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Archive for the ‘Kinase’ Category


New Studies toward Understanding Alzheimer Disease

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

There is no unifying concept of Alzheimer Disease beyond the Tau and beta amyloid roles.  Recently, Ingenbleek and Bernstein (journal AD) made the connection between the age related decline of liver synthesis of plasma transthyretin and the more dramatic decline of transthyretin at the blood brain barrier, and the relationship to inability to transfer vitamin A via retinol binding protein to the brain.  Related metabolic events are reported by several groups.

 

What else is New?

 

Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer’s disease.

Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD.

Sci Transl Med. 2016 May 25;8(340):340ra72.  http://dx.doi.org:/10.1126/scitranslmed.aaf1059

They show that Aβ oligomerization, a behavior traditionally viewed as intrinsically pathological, may be necessary for the antimicrobial activities of the peptide. Collectively, our data are consistent with a model in which soluble Aβ oligomers first bind to microbial cell wall carbohydrates via a heparin-binding domain. Developing protofibrils inhibited pathogen adhesion to host cells. Propagating β-amyloid fibrils mediate agglutination and eventual entrapment of unatttached microbes….Salmonella Typhimurium bacterial infection of the brains of transgenic 5XFAD mice resulted in rapid seeding and accelerated β-amyloid deposition, which closely colocalized with the invading bacteria.

This is quite interesting in that infection drives the production of acute phase reactants resulting in decreased production of transthyretin.  Whether this also has ties to chronic disease in the elderly and risk of AD is not known.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer’s disease.

Alfonso SI, Callender JA, Hooli B, Antal CE, Mullin K, Sherman MA, Lesné SE, Leitges M, Newton AC, Tanzi RE, Malinow R.

Sci Signal. 2016 May 10;9(427):ra47.  http://dx.doi.org:/10.1126/scisignal.aaf6209.

Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), they identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA(-/-) neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses.

 

Science Signaling Podcast for 10 May 2016: PKCα in Alzheimer’s disease.

Newton AC, Tanzi RE, VanHook AM.

Sci Signal. 2016 May 10;9(427):pc11. doi: 10.1126/scisignal.aaf9436.

Relevance of the COPI complex for Alzheimer’s disease progression in vivo.

Bettayeb K, Hooli BV, Parrado AR, Randolph L, Varotsis D, Aryal S, Gresack J,Tanzi RE, Greengard P, Flajolet M.

Proc Natl Acad Sci U S A. 2016 May 10;113(19):5418-23. http://dx.doi.org:/10.1073/pnas.1604176113

Inhibition of death-associated protein kinase 1 attenuates the phosphorylation and amyloidogenic processing of amyloid precursor protein.

Kim BM, You MH, Chen CH, Suh J, Tanzi RE, Ho Lee T.

Hum Mol Genet. 2016 Apr 19. pii: ddw114.

Extracellular deposition of amyloid-beta (Aβ) peptide, a metabolite of sequential cleavage of amyloid precursor protein (APP), is a critical step in the pathogenesis of Alzheimer’s disease (AD). While death-associated protein kinase 1 (DAPK1) is highly expressed in AD brains and its genetic variants are linked to AD risk, little is known about the impact of DAPK1 on APP metabolism and Aβ generation. This study demonstrated a novel effect of DAPK1 in the regulation of APP processing using cell culture and mouse models. DAPK1, but not its kinase deficient mutant (K42A), significantly increased human Aβ secretion in neuronal cell culture models. Moreover, knockdown of DAPK1 expression or inhibition of DAPK1 catalytic activity significantly decreased Aβ secretion. Furthermore, DAPK1, but not K42A, triggered Thr668 phosphorylation of APP, which may initiate and facilitate amyloidogenic APP processing leading to the generation of Aβ. In Tg2576 APPswe-overexpressing mice, knockout of DAPK1 shifted APP processing toward non-amyloidogenic pathway and decreased Aβ generation. Finally, in AD brains, elevated DAPK1 levels showed co-relation with the increase of APP phosphorylation. Combined together, these results suggest that DAPK1 promotes the phosphorylation and amyloidogenic processing of APP, and that may serve a potential therapeutic target for AD.

Recapitulating amyloid β and tau pathology in human neural cell culture models: clinical implications.

Choi SH, Kim YH, D’Avanzo C, Aronson J, Tanzi RE, Kim DY.

US Neurol. 2015 Fall;11(2):102-105.    Free PMC Article

The “amyloid β hypothesis” of Alzheimer’s disease (AD) has been the reigning hypothesis explaining pathogenic mechanisms of AD over the last two decades. However, this hypothesis has not been fully validated in animal models, and several major unresolved issues remain. Our 3D human neural cell culture model system provides a premise for a new generation of cellular AD models that can serve as a novel platform for studying pathogenic mechanisms and for high-throughput drug screening in a human brain-like environment.

The two key pathological hallmarks of AD are senile plaques (amyloid plaques) and neurofibrillary tangles (NFTs), which develop in brain regions responsible for memory and cognitive functions (i.e. cerebral cortex and limbic system) 3. Senile plaques are extracellular deposits of amyloid-β (Aβ) peptides, while NFTs are intracellular, filamentous aggregates of hyperphosphorylated tau protein 4.

The identification of Aβ as the main component of senile plaques by Drs. Glenner and Wong in 1984 5 resulted in the original formation of the “amyloid hypothesis.” According to this hypothesis, which was later renamed the “amyloid-β cascade hypothesis” by Drs. Hardy and Higgins 6, the accumulation of Aβ is the initial pathological trigger in the disease, subsequently leading to hyperphosphorylation of tau, causing NFTs, and ultimately, neuronal death and dementia 4,710. Although the details have been modified to reflect new findings, the core elements of this hypothesis remain unchanged: excess accumulation of the pathogenic forms of Aβ, by altered Aβ production and/or clearance, triggers the vicious pathogenic cascades that eventually lead to NFTs and neuronal death.

Over the last two decades, the Aβ hypothesis of AD has reigned, providing the foundation for numerous basic studies and clinical trials 4,7,10,11. According to this hypothesis, the accumulation of Aβ, either by altered Aβ production and/or clearance, is the initial pathological trigger in the disease. The excess accumulation of Aβ then elicits a pathogenic cascade including synaptic deficits, altered neuronal activity, inflammation, oxidative stress, neuronal injury, hyperphosphorylation of tau causing NFTs and ultimately, neuronal death and dementia 4,710.

One of the major unresolved issues of the Aβ hypothesis is to show a direct causal link between Aβ and NFTs 1214. Studies have demonstrated that treatments with various forms of soluble Aβ oligomers induced synaptic deficits and neuronal injury, as well as hyperphosphorylation of tau proteins, in mouse and rat neurons, which could lead to NFTs and neurodegeneration in vivo 1821. However, transgenic AD mouse models carrying single or multiple human familial AD (FAD) mutations in amyloid precursor protein (APP) and/or presenilin 1 (PS1) do not develop NFTs or robust neurodegeneration as observed in human patients, despite robust Aβ deposition 13,22,23. Double and triple transgenic mouse models, harboring both FAD and tau mutations linked with frontotemporal dementia (FTD), are the only rodent models to date displaying both amyloid plaques and NFTs. However, the NFT pathology in these models stems mainly from the overexpression of human tau as a result of the FTD, rather than the FAD mutations24,25.

Human neurons carrying FAD mutations are an optimal model to test whether elevated levels of pathogenic Aβ trigger pathogenic cascades including NFTs, since those cells truly share the same genetic background that induces FAD in humans. Indeed, Israel et al., observed elevated tau phosphorylation in neurons with an APP duplication FAD mutation 33. Blocking Aβ generation by β-secretase inhibitors significantly decreased tau phosphorylation in the same model, but γ-secretase inhibitor, another Aβ blocker, did not affect tau phosphorylation 33. Neurons with the APP V717I FAD mutation also showed an increase in levels of phospho tau and total tau levels 28. More importantly, Muratore and colleagues showed that treatments with Aβ-neutralizing antibodies in those cells significantly reduced the elevated total and phospho tau levels at the early stages of differentiation, suggesting that blocking pathogenic Aβ can reverse the abnormal tau accumulation in APP V717I neurons 28.

Recently, Moore et al. also reported that neurons harboring the APP V717I or the APP duplication FAD mutation showed increases in both total and phospho tau levels 27. Interestingly, altered tau levels were not detected in human neurons carrying PS1 FAD mutations, which significantly increased pathogenic Aβ42 species in the same cells 27. These data suggest that elevated tau levels in these models were not due to extracellular Aβ accumulation but may possibly represent a very early stage of tauopathy. It may also be due to developmental alterations induced by the APP FAD mutations.

As summarized, most human FAD neurons showed significant increases in pathogenic Aβ species, while only APP FAD neurons showed altered tau metabolism that may represent very early stages of tauopathy. However, all of these human FAD neurons failed to recapitulate robust extracellular amyloid plaques, NFTs, or any signs of neuronal death, as predicted in the amyloid hypothesis.

In our recent study, we moved one step closer to proving the amyloid hypothesis. By generating human neural stem cell lines carrying multiple mutations in APP together with PS1, we achieved high levels of pathogenic Aβ42 comparable to those in brains of AD patients 4446.

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Platform for AD drug screening in human neural progenitor cells with FAD mutations in a 3D culture system, which successfully reproduce human AD pathogenesis (amyloid plaques-driven tauopathy).

In addition to the impact on toxic Aβ species, our 3D culture model can test if these antibodies can block tau pathologies in 3D human neural cell culture systems 4446. Human cellular AD models can also be used to determine optimal doses of candidate AD drugs to block Aβ and/or tau pathology without affecting neuronal survival (Fig. 1).

While much progress has been made, many challenges still lie on the path to creating human neural cell culture models that comprehensively recapitulate pathogenic cascades of AD. A major difficulty lies in reconstituting the brain regions most affected in AD: the hippocampus and specific cortical layers. Recent progress in 3D culture technology, such as “cerebral organoids,” may also be helpful in rebuilding the brain structures that are affected by AD in a dish 52,53. These “cerebral organoids” were able to model various discrete brain regions including human cortical areas 52, which enabled them to reproduce microcephaly, a brain developmental disorder. Similarly, pathogenic cascades of AD may be recapitulated in cortex-like structures using this model. Adding neuroinflammatory components, such as microglial cells, which are critical in AD pathogenesis, will illuminate the validity of the amyloid β hypothesis. Reconstitution of robust neuronal death stemming from Aβ and tau pathologies will be the next major step in comprehensively recapitulating AD in a cellular model.

 

Family-based association analyses of imputed genotypes reveal genome-wide significant association of Alzheimer’s disease with OSBPL6, PTPRG, and PDCL3.

Herold C, Hooli BV, Mullin K, Liu T, Roehr JT, Mattheisen M, Parrado AR, Bertram L, Lange C, Tanzi RE.

