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In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 2655-2655

Abstract: Abstract 2655 Poster Board II-631 Introduction: The predictive power of measuring the effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients, called Individualized Tumor Response Testing (ITRT), has been recently further validated in a clinical trial, the UK's LRF CLL4 trial (Bosanquet ASH 2007). It predicts resistance better than sensitivity. We present a novel approach to ITRT based on measuring drug induced apoptosis of tumor cells in whole blood ex vivo (in vitro using freshly extracted samples). It uses a novel automated flow cytometry platform (ExviTech) capable of evaluating hundreds of drugs and drug combinations used in current treatment protocols, and can address the significant scaling of potential future protocols induced by a number of new drug approvals in each indication. Patients and Methods: We evaluated 47 samples of peripheral blood or bone marrow from patients diagnosed with hematological malignancies: 20 chronic Lymphocytic Leukemia (CLL), 14 Acute Lymphoblastic Leukemia (ALL), 7 Multiple Myeloma (MM), and 6 Acute Myeloblastic Leukemia (AML). After informed consent, samples, collected into heparin, were processed the same or the next day. Whole blood was diluted and incubated with drugs for 24 and 48 hours. Whole blood was used to retain erythrocytes and serum proteins enabling more clinically relevant physiological conditions. Three types of drugs were tested: 1) Approved drugs for each indication, including all possible pair wise combinations, and combinations administered within current and experimental protocols as advised by the PETHEMA groups in Spain. 2) Concomitant medicines (Con-Meds), including alternative drugs within the same class of antacids, antiemetics, etc… to test whether they may also induce apoptosis 3) Drugs in clinical trials, preferentially Phase III drugs, alone and in combination with approved drugs, which may form the basis of future treatment protocols. Drugs were plated at a final concentration equivalent to their reported plasma Cmax concentration. Synergistic drug combinations were identified as one drug potentiating the effect of the other. Results: The efficacy of each drug and combination tested was categorized as highly resistant, intermediate or highly sensitive. Highly resistant drug results were contraindicated. Among the highly sensitive treatments ex vivo, often those that effectively killed all malignant cells, we selected those whose drugs were significantly less toxic as treatment guidelines, highlighting those treatment protocols that act faster ex vivo (24 vs 48 hours) and/or show synergistic combinations. The final result was a set of multiple reasonable ex vivo options for hematologists. The efficacy of individual drugs varied notably from patient to patient, , as reported earlier by other methods. Drug-drug combinations show surprising results. Some combinations, effective at high doses, kill 80% of malignant cells when combined in low concentrations at which the individual drugs kill only 10%20% of these cells. On the contrary, many drug combinations were antagonistic, effectively turning them into cytoprotectors and the patient into potential resistance. Specific combinations that show consistent efficacy across samples are indicative of potential new protocols. Surprisingly, for a proportion of patients, some of the Con-Meds were highly efficient in killing malignant cells selectively. For example, in a particular CLL patient an antacid and an antiviral drug had similar efficacies as the best approved cytotoxic drugs. In other patients, drugs still in clinical trials showed high sensitivity and highly selective apoptosis – suggesting that those patients could be referred for inclusion into these trials, which could represent new alternatives especially for refractory patients with few therapeutic options available. Conclusions: We have developed a Personalized Medicine Multi-Drug ex vivo test, evaluating the efficacy of hundreds of drugs and drug combinations in whole blood. This scale could address the predictable expansion of multi-drug potential treatments as the existing extensive drug pipeline delivers new drug approvals, exploring hundreds of new protocols ex vivo. Promising results obtained ex vivo (in vitro using freshly extracted samples) need to be verified in clinical trials. Disclosures: Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech SL: Employment. Espinosa:Vivia Biotech SL: Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Gorrochategui:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. Gonzáalez:Vivia Biotech SL: Consultancy. Bosanquet:Vivia Biotech SL: Consultancy. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V114.22.2655.2655

