Oral MC4 Receptor Antagonists as Treatment for Cachexia

MC4 receptor modelCachexia affects many different chronically ill patient populations, including those with cancer. It results in loss of body weight, particularly of lean body mass (LBM), and is estimated to be responsible for over 20% of all cancer-related deaths. Currently, available drugs are ineffective, and new therapies are urgently needed. The anorexigenic peptide, α-melanocyte stimulating hormone (α-MSH), is believed to be crucially involved in the normal and pathologic regulation of food intake and it was speculated that blockade of its central physiological target, the melanocortin-4 receptor (MC4R), might provide a promising anti-cachexia treatment strategy. The idea is supported by animal studies with agouti-related protein (AgRP), the endogenous inverse agonist at the MC4 receptor, which was found to affect two hallmark features of cachexia: to increase food intake and to reduce energy expenditure.

In 1998, it was reported that MC4R mutations were associated with inherited human obesity. MC4R mutations have a prevalence of 1-2.5% in people with body mass indexes greater than 30, making it the most commonly known genetic defect predisposing people to obesity.

Researchers at Santhera Pharmaceuticals have now published results with orally available MC4R antagonists in an animal model of tumour-induced cachexia. Once daily oral administration of both compounds SNT207858 and SNT207707, starting the day after tumour implantation, significantly reduced the tumour induced weight loss. SNT207707 binds to the MC4 receptor with an affinity of 8 nM and shows a more than 200-fold selectivity vs. MC3 and MC5. SNT207858 is a 22 nM MC4 antagonist with a 170-fold selectivity vs. MC3 and a 40-fold selectivity versus MC5.

Full details of the study are published in the journal PLoS One.

Is the Cat Driving you Mad?

Cat's eyeToxoplasmosis is a parasitic infection caused by the protozoan, Toxoplasma gondii. The parasite is able to infect most warm-blooded animals, including humans, although the primary host is the cat family. Infection is via ingestion of infected meat, cat faeces or through mother-to-child transmission. The majority of infected humans display no symptoms, but those with suppressed immune systems, such as AIDS patients or pregnant women, are at greater risk of developing serious illness. It has been estimated that up to 30% of the World’s population is infected with the parasite.

Following the initial acute phase of infection, a latent phase ensues with the parasite forming cysts in muscle and brain. It is known that infection can modify an animal host’s behaviour and experiments in mice have demonstrated a reduced fear of predators. An increase in levels of the neurotransmitter, dopamine, has been observed in the brains of infected mice. Given the tropism of the parasite for brain tissue, it has long been speculated that Toxoplasmosis in humans may be linked with schizophrenia although no causal relationship has been established.

Scientists at Leeds University have now identified two nearly identical genes in the T. gondii genome with similarity to metazoan tyrosine hydroxylase. Unlike metazoan enzymes of this class, the T. gondii enzymes are bifunctional as they are able to utilize tyrosine and phenylalanine as substrates. Whilst the biological role(s) of a bifunctional tyrosine hydroxylase in T. gondii remain unclear, it is possible that the enzyme is required for supply of tyrosine for protein synthesis. Based on the higher substrate selectivity for tyrosine, however, synthesized tyrosine will be converted to L-DOPA. L-DOPA is the precursor to dopamine and it is plausible that the parasite tyrosine hydroxylase is responsible for the increased dopamine levels detected in the brains of infected rodents.

T. gondii’s ability to synthesise dopamine suggests a potential link with neurological conditions such as Schizophrenia, Parkinson’s disease, Tourette’s syndrome and attention deficit disorders.

The full paper is published in the March 11th issue of PLoS One.

Hitting Cancer with an Iron Fist?

