Drug Discovery in Neuroinflammations

Cutting Edge Readouts for Rheumatoid Arthritis Models (part 1)

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:

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

Preclinical Occlusion-induced ischemia reperfusion injury model

The preclinical occlusion-induced myocardial infarct model is a well-known technique for investigating the cardio-protection of a drug therapy in the event of ischemia/reperfusion injury. The advantage of the model is the ability to study the functional relevance of a drug treatment on the heart following direct coronary flow and the mechanisms by which the drug promotes myocardial protection.

Occlusion of the left coronary artery is performed to mimic myocardial infarction in humans that results from occlusion of arteriosclerotic plaques of coronary arteries. Using this model, scientists can get a better understanding of the functional, structural and molecular changes associated with clinical ischemic heart disease as well as investigate the cardio-protection of potential drug therapies. Following the 45 minutes of LAD occlusion, the LAD is opened and coronary blood flow is allowed. Therefore, any IV dosed drug will reach the heart tissue and the infarct area immediately.

 

Figure: Histology on four heart tissue samples from a vehicle treated group (A-B) and an active compound group (C-D) in the occlusion-induced ischemia reperfusion injury model. Occlusion duration was 45 minutes and reperfusion was over 48 hours. Slides A-D: transverse section of the heart at 0.5x magnification. Slides E-H: 4x magnification of slides A-D showing the scarred myocardium. 

MD Biosciences offers the occlusion-induced IR injury model as a contract research services. Study design can be customized to determine the duration of reperfusion following a 45-minute occlusion period. Readouts of the occlusion-induced ischemia-reperfusion injury model include histology of the heart as well as CPK and Troponin levels, which are relevant blood markers of myocardial infarction.  

Download the whitepaper to review details and further data on the model or speak to a scientist about evaluating a compound in the occlusion-induced IR model.

 

Targeting fibroblast-like synovioctyes: preclinical screening assays.

Rheumatoid arthritis (RA) is a chronic autoimmune joint disease characterized by inflammation of the synovium and destruction of cartilage and bone. During synovial inflammation, inflammatory cells (macrophages, mast cells, dentritic cells and lymphocytes) are recruited while resident cells (fibroblast synoviocytes, chondrocytes, osteoclasts, and osteoblasts) are altered to support the inflammatory process.  Together, these events create a pathological tissue response.
 
The synovium consists of two layers, the sublining and intimal lining.  In RA, the sublining becomes infiltrated with mononuclear cells, B lymphocytes produce autoantibodies, blood vessels proliferate, lymphoid aggregates form and the intimal lining shows increased cellularity.  Macrophages in the synovium produce pro-inflammatory cytokines, chemokines and growth factors which in turn activate fibroblast-like synoviocytes (FLS) to produce their own array of mediators (e.g. proteolytic enzymes, chemokines and cytokines).  This produces a paracrine/autocrine network that leads to synovitis, the recruitment of new cells and the destruction of the extracellular matrix.  Fibroblast-like synoviocytes have emerged as key pro-inflammatory cells promoting the disease, largely due to their ability to produce massive amounts of degradative enzymes.
 
The availability of biological therapies has improved clinical outcomes by decreasing inflammation and joint destruction, however only about half of the patients exhibit substantial efficacy. Targeting FLS may further improve clinical outcomes without suppressing systemic immunity.  In vitro FLS assays can be used to evaluate effective therapies for arthritis. Using FLS obtained from normal, RA and OA patients, we can evaluate a compound's effect on the production of pro-inflammatory mediators in a preclinical in vitro model. 

MD Biosciences preclinical services has established an in vitro cytokine-stimulated synoviocyte screening assay. Example data shown below is from normal, OA-postiive and RA-positive tissue (see more data on the assay page). Contact a scientist to establish a protocol relevant to your compound.

 

Effiacy of anti-CD20 therapy in Rhuematoid Arthritis

We read an interesting article published this week in Journal of Immunology (v184 Bottaro & co.) on the efficacy of anti-CD20 therapy in RA. The article highlights the continuing uncertaintity over the mode of action of B-cell directed therapy in Rheumatoid Arthritis (RA) [review of the differing theories is presented in Clin Exp Immunol. 2009 Aug;157(2):191-7].

