Quality of life is improved in breast cancer patients by Standardised Mistletoe Extract PS76A2 during chemotherapy and follow-up: a randomised, placebo-controlled, double-blind, multicentre clinical trial.

The objective of this randomised, multicentre, double-blind clinical trial was to investigate the impact of PS76A2, an aqueous mistletoe extract standardised to mistletoe lectins, on quality of life (QoL) in breast cancer patients. A total of 352 patients were randomly allocated to 2 groups receiving PS76A2 (15 ng mistletoe lectin/0.5 ml) or matching placebo twice weekly for 4 to 6 cycles of CMF (cyclophosphamide, methotrexate, fluorouracil) chemotherapy followed by 2 months follow-up. The primary efficacy end-point was the change from baseline of 3 FACT-G subscales (physical, emotional and functional well-being) during the fourth CMF cycle. Secondary measures included GLQ-8 (8 linear analogue self-assessment scales), Spitzer's uniscale and haematological variables. The main variables of safety analysis were adverse events, including injection site reactions and clinical laboratory tests. The results showed that physical, emotional and functional well-being improved upon PS76A2, but deteriorated following placebo. The treatment differences were statistically significant for the 3 subscales as well as for the summary score FACT-G, which was analysed as O'Brien's rank sum of its 3 subscales: The total score increased by 4.40 +/- 11.28, indicating a higher QoL after PS76A2, but decreased by 5.11 +/- 11.77 with placebo (p<0.0001). The GLQ-8 sum of 8 LASA scales was analysed as a summary score of GLQ-5 (sum of item nos. 1, 5, 6, 7, 8) and GLQ-3 (sum of item nos. 2, 3, 4). GLQ-5 characterises typical aspects of QoL, while GLQ-3 consists of 3 side-effects of CMF (feeling sick, numbness or pins and needles, loss of hair). GLQ-5 decreased by 42.9 +/- 125.0 upon PS76A2, indicating an improvement in QoL, but increased by 60.3 +/- 94.0 upon placebo (p<0.0001). GLQ-3 deteriorated in both groups (PS76A2: 13.9 +/- 52.4; placebo: 34.5 +/- 57.0), but the differences in favour of PS76A2 were, nevertheless, statistically significant (p=0.0007). The total score GLQ-8 improved by 28.9 +/- 154.6 after PS76A2 and deteriorated by 94.8 +/- 141.1 after placebo (p<0.0001). Spitzer's uniscale improved by 12.2 +/- 30.7 upon PS76A2 and deteriorated by 10.8 +/- 26.1 with placebo (p<0.0001). After follow-up without chemotherapy, a significant treatment difference in favour of PS76A2 was determined by means of FACT-G, GLQ-8 and Spitzer's uniscale. PS76A2 was well tolerated in this trial, with the exception of slight local reactions in 17.6% of the PS76A2 group. In conclusion, PS76A2 (15 ng mistletoe lectin/0.5 ml twice weekly) was shown to be safe and effective in improving QoL in breast cancer patients during chemotherapy and follow-up.

Recombinant mistletoe lectin (rML) is successful in treating human ovarian cancer cells transplanted into severe combined immunodeficient (SCID) mice.

The main active constituents carrying the anti-cancer activities of aqueous mistletoe extracts have recently been identified as the mistletoe lectins (MLs). Although three different isolectins have been isolated from plant extracts, molecular biological techniques have revealed the presence of one gene only. Subsequently, recombinant mistletoe lectin (rML) has become available and the aim of the present study was to analyse its anti-cancer potential. SoTü 3 human ovarian cancer cells (2x10(7)) were injected intraperitoneally into SCID mice, while rML treatment was started on the following day. Three experimental groups (n=20 SCID mice) each received every working day an intraperitoneal injection of 30, 150, 500 ng rML per kg body weight, respectively, while 20 SCID mice in the control group received the vehicle solution only. The survival of the animals was taken as the principal outcome measure. In addition, the peritoneal cavity was searched for the presence of tumour cells. Animals were sacrificed when the weight increase due to the development of ascites exceeded 120% of the initial body weight. The treatment continued until day 83 and the surviving animals were sacrificed 84 days after inoculation. In the control group, only two animals survived and were free of tumour at the end of the experiment at 84 days. In contrast, thirteen animals in the 500 ng/kg rML group were still alive and no evidence for the presence of tumour cells in the peritoneum was found. Both, number of animals surviving and survival time were larger in this treatment group. The 30 ng/kg rML group showed an increased number of survivors, whilst the 150 ng rML per kg body weight group revealed the worst survival rates. The results of the present study indicate that rML has potent anti-tumour activity, if administered locally into the peritoneum of a human ovarian cancer harbouring SCID mouse. RML is a macromolecule and its instillation into the peritoneal cavity seems to be particularly effective to inhibit intraperitoneal growth of cancer cells. One explanation might be that altered glycosylation of the cancer cells leads to an increased affinity of rML towards tumour cells. Clinical studies with post-operative instillation of rML in ovarian cancer patients should, therefore, be encouraged to provide clinical evidence for the effectiveness of rML treatment.

