Targeting Trends Newsletter 01q2

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Newsletter Highlights

  • Cytometry Research partners with Cytomation (page 2)
  • Suicide Transport Explained (page 5)
  • New Controls for Immunotoxins (page 7)

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Safety Studies Begin for Chronic Pain Therapeutic

San Diego Biotechnology Center Opens

Featured Neuroscience Antibodies

Recent Scientific References

Targeting Talk: Suicide Transport and Immunolesioning

  • What is immunolesioning?
  • What is suicide transport?
  • How do I administer a targeted toxin to achieve suicide transport?

Targeting Ticklers (Jokes)

Targeting Teaser Winners from last issue

Targeting Tools: Controls for Immunotoxins

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Controls for Immunotoxins

Advanced Targeting Systems announces two new control molecules for use with immunotoxins. We now offer mouse IgG (Cat. #IT-18) or rat IgG (Cat. #IT-17) conjugated to saporin. These new controls are the same molecular weight, consist of similar, comparable materials — saporin and a rat or mouse IgG — and are synthesized with the same protocols as the targeted immunotoxins. The difference is the cell- specific antibodies are replaced with “blanks,” antibodies that have no specificity, and no ability to target cells. In short, they are the perfect control molecules for behavioral experiments with Advanced Targeting Systems’ immunotoxins.

Controls are a vital part of the scientific procedure; without them it is difficult to isolate the specific effects from the non-specific or artifactual. With targeted toxin research, the same is true, and Advanced Targeting Systems often receives questions as to what makes the best control for a targeted toxin.

In the past, the response has been given according to what has been available. For an immunotoxin (a conjugate between an antibody and saporin), the suggested control is a mixture of the two components in their non-conjugated form. Of course, the lack of the conjugation process may detract from using this as a control, and there is always the question of how much antibody to mix with how much saporin.

Another suggestion for a control, since often saporin is connected to its targeting agent via a disulfide bond, is to reduce the disulfide bond. This method has some difficulties: 1) the reducing agent, if not removed, can have its own effect, and 2) usually the process is incomplete (unless carried out under drastic conditions), leaving a percentage of active material in the control. Finally, it’s expensive.

The new control immunotoxins avoid all of these difficulties. First of all, they are synthesized using the identical procedures that are used to synthesize the targeted immunotoxins, so there is no difference from the chemical point-of- view. They are very easy to use: they have the same molecular weight, you just use equal amounts of the control immunotoxin and the targeted immunotoxin. There are no complicated calculations to make. They are cost- effective. They are reasonably priced and time-saving because of the ease of preparation. As with all of the targeted immunotoxins, they are sterile-filtered and ready to go in phosphate-buffered saline at physiological pH.

In vitro data in the displayed graphs show that the control immunotoxins have orders of magnitude less cytotoxicity than the targeted immunotoxins. Their low toxicities are similar to that of saporin (on a molar basis), which is only taken into cells by bulk phase endocytosis, as opposed to antibody-mediated or receptor-mediated endocytosis of the targeted immunotoxins. These new molecules will make getting definitive data much easier.

Coming Soon: a new control peptide-toxin, that will use a randomly- generated, nonsense peptide conjugated with saporin. It will be the perfect control for SP-SAP, dermorphin-SAP and SSP-SAP.

 

Targeting Talk: Suicide Transport and Immunolesioning

What is immunolesioning?

Immunolesioning is a technique for making highly selective cellular lesions using immunotoxins. The immunotoxins consist of a monoclonal antibody to a cell surface molecule and a toxic effect or moiety such as saporin, a ribosome-inactivating protein. The selectivity of the lesion made with this technique depends on the selective expression of the target surface molecule on the cells of interest. Immunotoxins may be applied in a projection field where the toxin is taken up by axon terminals and retrogradely transported to the cell bodies resulting in destruction of an entire neuron. Other routes of application include: directly in vicinity of cell bodies, into CSF, and into culture supernatant.

What is suicide transport?

Suicide transport is an anatomically selective neural lesioning technique that relies on axonal uptake of a toxin that is retrogradely transported to the cell body resulting in destruction of the entire neuron. Examples include the toxic lectins [ricin, volkensin] and immunotoxins [192-Saporin (192-IgG-SAP): Cat. #IT-01, OX7-SAP: Cat. #IT-02, Anti-DBH-SAP: Cat. #IT-03]. The goal of using this technique is to selectively destroy a group of neurons based on where the corresponding axons project.

How do I administer a targeted toxin to achieve suicide transport?

Generally, precise control of dose and location of injection is important in suicide transport experiments. Consequently, pressure microinjection is the preferred method of toxin delivery. In the peripheral nervous system, subepineurial injection (inside the connective tissue sheath of a peripheral nerve) works well. Within the CNS, stereotactic techniques are typically used to deliver toxin to the desired target.