Mol Psychiatry. 2016 Feb 2. http://dx.doi.org:/10.1038/mp.2015.218.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer’s disease and APdE9 transgenic mouse brain and cell-based models.

Natunen T, Takalo M, Kemppainen S, Leskelä S, Marttinen M, Kurkinen KM, Pursiheimo JP, Sarajärvi T, Viswanathan J, Gabbouj S, Solje E, Tahvanainen E, Pirttimäki T, Kurki M, Paananen J, Rauramaa T, Miettinen P, Mäkinen P, Leinonen V, Soininen H, Airenne K, Tanzi RE, Tanila H, Haapasalo A, Hiltunen M.

Neurobiol Dis. 2016 Jan;85:187-205. http://dx.doi.org:/10.1016/j.nbd.2015.11.005.

Accumulation of β-amyloid (Aβ) and phosphorylated tau in the brain are central events underlying Alzheimer’s disease (AD) pathogenesis. Aβ is generated from amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aβ pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aβ40 and Aβ42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aβ accumulation.

Correction to Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 Sep 22;54(37):5781.  http://dx.doi.org:/10.1021/acs.biochem.5b00968. Epub 2015 Sep 8. No abstract available.

Massachusetts Alzheimer’s Disease Research Center: progress and challenges.

Hyman BT, Growdon JH, Albers MW, Buckner RL, Chhatwal J, Gomez-Isla MT, Haass C, Hudry E, Jack CR Jr, Johnson KA, Khachaturian ZS, Kim DY, Martin JB, Nitsch RM, Rosen BR, Selkoe DJ, Sperling RA, St George-Hyslop P, Tanzi RE, Yap L, Young AB, Phelps CH, McCaffrey PG.

Alzheimers Dement. 2015 Oct;11(10):1241-5. http://dx.doi.org:/10.1016/j.jalz.2015.06.1887. Epub 2015 Aug 19. No abstract available.

Alzheimer’s in 3D culture: challenges and perspectives.

D’Avanzo C, Aronson J, Kim YH, Choi SH, Tanzi RE, Kim DY.

Bioessays. 2015 Oct;37(10):1139-48. doi: 10.1002/bies.201500063. Epub 2015 Aug 7. Review.

Synaptotagmins interact with APP and promote Aβ generation.

Gautam V, D’Avanzo C, Berezovska O, Tanzi RE, Kovacs DM.

Mol Neurodegener. 2015 Jul 23;10:31. doi: 10.1186/s13024-015-0028-5.

Near-infrared fluorescence molecular imaging of amyloid beta species and monitoring therapy in animal models of Alzheimer’s disease.

Zhang X, Tian Y, Zhang C, Tian X, Ross AW, Moir RD, Sun H, Tanzi RE, Moore A, Ran C.

Proc Natl Acad Sci U S A. 2015 Aug 4;112(31):9734-9. doi: 10.1073/pnas.1505420112. Epub 2015 Jul 21.

A 3D human neural cell culture system for modeling Alzheimer’s disease.

Kim YH, Choi SH, D’Avanzo C, Hebisch M, Sliwinski C, Bylykbashi E, Washicosky KJ, Klee JB, Brüstle O, Tanzi RE, Kim DY.

Nat Protoc. 2015 Jul;10(7):985-1006. doi: 10.1038/nprot.2015.065. Epub 2015 Jun 11.

Cathepsin L Mediates the Degradation of Novel APP C-Terminal Fragments.

Wang H, Sang N, Zhang C, Raghupathi R, Tanzi RE, Saunders A.

Biochemistry. 2015 May 12;54(18):2806-16. doi: 10.1021/acs.biochem.5b00329. Epub 2015 Apr 28. Erratum in: Biochemistry. 2015 Sep 22;54(37):5781.

γ-Secretase modulators reduce endogenous amyloid β42 levels in human neural progenitor cells without altering neuronal differentiation.

D’Avanzo C, Sliwinski C, Wagner SL, Tanzi RE, Kim DY, Kovacs DM.

FASEB J. 2015 Aug;29(8):3335-41. doi: 10.1096/fj.15-271015. Epub 2015 Apr 22.

PLD3 gene variants and Alzheimer’s disease.

Hooli BV, Lill CM, Mullin K, Qiao D, Lange C, Bertram L, Tanzi RE.

Nature. 2015 Apr 2;520(7545):E7-8. doi: 10.1038/nature14040. No abstract available.

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New glucokinase activator

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RO-28-1675 for Type 2 Diabetes

by DR ANTHONY MELVIN CRASTO Ph.D

http://www.medchemexpress.com/product_pic/hy-10595.gif

 

RO-28-1675

  • (2R)-3-Cyclopentyl-2-[4-(methanesulfonyl)phenyl]-N-(thiazol-2-yl)propionamide
  • Ro 028-1675
  • Ro 0281675
  • Ro 28-1675

3-Cyclopentyl-2(R)-[4-(methylsulfonyl)phenyl]-N-(2-thiazolyl)propionamide

MW 378.51 .-70.4 °Conc 0.027 g/100mL; chloroform, 589 nm;  23 °C

 

Formula C18H22N2O3S2
CAS No 300353-13-3

Glucokinase Activators

Ro 28-1675 (Ro 0281675) is a potent allosteric GK activator with a SC1.5 value of 0.24± 0.0019 uM.

Roche (Innovator)

Hoffmann La Roche

PHASE 1    Type 2  DIABETES,
IC50 value: 0.24± 0.0019 uM (SC1.5) [1]
Target: Glucokinase activator
The R stereoisomer Ro 28-1675 activated GK with a SC1.5 of 0.24 uM, while the S isomer did not activated GK up to 10 uM. Oral administration of Ro 28-1675 (50 mg/Kg) to male C57B1/6J mice caused a statistically significant reduction in fasting glucose levels and improvement in glucose tolerance relative to the vehicle treated animals [1].
Comparison of rat PK parameters indicated that Ro 28-1675 displayed lower clearance and higher oral bioavailability compared to 9a.

Following a single oral dose, Ro 28-1675 reduced fasting and postprandial glucose levels following an OGTT, was well tolerated, and displayed no adverse effects related to drug administration other than hypoglycemia at the maximum dose (400 mg).

RO-28-1675 as glucokinase activator.

Joseph Grimsby et al., of Roche have recently discovered activators of glucokinase that increase kcat and decrease the S0.5 for glucose, and these may offer a treatment for type II diabetes. Glucokinase (GK) plays a key role in whole-body glucose homeostasis by catalyzing the phosphorylation of glucose in cells that express this enzyme, such as pancreatic β cells and hepatocytes.

By screening of a library of 120,000 structurally diverse synthetic compounds, they found one small molecule that increased the enzymatic activity of GK. Chemical optimization of this initial molecule led to the synthesis of RO-28-0450 as a lead GK activator which is a class of antidiabetic agents that act as nonessential, mixed-type GK activators (GKAs) that increase the glucose affinity and maximum velocity (Vmax) of GK. RO-28-0450 is a racemic compound.

Activation of GK was exquisitely sensitive to the chirality of the molecule: The R enantiomer, RO-28-1675, was found to be a potent GKA, whereas the S enantiomer, RO-28-1674, was inactive. RO-28-1675 also reversed the inhibitory action of the human glucokinase regulatory protein (GKRP). The activators binding in a glucokinase regulatory site originally was discovered in patients with persistent hyperinsulinemic hypoglycemi.

The result of RO-28-1675 as a potent small molecule GKA may shed light to the chemical biologists to devise strategy for developing activators. Thus for a success to this end we must focus on highly regulated enzymes, or cooperative enzymes such as glucokinase, where nature has provided binding sites that are designed to modulate catalysis.

 

SYNTHESIS

Paper

J. Med. Chem., 2010, 53 (9), pp 3618–3625
DOI: 10.1021/jm100039a
Abstract Image

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

Flash chromatography (Merck Silica gel 60, 70-230 mesh, 9/1, 3/1, and then 11/9 hexanes/ethyl acetate) afforded (2R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (2.10 g, 74%) as a white foam.   ….

PATENT

WO 2000058293

http://www.google.com/patents/WO2000058293A2?cl=en

 

Discovery, Structure−Activity Relationships, Pharmacokinetics, and Efficacy of Glucokinase Activator (2R)-3-Cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (RO0281675)

J. Med. Chem., 2010, 53 (9), pp 3618–3625   DOI:http://dx.doi.org:/10.1021/jm100039a
Abstract Image
Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

REFERENCES

[1]. Haynes NE, et al. Discovery, structure-activity relationships, pharmacokinetics, and efficacy of glucokinase activator (2R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (RO0281675).

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

http://www.nature.com/nrd/journal/v8/n5/fig_tab/nrd2850_T2.html

NMR…..http://www.medchemexpress.com/product_pdf/HY-10595/Ro%2028-1675-NMR-HY-10595-13569-2014.pdf

http://www.medchemexpress.com/product_pdf/HY-10595/Ro%2028-1675-Lcms_Ms-HY-10595-13569-2014.pdf

J Grimsby et al. Allosteric Activators of Glucokinase: Potential Role in Diabetes Therapy. Science Signaling 2003, 301(5631), 370-373.
T Kietzmann and GK Ganjam. Glucokinase: old enzyme, new target. Exp. Opin. Ther. Patents. 2005, 15(6), 705-713.

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P13K delta-gamma anticancer agent

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RP 6350, Rhizen Pharmaceuticals S.A. and Novartis tieup for Rhizen’s inhaled dual Pl3K-delta gamma inhibitor

by DR ANTHONY MELVIN CRASTO Ph.D

 

(A)           and                         (Al)                  and                (A2)

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (Compound A1 is RP 6350).

 

str1

 

RP 6350, RP6350, RP-6350

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

mw 415

Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation  In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PATENTS……..WO 11/055215 ,  WO 12/151525.

  • Antineoplastics; Small molecules
  • Mechanism of Action Phosphatidylinositol 3 kinase delta inhibitors; Phosphatidylinositol 3 kinase gamma inhibitors
  • Phase I Haematological malignancies
  • Preclinical Multiple myeloma

 

Swaroop K. V. S. Vakkalanka,
COMPANY Rhizen Pharmaceuticals Sa

https://clinicaltrials.gov/ct2/show/NCT02017613

 

PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

Company Rhizen Pharmaceuticals S.A.
Description Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Molecular Target Phosphoinositide 3-kinase (PI3K) delta ; Phosphoinositide 3-kinase (PI3K) gamma
Mechanism of Action Phosphoinositide 3-kinase (PI3K) delta inhibitor; Phosphoinositide 3-kinase (PI3K) gamma inhibitor
Therapeutic Modality Small molecule

 

Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
 Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
 1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

POSTER SESSIONS
Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
  • Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
  • RP6530, a novel, small molecule PI3K delta/gamma
  • Activity and selectivity of RP6530 for PI3K delta and gamma isoforms

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.

Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.

Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.

Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Disclosures:Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership.Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

 

PI3K Dual Inhibitor (RP-6530)


Therapeutic Area Respiratory , Oncology – Liquid Tumors , Rheumatology Molecule Type Small Molecule
Indication Peripheral T-cell lymphoma (PTCL) , Non-Hodgkins Lymphoma , Asthma , Chronic Obstructive Pulmonary Disease (COPD) , Rheumatoid Arthritis
Development Phase Phase I Rt. of Administration Oral

Description

Rhizen is developing dual PI3K gamma/delta inhibitors for liquid tumors and inflammatory conditions.

Situation Overview

Dual Pl3K inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases manifested by a reduction in neutrophilia and TNF in response to LPS. Scientific evidence for PI3-kinase involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches, which makes it a potential target for treatment of respiratory disease. Resistance to conventional therapies such as corticosteroids in several patients has been attributed to an up-regulation of the PI3K pathway; thus, disruption of PI3K signaling provides a novel strategy aimed at counteracting the immuno-inflammatory response. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ? and ? isoforms would be expected to attenuate the progression of immune response encountered in most variations of airway inflammation and arthritis.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.

 

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.png

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.pngtps:/

Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.

 

Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.

PATENT
WO2014195888, DUAL SELECTIVE PI3 DELTA AND GAMMA KINASE INHIBITORS

This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above

Figure imgf000094_0001

15 14 10 12 12a

REFERENCES
April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
  • 01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
  • 18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
  • 18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014

 

WO2011055215A2 Nov 3, 2010 May 12, 2011 Incozen Therapeutics Pvt. Ltd. Novel kinase modulators
WO2012151525A1 May 4, 2012 Nov 8, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO2013164801A1 May 3, 2013 Nov 7, 2013 Rhizen Pharmaceuticals Sa Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
US20110118257 May 19, 2011 Rhizen Pharmaceuticals Sa Novel kinase modulators
US20120289496 May 4, 2012 Nov 15, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO 2011055215

 

 

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Phase I/II Hepato-specific Glucokinase Activator

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Advinus Therapeutics announced that it has successfully completed a 14-day POC study in 60 Type II diabetic patients on its lead molecule, GKM-001, a glucokinase activator. The results of the trial show effective glucose lowering across all doses tested without any incidence of hypoglycemia or any other clinically relevant adverse events.

GKM-001 is differentiated from most other GK molecules that are in development, or have been discontinued, due to its novel liver selective mechanism of action.

GKM-001 belongs to a novel class of molecules for treatment of type II diabetes. It is an activator of Glucokinase (GK), a glucose-sensing enzyme found mainly in the liver and pancreas. Being liver selective, GKM-001 mostly activates GK in the liver and not in pancreas, which is its key differentiation from most competitor molecules that activate GK in pancreas as well.

GKM 001 in pipeline for Diabetes by Advinus

by DR ANTHONY MELVIN CRASTO Ph.D

ad 1
GKM 001

Advinus Therapeutics Private L,

A glucokinase activator for treatment of type II diabetes, currently in PI. Advinus is actively exploring partnership options to expedite further development and WW marketing of GKM-001.

Company Advinus Therapeutics Ltd.
Description Activator of glucokinase (GCK; GK)
Molecular Target Glucokinase (GCK) (GK)
Mechanism of Action Glucokinase activator
Therapeutic Modality Small molecule
Latest Stage of Development Phase I/II
Standard Indication Diabetes
Indication Details Treat Type II diabetes

PATENT

https://www.google.co.in/patents/WO2009047798A2?cl=en

Example Cl : (-)-{5-ChIoro-2-[2-(4-cyclopropanesulfonylphenyI)-2-(2,4- difluorophenoxy)acetylamino]thiazol-4-yl}-acetic acid, ethyl ester
1H NMR(400 MHz, CDCl3): δ 1.06-1.08 (m, 2H), 1.30 (t, J=7.2 Hz, 3H), 1.33-1.38 (m, 2H), 2.42-2.50 (m, IH), 3.73 (d, J=2 Hz, 2H), 4.22 (q, J=7.2 Hz ,2H), 5.75 (s, IH), 6.76- 6.77 (m, IH), 6.83-6.86 (m, IH), 6.90-6.98 (m, IH), 7.73 (d, J=8.4 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 9.96 (bs, IH). MS (EI) m/z: 571.1 and 573.1 (M+ 1; for 35Cl and 37Cl respectively).

Examples C2 and C3 were prepared in analogues manner of example (Cl) from the appropriate chiral intermediate:

Figure imgf000044_0002

Example Dl : (+)-{5-Chloro-2-[2-(4-cyclopropanesulfonylphenyl)-2-(2,4- difluorophenoxy)acetylamino]thiazol-4-yl}acetic acid, ethyl ester

Advinus’ GK-activator Achieves Early POC for Diabetes

November 29 2011

Partnership Dialog Actively Underway

Advinus Therapeutics, a research-based pharmaceutical company founded by globally experienced industry executives and promoted by the TATA Group, announced that it has successfully completed a 14-day POC study in 60 Type II diabetic patients on its lead molecule, GKM-001, a glucokinase activator. The results of the trial show effective glucose lowering across all doses tested without any incidence of hypoglycemia or any other clinically relevant adverse events.

The clinical trials on GKM-001 validate the company’s pre-clinical hypothesis that a liver selective Glucokinase activator would not cause hypoglycemia (very low blood sugar), while showing robust efficacy.

“GKM-001 is differentiated from most other GK molecules that are in development, or have been discontinued, due to its novel liver selective mechanism of action. GKM-001 has a prolonged pharmacological effect and a half-life that should support a once a day dosing as both mono and combination therapy.” said Dr. Rashmi Barbhaiya, MD & CEO, Advinus Therapeutics. He added that Advinus is actively exploring partnership options to expedite further development and global marketing of GKM-001.

GKM-001 belongs to a novel class of molecules for treatment of type II diabetes. It is an activator of Glucokinase (GK), a glucose-sensing enzyme found mainly in the liver and pancreas. Being liver selective, GKM-001 mostly activates GK in the liver and not in pancreas, which is its key differentiation from most competitor molecules that activate GK in pancreas as well. The resulting increase in insulin secretion creates a potential for hypoglycemia-a risk GKM-001 is designed to avoid. Advinus has the composition of matter patent on GKM-001 for all major markets globally. Both the Single Ascending Dose data, in healthy and type II diabetics, and the Multiple Ascending Dose Study in Type II diabetics has shown that the molecule shows effective glucose lowering in a dose dependent manner and has excellent safety and tolerability profile over a 40-fold dose range. The pharmacokinetic properties of the molecule support once a day dosing. GKM-001 has the potential to be “First-in-Class” drug to address this large, growing and yet poorly addressed market.

Advinus also has identified a clinical candidate as a back-up to GKM-001, which is structurally different. In its portfolio, the company has a growing pipeline for COPD, sickle cell disease, inflammatory bowel disease, type 2 diabetes, acute and chronic pain and rheumatoid arthritis in various stages of late discovery and pre-clinical development.

Advinus Therapeutics team discovers novel molecule for treatment of diabetes

  • The first glucokinase modulator discovered and developed in India 
  • A new concept for the management of diabetes for patients, globally 
  • 100 per cent ‘made in India’ molecule for the treatment of diabetes 
  • IND approved by DGCI, Phase I clinical trial shows excellent safety and tolerance profiles with efficacy

Bangalore: Advinus Therapeutics (Advinus), the research-based pharmaceutical company founded by leading global pharmaceutical executives and promoted by the Tata group, today, announced the discovery of a novel molecule for the treatment of type II diabetes — GKM-001.The molecule is an activator of glucokinase; an enzyme that regulates glucose balance and insulin secretion in the body.

GKM-001 is a completely indigenously developed molecule and the initial clinical trials have shown excellent results for both safety and efficacy.

“Considering past failures of other companies on this target, our discovery programme primarily focused on identifying a molecule that would be efficacious without causing hypoglycaemia; a side effect associated with most compounds developed for this target.

“Recently completed Phase I data indicate that Advinus’ GKM–001 is a liver selective molecule that has overcome the biggest clinical challenge of hypoglycaemia. GKM-001 is differentiated from most other GK molecules in development due to this novel mechanism of action,” said Dr Rashmi Barbhaiya, MD and CEO, Advinus Therapeutics.

He further added, “We are very proud that GKM-001 is 100 per cent Indian. Advinus’s discovery team in Pune discovered the molecule and entire preclinical development was carried out at our centre in Bangalore. The Investigational New Drug (IND) application was filed with the DGCI for approval to initiate clinical trials in India within 34 months of initiation of the discovery programme. Subsequent to the approval of the IND, we have completed the Phase I Single Ascending Dose study in India within two months.”

GKM-001 is a novel molecule for the treatment of type II diabetes. It is the first glucokinase modulator discovered and developed in India and has potential to be both first or best in class. The success in discovering GKM-001 is attributed to the science-driven efforts in Advinus laboratories and ‘breaking the conventional mold’ for selection of a drug candidate. Advinus has ‘composition of matter’ patent on the molecule for all major markets globally. Glucokinase as a class of target is considered to be novel as currently there is no product in the market or in late clinical trials. The strategy for early clinical development revolved around assessing safety (particularly hypoglycaemia) and early assessment of therapeutic activity (glucose lowering and other biomarkers) in type II diabetics. The Phase I data, in both healthy and type II diabetics, shows excellent safety and tolerability over a 40-fold dose range and desirable pharmacokinetic properties consistent with ‘once a day’ dosing. The next wave of clinical studies planned continues on this strategy of early testing in type II diabetics.

Right behind the lead candidate GKM-001, Advinus has a rich pipeline of back up compounds on the same target. These include several structurally different compounds with diverse potency, unique pharmacology and tissue selectivity. Having discovered the molecule with early indication of wide safety margins, desired efficacy and pharmacokinetic profiles, the company now seeks to out-licence GKM-001 and its discovery portfolio.

Kasim A. Mookhtiar, , Debnath Bhuniya, Siddhartha De, Anita Chugh, Jayasagar
Gundu, Venkata Palle, Dhananjay Umrani, Nimish Vachharajani, Vikram
Ramanathan and Rashmi H. Barbhaiya
Advinus Therapeutics Ltd, Hinjewadi, Pune – 411057, and Peenya Industrial Area,
Bangalore – 560058, India
REFERENCES

patent

wo 2008104994

wo 2008 149382

wo 2009047798
WO2008104994A2* 25 Feb 2008 4 Sep 2008 Advinus Therapeutics Private L 2,2,2-tri-substituted acetamide derivatives as glucokinase activators, their process and pharmaceutical application

///////GKM 001, pipeline, Diabetes, Advinus, type II diabetes, glucokinase modulator, Rashmi Barbhaiya

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Angiogenesis Inhibitors [9.5]

Writer and Curator: Larry H Bernstein, MD, FCAP

This article has the following structure:

9.5.1 Motesanib (AMG 706)

9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

9.5.1 Motesanib (AMG 706)

by DR ANTHONY MELVIN CRASTO Ph.D

http://newdrugapprovals.org/2015/05/15/motesanib-amg-706/

Motesanib (AMG 706) is an experimental drug candidate originally developed by Amgen[1] but is now being investigated by theTakeda Pharmaceutical Company. It is an orally administered small molecule belonging to angiokinase inhibitor class which acts as an antagonist of VEGF receptorsplatelet-derived growth factor receptors, and stem cell factor receptors.[2] It is used as the phosphate salt motesanib diphosphate.

