Graham Wells

Matrix metalloproteinases degrade myelin basic protein

Matrix metalloproteinases (MMPs) are a group of enzymes responsible for the degradation of interstitial connective tissue and basement membrane. The coding sequences for five of the human MMPs, viz. interstitial collagenase, 72 kDa gelatinase, stromelysin-1, matrilysin and 92 kDa gelatinase, were cloned and expressed in Chinese hamster ovary cells, and the proteins purified. The enzymes were compared for their ability to digest myelin basic protein, the major extrinsic membrane protein of central nervous system myelin. The most active on this substrate was 72 kDa gelatinase, followed by stromelysin-1; interstitial collagenase, matrilysin and 92 kDa gelatinase were of comparable but lesser activity. Production of these enzymes by glia or infiltrating inflammatory cells could therefore contribute to demyelination in neuroinflammatory disease.

Matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumour necrosis factor-α inhibitor

Matrix metalloproteinases (MMPs) are a large family of Zn2+ endopeptidases that are expressed in inflammatory conditions and are capable of degrading connective tissue macromolecules. MMP-like enzymes are also involved in the processing of a variety of cell surface molecules including the pro-inflammatory cytokine TNF-alpha. MMPs and TNF-alpha have both been implicated in the pathology associated with neuro-inflammatory diseases (NIDs), particularly multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have shown that BB-1101, a broad spectrum hydroxamic acid-based combined inhibitor of MMP activity and TNF processing, reduces the clinical signs and weight loss in an acute EAE model in Lewis rats. However, little is known about which MMPs are involved in the neuroinflammatory process. In order to determine the optimum inhibitory profile for an MMP inhibitor in the treatment of NID, we investigated the profile of MMP expression and activity during EAE. The development of disease symptoms was associated with a 3-fold increase in MMP activity in the cerebrospinal fluid (CSF), which could be inhibited by treatment with BB-1101, and an increase in 92 kDa gelatinase activity detected by gelatin substrate zymography. Quantitative PCR analysis of normal and EAE spinal cord revealed the expression of at least seven MMPs. Of these, matrilysin showed the most significant change, being elevated over 500 fold with onset of clinical symptoms and peaking at maximum disease severity. Of the other six MMPs detected, 92 kDa gelatinase showed a modest 5 fold increase which peaked at the onset of clinical signs and then declined during the most severe phase of the disease. Matrilysin was localised by immunohistochemistry to the invading macrophages within the inflammatory lesions of the spinal cord. Matrilysins potent broad spectrum proteolytic activity and its localisation to inflammatory lesions in the CNS suggest this enzyme could be particularly involved in the pathological processes associated with neuro-inflammatory disease.

Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS

In an experimentally-induced DTH model of MS, we examined mRNA and protein expression of a range of MMPs and of TNFalpha to establish the contribution that individual MMPs might make to the pathogenesis. In control rat brain, mRNA for all of the MMPs examined was detectable. However, by immunohistochemistry, only MMP-2 could be detected. In the DTH lesions, significant increases in the level of mRNA expression were observed for MMP-7, MMP-8, MMP-12, and TNFalpha. Where expression of MMP mRNA was increased, there was a corresponding increase in protein expression detected by immunohistochemistry. To determine whether the upregulated MMPs could invoke destructive events in the CNS, highly purified activated MMP-7, MMP-8, and MMP-9 were stereotaxically injected into the brain parenchyma. All provoked recruitment of leukocytes and BBB breakdown. In addition, MMPs 7 and 9 induced loss of myelin staining. In conclusion, specific MMPs are upregulated in DTH lesions; for the most part, measurement of mRNA was a predictor of increased protein expression. From our injections of MMPs, it is clear that the upregulated MMPs in the DTH lesions could participate in the disruption of the BBB, leukocyte recruitment, and tissue damage.

Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi‐competitor cDNA standard

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α). There is emerging evidence that MMPs have a role in inflammatory disorders of the central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)‐based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co‐amplified with the target cDNA in the same PCR reaction. Standard multi‐competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three‐fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT‐MMP‐1, 72 kDa gelatinase, and stromelysin‐3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin‐1 were strongly induced (1,000‐fold). Stromelysin‐2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.

