YIV-818-A: a novel therapeutic agent in prostate cancer management through androgen receptor downregulation, glucocorticoid receptor inhibition, epigenetic regulation, and enhancement of apalutamide, darolutamide, and enzalutamide efficacy.

Introduction: Prostate cancer is the second leading cause of cancer death among men in the United States. Castration-Resistant Prostate Cancer (CRPC) often develops resistance to androgen deprivation therapy. Resistance in CRPC is often driven by AR variants and glucocorticoid receptor (GR). Thus, drugs that target both could be vital in overcoming resistance. Methods: Utilizing the STAR Drug Discovery Platform, three hundred medicinal plant extracts were examined across 25 signaling pathways to identify potential drug candidates. Effects of the botanical drug YIV-818-A, derived from optimized water extracts of Rubia cordifolia (R.C.), on Dihydrotestosterone (DHT) or Dexamethasone (DEX) induced luciferase activity were assessed in 22RV1 cells harboring the ARE luciferase reporter. Furthermore, the key active compounds in YIV-818-A were identified through activity guided purification. The inhibitory effects of YIV-818-A, RA-V, and RA-VII on AR and GR activities, their impact on AR target genes, and their roles in modifying epigenetic status were investigated. Finally, the synergistic effects of these compounds with established CRPC drugs were evaluated both in vitro and in vivo. Results: YIV-818-A was found to effectively inhibit DHT or DEX induced luciferase activity in 22RV1 cells. Deoxybouvardin (RA-V) was identified as the key active compound responsible for inhibiting AR and GR activities. Both YIV-818-A and RA-V, along with RA-VII, effectively downregulated AR and AR-V proteins through inhibiting protein synthesis, impacted the expression of AR target genes, and modified the epigenetic status by reducing levels of Bromodomain and Extra-Terminal proteins (Brd2/Brd4) and H3K27Ac. Furthermore, these compounds exhibited synergistic effects with apalutamide, darolutamide, or enzalutamide, and suppressed AR mediated luciferase activity of 22RV1 cells. Co-administration of YIV-818-A and enzalutamide led to a significant reduction of 22RV1 tumor growth in vivo. Different sources of R.C. had variable levels of RA-V, correlating with their potency in AR inhibition. Discussion: YIV-818-A, RA-V, and RA-VII show considerable promise in addressing drug resistance in CRPC by targeting both AR protein and GR function, along with modulation of vital epigenetic markers. Given the established safety profile of YIV-818-A, these findings suggest its potential as a chemopreventive agent and a robust anti-prostate cancer drug.

YIV-906 enhances nuclear factor of activated T-cells (NFAT) activity of T cells and promotes immune checkpoint blockade antibody action and CAR T-cell activity.

YIV-906 is a systems biology botanical cancer drug, inspired by a traditional Chinese herbal formulation. Results from eight Phase I/II to II clinical studies demonstrated the potential of YIV-906 to prolong survival and improve the quality of life of cancer patients. As an immunomodulator in the tumor microenvironment, YIV-906 can turn cold tumors hot and potentiate anti-tumor activity for different classes of anticancer agents; and as a cytoprotector in the GI, YIV-906 can reduce non-hematological side effects and speed up damaged tissue recovery. YIV-906 enhanced anti-PD1 action against hepatoma in mice by stimulating both innate and adaptive immunity. In a Jurkat cell-staphylococcal superantigen E (SEE)-Raji cell culture model, YIV-906 promoted T cell activation with upregulation of CD69 by enhancing NFAT activity, with or without PD1-PD-L1 interaction. YIV-906 could trigger the phosphorylation of TCR downstream signaling cascades without the involvement of TCR. YIV-906 could inhibit SHP1 and SHP2 activities, which dephosphorylates TCR downstream proteins due to the PD1-PD-L1 interaction. Therefore, YIV-906 could enhance anti-PD1 action to rescue the depressed NFAT activity of Jurkat cells due to the PD1-PD-L1 interaction. In addition, YIV-906 enhanced the NFAT activity and killing capability of Jurkat cells expressing chimeric antigen receptor (CAR-CD19-CD3z) toward CD19 expressing cells, such as Raji cells, with or without PD1-PD-L1 overexpression. Ingredient herb S (Scutellaria baicalensis Georgi) of YIV-906 and some S compounds were found to play key roles in these activities. In conclusion, YIV-906 modulates adaptive immunity by activating T effector cells mainly through its action on SHP1/2. YIV-906 could also facilitate immune checkpoint blockade therapy or CAR-T cell therapy for cancer treatment.