References

  1. Wiley RG, Lappi DA (1994) Suicide Transport and Immunolesioning. Houston, R.G. Landes.
  2. Helke CJ, Charlton CG, Wiley RG (1985) Suicide transport of ricin demonstrates the presence of substance P receptors on medullary somatic and autonomic motor neurons. Brain Res 328:190-195.
  3. Contestabile A, Fasolo A, Virgili M, Migani P, Villani L, Stirpe F (1990) Anatomical and neurochemical evidence for suicide transport of a toxic lectin, volkensin, injected in the rat dorsal hippocampus. Brain Res 537:279-286.
  4. Panaglos MN, Francis PT, Pearson RCA, Middlemiss DN, Bowen DM (1991) Destruction of a sub-population of cortical neurons by suicide transport of volkensin, a lectin from Adenia volkensii. J Neurosci Meth 40:17-29.
  5. Wiley RG (1992) Neural lesioning with ribosomeinactivating proteins: suicide transport and immunolesioning. Trends in Neurosci 15:285-290.
  6. Roberts RC, Harrison MB, Francis SMN, Wiley RG (1993) Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons. Exp Neurol 124:242-252.
  7. Wiley RG, Stirpe F, Thorpe P, Oeltmann TN (1989) Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat. Brain Res 505:44-54.
  8. Wiley RG, Kline IR (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Meth 103(1):73-82.

 
See also: Targeted Toxins Catalog

Targeting Topics 01q2

Up-regulation of growth-associated protein 43 mRNA in rat medial septum neurons axotomized by fimbria-fornix transection.

Haas CA, Hollerbach E, Deller T, Naumann T, Frotscher M.

Eur J Neurosci 12(12):4233-4242, 2000. PMID: 11122335

Axonal growth and regeneration is limited in adult mammals, however if injured CNS neurons are in an environment permissive for growth, they can regenerate. Transection of septohippocampal fibers is a widely used method for studying CNS neuron response to injury. These fibers are composed of both cholinergic and GABAergic neurons. Haas et al. used a combination of cholinergic lesioning by 192-Saporin (Cat. #IT-01) and double staining to investigate whether both cell types were involved in neuron regeneration. The findings show that both transmitter phenotypes up-regulate mRNA levels of a protein associated with growth and synaptogenesis in developing neurons, and plasticity in adult neurons.

Baroreceptor sensitivity of rat supraoptic vasopressin neurons involves noncholinergic neurons in the DBB.

Grindstaff RJ, Grindstaff RR, Cunningham JT.

Am J Physiol Regul Integr Comp Physiol 279(5):R1934-43, 2000. PMID: 11049879

Baroreceptors are one component of the system that buffers acute changes in blood pressure. Part of this control stems from the baroreceptor ability to regulate vasopressin release from the neurohypophysis. Using 192-Saporin (Cat. # IT-01) to specifically eliminate cholinergic neurons in the diagonal band of Broca, Grindstaff et al. demonstrated that these neurons are not utilized in the pathway that relays baroreceptor information to the brain.

Dissociation of memory and anxiety in a repeated elevated plus maze paradigm: forebrain cholinergic mechanisms.

Lamprea MR, Cardenas FP, Silveira R, Morato S, Walsh TJ.

Behav Brain Res 117(1-2):97-105, 2000. PMID: 11099762

The septo-hippocampal pathway has been implicated in many behavioral processes such as learning, anxiety, and motivation. Using 192-Saporin (Cat. #IT-01) to lesion the cholinergic neurons of the medial septum of rats, the authors demonstrate changes in exploratory behavior associated with learning, but no changes in anxiety-associated behavior in their elevated plus maze paradigm.

Early migratory rat neural crest cells express functional gap junctions: evidence that neural crest cell survival requires gap junction function.

Bannerman P, Nichols W, Puhalla S, Oliver T, Berman M, Pleasure D.

J Neurosci Res 61(6):605-615, 2000. PMID: 10972957

Gap junctions are vital for intercellular communication, especially during development. Neural crest cells develop into several types of neural cells, often migrating as a mass of cells to their final destinations. Bannerman et al. use the anti-p75 antibody (Cat. #AB-N01) to confirm the presence of p75 in neural crest cells. The authors examine how crucial survival signals are communicated during migration and demonstrate that interfering with gap junction formation causes death of neural crest cells.

The molecular dynamics of pain control.

Hunt SP, Mantyh PW.

