HMG-CoA reductase inhibitors in osteoporosis: do they reduce the risk of fracture?

Osteoporosis affects a large number of people in industrialised countries. It has clinical and public-health impacts, most importantly due to subsequent fractures. Osteoporotic fractures are one of the most common causes of disability and are associated with enormous healthcare expenditure. The majority of existing treatment options for osteoporosis only inhibit bone resorption and prevent excessive bone loss but are not capable of stimulating bone formation. However, several recent in vitro and in vivo studies in animals demonstrated that HMG-CoA reductase inhibitors stimulate the production of bone morphogenetic protein (BMP-2), which is a potent regulating protein in osteoblast differentiation and activity. This suggests that HMG-CoA reductase inhibitors may have an anabolic effect on bones, making them a potentially interesting treatment option for osteoporosis. Additionally, several studies in humans showed that some HMG-CoA reductase inhibitors may have a beneficial effect on bone turnover and may lead to an increase in bone mineral density. Consequently, several observational studies tried to evaluate whether use of HMG-CoA reductase inhibitors is associated with a decreased risk of fractures. Even though not all results of these epidemiological studies, using different designs in different study populations, were entirely consistent, they provided substantial evidence that HMG-CoA reductase inhibitor use may decrease the bone fracture risk by approximately 50%. On the other hand, reanalysis of two randomised controlled trials of HMG-CoA reductase inhibitor therapy, designed to assess cardiovascular outcomes, could not show that patients treated with HMG-CoA reductase inhibitors had a lower fracture risk in comparison with placebo-treated patients. Therefore, to conclusively assess the potential of HMG-CoA reductase inhibitors in the prevention and treatment of osteoporosis, randomised controlled trials need to be performed to address this conflicting issue. Until the results of such trials are available, practitioners should prescribe the drugs that have been proven to reduce the risk of osteoporotic fractures.     

HMG-CoA reductase inhibitors: do they have potential in the treatment of polycystic ovary syndrome?

Many women of reproductive age are affected by polycystic ovary syndrome (PCOS), a heterogeneous endocrinopathy characterized by androgen excess, chronic oligo-anovulation and/or polycystic ovarian morphology. In addition, PCOS is often associated with insulin resistance, systemic inflammation and oxidative stress, which, on one hand, lead to endothelial dysfunction and dyslipidaemia with subsequent cardiovascular sequelae and, on the other hand, to hyperplasia of the ovarian theca compartment with resultant hyperandrogenism and anovulation. Traditionally, HMG-CoA reductase inhibitors (statins) have been used to treat dyslipidaemia by blocking HMG-CoA reductase (the rate-limiting step in cholesterol biosynthesis); however, they also possess pleiotropic actions, resulting in antioxidant, anti-inflammatory and anti-proliferative effects. Statins offer a novel therapeutic approach to PCOS in that they address the dyslipidaemia associated with the syndrome, as well as hyperandrogenism or hyperandrogenaemia. These actions may be due to an inhibition of the effects of systemic inflammation and insulin resistance/hyperinsulinaemia. Evidence to date, both in vitro and in vivo, suggests that statins have potential in the treatment of PCOS; however, further clinical trials are needed before they can be considered a standard of care in the medical management of this common endocrinopathy.     

Protein kinase Cδ mediates the activation of protein kinase D2 in platelets

Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCv in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a G_q inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is G_q-, but not G_12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ- or PKC?-deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and G_q-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets.     

Acetylcholinesterase inhibitors for the treatment of dementia in Alzheimer's disease: do we need new inhibitors?

Acetylcholinesterase inhibitors (AChEIs) have been shown to produce a small, but significant, improvement in cognition in patients with Alzheimer's disease. However, not all patients respond equally, and cognitive benefit may be of limited duration. Although new AChEIs continue to be developed, more recent studies have been aimed at developing inhibitors that have additional actions separate from AChE inhibition. Importantly, new treatments that target the underlying pathogenic mechanism of Alzheimer's disease (statins, secretase inhibitors, vaccination) may eventually emerge. These new treatments could make AChEI therapy less relevant for treatment of Alzheimer's disease.     