YIV-906 potentiated anti-PD1 action against hepatocellular carcinoma by enhancing adaptive and innate immunity in the tumor microenvironment.

YIV-906 (PHY906) is a standardized botanical cancer drug candidate developed with a systems biology approach-inspired by a traditional Chinese herbal formulation, historically used to treat gastrointestinal symptoms including diarrhea, nausea and vomiting. In combination with chemotherapy and/or radiation therapy, preclinical and clinical results suggest that YIV-906 has the potential to prolong survival and improve quality of life for cancer patients. Here, we demonstrated that YIV-906 plus anti-PD1 could eradicate all Hepa 1-6 tumors in all tumor bearing mice. YIV-906 was found to have multiple mechanisms of action to enhance adaptive and innate immunity. In combination, YIV-906 reduced PD1 or counteracted PD-L1 induction caused by anti-PD1 which led to higher T-cell activation gene expression of the tumor. In addition, YIV-906 could reduce immune tolerance by modulating IDO activity and reducing monocytic MDSC of the tumor. The combination of anti-PD1 and YIV-906 generated acute inflammation in the tumor microenvironment with more M1-like macrophages. YIV-906 could potentiate the action of interferon gamma (IFNg) to increase M1-like macrophage polarization while inhibiting IL4 action to decrease M2 macrophage polarization. Flavonoids from YIV-906 were responsible for modulating IDO activity and potentiating IFNg action in M1-like macrophage polarization. In conclusion, YIV-906 could act as an immunomodulator and enhance the innate and adaptive immune response and potentiate anti-tumor activity for immunotherapies to treat cancer.

A Phase II Clinical Trial on the Combination Therapy of PHY906 Plus Capecitabine in Hepatocellular Carcinoma.

LESSONS LEARNED: A PHY906 and capecitabine combination could be effective as a salvage therapy for patients with hepatocellular carcinoma (HCC) previously treated with multiple systemic therapies. This traditional Chinese medicine formulation can work with Western cancer chemotherapeutic agents to improve clinical outcomes or alleviate side effects for patients with advanced HCC. BACKGROUND: This study aimed to evaluate efficacy and safety of capecitabine combined with a PHY906 (a pharmaceutical-grade formulation of four traditional Chinese herbs) in the treatment of advanced hepatocellular carcinoma (HCC) in Asian patients who were positive for hepatitis B virus (HBV). METHODS: This study was an open-label, phase II safety and efficacy clinical trial of PHY906 and capecitabine in patients with advanced HCC. Patients received 750 mg/m2 capecitabine b.i.d. 14 days plus 800 mg of PHY906 b.i.d. on days 1-4 and days 8-11 every 21-day cycle. The primary endpoint was 6-month survival rate, and secondary endpoints were progression-free survival, overall survival, disease control rate, and safety. RESULTS: Thirty-nine subjects completed the study with a 46.2% stable disease rate. The median progression-free survival was 1.5 months, and median overall survival (mOS) was 6 months with a 51.3% 6-month survival rate. The most common adverse events included lower hemoglobin, diarrhea, pain, abdomen (not otherwise specified), fatigue, increased aspartate aminotransferase, and bilirubin. Patients who (a) had not received previous chemotherapies or targeted therapy or (b) had lower starting alpha-fetoprotein (AFP) levels or (c) had HBV infection showed better clinical outcome. CONCLUSION: Our data showed that PHY906 increases the therapeutic index of capecitabine by enhancing its antitumor activity and reduces its toxicity profile in advanced HCC.

Mechanism Based Quality Control (MBQC) of Herbal Products: A Case Study YIV-906 (PHY906).