Nat Rev Neurosci 2(2):83-91, 2001. PMID: 11252998

Over the last twenty years a great deal of progress has been made in the understanding of how pain is processed and transmitted by the CNS. The authors of this review highlight advances in systems neurobiology, behavioral analysis, genetics, and cell and molecular techniques. One method discussed is the use of the targeted toxin substance P-saporin (SP-SAP, Cat. #IT-07, also available with a more stable analog of substance P, SSP-SAP, Cat. #IT-11). This targeted toxin selectively lesions neurons expressing the NK1 receptor. Injection of SP-SAP into the spinal cord of rats dramatically attenuates the response to chronic pain stimuli, yet leaves acute pain response intact.

Regulation of sympathetic tone and arterial pressure by rostral ventrolateral medulla after depletion of C1 cells in rat.

Schreihofer AM, Stornetta RL, Guyenet PG.

J Physiol 529 Pt 1:221-236, 2000. PMID: 11080264

The rostral ventrolateral medulla (RVLM) controls and maintains basal sympathetic vasomotor tone, and is also vital to many sympathetic reflexes. Sympathetic nerve activity and arterial pressure correlate with the C1 adrenergic neurons in the RVLM, but there are also non-catecholaminergic neurons present. Schreihofer et al. used anti-DBH-SAP (Cat. # IT-03) to eliminate the C1 cells of the RVLM to investigate the non- catecholaminergic neuron contribution to vasomotor tone. Their data indicate C1 cells are necessary for full expression of sympathoexcitatory responses generated by the RVLM.

Neuronal lesioning with axonally transported toxins.

Wiley RG, Kline IVRH.

J Neurosci Methods 103(1):73-82, 2000. PMID: 11074097

Functional neuroanatomy studies have long utilized lesioning. Given the complexity of heterogeneous neuron populations, conventional lesioning methods have proved relatively crude and have provided limited information. Wiley and Kline detail some of the immunotoxins utilizing saporin as well as neuropeptide-saporin conjugates that have found use in recent neurological research. These products include SP- SAP (Cat. #IT-07), which eliminates neurons expressing the neurokinin 1 receptor, 192-Saporin (Cat. #IT-01), which eliminates neurons expressing the p75 receptor in rats, anti-DBH-SAP (Cat #IT-03), which destroys noradrenergic and adrenergic neurons, and OX7-SAP (Cat. #IT-02), which is a suicide transport agent targeting all rat neurons. The authors also discuss some of the protocols and methods utilized with these compounds.

Non-linear cortico-cortical interactions modulated by cholinergic afferences from the rat basal forebrain.

Villa AE, Tetko IV, Dutoit P, Vantini G.

Biosystems 58(1-3):219-228, 2000. PMID: 11164650

Elimination of the cholinergic neurons of the basal forebrain (BF) is an excellent model for some aspects of Alzheimer’s Disease (AD). 192-Saporin (Cat. #IT- 01) is a very effective tool for elimination of cholinergic neurons in the BF. Villa et al. investigate whether field potential changes in the brains of lesioned animals mimic changes observed in the brains of human AD patients. The data presented indicate depletion of cholinergic neurons from the BF of both rats and humans produces similar changes in field potential.

Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of [alpha]2B adrenoceptors.

Sawamura S, Kingery WS, Davies MF, Agashe GS, Clark JD, Kobilka BK, Hashimoto T, Maze M.

J Neurosci 20(24):9242-9251, 2000. PMID: 11125002

Nitrous oxide has been used extensively in surgical anesthesia for more than 150 years, but the molecular mechanism of action has not yet been defined. Sawamura et al. investigate whether noradrenergic neurons in the brainstem are involved in the analgesic action of nitrous oxide. The authors injected rats with anti-DBH-SAP (Cat. #IT-03) to destroy pontine noradrenergic neurons. The treated rats demonstrated the usual sedative effects of nitrous oxide, but the analgesic effects were reduced or blocked. Coupled with data from null mice for the α2B adrenoceptor, the data indicates that α2 adrenoceptor subtypes and ligands are involved in the analgesic but not sedative effects of nitrous oxide.

Cortical cholinergic inputs mediate processing capacity: effects of 192 IgG-saporin-induced lesions on olfactory span performance.

Turchi J, Sarter M.

Eur J Neurosci 12(12):4505-4514, 2000. PMID: 11122361

Many experiments support the theory that the basal forebrain (BF) is involved in major aspects of attention that influence learning and memory. Elimination of cholinergic neurons in the BF by 192-Saporin (Cat. #IT-01) has been shown to reduce the ability of rats to perform a task while paying attention to more than one thing. The authors tested the treated rat’s ability to identify one olfactory stimuli from an increasing number of such stimuli. While the performance of the treated rats returned to control levels within four weeks post- lesion, their performance reflected increased time between tests. These data indicate that cholinergic neurons of the BF play a role in attentional capacities.

Safety Studies Begin

Early last month (March 2001), Advanced Targeting Systems (ATS) received funding from the National Institute of Mental Health (NIMH) to begin toxicology/safety studies of Substance P-Saporin (SP-SAP), a potential therapeutic for the treatment of chronic pain. The studies will be completed under the direction of three scientists who are experts in their respective fields.