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

1. The role of protein kinase C (PKC) in mediating the action of κ-receptor stimulation on intracellular Ca²⁺ and cyclic AMP production was determined by studying the effects of trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.
2. U50,488H at 2.5–40 μM decreased both the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the κ-agonist, that were blocked by a selective κ-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ-receptor stimulation.
3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca²⁺]_i transient induced by caffeine, that depletes the SR of Ca²⁺, was used as an indicator of Ca²⁺ content in the SR. The caffeine-induced [Ca²⁺]_i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine-induced [Ca²⁺]_i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca²⁺ from the SR. When the increase in IP₃ production in response to κ-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ-receptor stimulation does not involve IP₃. The observations suggest that PKC may act directly at the SR.
4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ-receptor stimulation on Ca²⁺ in the SR and cyclic AMP production, both of which play an essential role in Ca²⁺ homeostasis in the heart.

     

The cardiac effects of mitoxantrone: do the benefits in multiple sclerosis outweigh the risks?

Mitoxantrone, an immunosuppressant agent with potent anti-inflammatory activity, has been used to treat patients with multiple sclerosis (MS) who have worsening relapsing-remitting (RRMS) or secondary progressive multiple sclerosis (SPMS) despite prior therapy with interferons or glatiramer acetate. From previous experience of treating cancer with mitoxantrone, it was expected that cardiotoxic effects and occasional malignancy would develop in some patients treated with this agent. From the earliest trials, reduction of left ventricular ejection fraction (LVEF) was seen in 2-3% of cases, and in some this effect may persist and less commonly there can be congestive heart failure and even death. There are also occasional reports of leukaemia developing in MS patients treated with this agent. Mitoxantrone has been shown to reduce relapses, the number of new lesions visualised on magnetic resonance imaging and stop or reduce the progression of the disease in many patients treated. The drug has found a place in MS therapy because in this progressing group of MS patients who are failing on the disease-modifying therapies with interferons or glatiramer acetate, trials have shown that mitoxantrone may arrest or even improve many patients. Recognising the risks, mitoxantrone therapy is a reasonable option for MS patients with RRMS and SPMS who are progressing despite current disease-modifying therapy.     

A preclinical view of cholinesterase inhibitors in neuroprotection: do they provide more than symptomatic benefits in Alzheimer's disease?

The prevalence of Alzheimer's disease (AD), a neurodegenerative condition whose greatest risk factor is old age, is expected to rise dramatically during the next five decades, along with the trend for increased longevity. Early diagnosis and intervention with therapies that halt or slow disease progress are likely to represent an important component of effective treatment. Although much progress has been made in this area, there are currently no clinically approved interventions for AD that are classed as disease modifying or neuroprotective. Cholinesterase inhibitors are a drug class used for the symptomatic treatment of AD. Recent evidence from preclinical studies indicates that these agents can attenuate neuronal damage and death from cytotoxic insults, and therefore might affect AD pathogenesis. The mechanisms by which these actions are mediated might or might not be directly related to their primary mode of action.     

Mechanisms to inhibit matrix metalloproteinase activity: where are we in the development of clinically relevant inhibitors?

Matrix metalloproteinases (MMPs) are involved in numerous pathophysiological processes, including cancer and cardiovascular disease. MMPs proteolyze multiple targets, including extracellular matrix, cytokines, and growth factors. Due to a high clinical relevance, MMPs have long been a target for pharmaceutical intervention. Although numerous drug therapies to inhibit MMPs have been explored, only one agent (doxycycline hyclate) is currently approved for clinical use. Multiple reasons potentially explain the lack of success in developing MMP inhibitors, including issues with selectivity and specificity and the presence of multiple substrates with conflicting functions. Major recent advances in the MMP field include an increased understanding of MMP biology, the improved establishment of parameters to adequately evaluate efficacy, and methods to enhance inhibitor design. This review will explore the latest research and patents targeted at MMP inhibition, and will focus on both direct and indirect mechanisms to block MMPs.     

Role for protein kinase C delta in the functional activity of human UGT1A6: implications for drug–drug interactions between PKC inhibitors and UGT1A6

1. Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug–drug interactions.

2. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems.

3. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC δ selective inhibitor; IC₅₀?=?9.0?±?1.2 μM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%.

4. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system.

5. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC δ increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% ± 4%).

6. These results identify an important role for PKC δ in UGT1A6-mediated glucuronidation and suggest that PKC δ inhibitors could interfere with glucuronidation of UGT1A6 substrates.

     

Can optical recordings of membrane potential be used to screen for drug-induced action potential prolongation in single cardiac myocytes?

INTRODUCTION: Potential-sensitive dyes have primarily been used to optically record action potentials (APs) in whole heart tissue. Using these dyes to record drug-induced changes in AP morphology of isolated cardiac myocytes could provide an opportunity to develop medium throughout assays for the pharmaceutical industry. Ideally, this requires that the dye has a consistent and rapid response to membrane potential, is insensitive to movement, and does not itself affect AP morphology. MATERIALS AND METHODS: We recorded the AP from isolated adult guinea-pig ventricular myocytes optically using di-8-ANEPPS in a single-excitation dual-emission ratiometric system, either separately in electrically field stimulated myocytes, or simultaneously with an electrical AP recorded with a patch electrode in the whole-cell bridge mode. The ratio of di-8-ANEPPS fluorescence signal was calibrated against membrane potential using a switch-clamp to voltage clamp the myocyte. RESULTS: Our data show that the ratio of the optical signals emitted at 560/620 nm is linearly related to voltage over the voltage range of an AP, producing a change in ratio of 7.5% per 100 mV, is unaffected by cell movement and is identical to the AP recorded simultaneously with a patch electrode. However, the APD90 recorded optically in myocytes loaded with di-8-ANEPPS was significantly longer than in unloaded myocytes recorded with a patch electrode (355.6+/-13.5 vs. 296.2+/-16.2 ms; p<0.01). Despite this effect, the apparent IC50 for cisapride, which prolongs the AP by blocking IKr, was not significantly different whether determined optically or with a patch electrode (91+/-46 vs. 81+/-20 nM). DISCUSSION: These data show that the optical AP recorded ratiometrically using di-8-ANEPPS from a single ventricular myocyte accurately follows the action potential morphology. This technique can be used to estimate the AP prolonging effects of a compound, although di-8-ANEPPS itself prolongs APD90. Optical dyes require less technical skills and are less invasive than conventional electrophysiological techniques and, when coupled to ventricular myocytes, decreases animal usage and facilitates higher throughput assays.     

Oral targeting of protein kinase C receptor: promising route for diabetic retinopathy?

In patients with diabetes, hyperglycemia is known to promote high levels of diacylglycerol which activates protein kinase C (PKC) in the vascular tissues and leads to the production of vascular endothelial growth factor (VEGF) in the retina. PKC activation and increased concentration of VEGF are likely to play a key role in diabetic microvascular complications, particularly change in vascular permeability, inflammation, fluid leakage and ischemia in the retina. PKC comprises a super family of isoenzymes that is activated in response to various stimuli. The PKC family consists of 12 isomers that possess distinct differences in structure, substrate requirement, expression and localization. PKC isomer selective inhibitors and VEGF trap are likely to be new therapeutics, which can delay the onset or stop the progression of diabetic vascular disease. A new promising therapy for diabetic retinopathy is undergoing Phase III trials, in which they proposed to target PKC βII isomer using Ruboxistaurin by oral administration. Besides retina, PKC βII isomer is found in higher concentration in brain, spleen, etc. So, oral targeting may be a questionable approach since generalized inhibitors may prove toxic in the treatment of diabetic retinopathy and ocular delivery may be a better alternative approach.     

Anacetrapib for the treatment of dyslipidaemia: the last bastion of the cholesteryl ester transfer protein inhibitors?

Introduction: Inhibition of cholesteryl ester transfer protein (CETP) has emerged as a potential way to decrease cardiovascular risk by raising high density lipoprotein (HDL) cholesterol and lowering low density lipoprotein (LDL) cholesterol concentrations. However, high profile withdrawals of several CETP inhibitors have cast doubt over this hypothesis. Despite this concern, anacetrapib appears to be safe, well-tolerated and delivers a substantial increases in HDL cholesterol and reductions in LDL cholesterol as monotherapy and when combined with a statin.

