Preclinical Drug Discovery Blog

Inflammation after acute ischemic stroke- preclinical models

MD Biosciences — Tue, 14 Feb 2012 21:00:00 GMT

The most common form of stroke is acute ischemic stroke (approximately 85% of cases), which is caused by either an atherothrombosis in a major cervical or intracranial artery or an embolism that travels from the heart. The resulting occlusion causes a sudden deficiency of oxygen and glucose in the brain region normally serviced by the blocked artery. Victims of large-vessel ischemic strokes lose on the order of 100 million neurons per hour prior to treatment, causing immediate, permanent neural damage in the infarct area, termed the ischemic core. Further neural damage occurs in the areas surrounding the core, called the penumbra, where the tissue becomes highly inflamed and slowly dies. Stroke sufferers experience a range of neurological deficits including partial paralysis, impaired memory, loss of speech, and/or decreased cognition and many become permanently disabled, requiring institutional care. [1-4]

Unfortunately, current stroke therapies approved for human use are very limited. The only drug clinically available in the US is intravenous recombinant tissue plasminogen activator (rt-PA), a thrombolytic agent that has been shown to improve stroke patient functional outcomes. However, rt-PA is only effective if administered within the first 3 hours after symptom onset and carries with it a significant risk of intracranial hemorrhage. Consequently, only about 5 to 10% of patients can receive this therapy. Over 1,000 other potential stroke therapeutics have been tested in preclinical cerebral stroke models and approximately one tenth of these have made it to clinical trials. However, the majority of these efforts have already failed. It is likely that the most effective way to improve outcomes is rapid reperfusion of the ischemic area using thrombolytic means in combination with neuroprotective strategies to salvage cells within the penumbra and prevent them from becoming part of the ischemic core. To this end, researchers have been increasingly focused on post-stroke neuroinflammation and the role it plays in neurotoxicity and neuroprotection. [1-4]

References

Candelario-Jalil, E. (2009). Injury and repair mechanisms in ischemic stroke: considerations for the development of novel neurotherapeutics. Current Opinion in Investigational Drugs 10(7):644-654.
Ceulemans, A.-G., Zgavc, T., Kooijman, R., Hachimi-Idrissi, S., Sarre, S., and Michotte, Y. (2010). The dual role of the neuroinflammatory response after ischemic stroke: modulatory effects of hypothermia. Journal of Neuroinflammation 7:74.
Downes, C.E. and Crank, P.J. (2010). Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS? British Journal of Pharmacology 160:1872-1888.
Lakhan, S.E., Kirchgessner, A., and Hofer, M. (2009). Inflammatory mechanisms in ischemic stroke: therapeutic approaches. Journal of Translational Medicine 7:97.
Bacigaluppi, M., Comi, G. and Hermann, D.M. (2010). Animal models of ischemic stroke. Part two: Modeling cerebral ischemia. The Open Neurology Journal 4:34-38.

Microglial involvement in Neuropathic Pain: 5 activation pathways

MD Biosciences — Wed, 26 Oct 2011 14:25:00 GMT

Neuroinflammation is a common thread in neuropathic pain (NP), regardless of the conditions under which neuropathic pain develops. This opens up a whole new avenue for investigations into neuropathic pain pathology. Since the primary cell type responsible for immune-like functions in the CNS is microglia, many researchers have turned their attention toward working to better understand microglial physiology and its potential involvement in neuropathic pain.

[short overview of microglial cells can be found here]

Microglial participation in NP pathophysiology has been investigated using a wide variety of experimental preclinical models. Some of the most common models used are the CCI, SNL and STZ-induced Diabetic neuropathy model. Several lines of evidence compiled using these models have demonstrated the intimate involvement of microglial cells in the establishment of neuropathic pain. More specifically, the process of microglial activation is now thought to be both necessary and sufficient for neuropathic pain initiation. Although there is some variability between results obatined using the different neuropathic pain models, generally microglial cells in the ipsilateral dorsal horn of the spinal cord become activated within approximately 4 hours, increase 2- to 4-fold in number by day 2 and remain active for several months after peripheral nerve injury. These effects can be suppressed by non-specific microglial inhibitors in these preclinical models. In the context of neuropathic pain, local, responding microglial cells are known to be activated by a broad range of stimuli, five predominant activation pathways appear to be most critical and are identified by their major ligand receptor.

TLR4 (toll-like receptor family member 4)
P2X4 (purinociceptor 4)
INF-g and CB2
MCP-1
Fractalkine

These mechanisms have emerged as exciting new focal points for assessing opportunities for the future development of pharmacotherapies, gene therapies or cell-based therapies for neuropathic patients. You can read more about the activation pathways in our new eBook.

Overview of microglial cells in the CNS

MD Biosciences — Mon, 24 Oct 2011 20:56:00 GMT

Of the roughly 70% of cells in the central nervous system (CNS) that are glia, appromixately 5-10% are microglial cells. Microglial cells are derived from peripheral myeloid progenitor cells that enter the CNS during embryonic development. Though ubiquitous in the CNS, microglial cell densities vary by region. They function to provide structural and trophic support to neurons and serve as the resident immune-competent cells of the CNS, tasked with:

detection of infections and injuries
protection of healthy tissues
elimination of disturbances
restoration of homeostatic conditions

Normally, microglial morphology is characterized by small soma with many thin, branded processes. Microglial processes come in contact with neurons, endothelial cells and astrocytes but not other microglial cells. In fact, each cell appears to be responsible for a distinct territory, within which it contantly samples the extracellular microenvironment by sweeping its processes through the tissue without disrupting neuronal connectivity.

Microglial cells have a very low threshold for activation and can be activated by a wide vavriety of stimuli. Once activated, they undergo morphological and phyiological changes and they mobilize and proliferate. Activated cells display enlarged soma with shorter processes or even amoeba-like shapes and drmatically altered gene expression profiles. They home to injured areas, perform phagocytic and antigen presentation functions, and re-enter the cell cycle to increase their number. As microglial cells are not electrically coupled with other cells, they act solely via the release of diffusible mediators to communicate with neighboring cells in a paracrine fashion. Microglial phenotypes are extremely plastic. The process of microglial activation is neither an "all-or-none" committment, nor a linear path, which allows for creation of a wide range of activated phenotypes to achieve very graded responses to real or perceived threats to the CNS. Taken together with evidence of microglial populations haven already "built-in" heterogeneity and the possibilitiy that when individual cells are activated once, they may respond differently when activated again through potentially long-lasting epigenetic mechanismsm, the picture of microglial activities in the CNS becomes extremely complex.

Behavior based findings in preclinical nerve injury models

MD Biosciences — Wed, 24 Aug 2011 20:00:00 GMT

We get a lot of questions on the various neuropathic pain models and how to choose the one that's most appropriate or a comparison of what's involved with each model (e.g. surgery, behaviors, centralization, peripheral vs central involvement etc). We thought it may be helpful to discuss the various aspects of these models to assist with the selection and understanding of the mechanisms and behaviors. Of course, it ultimately depends on the drug target and the pathway involved and we can certainly discuss individual specifics with you.

Neuropathic Pain Models

Preclinical models in pain research offer great promise for both the identification of pain mechanism and the investigation of possible therapeutic applications. Since there is not one mechanism that is responsible for generation and maintenance on neuropathic pain (NPP), it is essential to select the best model for each specific research interest or a combination of appropriate but distinct models.

At present, preclinical models for NPP cover various etiologies and are related to symptoms leading to an extensive picture of clinical NPP manifestations. The majority of these preclinical models of NPP involve traumatic injuries to peripheral nerves, nerve roots or spinal cord by transaction, ligation or compression (Fig 1). Other models are related to direct or indirect nerve inflammation, ischemia, drug toxicity or systemic metabolic disorder leading to nerve ending damage.

All of these models have been characterized by precise behavior-based evaluation using different methods of sensory stimulation. Many molecular, physiological and structural modifications have been described in these models. Most of the rodent models are described also with actual electrophysiology measurement and imaging techniques as well as genomic and proteomic screening. Over the coming weeks, we will discuss these various findings in nerve injury models.

Behavior based findings in two common preclinical Nerve Injury Models

Spinal Nerve Ligation (SNL)

Surgical procedures on selective spinal nerves such as the SNL model allows the direct access to sensory and motor fibers and clear segment location of injured (L5/L6) and non injured DRG (L4) and afferents. In this model the tight ligation of the L5/L6 spinal nerves results in robust and consistent sympathetic related neuropathic pain behavior including indirect signs of spontaneous pain, heat hyperalgesia, mechnical allodynia and cold allodynia.

Chronic constriction injury (CCI, also Bennet and Xie)

The CCI model is induced by applying 4 catgut loosely around the sciatic nerve. This model allows sensory testing in the hind paw as not all the sensory nerved are damages. Moderate autotomy, guarding and excessive grooming of the injured limb are reported as well as thermal hyperalgesia and mechanical allodynia were recorded. The main challenges in this model is the standardization of the loose but constrictive ligature.

The following table summarizes the behavior based response following each of the surgical-induced preclinical neuropathic pain models.

Parameter	CST	TST	CCI	SNL	PSL
Autotomy	65%	-	~10%	~10%	-
Natural Pain Behavior	Low	0	Significant peak at day 9	Moderate Peak at day 16	Low
Response to hot plate (duration of lifting time)	Low	Low	Significant, yet reversable with a peak at days 3-9 post surgery	Moderate persistance until day 28	Minimal only at day 3
Response to VF	Significant	Significant	Moderate	Significantly higher than other models	Significant
Pin Prick	Significant	Significant	Significant	Significantly higher than other models	Significant
Acetone Test	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1	Significant starting from day 1
Cold Plate	Moderate from day 21	Minor from day 21	Significant from day 1	Moderate from day 1	Significant peak at day 14

Abbreviations: CST - complete sciatic transection; TST - tibial and sural transection; CCI - chronic constriction injury; SNL - spinal nerve ligation; PSL - partial nerve ligation; VF - von frey

Hopefully this comparison of the behavioral findings for each spinal nerve injury model helps sort out the differences and similarities between models. If you would like to speak more about the models as it relates specifically to your compound, please contact us. Our scientists love talking about this stuff.

New eBook: The link b/t pain & inflammation, targets in the overlap?

MD Biosciences — Tue, 19 Jul 2011 17:34:00 GMT

Neuropathic pain presents a wide variety of challenges to researchers, not the least of which is the simple fact that neuropathic pain, by definition, requires neuronal damage, which in turn automatically initiates immune response that often inflicts further neuronal damage. The interactions between the nervous system and immune system in the case of neuropathic pain make for a very complex story that is only beginning to unfold:

At the anatomical-level, neuro-immune interactions have been shown to take place all along the pain processing pathway. This is partially facilitated by increased permeability of the blood-brain barrier following SCI or peripheral nerve injury.

At the cellular level, neuro-immune interactions involve a variety of cells including mast cells, neutrophils, macrophages and T cells as well as glial cells with immune-like functions.

At the biochemical level, factors either directly produced by involved leukocytes and immune system factors released by glial cells expose prominent potenital therapeutic targets.

As neuroimmunologists find interactions between the nervous and immune systems, well-kown disorders may be found in the overlap. This eBook explores the immune system, inflammation, pain processing as well the various cells involved in the neuro-inflammation aspect of neuropathic pain and the potential inflammation-related drug targets.

