metabolism – Drug Discovery Opinion

Illuminating the Link between Bone and Metabolism

Bone Tree — Image: Flickr – Livin-Lively

Two back-to-back studies published in the July 23^rd issue of Cell, one from Columbia University Medical Center and the other from Johns Hopkins researchers, further the hypothesis that metabolic control and bone remodelling are inextricably linked. Both studies point to osteocalcin, a hormone released by bone, as a key mediator of this link.

The Johns Hopkins study used a conditional knock-out in mice to specifically suppress the expression of the insulin receptor in osteoblasts, the bone-forming cells of the skeletal system. As the mutant mice aged they became fat, had elevated blood sugar, and were glucose intolerant and resistant to insulin, mirroring the picture of diabetes in humans. The researchers found that the mutant mice had fewer osteoblasts, reduced bone formation and lower levels of circulating undercarboxylated osteocalcin (the active form of the hormone). The study showed that signalling via the insulin receptor in osteoblasts suppressed Twist2, an inhibitor of osteoblast development, and enhanced expression of osteocalcin, a mediator of insulin sensitivity and secretion.

The Columbia study links the complete bone remodelling process to energy regulation. Osteocalcin is released from osteoblasts predominantly in an inactive, carboxylated form. The researchers demonstrated that insulin signalling in osteoblasts stimulates release of inactive osteocalcin and activates osteoclasts, which activate the osteocalcin via decarboxylation in a bone-resorption-dependent manner.

The studies clearly have potential impact on human therapy, although significant questions remain. As yet the receptor for undercarboxylated osteocalcin is unknown, so the mechanism by with the hormone stimulates insulin release is unclear. Further work will be necessary to understand the interplay between skeletal- and metabolic-homeostasis in humans.

The two papers are previewed in Cell.

Mice Can Do What Poppies Can

poppies and mouse — *Papaver somniferum*: Flickr - Alastair Rae; Mouse: Flickr - Sergey Yeliseev

Preparations from the opium poppy, papaver somniferum, have been used for thousands of years to relieve pain. In the early 1800s, Sertürner isolated one of the constituent alkaloids, morphine, which was later shown to be almost entirely responsible for the analgesic activity of crude opium extracts. Since then, morphine and other opiates have been shown to bind to specific receptors, leading to speculation that mammals may be able to biosynthesise morphine. It has also been recognised that humans excrete small but steady amounts of morphine in their urine, but it has been unclear whether this comes from dietary sources or whether the presence of morphine in urine provides evidence that people can actually biosynthesise morphine. The discovery of the simple isoquinoline alkaloid, tetrahydropapaveroline (THP), which can be formed from catecholamines, in both human and rodent brain and in human urine also suggested a possible biosynthetic linkage.

A team led by Nadja Grobe from the Donald Danforth Plant Science Center and Marc Lamshöft from the Institute of Environmental Research, University of Technology Dortmund have now shown conclusively for the first time that mammals are able to synthesise morphine. When either unlabelled or deuterated THP, a potential precursor of morphine, was administered to mice, analysis of their urine showed that the compound had been extensively metabolised. Salutaridine, a known biosynthetic precursor of morphine in the opium poppy, was found amongst the metabolites and when deuterated salutaridinol, the biosynthetic reduction product of salutaridine, was administered to the mice, it was shown to be converted to deuterated thebaine, which was excreted in their urine. Deuterated thebaine was also administered and deuterated morphine together with the related compounds, codeine and oripavine, was recovered in urine.

The study, which is published in the journal PNAS, provides the first evidence that mammals have the capacity to convert THP, known to be present in the brain, into morphine. Although the pathway has now been shown to exist, it is still not clear whether mammals naturally produce morphine as an analgesic.

How Small Molecule Restores Aldehyde Dehydrogenase Function

Human Aldehyde Dehydrogenase, apo form (PDB ID = 1O05)

The lower alcohol tolerance of some Asian groups compared with people of European descent is caused, in part, by a mutant copy of the aldehyde dehydrogenase gene, ALDH2. As well as carrying out the second step in the oxidative metabolism of alcohol, the conversion of acetaldehyde to acetic acid, ALDH2 metabolises toxic species created by lack of oxygen in the wake of a heart attack and is involved in the metabolism of nitroglycerine which is used to treat angina. People with a deficiency in the activity of ALDH2 are at increased risk of cardiovascular damage and scientists at Indiana University and Stanford University reported in 2008 that a small molecule activator of ALDH2, Alda-1, could reduce infarct size in rats if administered before ischaemic damage. In vitro, Alda-1 was found to be a particularly effective activator of the inactive form of the enzyme found in some East Asian populations, suggesting that treatment with Alda-1 could be of benefit to individuals with either wild-type or mutant ALDH2 who are subjected to cardiac ischaemia by a heart attack or by procedures such as coronary bypass surgery.

Writing in Nature Structural & Molecular Biology, the team have now described mechanistic details of the activation of ALDH2 by Alda-1, a discovery which should lead to more potent and selective analogues of Alda-1 with better promise as drug candidates to minimize ischaemic heart damage. The structures of bound Alda-1 reveal how the compound activates the wild-type enzyme and how it restores the activity of the inactive form by acting as a structural chaperone.

