Background: Neurokinin-1 receptors (NK1-rs) located on superficial dorsal horn neurons are essential for integration of nociceptive input. Intrathecal injection of substance P-saporin (SP-SAP) leads to local loss of spinal NK1-r (+) neurons suggesting its potential as a therapeutic agent for chronic pain. The authors determined, in a canine model, effects of lumbar intrathecal SP-SAP. Methods: Distribution of SP-SAP and Saporin was determined in plasma, lumbar cerebrospinal fluid, and tissue. Safety of intrathecal SP-SAP was determined in four groups (six dogs each) administered 0 (0.9% saline), 1.5, 15, or 150 µg SP-SAP through lumbar intrathecal catheters. Behavioral, physiologic, and biochemical variables were assessed. Spinal tissues were collected at 7 and approximately 90 days, or earlier if significant morbidity developed, and analyzed for NK1-r (+) neuron loss and histopathology. Results: SP-SAP and Saporin were detectable in lumbar cerebrospinal fluid for up to 4 and 24 h, respectively. Animals receiving intrathecal saline, 1.5, or 15 µg of SP-SAP showed no persistent neurologic deficits. Three animals receiving 150 µg of SP-SAP developed pelvic limb paraparesis and were euthanized prematurely. Immunohistochemistry and in situ hybridization cell counts confirmed a significant reduction in NK1-r (+) in superficial dorsal horn neurons from lumbar spinal cord after intrathecal administration of 15 and 150 µg of SP-SAP. A significant loss of NK1-r neurons in the lumbar ventral horn occurred only with 150-µg SP-SAP. Conclusion: Intrathecal 15-µg SP-SAP reduced dorsal, but not ventral, NK1-r (+) neurons at the spinal level of delivery with minimal side effects, whereas 150-µg SP-SAP resulted in motor neuron toxicity.

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed ‘molecular surgery’, with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer’s Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Streptavidin-Saporin can be considered a type of ‘secondary’ targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the ‘Molecular Surgery’ capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Cancer pain is a great fear, at times greater than even death, in the progression of the disease. 10-15% of cancer patients have pain that is refractory to opioid treatment. There is a common fear among terminal patients that pain treatments will leave them unable to function normally at a time when that is extremely important for them. Substance P-Saporin (SP-SAP), targets with Substance P (SP), a neuromodulator that binds to receptors on laminae I and X of the dorsal horn of NK1r-bearing neurons. These neurons are critical components of the high level, intense pain pathway. The toxic portion of the drug is Saporin (SAP), a ribosome-inactivating protein that, once assisted inside a cell by Substance P, ablates neurons that express NK1r. A single intrathecal infusion of SP-SAP alleviated pathologic pain perception without affecting other sensory signal pathways in preclinical studies conducted in rats. Studies published in the journal Science show pathological pain relief is permanent with no long-term side effects such as central sensitization. A veterinary single-blind clinical trial for the treatment of pain associated with bone tumors in canine companion animals showed that after one injection of SP-SAP, dogs had a much higher quality of life throughout the cancer. Patients in the ongoing SP-SAP phase I clinical trial (NCT02036281) are treated at University of Texas Southwestern. Eligibility: 18 years or older, signed consent, able to verbally report pain, able to complete pain surveys, perform motor/sensory tests, able to undergo intrathecal catheter placement, other therapeutics and palliative options have been exhausted, and have been diagnosed with advanced cancer leading to intractable pain with minimum expected survival of one month. Treatment: a percutaneous intraspinal catheter is placed at the L5-S1 interspace and the advanced 4-5 cm into the intrathecal space under fluoroscopic guidance. 1 mL SP-SAP is mixed with 1 mL patient CSF fluid and administered via the catheter. Subjects are monitored for four hours in the recovery room before catheter is removed, monitored in hospital 24 hours, and discharged. The starting dose of SP-SAP was 1 mcg in 1mL, 1/10th the dog no observable adverse effect level. Dose: Single subject cohorts are at doses of 1, 2, 4, 8, 16, 32, 64, and 90 mcg. If a drug-related dose-limiting toxicity is observed in two or more patients in a cohort, the maximal tolerated dose (MTD) has been exceeded, and the next lower dose level will be the MTD. An additional 15 patients will be treated at the MTD to confirm safety. Endpoints: Response measurements include pain level measurements, Brief Pain Inventory (BPI), VAS “pain bothersomeness,” VAS “pain intensity,” ODI, SF-36, EQ-5D, BDI, and Medication Use logs in patient diary. Primary endpoints for the trial are either a 20% reduction in pain scores or a 20% reduction in pain medications taken by the patient. Citation Format: Arthur E. Frankel, Douglas A. Lappi, Carl Noe, Denise Higgins, Brian J. Russell. Preliminary results from a phase I study of Substance P-Saporin in advanced cancer patients with intractable pain. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT030.

191 Background: For 10-15% of cancer patients, pain cannot be controlled using existing therapies (Zech et al., Pain, 63, 65-76, 1995). SP-SAP is the first targeted toxin to undergo phase I testing for pain. SP-SAP (Wiley and Lappi, Neurosci Lett, 230, 97-100, 1997) covalently linkes substance P (SP), a neuromodulator that binds to NK1 receptors on laminae I and X of the dorsal horn, and saporin (SAP), a ribosomal toxin that ablates cells that express NK1 and endocytose the toxin. Intrathecal injections of SP-SAP in rats reduce chemically or thermally induced pain, thermal hyperalgesia, and mechanical allodynia, with no signs of motor, sensory, or behavioral dysfunction (Mantyh et al., Science, 278, 275-9, 1997; Nichols et al., Science, 286, 1558-61, 1999; Suzuki et al., Nature Neurosci, 5, 139-26, 2002; Vierck et al., Neurosci, 199, 223-32, 2003). In canines, intrathecal injections into the lumbar region – 45 mcg for 10-15 kg (Allen et al., Toxicol Sci, 91, 286-98, 2006) or 15 mcg for 8-12 kg (Wiese et al., Anesthesiol, 119, 1163-77, 2013) – produce no neurological, behavioral, or histological deficits. A double-blinded RCT of SP-SAP for the treatment of bone pain in canines (Brown and Agnello, Anesthesiol, 199, 1178-85, 2013) showed that a single injection of 60 mcg (above 30 kg) or 40 mcg (below 30 kg) significantly reduced pain as measured by time to owner unblinding (p=0.002) or number of canines unblended (p=0.001). Methods: A single intrathecal injection of SP-SAP is made at the L5-S1 interspace of cancer patients with intractable sub-umbilical pain. Single subject cohorts are at doses of 1, 2, 4, 8, 16, 32, 64, and 90 mcg. Primary outcomes are assessed by pain survey (VAS “bothersome pain”, VAS “pain intensity”, ODI, EQ-5D, and BDI) and medication use log. Patients are monitored for toxicity, neurological and cardiac abnormalities, and SP-SAP antibodies for 6 months. Results: This report presents the results from the first three subjects of the phase I study. No evidence of toxicity or neurological or cardiac abnormalities were found. No clear evidence for pain reduction was observed. Conclusions: At existing doses (< 4 mcg), SP-SAP appears safe for human use. The study is continuing with dose escalation. Clinical trial information: NCT02036281.