median percent reduction in hot flushes that woke participants from their sleep was 54% (p < 0.001). Low-density lipoprotein (LDL-C) and total cholesterol both declined significantly from baseline. There were no serious adverse events, reports of abnormal uterine bleeding, or significant changes in double-wall endometrial thickness. CONCLUSIONS: Treatment with MF102 resulted in a marked decrease in the frequency of moderate to severe hot flushes, was well-tolerated, and demonstrated no safety concerns.

Bezielle selectively targets mitochondria of cancer cells to inhibit glycolysis and OXPHOS.

Bezielle (BZL101) is a candidate oral drug that has shown promising efficacy and excellent safety in the early phase clinical trials for advanced breast cancer. Bezielle is an aqueous extract from the herb Scutellaria barbata. We have reported previously that Bezielle was selectively cytotoxic to cancer cells while sparing non-transformed cells. In tumor, but not in non-transformed cells, Bezielle induced generation of ROS and severe DNA damage followed by hyperactivation of PARP, depletion of the cellular ATP and NAD, and inhibition of glycolysis. We show here that tumor cells' mitochondria are the primary source of reactive oxygen species induced by Bezielle. Treatment with Bezielle induces progressively higher levels of mitochondrial superoxide as well as peroxide-type ROS. Inhibition of mitochondrial respiration prevents generation of both types of ROS and protects cells from Bezielle-induced death. In addition to glycolysis, Bezielle inhibits oxidative phosphorylation in tumor cells and depletes mitochondrial reserve capacity depriving cells of the ability to produce ATP. Tumor cells lacking functional mitochondria maintain glycolytic activity in presence of Bezielle thus supporting the hypothesis that mitochondria are the primary target of Bezielle. The metabolic effects of Bezielle towards normal cells are not significant, in agreement with the low levels of oxidative damage that Bezielle inflicts on them. Bezielle is therefore a drug that selectively targets cancer cell mitochondria, and is distinguished from other such drugs by its ability to induce not only inhibition of OXPHOS but also of glycolysis. This study provides a better understanding of the mechanism of Bezielle's cytotoxicity, and the basis of its selectivity towards cancer cells.

Identification and analysis of the active phytochemicals from the anti-cancer botanical extract Bezielle.

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it.

Estrogenic plant extracts reverse weight gain and fat accumulation without causing mammary gland or uterine proliferation.

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women.

Bezielle (BZL101)-induced oxidative stress damage followed by redistribution of metabolic fluxes in breast cancer cells: a combined proteomic and metabolomic study.

Bezielle is an orally administered aqueous extract of Scutellaria barbata for treatment of advanced and metastatic breast cancer. Phase I trials showed promising tolerability and efficacy. In our study, we used a combined proteomic-metabolomic approach to investigate the molecular pathways affected by Bezielle in ER-positive BT474 and ER-negative SKBR3 cell lines. In both, Bezielle inhibited cell proliferation, induced cell death and G2 cycle arrest by regulating the mediator proteins Jab1, p27(Kip1) and p21(Cip1) . In addition, it stimulated reactive oxygen species production, hyperactivation of PARP and inhibition of glycolysis. Bezielle's ability to induce oxidative stress was associated with the changes in expression of redox potential maintaining enzymes: glutathione- and thioredoxin-related proteins and peroxiredoxins. In regards to cell metabolism, decreased expression of α-enolase was associated with a reduction of de novo (13) C-lactate formation. Reduced Krebs cycle activity as evidenced by the reduced expression of α-ketoglutarate dehydrogenase and succinyl-CoA synthetase led to decreased intracellular succinate concentrations. By inhibiting glucose metabolism, cells reacted by lowering the expression of glucose transporters and resulting in decreased intracellular glucose concentration. Decreased expression of fatty acid synthase and reduced concentration of phosphocholine indicated considerable changes in phospholipid metabolism. Ultimately, by inhibiting the major energy-producing pathways, Bezielle caused depletion of ATP and NAD(H). Both cell lines were responsive, thus suggesting that Bezielle has the potential to be effective against ER-negative breast cancers. In conclusion, Bezielle's cytotoxicity toward cancer cells is primarily based on inhibition of metabolic pathways that are preferentially activated in tumor cells thus explaining its specificity for cancer cells.

Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2)) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E(2). The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

A phase 1B dose escalation trial of Scutellaria barbata (BZL101) for patients with metastatic breast cancer.