Download the complimentary eBook: The Link between Pain and Inflammation

The ideal preclinical model system: large vs small species.

MD Biosciences — Wed, 08 Jun 2011 14:46:00 GMT

Common models for preclinical efficacy often use rodents as they are readily available, cost effective, easy to handle and most familiar to investigators. In choosing a preclinical model, one also needs to consider the anatomical/functional similarity to humans and there are cases where moving onto a larger species is more relevant to the clinic and human condition. Two of those cases are described below:

Post-operative pain and wound healing:

Small animals such as the rat and mouse different from humans in that they have a dense layer of hair on the body, a thin epidermis and they heal primarily through wound contraction as opposed to re-epithelization as in the swine and human. Anatomically and physiologically, swine skin is more similar to human skin:

Skin has thick epidermis
Well developed rete-ridges, dermal papillary godies and adundant subdermal adipose tissue
Swine dermal collagen is biochemically similar to human dermal collagen
Size, orientation and distribution of blood vessels in the dermis or porcine skin is similar to human skin
Sparse body hair which progresses through the hair cycle independently of neighboring follicles, which is importnat since they play a role in re-epithelialization
Overall physiology of porcine is sinmilar to human physiology with most organ systems being similar in anatomy and funtion

Acute myocardial infarct (AMI) and ischemic reperfusion (IR) injury

Anatomically, swine hearts are very similar in size and gross structure to human hearts and at the level of coronary vasculature are nearly identical - blood supply is right side dominat and pre-formed collaterals are absent. Physiologically, the baseline heart rate and blood pressure of swine are similar to humans.

AMI induction in swine is relatively easy by a variety of means and produces infarcts similar to those observed in humans with predictable sizes, locations and time courses. All cardioprotective schemes so far identified for humans have been described in swine after ischemia and reperfusion, namely hibernation ad ischemic pre- and post-conditioning.

Overall advantages

Experimentally, porcine are capable of tolerating long and complex protocols, medical device implantation and repeated surgeries. In the post-operative pain model, pain can be evaluated up to 12 days and wound healing/inflammation can be observed simultaneously with pain. In the cardiac models, hearts are large enough to allow myocardial biopsies to be taken both from infarct area and an unaffected area, providing a conventional internal control. Additionally, intracoronary drug delivery and implantation of devices or microdialysis probes enables measurement of small, diffusible bioactive molecules.

For information on either the post-operative pain model or acute myocardial infarct in swine, download the following whitepapers:

		Myocardial Infarct Models: Evaluating the myocardial protection of potential drug therapies or devices.
		A model of post-operative pain: assessment of analgesic affects of local treatment strategies.

References

Sullivan, T.P. et al., (2001) Wound Rep Reg. 9:66
Klocke, R., Tian, W. Kuhlmann, M.T., and Nikol, S. (2007). Cardiovascular Research, 74, 29‐38.
Dixon, J.A. and Spinale, F.G. (2009). Circulation: Heart Failure, 2(3), 262‐271.
Swindle, M.M., Makin, A., Herron, A.J., Clubb, F.J., and Frazier, K.S. (2011). Veterinary Patholology, Mar 25 [Epub ahead of print].
Heusch, G., Skyschally, A., and Schulz, R. (2011). Journal of Molecular and Cellular Cardiology, Mar 5 [Epub ahead of print].

Receptor & Receptor Ion Channels involved in neuropathic pain.

MD Biosciences — Tue, 07 Jun 2011 14:58:00 GMT

This post continues on our discussion of potential inflammation-related drug targets for the treatment of neuropathic pain. See also Pro-inflammatory cytokines and anti-inflammatory cytokines as targets in neuropathic pain.

TLR4 and its relevance to neuropathic pain

Toll-like receptors (TLRs) are a family of 13 pattern recognition receptors expressed by leukocytes that are responsible for identifying foreign toxins and microbes and initiating inflammation as a part of the innate immune response. TLR4 is expressed on macrophages, microglia and Schwann cells.

TLR4 is thought to be activated by necrotic cells, injured axons, and extracellular matrix components. Elimination or modification of TLR4 function at either the receptor itself (via knockout, point mutation, antisense oligoneucleotide or antagonist treatment) or its associated signal transduction cascade reduces or completely prevents microglial activation and associated cytokine release. This leads to further macrophage recruitment, microglial activation, pain hypersensitivity, hyperalgesia and allodynia.

P2X4R and its relevance to neuropathic pain

Purigenic receptors are a large family of receptors that bind various forms of adenosine nucleotides. The P2X subtype receptors (of which there are 7 currently known) are extracellular ARP- or ADP-sensitive ligand-gated ion channels found on a variety of neuronal and glial cell types.

P2X4R expression is up-regulated in spinal cord microglia upon nerve damage, the inhibition of which prevents allodynia. Further, intrathecal application of ATP activates microglia and intiates allodynia in rats. Pharmacological inhibition of P2X4R reduces allodynia and lack of P2X4R prevents allodynia development after nerve damage. Presumably, the mechanism by which P2X4 is functioning in the development of hyperalgesia and allodynia in animals models of neuropathic pain involves ATP released from activated astrocytes.

TRPV1 (vanilloid receptor) and its relevance to neuropathic pain

TRPV1 is a ligand-gated ion channel expressed in nociceptors and is a member of the transient receptor potential (TRP) family of ion channels. TRPV1 can be activated by capsaicin, low pH, nixious heat, spider toxins, and the endocannabinoid, AEA.

The expression and function of TRPV1 is altered under inflammatory conditions by a variety of mechanisms. Inflammatory mediators including TNF, PGE2, and Bradykinin alter expression of TRPV1 in nociceptor cell bodies in the DRG, trafficking of TRPV1 to peripheral terminals, and activity of TRPV1 once inserted in the membrane. In models of neuropathic pain, TRPV1 antagonists reduce pain hypersensitivity.

Choosing the appropriate neuropathic pain model is dependent upon the target and mechanism of the compound. For further information on neuropathic pain models, download the whitepaper: Periphery Nerve Injury Models: Understanding underlying mechanisms of neuropathic pain.

References

Austin, P.J. and Moalem-Taylor, G (2010) Nociceptors: the sensors of the pain pathway. Journal of Clinical Investigation. 120(11):823
Smith, H.S. (2010) Activated microglia in nociception. Pain Physician. 13:295
Leung, L and Cahill, C.M. (2010) TNF-alpha and neuropathic pain - a review. Journal of Neuroinflammation. 7(1):27
Stein, C. et al. (2009) Peripheral mechanisms of pain and analgesia. Brain Research Reviews. 60(1):90
Schlosburg, J.E. et al., (2009) Targetting fatty acide amid hydrolase (FAAH) to treat pain and inflammation. The American Association of Pharmaceutical Scientists Journal 11(1):39
Patapoutian, A. et al., (2009) Transient receptor potential channels: targeting pain at the source. Nature Reviews Drug Discovery. 8(1):55

Preclinical contact hypersensitivity models - DNCB or FITC?

MD Biosciences — Wed, 01 Jun 2011 14:37:00 GMT

Contact hypersensitivity dermatitis occurs when the immune system mounts a response to chemicals the body comes into contact with via the skin. Alone these chemicals would be too small for the immune system to respond to, but they are all capable of binding to proteins within the body, a process termed haptenation. In most individuals this is harmless however in some individuals an immune response against chemicals bound to self proteins is mounted leading to inflammation of the skin at the contact site. Many commonly encountered chemicals are capable of acting as contact sensitizers, these include petrochemicals, heavy metal ions (i.e Nickel) and some plant extracts (i.e urushiol from poison ivy).

DNCB-induced Contact Dermatitis

The 2,4-Dinitrochlorobenzene (DNCB) induced contact dermatitis model is Th1 mediated with IFN-g production by both CD4+ and CD8+ cells observed and increased IL-12p40 mRNA observed in the draining lymph node. Cell mediated responses are thought to be of particular importance in the pathology associated with challenge of sensitised individuals.

FITC-induced Contact Dermatitis

The Fluorescein isothyocyanate (FITC) induced contact dermatitis model is mediated by the Th2 pathway. Unlike many contact hypersensitivity reactions which induce strongly cytotoxic T cell mediated responses; the response to FITC challenge exhibits many of the hallmarks of atopic dermatitis; Local eosinophilia, mast cells infiltration, Anti-FITC IgE and IL-4 and IL-10 production by CD4+ cells are observed following sensitisation. In addition immediate and late phase responses are observed. Work to dissect the pathological mechanism has taken place, yielding the following results; The passive transfer of immune sera results in a rapid transient response to FITC challenge (peaking 15-30 minutes, returning to normal by 24hrs), The adoptive transfer of LN cells from sensitised mice results in a more delayed and sustained ear swelling, The depletion of CD4+ cells prior to adoptive transfer prevents ear swelling following application of FITC. These results suggest that IgE is responsible for the immediate phase of the response to FITC application while CD4+ cells sustain the response.

It is becoming apparent that a role for the Th17 pathway may also be important in the development of contact hypersensitivity and allergic responses, and has been implicated in the FITC induced contact dermatitis model. The Aryl hydrocarbon receptor (AhR) activation is a cofactor in the development of Th17 responses and AhR null mice have been shown have impaired Langerhans cell maturation and as a result impaired responses to FITC sensitisation.

Learn more about the DNCB and FITC-induced Contact Dermatitis preclinical efficacy models.

References:

Dearman RJ, and Kimber I. Role of CD4(+) T helper 2-type cells in cutaneous inflammatory responses induced by fluorescein isothiocyanate. Immunology. 2000;101(4):442-51.
Dearman RJ, Humphreys N, Skinner RA, and Kimber I. Allergen-induced cytokine phenotypes in mice: role of CD4 and CD8 T cell populations. Clin Exp Allergy. 2005;35(4):498-505.
Takeshita K, Yamasaki T, Akira S, Gantner F, and Bacon KB. Essential role of MHC II-independent CD4+ T cells, IL-4 and STAT6 in contact hypersensitivity induced by fluorescein isothiocyanate in the mouse. Int Immunol. 2004:16(5):685-95.
Hayashi M, Higashi K, Kato H, Kaneko H. Assessment of preferential Th1 or Th2 induction by low-molecular-weight compounds using a reverse transcription-polymerase chain reaction method: comparison of two mouse strains, C57BL/6 and BALB/c. Toxicol Appl Pharmacol. 2001:177(1):38-45.
Cowden JM, Zhang M, Dunford PJ, and Thurmond RL. The Histamine H4 Receptor Mediates Inflammation and Pruritus in Th2-Dependent Dermal Inflammation. J. Inv. Dermatology 2010: 130, 1023–1033.
Jux B, Kadow S, and Esser C. Langerhans Cell Maturation and Contact Hypersensitivity Are Impaired in Aryl Hydrocarbon Receptor-Null Mice. J Immunol. 2009;182;6709-6717.

Inflammatory events underlying cardiovascular disease.

MD Biosciences — Tue, 10 May 2011 19:51:00 GMT

Cardiovascular disease (CVD) including heart disease, vascular disease and atherosclerosis are the most critical global health threats.