YIV-906 (PHY906), a four-herb Chinese medicine formulation, is inspired by an 1800 year-old Chinese formulation called Huang Qin Tang which is traditionally used to treat gastrointestinal (GI) symptoms. In animal studies, it could enhance anti-tumor activity of different classes of anticancer agents and promote faster recovery of the damaged intestines following irinotecan or radiation treatment. Several clinical studies have shown that YIV-906 had the potential to increase the therapeutic index of cancer treatments (chemotherapy, radiation) by prolonging life and improving patient quality of life. Results of animal studies demonstrated five clinical batches of YIV-906 had very similar in vivo activities (protection of body weight loss induced by CPT11 and enhancement of anti-tumor activity of CPT11) while four batches of commercial-made Huang Qin Tang, HQT had no or lower in vivo activities. Two quality control platforms were used to correlate the biological activity between YIV906 and HQT. Chemical profiles (using analysis of 77 peaks intensities) obtained from LC-MS could not be used to differentiate YIV-906 from commercial Huang Qin Tang. A mechanism based quality control (MBQC) platform, comprising 18 luciferase reporter cell lines and two enzymatic assays based on the mechanism action of YIV-906, could be used to differentiate YIV-906 from commercial Huang Qin Tang. Results of MBQC could be matched to their in vivo activities on irinotecan. In conclusion, the quality control of an herbal product should be dependent on its pharmacological usage. For its specific usage appropriate biological assays based on its mechanism action should be developed for QC. Chemical fingerprints comparison approach has limitations unless irrelevant chemicals have been filtered out. Additionally, using a similarity index is only useful when relevant information is used. A MBQC platform should also be applied on other herbal products.

PHY906(KD018), an adjuvant based on a 1800-year-old Chinese medicine, enhanced the anti-tumor activity of Sorafenib by changing the tumor microenvironment.

PHY906 (KD018) is a four-herb Chinese Medicine Formula. It has been shown to potentially enhance the therapeutic indices of different class anticancer agents in vivo. Here, PHY906 is reported to enhance the anti-tumor activity of Sorafenib in nude mice bearing HepG2 xenografts. Among the four herbal ingredients of PHY906, Scutellaria baicalensis Georgi (S) and Paeonia lactiflora Pall (P) are required; however, S plays a more important role than P in increasing tumor apoptosis induced by Sorafenib with an increase of mouse(m)FasL and human(h)FasR expression. PHY906 may potentiate Sorafenib action by increasing hMCP1 expression and enhancing infiltration of macrophages into tumors with a higher M1/M2 (tumor rejection) signature expression pattern, as well as affect autophagy by increasing AMPKα-P and ULK1-S555-P of tumors. Depletion of macrophage could counteract PHY906 to potentiate the anti-tumor activity of Sorafenib. It was reported that tumor cells with higher levels of ERK1/2-P are more susceptible to Sorafenib, and the S component of PHY906 may increase ERK1/2-P via inhibition of ERK1/2 phosphatase in HepG2 tumors. PHY906 may potentiate the anti-hepatoma activity of Sorafenib by multiple mechanisms targeting on the inflammatory state of microenvironment of tumor tissue through two major ingredients (P and S) of PHY906.

Safety surveillance of traditional Chinese medicine: current and future.

Herbal medicine, including traditional Chinese medicine, has been used for the prevention, treatment, and cure of disorders or diseases for centuries. In addition to being used directly as therapeutic agents, medicinal plants are also important sources for pharmacological drug research and development. With the increasing consumption of herbal products intended to promote better health, it is extremely important to assure the safety and quality of herbal preparations. However, under current regulation surveillance, herbal preparations may not meet expectations in safety, quality, and efficacy. The challenge is how to assure the safety and quality of herbal products for consumers. It is the responsibility of producers to minimize hazardous contamination and additives during cultivation, harvesting, handling, processing, storage, and distribution. This article reviews the current safety obstacles that have been involved in traditional Chinese herbal medicine preparations with examples of popular herbs. Approaches to improve the safety of traditional Chinese medicine are proposed.

