Clinical Chemistry
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Clinical Chemistry 53: 148-149, 2007; 10.1373/clinchem.2006.080473
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(Clinical Chemistry. 2007;53:148-149.)
© 2007 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Ex Vivo Simulation of Drug Action: Quantification of Drug-Induced mRNA as a Bridge Between Preclinical and Clinical Trials

Masato Mitsuhashi

Hitachi Chemical Research Center, 1003 Health Sciences Road, Irvine, CA 92617, Fax: 949-725-2727, E-mail mmitsuhashi{at}hcrcenter.com.


To the Editor:

The recent tragedy, in which all active drug recipients developed life-threatening multiple organ failure during the phase I clinical trial of superagonistic anti-CD28 monoclonal antibody (TGN1412) (1), raised serious questions regarding the safety of human drug-trial participants. Results obtained in animal models do not always correspond to those observed in humans, and human cells placed in an in vitro culture environment may no longer exhibit physiologic functions that occur under natural conditions. An ideal precautionary method for investigational drugs designed to be administered intravenously is to mix each drug candidate with whole blood in test tubes and evaluate both desired function and unexpected adverse reactions. Such an ex vivo test is completely safe and applicable to drugs acting on leukocytes. The biggest technical challenge, however, is to identify drug actions in whole blood in a short period of time, because longer incubations might be associated with secondary artifacts. Gene expression analysis is an interesting candidate method, because mRNA transcription occurs earlier than protein synthesis and final biological outcomes.

In a recent issue of Clinical Chemistry (2), my group reported a novel assay system that quantified absolute amounts of mRNA in human whole blood by assessing the recovery of purified mRNA and the efficiency of cDNA synthesis in each sample. In this system, which used 50 µL of whole blood, small detectable variations among triplicate blood aliquots allowed us to identify increases as minute as 1.5–2-fold with statistical significance(2). Using this system, our group evaluated the effect of mouse antihuman CD28 monoclonal antibody because the humanized version used in the clinical trial(1) was not freely available.

We obtained anonymized blood samples from 6 healthy adult volunteers (Apex Research), after receiving institutional review board approval. Triplicate 60-µL aliquots of each heparinized whole blood sample were incubated with either 1 mg/L of mouse antihuman CD28 monoclonal antibody IgG1{kappa} or control purified mouse IgG1{kappa} (BioLegend) at 37 °C for 2 h. The concentrations of various mRNAs were quantified with SYBR green real-time PCR as described previously (2). Increases were calculated by the difference from control IgG. As shown in Fig. 1 , after addition of mouse antihuman CD28 monoclonal antibody and incubation at 37 °C for only 2 h, whole blood from 1 healthy individual showed significant induction of inflammation- and apoptosis-related mRNA, such as interleukin-2 (IL-2), tumor necrosis factor superfamily (TNFSF)-1 (lymphotoxin {alpha}), TNFSF-5 (CD40 ligand), CCL chemokine-8, CCL chemokine-20, and CXCL chemokine-10. The huge induction of IL-2 mRNA is reminiscent of the failure of systemic IL-2 therapy decades ago(3). In blood from other healthy individuals, we observed significant reductions in the concentrations of IL-6, TNFSF-2 (TNF{alpha}), CCL-8, CXCL-1, and CXCL-10 mRNA. The identification of these sensitive individuals serves as a warning of the potential toxicity of superagonistic anti-CD28 antibody as a drug candidate. The results also demonstrate that ex vivo mRNA analysis is an interesting assay model that can serve as a bridge from preclinical research to clinical trials.


Figure 1
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  		Figure 1. Drug-induced leukocyte mRNA in whole blood.

Open circles (nonresponders) and open triangles (responder) indicate the mean values from each individual. * indicates the individuals with P <0.05 between control and anti-CD28. Dashed box shows the range of 0.5–2 fold increase, at which the majority of responses were not significant.


Acknowledgments

This study was financially supported by Hitachi Chemical Research Center.


References

  1. Mayor S. Severe adverse reactions prompt call for trial design changes. BMJ 2006;332:683.[Free Full Text]
  2. Mitsuhashi M, Tomozawa S, Endo K, Shinagawa A. Quantification of mRNA in whole blood by assessing recovery of RNA and efficiency of cDNA synthesis. Clin Chem 2006;52:634-642.[Abstract/Free Full Text]
  3. Rosenstein M, Ettinghausen SE, Rosenberg SA. Extravasation of intravascular fluid mediated by the systemic administration of recombinant interleukin 2. J Immunol 1986;137:1735-1742.[Abstract]



The following articles in journals at HighWire Press have cited this article:


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M. Mitsuhashi, K. Endo, K. Obara, H. Izutsu, T. Ishida, N. Chikatsu, and A. Shinagawa
Ex Vivo Simulation of the Action of Antileukemia Drugs by Measuring Apoptosis-Related mRNA in Blood
Clin. Chem., April 1, 2008; 54(4): 673 - 681.
[Abstract] [Full Text] [PDF]

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