Ganglioside treatment and nerve regeneration: a morphological study after nerve crush in rats.

The left sciatic nerve of male Wistar rats was crushed by a standardized technique. From the day of the nerve crush onwards the rats were given a daily subcutaneous injection of 50 mg/kg of a ganglioside mixture. The controls were given an equal volume of phosphate buffer. Morphometric analysis of the sciatic nerve was carried out on days 11 and 15 after the nerve crush. The number of nerve fibres in the rats treated with gangliosides was larger than in the controls on both days and cross-sections through the regenerating fibres showed larger areas of axon and myelin. These differences were statistically significant (P less than 0.05) for most parameters.

Assessment of the genotoxic risk from laxative senna products.

Laxative senna products and several of their specific components have been submitted to a large number of genetic tests. While most studies gave negative responses, results from some of the studies suggest that components of senna products, particularly emodin and aloe-emodin, have genotoxic activity. Assessment of the genotoxicity profile of these substances, in light of other data from animal and human metabolism or kinetic studies, human clinical trials and rodent carcinogenicity studies do not support concerns that senna laxatives pose a genotoxic risk to humans when consumed under prescribed use conditions.

Cellular immunomodulation and safety of standardized aqueous mistletoe extract PS76A2 in tumor patients treated for 48 weeks.

Non-selected tumor patients (n=12) with various solid carcinomas were treated continuously twice weekly over 48 weeks with the aqueous mistletoe extract PS76A2, standardized to active mistletoe lectin. The preparation was applied subcutaneously at a concentration of 15 ng mistletoe lectin per 0.5 ml. Cellular immune response and safety were determined at various times during and after the therapy. In the course of treatment, virtually all the investigated immunoparameters were raised compared to the baseline values at the start of treatment. The statistically significant rises in the cell count of total lymphocytes, monocytes and natural killer (NK) cells was noteworthy. The differences in comparison with the baseline values at the various measuring times during treatment were up to 35%. In the first weeks of treatment at least, the raised cell count of NK cells correlated with the significantly increased cytotoxic activity versus tumor cells ex vivo. The NK factor (product of NK cells and ex vivo activity) was determined to assess the total NK activity more accurately, which rose up to 50% compared to the baseline value. Other lymphatic subpopulations, for instance CD3+, CD8+, CD3+CD4+ and CD3+CD8+ cells, also revealed distinct rises in cell count in the course of treatment. Within 6 weeks after completion of treatment, the overall values dropped again; but for a series of immunoparameters--in particular for the NK cells--they were still raised in comparison to the baseline values. The extensive laboratory diagnostics (haematology, clinical chemistry) showed that treatment with the standardized mistletoe extract PS76A2 was well tolerated by all patients. In single cases, local reactions at the injection sites were of a minor nature and reversible within two days. Summarizing, it can be stated that the standardized mistletoe extract PS76A2 significantly improved the immune status of tumor patients and was administered safely over a long period.

Antitumoral effects of an intravesically applied aqueous mistletoe extract on urinary bladder carcinoma MB49 in mice.