The number of intestinal bacteria is not critical for the enhancement of antitumor activity and reduction of intestinal toxicity of irinotecan by the Chinese herbal medicine PHY906 (KD018).

BACKGROUND: The four-herb Chinese medicine PHY906(KD018) has been shown to both enhance the in vivo antitumor activity of irinotecan (CPT-11) against colon cancer tumor allografts and alleviate intestinal toxicity caused by CPT-11. METHODS: Since intestinal bacteria can metabolize CPT-11 and PHY906, we investigated whether intestinal bacteria play a critical role in the in vivo activity of PHY906 in murine Colon-38 tumor-bearing mice. Intestinal bacteria were depleted using streptomycin/neomycin for 10 days before and during treatment with PHY906 and/or CPT-11. qPCR using 16S DNA group-specific primers was used to quantify the levels of the major intestinal bacteria. RESULTS: Both PHY906 and antibiotic treatment changed the profile of intestinal bacteria species: Lactobacillus/Enterococcus, Bacteroides, Clostridium leptum, and E. rectale/C. coccoides. Antibiotic treatment did not alter the ability of PHY906 to enhance the antitumor activity of CPT-11. Antibiotic treatment alone partially reduced animal body weight loss in CPT-11-treated mice. However, PHY906 treatment was able to protect against the body weight loss in the CPT-11/antibiotic treatment group. H&E and PCNA staining of intestine showed that antibiotic treatment partially reduced the intestinal damage caused by CPT-11 but not as effectively as PHY906 treatment. Antibiotic treatment plus PHY906 conferred the most effective protection of intestine histological structure against damage by CPT-11. Both PHY906 and antibiotic treatment inhibited CPT-11-associated inflammatory processes, including infiltration of the intestine by neutrophils, MCP1 and TNF-alpha mRNA expression in the intestine, and expression of pro-inflammatory cytokines G-CSF and MCP1 proteins in the plasma. However, whereas antibiotic treatment suppressed the mRNA expression of two important intestinal progenitor/stem cell markers, Olfm4 and Lgr5, PHY906 treatment resulted in enhanced expression of these two stem cell markers. CONCLUSIONS: Alterations in the population of intestinal bacteria did not affect the abilities of PHY906 to enhance CPT-11 antitumor activity or reduce the intestinal toxicity associated with CPT-11 treatment. The major species of intestinal bacteria do not appear to play a role in PHY906's enhancement of the therapeutic index of CPT-11 in tumor-bearing mice. Thus, patients with different intestinal bacterial profiles may still benefit from PHY906 treatment alongside CPT-11.

First-in-human phase II trial of the botanical formulation PHY906 with capecitabine as second-line therapy in patients with advanced pancreatic cancer.

BACKGROUND: Preclinical studies showed a Chinese botanical formula, PHY906, has synergistic anti-tumor activity with capecitabine. Our phase I study determined maximal tolerated dose of capecitabine 1,500 mg/m(2) BID day 1-7 and PHY906 800 mg BID day 1-4 every 2 weeks. We conducted this phase II study to explore the efficacy of capecitabine and PHY906 in patients with advanced pancreatic cancer who were previously treated with gemcitabine-based regimens. METHODS: Patients with pancreatic cancer and an Eastern Cooperative Oncology Group performance status of 0-2 received PHY906 and capecitabine. Toxicity was assessed per NCI-CTCAE v3.0 and response per response evaluation criteria in solid tumors q 6 weeks. Correlative studies of cytokines, chemokines and growth factors were tested using a cytometric bead array. Quality of life was assessed by utilizing Edmonton symptom assessment system. The primary objective was overall survival. RESULTS: The study enrolled 25 patients. Median progression-free survival (mPFS) was 10.1 weeks (range 0.4-54.1) and median overall survival (mOS) was 21.6 weeks (range 0.4-84.1). Eighteen patients received at least 2 cycles, and achieved mPFS of 12.3 weeks and mOS of 28 weeks. Six-month survival rate was 44 % (11/25). Unsupervised clustering of patients grouped those with shortened survival together by their cytokine profile showed that only IL-6 had a significant difference (p < .001) between short- and long-term survivors. CONCLUSIONS: Capecitabine plus PHY906 provides a safe and feasible salvage therapy after gemcitabine failure for APC. Role of IL-6 in tumor progression and tumor cachexia needs to be investigated with respect to its relation to pathophysiology of pancreatic cancer and development of anti-IL-6 therapeutics.