An estimated 26 million people are living with the effects of heart disease and is a major cause of death in western society. Until recently the widely held belief was that the CVD is simply the process as a build up of fat on the surface of artery walls. Eventually, this build up of fat blocks the artery and a heart attack or stroke occurs. However, the process has now been identified as a disease of the inner artery wall (intima) and inflammation is a key factor in its progression.

The source of inflammation in CVD is not completely understood. However, numerous factors are thought to initiate the complex inflammatory process such infectious agents for example herpes viruses and Chlamydia pneumoniae. Other promoters and stimulators of inflammation leading to endothelial injury include smoking, hyperglycaemia, oxidised low-density lipoprotein (LDL) or sheer stress on the vessel wall by hypertension. Genetic factors may also play a role in the degree and duration of the inflammatory response, although this still needs to be fully explored.

Once stimulated by a promoter or stimulator (including those mentioned above), endothelial cells of the intima interpret their presence as unwanted and activate the immune system to deal with the problem. The gene transcription factor NF-kB is released, serving as a promoter of early cytokines such as TNF-α and IL-6, chemokines such as MCP-1 and adhesion molecules. The chemokines attract monocytes and T lymphocytes (T cells) from the blood stream allowing monocytes to travel across the endothelial barrier and become macrophages. Entry of monocytes into the vessel wall is a key factor in the development of atherosclerosis, as blocking monocyte migration has ameliorated atherosclerosis in in vivo models (1). Once inside the intima, these mononuclear cells produce pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α to stimulate the inflammatory cascade. Metalloproteinases are also released, promoting smooth muscle cell proliferation and uptake of LDL by these macrophages to form foam cells.

Through uptake of LDLs, a fatty streak can develop into a necrotic plaque that is sealed off from the blood flow by the fibrous cap and is held in balance by collagen deposition and degredation. Fissuring or rupturing of this cap can occur when the balance is disrupted by increased inflammation leading to thinning of the collagen cap. The plaque rupture exposes thrombotic substances to the blood, leading to local thrombus formation and downstream microemobolization (2). Furthermore, inflammatory cytokines activate platelets expressing P-selectin and CD40, thus increasing platelet-platelet adhesiveness (3). Cytokines also signal the production of acute phase proteins such as fibrinogen serum amyloid A and C-reactive protein. These are systemic downstream markers which can be useful in assessing cardiovascular risk in patients.

The role of inflammation in cardiovascular disease is not strictly limited to the innate inflammatory response. The adaptive immune response particularly lymphocytes are also involved in CVD. Flow cytometry based methods have quantitatively investigated the cell composition of a normal aortas (4, 5). These have demonstrated that both T and B lymphocytes, macrophages and dendritic cells reside within a major site of the arterial wall (lamina adventitia) of non inflamed aortas. To further visualise the induction of the immune response and investigate the relationship between the immune and cardiovascular systems, multiphoton laser-scanning microscopy (MPLSM) could be used, however this is still at a method development stage (6).

Prevention of the initial development of CVD and progression over time is the goal of any prevention program. With increasing knowledge, the approach to identifying the underlying causes of heart disease is changing rapidly. Much research has identified inflammation as an underlying or active factor in the development of the disease. For the past two decades, clinical trials of antiatherosclerotic drug therapies have sought to reduce CVD morbidity and mortality. This includes the use of a group of drugs called statins (atorvastatin and rosuvastatin) (7) to treat high cholesterol levels which have been shown in large randomised trials, to reduce cardiovascular events in risk patients (8). Research has demonstrated that at higher doses, statins slow or even reverse plaque progression as demonstrated during intravascular ultrasound (9). Recently however, clinical findings have indicated that statins may slow progression of disease at a rate and to an extent that cannot be attributed to lower LDL alone. The proposed mechanisms for such pleiotropic actions include endothelial-dependent nitric oxide bioavailability, inhibition of oxidative stress and anti-inflammatory activity. In particular a number of clinical trials have shown that statins reproducibly lower circulating levels of C reactive protein (CRP) an inflammatory biomarker associated with acute coronary syndromes (10). Reducing inflammation may therefore be a key mechanism by which statins alter the biology of the plaque and slow down disease progression.

Although statins are currently the most popular and widely prescribed drugs to help treat CVD, evidence indicates side effects such as a higher risk of drug interactions in elderly, muscle pain or memory related problems are linked to their use. It is therefore necessary to continue the investigation into inflammation and in inflammatory cell-cell interactions to help develop more effective therapies.

References

Stewart SH, Mainous AG III, Gilbert G. J Am Board Fam Pract 2002;15:437-442.
Taylor, Marcia L. Southern Medical Journal 2004.
Mainous AG, Pearson WS. Fam Med 2003;35:112-118.
Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. J Exp Med. 2006; 203: 1273–1282.
Jongstra-Bilen J, Haidari M, Zhu SN, Chen M, Guha D, Cybulsky MI. J Exp Med. 2006; 203: 2073–2083.
Owain R. Millington, James M. Brewer, Paul Garside and Pasquale Maffia. Methods In Molecular Biology. 2010; 616: part 3 193-206.
http://en.wikipedia.org/wiki/Statin.
Jain MK, Ridker PM: Nat Rev Drug Discov, 2005; 4: 977-987.
Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM, Jr., Kastelein JJ, Koenig W, Libby P, Lorenzatti AJ, MacFadyen JG, Nordestgaard BG, Shepherd J, Willerson JT, Glynn RJ: N Engl J Med, 2008; 359: 2195-2207.
Nissen SE, Tuzcu EM, Schoenhagen P, Crowe T, Sasiela WJ, Tsai J, Orazem J, Magorien RD, O'Shaughnessy C, Ganz P: N Engl J Med, 2005; 352: 29-38.

Inflammation & Pain processing: Relevant preclinical efficacy models

MD Biosciences — Wed, 04 May 2011 18:46:00 GMT

Chronic, destructive inflammation is at the core of a wide variety of diseases and conditions.

Inflammation, whether acute or chronic, is very often associated with pain. Similar to inflammation, pain can be physiological (an adaptive means of protecting tissues from real or perceived danger) or pathological (chronic, and often debilitating despite resolution of the original stimulus). Chronic pain can be caused by a variety of situations including inflammatory diseases such as osteo‐ and rheumatoid arthritis (inflammatory pain), tumor formation (cancer pain), and nerve injury (neuropathic pain).

Pain Processing

While the process of physiological nociception and pain perception is very complex, depending on the quality, intensity, and locality of the stimulus and the species, developmental age, and psychological state of the subjects (i.e., stress level, anticipation, emotional state, etc.), the general pathway for transmitting pain information to the brain is well documented. Nociceptors are pseudounipolar neurons with unencapsulated peripheral terminals the skin, muscles, joints, or viscera; cell bodies residing in the dorsal root ganglion (DRG); and central terminals in the dorsal horn of the spinal cord. There are generally two types of nociceptors – A‐fibers are fast‐conducting with myelinated axons and have small receptive fields for stimulus localization while C‐fibers are slower with unmyelinated axons that are bundled into fascicles wrapped by Schwann cells and have broad receptive fields. Nociceptors normally are electrically silent and have a high threshold compared to somatosensory neurons involved in, for example, vision or hearing. Once stimulated, nociceptors produce all or nothing action potentials releasing glutamate as their primary neurotransmitter and having excitatory effects on postsynaptic cells in the dorsal horn. In the dorsal horn, primary afferent neurons either synapse directly with projection neurons or, more commonly, first with a variety of excitatory and inhibitory interneurons for signal modification. Ascending projection neurons extend, mostly contralaterally, to supraspinal targets including the caudal ventrolateral medulla, the nucleus of the solitary tract, the lateral parabrachial area, the periaqueductal grey matter, and the thalamus. Descending pathways projecting from the nucleus raphe magnus and the locus coeruleus release serotonin and norepenephrin, respectively, via volume transmission in the DRG to further modify pain processing. All along the pain processing pathway, from the primary afferent nociceptors, to the dorsal horn of the spinal cord, to the supraspinal processing centers and including descending projections that further modify processing, there is a delicate balance of excitation and inhibition that is important for properly representing the pain stimulus. Miss‐communication at any of these locations can result in chronic pain.

Selecting Relevant Preclinical Models

Pain therapies can provide relief either through targeting sensitizing agents or by inhibiting the activity of neurons involved in the pain processing directly. Choosing the appropriate pain model should be based off the primary mechanism, site of action, drug class, and required behavioral readouts. Additionally, pain models themselves can be highly customized once the appropriate model has been selected based on the mode of delivery and target. MD Biosciences has extensive experience working with a wide range of drug classes as well as customized applications for route of delivery. We can help choose the appropriate model and approach for your pain therapeutics program. Read a case study covering customized approaches in pain therapies and contact us if you would like to discuss your program.

The link between TH17 & osteoclast function in RA

MD Biosciences — Tue, 19 Apr 2011 16:33:00 GMT

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects approximately 1% of the population, and in 2010 cost the US alone $39.2 billion (1,2). The disease is characterized by bone erosion, cartilage damage, synovial hyperplasia and cellular infiltration, all of which result in debilitating joint pain and stiffness (1,3,4). Studying preclinical models such as the collagen-induced arthritis (CIA) model and the anti-collagen antibody induced arthritis (ACAIA) model, which show the above hallmarks of disease has allowed the identification of the cells and cytokines involved in the pathogenesis of the disease (5,6).

Many of the current therapies designed for RA focus on reducing the inflammation present within the joints but do not impact on the process of bone erosion; therefore one of the current goals in RA research is to inhibit the bone destruction that occurs (7). This exciting new field of research is known as osteoimmunology and it is beginning to highlight the link between the immune system and the skeletal system in the development, progression and establishment of RA (8).

The skeletal system consists of bone, cartilage and the connective tissues that connect the bones. Bone comprises of a solid matrix containing hydroxyapatite crystals, collagen fibres and cells. The main types of cells present within the bone are osteocytes, osteoblasts, osteoprogenitor cells and osteoclasts. Osteocytes are mature cells that maintain the bone matrix by dissolving and rebuilding it; osteoprogenitor cells are mesenchymal stem cells that differentiate into osteoblasts; osteoblasts are immature cells that produce new bone matrix, a process known as osteogenesis; and osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone using hydrochloric acid and enzymes such as cathepsin K and matrix metalloproteinases in a process known as bone resorption (9). In a healthy individual there is a delicate balance between the number and function of osteoblasts and osteoclasts present within the joint, ensuring that in the normal process of bone remodeling the bone that is degraded is replaced. In an individual affected by RA several factors result in an increase in the number and function of osteoclasts, offsetting this balance and causing destructive bone erosion (10).

Interestingly, this field of osteoimmunology is beginning to pinpoint the inflammatory processes present within the arthritic joint, which are driving the osteoclast differentiation and activation. RA was previously thought to be a Th1 mediated disease; however, research has shown that it is most likely that Th17 cells are involved in the pathogenesis of RA, and it has now been established that there is a link between the Th17 cells which are found in the joint and osteoclast function (10,11).

