Actions of intrathecal diphtheria toxin-substance P fusion protein on models of persistent pain Rafael Benoliel a,b , Eli Eliav b , Andrew J. Mannes a,c , Robert M. Caudle a , Susan Leeman d , Michael J. Iadarola a, * a Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA b The Department of Oral Diagnosis, Oral Medicine, Oral Radiology, The Hebrew University, Hadassah School of Dental Medicine, Jerusalem, Israel c Department of Anesthesiology, Hospital of the University of Pennsylvania, 4 Ravdin, 3400 Spruce Street, Philadelphia, PA 19104, USA d Department of Pharmacology, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118, USA Received 20 February 1998; received in revised form 5 August 1998; accepted 24 August 1998 Abstract Substance P (SP) plays a central role in the transduction of second messenger signals from primary afferent nociceptive terminals to second-order neurons in the spinal cord. We have tested a recombinant engineered diphtheria toxin/SP fusion protein (DAB389SP) in acute and chronic pain models in the rat. DAB389SP binds to the SP receptor (SPR) and is internalized and kills SPR-expressing cells by blocking cellular protein synthesis. DAB389SP delivery was by intrathecal infusion, of varying duration, at the lumbar level. In the chronic constriction injury model of neuropathic pain a significant reduction in mechanically induced hyperalgesia was obtained. This effect was less marked in an acute carageenan inflammation model. Although other pain characteristics (mechano-allodynia, cold-allodynia, and heat- hyperalgesia) showed some improvement, these were less pronounced. Immunocytochemistry revealed a toxin-induced reduction in lamina I, of SPR and of NMDA NR1 subunit receptor expressing neurons, and of c-Fos, an inducible molecular marker of persistent nociceptive activity. The use of cytotoxic fusion proteins to target specific cell types may be of considerable benefit in the study of nociception and the treatment of chronic pain.  1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Cytotoxins; Immunotoxins; Substance P 1. Introduction Substance P (SP) is an 11 amino acid peptide present in neuronal systems throughout the central nervous system (CNS). Direct tests of neuronal excitability demonstrate that SP is a potent neuroactive peptide that acts primarily on SP receptor (SPR)-expressing neurons. This receptor, classified as the neurokinin 1 (usually abbreviated as NK1) receptor, is a member of the seven transmembrane G-protein coupled receptor superfamily (Watling et al., 1995). Based on anatomical, electrophysiological, and phar- macological studies, SP has been shown to be involved in diverse motor, autonomic and sensory system functions. Although the exact role that SP and its receptor play in signaling nociception is unclear, they are particularly abun- dant in lamina I of the spinal cord (Brown et al., 1995). Moreover, electrical stimulation of peripheral nociceptors at C-fiber intensity induces the release of SP in the spinal cord (Duggan et al., 1988). When SP binds to its receptor, one or more cascades of second messenger signaling path- ways are activated (see review by Krause et al., 1993) even- tually triggering endocytosis of the SP/SPR complex. Within the endosome SP is degraded, and the SPR recycles to the surface (Grady et al., 1995). This SP/SPR internaliza- Pain 79 (1999) 243–253 0304-3959/99/$ - see front matter  1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S0304-3959(98)00170-5 * Corresponding author. Pain and Neurosensory Mechanisms Branch, NIDR, NIH, 49 Convent Drive, Room 1A11, MSC 4410, Bethesda MD, 20892–4410, USA. Tel.: +1-301-496-2758; fax: +1-301-402-0667; e-mail: iadarola@yoda.nidr.nih.gov