Extravasation can present serious accidental complication of intravenous drug application. While monoclonal antibodies do not show the necrotic potential of cytotoxic chemotherapy drugs, considerable inflammatory toxicity can occur, necessitating standardized operating procedures for the management of their extravasation. Here, we report the clinical course and management of dinutuximab beta extravasation in a 3-year-old child. Dinutuximab beta is a chimeric monoclonal antibody targeting the GD2 disialoganglioside on the surface of neuroblastoma cells that has in recent years gained significant importance in the treatment of high-risk neuroblastoma, now contributing to both first- and second-line therapy protocols. The dinutuximab beta extravasation reported here occurred when the patient received the antibody cycle as a continuous infusion over a 10-day period after haploidentical stem cell transplantation for relapsed high-risk neuroblastoma. The extravasated dinutuximab beta caused local pain, swelling, and hyperemia accompanied by fever and an overall deterioration in the general condition. Laboratory diagnostics demonstrated an increase in C-reactive protein level and total white blood cell count. Clinical complication management consisted of intravenous fluid therapy, local dabbing with dimethyl sulfoxide (DMSO), analgesia with dipyrone, as well as application of intravenous antibiotics to prevent bacterial superinfection in the severely immunocompromised host. The patient considerably improved after six days with this treatment regimen and fully recovered by day 20.

Background: The use of anthroposophic medicine (AM) is popular in Central Europe, especially in German-speaking countries. Although these therapies are judged to be beneficial by many patients, there are few data with regard to the safety and efficacy in pediatric oncology. Several theoretical concerns have been published with regard to tumor enhancement or promotion of metastatic dissemination due to mistletoe. To test the indirect safety of supportive anthroposophic treatment accompanying the first-line treatment in children with medulloblastoma in this respect we performed a retrospective matched-pair analysis of patients with medulloblastoma treated by standard first-line radiochemotherapy with or without a concomitantly applied panel of AM including mistletoe. The question was whether the effectiveness of the first-line therapy is altered by AM.Procedure: Seventeen patients with AM were matched in a 1: 2 ratio with 34 patients from the database of the German HIT study group with regard to the criteria of diagnosis, age, status of metastatic dissemination, resection status, and first-line therapy.Results: The overall survival after 10 years was 58.33% for the AM group and 57.14% for the control group, that is, showing no statistically significant difference (stratified Cox regression; P = 0.6023). Event-free survival (including metastases) also did not differ between the groups (stratified Cox regression; P = 0.4275).Conclusions: AM consisting of different combinations of specific pharmacologic and nonpharmacologic interventions seems to be safe with respect to any potential negative impact on the first-line therapy. There is no evidence with regard to tumor enhancement. The effectiveness of the supportive AM cannot be assessed on the basis of these data.

PURPOSE:Due to the severe problems accompanied with multiple drug resistance (MDR), agents that can induce apoptosis independently of death-suppressing proteins are required. Here, we show that the ferrocene derivative HUNI 068 is active against cancer cells and overcomes different mechanisms of multiple drug resistance (MDR). METHODS:Proliferation inhibition was determined by using a CASY(®)CellCounter. DNA fragmentation assay and annexin-V/PI binding assays measured apoptosis, and necrosis was excluded by LDH-release assay. Drug-resistant cell lines were generated to test the ability to overcome MDR. By real-time PCR, alterations in gene expression of treated cells were analyzed. The apoptosis pathway was investigated by immunoblotting and measurement of mitochondrial membrane permeability transition. RESULTS:HUNI 068 leads to proliferation inhibition and apoptosis mediation, but only minimal necrosis induction. Healthy leukocytes seem to be less affected than cancer cells. The compound overcomes drug resistance to vincristine and daunorubicin. Independence of p-glycoprotein and Bcl-2 overexpression is probable, and upregulation of the anti-Bcl-2 protein harakiri was seen. Combined treatment with vincristine leads to synergistic effects. In different primary tumor cells, HUNI 068 achieved acceptable effects where tolerance to some conventional drugs was shown. Induction of apoptosis is FADD-independent, but associated with a reduced mitochondrial membrane potential and activation of caspase-9, indicating the intrinsic apoptosis pathway via mitochondria. CONCLUSIONS:HUNI 068 is a promising new compound with activity even against MDR tumor cells. Further investigations into the class of ferrocene-derived agents might reveal compounds with improved activity for a more specific and safe anti-cancer therapy.