IL-6 along with TGF-β in the presence of IL-23 induces Th17 cell differentiation. IL-6, IL-23 and TGF-β are all produced by macrophages; IL-23 is also produced by activated dendritic cells and TGF-β by synovial fibroblasts (1,3). Th17 cells produce several cytokines including IL-17A, IL-17F, IL-21 and IL-22 (8). IL-17A, which has been found in high concentrations in the synovium and synovial fluid of patients with RA, has multiple functions. It indirectly induces RANKL (receptor activator of NF- κB ligand) expression by synovial macrophages to produce IL-1 and TNF-α, and directly induces expression of RANKL on synovial fibroblasts and osteoblasts (12). RANKL binds to RANK on osteoclast precursor cells and allows these cells to differentiate into mature osteoclasts. Th17 cells also express RANKL, however the current literature shows that Th17 cells alone cannot induce osteoclastogenesis, osteoblasts are also required (11). This may be due to the fact that Th17 cells also produce a low amount of IFNγ, which is known to inhibit the differentiation of osteoclast precursor cells into mature osteoclasts (11).

Along with T cells, macrophages, neutrophils, mast cells and B cells are known t infiltrate the joint and contribute to the ongoing inflammation (1,4). Synovial macrophages express IL-1, IL-6 and TNF-α which are involved in the process of bone resorption (see Figure 1). IL-1 binds to the IL-1 receptors present on mature osteoclasts and TNF-α binds to TNF receptors present on osteoclast precursor cells. Both cytokine-receptor interactions trigger the expression of the transcription factor NF-κB, which allows the activation of osteoclasts and differentiation of precursor cells, respectively (12). TNF-α also induces the expression of RANKL on synovial fibroblasts and osteoblasts, and TNF receptors on osteoclast precursor cells, both of which are important in the differentiation and activation of osteoclasts (8,11,12).

Several interesting developments have already been made within this exciting new field of research. Lubberts et al showed that use of anti-IL-17A in the collagen induced murine model of RA decreased RANKL expression on synovial fibroblasts and osteoblasts and also decreased clinical arthritis scores observed (13); and Sato et al have shown that there is a positive correlation between IL-23 and RANKL expression in the synovium of patients with RA(11). Both studies therefore positively maintain the theory that Th17 cells represent a target for further therapeutic studies in RA. As this new cross-over field highlights further links between the bone remodeling process and the immune system, the prospect for new therapies which aim to tackle the inflammation and the bone erosion in RA looks promising.

References

McInnes, I.B. and Schett, G. Nature Reviews Immunology. 7, 429-442 (2007).
Birnbaum, H. et al. Current Medical Research and Opinion. 26(1), 77-90 (2010).
Brennan, F.M. and McInnes, I.B. The Journal of Clinical Investigation. 118(11), 3537-3545 (2008).
Cascão, R. et al. Neutrophils in rheumatoid arthritis: Autoimmunity Reviews. 9, 531-535 (2010).
Brand, D.D. et al. Springer Seminars in Immunopathology. 25, 3-18 (2003).
Nandakumar, K.S. and Holmdahl, R. Arthritis Research and Therapy. 8, 223 (2006).
van Vollenhoven, R.F. Nature Reviews Rheumatology. 5, 531-541 (2009).
Okamoto, K. and Takayanagi, H. International Immunopharmacology (2010). doi: 10.1016/j.intimp.2010.11.010
Martini, F.H. Fundamentals of Anatomy and Physiology, 7^th Edition.
Sato, K. Allergology International. 57, 109-114 (2008).
Sato, K. et al. The Journal of Experimental Medicine. 203 (12), 2673-2682 (2006).
Adamopoulos, I.E. and Bowman, E.P. Arthritis Research and Therapy. 10, 225 (2008).
Lubberts, E. et al. Arthritis and Rheumatism. 50(2), 650-659 (2004).

Anti-inflammatory cytokines as potential targets for neuropathic pain

MD Biosciences — Fri, 15 Apr 2011 14:29:00 GMT

We are continuing our series on the immune system, inflammation related factors and potential drup targets that fall in the overlap of the immune and nervous system. Our last discussion covered the pro-inflammatory cytokines and their relevance to neuropathic pain. This week we will cover anti-inflammatory cytokines.

IL-4

IL‐4 is released from activated mast cells and T cells and exerts anti-inflammatory effects by inhibiting release of IL‐1β, IL‐6, and TNF, promoting T cell differentiation into the anti‐inflammatory Th2 phenotype, and preventing macrophage and microglia activation. IL‐4 acts via two different heterodimers of IL‐4R. Using the spinal nerve ligation (SNL, Chung) model, pre‐treatment with IL‐4 delays onset of pain hypersensitivity and post‐treatment eliminates pain. These effects are associated with reductions in IL‐1β and microglial activation (1).

IL-10

IL‐10 is released from activated lymphocytes, macrophages, and mast cells and is a potent anti-inflammatory cytokine, known to inhibit the release of IL‐1β, IL‐6, and TNF. IL‐10 functions through binding its heterodimeric IL‐10Rα/β complex. In several preclinical neuropathic pain models, intrathecal administration of IL‐10 prevents or reverses pain hypersensitivity. IL‐10 is thought to act via inhibition of pro‐inflammatory cytokine release and thus reduction in recruitment and activation of other immune and immune‐like glial cells (1).

TGF‐β

TGF‐β is a pleiotropic, anti-inflammatory cytokine that acts via the TGF‐βRIII/RII heterodimeric complex. Intrathecal application of TGF‐β decreases development of pain hypersensitivity and eliminates existing pain in several preclinical models of neuropathic pain. These effects were associated with reductions in microglial proliferation, microglial and astrocytic activation, and neuronal expression of MCP‐1 (1).

Reference

Austin, PJ and Moalem-Taylor, G (2010) The neuro-immune balance in neuropathic pain: involvement of inflammatory immune cells, iimmune-like glial cells and cytokines. Journal of Neuroimmunology, 229 (1-2):26.

Adipose tissue - a site of inflammation?

MD Biosciences — Tue, 12 Apr 2011 16:41:00 GMT

Inflammatory events associated with Obesity

There is an increasing problem with obesity worldwide. In 2008 the World Health Organisation reported that 1.5 billion adults (+20yrs) worldwide are overweight (defined by a body mass index of equal to or greater than 25). Of these figures around 500 million were classed as obese with a BMI greater than 30 (1). Obesity has been associated with an increased risk in chronic diseases including cardiovascular disease, diabetes and some cancers. It has also been suggested that some forms of obesity are associated with low grade chronic inflammation. What remains to be elucidated is the primary trigger that is responsible for initiating the inflammatory cascade within adipose tissue. Here, we explore the cell interactions and cytokines associated with adipose tissue and their impact on inflammatory disease.

Adipose tissue - a site of inflammation?

The function of adipose tissue is to store energy in the body for times of calorific restriction but it is apparent that fat is not just simply an inert storage organ. Recent studies have demonstrated a crucial link between fat tissue and inflammation and revealed close interactions between adipocytes and cells of the innate and adaptive immune response. Adipocytes release bio-active molecules known as adipokines as well as secreting pro-inflammatory cytokines like IL-6, IL-8 and TNF (2). In particular, the key adipokine known as adiponectin is found at a high plasma concentration and can enhance insulin sensitivity as well as having anti-inflammatory properties. Adiponectin deficient mice have increased expression of endothelial adhesion molecules as well as increased leukocyte binding (2). Furthermore, adipocytes release chemoattractants such as monocyte chemotactic protein (MCP-1) that are able to draw monocytes from the blood into adipose tissue. Elevated MCP-1 levels correlate with weight gain and are over-expressed in adipose tissue (3) resulting in increased numbers of adipose tissue macrophages (ATM), a phenomenon which has been demonstrated in rodent models and in humans (4). These adipose resident macrophages shift from a protective alternatively activated (‘M2’) macrophage state to a more classical pro-inflammatory ‘M1’ state due to pro-inflammatory cytokines involved in activation of macrophages like TNF-α and IL-6 (5), a crucial process in inflammation associated with obesity.

ATMs have been well characterised in their involvement in insulin resistance in obese individuals. Using leptin (ob/ob) or leptin receptor (db/db) deficient mice and diet induced obesity (DIO) in C57BL/6 mice Xu et al demonstrated that the expression of genes related to inflammation are restricted to macrophages in the white adipose tissue (WAT). Using PPARγ agonists such as Rosiglitazone, which regulate adipocyte differentiation and are used therapeutically for treating insulin sensitivity, the mRNA levels for inflammation genes such as MIP-1α, MCP-1, ADAM can be down regulated in ob/ob mice. (6). Furthermore, upregulation of genes associated with inflammation were not found to be abundant in muscle, liver or spleen of obese mice. The expression of inflammatory specific genes in adipose tissue macrophages is reported to occur before the increase in insulin resistance. This suggests that inflammation associated with obesity may be crucial in the development of obesity related insulin resistance which can lead to type II diabetes (6). These findings suggest a strong correlation with inflammation and white adipose tissue in obesity. Further to this, there is a strong correlation with macrophages in adipose tissue and insulin resistance in mouse models, demonstrated by evaluating the temporal expression of macrophage marker levels in the WAT of mice fed a high fat diet. It was found that the expression of macrophage markers were up-regulated before the increase in circulating insulin levels at 16 weeks of high fat diet. These results suggest that macrophage activity occurs after the increase in adipose tissue but before the insulin resistance (5). Interestingly, lymphocyte infiltration to white adipose tissue has been shown to precede the activation of macrophages in white adipose tissue (7).

Furthermore there is compelling evidence to suggest that IL-17 could be a key link between adipose tissue and inflammation. IL-6 production in association with adipose tissue has led investigation into Th-17 differentiation and IL-17 associated autoimmunity in obesity. T-cells from diet induced obese (DIO) mice expand TH-17 cells and produce higher levels of IL-17 than lean controls in an IL-6 dependent manner (8). This bias towards a TH-17 like state may be linked to exacerbation of autoimmune diseases. However, it is unclear how this relates to macrophage activation in white adipose tissue, but it is clear that the process is much more dynamic than involving only a few cell subtypes.

References

World Health Organisation
Vachharajani, V et al. (2009) Life. 61(4), 424-430
Rasouli, N. (2008) J Clin. Endocrinol. Metab. 93: s64-s73
Nishimura s. et al (2009) Inflammation and Regeneration 29, 118-122
Lumneg, C.N. (2007) The Journal of Clinical Investigation. 117, 178-184
XU, H et al. (2003) J. Clin. Invest. 112:1821-1830
Caspar-Baguil. S et al (2009) Journal of physiology and biochemistry. Volume 65, Number 4, 423-436
Ahmed, M. (2010) Cytokine and Growth Factor Reviews. 449-453
Wellen, K. E et al (2003) The journal of clinical investigation. 112, 1785-1788

The need for novel asthma treatments & suitable preclinical models.

MD Biosciences — Wed, 06 Apr 2011 16:08:00 GMT

There is a major unmet need in the treatment of asthma which is growing in incidence and prevalence in industrialized countries. The prevalence of asthma has doubled in the Western world over the previous 20 years. In addition to the estimated 180,000 asthma related deaths per year, there is a substantial economic burden due to lost school/work days and increased medical costs.

Allergic asthma is typically triggered by allergens in the air such as pollen, mold, dust mites etc and is commonly characterized by reversible airway destruction, elevated levels of IgE causing mast cell activation, chronic airway inflammation and airway hyper-responsiveness (AHR). The immunological processes involved are characterized by proliferation and activation of Th2 lymphocytes, setting off an allergic cascade. Treatments currently available act by dampening inflammation or relaxing airways but do not alter underlying pathology and disease exacerbations still occur. These facts highlight the need for novel treatments, which in turn require suitable efficacy models.