Therapies such as rituximab (anti-CD20) may be involved in one or more of the following:

• remove plasma cell precursors thereby decreasing auto-reactive monoclonal antibodies
• deplete the B cells that act as antigen presenting cells to auto-reactive T cells
• remove a cytokine producing B-cell population
• disrupt peripheral lymphoid tissue
• or a combination of the above.

The authors previously used MRI to measure the synovial volume in the TNF-Tg mice and demonstrated a relationship to popliteal lymph node (PLN) volume. They observed that synovial volume is relatively constant while PLN volume increases however when the PLN "collapses" then synovial volume increases dramatically. This lead to the question of what happens when the PLN collapses that it appears to induce the pathological synovial changes.

In this paper the authors show, by immunohistochemistry, huge changes in the PLN architecture after collapse, characterised by influx of B cells into the paracortical sinuses and T cell area. The authors characterise these B cells as a unique population of previously undefined B cells which are also present in the KBxN mouse model of RA. The authors then to go on to show that this population of B cells are depleted by anti-CD20 therapy which is also surprisingly efficacious in the TNF-Tg model despite its previous appearance of being T and B cell independent model. The overall message from the paper is definition of a unique B cell population that may be the target of anti-CD20 therapy.

 

Pre-clinical efficacy models of Rheumatoid Arthritis:

Collagen-induced arthritis
Collagen-antibody induced arthritis
Adjuvant-induced arthritis

 

Download the Whitepaper:
Collagen-antibody induced arthritis: A short, synchronized and rapid alternative to the Collagen-induced arthritis model.

Determine immunomodulatory mechanism of action for compounds

Determine immunomodulatory mechanism of action for compounds in indication discovery or respositioning of approved therapies.

The ability to determine the immunomodulatory mechanism of action and target disease choice for compounds simultaneously is critical for decreasing discovery timelines, reducing late-phase failures and maximizing the therapeutic potential. The Senerga® Mode of Action Program is a more systematic, efficient and focused progression towards clinical studies resulting in the avoidance of expensive and time-consuming screens of compounds in a range of disease models.

The technology behind the Senerga® Mode of Action Program enables tracking of the key events that are common to all adaptive immune responses. By examining the common events that underlie many diseases that result from an inappropriate immune response, not only are multiple potential target diseases effectively screened simultaneously, but the mode of action is also discovered in the process. Traditional models offer post-event analysis, whereas Senerga® offers event analysis as it happens along with precise mechanistic dissection.

Implementing a program such as Senerga® has an integral place in a research strategy. Compounds can be evaluated in the Senerga® Program in as little as two weeks, which provides a great advantage to the drug discovery process for new chemical entities as well as respositioning strategies.

Contact a scientist to discuss the Senerga® Mode of Action Program

Parameters for evaluating neuroprotective treatments in EAE models

Cognitive impairment is common in multiple sclerosis (MS), occuring at all stages of disease. It is a main source of disability, social impairment and has a great impact on an individuals quality of life. In the clinic, factors that can affect MS-related cognitive impairment are disease course, fatigue, and affective disturbance.  While the neurochemical basis underlying motor and cognitive defects in patients with MS is unclear, it appears that a balance of tissue destruction, tissue repair and adaptive function reorganization are related to the degree of impairment.

Its most commonly believed that MS is an autoimmune disease in which the body's own immune system recognizes myelin proteins or myelin related proteins as foreign and marks them for destruction. In the body's periphery, major histocompatibility complex (MHC) Class II proteins expressed on the surface of antigen presenting cells (APC) mistakenly bind to these proteins. This causes a naive-T (Th0) to bind to the antige and undergo activation and differentiation. Adhesion molecules and matrix metalloproteinases (MMPs) help T-helper 1 (TH1) cells stick and penetrate the blood brain barrier (BBB) into the CNS, they engage antigen-MHC complexes and produce pro-inflammatory cytokines leading to damage in the CNS. 

Although no animal model thus far establishes all facets of human MS, experimental autoimmune encephalomyeltis (EAE) models are most studied for the disease. Although Th1 cells are an important component in the pathology of the disease, more recent findings suggest that a proinflammatory cascade of TH17 cells, IL-6 and TGF-beta in the nervous system may play a critical role in the pathogenesis of EAE and MS. 