The objective of the present study was to investigate the effects of a locally applied aqueous mistletoe extract (AME) on the growth of urinary bladder carcinoma MB49 in an orthotopic murine model. On day 1, a total of 4 x 10(4) tumor cells was implanted into the bladder of female C57BL/6J mice. The animals were then randomly allocated to three groups of 13 mice each. From day 11 onwards, AME was given intravesically 3 days a week for 4 consecutive weeks at concentrations related to 30 or 300 ng bioactive mistletoe lectin (ML)/ml. The animals received a total volume of 0.1 ml. In the control group, 39% of the mice survived to the end of the scheduled study period in comparison to 69% and 85% in the groups treated with 30 or 300 ng ML/ml, respectively. At necropsy, 80% of the surviving control animals showed a visible solid bladder tumor, whereas only 56% and 18% had tumors in the treated groups. In both cases, the differences were statistically significant at the high concentration in comparison to controls (p < 0.05). A non-significant effect was observed regarding the formation of multiple metastases (40% in controls vs 33% and 18% in the treated groups). From the results, it was concluded that under the conditions described, AME shows antitumoral activity which is considered to be mainly due to the cytotoxic properties of mistletoe lectins, the main effective constituents of mistletoe extracts.

Mistletoe extracts standardized to mistletoe lectins in oncology: review on current status of preclinical research.

Since the identification and characterization of mistletoe lectins as pharmacologically active constituents at the end of the 1980s, research on mistletoe has made substantial advances. Mistletoe extracts are now available that are standardized in terms of the active mistletoe lectins (measured as mistletoe lectin I, ML I). This constitutes an indispensable precondition for reproducible investigations. Preclinical studies have shown that mistletoe extracts standardized in terms of ML I or pure ML I itself have highly potent cytotoxic and immunostimulating effects, predominantly on the cellular immune system. The immunostimulating effect is correlated with the apoptosis of immunologically active cells at low concentrations. Cytotoxic effects on tumor cells are likewise apoptosis-related, but at higher levels necrotic cell death predominates. Due to these properties, mistletoe extracts or pure ML I showed antitumoral activities in different animal models. The objective of this review is to present the current state of preclinical research on standardized mistletoe extracts which hence may be included in the category of rationalphytotherapy.

Anticancer activity of an aqueous mistletoe extract (AME) in syngeneic murine tumor models.

Mistletoe extracts have been used for decades for non-specific stimulation of the immune system in cancer therapy. Mistletoe lectins (ML) have been identified as the active principle with cytotoxic and immunomodulatory potencies. In the present in vivo experiments, the anticancer effects of an aqueous mistletoe extract (AME) were investigated in different subcutaneously growing syngeneic murine tumors such as Renca renal cell carcinoma, C8 colon 38 carcinoma, F9 testicular carcinoma, B16 melanoma and Lewis lung carcinoma. The animals used were immunocompetent mice of different strains (C57BL/6, BALB/c), depending on the type of tumor tested. After tumor transplantation, the mice were treated with AME at dose levels corresponding to 0, 0.3, 3, 30 or 300 ng ML/kg/d by the i.p. or s.c. route for a maximum of 4 consecutive weeks. The tumor volume was determined by serial caliper measurements and expressed relative to controls. Significant tumor growth inhibition was observed with the Renca , C8 colon 38 and F9 testicular carcinomas at 30 and 300 ng ML/kg/d. These findings were confirmed in independent repeat experiments. No inhibitory effects were seen with the Lewis lung carcinoma and B16 melanoma under the conditions described above. In conclusion, AME showed in vivo anticancer activity in different transplantable syngeneic murine tumor models following repeated parenteral treatment. In view of the low dose levels used, the effects are most likely due to the immunostimulatory rather than to the cytotoxic potencies of AME.

The standardised mistletoe extract PS76A2 improves QoL in patients with breast cancer receiving adjuvant CMF chemotherapy: a randomised, placebo-controlled, double-blind, multicentre clinical trial.