We demonstrate the cytotoxic potential of the Schiff base iron complex [FeIII(salophene)Cl] in vitro and ex vivo and illustrate its ability to overcome multiple drug resistance in vincristine and daunorubicine resistant leukemic cells (Nalm-6). Treatment of lymphoma cells (BJAB) with [FeIII(salophene)Cl] led to the exclusion of unspecific necrosis, a concentration-dependent inhibition of proliferation and a specific apoptotic cell death. We further detected a significant loss of the mitochondrial membrane potential in lymphoma cells and an up- and downregulation of various apoptosis relevant genes, respectively, indicating the involvement of the intrinsic mitochondrial pathway.

The novel luminescent gold(I) complex [N-(N',N'-dimethylaminoethyl)-1,8-naphthalimide-4-sulfide](triethylphosphine)gold(I) was prepared and investigated for its primary biological properties. Cell culture experiments revealed strong antiproliferative effects and induction of apoptosis via mitochondrial pathways. Biodistribution studies by fluorescence microscopy and atomic absorption spectroscopy showed the uptake into cell organelles, an accumulation in the nuclei of tumor cells, and a homogeneous distribution in zebrafish embryos. In vivo monitoring of vascularisation in developing zebrafish embryos revealed a significant anti-angiogenic potency of the complex. Mechanistic experiments indicated that the inhibition of thioredoxin reductase (based on the covalent binding of a gold triethylphosphine fragment) might be involved in the pharmacodynamic behavior of this novel gold species.

BACKGROUND:Helleborus niger is used in the adjuvant treatment of different tumors in anthroposophical medicine. Indications include various types of brain tumors in children, as well as prostate cancer, leukemia and lymphoma. Our aim was to investigate the therapeutic effects of these extracts apart from the traditional use. PROCEDURES:: We used an aqueous whole plant extract of H. niger in different cancer and leukemia cell lines and primary cells of patients with childhood ALL and AML and identified the main mechanisms of action. RESULTS:A strong inhibition of proliferation is caused by specific apoptosis induction, which is executed via the mitochondrial pathway and caspase-3 processing. Apoptosis could be detected in lymphoma (BJAB), leukemia (Reh, Nalm6, Sup-B15) and melanoma (Mel-HO) cells and overcomes a Bcl-2-mediated block of apoptosis. In primary cells of patients with childhood ALL and AML, which were partly poor responding to doxorubicin and daunorubicin, a strong apoptosis induction was determined. In combination with the vinca alkaloid vincristine, strong synergistic effects were detected in BJAB cells. CONCLUSION:We demonstrate in vitro efficacy of H. niger extract in cells of hematological malignancies; these studies should encourage in vivo experiments.

Viscum album (Mistletoe) is one of the most widely used alternative cancer therapies. Aqueous mistletoe extracts (MT) contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. Although MT is widely used, there is a lack of scientifically sound preclinical and clinical data. In this paper, we describe for the first time the in vivo efficacy and mechanism of action of MT in lymphoblastic leukemia. For this purpose, we first investigated both the cytotoxic effect and the mechanism of action of two standardized aqueous MTs (MT obtained from fir trees (MT-A); MT obtained from pine trees (MT-P)) in a human acute lymphoblastic leukemia (ALL) cell line (NALM-6). MT-A, MT-P and ML-I inhibited cell proliferation as determined by Casy® Count analysis at very low concentrations with MT-P being the most cytotoxic extract. DNA-fragmentation assays indicated that dose-dependent induction of apoptosis was the main mechanism of cell death. Finally, we evaluated the efficacy of MT-A and MT-P in an in vivo SCID-model of pre-B ALL (NALM-6). Both MTs significantly improved survival (up to 55.4 days) at all tested concentrations in contrast to controls (34.6 days) without side effects.