Using a combination of parameters in models of EAE such as T cell infiltration, microglial activation, demyelination/ remyelination, EAE scores and cognitive function provides a useful method for testing potential neuroprotective treatments.

Data: correlation between rotarod and clinical scores in the MOG-induced EAE model

 

References
Reuter, F et al (2009) Rev. Neurol 165:4
Jones, M et al (2008) J Neuroimmunol 199:83

 

Whitepaper: Myelin mediated models of EAE for the study of MS 

Why is post-operative pain under treated?

Adequate pain relief following surgical procedures is well-documented to improve the degree and time course of patient recovery. Nontheless, post-operative pain remains grossly under treated, with up to 70% of patients reporting moderate to severe pain following surgery (1). Perhaps the biggest underlying contributor to the under treatment of post-operative pain is simply a lack of information, both on the part of basic scientists as well as clinicians. Scientists are in the relatively early stages of investigation into the specific mechanisms contributing to the development of incisional pain, which may differ from those mediating acute pain induced by chemical or inflammatory algesic agents. Currently, clinicians essentially rely on treatments that have been developed for other painful conditions, most notably opioids, the side effects of which can hinder rehabilitation and recovery. 

Because opioids are the mainstay treatment for post-operative pain, as well as many other painful conditions, a lack of education regarding the incidence of side effects and abuse potential of this class of drugs can also contribute to under treatment. Proper use of adjuvants to opioid therapy and other currently available tretments is imperative for improving post-operative pain in the short-term. 

A better understanding of the mechanisms of post-operative pain will undoubtedly improve pain management following surgery and allow clinicians to better tailor treatment to individual patients and procedures. The development of novel alternatives to conventional opioid-based analgesia as well as new devices and delivery methods will be essential for improving post-operative pain relief.  

Current Treatments for Post-operative Pain

Opioid drugs such as morphine are widely used in the treatment of post-operative pain, particularly following major surgery. Although they are typically efficacious with regard to pain relief itself, the adverse side effects that accompany opioid administration often limit the utility of this class of drugs. As such, other treatments are often used in conjunction with opioid administration in order to achieve analgesia while reducing opioid treatments. For example chlonidine, an a2-adrenergic receptor agonist, sometimes accompanies fentanyl or other opioids given via epidural administration. In addition, opioids are typically more effective for treating pain occuring at rest rather than evoked pain caused by movement or coughing; therefore, combination therapy can also be useful for improving and hastening recovery and rehabilitation (3). This is an important aspect of post-operative pain treatment to consider, as severe pain and lack of mobility following surgery are risk factors for the development of chronic pain syndromes (4). 

Non-steroidal anti-inflamatory drugs (NSAIDs) are a second class of drugs that are typically given to patients following surgery. In fact, nearly all patients receive some kind of NSAID to treat post-operative pain (4). They may not provide sufficient pain relief on their own, especially after major surgery, but can be combined with opioids or other interventions to improve analgesia. 

Afferent neutral blockage, although not employed with particularly high frequency in the clinic, is an analgesic treatment that has gained more attention from preclinical researcher in recent years. One important benefit of local anasethetics, such as lidocaine, is that they are effective for treating mobilization-evoked pain and may improve long-term patient outcomes (4). In addition, this class of drugs is relatively safe and well tolerated. However, these drugs also have non-selective effects on neuronal transmission, so normal sensory perception is affected. In addition, there are some associated cardiac and neurological risks, especially with systemic delivery.

 

References:
1. Pyati, S. and Gan T.J. (2007) Perioperative pain management. CNS Drugs. 21(3):185 - 211.

2. Grinstein-Cohen, O. et al. (2009) Improvements and difficulties in postoperative pain management. Orthopaedic Nursing, 28(5):232-239

3. Brennan, T.J., et al. (2005) Mechanisms of incisional pain. Anesthesiology Clin N Am, 23:1-20

4. Breivik, H. and Stubhaug, A (2008) Management of Acute postoperative pain: still a long way to go! Pain. 137:233-234

 

Speak to a scientist

Review models of pain

 

Resources:

 

Download PAIN PROCESSING AND PATHWAYS eBook:
Choosing suitable behavior tests for common drug classes based on the
primary mechanism and site of action.