The energy demands of rapidly proliferating cancer cells require high levels of nutrients, including iron. Since cells are sensitive to free iron, they utilise iron storage proteins to protect themselves. Scientists at the German Cancer Research Center (DFKZ) and University Medical Center Mannheim, studying Sézary’s disease, have now shown that manipulation of free intracellular iron can induce apoptosis in T-cell lymphomas.

iron-rich riverSézary’s disease is an aggressive and ultimately fatal type of cutaneous T-cell lymphoma that is resistant to currently available treatments. Apoptosis resistance in leukemias and lymphomas is mediated by aberrant signalling of the NF-κB pathway. The researchers have demonstrated that cell death of cutaneous T-cell lymphoma cell lines induced by inhibition of the NF-κB pathway is a result of increased free intracellular iron and reactive oxygen species (ROS). Using T-cells from Sézary patients they show that inhibition of constitutively active NF-κB causes down-regulation of ferritin heavy chain (FHC) that leads to an increase of free intracellular iron, which, in turn, induces massive generation of ROS. The involvement of FHC was confirmed by direct down-regulation using siRNA.

Importantly, T cells isolated from healthy donors do not display down-regulation of FHC and, therefore, do not show an increase in iron and cell death upon NF-κB inhibition.

The work, published in the journal Cancer Research, suggests FHC as a novel target for therapeutic intervention in lymphoma.

Towards the Virtual Liver

Drug-induced liver injury (DILI) is the most frequent cause of acute liver failure in the US and a major obstacle in the development of new medicines. It is therefore not surprising that prediction of liver toxicity is of considerable interest to healthcare professionals and drug companies alike.

Now The Hamner Institutes for Health Sciences and Entelos, Inc. have announced a partnership to create a computer model of liver function in virtual patients to improve understanding of how drugs can sometimes damage the liver.

The partnership supports the FDA’s Critical Path Initiative, which aims to reduce the time taken to develop and approve safe and effective medicines, and two FDA scientists will join the scientific advisory board for the project.

liverEntelos will use its clinically validated PhysioLab biosimulation platform to build a mathematical model of liver function using information from a variety of sources, incorporating The Hamner’s expertise in liver injury and systems biology. Additional important information will be supplied through Hamner research programs employing novel liver-derived cell models and special metabolism studies made possible by a new Hamner metabolomics laboratory. The objective of the collaboration is to develop a virtual liver that will account for the effects of genetic variations and other factors, such as patient sex, age, behavioural characteristics and environmental influences. In parallel, virtual rodents will be developed that will provide an improved means by which to evaluate preclinical drug effects and mechanisms of liver injury across species.

If successful, the platform will have many potential uses, including guiding the development of new diagnostic tests and new ways to test drug safety in the laboratory.

Candidate Drug for Henipavirus Infection

Two viruses from the Henipavirus genus, Nipah (NiV) and Hendra (HeV), are recently emerged zoonitic (transmissible from animals to humans) paramyxoviruses that cause encephalitis in humans. HeV, previously known as equine morbillivirus, emerged as the causative agent of an outbreak of fatal respiratory disease in horses and man in Australia in 1994. NiV emerged in 1998/1999 in Malaysia and Singapore causing fatal encephalitis in humans. Human fatality rates can be as high as 75%.
Henipavirus structure
chloroquine structureResearchers at Weill Medical College of Cornell University, Australian Animal Health Laboratory, University of Tennessee Health Science Center and Rockefeller University have now developed a high-throughput assay that is able to identify inhibitors that target several stages of the viral life cycle. Their initial screen showed that chloroquine, approved for malaria treatment, inhibited infection with live HeV and NiV at a concentration of 1µM in vitro (IC50=2µM), lower than the plasma concentrations present in humans receiving chloroquine treatment for malaria.

The scientists speculate that the mechanism of action of chloroquine is likely to be inhibition of cathepsin L, a host enzyme essential for processing of the viral fusion glycoprotein and maturation of newly budding virions. In the absence of this processing step, virions are not infectious. Chloroquine has previously been shown to suppress the activity of cathepsin L.

The authors of the current study, published online ahead of print in the Journal of Virology, suggest that the established safety profile and broad experience with chloroquine in humans should provide an option for treating individuals infected by these deadly viruses.