Motesanib, also known as AMG-706, is an orally administered multikinase inhibitor that selectively targets VEGF receptors, platelet-derived growth factor receptors, and Kit receptors.

N-(3,3-Dimethylindolin-6-yl){2-[(4-pyridylmethyl)amino](3-pyridyl)}carboxamide

motesanib-amg-706-a10608

motesanib-amg-706-a10608

http://www.adooq.com/media/catalog/product/cache/1/image/9df78eab33525d08d6e5fb8d27136e95/m/o/motesanib-amg-706-a10608.gif

http://www.chemblink.com/products/453562-69-1.htm

9.5.2 Drugs that block cancer blood vessel growth (anti angiogenics)

http://www.cancerresearchuk.org/about-cancer/cancers-in-general/treatment/biological/types/drugs-that-block-cancer-blood-vessel-growth

When it has reached 1 to 2mm across, a tumor needs to grow its own blood vessels in order to continue to get bigger. Some cancer cells make a protein called vascular endothelial growth factor (VEGF). The VEGF protein attaches to receptors on cells that line the walls of blood vessels within the tumour.

Drugs that block blood vessel growth factor

Some drugs block vascular endothelial growth factor (VEGF) from attaching to the receptors on the cells that line the blood vessels. This stops the blood vessels from growing.

A drug that blocks VEGF is bevacizumab (Avastin). It is also a monoclonal antibody.

Drugs that block signalling within the cell

Some drugs stop the VEGF receptors from sending growth signals into the blood vessel cells. These treatments are also called cancer growth blockers or tyrosine kinase inhibitors (TKIs).

Sunitinib (Sutent) is a type of TKI that blocks the growth signals inside blood vessel cells. It is used to treat kidney cancer and a rare type of stomach cancer called gastrointestinal stromal tumour (GIST).

Drugs that affect signals between cells

Some drugs act on the chemicals that cells use to signal to each other to grow. This can block the formation of blood vessels. Drugs that works in this way include thalidomide and lenalidomide (Revlimid).

Each drug has different side effects. You can look up the name of your drug in our cancer drug section to find out about the side effects you may have.

To find trials using anti angiogenesis treatment go to our clinical trials database and type ‘angiogenesis’ into the search box.

http://www.cancer.gov/about-cancer/treatment/types/immunotherapy/angiogenesis-inhibitors-fact-sheet

Tumors can cause their blood supply to form by giving off chemical signals that stimulate angiogenesis. Tumors can also stimulate nearby normal cells to produce angiogenesis signaling molecules. The resulting new blood vessels “feed” growing tumors with oxygen and nutrients, allowing the cancer cells to invade nearby tissue, to move throughout the body, and to form colonies of cancer cells, called metastases. Because tumors cannot grow beyond a certain size or spread without a blood supply, scientists are trying to find ways to block tumor angiogenesis.

Angiogenesis requires the binding of signaling molecules, such as vascular endothelial growth factor (VEGF), to receptors on the surface of normal endothelial cells. When VEGF and other endothelial growth factors bind to their receptors on endothelial cells, signals within these cells are initiated that promote the growth and survival of new blood vessels.

Angiogenesis inhibitors interfere with various steps in this process. For example, bevacizumab (Avastin®) is a monoclonal antibody that specifically recognizes and binds to VEGF (1). When VEGF is attached to bevacizumab, it is unable to activate the VEGF receptor. Other angiogenesis inhibitors, including sorafenib and sunitinib, bind to receptors on the surface of endothelial cells or to other proteins in the downstream signaling pathways, blocking their activities (2).

The U.S. Food and Drug Administration (FDA) has approved bevacizumab to be used alone forglioblastoma that has not improved with other treatments and to be used in combination with other drugs to treat metastatic colorectal cancer, some non-small cell lung cancers, and metastatic renal cell cancer. Bevacizumab was the first angiogenesis inhibitor that was shown to slow tumor growth and, more important, to extend the lives of patients with some cancers.

The FDA has approved other drugs that have antiangiogenic activity, including sorafenib (Nexavar®), sunitinib(Sutent®), pazopanib (Votrient®), and everolimus (Afinitor®). Sorafenib is approved for hepatocellular carcinoma and kidney cancer, sunitinib and everolimus for both kidney cancer and neuroendocrine tumors, and pazopanib for kidney cancer.

Angiogenesis inhibitors are unique cancer-fighting agents because they tend to inhibit the growth of blood vessels rather than tumor cells. In some cancers, angiogenesis inhibitors are most effective when combined with additional therapies, especially chemotherapy. It has been hypothesized that these drugs help normalize the blood vessels that supply the tumor, facilitating the delivery of other anticancer agents, but this possibility is still being investigated.

Angiogenesis inhibitor therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period.

Initially, it was thought that angiogenesis inhibitors would have mild side effects, but more recent studies have revealed the potential for complications that reflect the importance of angiogenesis in many normal body processes, such as wound healing, heart and kidney function, fetal development, and reproduction. Side effects of treatment with angiogenesis inhibitors can include problems with bleeding, clots in the arteries (with resultant stroke or heart attack), hypertension, and protein in the urine (35). Gastrointestinal perforation and fistulas also appear to be rare side effects of some angiogenesis inhibitors.

In addition to the angiogenesis inhibitors that have already been approved by the FDA, others that target VEGF or other angiogenesis pathways are currently being tested in clinical trials (research studies involving patients). If these angiogenesis inhibitors prove to be both safe and effective in treating human cancer, they may be approved by the FDA and made available for widespread use.

In addition, phase I and II clinical trials are testing the possibility of combining angiogenesis inhibitor therapy with other treatments that target blood vessels, such as tumor-vascular disrupting agents, which damage existing tumor blood vessels (6).

9.5.3 Recent Advances in Anti-Angiogenic Therapy of Cancer

Rajeev S. Samant and Lalita A. Shevde
Oncotarget. 2011 Mar; 2(3): 122–134.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260813/

More than forty anti-angiogenic drugs are being tested in clinical trials all over the world. This review discusses agents that have approved by the FDA and are currently in use for treating patients either as single-agents or in combination with other chemotherapeutic agents.

Tumor angiogenesis is generation of a network of blood vessels within the cancerous growth. This process can occur two ways: The more accepted model involves the release of signaling molecules by the tumor cells; these molecules activate the surrounding tissue to promote growth of new blood vessels. This stimulates vascular endothelial cells to divide rapidly [910]. The other model proposes the generation of new vasculature by vasculogenic mimicry. This model argues that the tumor cells trans-differentiate in endothelial-like cells and create structures from inside of the tumor tapping into a nearby blood vessel [4].

Escape of the tumor cell from the confines of the primary tumor to distant body parts is the pre-requisite for hematogenous metastasis. This escape route is provided by the tumor vasculature. Thus, it was envisioned that inhibition of angiogenesis will also lead to inhibition of metastasis. This phenomenon was demonstrated by very elegant mouse model studies using angiostatin [1112]. Angiostatin was also demonstrated to be secreted by some primary tumors leading to restricted growth of the metastasis leading to “dormancy” of the metastasis. Mice deficient in angiogenesis (Id1 & Id3 deficient) showed significantly less tumor take rates [13]. Independent studies showed absence of metastasis in angiogenesis deficient mice [1415]. Defective angiogenesis was attributed to impaired VEGF-dependent recruitment of precursor endothelial cells from the bone marrow to the newly developing tumor vasculature [16].

Metastasis of malignant tumors to regional lymph nodes is one of the early signs of cancer spread in patients, and it occurs at least as frequently as hematogenous metastasis [17]. Particularly, in cancers, such as breast cancer, lymphatic metastasis is a predominant route for tumor spread. The contribution of lymphatic system to the tumor growth is an area that is relatively less studied. However, lymphatic vessels are speculated to contribute to tumor growth and metastasis in a variety of ways. The VEGF, FGF2 and PDGF produced by vascular endothelial cells are proposed to be involved in the activation of lymphatic endothelial cells, which in turn produce matrix metalloproteases and urokinase plasminogen activator (uPA) that can promote malignant tumor growth. Thus, there exists a synergistic crosstalk between the tumor and the lymphatic vessels and blood vessels.

Angiogenesis is a complex and intricately regulated process. Like all other regulated biological phenomena, angiogenesis has activators or pro-angiogenic factors and inhibitors or anti-angiogenic factors [9].

The Activators

Tumor cells activate signaling pathways that promote uncontrolled proliferation and survival. These include the PI3K/AKT/mTOR pathway, Hedgehog pathway and, Wnt pathway [1824] that produce pro-angiogenic signaling intermediates [2526]. Among the several reported activators of angiogenesis present in cells two proteins appear to be the most important for sustaining tumor growth: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). VEGF and bFGF are secreted by the tumor into the surrounding tissue. They bind to their cognate receptors on endothelial cells. This activates a signaling cascade that transmits a nuclear signal prompting target genes to activate endothelial cell growth. Activated endothelial cells also produce matrix metalloproteinases (MMPs). These MMPs break down the extracellular matrix and allow the migration of endothelial cells. The division and migration of the endothelial cells leads to formation of new blood vessels [2728].

The Inhibitors

If angiogenesis is so critical for the tumor growth, then agents that inhibit angiogenesis would have great therapeutic value. With the discovery of endostatin, the concept of anti-angiogenic therapy was launched and popularized by Dr. Folkman [29]. Angiogenesis inhibitors have been discovered from a variety of sources. Some are naturally present in the human body e.g. specific fragments of structural proteins such as collagen or plasminogen (angiostatin, endostatin, tumstatin) [30]. Others are natural products in green tea, soy beans, fungi, mushrooms, tree bark, shark tissues, snake venom etc. [31]. A plethora of synthetic compounds are also characterized to have anti-angiogenic properties [32].