Ovalbumin-induced Asthma Model
The immune response during asthma is well preserved between mice and humans. In human asthma, eosinophils and lymphocytes are found to infiltrate the bronchial mucosa. Increased mucus secretion and production of Th2 associated cytokines such as IL-4, IL-5 and IL-13 are also found. IL-4 induces differentiation of CD4 T cells into Th2 cells, induces the proliferation of activated B cells and is the major cytokine involved in B cell class switching to IgE (the antibody isotype most associated with human asthma). IL-5 is involved in eosinophil activation and also facilitates B cell growth and antibody production. The activities of IL-13 and IL-4 show a high level of overlap, although it is thought that IL-4 acts primarily in the initial sensitisation, with IL-13 more important during secondary exposure to the allergen. In addition to inducing IgE production, IL-13 can induce AHR, goblet cell metaplasia and airway glycoprotein hypersecretion, which all contribute to airway obstruction. Mast cells are also central to the development of asthma due to their ability to release an array of preformed and newly synthesized inflammatory mediators such as cytokines, leukotrienes and prostaglandins. Mast cells are also thought to be involved in the tissue remodelling that occurs later in asthma.

While we know many factors and events that play an important role in the initiation, progression and persistence of allergic asthma, there is still a lot to be understood about the immunoregulatory mechanisms. The murine OVA-induced asthma model is a widely used model that results in the characteristic features of asthma allowing the study and assessment of novel treatments.

The study of lung function
In recent years there has been much discussion as to whether methods of studying lung function in pre-clinical models of allergic lung inflammation are worthwhile. Some researchers maintain that the physiological differences in rodent lung function versus human lungs mean that lung function studies in rodents are meaningless. Despite this, the most consistent diagnostic feature of asthma is airway hyperresponsiveness (AHR) in response to chemicals such as Methacholine or Adenosine. For this reason, many researchers feel that in order for an asthma therapeutic to be efficacious, it must be shown to affect AHR.

Although much research on this topic has been carried out, we still do not fully understand why the AHR response occurs. Airway inflammation involving cytokines such as IL-4, IL-5 and IL-13 and cells such as mast cells and eosinophils as well as neurogenic abnormalities are believed to contribute to AHR.

Analysis of BALF fluid for cell composition and cytokine excretion
In a BALF sample from a normal lung, macrophages make up greater than 90 percent of the cells present. In a normal lung the lymphocytes represent 10% of the cells, however in the OVA preclinical model, there is an increase in number of lymphocytes even if the % remains static. Cytokines excretion is also elevated in the BALF of an asthmatic mouse.

Conclusion
Preclinical asthma models remain an important tool for the industry as they are well-characterized and offer well-established readouts such as pulmonary cell influx and antibody levels, which have good correlation with human disease. The wide range of readouts allows the design of experiments with the greatest potential to identify any anti-asthmatic activities possessed by test compounds.

Pro-inflammatory cytokines as potential targets in neuropathic pain.

MD Biosciences — Mon, 04 Apr 2011 18:06:00 GMT

Over the past few weeks we have been reviewing the overlap between the nervous and immune systems. We recently discussed the various cell types involved and are going to move into some of the various inflammation related drug targets that may have potential involvement in neuropathic pain. We'll start with pro-inflammatory cytokines.

IFN-gamma:

Interferon gamma is a type II interferon in which the active form is a soluble homodimer with potent pro-inflammatory properties. It is mainly produced by T-cells. IFN-γ binds the cell surface receptor IFN-γR to initiate signaling.

In the spinal cord, IFN-γ is produced by infiltrating Th1 cells as well as astrocytes and damaged neurons. It has been shown to recruit and activate microglia. Intrathecal application of IFN-γ induces pain hypersensitivity in intact animals and not in IFN-γR^-/- animals. IFN-γ also increases neuron excitability and spontaneious firing in the dorsal horn (1, 2).

IL-1β

IL-1β is a power anti-inflammatory cytokine with pleiotropic effects on mny cell types via binding cell-surface IL-1R1 receptor. IL-1b activity is modulated by both the natural receptor agonist, IL-1ra, and the IL-1RII decoy receptor.

IL-1β production is up-regulated upon peripheral nerve damage at the site of injury, the DRG, and the spinal cord. IL-1b increases neuronal excitability and has direct algesic effects when applied intraneurally (sciatic nerve) or intrathecally in rats. In CCI-injury, IL-1R neutralizing antibody applied to the wound or IL-1b neutralizing antibody delivered intrathecally reduces allodynia, Further both IL-1RI knock-out mice and mice over-expressing IL-1ra show decreased hyperalgesia and allodynia after SNL surgery.

IL-6

IL-6 is a predominantly pro-inflammatory cytokine, though in some contexts can have anti-inflammatory effects. It is produced by a variety of leukocytes and exerts its biological activity by binding the IL-6R, which heterodimerizes with its gp130 signaling component.

Nerve damage results in a local up-regulation of both IL-6 and IL-6R expression as well as an increase in IL-6 found in the DRG. Peripheral nerve injury also induces an increase in IL-6 expression in the dorsal horn of the spinal cord in microglia and neurons. Intrathecal delivery of IL-6 results in pain hypersensitivity that can be blocked by IL-6 neutralizing antibodies. Il-6 mediated signaling induces CX3CR1 expression by microglia following CCI in rats (1)

TNF

Tumor necrosis factor (TNF) us a small diffusible homotrimeric cytokine with pro-inflammatory properties and two receptors (TNFR1 and TNFR2) expressed by a variety of cells. TNFR1 is constitutively expressed and when activated results in ligand/receptor complex internalization. TNFR2 is inducibly expressed and when bound results in ligand/receptor complex shedding from the cell surface.

Peripherally, nerve damage results in TNF release from resident mast cells and macrophages, Schwann cells, and infiltrating neutrophils and macrophages locally and from satellite glial cells, neurons and infiltrating macrophages in the DRG. Intraneural injection of TNF in intact rats results in pain hypersensitivity that is reversible with neutralizing antibody. Centrally, TNF is increased, likely as a result of microglia and astrocyte activity, in both the spinal cord and supraspinal targets. TNF encourages neuronal hyperexcitability (via its effects on neuronal cation channels) and inflammation at all locations along the pain processing pathway (1, 4).

References

Austin, P.J. and Moalem-Taylor, G. (2010) Journal of Neuroimmunolgy. 229(1-2), 26.
Smith, H.S. (2010) Pain Physician. 13:295
Ren, K and Torres, R (2009) Brain Research Reviews. 60:57
Leung, L and Cahill, C.M. (2010) Journal of Neuroinflammation. 7(1):27.

T cells in Rheumatoid Arthritis: where, why and when?

MD Biosciences — Mon, 28 Mar 2011 17:02:00 GMT

The role of T-cells and their actions in rheumatoid arthritis (RA) has been the focus of a great deal of research for some time [1], mainly as a result of many observations in human patients and experimental animal models. The association of Human Leukocyte Antigen (HLA) DR, a MHC class II cell surface receptor, in RA provides the strongest evidence that CD4+ T-cells are involved in the development of disease [2, 3, 6]. Many other types of T-cells, including CD8+, regulatory T-cells and γδ T-cells have been shown to play different roles in the progression of RA [1, 2, 3, 8]. The mechanisms of disease involved in RA are still unknown; however the main hypothesis theorizes that auto antigens are presented to auto reactive T helper cells, which then orchestrate the inflammatory processes which are characteristic of the disease [3]. The nature of the antigens involved is unknown however several candidates have been suggested, most recently, citrullinated proteins [5, 6].

Work in animal models of arthritis has provided valuable information on the role of T-cells in this complex disease [9]. The importance of T-cells can be shown in the transfer of CD4+ T-cells from an arthritic animal to a healthy animal, which leads to induction of tissue damage in the recipient [2]. Interestingly, recent research has delivered surprising results on the location of T-cells in models of arthritis [4]. In the model of proteoglycan induced arthritis (PGIA), fluorescence-labelled donor T-cells were tracked in SCID mice and the majority were found in the lymph nodes with unexpectedly few found in the joint [5]. In addition, this study also demonstrated that inhibition of T-cell movement from the lymph tissue, using the drug FTY720, did not prevent development or reduce severity of disease in the PGIA model, suggesting that T-cells do not have to be present in the joint for the induction of arthritis in this model [5].

In another study, this time using humanized DR-4 transgenic mice in the collagen induced arthritis (CIA) model, pro-inflammatory T-cells were tracked using a tetramer and were found in the blood and joints during early stages of the disease [3]. However, as disease progressed, the T-cells became undetectable in the joint, rare in the blood and decreased in the lymph tissues. The authors of this study conclude that there may be a “threshold” of activated T-cells circulating that is required for the onset of disease, and once reached the number of active T-cells decreases in the blood, lymph nodes and synovial fluid [3]. These findings raise interesting questions on the link between the location and role of T-cells in models of arthritis.

With all of the evidence for the involvement of T-cells in the development of RA, there is potential for T-cell specific therapies to be the next line in defense against the disease [2]. The most recent T-cell targeted therapy is Abatacept (Orencia®), a fusion protein of the extracellular portion of CTLA4 and human IgG1Fc, which interrupts the “second signal” between the CD28 ligand on the T-cell by binding the CD80/86 ligand on the antigen presenting cell [6, 7, 10]. Clinical trials have shown that Abatacept improves swollen and tender joint complaints and radiological results in patients with disease reduction comarable to some present anti-TNF agents while providing a better safety profile than those drugs [7, 10]. The impressive clinical data for this drug suggests that T-cell specific therapies are promising new agents in the treatment of RA.

The importance of T-cells in RA has been highlighted through a focus of research on the subject, however many questions still remain over their exact function in the pathogenesis of disease. The questions surrounding the location of T-cells during disease, the still unidentified auto antigens that cause the disease and the failure of T-cells to function properly are all areas that demand further research to advance our knowledge of the most common inflammatory condition in humans.

References:

Fournier, C. (2005). Joint Bone Spine, 72: 527-532
Skapenko, A. et al (2005). Arthritis Research & Therapy, 7(Suppl 2): S4-S14
Svendsen, P. et al (2004). The Journal of Immunology, 173: 7037-7045
Kamradt T, Frey O. (2010). Arthritis Research & Therapy, 12:122
Angyal, A. et al (2010). Arthritis Research & Therapy, 12:R44
Andersson, A. et al (2008). Arthritis Research & Therapy 10:204
Solomon, GE (2010). Bulletin of the NYU Hospital for Joint Diseases, 68(3):162
Roark, CL. et al (2007). The Journal of Immunology, 179: 5576-5583
Goronzy JJ, Weyland, CM (2009). Arthritis Research & Therapy, 11:249
Van Vollenhoven, R.F. (2009) Nature Reviews Rheumatology, 5: 531-541

Cells involved in the neuro/immune interactions along pain processing pathway.

MD Biosciences — Tue, 15 Mar 2011 19:19:00 GMT

Previously we discussed various interactions that occur between the immune and nervous systems that are potential contributors to neuroinflammatory disorders. In this post, we will call out some of the specific cells involved in these interactions.