More on the Mechanism of Glatiramer Acetate in Multiple Sclerosis

Approved in 1995, glatiramer acetate (Copaxone®, copolymer-1) is a disease-modifying drug that has been demonstrated to reduce the relapse rate and progression of disability in relapsing-remitting multiple sclerosis (RRMS) patients. The compound is a mixture of synthetic peptides (50-90 amino acids) composed of alanine, glutamic acid, lysine and tyrosine. Originally developed to mimic myelin basic protein, a major component of the neuronal myelin sheath, it was intended for use as an inducer of experimental autoimmune encephalitis (EAE). The unexpected inhibition of EAE that was observed with glatiramer acetate led to clinical trials and subsequent approval for RRMS.

The efficacy of glatiramer acetate has been ascribed to an effect on the adaptive immune response, shifting towards a Th2 polarisation of myelin-specific T-cells. Further studies have demonstrated an immunomodulatory effect on monocytes, macrophages and dendritic cells. However, the full mechanistic picture is still unclear.
Collaborating scientists from the University of Geneva, Technische Universität München and University of California, San Francisco, have now demonstrated an effect of glatiramer acetate on the IL-1 system. Their research has shown that treatment with the polymer increases blood levels of secreted IL-1 receptor antagonist (sIL-1Ra), a natural inhibitor of IL-1β, both in RRMS patients and in EAE mice. In the same subjects, levels of IL-1β were undetectable. Additional in vitro experiments with T-cell contact-activated monocytes, a model relevant to chronic inflammation, showed that glatiramer acetate strongly reduced expression of IL-1β, whilst enhancing expression of sIL-1Ra. This is in contrast to effects in monocytes subjected to acute inflammatory conditions (stimulation with LPS), where glatiramer acetate increased production of both sIL-1Ra and IL-1β. The authors conclude that the effects on the IL-1 system in chronic inflammatory conditions contribute to the therapeutic effects of glatiramer acetate in RRMS.

The study is published in the online early edition of the journal PNAS.

Inhibition of PKC-βII for Chemoprevention

precancerous adenomaInappropriate activation of PKC isozymes has been implicated in many forms of cancer and researchers at the Mayo Clinic, Florida Campus, have been elucidating the roles of the isozymes in colon carcinoma using transgenic mice. Their earlier studies, reported in the January 15th issue of Cancer Research, demonstrated a requirement for PKC-βII in the initiation of colon cancer in mice exposed to a carcinogen. The same study also showed that PKC-ι/λ was required for cancer progression.

Now the scientists have shown that daily administration of the selective PKC-β inhibitor, Enzastaurin, provides a degree of protection to mice administered a carcinogen known to cause colon tumours. Since colon cancer develops over a 10-15 year period in humans, there is a large window of opportunity for the use of chemopreventative drugs. The authors suggest that Enzastaurin could represent a good candidate for clinical study in this setting because it has few side-effects.

enzastaurinEnzastaurin is currently in Phase III clinical trials for the treatment of B-cell lymphoma and high-grade brain gliomas. Although Enzastaurin is selective for PKC-β, it also significantly inhibits other PKC isozymes.

The full study with Enzastaurin is published in the February 15th issue of Cancer Research.

New Insights into DJ-1/PARK7 Oncogenic Mechanism

Loss-of-function mutations of human DJ-1/PARK7 are associated with autosomal recessive, early onset Parkinson’s disease. In contrast, amplification of DJ-1 has been observed in numerous tumours and its expression correlated with poor prognosis. Experimental data suggest that DJ-1 contributes to cell survival by enhancing the phosphorylation of Akt, resulting in inhibition of PTEN function. Further data support a role in protection from oxidative stress – knock-out of DJ-1 enhances hydrogen peroxide mediated cytotoxicity.

Hela cells stained for PARK7Researchers have now identified DJ-1 as an upstream activator of hypoxia inducible factor-1(HIF1) function in cancer cells, conferring resistance to hypoxia-induced apoptosis. HIF1 is a transcription factor that plays a key role in a tumour’s ability to adapt under hypoxic conditions, critical for survival and progression. The authors’ experiments demonstrate that loss of DJ-1 in human cell lines decreases transcription of all HIF1-responsive genes examined and that expression of DJ-1 is critical to the activities of Akt and mTOR that are required to maintain HIF1 stability. In addition, DJ-1 has been shown to regulate the activity of the metabolic sensor, AMPK.