Patients with breast cancer receiving adjuvant chemotherapy frequently suffer from a restricted quality of life (QoL) due to the side-effects of chemotherapy and the consequences of coping with the diagnosis. Therefore, the objective of this clinical study was to investigate the impact of PS76A2, an aqueous mistletoe extract standardised to the galactoside-specific mistletoe lectin, on QoL by performing a placebo-controlled trial. Overall, 272 patients with breast cancer receiving adjuvant CMF chemotherapy (cyclophosphamide-methotrexate-fluorouracil) were enrolled and randomised to groups receiving placebo or PS76A2 at concentrations of 10, 30 or 70 ng mistletoe lectin (ML) per ml. The patients received 0.5 ml study medication twice weekly subcutaneously for 15 consecutive weeks (4 CMF cycles). Primary variables were the self-assessment QoL scores GLQ-8 (Global Life Quality) and Spitzer's uniscale. As a result, statistically significant effects on QoL were obtained with the medium dose (15 ng ML/0.5 ml). The treatment difference between the medium dose and placebo with regard to the GLQ-8 sum was 60.8 mm (95% confidence interval: 19.3 to 102.0 mm). The treatment effect for Spitzer's uniscale between the medium dose and placebo was 16.4 mm (95% confidence interval: 6.3 to 26.6 mm). The results on QoL were supported by an increase of T helper lymphocytes (CD4+) and the CD4+/CD8+ ratio (p<0.05). Overall, PS76A2 was well tolerated. Local reactions at the injection sites occurred dose-dependently, but were mild at the low and medium dose levels.

No evidence of stimulation of human tumor cell proliferation by a standardized aqueous mistletoe extract in vitro.

The in vitro antiproliferative or stimulatory activity of an aqueous mistletoe extract (AME) with a defined content of bioactive mistletoe lectin (ML) was investigated in 6 human tumor cell lines, including two melanomas and leiomyosarcomas, each of which had previously been reported to show evidence of growth stimulation if treated with low concentrations of isolated ML. The effects of AME were compared to that of the standard cytotoxic agent adriamycin (ADR) using the well established propidium iodide and sulforhodamin B proliferation assays. The AME concentrations used ranged from 0.5 pg to 5 ng (0.82 fMol-85 pM) bioactive ML/ml in melanoma (HT-144, SK-MEL-28) and leiomyosarcoma (SK-MLS-1, S-UT-1B) cell lines and from 0.1-100 ng ML/ml (1.7 pM-1.7 nM) in MCF-7 breast cancer and SW620 colon carcinoma cell lines, respectively. The influence of AME on cell growth was determined at various time-points from 24 hours to 6 days of exposure. We found a time- and cell line-dependent inhibition of tumor cell growth, but no reproducible stimulation of tumor cell proliferation. Inhibitory concentrations 50% (IC50) for e.g. the SK-MEL-28 melanoma cell line, decreased from 4.1 ng ML/ml at 24 hours to 0.16 ng ML/ml at 72 hours and 0.18 ng ML/ml at 5 days. Our data clearly demonstrate that, by applying scientifically valid methods and procedures, the standardized AME did not stimulate tumor cell proliferation but showed time- and concentration-dependent antiproliferative effects.

Anticancer activity of rViscumin (recombinant mistletoe lectin) in tumor colonization models with immunocompetent mice.

The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe lectin) were investigated in two mouse tumor models. After intravenous inoculation with RAW-117-P or L-1 sarcoma cells in Balb/c mice, rViscumin was given s.c. at non-toxic doses ranging from 0.3 to 150 ng rViscumin/kg. One set of experiments was performed to investigate the survival of rViscumin-treated animals. Another set was carried out to analyze the effect of rViscumin treatment on the number of tumor colonies in infiltrated lungs (RAW-117P) or liver (L-1) and the activation of immune cell subsets, respectively. An overall prolonged survival time after treatment with rViscumin and a reduction in the number of tumor colonies after administration of certain rViscumin doses was observed. Immunophenotyping of the peripheral leukocytes of treated mice revealed increased numbers of T-lymphocytes, pan-NK cells and activated monocytes. The results indicate that rViscumin has antineoplastic properties and might therefore be a promising candidate in cancer therapy.

Lack of emodin genotoxicity in the mouse micronucleus assay.

The study was performed to investigate the potential of emodin (1,3,8-trihydroxy-6-methylanthraquinone) to induce micronuclei in polychromatic erythrocytes (PCEs). Mice of both genders received a single oral dose of 2000 mg emodin/kg and were killed 24 and 48 h later. Bone marrow cells were collected from 5 males and 5 females and 2000 PCEs per animal were scored for the presence of micronuclei. There was no enhancement in the frequency of micronuclei at both preparation intervals when compared to the negative controls. Blood level examinations confirmed the systemic availability of emodin. Plasma levels of up to 190 micrograms emodin/ml represented concentrations being in the concentration range that induced positive responses in several genotoxicity cell culture assays.