Areas covered: We discuss the role of CETP and HDL cholesterol as therapeutic targets, describe the pharmacokinetics and pharmacodynamics of anacetrapib, as well as report on the recent clinical trials.

Expert opinion: The focus of CETP inhibition has shifted from HDL cholesterol-raising to LDL cholesterol-lowering. Although anacetrapib appears to be safe and is effective in altering lipid-related biochemical parameters of interest, its effect on cardiovascular outcomes remains unknown. Extrapolation of LDL cholesterol lowering to improved cardiovascular outcomes is not possible, because LDL and HDL functionality in the setting of anacetrapib treatment is unclear. The results of the phase III REVEAL randomised controlled trial will be critical for anacetrapib to establish a place in clinical care.     

Regulation of cardiac nerves: a new paradigm in the management of sudden cardiac death?

The heart is extensively innervated, and its performance is tightly regulated by the autonomic nervous system. To maintain cardiac function, innervation density is stringently controlled, being high in the subepicardium and the central conduction system. In diseased hearts, cardiac innervation density varies, which in turn leads to sudden cardiac death. After myocardial infarction, sympathetic denervation is followed by reinnervation within the heart, leading to unbalanced neural activation and lethal arrhythmia. Diabetic sensory neuropathy causes silent myocardial ischemia, characterized by loss of pain perception during myocardial ischemia, which is a major cause of sudden cardiac death in diabetes mellitus (DM). Despite its clinical importance, the molecular mechanism underlying innervation density remains poorly understood. We found that cardiac sympathetic innervation is determined by the balance between neural chemoattraction and chemorepulsion, both of which occur in the heart. Nerve growth factor (NGF), which is a potent chemoattractant, is synthesized abundantly by cardiomyocytes and is induced by endothelin-1 upregulation in the heart. In contrast, Sema3a, which is a neural chemorepellent, is expressed strongly in the trabecular layer in early stage embryos and at a lower level after birth, leading to epicardial-to-endocardial transmural sympathetic innervation patterning. We also found that cardiac NGF downregulation is a cause of diabetic neuropathy, and that NGF supplementation rescues silent myocardial ischemia in DM. Both Sema3a-deficient and Sema3a-overexpressing mice showed sudden death or lethal arrhythmias due to disruption of innervation patterning. The present review focuses on the regulatory mechanisms involved in neural development in the heart and their critical roles in cardiac performance.     

Organophosphorus pesticides: do they all have the same mechanism of toxicity?

Organophosphorus (OP) pesticides are used extensively to control agricultural, household and structural pests. These pesticides constitute a diverse group of chemical structures exhibiting a wide range of physicochemical properties, with their primary toxicological action arising from inhibition of the enzyme acetylcholinesterase (AChE, EC 3.1.1.7). Historically, risk characterizations for these toxicants have been based on hazard and exposure data pertaining to individual chemicals. The Food Quality Protection Act of 1996 now requires, however, that combined risk assessments be performed with pesticides having a common mechanism of toxicity. It is therefore critical to consider whether OP pesticides all exert toxicity through a common mechanism. This brief review evaluates the comparative toxicity of the 38 OP AChE inhibitors currently registered for use as pesticides in the United States and examines the data which suggest that some OP pesticides have toxicologically relevant sites of action in addition to AChE. It is concluded that all OP anticholinesterases potentially have a mechanism of toxicity in common, that is, phosphorylation of AChE causing accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequent clinical signs of cholinergic toxicity. Additional macromolecular targets for some OP pesticides, however, may alter the cascade of events following AChE phosphorylation and thereby modify that common mechanism. Furthermore, other macromolecular targets of some OP pesticides appear capable of altering noncholinergic neurochemical processes. These additional actions may contribute to qualitative and quantitative differences in toxicity sometimes noted in the presence of similar levels of AChE inhibition induced by different OP pesticides. Further investigation of these additional sites of action may allow subclassification and influence the decision to perform combined risk assessments on this class of pesticides based on common mechanism of toxicity.     