ANTI-ANGIOGENIC TREATMENT OF CANCER

Since angiogenesis is an event critical to primary tumor growth as well as metastasis, anti-angiogenic treatment of tumors is a highly promising therapeutic avenue [33]. Thus, for over last couple of decades, there has been a robust activity aimed towards the discovery of angiogenesis inhibitors [3435]. More than forty anti-angiogenic drugs are being tested in human cancer patients in clinical trials all over the world. From the several anti-angiogenic agents reported, we have focused this review on discussing those agents that have received FDA approval in the United States and are currently in use for treating patients either as a single-agent or in combination with other chemotherapeutic agents (Figure ​(Figure1).1). Based on functionality, the anti-angiogenic drugs can be sub-divided into three main groups:

angiogenesis inhibitors oncotarget-02-122-g001

angiogenesis inhibitors oncotarget-02-122-g001

Figure 1

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260813/bin/oncotarget-02-122-g001.jpg

Targets of FDA-approved angiogenesis inhibitors: Angiogenesis inhibitors impact both, the tumor as well as the endothelial cells resulting in the disruption of the effects of the microenvironment in promoting tumor growth and angiogenesis

Drugs that inhibit growth of endothelial cells

e.g. Endostatin and combretastatin A4, cause apoptosis of the endothelial cells [36]. Thalidomide is also a potent inhibitor of endothelial cell growth [37].

Drugs that block angiogenesis signaling

e.g. anti-VEGF antibodies (Avastin, FDA approved for colorectal cancer), Interferon-alpha (inhibits the production of bFGF and VEGF) [36].

Drugs that block extracellular matrix breakdown

e.g. inhibitors of MMPs [38].

ANTI-ANGIOGENIC THERAPIES THAT HAVE RECEIVED USA-FDA APPROVAL

Conventional chemotherapy is usually a systemic therapy that tries to capture a narrow therapeutic window offered by rapid proliferation of tumor cells compared to the normal cells. Chemotherapy has significant side effects such as hair loss, diarrhea, mouth ulcer, infection, and low blood counts. Anti-angiogenic therapy has several advantages over chemotherapy as it is mostly not directed towards directly killing cells but stopping the blood vessel formation, an event that is rare in tissues other than growing tumor. Hence it is well tolerated by the patients and has fewer side effects [29]. There are currently seven approved anti-cancer therapies in two primary categories:

  1. Monoclonal antibodies directed against specific pro-angiogenic growth factors and/or their receptors
  2. Small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors.

Besides these, inhibitors of mTOR (mammalian target of rapamycin), proteasome inhibitors and thalidomide have also been reported to indirectly inhibit angiogenesis through mechanisms that are not completely understood.

MONOCLONAL ANTIBODY THERAPIES

Four monoclonal antibody therapies are approved to treat several tumor types:

Bevacizumab (Avastin®)

The first FDA approved angiogenesis inhibitor, Avastin is a humanized monoclonal antibody that binds biologically active forms of vascular endothelial growth factor (VEGF) and prevents its interaction with VEGF receptors (VEGFR-1 and VEGFR-2), thereby inhibiting endothelial cell proliferation and angiogenesis. Bevacizumab has been tested in phase I studies in combination with chemotherapy with a good safety profile [39]. This treatment is approved for metastatic colorectal cancer and non-small cell lung cancer [4043]. Bevacizumab has also evolved as a first line of treatment in combination with paclitaxel in breast cancer patients by virtue of its ability to double median progression-free survival (PFS) [44]. In combination with chemoendocrine therapy (including capecitabine and vinorelbine, and letrozole) bevacizumab treatment significantly decreased the percentage of viable circulating endothelial cells and prevented the chemotherapy-induced mobilization of circulating progenitors [45]. In combination with irinotecan, bevacizumab significantly increased PFS in glioma patients [4647]. VEGF has emerged as a compelling therapeutic target for leukemias. Inhibition of angiogenesis in hematological malignancies interdicts the angiogenesis within the bone marrow ecosystem comprised of multiple cell types, including fibroblasts, endothelial progenitor cells, endothelial cells, dendritic cells and, malignant cells, blocking the availability of nutrients to cancer cells and disrupting crosstalk between the various cell types to curtail the malignant phenotype [48].

Cetuximab (Erbitux®)

This is a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), preventing ligand binding and activation of the receptor resulting in internalization and degradation of the receptor culminating in inhibition of cell proliferation and angiogenesis. Cetuximab downregulated VEGF expression in a dose-dependent manner in a human colorectal carcinoma (CRC) cell line and in human CRC mouse xenografts [49]. The xenografts also showed a significant reduction in blood vessel counts following several rounds of cetuximab treatment [49], indicating that the tumor-promoting effects of EGFR overexpression may be mediated through VEGF stimulation and tumor angiogenesis. This treatment is approved for metastatic CRC and head and neck cancer [50] in patients who are refractory to irinotecan-based chemotherapy. In combination with irinotecan (an inhibitor of topoisomerase I), cetuximab is the first monoclonal antibody that has been approved by the FDA as second-line treatment for metastatic colorectal cancer [5152]. In Phase I and Phase III trials [5354] cetuximab significantly improved the effects of radiotherapy in patients with unresectable (cannot be removed by surgery) squamous cell carcinoma of the head and neck (SCCHN). Cetuximab has also been shown to sensitize cells to radiation and chemotherapy, potentially through blocking EGFR nuclear import and the associated activation of DNA protein kinase enzymes necessary for repairing radiation- and chemotherapy-induced DNA damage [55]. Compared to radiation alone, cetuximab plus radiation therapy can nearly double the median survival in patients with a certain kind of head and neck cancer that has not spread to other parts of the body [54] making cetuximab the only drug achieving interesting response rate in second line treatment of advanced SCCHN [56]. Cetuximab was also found to be tolerated well in combination with cisplatin, or carboplatin, and fluorouracil [5758].

Panitumumab (Vectibix™)

It is a fully humanized anti-EGFR monoclonal antibody that binds specifically to the human EGFR. Panitumumab is a recombinant human monoclonal antibody [59]; therefore, the risk of an infusion reaction is minimized. Vectibix® is indicated as a single agent for the treatment of EGFR-expressing, metastatic colorectal carcinoma with disease progression on or following fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens [6062]. The effectiveness of Vectibix® as a single agent for the treatment of EGFR-expressing, metastatic CRC is based on progression-free survival [6364]. Panitumumab is used in patients who are not responding to regimens containing fluorouracil, oxaliplatin, and irinotecan [60]. Patients often receive panitumumab after receiving bevacizumab or cetuximab. Panitumumab can be given with FOLFOX (oxaliplatin, leucovorin, and fluorouracil) or FOLFIRI (irinotecan, leucovorin, and fluorouracil) regimens, or as a single agent. Currently no data are available that demonstrate an improvement in disease-related symptoms or increased survival with Vectibix® in colon cancer [65]. This drug is also being tested for aerodigestive track and head and neck cancer [6667].

Trastuzumab (Herceptin®)

Is a humanized monoclonal antibody that binds the extracellular domain of HER-2, which is overexpressed in 25-30% of invasive breast cancer tumors [68]. HER2-positive breast cancer is highly aggressive disease with high recurrence rate, poorer prognosis with decreased survival compared with HER2-negative breast cancer [69]. Herceptin® is designed to target and block the function of HER2 protein overexpression. This is the first humanized antibody is approved for Breast cancer [70]. Herceptin® is approved by the FDA to treat HER2 positive breast cancer that has metastasized after treatment with other anticancer drugs [71]. It is also approved to be used with other drugs to treat HER2-positive breast cancer that has spread to the lymph nodes to be used after surgery. The FDA first approved Herceptin in September 1998 [7173]. In November 2006, the FDA approved Herceptin as part of a treatment regimen containing doxorubicin, cyclophosphamide and paclitaxel, for the adjuvant treatment of patients with HER2-positive, node-positive breast cancer (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/default.htm). In January 2008, the FDA approved Herceptin as a single agent for the adjuvant treatment of HER2-overexpressing node-negative (ER/PR-negative or with one high-risk feature) or node-positive breast cancer, following multi-modality anthracycline-based therapy (http://biopharminternational.findpharma.com/biopharm/News/FDA-Approves-Expanded-Adjuvant-Indications-for-Her/ArticleStandard/Article/detail/518867). Trastuzumab is also being studied in the treatment of other types of cancers such as pancreatic [74], endometrial [75], lung [76], cervical [77] and ovarian cancer [78]

SMALL MOLECULE TYROSINE KINASE INHIBITORS (TKIs)

Protein tyrosine kinases have emerged as crucial targets for therapeutic intervention in cancer especially because they play an important role in the modulation of growth factor signaling. As per ClinicalTrials.gov (www.clinicaltrials.gov), there are 43 ongoing studies on tyrosine kinase inhibitors in angiogenesis. Since discussing all of them is beyond the scope of this article, we have focused our discussion on the three TKIs that are currently approved as anti-cancer therapies:

Erlotinib (Tarceva®)

Erlotinib hydrochloride (originally coded as OSI-774) is an orally available, potent, reversible, and selective inhibitor of the EGFR (ErbB1) tyrosine kinase activity. Erlotinib hydrochloride has been approved by FDA for treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen [7980]. Interesting recent studies have demonstrated that since Erlotinib and Bevacizumab act on two different pathways critical to tumor growth and dissemination, administering these drugs concomitantly may confer additional clinical benefits to cancer patients with advanced disease. This combination therapy may prove to be a viable second-line alternative to chemotherapy in patients with NSCLC [81]. Also, for patients with locally advanced, unresectable or metastatic pancreatic carcinoma, Erlotinib has received FDA approval for the treatment in combination with gemcitabine [8283]. Erlotinib is also being studied in the treatment of other types of cancers. For example combination of Erlotinib with Bevacizumab has been evaluated in metastatic breast cancer [84], hepatocellular carcinoma [85] and in metastatic renal cancer [86] as phase II trials. Outcomes for prostate, cervical and colorectal cancers treated with Erlotinib are cautiously optimistic [8789].

Sorafenib (Nexavar®)

Sorafenib is an orally active inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-β, and Raf-1 tyrosine kinase activity [90]. It has received the approval of FDA for the treatment of patients with unresectable hepatocellular carcinoma [91] and advanced renal cell carcinoma [92]. However, not all advanced hepatocellular carcinoma patients were able to tolerate sorafenib and some patients experienced tumor progression [91]. Sorafenib has shown improvements in PFS in patients with renal cell carcinoma [93]. It is one of the aggressively studied drugs. According to the NCI clinical trials search results, there are about 168 active clinical trials involving sorafenib in a variety of cancers.

Sunitinib (Sutent®)

Sunitinib targets activity of multiple tyrosine kinases such as VEGFR-1, VEGFR-2, VEGFR-3, PDGFR- β, and RET [94]. It is approved by FDA as Sunitinib malate for treating advanced (metastatic) renal cell carcinoma [95]. It is also approved by FDA for gastrointestinal stromal tumor (GIST) in patients whose disease has progressed or who are unable to tolerate treatment with imatinib (Gleevec), the current treatment for GIST patients [9596]. Sunitinib has shown early evidence of anti-tumor activity in Phase II trials in US, European and Asian patients with locally advanced, unresectable and metastatic hepatocellular carcinoma. A Phase III trial of sunitinib in hepatocellular carcinoma is ongoing [97]. According to the NCI clinical trials search results, Sunitinib is currently evaluated in about 150 active clinical trials. It is evaluated for ovarian [98], breast [99] and non small cell lung cancer [100] among others [101].