At the anatomical level, neuro‐immune interactions have been shown to take place all along the pain processing pathway. This is partially facilitated by increased permeability of the blood‐brain barrier following SCI or peripheral nerve injury [1]. At the cellular level, neuro‐immune interactions involve leukocytes including mast cells, neutrophils, macrophages, and T cells as well as glial cells with immunelike functions including Schwann cells and satellite glial cells in the PNS and microglia and astrocytes in the CNS. Alterations in glial cell function other than those associated with immune cells, immune system signaling molecules, or immune‐like functions of glial cells (i.e., alterations in neurotrophic factor signaling, potassium ion buffering, neurotransmitter re‐uptake, or gap junction maintenance) are outside the scope of this review and are not included here.

Mast Cells: Resident tissue mast cells become degranulated at the site of nerve injury and release histamine, serotonin, proteases, prostaglandins, and a variety of cytokines. Some of these are capable of sensitizing (histamine and TNF) or directly activating (serotonin) nocicpetors, and recruiting neutrophils and macrophages to the wound (histamine and TNF), initiating inflammation and resulting in hyperalgesia. In rats following PNL, these effects can be significantly reduced by chemical prevention of mast cell degranulation. The mechanism by which mast cells are triggered is yet unknown. [2]

Neutrophils: Neutrophils rapidly infiltrate the site of nerve injury (peaking at approximately 24h), responding to chemoattractants such as NGF and MCP‐1, and promote the inflammation process by releasing pro‐inflammatory cytokines (IL‐1β, IL‐6, and TNF) and macrophage‐attracting chemokines (MIP‐1α and MIP‐1β). Additional neutrophil activities, particularly within the DRG, have been suggested, but are yet controversial. Nevertheless, the importance of a role for neutrophils is highlighted by evidence that either depletion of circulating neutrophils or prevention of neutrophil infiltration immediately following nerve injury results in the reduction of hyperalgesia and allodynia in rats. [2]

Macrophages: After nerve injury, resident tissue macrophages become active and circulating macrophages are recruited to the wound (beginning at approximately 24h), to the DRG (one week after CCI) and to the spinal cord (after SCI). Knocking down activity of macrophage chemoattractants, MIP‐1α and MIP‐1β , results in a reduction in macrophage infiltration and Wallerian degredation (phagocytosis of dead or dying Schwann cells, axotomized axons, and other cellular debris). Macrophage depletion by a variety of means can result in reductions of hyperalgesia in several neuropathic pain models, though there are some conflicting reports. Further, once activated, macrophages release a variety of signaling molecules that contribute to neuropathic pain (i.e., IL‐1β, IL‐6, TNF, reactive oxygen species, prostaglandins, and Cathepsin S) and pharmacological suppression of activation results in alleviation of
symptoms. For example, suppression prevents up‐regulation of sciatic nerve IL‐1β and associated hyperalgesia in mice following PNL. [2]

T Cells: T cells enter sites of nerve injury (onset at 3 days, peak at 3 weeks), the DRG, and the spinal cord (peak at 7 days) and have been demonstrated to have a significant role in the development of neuropathic pain as hyperalgesia either does not develop or is significantly reduced in murine models of T lymphocyte dysfunction. T cells engage in killing activity (Tc cells), interact with glia to increase inflammation and further sensitize neurons, and release pro‐inflammatory cytokines themselves (Th1 and Th17 cells). Giving Th1 cells to CCI‐treated rats lacking functional T cells increases pain sensitivity while giving Th2 cells decreases it, thus implicating the involvement of T lymphocyte derived proinflammatory cytokines IFN‐γ, TNF, and IL‐17. [2]

Schwann Cells & Satellite Glial Cells: Within hours of nerve injury, Schwann cells regain the ability to proliferate and migrate, initiate or up‐reguate the release of a variety of cytokines, chemokines, neurotrophic factors, and other signaling molecules, and begin demyelination. Pro‐inflammatory cytokines are liberated, some of which (IL‐1β and TNF) are shown to directly contribute to hyperexcitability of neurons in addition to their effects on promoting inflammation. Chemokines involved in recruiting macrophages (LIF and MCP‐1) are released. Similar to Schwann cells in peripheral nociceptive nerves, satellite cells in the DRG become activated and begin to proliferate and release proinflammatory cytokines IL‐1β and TNF. It is thought that satellite cells are activated by ATP from damaged neurons via purinergic receptors. [2]

Microglia & Astrocytes: Resident and infiltrating microglia are very rapidly activated (within 4h of injury) by cytokines (IFN‐γ among others), chemokines (Fractalkine and MCP‐1), toll‐like receptor (TLR) activity, ATP (via P2 purinergic receptors), glutamate, and neuropeptides including Substance P from both neuronal and glial sources in the spinal cord and supraspinal targets. Blocking microglial spinal cord infiltration, by MCP‐1 neutralizing antibody or lack of CCR2, result in reduction in pain hypersensitivity in post‐PNL mice. Further, a growing body evidence demonstrates that microglial activation is both necessary and sufficient for induction of neuropathic pain. Peripheral nerve damage induces microglial activation and pharmacological inhibition of that activation reduces both developing and existing neuropathic pain in animal models. Once activated, microglia release pro‐inflammatory and pronociceptive molecules including IL‐6, TNF, nitric oxide (NO), and prostaglandin E2 (PGE2), promoting inflammation and sensitizing nearby neurons.[1-3] Astrocytes in the spinal cord become activated after microglia, beginning at 4 days post‐injury and lasting 3 months or more, suggesting astrocyte participation in maintenance of neuropathic pain. Once activated, astrocytes are known to increase the sensitivity of or directly excite neighboring neurons (via release of reactive oxygen species, prostaglandins, excitatory amino acids, and ATP) and promote inflammation (via the pro‐inflammatory cytokine signaling of IL‐1β, IL‐6, and TNF). Evidence of astrocyte activation post‐injury appears in supraspinal targets of pain processing as well. While specific pharmacological inhibition of astrocyte activation is difficult, use of general glial inhibitors results in reductions in neuropathic pain in animal models. [2]

Would you like to discuss potential targets that would be relevant in either neuropathic pain or inflammatory diseases? Our scientists are specialized in both areas and would be happy to speak with you. Contact us today.

References

Saab, C.Y. and Hains, B.C. (2009). Remote neuroimmune signaling: a long‐range mechanism of nociceptive network placticity. Trends in Neuroscience, 32(2), 110‐117.
Austin, P.J. and Moalem‐Taylor, G. (2010). The neuro‐immune balance in neuropathic pain: involvement of inflammatory immune cells, immune‐like glial cells and cytokines. Journal of Neuroimmunology, 229(1‐2), 26‐50.
Smith, H.S. (2010). Activated microglia in nociception. Pain Physician, 13, 295‐304.

Case study: 25 preclinical CAIA studies, 18 months, 1 out-licensed compound

MD Biosciences — Mon, 07 Mar 2011 20:14:00 GMT

With the number of blockbuster drugs approaching patent expiration and pharma companies struggling to maintain pipeline and portfolios with in-house programs, companies are increasingly turning to licensing, aquisitions and partnerships. Early-stage licensing deals tend to carry more risk for pharma companies in-licensing. To offset this risk, additional data may be required from the pharma partner to confirm any internal research performed by the biotech/out-licensing company.

Mini-case study. MD Biosciences helps a medium-sized biotech company to develop Rheumatoid Arthritis drug for out-licensing in under two years.

A medium-sized biotech company approached us with a compound that showed some efficacy in rheumatoid arthritis (RA) with a business goal of out-licensing the potential drug within two years. In order to push this compound through in approximately 18 months, a strategic and precisely timed development program needed to be established. MD Biosciences was able to present the sponsor with a program that entailed 25 preclinical studies over the course of 18 months. This was made possible by modifying models such as the rapid 10-day Collagen Antibody-induced arthritis (CAIA) model. Each study was customized specifically to address the appropriate questions, building from one study to the next.

The outcome was a successful preclinical program in which 25 carefully designed studies were executed with rapid turn-around times and quality, consistent results. This is critical to allow data comparison over the course of the development period. After 18 months, the compound was developed to an appropriate stage and the company succeeded in out-licensing in a multi-billion dollar deal, indicating this development program as central to the success.

About MD Biosciences

MD Biosciences approaches every preclinical study with the end result in mind. Careful study design is critical to deliver data that will inform about next steps. Often standard models don't address the questions properly and through either minor customizations or a custom developed model, a sponsor can be have access to data that is more informative. If you would like to speak with a scientist on a specific study design, please contact us.

Interaction between nervous & immune systems. Preclinical targets.

MD Biosciences — Thu, 03 Mar 2011 20:28:00 GMT

Some of the most interesting and rapidly developing areas inbiomedical science are those being built between the lines previously drawn around classical fields of study. Neuroimmunology is jsut one of the many examples and is a field that is growing as researchers find interactions between the nervous and immune systems not previously known, and discover that some well-known disorders perhaps fall into this overlap category.

Immune System

As a result of constant pressure from environmental toxins and microbes, the immune system has evolved into a very complex physiology that functions to distinguish self vs. non‐self and mount specific and aggressive attacks when foreign material is detected. The innate immune system serves as the front line for host defense and includes anatomical barriers (i.e., tight cell‐to‐cell contacts and secreted mucous), cellular surveillance mechanisms (i.e., early identification and phagocytosis of foreign microbes by immune cells), and a protective biochemical environment (i.e., altered pH, bioactive small molecules, and soluble proteins in biological fluids such as mucous, saliva, tears, etc.). In addition to the constitutive presence of these defenses, the innate immune system responds to signs of microbe invasion by rapidly up‐regulating various diffusible signaling factors and recruiting additional immune cells as well as activating the adaptive immune response by initiating inflammation, presenting foreign antigen, and guiding effector lymphocytes to the site of infection. Protection of the host from microbial invasion is not the only task the immune system must manage – it is also responsible for wound healing. The wound repair process is activated immediately upon detection of tissue damage and proceeds through several overlapping phases.

Inflammation, whether acute or chronic, is very often associated with pain. Similar to inflammation, pain can be physiological (an adaptive means of protecting tissues from real or perceived danger) or pathological (chronic, and often debilitating despite resolution of the original stimulus). Chronic pain can be caused by a variety of situations including inflammatory diseases (inflammatory pain), tumor formation (cancer pain), and nerve injury (neuropathic pain).

Pain processing

Stay tuned as we continue to explore in the coming weeks Neuropathic pain, Cell Types involved in the Neuro-inflammation aspect or neuropathic pain, Relevant preclinical models, and Potential inflammation-related drug targets for treatment of neuropathic pain.

About MD Biosciences
MD Biosciences is a preclinical CRO offering services in inflammations, neurology, pain and cardiac. If you would like to speak with a scientist, please contact us now.