Despite advances in chemotherapeutic treatment of hematological diseases in childhood, there is significant rate of relapse with high death rates, so that research in new therapeutic molecules is on demand to combat resistance. Search for low molecular weight inhibitors in plant material therefore offers a wide spectrum of new anticancer compounds. Tryptanthrin is found in several plants like Isatis tinctoria or Polygonum tinctorium. Our experiments revealed that NT1, the modified 8-Nitro-3-chloro-derivative of tryptanthrin, specifically induced apoptosis in BJAB, Nalm6, Reh, Sup-B15 and p388 cells, as evidenced by DNA fragmentation, Annexin/PI staining, dissipation of the mitochondrial membrane potential measured via flow cytometry and processing of caspase-3 by Western Blot analysis. These events occurred in the absence of cellular lysis, i. e. in the absence of release of lactate dehydrogenase from the cells, thereby excluding NT1-induced necrosis. A dose-dependent inhibition of proliferation up to 96% hereby precedes the DNA fragmentation and apoptosis. In contrast, healthy leucocytes were not affected by treatment with NT1. NT1-induced cell death was furthermore functionally characterized by the use of different cellular model systems. Investigating control vector-(pcDNA3-mock-transfected) and pcDNA3-FADDdn-transfected BJAB cells, which were stably transfected with a dominant-negative FADD mutant lacking the N-terminal death effector domain, revealed that NT1-induced apoptosis is mainly executed via the CD95-dependent cascade. These findings were confirmed by real-time PCR, which unveiled an up to 8-fold increase of caspase-8, 20-fold upregulation of the FAS ligand and the members 9, 11B and 25 of the TNF receptor superfamily during NT1-induced apoptosis. The strong inhibitor of apoptosis Bcl-2 often leads to severe drug resistance in treatment of malignancies. Melanoma cells transfected with the pIRES plasmid (MelHO/pIRES) and the Bcl-2 cDNA in pIRES (Mel-HO/Bcl-2), which in consequence 30-fold overexpress the Bcl-2 protein, were incubated with NT1 for 72h. Apoptosis showed to be independent on increased Bcl-2 levels, which is in line with an upregulation of the anti-Bcl-2 protein Harakiri during NT1 treatment measured by real-time PCR. Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in pediatric leukemia. Recent studies have demonstrated that members of the inhibitor of apoptosis (IAP) family proteins like survivin or XIAP are upregulated in pediatric ALL or AML. XIAP mRNA expression was found to be 160-fold down regulated in BJAB cells treated with NT1 as compared to untreated cells. Thus, NT1 is able to sensitize resistant tumor cells to cytostatic therapy in combined treatment with other drugs as shown in further experiments. Analyzing primary leukemia cells from 24 children with de novo ALL, 4 with relapsed ALL and 6 with newly diagnosed AML, we could demonstrate that NT1 overcomes drug resistance against daunorubicin (p<0.05) ex vivo. First in vivo experiments in a lymphoma NOD/SCID mouse model showed a significant decrease in tumor growth after two 5-day blocks of oral NT1 monotherapy (200mg/kg/BW) (p<0.05). No bodyweight loss or severe side effects could be detected. Taken together, the tryptanthrin derivative NT1 comprises a totally new, very promising class of cytostatic agents for lymphoma and leukemia therapy. Future studies will aim to elucidate the target structures of this compound.

Helleborus niger, also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In complementary medicine Helleborus niger is used as adjuvant drug in the treatment of non-metastasised and metastasised forms of bronchial cancer, abdominal tumours and prostate cancer. It is also applied in myeloproliferative diseases like Hodgkin and Non-Hodgkin lymphoma, leukaemic disorders and AIDS- related diseases like the Kaposi sarcoma. Until now, there is no clinical or preclinical data regarding the effects of Helleborus niger in vivo, ex vivo or in vitro. For this purpose, we investigated the cytotoxic effects of four different standardized aqueous Helleborus niger extracts from the companies Hiscia and Helixor on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of Helleborus niger. After 4h of treatment with the extracts no significant LDH release was measured, thus excluding an unspecific, necrotic damage of the cell membrane. After 24h a dose dependent inhibition of proliferation up to 69% could be found and after 48h a distinction into early (45,2%) and late apoptotic (45,5%) cells was detected via Annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA fragments after 72h, once more identifying apoptosis as cause of the cell death. In the Western Blot analysis a processing of Caspase-3 could be found after 36 h incubation with the extract. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the three human acute lymphoblastic leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. The apoptosis induction caused by the root extract was higher than the apoptotic cell death in the other extracts. There are two major pathways of apoptosis, the extrinsic pathway via death receptors like FADD and the intrinsic pathway via the mitochondria. In BJAB cells a breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition was detected after 48h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we found a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency of Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. In contrast, apoptosis induction by Helleborus niger seems to be independent of Smac overexpression, which could be shown in Jurkat cells. In combination with the vinca alkaloid vincristine, which is used in the treatment of ALL, a synergistic effect could be detected. The apoptosis induction was up to 16% higher in combination than in the single treatment. Finally, we evaluated the effect on primary leukemia cells ex vivo. Interestingly, we could show a significant apoptosis induction in primary leukemia cells from 2 patients with ALL or AML in childhood, which were resistant to the treatment with the anthracycline doxorubicin. For the first time, we were able to show that extracts of Helleborus niger induce apoptosis in different cancer cell lines and primary leukemia cells. Apoptosis is executed via the intrinsic pathway and is independent of Smac overexpression. Thus, we present an interesting baseline for the design of upcoming in vivo experiments or clinical trials.