The authors conclude that their results strengthen the case for therapeutic intervention at the level of DJ-1 in cancer cells. It will be necessary, however, to consider the loss-of-function consequences. The full study is reported in the January 27th issue of PNAS.

Exploiting the Kinetic Isotope Effect in Drug Discovery

kinetic isotope effectThe Kinetic Isotope Effect (KIE) refers to the dependence of rate of reaction on the isotopic identity of an atom participating in the reaction. The largest effect is observed when the isotopic replacement is made to an atom involved in bond breaking or formation in the rate-limiting step – a primary isotope effect. When the isotopic substitution is made to an atom that does not participate in the rate-determining step, the magnitude of the effect is smaller – a secondary isotope effect. This property has long been used in the elucidation of reaction mechanisms.

The KIE is most pronounced when the relative mass change of the substituted isotope is large. For example, a change from hydrogen to deuterium represents a 100% increase in mass, whilst a 12C to 13C change represents only an 8% increase. Reactions involving C-H bonds are typically 6-10 times faster than the corresponding C-D bonds, whereas a reaction involving 12C is only marginally faster than the corresponding 13C reaction.

In addition to its utility in elucidation of reaction mechanisms, deuterium has also found application as a non-radioactive isotopic tracer for studying metabolic pathways. Because of the KIE, however, care has to be taken that the substitution does not modify metabolism. Turning this property to advantage, two US companies are targeting deuterated versions of clinically proven drugs to identify analogues with improved properties.

venlafaxineIn October 2008, Auspex Pharmaceuticals announced positive results from its Phase 1 clinical trial of SD-254, claiming the first validation in humans of targeted deuterium substitution. SD-254 is a deuterium substituted version of venlafaxine, a selective serotonin-norepinephrine reuptake inhibitor (SNRI). The trial indicated a superior pharmacokinetic profile for SD-254 compared to venlafaxine.

paroxetineAt around the same time, Concert Pharmaceuticals reported preclinical data on CTP-347, a deuterium substituted version of the selective serotonin reuptake inhibitor (SSRI), paroxetine. CTP-347 is being developed to treat vasomotor symptoms (hot flashes) caused by tamoxifen treatment or the menopause. CTP-347 does not cause mechanism-based inactivation of CYP2D6 at clinically relevant concentrations and may be more suitable for co-administration with tamoxifen which is activated by metabolism by CYP2D6. CTP-347 is currently in Phase I clinical testing.

Both companies have numerous other deuterated compounds in their pipelines targeting a wide range of therapeutic indications.

Specificity or Polypharmacology?

As a target class, kinases have received considerable attention over the last 20 years. There are now 8 kinase inhibitors on the market, approved for use in a number of cancers, with many more in clinical development. Targeting of the catalytic ATP-binding site has proved the most fruitful in drug discovery, but the conserved nature of the site has presented challenges to identification of specific inhibitors. In fact all of the approved compounds inhibit more than one tyrosine kinase, although they maintain selectivity over the serine/threonine and phosphoinositide (PI) kinase classes. This multiple target activity has, however, proven advantageous in an oncology setting.

co-crystal structuresA collaborative group of researchers has recently reported the identification of dual inhibitors of tyrosine and PI kinases. Their rationale for searching for such compounds was the knowledge that reactivation of PI3 kinase signalling is a common mechanism of resistance to tyrosine kinase inhibitors. Furthermore, preclinical studies combining tyrosine and PI kinase inhibitors have demonstrated efficacy. Despite the sequence dissimilarity between tyrosine and PI kinases, the scientists were able to identify a number of molecules with novel selectivity profiles. In addition, one compound was discovered with unexpected specificity for mTOR compared to other members of the PI kinase family. Full details are reported in Nature Chemical Biology.