Ganoderma tsugae Induces S Phase Arrest and Apoptosis in Doxorubicin-Resistant Lung Adenocarcinoma H23/0.3 Cells via Modulation of the PI3K/Akt Signaling Pathway.

Ganoderma tsugae (GT) is a traditional Chinese medicine that exhibits significant antitumor activities against many types of cancer. This study investigated the molecular mechanism by which GT suppresses the growth of doxorubicin-resistant lung adenocarcinoma H23/0.3 cells. Our results reveal that GT inhibits the viability of H23/0.3 cells in vitro and in vivo and sensitizes the growth suppression effect of doxorubicin on H23/0.3 cells. The data also show that GT induces S phase arrest by interfering with the protein expression of cyclin A, cyclin E, CDK2, and CDC25A. Furthermore, GT induces cellular apoptosis via induction of a mitochondria/caspase pathway. In addition, we also demonstrate that the suppression of cell proliferation by GT is through down-regulation of the PI3K/Akt signaling pathway. In conclusion, this study suggests that GT may be a useful adjuvant therapeutic agent in the treatment of lung cancer.

Old formula, new Rx: the journey of PHY906 as cancer adjuvant therapy.

ETHNOPHARMACOLOGICAL RELEVANCE: PHY906, is a decoction of a mixture of the four herbs Scutellaria baicalensis Geori, Glycyrrhiza uralensis Fisch, Paeonia lactiflora Pall, and Ziziphus jujuba Mill. A combination of these four herbs has been in continuous use in traditional Chinese medicine for over 1800 years for treating a variety of gastrointestinal distress such as diarrhea, cramps, nausea, vomiting etc. AIM OF THE STUDY: Preclinical and clinical studies to find PHY906 enhances the therapeutic indices of a broad spectrum of anticancer agents. MATERIALS AND METHODS: Using various mouse tumor xenograft and allograft models, PHY906 has been shown to enhance the chemotherapeutic efficacy of a variety of anticancer agents in various cancers. The PHY906 clinical program consists of five trials in three different types of cancers in both the United States and Taiwan. To date, approximately 150 subjects have received PHY906 in combination with chemotherapy in these five clinical studies. RESULTS: Preclinical studies have shown that PHY906 enhances the therapeutic indices of a broad spectrum of anticancer agents. These findings have been examined in clinical studies for colorectal, liver, and pancreatic cancers when PHY906 is used as an adjuvant to chemotherapy and the results were promising; i.e. PHY906 could reduce chemotherapy-induced toxicities and/or increase chemotherapeutic efficacy. Furthermore, PHY906 did not affect the pharmacokinetics of the chemotherapeutic agents used. Some information has been obtained regarding the mechanism of action of PHY906 in preclinical studies. A comprehensive platform, PhytomicsQC that integrates chemical and biological fingerprints together with a novel biostatistical methodology has been developed to assess the quality of different batches of PHY906. CONCLUSIONS: Over a ten-year period, the multiplex technology "PhytomicsQC" has been used to show batch-to-batch consistency of PHY906 production. Advanced clinical trials are ongoing to demonstrate the effectiveness of PHY906 as adjuvant therapy for cancer patients undergoing chemotherapy.

A phase I study of the chinese herbal medicine PHY906 as a modulator of irinotecan-based chemotherapy in patients with advanced colorectal cancer.