Download A new animal model of Post-Operative Pain Whitepaper
An optimal model for assesssment of analgesic effect of various local treatment strategies such as new implants, patches, medical devices and creams

Pain Processing: Cation Channel Blockers. Choosing models of pain.

Sodium and calcium cation channel blockers.

There are several types of drugs that have been developed to decrease the firing rate of nociceptive neurons by blocking cation channels. Among the most commonly known are lidocaine and bupivacaine, typically used as local anesthetics, which form an intracellular block of the voltage gated sodium channels (VGSCs) that are necessary for action potential generation. Without action potential firing, nociceptors are unable to propagate their message, and pain is thereby blocked. The main disadvantage of this class of drugs is that without selectivity for nociceptive sensory neurons, tactile input is also lost, leading to the numbness that accompanies local anesthetic administration.

Sodium channel blockers are most commonly used to treat neuropathic and other types of chronic pain; as such, models of neuropathic pain, particularly peripheral neuropathy models, are an excellent option for testing novel compounds of this drug class. Their analgesic efficacy may be more widespread, however, as they have shown to be useful against inflammatory pain and in some post-operative pain models. Notably, they are also among the few drug types shown to be effective in models of visceral pain.

Voltage gated calcium channels (VGCC) are another pharmacological target for pain relief. Gabapentin and pregabalin fall under the classification of gabapentinoids, which, while structurally similar to the endogenous neurotransmitter GABA, do not function as such. Instead, they bind to the α2-δ subunit of VGCC to reduce calcium influx into nerve terminals and thereby decrease neurotransmitter release. The α2-δ subunit of VGCC is highly expressed in the dorsal horn of the spinal cord, and decreasing the release of glutamate and substance P from nociceptive primary afferent neurons here is likely the main mechanism of action for drugs of this type. However, disinhibition of endogenous descending inhibitory pathways at supraspinal sites may also contribute to their analgesic effects (1). Gabapentinoids are tested primarily in models of neuropathic pain, including both nerve injury and neuropathy models, which reflects their clinical utility. 

TRPV1 ligands

The development of more selective cation channel blockers as a solution to avoiding the side effects that accompany a general neuronal blockade has been the subject of much investigation recently. Transient receptor potential (TRP) channels are attractive targets, as they are predominantly expressed in nociceptive DRG neurons. Activation of TRP channels, therefore, has little or no effect on normal mechanical sensation, and drugs that target these channels could potentially avoid centrally-mediated side effects as well.

TRPV1 channels, in particular, are widely studied as a potential therapeutic target. TRPV1 is a non-selective cation channel is activated by capsaicin, the active ingredient in chili peppers, as well as heat. The function of TRPV1 is also modulated by a variety of sensitizing agents released after injury, including protons. Inflammation resulting from injury can reduce tissue pH, thereby activating TRPV1, causing an increase in sodium and calcium influx into the cell, and thereby contributing to the sensitization of nociceptors under these conditions (2). TRPV1 can be targeted through either antagonists to block activation directly or with agonists, which work by causing desensitization of the receptor following robust activation.

Given the effects of inflammation on TRPV1 function, it is not surprising that ligands for this receptor have shown efficacy in a variety of inflammatory pain models, including post-surgical and arthritic pain as well as standard inflammatory pain models (2). Similar to other cation channel blockers, they are also effective in models of neuropathic pain, particularly peripheral neuropathy models, and in some models of visceral pain.

Efficacy Models of Pain

Speak with a scientist about evaluating a compound in a model of pain

Whitepapers: Peripheral Nerve Injury models and Post-operative pain models

 

References:

1. Tanabe, M., et al., Pain relief by gabapentin and pregabalin via supraspinal mechanisms after peripheral nerve injury. J Neurosci Res, 2008. 86(15): p. 3258-64.

2. Patapoutian, A., S. Tate, and C.J. Woolf, Transient receptor potential channels: targeting pain at the source. Nat Rev Drug Discov, 2009. 8(1): p. 55-68 

Cannabinoid System as a target for pain relief.