How to Keep Hepatocytes Happy

The liver plays a central role in metabolising and clearing drugs and other xenobiotics from the body and, because of this role, is particularly susceptible to damage. More than 900 drugs have been linked to more or less severe liver damage, and hepatotoxicity is the most common reason for drugs being withdrawn from the market. Animal studies and even early stage clinical trials often fail to pick up liver toxicity and, although many drugs cause low level injury that shows up only in biochemical tests for liver function, drug-induced liver injury accounts for 5% of hospital admissions and more than 50% of cases of acute liver failure. As well as imposing a risk to patients, the recall of marketed products results in substantial financial costs to the pharmaceutical industry. As an ageing population uses more and more prescription drugs, as well as over-the counter medicines, the chances of drug-induced liver injury are likely to increase.

Both in vitro and in vivo tests to evaluate the potential for liver toxicity are widely used and a team led by researchers at Massachusetts General Hospital have now developed an innovative method of culturing liver cells that they believe will be more predictive than existing in vitro methods. Freshly cultured liver cells rapidly lose their metabolic competence under standard culture conditions and earlier studies had suggested that animal-derived serum, which is commonly used in cell cultures, may interfere with the metabolism of cultured hepatocytes. Since one of the key stresses involved in moving cells from an in vivo environment into cell culture is a ten-fold drop in oxygen levels, the researchers hypothesised that a high-oxygen, serum-free culture medium might provide a better environment for growing hepatocytes. Experiments showed that both human and rat hepatocytes grown with endothelial cells in a serum-free culture with 95% oxygen quickly resumed normal metabolic activity, including gene expression and cell function. These cultured cells successfully predicted the clearance rates for both rapidly and slowly cleared drugs and maintained a high level of metabolic activity for several weeks. Although oxygen levels had been thought to affect cell survival, they had not previously been recognised to influence gene expression or metabolic function of cultured hepatocytes. The authors believe that the new culture method provides an environment that is more similar to that of hepatocytes in an intact liver and will keep cells viable for longer, thus providing a more predictive system for evaluating hepatotoxicity.

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

Deuterium is Go

Concert Pharmaceuticals and GlaxoSmithKline recently announced a collaboration to develop deuterium-containing medicines, including CTP-518, a partially deuterated version of the HIV protease inhibitor, atazanavir (Reyataz™), marketed by Bristol-Myers Squibb. Reyataz™ is used in combination therapy to treat HIV/AIDS and, for most patients, the recommended dose is one 300mg tablet daily taken with ritonavir (Norvir™). Ritonavir was originally developed as a ‘stand-alone’ HIV protease inhibitor but is now primarily used, not for its antiviral activity, but to ‘boost’ levels of other protease inhibitors by inhibiting their metabolism. Despite its marked benefits as part of combination therapy, ritonavir is poorly tolerated by some patients and also influences the metabolism of concurrently administered drugs, especially those metabolised by CYP 3A4.

Concert is pioneering the modification of existing medicines by selectively replacing hydrogen atoms with deuterium atoms in the expectation that the modified compounds will have similar activity at the target enzyme or receptor, together with improved ADME properties. CTP-518 has been shown to have similar antiviral potency to atazanavir but slower hepatic metabolism, leading to the hope that it could be used clinically without the need for ‘boosting’ by ritonavir. This could lead to better safety and tolerability for patients and also allow for the inclusion of CTP-518 in fixed dose regimens. CTP-518 is expected to enter Phase I clinical trials in the second half of 2009.

Concert has filed a patent application (WO20081566632) claiming derivatives of atazanavir, including compounds 120 and 122.
Deuterated atazanavir
The antiviral activities of compounds 120 and 122 were shown to be similar to, or slightly better than, that of atazanavir, both in the presence and absence of serum proteins. In human liver microsomes, compounds 120 and 122 showed an approximately 50% increase in half life compared with atazanavir. Following oral co-dosing in rats, compound 122 showed a 43% increase in half life, a 67% increase in Cmax and an 81% increase in AUC compared with atazanavir. When administered to chimps, both compounds showed around 50% increases in half life compared with atazanavir and about 2-fold increases in urine concentration.

Eat Less, Live longer, Remember More?

breaking bread The practice of calorific restriction as a means to health, improved mental faculties and a longer life is controversial and two recent studies have contributed more fuel to the debate. A study carried out by scientists at the University of Southern California, and published online on January 13^th in The Journal of Nutrition, compared metabolic rates in two strains of mice, one genetically engineered to be fat and the other lean. Lifespan of the ‘fat’ strain is increased by calorie restriction whereas that of the ‘lean’ strain is not. When both groups of mice were allowed to eat as much as they wanted, they ate similar amounts and had similar body weights at 4 months old. Later in life, however, the ‘fat’ strain mice were significantly heavier than the ‘lean’ strain mice, a difference linked to lower metabolic rate in the ‘fat’ mice. Only the mice with the lower metabolic rate benefitted from a reduced calorie diet. The study authors conclude that calorie restriction may be pointless – and possibly even dangerous – for non-obese individuals and that, ideally, energy expenditure and energy intake should be in balance.