PHY906 is a novel Chinese herbal preparation that has been used in the Orient for over 1800 years to treat a wide range of gastrointestinal side effects including diarrhea, abdominal cramps, vomiting, fever, and headache. Preclinical and clinical studies were conducted to further investigate the biologic and clinical activities of this herbal medicine. To ensure standardization and maintain interbatch reliability of PHY906, high performance liquid chromatography (HPLC) was used to establish a "chemical fingerprint" of PHY906. In vivo preclinical studies using the murine Colon 39 tumor model showed that PHY906 protected against the weight loss associated with irinotecan treatment. In the presence of PHY906, mice were able to tolerate otherwise lethal doses of irinotecan. Significantly improved antitumor activity and overall survival were observed in animals treated with the combination of irinotecan and PHY906 versus irinotecan alone. The combination of PHY906 with irinotecan, 5-fluorouracil (5-FU), and leucovorin (LV) also resulted in at least additive antitumor activity with no increased host toxicity. Based on these in vivo studies, a phase I multicenter, double-blind, randomized, placebo-controlled, dose escalation, cross-over study of PHY906 as a modulator of the weekly, bolus regimen of irinotecan, 5-FU, and LV (IFL) in the first-line treatment of patients with advanced colorectal cancer (CRC) was conducted. The specific objectives of this clinical trial were to determine the safety and tolerability of PHY906 when administered concomitantly with the bolus, weekly IFL regimen. Treatment with PHY906 did not alter the pharmacokinetics of 5-FU, irinotecan, or the irinotecan metabolite SN-38.

A comprehensive platform for quality control of botanical drugs (PhytomicsQC): a case study of Huangqin Tang (HQT) and PHY906.

BACKGROUND: Establishing botanical extracts as globally-accepted polychemical medicines and a new paradigm for disease treatment, requires the development of high-level quality control metrics. Based on comprehensive chemical and biological fingerprints correlated with pharmacology, we propose a general approach called PhytomicsQC to botanical quality control. METHODS: Incorporating the state-of-the-art analytical methodologies, PhytomicsQC was employed in this study and included the use of liquid chromatography/mass spectrometry (LC/MS) for chemical characterization and chemical fingerprinting, differential cellular gene expression for bioresponse fingerprinting and animal pharmacology for in vivo validation. A statistical pattern comparison method, Phytomics Similarity Index (PSI), based on intensities and intensity ratios, was used to determine the similarity of the chemical and bioresponse fingerprints among different manufactured batches. RESULTS: Eighteen batch samples of Huangqin Tang (HQT) and its pharmaceutical grade version (PHY906) were analyzed using the PhytomicsQC platform analysis. Comparative analysis of the batch samples with a clinically tested standardized batch obtained values of PSI similarity between 0.67 and 0.99. CONCLUSION: With rigorous quality control using analytically sensitive and comprehensive chemical and biological fingerprinting, botanical formulations manufactured under standardized manufacturing protocols can produce highly consistent batches of products.

The four-herb Chinese medicine PHY906 reduces chemotherapy-induced gastrointestinal toxicity.

PHY906, a four-herb Chinese medicine formula first described 1800 years ago, decreases gastrointestinal toxicity induced by the chemotherapeutic drug CPT-11 (irinotecan), as shown in a phase I/II clinical study. Similarly, in a murine colon 38 allograft model, PHY906 increased the antitumor activity of CPT-11 while decreasing animal weight loss caused by CPT-11. Here, we have further examined the effect of PHY906 on the intestinal toxicity caused by CPT-11 in mice. PHY906 did not protect against the initial DNA damage and apoptosis triggered by CPT-11 in the intestine, but by 4 days after CPT-11 treatment, PHY906 had restored the intestinal epithelium by promoting the regeneration of intestinal progenitor or stem cells and several Wnt signaling components. PHY906 also potentiated Wnt3a activity in human embryonic kidney-293 cells. Furthermore, PHY906 exhibited anti-inflammatory effects in mice by decreasing the infiltration of neutrophils or macrophages, tumor necrosis factor-alpha expression in the intestine, and proinflammatory cytokine concentrations in plasma. Chemical constituents of PHY906 potently inhibited nuclear factor kappaB, cyclooxygenase-2, and inducible nitric oxide synthase. Our results show that the herbal medicine PHY906 can counteract the toxicity of CPT-11 via several mechanisms that act simultaneously.

Phase I/II study of PHY906/capecitabine in advanced hepatocellular carcinoma.