Inflammatory mediators and neuropathic pain: preclinical models

MD Biosciences — Mon, 24 Jan 2011 11:40:00 GMT

Neuropathic pain is a chronic pain condition caused by lesion or inflammation affecting the nervous system. It is relatively common, can be severely debilitating and clinically significant relief is often difficult to achieve in part because conventional opioid therapy is typically less effective for neuropathic pain. The common symptoms of neuropathic pain include allodynia (pain resulting from normally innocuous stimulus), hyperalgesia (increased sensitivity to painful stimuli) and spontaneous pain. It has been widely known that a number of mechanisms are involved such as ectopic excitability of sensory neurons, altered gene expression of sensory neurons, and sensitization of neurons in the dorsal horn of the spinal cord. However, increasing evidence and research points to the interaction between the immune system and the nervous system playing a crucial role in the the underlying mechanisms of neuropathic pain (1). Following nerve damage, an inflammatory response is initiated: complement system is activated, a variety of inflammatory cells are recruited to the site of nerve injury, dorsal root ganglia (DRG) and to the spinal dorsal horn. Activation of immune-like glial cells and an upregulation of inflammatory mediators all contribute to neuropathic pain (1-10).

Immune-like glial cells implicated in the pathogenesis of neuropathic pain are mast cells, neutrophils, macrophages and T cells in the peripheral nervous system and microglia and astrocytes in the central nervous system (1). Inflammatory mediators implicated include cytokines, chemokines, prostaglandins, histamine, opioid peptides etc (1). Of the cytokines, it has been reported that TNFa, IL-1, IL6 and IL-10 are involved in neuropathic pain (4). More recent research also points to a possible involvement of IL-17 and TH17, which is already indicated in numerous inflammatory disorders such as multiple sclerosis, arthritis, and psoriasis (11) and continuing research. Research has demonstrated that T cells in the injured peripheral nerve and dorsal spinal cord contribute to neuropathic pain and T-cell infiltration after nerve injury was associated with IL-17 expression (12). While IL-17 is clearly not the only inflammatory mediator involved, ongoing research suggests cross-communication between IL-17 and other cytokine signaling systems is potentially involved in the pathogenesis of neuropathic pain (12). An understanding of these mechanisms that underly neuropathic pain is crucial to developing more effective therapies. The use of relevant preclinical models of neuropathic pain such as chronic constriction injury and spinal nerve ligation will play a central role in the research and development of new therapies.

References

Moalem, G and Tracey, DJ (2006) Brain Res Rev 51:240.
Griffin RS, et al (2007) J Neurosci 27:8699
Li M, et al (2007) Eur J Neurosci 26:3486-3500
Cui JG,et al (2000). Pain 88:239
Hu P, et al (2007) Brain Behav Immun 21:599
Sweitzer SM, et al (2002) Pain 100:163
Hanani M, et al (2002) Neuroscience 114:279-283, 2002
Colburn RW, Rickman AJ, DeLeo JA: (1999) Exp Neurol 157:289
Milligan ED, et al (2003) J Neurosci 23:1026
Watkins LR, Maier SF (2002) Physiol Rev 82:981
Kim, CF and Moalem-Taylor, G (2010) Journal of Pain doi:10.1016/j.jpain.2010.08.003
Kleinschnitz C, et al (2006) Exp Neurol 200:480

The link between pain & inflammation - relevant preclinical models?

MD Biosciences — Wed, 15 Sep 2010 19:12:00 GMT

Considering the close link between inflammation and the pain process, preclinical efficacy models that allow the evaluation of both pain and inflammation are crucial to developing new therapies.

One of the prominent features of inflammatory conditions is that normally innocuous stimuli produce pain. The pain process involves several areas which include nociception, pain perception and pain behavior. After tissue injury or nerve damage, neurons along the nociceptive pathway may display enhanced sensitivity and responsiveness. A variety of events and agents can contribute to this sensitization, including the release of inflammatory mediators (such as cytokines or prostaglandins) or the release of algesic substances from damaged cells. Cytokines and prostaglandins are important mediators of inflammation that also have an effect on pain and nociceptors. Cytokines have influence over sensory neurons and may act directly upon nociceptors or indirectly by stimulating the release of prostaglandins, which are considered sensitizing agents and in some cases directly activate nociceptors.

What would efficacy and mechanistic models that allow you to evauate the effects on pain as well as the contributing inflammatory conditions mean for your therapy?

Download a complimentary whitepaper and eBook to learn:

Pain processing and pathways
Behavior tests for common drug classes based on primary mechanism and site of action
How to discover or confirm how and where a compound acts upon the adaptive immune response
Interactions of cells that are crucial to all immune responses
Models that enable the evaluation of effects on pain and the contributing inflammatory conditions

The EAE model is associated with all Seven FDA Approved MS Drugs

MD Biosciences — Tue, 24 Aug 2010 14:54:00 GMT

The Experimental Autoimmune Encephalomyelitis (EAE) model is associated with all Seven FDA Approved MS Drugs and Fingolimod

Introduction:
Multiple Sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that results in motor, sensory and cognitive impairment. MS is one of the most common disabling neurological diseases in young adults and is more prevalent in Caucasians of northern European ancestry. The disease course is unpredictable and life-long, and affects women more commonly than men. The main characteristics of this disease are focal areas of demyelination and infiltration of inflammatory cells in the CNS. Despite numerous studies and experimental trials a complete understanding of the pathogenesis still remains unclear. The etiology of the disease seems to be dependent on genetic and environmental factors, which result in substantial observed variations throughout the course of the disease. Today, new treatments and medications are advancing hope for people affected by the disease, and the experimental autoimmune encephalomyelitis (EAE) model continues to play an essential role in MS drug development.

On January 20, 2010, Novartis reported that its oral drug Fingolimod (FTY720) had positive results after phase III clinical trial testing and that Novartis is submitting formal documents for drug approval by the Food & Drug Administration (FDA). Fingolimod would be the first FDA approved oral medication for the treatment of MS. Similar to the current FDA approved, disease-modifying drugs for MS, Fingolimods success has been associated with research conducted in models of EAE. Although no animal model thus far establishes all facets of human MS, EAE models are the most commonly used disease models and are an important tool for scientists testing MS drug candidates.

Current drugs for MS and Fingolimod:
Three of the seven FDA approved drugs for MS, Tysabri® (Natalizumab), Copaxone® (Glatiramer Acetate) and Novantrone® (Mitoxantrone), were clinically developed as a direct result of initial discoveries made using an EAE model. Glatiramer Acetate was first modeled in 1971 as a mixture termed Copolymer 1, consisting of glutamate, tyrosine, alanine and lysine. It was first tested in an acute model of EAE and then subsequently in guinea pig and nonhuman primate models of relapsing EAE where in both instances it suppressed disease. Following the initial success in the EAE model it was clinically tested and found to be effective in the treatment of relapsing-remitting MS. The entire development process from the modeling in EAE to FDA approval took 25 years.

Mitoxantrone was first modeled in EAE using rats because it belonged to a class of drugs that had cytotoxic effects, but had previously shown potential for improving MS. Using this model it was found to reverse paralysis. The preliminary work was done in the mid-1980s and progressed into clinical testing that demonstrated positive results leading to its approval by the FDA in 2000 for the treatment of progressive-relapsing, secondary-progressive and worsening relapsing-remitting MS.

Natalizumab is another drug developed as the result of EAE findings. It was first modeled using rats where it inhibited paralysis in an acute model of EAE. This work was done in the early 1990s and similar to the other drugs was tested in clinical trials, which resulted in its FDA approval for the treatment of relapsing MS in 2004. Soon after its approval, it was pulled from the market as a result of two deaths where patients developed Progressive Multifocal Leukoencephalopathy (PML). In 2006, Natalizumab was re-approved by the FDA after a panel voted unanimously that the benefits outweighed the risks.

The other four drugs, in the category known as b-interferons, were tested in EAE models as an additional assessment to further characterize their mechanism of action, and were not clinically developed as a direct result of an initial EAE investigation. b-interferons were developed based on the thought that MS was a virally mediated disease, and that the known immunomodulatory effects of b-interferon may be used to treat MS. The two b-interferons used for the treatment of MS are INFb-1a and INFb-1b. INFb-1a is identical to endogenous IFN-b and IFNb-1b differs in structure by two amino acids and is not glycosylated. The active ingredient in the drugs Avonex® and Rebif® is INFb-1a, while INFb-1b is the active ingredient in Betaseron® and Extavia®. b-interferons have been shown to reduce the progression of disease and the extent of demyelination in a PLP-induce EAE mouse model. Not only did this coincide with clinical trial test results, it was also an effective means to further characterize the drug pathway.

As mentioned in the introduction, oral Fingolimod is the latest drug that is currently pending approval by the FDA. It would be the first oral disease-modifying drug for MS as the others are delivered by injection or IV infusion. Fingolimod modulates sphingosine-1 phosphate receptors. It’s thought that the drug causes T cells and B cells (immune cells) to be immobilized in the lymph nodes obstructing their circulation to the central nervous system, thus alleviating the inflammatory aspects of MS and concomitant damage to neurons and myelin. Before Fingolimod was pursued in the treatment of MS, it had previously been used as an immunomodulating agent for islet transplantation and kidney transplantation. Its advantage over traditional immunosuppressive agents was its ability to not inhibit T cell activation and to not hinder viral immunity. Comparisons were drawn and scientists took note of its potential ability as a therapeutic for MS. Tests were first conducted using an EAE model in Lewis rats that indicated that oral Fingolimod reduced lymphocyte trafficking and CNS inflammation. This provided intriguing data to move forward with the clinical development of Fingolimod as a therapeutic agent for MS. Results from recent phase III clinical trials were published in January of 2010. They indicated positive results with regards to reducing relapse rates of MS and slowing the progression of disability.

Conclusion:

The EAE model plays an integral role in the development and understanding of drugs for MS and future discoveries will no doubt rely on EAE for efficacy and safety testing. The 7 drugs currently approved by the FDA and Fingolimod, waiting approval, are important steps forward in the treatment of MS.

Whitepaper: Experimental Autoimmue Encephalomyeletis (EAE)
Myelin mediated disease models for studying acute, chronic and remitting-relapsing disease courses in MS.

References:

Sriram S, Steiner I. (2005) Ann Neurol. 58: 939-945.
Steinman L, Zamvil SS. (2006) Ann Neurol. 60:12-21.
Friese MA, Montalban X, Willcox N, et al. (2006) Brain. 129: 1940-1952.
Emerson MR, Gallagher RJ, Marquis JG, et al. (2009) AALAS J. 59: 112-128.
Virley DJ. (2005) Journ Amer Soc Experim NeuroTherapeutics. 2: 638-649.
Gold R, Linington C, Lassmann H. (2006) Brain. 129: 1953-1971.
The National Multiple Sclerosis Society. (2009) http://www.nationalmssociety.org/index.aspx

What in vivo models of PD have revealed about pathogenesis and treatment

MD Biosciences — Wed, 11 Aug 2010 13:00:00 GMT

Parkinson’s Disease (PD) is typically an adult-onset progressive neurodegenerative movement disorder that affects millions of people worldwide. Pathologically, PD is characterized by the profound and specific loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Other areas interconnected with the SNpc, the caudate and putamen, collectively known as the striatum, also show remarkable loss of their projection fibers. In accordance with insult to brain regions involved in controlling coordinated movements, the cardinal symptoms of PD include bradykinesia, resting tremor, rigidity, and postural instability (1). To date, research into the etiology of
PD has revealed that most cases are sporadic, though some thirteen genetic loci have been identified to be disease-related (2). Examination of the biochemical properties of these mutant proteins and the pathways in which they are involved has led to the uncovering of three basic pathogenetic pathways common to both heritable and idiopathic forms of PD: (i) abnormal protein control, (ii) mitochondrial dysfunction, and (iii) altered kinase activity (2). Progress towards the identification of disease-related genes has thus led to the expansion of animal models of PD from the classic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6-hydroxydopamine (6-OHDA)-induced neurotoxin models to genetic models of the disease. Due to its complex pathology, however, no animal disease model has yet to faithfully replicate all aspects of human PD. With the evolution of such models, though, converging lines of evidence from toxin-induced and genetic models have continued to further our understanding of the pathological processes underlying PD and lend themselves as useful systems for the examination of therapeutic interventions.