Drug Targeting to Monocytes and Macrophages Using Esterase-Sensitive Chemical Motifs

The therapeutic and toxic effects of drugs are often generated through effects on distinct cell types in the body. Selective delivery of drugs to specific cells or cell lineages would, therefore, have major advantages, in particular, the potential to significantly improve the therapeutic window of an agent. Cells of the monocyte-macrophage lineage represent an important target for many therapeutic agents because of their central involvement in a wide range of diseases including inflammation, cancer, atherosclerosis, and diabetes. We have developed a versatile chemistry platform that is designed to enhance the potency and delivery of small-molecule drugs to intracellular molecular targets. One facet of the technology involves the selective delivery of drugs to cells of the monocyte-macrophage lineage, using the intracellular carboxylesterase, human carboxylesterase-1 (hCE-1), which is expressed predominantly in these cells. Here, we demonstrate selective delivery of many types of intracellularly targeted small molecules to monocytes and macrophages by attaching a small esterase-sensitive chemical motif (ESM) that is selectively hydrolyzed within these cells to a charged, pharmacologically active drug. ESM versions of histone deacetylase (HDAC) inhibitors, for example, are extremely potent anticytokine and antiarthritic agents with a wider therapeutic window than conventional HDAC inhibitors. In human blood, effects on monocytes (hCE-1-positive) are seen at concentrations 1000-fold lower than those that affect other cell types (hCE-1-negative). Chemical conjugates of this type, by limiting effects on other cells, could find widespread applicability in the treatment of human diseases where monocyte-macrophages play a key role in disease pathology.

Reduction of excitotoxicity and associated leukocyte recruitment by a broad-spectrum matrix metalloproteinase inhibitor.

An important step in the cascade leading to neuronal cell death is degradation of laminin and other components of the brain extracellular matrix by microglia‐derived proteases. Excitotoxic cell death of murine hippocampal neurones in vivo can be prevented by inhibitors of tissue plasminogen activator (tPA) or by inhibitors of plasmin. Plasmin is a potent activator of the matrix metalloproteinases (MMPs), which are made by resident and recruited leukocytes following CNS injury. In this study, we show, using Taqman RT‐PCR, that MMP mRNAs, but not other calcium‐dependent proteases such as calpain mRNAs, are acutely up‐regulated after an excitotoxic challenge in vivo. α2‐antiplasmin or BB‐3103, a broad‐spectrum inhibitor of the MMPs, co‐injected with kainic acid into the striatum, inhibits excitotoxic cell death in the rat striatum, and reduces both the number of recruited macrophages and the size of the lesion. We also show that leukocyte populations differentially express MMPs, which may account, in part, for the expression profile we observe in the challenged brain. Our results show that inhibition of the MMPs in the rat will prevent kainic acid‐induced cell death in the brain. These studies suggest that MMP inhibitors have therapeutic potential for use in stroke, and support the increasing evidence that microglial activation may contribute to neuronal cell death.

A phase I first-in-human study with tefinostat – a monocyte/macrophage targeted histone deacetylase inhibitor – in patients with advanced haematological malignancies

Tefinostat (CHR‐2845) is a monocyte/macrophage targeted histone deacetylase inhibitor (HDACi). This first‐in‐human, standard 3 + 3 dose escalating trial of oral, once daily tefinostat was conducted to determine the safety, tolerability, pharmacokinetic and pharmacodynamic profile of tefinostat in relapsed/refractory haematological diseases. Eighteen patients were enrolled at doses of 20–640 mg. Plasma concentrations of tefinostat exceeded those demonstrated to give in vitro anti‐proliferative activity. Flow cytometric pharmacodynamic assays demonstrated monocyte‐targeted increases in protein acetylation, without corresponding changes in lymphocytes. Dose‐limiting toxicities (DLTs) were not observed and dose escalation was halted at 640 mg without identification of the maximum tolerated dose. Drug‐related toxicities were largely Common Toxicity Criteria for Adverse Events grade 1/2 and included nausea, anorexia, fatigue, constipation, rash and increased blood creatinine. A patient with chronic monomyelocytic leukaemia achieved a bone marrow response, with no change in peripheral monocytes. An acute myeloid leukaemia type M2 patient showed a >50% decrease in bone marrow blasts and clearance of peripheral blasts. In conclusion, tefinostat produces monocyte‐targeted HDACi activity and is well tolerated, without the DLTs, e.g. fatigue, diarrhoea, thrombocytopenia, commonly seen with non‐targeted HDACi. The early signs of efficacy and absence of significant toxicity warrant further evaluation of tefinostat in larger studies. (clinicaltrials.gov identifier: NCT00820508).