PHY906 is a Chinese medicine formula with claims for the treatment of severe gastrointestinal distress. PHY906 enhanced the therapeutic index of various chemotherapeutic agents in human hepatocellular carcinoma xenografts. Accordingly, here a phase I/II clinical study was conducted with the combination of capecitabine in patients with advanced, unresectable hepatocellular carcinoma. More than 60% of patients had either stable disease or better after two treatment cycles. Median overall survival was 9.2 months. Asian patients had a higher median overall survival (16.5 months) than non-Asian patients (6.2 months, p=0.03). Patients' quality of life did not deteriorate significantly during treatment. This finding supported further investigation of PHY906 as an adjuvant therapy of capecitabine in a larger hepatocellular cancer population.

Liquid chromatography/mass spectrometry analysis of PHY906, a Chinese medicine formulation for cancer therapy.

PHY906 is a Chinese medicine formulation prepared from four medicinal herbs for adjuvant cancer chemotherapy. In this paper, liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOFMS) was used to clarify the chemical composition of PHY906. The aqueous extract of PHY906 was separated on a Waters Atlantis C(18) column, and was eluted with acetonitrile/0.05% (v/v) formic acid. The separated compounds were identified with pure standards, or tentatively characterized by analyzing their mass spectra recorded in both negative and positive ion polarity modes. Further structural information was obtained from in-source fragmentation. Based on the LC/MS analysis, we proposed the structures for 64 bioactive compounds, including flavonoids, triterpene saponins, and monoterpene glycosides. All the compounds identified from PHY906 were further assigned in the four individual herbs, and some of them are reported for the first time.

Increased anti-P-glycoprotein activity of baicalein by alkylation on the A ring.

The aqueous extract of Scutellariae baicalensis Georgi has inhibitory activity against P-gp 170, a multiple drug resistant gene product. Baicalein, one of the major flavones, was found to be responsible for this activity. The hydroxyl groups of the A ring of baicalein were systematically alkylated in order to assess the effect of such modifications on the activity against P-gp 170. The impact of the baicalein modifications on activity against the growth of a human nasopharyngeal cancer cell line KB and its P-gp 170 overexpressing cell line KB/MDR were also examined. The results indicate that alkylation of R5 of baicalein does not have a major impact on the interaction with P-gp 170, whereas alkylation of R6 or R7 alone or both, could enhance the interaction of baicalein with P-gp 170 as well as the amount of intracellular accumulation of vinblastine, a surrogate marker for the activity of P-gp 170 pump of KB/MDR cells. In this case, the optimal linear alkyl functionality is a propyl side chain. These modifications could also alter the activity of compounds inhibiting cell growth. Among the different compounds synthesized, the most potent molecule against P-gp 170 is 5-methoxy-6,7-dipropyloxyflavone (23). Its inhibitory activity against P-gp 170 is approximately 40 times better, based on EC50 (concentration of the compound enhancing 50% of the intracellular vinblastine accumulation in the KB/MDR cells) and 3 times higher, based on Amax (the intracellular vinblastine accumulation of the KB/MDR cells caused by the compound) as compared to baicalein. Compound 23 is also a more selective inhibitor than baicalein against P-gp 170, because its cytotoxicity is less than that observed for baicalein. The growth inhibitory IC50 of compound 23 against KB and KB/MDR cells are about the same, suggesting that compound 23 is unlikely to be a substrate of P-gp 170 pump. Acetylation of R6, R7 or both could also decrease EC50 and increase Amax. Acetylated compounds are more toxic than baicalein, and their potency against cell growth is compromised by the presence of P-gp 170, suggesting that these compounds are substrates of P-gp 170. Benzylation of R6 or R7 but not both also enhanced anti-P-gp170 activity and potency against cell growth; however, the presence of P-gp 170 in cells did not have an impact on their sensitivity to these molecules, suggesting that the benzylated compounds are inhibitors but not substrates of P-gp 170, and perhaps have a different mechanism of action. In conclusion, the substitutions of R6 and R7 hydroxyl groups by alkoxy groups, acetoxy groups, or benzyloxy groups could yield compounds with different modes of action against P-gp 170 with different mechanisms of action against cell growth.