A second study, published in the Proceedings of the National Academy of Sciences, looked at the effect of calorie restriction and intake of unsaturated fatty acids on cognitive performance in older people. Fifty healthy, normal to overweight subjects, with an average age of sixty, were assigned to one of three groups: 30% calorie reduction, increased intake of unsaturated fatty acids or control. Memory performance was measured before the trial began and after three months. A significant improvement (20%) in verbal memory scores was seen in the group eating fewer calories whereas no significant changes were seen in the other two groups. The improvements in memory correlated with decreases in fasting plasma insulin levels and a marker of inflammation, and were most pronounced in individuals who stuck strictly to the diet. The researchers plan to repeat the study in a larger group of people and also to study the effects of calorie restriction in patients with mild cognitive impairment. It is not clear whether reducing calorie intake would improve memory in lower weight individuals.

New Approach could Starve Tumour Cells to Death

Tumours are heterogeneous and contain both oxygenated and hypoxic regions. Cells in regions with low oxygen levels mainly use glucose for glycolytic energy production and release lactic acid in the process. It had been thought that tumour cells with an ample oxygen supply primarily used glucose for oxidative energy production, but a new study published in the Journal of Clinical Investigation shows that lactate plays a major role in fuelling the oxidative metabolism of these cells. Cells in different regions of a tumour are thus able to mutually regulate their access to energy metabolites, reserving glucose for use by cells in hypoxic regions and recycling their waste product. The study also identified the monocarboxylate transporter 1 (MCT1) as the main route of lactate uptake and, using three different tumour models, showed that blocking MCT1 with α-cyano-4-hydroxycinnamate or siRNA caused a switch from lactate-fuelled respiration to glycolysis. This switch in metabolism of oxygenated cells induces necrosis of distant hypoxic cells by effectively starving them of glucose. These hypoxic cells are known to be very aggressive and difficult to kill with conventional treatments. The reduced oxygen consumption by surviving tumour cells after MCT1 inhibition also rendered the tumours more sensitive to the effects of radiotherapy.

lactic acid Since MCT1 is expressed in a variety of primary human tumours, the study demonstrates the therapeutic potential of MCT1 inhibitors, as well as the likely benefit of combining these with radiotherapy.

New Mechanism for Treatment of Tumours

More than 80 years ago, Nobel laureate Otto Heinrich Warburg pointed to a difference in mitochondrial energy metabolism between tumour cells and normal healthy cells. This observation led to significant advances in cancer imaging using positron emission tomography (PET) and, because the altered energy metabolism is common to many types of cancer cells but not normal cells, it is also an attractive target for therapy. Now, Cornerstone Pharmaceuticals has announced the start of a clinical trial with a ‘thioctan’, CPI-613, the first example of an altered energy metabolism-directed (AEMD) compound.

In laboratory tumour models and animal studies, the new class of compounds were effective, even against difficult to treat tumours such as those of the lung, colon and pancreas, and showed very few adverse effects.

The AEMD technology platform being developed by Cornerstone is based upon the research of Paul M. Bingham, Ph.D. and Zuzana Zachar, Ph.D., Stony Brook University, Stony Brook, NY. These scientists disclosed ‘Lipoic acid derivatives and their use in treatment of disease’ in a patent filed in 1999.

The inventors describe key differences between metabolism in normal cells compared to that in cancerous cells:

The vast majority of normal cells utilize a single metabolic pathway to metabolize their food. The first step in this metabolic pathway is the partial degradation of glucose molecules to pyruvate in a process known as glycolysis or glycolytic cycle. The pyruvate is further degraded in the mitochondrion by a process known as the tricarboxylic acid (TCA) cycle to water and carbon dioxide, which is then eliminated. The critical link between these two processes is a large multi-subunit enzyme complex known as the pyruvate dehydrogenase (“PDH”) complex (“PDC”). PDC functions as a catalyst which funnels the pyruvate from the glycolytic cycle to the TCA cycle.

Most cancers display profound perturbation of energy metabolism. This change in energy metabolism represents one of the most robust and well-documented correlates of malignant transformation.

Because tumor cells degrade glucose largely glycolytically, i.e. without the TCA cycle, large amounts of pyruvate must be disposed of in several alternate ways. One major pathway used for disposal of excess pyruvate involves the joining of two pyruvate molecules to form the neutral compound acetoin. This generation of acetoin is catalyzed by a tumor-specific form of PDC. Although the TCA cycle still functions in cancer cells, the tumor cell TCA cycle is a variant cycle which depends on glutamine as the primary energy source. Tumor-specific PDC plays a regulatory role in this variant TCA cycle. Thus, inhibition or inactivation of a single enzyme, namely tumor-specific PDC, can block large scale generation of ATP and reducing potential in tumor cells.