The purpose of this study was to determine the safety and maximum tolerated dose (MTD) of BZL101 (FDA IND# 59,521), an orally delivered aqueous extract from the herb Scutellaria barbata, in women with metastatic breast cancer (MBC). The trial was an open-label, phase 1B, multicenter, dose escalation study. Eligible patients had histologically confirmed breast cancer and measurable stage IV disease. The standard phase 1 "3 + 3" study design was used to determine the MTD. Primary endpoints were toxicity and MTD of BZL101. Secondary outcomes included efficacy based on RECIST criteria. A total of 27 women with a median of 2 prior chemotherapy treatments for metastatic disease were treated in four different dose cohorts. Grade 3 and 4 adverse events (AEs) were uncommon. Dose-limiting toxicities included the following: grade 4 AST elevation, grade 3 diarrhea, grade 3 fatigue, and grade 3 rib pain. Fourteen patients were evaluable according to Response Evaluation Criteria in Solid Tumors. Investigator assessment classified three patients with stable disease for >120 days (21%). One patient was on BZL101 for 449 days and remains stable for 700 + days. Independent radiology review identified three patients with objective tumor regression (>0% and <30%). The MTD was not reached, thus per protocol, the MTD was defined as the maximum administered dose of BZL101 40 g/day. In conclusion, oral administration of BZL101 was safe, well tolerated, and showed promising clinical evidence of anticancer activity in this heavily pretreated population of women with MBC.

Is a shorter hot flash diary just as good as a 7-day diary?

OBJECTIVE: In randomized trials, the most common way to measure the effect of treatment on the frequency and severity of menopausal hot flashes is a 7-day self-reported diary. However, adherence with completing the hot flash diary in real time may be poor, and completing a diary is cumbersome for study participants. Our objective was to determine if a shorter diary for recording self-reported hot flashes is as accurate and precise as the traditional 7-day diary. METHODS: Using cross-sectional data from a multicenter randomized clinical trial of an herbal preparation (MF101, an estrogen receptor beta-selective agonist for treatment of menopausal hot flashes), we compared findings based on shorter diaries with findings based on a 7-day diary. RESULTS: With 3 days of diary keeping, the mean number of hot flashes per day and mean severity were almost identical to the means based on the 7-day diary, the SDs of the means were almost identical, and the intraclass correlations were almost perfect. The difference in the mean number of hot flashes per day compared with the 7-day diary was only 12% of one hot flash. Data from a different clinical trial revealed similar correspondence between the findings of a 3- and 7-day diary. CONCLUSIONS: In our study, the optimal duration of diary keeping to record menopausal hot flashes seems to be 3 days. In addition to being as good as a 7-day diary, a 3-day diary would be less burden on study participants and research staff and less expensive.

MF101, a selective estrogen receptor beta modulator for the treatment of menopausal hot flushes: a phase II clinical trial.

OBJECTIVE: To determine the optimal dose, safety, and efficacy of an estrogen receptor beta selective Chinese herbal extract, menopausal formula 101 (MF101), for treating hot flushes. METHODS: A randomized, blinded trial in 217 postmenopausal women with hot flushes randomized to 5 or 10 g/day of MF101 or placebo for 12 weeks. RESULTS: The effects of 5 g/day of MF101 did not differ from those of placebo. After 12 weeks, the mean percent decrease in frequency of hot flushes in the 10 g/day group was 12.9% greater than that in the placebo group (P = 0.15), the median percent decrease was 11.7% greater than that in the placebo group (P = 0.05), and the proportion of women with at least a 50% reduction in hot flushes was 16.2% greater than that in the placebo group (P = 0.03). CONCLUSIONS: Treatment with 10 g/day of MF101 reduces the frequency of hot flushes. Trials with higher doses are planned.

Molecular mechanisms underlying selective cytotoxic activity of BZL101, an extract of Scutellaria barbata, towards breast cancer cells.

We studied the mechanism of the cytotoxic activity of BZL101, an aqueous extract from the herb Scutellaria barbata D. Don, which is currently in phase II clinical trial in patients with advanced breast cancer. The phase I trial showed favorable toxicity profile and promising efficacy. We report here that BZL101 induces cell death in breast cancer cells but not in non-transformed mammary epithelial cells. This selective cytotoxicity is based on strong induction by BZL101 of reactive oxygen species (ROS) in tumor cells. As a consequence, BZL101 treated cancer cells develop extensive oxidative DNA damage and succumb to necrotic death. Data from the expression profiling of cells treated with BZL101 are strongly supportive of a death pathway that involves oxidative stress, DNA damage and activation of death-promoting genes. In breast cancer cells oxidative damage induced by BZL101 leads to the hyperactivation of poly (ADP-ribose) polymerase (PARP), followed by a sustained decrease in levels of NAD and depletion of ATP, neither of which are observed in non-transformed cells. The hyperactivation of PARP is instrumental in the necrotic death program induced by BZL101, because inhibition of PARP results in suppression of necrosis and activation of the apoptotic death program. BZL101 treatment leads to the inhibition of glycolysis selectively in tumor cells, evident from the decrease in the enzymatic activities within the glycolytic pathway and the inhibition of lactate production. Because tumor cells frequently rely on glycolysis for energy production, the observed inhibition of glycolysis is likely a key factor in the energetic collapse and necrotic death that occurs selectively in breast cancer cells. The promising selectivity of BZL101 towards cancer cells is based on metabolic differences between highly glycolytic tumor cells and normal cells.

Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist.

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Selective activation of estrogen receptor-beta transcriptional pathways by an herbal extract.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

Phase I trial and antitumor effects of BZL101 for patients with advanced breast cancer.

BACKGROUND: Botanical therapies are often used by breast cancer patients yet few clinical trials have evaluated their safety and efficacy. We studied mechanisms of activity and performed a phase I clinical trial in patients with advanced breast cancer to evaluate BZL101, an aqueous extract from Scutellaria barbata. METHODS: Preclinical studies were conducted in vitro to characterize cell death induced by BZL101. In a phase I trial, eligible patients had histologically confirmed, measurable metastatic breast cancer. Treatment consisted of 350 ml per day of oral BZL101, administered as sole cancer therapy until disease progression, toxicity or personal preference to discontinue. Primary endpoints were safety, toxicity and tumor response. RESULTS: BZL101 extract induced strong growth inhibition and apoptosis of breast cancer cell lines. In the phase I trial, 21 patients received BZL101. Mean age was 54 years (30-77) and mean number of prior treatments for metastatic disease was 3.9 (0-10). There were no grade III or IV adverse events (AEs). The most frequently reported BZL101-related grade I and II AEs included: nausea (38%), diarrhea (24%), headache (19%) flatulence (14%), vomiting (10%), constipation (10%), and fatigue (10%). Sixteen patients were evaluable for response. Four patients had stable disease (SD) for >90 days (25%) and 3/16 had SD for >180 days (19%). Five patients had objective tumor regression, one of which was 1 mm short of a PR based on RECIST criteria. CONCLUSIONS: BZL 101 inhibits breast cancer cell lines by inducing apoptosis. In a phase I clinical trial, BZL101 was safe and had a favorable toxicity profile. BZL101 demonstrated encouraging clinical activity in this heavily pretreated population.

Traditional Chinese medicine in the treatment of breast cancer.

The treatment of breast cancer involves multiple disciplines, and current recommendations are based on modern tenets of biology and pharmacology along with a growing body of evidence-based literature. Despite advances in screening, surgery, adjuvant radiation, and systemic therapy, as well as novel biologically targeted therapies, there are limitations to their benefits, especially in advanced disease. Complementary therapies including Oriental medicine have enjoyed a growing popularity as a less intensive and more "natural" approach to achieving health or improving quality of life. However, definitive literature in this area is scant and therefore has not been integrated into the mainstream medical community. Traditional Chinese medicine (TCM) may serve as a useful model for scientific inquiry since there is a standardized system of diagnostics and therapies, and this discipline is practiced worldwide. Still, the holistic and individualized nature of TCM presents challenges to rigorous clinical testing, and as a result, most published work in this field is in the form of anecdotal reports or uncontrolled series. Among the components of TCM, herbal or botanical agents possess complex biological activities that could affect many aspects of carcinogenesis such as cell growth and proliferation, apoptosis, host-tumor interactions, and immune function and differentiation. Despite a fairly extensive series of laboratory studies detailing many biological effects of botanical agents, few clinical trials have been completed to test specific hypotheses regarding the mode of action of TCM. Most TCM therapies have been empirically applied in a series of patients, with descriptive results provided. The summaries of a few studies highlighted in this review can provide some evidence of safety but generally do not possess the design and results to verify clinical effectiveness. Nevertheless, the theory of TCM coupled with laboratory studies and safety information can serve as a basis for the design of more definitive trials of TCM for specific indications in breast cancer. There are increasing interest and growing opportunities for investigative approaches that could ultimately verify or reject TCM and specific botanical agents. Some of these initiatives in the area of breast cancer prevention and treatment are outlined.

Antiproliferative activity of Chinese medicinal herbs on breast cancer cells in vitro.

Chinese medicinal herbs are traditionally used to prevent and treat a variety of diseases, including cancer. These herbal preparations are purported to have many biological effects including direct antiproliferative effects on cancer cells, anti-mutagenic activity, and stimulatory or suppressive effects on immune responses. The present study investigates the effects of aqueous extracts from seventy-one Chinese medicinal herbs on the growth of five breast cancer cell lines (SK-BR-3, MCF7, MDA-MB-231, BT-474 and MCNeuA). Twenty-one percent (15 out of 71) of the extracts demonstrated greater than 50% growth inhibition on at least 4 of the 5 cell lines. Dose-response curves were obtained for several of the most potent crude extracts and demonstrated IC50 values ranging from < 10 micrograms/ml to > 1 mg/ml. Six of seven herbs tested induced high molecular weight DNA fragmentation, an early marker of apoptosis, while one of these also induced low molecular weight DNA fragmentation. Flow cytometric analysis of breast cancer cells exposed to one of these herbs (Rheum palmatum) suggested that it arrests cells in the G2/M phase of the cell cycle. These results indicate that many of the herbs used in traditional Chinese medicine for the treatment of cancer have significant growth inhibitory effects on breast cancer cells in vitro.