Is inositol 145-trisphosphate a second messenger coupled to kappa-opioid receptor in cardiac myocytes of the rat?

AIM: To elucidate whether inositol 1,4, 5-trisphosphate (IP_3) is a second messenger linking activation of kappareceptor to the release of Ca~(2 ) from sarcoplasmic reticulum in rat cardiac myocytes. METHODS: Fura-2 fluorimetric     

Drug transporters in the lung—do they play a role in the biopharmaceutics of inhaled drugs?

The role of transporters in drug absorption, distribution and elimination processes as well as in drug–drug interactions is increasingly being recognised. Although the lungs express high levels of both efflux and uptake drug transporters, little is known of the implications for the biopharmaceutics of inhaled drugs. The current knowledge of the expression, localisation and functionality of drug transporters in the pulmonary tissue and the few studies that have looked at their impact on pulmonary drug absorption is extensively reviewed. The emphasis is on transporters most likely to affect the disposition of inhaled drugs: (1) the ATP-binding cassette (ABC) superfamily which includes the efflux pumps P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs), breast cancer resistance protein (BCRP) and (2) the solute-linked carrier (SLC and SLCO) superfamily to which belong the organic cation transporter (OCT) family, the peptide transporter (PEPT) family, the organic anion transporter (OAT) family and the organic anion transporting polypeptide (OATP) family. Whenever available, expression and localisation in the intact human tissue are compared with those in animal lungs and respiratory epithelial cell models in vitro. The influence of lung diseases or exogenous agents on transporter expression is also mentioned. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2240–2255, 2010     

Renaissance of NMDA receptor antagonists: do they have a role in the pharmacotherapy for alcoholism?

Long-term alcohol exposure leads to the development of alcohol dependence, which is possibly induced by changes in specific neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptor is a particularly important site of action for ethanol. Ethanol potently and selectively inhibits NMDA receptors (NMDARs) and prolonged ethanol exposure produces a compensatory 'upregulation' of NMDAR functions. These changes are believed to underlie the development of ethanol tolerance and dependence as well as acute and delayed signs of withdrawal. Therefore, negative modulators of NMDARs may be useful agents for the pharmacotherapy of alcoholism. NMDAR antagonists attenuate not only the physical symptoms but also some affective and motivational components of alcohol withdrawal. Encouraging experimental results have been obtained with novel uncompetitive (memantine and neramexane (Merz & Co GmbH/Forest Laboratories Inc)), glycine site and NR2B subunit-selective NMDA antagonists (SSNAs). Recently emerged NR2B SSNAs (CP-101606 (Pfizer Inc), Co-101244 (Pfizer Inc/Purdue Neuroscience Corp/Senju Pharmaceutical Co Ltd), CI-1041 (Purdue Neuroscience Corp/Pfizer Inc) and indole-2-carboxamide derivatives) have demonstrated excellent in vitro potency against withdrawal-induced cytotoxicity. Although in vivo data are few, according to their in vitro efficacy and good tolerability, novel NMDA antagonists, especially the NR2B-selective antagonists, may offer a preferable alternative to the presently available pharmacotherapies for treating alcoholism.     

Inhibition of protein kinase C,but not of protein kinase A,blocks the development of acute antinociceptive tolerance to an intrathecally administered μ-opioid receptor agonist in the mouse

A specific protein kinase C inhibitor, calphostin C, which injected alone had no effect on the antinociception induced by intrathecal (i.t.) administration of a selective μ-opioid receptor agonist, [d-Ala²,NMePhe⁴,Gly(ol)⁵]enkephalin (DAMGO), dose-dependently attenuated the development of acute tolerance to the i.t. DAMGO-induced antinociception in male ICR mice. On the other hand, a selective protein kinase A inhibitor, KT5720, did not have any effect on the development of acute tolerance to DAMGO antinociception. These findings suggest that protein kinase C, but not protein kinase A, plays an important role in the development of acute tolerance to the μ-opioid receptor agonist-induced antinociception.