Animal Models of Parkinson’s Disease
Until the discovery of a human gene that could be undeniably linked to PD, the first animal models of PD were generated by acute exposure to neurotoxins. The accidental discovery that acute
exposure to MPTP can cause severe degeneration of DAergic neurons in the SNpc and lead to a PD-like symptoms in humans (3) spawned the first generation of animal models of PD. Over time, researchers discovered several more compounds able to cause PD-like disease in animals, including 6-hydroxydopamine (6-OHDA), rotenone, paraquat, and epoxomicin (4). With the exception of epoxomicin, which inhibits proteosome function, these toxins act by impairing mitochondrial function, an event also common to both human PD and genetic animal models. Of the toxin-induced animal models, the two most commonly used involve the administration of MPTP and 6-OHDA. These “classical” models of PD have been well characterized, with the underlying mechanisms of toxicity well understood, making them useful tools for the development of new therapies and interventions.

Download the full whitepaper: What animal models of PD have revealed about pathogenesis and treatment.

References:

D. J. Moore, A. B 1. . West, V. L. Dawson, T. M. Dawson, Annu. rev. Neurosci. 28, 57 (2005).
J. B. Schulz, J Neurol 255 Suppl 5, 3 (Sep 1, 2008).
J. W. Langston, P. Ballard, J. W. Tetrud, I. Irwin, Science 219, 979 (Feb 25, 1983).
M. Terzioglu, D. Galter, FEBS J 275, 1384 (Apr 1, 2008).

Cutting edge Readouts for Rheumatoid Arthritis Model (part 2)

MD Biosciences — Tue, 10 Aug 2010 12:54:00 GMT

Continuing the discussion of imaging technologies, this week we will cover biofluorescence and bioluminescence as readouts for RA models.

Biofluorescence

Traditional extrinsic fluorescent dyes fluoresce within the range of 300-500nm. Unfortunately, at these wavelengths, biological samples autofluoresce. Recent advancement in technology and development of dyes that fluoresce close to the near infrared (NIR) range (700-900nm) has produced dyes better suited for in vivo studies. A charged-coupled detector (CCD) is used to detect the photons emitted from these dyes after appropriate excitation. Initially these studies were limited due to interference from thermal energy. However, cooling of the chip markedly increased the quality of the image (1). Imaging using these techniques, coined fluorescence molecular tomography (FMT) or optical fluorescence tomography (OFT) in some studies, has been used to follow therapeutic agents (2), assess cell surface molecule expression (3) and to determine disease state by quantifying enzymatic activity (4, 5). Enzymatic activity is assessed using activity based probes (APBs). ABPs consist of a dye and quencher attached to opposite ends of a peptide linker. When the peptide is cleaved by a protease the signal is released. Commercially available APBs Prosense750 (which detects Cathepsin activity) and ProSense680 (which detects MMP activity) were used by Peterson et al (5) in their study assessing the performance of different therapeutic agents. There are also non-specific dyes which accumulate due to increased vascular leakage (6).

Bioluminescence

Like fluorescence imaging, bioluminescence imaging offers a non-toxic, non-invasive means of following specific events longitudinally in live animals. However, unlike fluorescence imaging, bioluminescence imaging requires no exogenous excitation. The most commonly used bioluminescence assay involves the luciferase gene. The luciferase gene, found naturally in glow worms and fireflies, oxidises Luciferin. This reaction results in the release of a photon, which can be quantified and localised with imaging systems. Luciferase-expressing cells can be transferred into hosts or Tg mice expressing luciferase under specific promoters of interest can be used in bioluminescence studies. After injection of the substrate luciferin the cells of interest can be imaged. Nakajima et al (7) used luciferase bioluminescence to show that adoptively transferred CII-specific T cells home to the joints of CIA arthritic mice. In other studies mice expressing luciferase under the Nf-kB (8) or human IL-1b (9) reporter were used to investigate arthritis models and the animal’s response to therapy. One group used bioluminescence in the CIA model to directly visualise their novel therapy (a cytolytic adenovirus) (10). Luminol is also a bioluminescent agent. When exposed to an appropriate oxidising species, luminol emits a blue luminescence. MPO activity at sites of inflammation can generate the necessary oxidative species to catalyse this reaction. Investigators have exploited this using a model of LPS-induced arthritis where luminol bioluminescence was shown to co-localise with sites of inflammation (11).

View pre-clinical models of Rheumatoid Arthritis

References

Spibey CA, Jackson P, Herick K. Electrophoresis. 2001 22(5):829-36.
Paiframan R, Airey M, Moore A, Vulger A, Nesbitt A. J Immunol Methods. 31;348(1-2):36-41.
Hansch A, Frey O, Sauner D, Hilger I, Haas M, Malich A, Bräuer R, Kaiser WA. Arthritis Rheum. 2004 50(3):961-7.
Wunder A, Tung CH, Muller-Ladner U, Weissleder R, Mahmood U. Arthritis Rheum. 2004 50(8):2459-65.
Peterson JD, Labranche T, Vasquez KO, Kossodo S, Melton M, Rader R, Listello JT, Abrams MA, Misko TP. Arthritis Res Ther. 2010 12(3):R105.
Hansch A, Frey O, Hilger I, Sauner D, Haas M, Schmidt D, Kurrat C, Gajda M, Malich A, Brauer R, Kaiser WA. Invest Radiol. 2004 39(10):626-32.
Nakajima A, Seroogy CM, Sandora MR, Tarner IH, Costa GL, Taylor-Edwards C, Bachmann MH, Contag CH, Fathman CG. J Clin Invest. 2001 107(10):1293-301.
Carlsen H, Moskaug JO, Fromm SH, Blomhoff R. J Immunol 2002 168(3):1441.
Li L, Fei Z, Ren J, Sun R, Liu Z, Sheng Z, Wang L, Sun X, Yu J, Wang Z, Fei J. BMC Immunol. 2008 9:49.
Chen SY, Shiau AL, Shieh GS, Su CH, Lee CH, Lee HL, Wang CR, Wu CL. Arthritis Rheum. 2009 60(11):3290-302.
Gross S, Gammon ST, Moss BL, Rauch D, Harding J, Heinecke JW, Ratner L, D. Nat Med. 2009 15(4):455-61.

Cutting Edge Readouts for Rheumatoid Arthritis Models (part 1)

MD Biosciences — Mon, 05 Jul 2010 15:10:00 GMT

Rheumatoid arthritis is a chronic and progressive inflammatory condition estimated to affect between 0.5% and 1% of the world’s population, with more women being affected than men. RA is a systemic disease manifesting mainly as a disabling destruction of the synovial joints of the hands and feet. In addition to the disability and decreased quality of life caused by RA, patients are at increased risk of developing cardiovascular disease. Joint destruction is induced by dysregulated immune activation of both the innate and adaptive immune responses resulting in alterations in the synovium, cartilage and bone. The normal joint has a thin synovial lining (intimal lining layer), 1-3 cells thick. Beneath this is a sub-lining layer of connective tissue scattered with immune cells, blood vessels and nerve cells. Together these layers form the synovium, which produces the synovial fluid that serves to lubricate the joint. Disease initiation results in profound changes in the structure and composition of the synovium and synovial fluid; with the infiltration of inflammatory cells, synovial cell hyperplasia, increased angiogenesis, fibroblast proliferation and extracellular matrix production. This increase in synovial cell proliferation can result in the lining increasing up to five times its original size and can result in pannus formation. The culmination of these events is bone and cartilage erosion and loss of joint function.

Extensive research spanning five decades has failed to elucidate the precise aetiology of RA. However, it is clear that the disease is complex, heterogenous and can probably be initiated by several mechanisms. The strongest association is with HLA II, although both genetic and environmental factors have been implicated in disease. Several animal models have been developed to study the mechanisms of disease and to screen potentially therapeutic agents. There are several commonly used induced models including Collagen-Induced Arthritis (CIA), Collagen-Antibody Induced Arthritis (CAIA), and Zymosan-induced arthritis. As well as several spontaneously arthritic mouse models: TNFa over-expressing transgenic (Tg) mice, K/BxN mice, SKG mice, Human DR4-CD4 mice, IL-1Ra^-/- mice. However, it is recent advances in imaging technology that has allowed these models to provide significantly better information about disease and potential therapies. Here, we discuss state of the art imaging modalities paying particular attention to the advantages and disadvantages of using these new technologies in RA models.

Magnetic Resonance Imaging (MRI)

MRI employs powerful magnets and radiowaves to create excellent 3D images with superb spatial resolution. Furthermore, information about metabolic processes, physiology and tissue status can be obtained with MRI scanning. The magnetic field created by the scanner causes the body’s hydrogen atoms to line up in a specific orientation. Radiowaves are then sent towards these atoms and a computer records the signals that return. Bone erosion, synovitis, tendonopathy, and bone oedema can all be detected using this technique. In contrast to CT, MRI has improved soft tissue contrast and does not expose animal to low dose radiation. In addition, MRI does not always require contrast enhancing agents, minimising side effects on subjects. However, contrast enhancing agents such as gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) and ultra-small super paramagnetic iron oxide (USPIO) particles can be used to maximise the information retrieved by MRI. Gd-DTPA can generate information about vascular flow and permeability as well as information about intra-articular extracellular space, whereas, USPIO particles can generate information about articular content. Several studies that used this technique have shown that MRI technology can follow disease progression using synovial inflammation and draining lymph node volume as biomarkers. Importantly, these biomarkers respond to therapy and thus can be used to screen new potential therapies^1-4. IV injection of USPIO particles leads to their accumulation within macrophages of the endoreticular system. These macrophages can be tracked and are recruited to the joint during disease⁵. MR technology has also been used to follow T cell fate in vivo. In these studies T cells are loaded ex vivo and reintroduced into the mouse which is then scanned to detect where the T cell localise ^{6, 7}. MR scanning can detect disease before irreversible damage occurs. This in conjunction with the ability to image the same animal repeatedly results in MR scanning being an extremely powerful technique allowing longitudinal studies in the same animal where early disease can be followed and the response to therapy assessed.

Download whitepaper: Collagen antibody-induced arthritis. A short, more synchronized alternative to the CIA model.

References:

Dardzinski BJ et al., Magn Reson Imaging. 2001 (9):1209-16.
Proulx ST, et al., Arthritis Rheum. 2007 56(12):4024-37.
Guo R, et al., Arthritis Rheum. 2009 60(9):2666-76.
Lee SI et al., J Radiol. 2009 10(6):651.
Beckmann N et al., Magn Reson Med. 2003 49(6):1047-55.
Dodd SJ et al., Biophys J. 1999 76(1 Pt 1):103-9.
Josephson L et al., Bioconjug Chem. 2002 13(3):554-60.