Inhibitors of mTOR

mTOR plays a part in the PI3 kinase/AKT pathway involved in tumor cell proliferation and angiogenesis [102]. Rapamycin and related mTOR inhibitors inhibit endothelial cell VEGF expression, as well as VEGF-induced endothelial cell proliferation [103]. Inhibitors of mTOR are an important class of anti-angiogenic agents. These include: deforolimus, everolimus, rapamycin (sirolimus), and temsirolimus [104105]. Temsirolimus (Toricel™) is a small molecule inhibitor of mTOR, approved for treating advanced renal cell carcinoma [106]. It is a type of rapamycin analog and a type of serine/threonine kinase inhibitor, it is also called CCI-779. In pre-clinical models combination therapy for treating breast cancer using anti-estrogen, ERA-923, and temsirolimus has been successfully tested [107]. It is found to be highly effective against human melanoma when tested in combination with cisplatin and DTIC (in independent studies) in a SCID mouse xenotranplantation model [108109]. There are over 41 active studies of Temsirolimus for a variety of solid tumors [110]. mTOR inhibition has also been strongly advocated in as a putative cancer therapeutic strategy for urologic malignancies [111]. In a pilot study (6 patients) with imatinib-resistant CML, rapamycin induced major and minor leukocyte responses, with an observed decrease in the mRNA levels of VEGFA in circulating leukaemic cells [112]. Combination treatments for breast cancer with aromatase inhibitor [113] and letrozol [114] are also being evaluated. Rapamycin treatment brought partial responses (>50% reduction in the absolute number of blood blasts) and stable disease in adult refractory/relapsed AML [115]. In a recent report, Deforolimus was studied in a Phase 2 trial in pretreated patients with various hematological malignancies, including ALL, AML, CLL, CML, MDS, agnogenic myeloid metaplasia, mantle cell lymphoma and T-cell leukemia/lymphoma [116]. Overall, 40% of deforolimus-treated patients experienced hematological improvement or stable disease.

OTHER ANGIOGENIC AGENTS

Bortezomib (Velcade®)

Is a proteasome inhibitor that disrupts signaling of cancer cells, leading to cell death and tumor regression. It is the first compound in its class to be used in clinical practice. It has indirect anti-angiogenic properties [117]. While its exact mechanism is not understood, it induces the pro-apoptotic BH3-only family member NOXA in a p53 independent fashion triggering of a caspase cascade culminating in apoptosis in melanoma and myeloma cells [118]. It is FDA-approved for the treatment of myeloma that has relapsed after two prior treatments (or where resistance has developed following the last treatment). It was also found to induce high quality responses as third line salvage therapy with acceptable toxicity in a significant proportion of homogeneously pre-treated myeloma patients with progressive disease after autologous transplantation and thalidomide. [119]. In a Phase 3 trial involving 669 myeloma patients treated with at least one prior therapy, bortezomib increased median, improved overall survival, and increased response rate, compared with high-dose dexamethasone [120]. In combination with doxorubicin and gemcitabine, bortezomib was also found to be effective in heavily pretreated, advanced Cutaneous T cell Lymphomas (CTCL) [121]. Bortezomib was also reported to be active as a single agent for patients with relapsed/refractory CTCL and Peripheral T Cell Lymphoma (PTCL) with skin involvement [122]. On the contrary, the use of bortezomib was discouraged after a phase II study revealed that found in combination with dexamethasone, bortezomib is not active in heavily pre-treated patients with relapsed Hodgkin’s lymphoma [123124].

Thalidomide (Thalomid®)

Possesses immunomodulatory, anti-inflammatory, and anti-angiogenic properties, although the precise mechanisms of action are not fully understood. Thalidomide was the first angiogenesis inhibitor to demonstrate clinical efficacy in multiple myeloma [37125]. Specifically in myeloma, thalidomide down-regulated VEGF secretion from bone marrow endothelial cells obtained from patients with active disease. In a landmark Phase 2 clinical trial, 169 previously treated patients with refractory myeloma received thalidomide monotherapy [126]. Partial response, was achieved in 30% of patients, and 14% achieved a complete or nearly complete remission. The survival rate at 2 years was 48%. These results led to many subsequent clinical studies of thalidomide in myeloma, leading ultimately to FDA approval of the drug in 2006, for the treatment of newly diagnosed multiple myeloma, in combination with dexamethasone. In the pivotal Phase 3 trial, the response rate in patients receiving thalidomide plus dexamethasone was 63% compared to 41% with dexamethasone alone [127]. Long-term outcome measures, including time-to-progression (TTP) and PFS, were recently reported for a 470 patient randomized, placebo-controlled Phase 3 clinical trial of a similar protocol in newly diagnosed multiple myeloma, with comparable overall response rates [128]. Significant increases resulted in both median TTP and median PFS for the thalidomide plus dexamethasone group versus dexamethasone alone.

Thalidomide was found to be moderately tolerated and minimally effective in patients with histologically proven advanced hepatocellular carcinoma [129]. Thalidomide provided no survival benefit for patients with multiple, large, or midbrain metastases when combined with WBRT (whole-brain radiation therapy) [130]. On the contrary, thalidomide did not significantly add to the efficacy of the fludarabine, carboplatin, and topotecan (FCT) regimen in poor prognosis AML patients [131] and was also ineffective in improving prognosis or decreasing plasma VEGF levels in patients with persistent or recurrent leiomyosarcoma of the uterus [132].

METRONOMIC THERAPY

While conventional anti-angiogenic therapy is based on Maximum Tolerated Doses (MTD), the cells involved in angiogenesis may regenerate during the three- to four-week interval between cycles of the chemotherapy. Taking advantage of the fact that endothelial cells are about 10–100 times more susceptible to chemotherapeutic agents than cancer cells, therapy based on daily, oral, low-dose chemotherapeutic drugs was designed. Metronomic chemotherapy refers to the close, rhythmic administration of low doses of cytotoxic drugs, with minimal or no drug-free breaks, over prolonged periods. Metronomic therapy appears promising mainly due to the fact that its anti-angiogenic and anti-tumorigenic effects are accompanied by low toxicity, limited side effects, no need for hospitalization and allowing for feasible combinations with selective inhibitors of angiogenesis. There are several foreseeable advantages and opportunities for metronomic chemotherapy: activity against the parenchymal and stromal components, pro-apoptotic activity, reduction of the likelihood of emergence of acquired resistance, feasibility of long term administration and acceptable systemic side effects [133]. In a pilot phase II study conducted by Correale et al [134] to investigate the toxicity and activity of the novel metronomic regimen of weekly cisplatin and oral etoposide in high-risk patients with NSCLC, the objective response rate was 45.2%, disease control was 58.1%, meantime to progression and survival were 9 and 13 months, respectively. Pharmacokinetic analysis showed that this regimen allowed a greater median monthly area under the curve of the drugs than conventional schedules. In a Phase I trial of metronomic dosing of docetaxel and thalidomide, of the 26 patients with advanced tumors enrolled, prolonged freedom from disease progression was observed in 44.4% of the evaluable patients [135].

Circulating endothelial progenitor cells (EPCs) also participate in tumor angiogenesis. In a study comparing the effects of metronomic chemotherapy over conventional dose-dense chemotherapy, it was found that the numbers of circulating EPCs and the plasma levels of VEGF increased sharply, doubling pre-therapeutic levels at day 21 after conventional chemotherapy, whereas under low-dose metronomic chemotherapy, the numbers of circulating EPCs decreased significantly and VEGF plasma concentrations remained unchanged. These observations provide evidence that conventional dose-dense chemotherapy leads to rebound EPC mobilization even when given with adjuvant intention, while low-dose metronomic scheduling of cytotoxic substances such as trofosfamide may sharply reduce EPC release into the circulation. [136].

Combined bevacizumab and metronomic oral cyclophosphamide was also discovered to be a safe and effective regimen for heavily pre-treated ovarian cancer patients [137]. Treatment with metronomic capecitabine and cyclophosphamide in combination with bevacizumab was shown to be effective in advanced breast cancer and additionally was minimally toxic [138]. Metronomic treatment with carboplatin and vincristine associated with fluvastatin and thalidomide significantly increased survival of pediatric brain stem tumor patients. Tumor volume showed a significant reduction accompanied by increased quality of life [139]. Thus, given the fact that the most evident effect of selective anti-angiogenic agents (i.e. bevacizumab) is the significant prolonging of the duration of response obtainable by chemotherapy alone, with minimal possible side effects of cytotoxic agents given in association metronomic chemotherapy should be considered both as novel up-front or maintenance treatment in patients with biologically poorly aggressive advanced cancer diseases [140].

Overall, metronomic chemotherapy was able to induce tumor stabilization and prolong the duration of clinical benefit, without much associated toxicity. Emerging evidence suggests that metronomic chemotherapy could also activate the host immune system and potentially induce tumor dormancy [141143].

CONCLUSIONS AND FUTURE PERSPECTIVES

While angiogenesis as a hallmark of tumor development and metastasis is now a validated target for cancer treatment, the overall benefits of anti-angiogenic drugs from the perspective of impacting survival have left much to desire, endorsing a need for developing more effective therapeutic regimens e.g., combining anti-angiogenic drugs with established chemotherapeutic drugs [144145]. There are now several agents that target the tumor vasculature through different pathways, either by inhibiting formation of the tumor neovasculature or by directly targeting the mature tumor vessels. The main body of evolving evidence suggests that their effects are compounded by their synergistic use with conventional chemotherapy rather than individual agents. Anti-angiogenic drugs such as bevacizumab can bring about a transient functional normalization of the tumor vasculature. This can have an additive effect when co-administered with chemo/radiotherapy. But long term inhibition of angiogenesis reduces tumor uptake of co-administered chemotherapeutic agents. This underscores the need for discovering new targets for anti-angiogenic therapy in order to effectively prohibit angiogenesis and circumvent mechanisms that contribute to resistance mechanisms that emerge with long term use of anti-angiogenic therapies. It also warrants a need to define reliable surrogate indicators of effectiveness of the anti-angiogenic therapy as well as dependable markers for identifying the patients who are most likely to benefit from the combination of anti-angiogenic therapy and conventional chemotherapy.

Several new frontiers are emerging. New advances in understanding endothelial cells, which constitute the tumor vasculature, towards developing antiangiogenic strategies are one of the important ones [146147]. Novel cellular targets such as integrins and microRNAs and novel treatment options such as possible use of pharmaconutrients to modulate angiogenic pathways need careful testing and evaluation [148151]. Finally, the administration of these drugs in a metronomic schedule is likely to improve the overall response to anti-angiogenic drugs making it feasible to administer them with conventionally toxic chemotherapeutic drugs, thus increasing the armamentarium of drug combinations that can be employed for treatment.