Genotoxicity of aloeemodin in vitro and in vivo.

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot text [DBA/2JxNMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.

No induction of chromosomal aberrations in Chinese hamster ovary cells by chrysophanol.

Chrysophanol is an anthraquinone which occurs in several herbal drugs, e.g. senna, a commonly used laxative. As there are only limited data on its clastogenic potential we have investigated its capability to cause chromosomal aberrations in the Chinese hamster ovary cell assay. There were no significant increases in chromosomal aberrations when chrysophanol was tested up to its limit of solubility with or without metabolic activation. We conclude that chrysophanol had no clastogenic activity under the conditions described.

No clastogenic activity of a senna extract in the mouse micronucleus assay.

In previous studies, an analytically well-defined senna extract, commonly used as a laxative, gave positive responses in vitro in the Ames test and in the CHO assay. Therefore, the objective of this study was to investigate the genotoxic activity of the same senna extract in an in vivo genotoxicity assay by means of the generally acknowledged MNT. After administration of an oral dose of 2000 mg senna extract/kg to NMRI mice of both genders, which is equivalent to 119 mg potential rhein/kg, 5.74 mg potential aloeemodin/kg and 0. 28 mg potential emodin/kg, there were no elevated levels of micronuclei in bone marrow cells. Kinetic studies were performed in parallel to demonstrate target organ availability. Highest concentrations in the plasma were reached after 1 h with 3.4 microg rhein/ml and 0.065 microg aloeemodin/ml. In all cases, emodin was below the limit of quantification. From the results, the in vitro clastogenic activity of the senna extract could not be confirmed in the mouse micronucleus assay. Together with further negative in vivo genotoxicity studies with anthranoids, the conclusion can be drawn that there is no indication so far demonstrating a genotoxic risk for patients taking senna laxatives.

An oral carcinogenicity and toxicity study of senna (Tinnevelly senna fruits) in the rat.

Senna (Tinnevelly senna fruits), a known laxative derived from plants, was administered by gavage to Sprague-Dawley (Crl:CD (SD) BR) rats once daily at dose levels of 0, 25, 100 and 300 mg/kg/day for up to 104 consecutive weeks. Based upon clinical signs related to the laxation effect of senna, the highest dose (300 mg/kg/day) was considered to be a maximum tolerated dose. Sixty animals per sex were assigned to the control and dose groups. Assessments included clinical chemistry, hematology, full histology (control and high-dose groups; in addition, low and mid dose: intestinal tract, adrenals, liver, kidneys, brain and gross lesions) and toxicokinetics. The primary treatment-related clinical observation was mucoid feces seen at 300 mg/kg/day. When compared to controls, animals administered 300 mg/kg/day had slightly reduced body weights, increased water consumption and notable changes in electrolytes in serum (increases in potassium and chloride) and urine (decreases in sodium, potassium and chloride). The changes in electrolytes are most likely physiologic adaptations to the laxative effect of senna. At necropsy, dark discoloration of the kidneys was observed in animals in all treated groups. Histological changes were seen in the kidneys of animals from all treated groups and included slight to moderate tubular basophilia and tubular pigment deposits. In addition, for all treated groups, minimal to slight hyperplasia was evident in the colon and cecum. These histological changes, together with the changes seen in the evaluation of clinical chemistry and urine parameters, have been shown to be reversible in a previous 13-week rat study of senna. No treatment-related neoplastic changes were observed in any of the examined organs. Based upon these data, it is concluded that senna is not carcinogenic even after daily administration for 2 years at dosages of up to 300 mg/kg/day in Sprague-Dawley rats.

Acute toxicity of aristolochic acid in rodents.

The acute toxic effects of aristolochic acid (AA) were tested in rats and mice of both sexes. Oral or intravenous administration in high doses was followed by death from acute renal failure within 15 days. Histologically, the predominant features were severe necrosis affecting the renal tubules, atrophy of the lymphatic organs and large areas of superficial ulceration in the forestomach, followed by hyperplasia and hyperkeratosis of the squamous epithelium. The LD50 ranged from 56 to 203 mg/kg orally or 38 to 83 mg/kg intravenously, depending on species and sex.