Dr. Douglas Lappi (President and Chief Scientific Officer of ATS) is principal investigator for the project and will oversee the various aspects of the studies. He is an expert in the design, construction and testing of targeted toxins. His laboratory will be producing the drug and performing quality control assays throughout the project.

Dr. Tony Yaksh (Professor of Anesthesiology and Pharmacology at the University of California, San Diego School of Medicine) will direct the administration of the drug. He is the leading expert in spinal cord delivery of experimental agents. The dog is one of the species routinely used to satisfy most regulatory requirements for drug safety evaluation. The studies will assess safety from four points: 1) intrathecal dose ranging to determine the maximum tolerated dose, 2) kinetics of cerebral spinal fluid to determine how the drug penetrates spinal tissue, is redistributed and eliminated, 3) histopathology to determine impact of drug on organs and tissue, and 4) spinal GLP safety studies to determine physiological (heart and respiratory rate, blood pressure) and behavioral (arousal, muscle tone, coordination) impacts of drug administration (4).

Dr. Patrick Mantyh (Professor, University of Minnesota, Minneapolis) has established the efficacy of SP-SAP in rats and is internationally acclaimed for his immunohistochemical analysis. His laboratory will measure parameters involving the efficacy and specificity of the SP-SAP treatment. Immunohistochemistry will help in determining where the drug travels and what impact, if any, it has on spinal cord neurons (See Figure).

spsap_dog

Figure Legend: This figure shows the staining of NeuN, CGRP and IB4 in the canine spinal cord. Immunostaining is one of the tests that will be used for determination of specificity and evaluation of bystander effects by SP-SAP. Confocal photomicrographs show the pattern of immunohistochemical labeling of the neuronal nuclear marker, NeuN, a peptidergic sensory nerve fiber marker, CGRP, and the non-peptidergic sensory nerve fiber marker, IB4, in the dog spinal cord. The NeuN staining is distributed throughout the entire gray matter, while the staining for the sensory fibers (CGRP, IB4) is localized to the dorsal horn (Photo supplied by Dr. Patrick Mantyh).

The development of SP-SAP was first published in 1997 (1) by Drs. Ronald G. Wiley and Douglas Lappi, two of the founders of ATS. Their collaboration with Dr. Patrick Mantyh led to two publications in the journal Science (2, 3). These three articles describe the results of experiments with SP-SAP in the rat.

SP-SAP is a targeted toxin that permanently eliminates cells that bear the Substance P receptor. This receptor is one of many involved in the transmission of pain signals to the brain. There are two general categories of pain to be considered in this process: 1) acute pain, a physiologically important survival tool (rose thorn pricking finger, cat claws scratching cheek), and 2) chronic or noxious pain (pain that persists beyond normal healing time), often the cause of severe pathological states. The scientists used standard models of chronic pain to determine that the perception of noxious pain in the models was greatly reduced in those animals who received injections of SP-SAP. But probably just as important, the perception of acute pain was left intact. This was an extraordinarily important finding and led ATS to the decision to begin the process of developing SP-SAP as a drug.

Over the next few months, ATS will be interacting with the Center for Drug Evaluation and Research. This organization is part of the U.S. Food and Drug Administration and will evaluate the drug development plan and make determinations about the composition and guidelines for the initial clinical trials in humans. Their preliminary feedback has been a recommendation to begin clinical trials in patients with terminal cancer whose pain is no longer treatable with opioid-based drugs such as morphine. The size of this patient population may qualify SP-SAP for development as an orphan drug.

ATS is optimistic about the therapeutic possibilities of SP-SAP. The funding from NIMH is an important first step in getting the drug development process under way. The process has already begun to obtain the additional funding needed to complete the toxicology tests required by the FDA. The present goal is to be able to begin the first clinical trials in humans before the end of 2002. Progress reports will be printed in our newsletter and on the ATS website.

References

  1. Wiley RG, Lappi DA (1997) Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin. Neurosci Lett 230:97-100.
  2. Mantyh PW, Rogers S, Honore P, Allen B, Ghilardi JR, Li J, Daughters RS, Vigna SR, Lappi DA, Wiley RG, Simone DA (1997) Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279.
  3. Nichols ML, Allen BJ, Rogers SD, Ghilardi JR, Honore P, Li J, Lappi DA, Simone DA, Mantyh PW (1999) Transmission of chronic nociception by spinal neurons expressing the substance P receptor. Science 286:1558-1561.
  4. Yaksh TL, Rathbun ML, Provencher JC. (1999) Preclinical safety evaluation for spinal drugs. In: Spinal Drug Delivery, Yaksh TL (ed.), Elsevier Science B.V., Amsterdam, pp. 417-437.