9.5.4 Angiogenesis inhibitors in cancer therapy: mechanistic perspective on classification and treatment rationales

El-Kenawi AE1, El-Remessy AB.
Br J Pharmacol. 2013 Oct; 170(4):712-29.
http://dx.doi.org:/10.1111/bph.12344

Angiogenesis, a process of new blood vessel formation, is a prerequisite for tumor growth to supply the proliferating tumor with oxygen and nutrients. The angiogenic process may contribute to tumour progression, invasion and metastasis, and is generally accepted as an indicator of tumor prognosis. Therefore, targeting tumor angiogenesis has become of high clinical relevance. The current review aimed to highlight mechanistic details of anti-angiogenic therapies and how they relate to classification and treatment rationales. Angiogenesis inhibitors are classified into either direct inhibitors that target endothelial cells in the growing vasculature or indirect inhibitors that prevent the expression or block the activity of angiogenesis inducers. The latter class extends to include targeted therapy against oncogenes, conventional chemotherapeutic agents and drugs targeting other cells of the tumor micro-environment. Angiogenesis inhibitors may be used as either monotherapy or in combination with other anticancer drugs. In this context, many preclinical and clinical studies revealed higher therapeutic effectiveness of the combined treatments compared with individual treatments. The proper understanding of synergistic treatment modalities of angiogenesis inhibitors as well as their wide range of cellular targets could provide effective tools for future therapies of many types of cancer.

Two major processes of blood vessel formation are implicated in the development of vascular system: vasculogenesis and angiogenesis. Vasculogenesis prevails in the embryo and refers to the formation ofde novo blood vessels by in situ differentiation of the mesoderm-derived angioblasts and endothelial precursors. Angiogenesis is the formation of new capillaries from pre-existing vessels and circulating endothelial precursors (Polverini, 2002; Chung et al., 2010; Ribatti and Djonov, 2012). Angiogenesis is a tightly controlled dynamic process that can occur physiologically in those tissues that undergo active remodeling in response to stress and hypoxia (Carmeliet, 2003; Folkman, 2007). However, it can be aberrantly activated during many pathological conditions such as cancer, diabetic retinopathy as well as numerous ischemic, inflammatory, infectious and immune disorders (Carmeliet, 2003; Ali and El-Remessy, 2009; Willis et al., 2011). Although the concept of proposing angiogenesis inhibitors as anticancer drugs received considerable skepticism when first presented by Dr. Folkman in the early 1970s (Folkman, 1971), active research in the field and subsequent clinical trials eventually resulted in US Food and Drug Administration (FDA) approval of bevacizumab for colorectal cancer in 2004 (Cohen et al., 2007). Since then, several angiogenic inhibitors have been identified. This review will provide an overview of the key mechanisms involved in tumor angiogenesis, classification of angiogenesis inhibitors as well as treatment rationales from the mechanistic point of view.

Sustained angiogenesis as a hallmark of cancer

Proliferating tumours tend to activate an angiogenic phenotype to fulfil their increased demand of oxygen and nutrients (Hanahan and Folkman, 1996; Carmeliet, 2005). Additionally, paracrine release of anti-apoptotic factors from activated endothelial cells in the newly formed vasculature supplies tumour cells with a survival privilege (Folkman, 2003). Consequently, in order to progress, tumors tend to activate an event called ‘angiogenic switch’ by shifting the balance of endogenous angiogenesis inducers and inhibitors towards a pro-angiogenic outcome. As a result, dormant lesion progresses into outgrowing vascularized tumor and eventually into a malignant phenotype (Hanahan and Folkman, 1996; Baeriswyl and Christofori, 2009). Hypoxia drives such imbalance through up-regulation of the transcription factor hypoxia inducible factor-1α (HIF-1α), which in turn increases the expression of many angiogenesis inducers as well as suppresses the expression of endogenous angiogenesis inhibitors (Pugh and Ratcliffe, 2003). In spite of that, accumulating evidence indicates that angiogenic cascade can be also driven by alternative HIF-1-independent pathways (Mizukami et al., 2007; Arany et al., 2008; Lee, 2013).

As summarized in Table 1, the angiogenesis inducers are a wide range of mediators that include many growth factors, a plethora of cytokines, bioactive lipids, matrix-degrading enzymes and a number of small molecules (Folkman, 1995; Folkman, 2003; Lopez-Lopez et al., 2004; Bouis et al., 2006; El-Remessy et al., 2007; Bid et al., 2011; MacLauchlan et al., 2011; Murakami, 2011; Fagiani and Christofori, 2013; Qin et al., 2013). Pro-angiogenic growth factors mostly activate a series of surface receptors in a series of paracrine and autocrine loops with the VEGF-A signaling representing the critical rate-limiting step, physiologically and pathologically. VEGF-A (traditionally known as VEGF) is the most potent VEGF isoform that acts mainly on VEGF receptor 2 (VEGFR2) to mediate vascular permeability, endothelial proliferation, migration and survival (Takahashi and Shibuya, 2005; Bouis et al., 2006). In spite of the well-established master roles of VEGF signaling in literature, those processes are probably accomplished through a highly regulated interplay between VEGF and other pro-angiogenic factors. In this context, basic fibroblast growth factor (bFGF) activation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation (Murakami et al., 2011). Similarly, sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid that can directly contribute to tumor angiogenesis (reviewed in Sabbadini, 2011), is needed for VEGF-induced blood vessel formation, indicating the cooperation between S1P and VEGF in tumor angiogenesis (Visentin et al., 2006). As a net result, the pro-angiogenic interplay of those ligands and others dominates over the activities of two dozen endogenous angiogenesis inhibitors that can be either matrix-derived inhibitors or non–matrix-derived inhibitors (Nyberg et al., 2005).

Table 1. Pro-angiogenic mediators implicated in tumor angiogenesis

Category Examples References
Growth factors VEGFs Bouis et al., 2006
FGFs Ibid
TGFs Ibid
PDGFs Ibid
Insulin-like growth factors Lopez-Lopez et al., 2004; Bid et al., 2011
ANGs Fagiani and Christofori, 2013
Cytokines IL-8 Strieter et al., 2004
CSF-1 Lin et al., 2006
Bioactive lipids PGE2 Wang and Dubois, 2010
S1P Murakami, 2011
Matrix-degrading enzymes MMPs Bourboulia and Stetler-Stevenson, 2010
Heparanases Vlodavsky and Friedmann, 2001
Small mediators NO MacLauchlan et al., 2011
Peroxynitrite El-Remessy et al., 2007
Serotonin Qin et al., 2013
Histamine Qin et al., 2013

The multistep angiogenic process starts with vasodilation and increased permeability of existing vessels in response to tumor cell-secreted VEGF. This is accompanied by loosening of pericytes covering mediated by angiopoietin-2 (ANG2), a ligand of tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2) receptor (Bergers and Benjamin, 2003; Jain, 2003; Fagiani and Christofori, 2013). Meanwhile, many secreted matrix-degrading enzymes, such as MMPs and heparanases, function in concert to dissolve the basement membrane and to remodel the extracellular matrix (ECM) as well as to liberate more pro-angiogenic growth factors (bFGF and VEGF) from matrix heparan sulfate proteoglycans (HSPGs) respectively (Houck et al., 1992; Whitelock et al., 1996; Vlodavsky and Friedmann, 2001; Tang et al., 2005; van Hinsbergh and Koolwijk, 2008). The overall chemotactic angiogenic stimuli guide endothelial cells to migrate, to align into tube-like structures and to eventually form new blood vessels. However, such blood vessels are characterized by being disorganized, chaotic, hemorrhagic and poorly functioning (Bergers and Benjamin, 2003).

The angiogenic phenotype in tumor micro-environment can further be sustained and extravagated by the recruitment of other types of stromal cells. Stromal cells such as fibroblasts, mesenchymal stem cells and various bone marrow-derived myeloid cells including macrophages, TIE2-expressing monocytes, neutrophils and mast cells contribute to tumor angiogenesis through their production of growth factors, cytokines and proteases (Murdoch et al., 2008; Joyce and Pollard, 2009; Cirri and Chiarugi, 2011). For example, in response to cancer cell-derived TGF-β, PDGF or bFGF, fibroblasts are transformed to an activated phenotype with a higher proliferative activity and myofibroblastic characteristics (Kalluri and Zeisberg, 2006; Cirri and Chiarugi, 2011). Such carcinoma-associated fibroblasts (CAFs) were shown to promote angiogenesis and metastasis by secreting large amounts of MMP-2 and MMP-9 as well as by expressing many cytokines and chemokines that resulted in immune cell infiltration (Gerber et al., 2009; Giannoni et al., 2010). Furthermore, it has been shown that PDGF-C produced by CAFs is able to elicit VEGF production from tumor cells, thereby sustaining the angiogenic shift (Crawford et al., 2009). Similarly, tumor-associated macrophages (TAMs), one of the bone marrow myeloid-derived cells, are induced to develop into polarized type II (alternatively activated or M2 macrophages), upon exposure to tumor hypoxia and tumor cell-derived cytokines (Leek et al., 2002; Rogers and Holen, 2011). M2 macrophages tend to produce many pro-angiogenic growth factors, cytokines and matrix-degrading enzymes such as VEGF, PDGF, bFGF, TNF-α, COX-2, MMP-9, MMP-7 and MMP-12 (Lewis and Pollard, 2006).

From another perspective, angiogenesis may be dispensable for progression of some malignancies. For example, some tumours may co-opt pre-existent vessels as an alternative way to obtain blood supply. Vessel co-option was first described in the brain, one of the most densely vascularized organs, in which tumours may develop in earlier stages without the activation of angiogenic response (Holashet al., 1999; Leenders et al., 2002; Bergers and Benjamin, 2003; Hillen and Griffioen, 2007). In another example, hypovascularized tumors such as pancreatic ductal adenocarcinoma may involve certain adaptation to flourish in the absence of prominent angiogenesis (Bergers and Hanahan, 2008). Obviously, in both cases, tumors may be intrinsically indifferent to angiogenesis inhibitors. However, in most other cases, therapy directed towards the vasculature of solid tumors is being considered as an important direction in cancer treatment.

Classification of angiogenesis inhibitors

Growth of newly formed vessels in tumor micro-environment can be inhibited directly by targeting endothelial cells in the growing vasculature or indirectly by targeting either tumor cells or the other tumor-associated stromal cells. Therefore, angiogenesis inhibitors can be classified into direct and indirect inhibitors (Kerbel and Folkman, 2002; Folkman, 2007).