Language: English

Publisher: American Society of Hematology

Publication Date: 2009

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 4414-4414

Abstract: Abstract 4414 Introduction Discovery of novel non cytotoxic drugs for cancer focuses on targets selectively expressed in malignant cells, only testing at the end if they are toxic to patients. We have developed a novel approach to discover these drugs starting at the end; we screen 2.000 approved drugs with proven safety, directly on freshly extracted (ex vivo) blood samples of patients with Chronic Lymphocytic Leukemia (CLL). These screens are enabled by a novel technology platform based on automated flow cytometry we call ExviTech for ex vivo technology. Patients and Methods All screening studies were performed directly on either peripheral blood or bone marrow samples from 44 patients diagnosed with various subtypes of B-cell malignancies, after informed consent. Patient samples were diluted and plated with each of the 2.000 drugs individually, retaining the erythrocyte population and serum proteins to enable clinically relevant concentrations. The experimental assay was setup the same or a day after sample extraction. Each sample was diluted to achieve a leukemic cell concentration of approximately 3,000 cells/μl; then 45μl of the suspension is added to each well of 96-well plates that contain the pharmacological agents (final concentration of 30μM). The compound plates were then sequentially incubated for 24 hours at 37°C with 5% CO2 for screening (sterile conditions). After incubation, the erythrocytes were lysed and the leucocytes incubated with Annexin V-FITC, anti-CD45-APC and anti-CD19-PE added to each well. The plates were then transferred to an automated flow cytometry system where the contents of each well were aspirated and analyzed by a CyAn flow cytometer. Candidates from the primary screens were validated in additional samples with dose-responses, combinations with approved drugs, multiple incubation times, etc… Results Analyzing primary screens from 24 CLL patients, three related compounds (Vivia007, Vivia008 and Vivia009) were found to consistently induce apoptosis of nearly all leukemic B-cells from most of the patient samples diagnosed with B-cell chronic lymphocytic leukemia at levels equal to or greater than known CLL active cytotoxic agents. Notably, these candidates are equally effective against samples of p53 mutated patients. These 3 drugs are pharmacologically me-too drugs sharing the same target and mechanism of action, and are non cytotoxic drugs with a known and good safety profile, administered to millions of patients over many years. Validation experiments were done on 20 additional CLL patients and Vivia009 emerged as the most effective agent with an average EC50 of 18.2μM. The mechanism of action is different than the known mechanism of Vivia009 and its class members for their approved indications. Consistent with this observation, only 3 of 15 members of the same pharmacological drug class were efficacious against CLL malignant cells. All 3 Vivia′s candidates were equally efficacious against other B-Cell Malignancies such as B-ALL (pediatric and adult), and Multiple Myeloma. These drugs are not effective in their current oral formulation and require a novel intravenous formulation. Interestingly, kinetics of induction of apoptosis were faster for Vivia009 than for fludarabine, cyclophosphamide and mitoxantrone. Vivia009 requires only 1 hour of incubation with fresh cells to induce maximal apoptosis. This timeline is less than the 3 hours in which Vivia009 was found present at high concentrations in bone marrow of rats using a single intravenous bolus. Thus, Vivia009 seems to fulfill the pharmacokinetic criteria to eliminate all leukemic cells with a single intravenous bolus, which would be a major advantage over current treatments (5-days fludarabine or 3 days FCR). Animal models are ongoing to confirm the non cytotoxic nature of the candidates in the novel IV formulation and the fewer days needed to reach remission, both compared with fludarabine monotherapy. Conclusions In summary, our results demonstrate the potential of the ExviTech technology platform as a successful model for the systematic search of new uses for already existing approved drugs directly on patient samples of hematological malignancies. A new drug candidate with excellent safety profile has been identified with similar efficacy ex vivo as the best approved cytotoxic drugs, which is a non-cytotoxic drug with fast kinetics that might enable significantly safer and shorter treatments. Disclosures: Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Ana Espinosa, Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. González:Vivia Biotech SL: Consultancy. Dominguez-Gil:Vivia Biotech SL: Consultancy. Troconiz:Vivia Biotech SL: Consultancy. Rodriguez de Fonseca:Vivia Biotech SL: Consultancy. Saunders:Vivia Biotech: Consultancy. Montejo:Vivia Biotech SL: Consultancy. Caveda:Vivia Biotech SL: Employment. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V114.22.4414.4414

Language: English

Publisher: American Society of Hematology

Publication Date: 2009

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7