[reaction: see text] As a contribution to bioorganometallic chemistry, an enantioselective synthesis of novel carbocyclic nucleoside analogues with a ferroceno-cyclopentene backbone was developed. Diastereoselective cuprate 1,4-addition or Mukaiyama-Michael addition to a planar-chiral enoate (ethyl (E)-2-[2-methoxycarbonyl-ferrocenyl]-acrylate) allowed for the introduction of different side chains (RCH(2)). Other important steps include a Dieckmann cyclization and the attachment of the nucleobase (NB) in an iron-assisted S(N)1 reaction. Some of the target compounds were shown to exhibit significant apoptosis-inducing activity (LD(50) = 10-20 microM) against tumor cells.

In spite of the relatively good prognosis for de novo acute lymphoplastic leukemia (ALL), the survival rate for relapsed ALL is only 40%. The therapy of relapsed ALL and secondary leukemias with the common cytostatic drugs often show a poor response. Therefore new substances are required to lead multiple drug resistant leukemia cells into cell death. Nucleoside analogs play a very important role in anti-leukemic therapy. Here we present a new, own-synthesized class of Ferrocenyl Nucleosides, a type of organometallic sandwich compounds, where two cyclopentadenyl rings bind to a central Fe2+ ion. We could show anti-leukemic and anti-cancer activity in different leukemia and tumor cell lines and primary cells from children with primary ALL and AML. The apoptosis induction was efficient in low micro molar concentrations (LC50 = 10–20μM). The DNA fragmentation has been extraordinary high (LC90 = 30–50μM). Ex vivo experiments showed that some Ferrocenyl Nucleoside Analogs, like PJ184, are able to overcome resistance against anthracyclines like Doxorubicin (p<0,001), Daunorubicin (p<0,001) and Idarubicin (p<0,001) or alkaloids like Vincristin (p<0,001). Furthermore Ferrocenyl Cytosin Analog (PJ184) mediated apoptosis induction is significant higher than apoptosis induced by the clinical used nucleoside analogs like Fludarabin (p<0,001), Cytarabine (p<0,001) and Cladribine (p<0,001; all p-values by ANOVA test). These results have been achieved by testing primary lymphoblasts of seven primary ALL patients after incubation for 60h with LC50 determined in leukemic cell lines. The testing of one AML revealed similar results. Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation, mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. The cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. In order to characterize the Ferrocenyl Nucleoside apoptosis pathway we used cellular model systems with deficiencies in different molecular parts of the apoptosis cascade. Apoptosis reveals to be independent of CD95 receptor and the pro-apoptotic factor SMAC, but shows a dependency of the apoptosis inhibitor Bcl-2. We also investigated a loss of mitochondrial membrane potential, which indicates the involvement of the mitochondrial apoptosis machinery. Taken together, these new class of Ferrocenyl Nucleoside Analogs revealed very promising in vitro and ex vivo results and may lead to a new approach in antileukemic therapy. In vivo experiments will be performed soon.

Mistletoe (Viscum album) is one of the most used alternative cancer therapies applied as monotherapy or in combination with conventional therapies. Anti-tumor effects of mistletoe (MT) extracts were related to cytostatic and immunomodulatory effects observed in vitro. Aqueous MT extracts contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. The MT lectins inhibit protein biosynthesis by inactivating the 60S ribosomal subunit. Mistletoe lectin-I (ML-I) is one important apoptosis inducing compound. It is a heterodimer that consists of a cytotoxic A-chain (ribosome inactivating protein, RIP type 1) linked by a carbohydrate binding B-chain for cellular lectin uptake. However, although MT is widely used, there is a lack of scientific preclinical and clinical data. Here, we describe for the first time efficacy and mechanism of MT extracts against lymphoblastic leukemia in vitro and in vivo.