The body's cannabinoid system consists of two cannabinoid receptors, CB1 and CB2, their endogenous ligands, which include 2-arachidonoyl glycerol (2-AG) and anandamide (AEA), and the enzymes that regulate the synthesis and degradation of these ligands. While the endogenous cannabinoid system serves naturally to modulate pain transmission, it can be exploited to provide more robust relief, either through administration of agonists at CB1 or CB2 receptors or through inhibition of degrading enzymes to increase endogenous cannabinoid levels.

CB1 receptors are expressed in neurons throughout the central and peripheral nervous system, including in the DRG, where noiciceptor cell bodies reside, the dorsal horn of the spinal cord, and the PAG, all of which are important sites for modulation of pain transmission. CB2 receptors, on the other hand, are not found in the CNS under normal conditions (although they may be upregulated in nociceptive neurons after injury) and are instead expressed in a variety immune cells and microglia. Although activation of either receptor can promote pain relief, CB1 receptors are responsible for the centrally-mediated psychomimetic side effects that sometimes accompany administration of cannabinoid receptor agonists such as tetrahydrocannabinol (THC).

Both CB1 and CB2 are GPCRs that signal predominantly through Gi/o to decrease VGCC conductance and activate GIRKs to hyperpolarize cells. Therefore, ligand binding to cannabinoid receptors results in decreased release of excitatory neurotransmitters from nociceptive neurons and post-synaptic cells exhibiting decreased excitability for signals they do receive. Activation of cannabinoid receptors on immune cells can similarly inhibit their function and thereby indirectly modulate pain processing. Since CB2 receptors are found primarily on immune cells and microglia, this indirect, anti-inflammatory effect is the primary mechanism by which CB2-selective agonists modulate pain responses.

Cannabinoid agonists have shown efficacy in acute models such as tail flick and capsaicin injection, as well as carrageenan and CFA inflammatory pain models. Translation from animal models to the human condition has been documented for a variety of neuropathic conditions as well as for post-operative pain relief; therefore, both neuropathic and post-operative pain models would be appropriate for testing novel compounds designed to target the cannabinoid system as well.

α2-adrenergic Agonists and Tricyclic Antidepressants Evaluating Compounds in Relevant Models of Pain

α2-adrenergic receptors (α2ARs) are found in many areas in throughout the nervous system, but the α2ARs on pre- and post-synaptic neurons in the dorsal horn of the spinal cord are the main target for both endogenous and exogenous analgesia. One of the major descending inhibitory pain pathways involves the projection of noradrenergic neurons in the locus ceruleus back down to the spinal cord to activate α2ARs at this site. These receptors can also be targeted pharmacologically through administration of selective agonists or through the inhibition of noradrenaline (also known as norepinephrine) reuptake by drugs such as tricyclic antidepressants.

α2ARs are divided into three subtypes, the α2A-, α2B- and α2C-ARs. All three are Gi/o coupled GPCRs. α2AARs are expressed mostly on the central, pre-synaptic terminals of nociceptors and inhibit VGCC on these terminals to reduce the release of excitatory neurotransmitters such as glutamate and substance P. At the same time, α2CARs, expressed primarily on the second order neurons in the dorsal horn, reduce excitability of these neurons by increasing conductance through GIRK channels [1].

Tricyclic antidepressants (TCAs) are used clinically for the treatment of various neuropathic pain conditions, including nerve injury and diabetic neuropathy. Importantly, their analgesic efficacy is independent of the co-existence of depression in patients. Most TCAs have some action on both serotonin and norepinephrine reuptake, but their analgesic actions are largely mediated by increasing spinal noradrenergic tone coming from descending pathways, which then increases activation of α2ARs to produce pain relief as described above.

In accordance with their clinical usage, animal models of neuropathic pain are widely used to test novel TCAs. In fact, TCAs show little efficacy in animal models of acute or inflammatory pain. Although neuropathic and other forms of chronic pain are common indications for the clinical use of α2AR agonists as well, they show robust antinociception in a much wider variety of animal models, including acute and inflammatory ones. Also, they are used both clinically for and in animal models of postoperative pain [1].

Reference

1. Pan, H.L., et al., Modulation of pain transmission by G-protein-coupled receptors. Pharmacol Ther, 2008. 117(1): p. 141-61.