Direct endogenous inhibitors of angiogenesis

Direct endogenous inhibitors of angiogenesis, such as angiostatin, endostatin, arrestin, canstatin, tumstatin and others, are fragments released on proteolysis of distinct ECM molecules. Endogenous inhibitors prevent vascular endothelial cells from proliferating, migrating in response to a spectrum of angiogenesis inducers, including VEGF, bFGF, IL-8 and PDGF (Kerbel and Folkman, 2002; Abdollahi et al., 2004; Mundel and Kalluri, 2007; Ribatti, 2009). This direct anti-angiogenic effect may be mediated by interference with endothelial integrins along with several intracellular signaling pathways (Mundel and Kalluri, 2007). For example, the ability of tumstatin-derived active peptide to inhibit angiogenesis and tumour growth is associated with the expression of the adhesion receptor, αvβ3 integrin, on tumor endothelial cells (Eikesdal et al., 2008). Through binding αvβ3 integrin, full tumstatin was found to inhibit endothelial cell activation of focal adhesion kinase, PI3K, Akt, mammalian target of rapamycin (mTOR) and others (Maeshima et al., 2002). Direct targeting of those signaling pathways by endogenous inhibitors was thought to be the least likely to induce acquired drug resistance because they target endothelial cells with assumed genetic stability rather than unstable mutating tumour cells (Kerbel and Folkman, 2002). However, endostatin has not yet led to any documented benefit to patients in randomized phase III trials, or even modest activity in phase II trials (Ellis and Hicklin, 2008).

Indirect inhibitors of angiogenesis

Indirect inhibitors of angiogenesis classically prevent the expression or block the activity of pro-angiogenic proteins (Folkman, 2007). For example, Iressa, an EGF receptor (EGFR) TK inhibitor (TKI), blocks tumour expression of many pro-angiogenic factors; bevacizumab, a monoclonal antibody, neutralizes VEGF after its secretion from tumour cells whereas sunitinib, a multiple receptor TKI, blocks the endothelial cell receptors (VEGFR1, VEGFR2 and VEGFR3), preventing their response to the secreted VEGF (Folkman, 2007; Roskoski, 2007). In addition, this class extends to include conventional chemotherapeutic agents, targeted therapy against oncogenes and drugs targeting other cells of the tumor micro-environment (Kerbel et al., 2000; Ferrara and Kerbel, 2005).

Conventional chemotherapeutic agents

Conventional chemotherapeutic agents have been shown to have anti-angiogenic properties in addition to the ability to induce direct cancer cell death. Such chemotherapeutic agents can affect the endothelial cell population in the tumour bed during treatment cycles because they have significantly higher proliferation rates than resting endothelium outside a tumor, making them more susceptible to cytotoxic effect (Kerbel et al., 2000; Folkman, 2003). However, the cyclic treatment rationale of cytotoxic drugs allows the potential damage to the tumour vasculature to be repaired during the long breaks. Thus, continuous low doses of chemotherapeutic agents were suggested as a way to reduce side effects and drug resistance (Drevs et al., 2004). This modality is termed metronomic therapy, and clinically, it refers to the daily administration of 5–10% of the phase II-recommended dose of the chemotherapeutic agent (Penel et al., 2012). The extended use of such low doses of cytotoxic agents elicits an anti-angiogenic activity through induction of endothelial cell apoptosis and decreasing the level of circulating endothelial precursors (Hamano et al., 2004; Shahrzad et al., 2008). In clinical investigations, metronomic dosing of cyclophosphamide and others showed promising efficacy in patients with advanced, multiple metastasized and/or multiple pretreated solid tumours (Lord et al., 2007; Fontana et al., 2010; Nelius et al., 2011; Gebbia et al., 2012; Briasoulis et al., 2013; Navid et al., 2013).

VEGF-targeted therapy

VEGF-targeted therapy includes neutralizing antibodies to VEGF (e.g. bevacizumab) or VEGFRs (e.g. ramucirumab), soluble VEGFR/VEGFR hybrids (e.g. VEGF-Trap) and TKIs with selectivity for VEGFRs (e.g. sunitinib and sorafenib; Baka et al., 2006; Ellis and Hicklin, 2008; Hsu and Wakelee, 2009). Bevacizumab, a humanized monoclonal antibody against all isoforms of VEGF-A, has been approved for the treatment of colorectal, lung, glioblastoma and renal cell carcinoma (Hsu and Wakelee, 2009). Many other clinical trials with promising efficacy were also conducted in other cancers such as head and neck cancer, hepatocellular carcinoma, ovarian cancer, metastatic melanoma and gastric cancer (Argiris et al., 2011; 2013; Burger et al., 2011; Ohtsu et al., 2011; Fang et al., 2012; Minor, 2012; Schuster et al., 2012; Van Cutsem et al., 2012). However, for metastatic breast cancer, bevacizumab had been initially granted an accelerated FDA approval, which was later withdrawn due to lack of improvement evidence in disease-related symptoms or overall survival (Burstein, 2011; Montero et al., 2012). Similarly, clinical trials showed that the addition of bevacizumab to the treatment regimens of advanced pancreatic cancer did not extend overall survival (Chiu and Yau, 2012). The neutralization of VEGF-A can also be achieved by soluble receptor construct (VEGF-Trap) that monomerically ‘traps’ the different isoforms of VEGF-A, in addition to VEGF-B and placental growth factor (Rudge et al., 2007). VEGF-Trap showed clinical benefit in a phase III trial of oxaliplatin pretreated metastatic patients with colorectal cancer, and is currently being investigated in a prostate cancer phase III trial (Gaya and Tse, 2012). TKIs are small molecules with different chemical structures that have the ability to interact physically with the highly conserved kinase domain shared by different VEGFRs as well as PDGF receptors (PDGFRs), FGF receptors (FGFRs), EGFR, Raf kinases and c-Kit (a receptor of the pluripotent cell growth factor, stem cell factor). Such interaction directly inhibits tyrosine phosphorylation and the subsequent many downstream pro-angiogenic signaling networks (Baka et al., 2006; Ivy et al., 2009). Those multi-targeted TKIs demonstrated efficacy against various solid malignancies in different clinical trials, some of which have lead eventually to FDA approval of sunitinib and sorafenib. Sunitinib, known to inhibit several receptor TKs (RTKs) including VEGFR1–3, PDGFR-α, PDGFR-β, c-Kit, colony-stimulating factor-1 receptor (CSF-1R) and Flt-3, was approved for the treatment of renal cell carcinoma and gastrointestinal stromal cell tumours. Sorafenib that acts also by inhibiting VEGFR1–3 and PDGFR-β in addition to the serine–threonine kinases Raf-1, B-Raf, was approved for hepatocellular carcinoma in addition to renal cell carcinoma (Llovet et al., 2008; Ivy et al., 2009; Huang et al., 2010).

FGF-targeted therapies

FGF-targeted therapies were recently reconsidered as promising anti-angiogenic and anti-tumor agents after a long period of little attention for drug development, partly due to redundancy (Bono et al., 2013). The FGFR superfamily with its 18 ligands and four receptors has been involved in endothelial cell migration, proliferation and differentiation (Presta et al., 2005). Therapeutic targeting of FGF/FGFR signalling was accomplished by either monoclonal antibodies that inhibit FGFs binding, small molecules that inhibit FGFR TK activity or allosteric modulators that bind the extracellular FGFR domain. Monoclonal antibodies against bFGF displayed potent anti-tumor and anti-angiogenic effects in different preclinical cancer models, which warrant further clinical evaluation (Zhao et al., 2010; Wang et al., 2012). Pan inhibitors of the FGFR TKs such as AZD4547 (blocks the activity of FGFR1–3) and ponatinib (blocks all the FGFR isoforms) elicited potent anti-tumor activities in preclinical investigations so they are currently being evaluated in clinical trials. Those inhibitors displayed the greatest potency in FGFR-driven cancer models, which may be attributed to the interference with the oncogenic functions of either amplified or constitutively active FGFR (Dutt et al., 2011; Zhao et al., 2011; Gavine et al., 2012; Gozgit et al., 2012). Accordingly, further studies are needed to evaluate the relative contribution of angiogenic versus oncogenic inhibitory mechanisms towards the overall anti-tumor activity. The allosteric antagonist of the FGFR, SSR128129E, showed a strong anti-angiogenic activity in addition to tumour growth and metastasis inhibitory effects in animal models of arthritis and cancer respectively. Because allosteric modulators leave a residual level of baseline signalling, they have the ability to fine-tune target biological responses. As a result, allosteric multi-FGFR inhibitors may have an improved benefit/risk ratio that is not attainable with the other TKIs (Bonoet al., 2013; Herbert et al., 2013). However, preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional pro-angiogenic RTKs that may also reduce the risk of drug resistance (Hilberg et al., 2008). For example, dual inhibition of VEGFRs and FGFRs using brivanib produced enduring tumour stasis and angiogenic blockade following the failure of VEGF-targeted therapies (Allen et al., 2011). Furthermore, triple inhibition of FGFRs, VEGFRs and PDGFR(s) using dovitinib (TKI258) or nintedanib (BIBF 1120) displayed broad-spectrum anti-tumour activities in several tumour xenograft models as well as promising data in clinical trials. Combined inhibition of FGFR/VEGFR/PDGFR targets not only tumour cells, but also endothelial cells, pericytes and smooth muscle cells, resulting in an effective inhibition of tumour growth, angiogenesis and metastasis even in advanced tumour stages (Hilberg et al., 2008; Ledermann et al., 2011; Taeger et al., 2011; Chenet al., 2012; Angevin et al., 2013).

Oncogene-targeted therapy

Oncogenes, genes that cause the transformation of normal cells into cancerous cells, are thought to up-regulate many pro-angiogenic proteins. Therefore, anticancer drugs that were developed for their capacity to block an oncogene also have an indirect anti-angiogenic activity (Kerbel et al., 2000; Bergers and Benjamin, 2003; Folkman, 2003). For example, dasatinib and other inhibitors of sarcoma (Src), an aberrantly activated non-RTK associated with many human malignancies, showed potent anti-angiogenic effects through the down-regulation of VEGF and IL-8 (Summy et al., 2005; Han et al., 2006; Haura et al., 2010). Another example is to target the oncogenic Ras using farnesyl transferase (FT) inhibitors, which inhibit post-translational farnesylation of Ras that governs the latter’s activity (Awada et al., 2002). FT inhibitors were found to inhibit tumor VEGF expression and block FTase-dependent Ras activation, which is critically involved in VEGF-elicited angiogenic signal transduction and angiogenesis (Han et al., 2005; Izbicka et al., 2005; Kim et al., 2010). In addition to classical oncogenes inhibition, interference with other tumor-deregulated signaling pathways would offer another approach in targeting angiogenesis. For example, inhibitors of heat shock protein 90 (HSP90), a chaperone molecule known to protect oncoproteins from misfolding and degradation in the protein-rich intracellular environment, were found to prevent VEGF production and to disrupt multiple pro-angiogenic signalling pathways in numerous cancer cells. They were also shown to inhibit tumour growth and vascularity of different human tumor xenografts (Sanderson et al., 2006; Langet al., 2007; Eccles et al., 2008; Trepel et al., 2010; Moser et al., 2012). Proteasome inhibitors, such as bortezomib (PS-341) or MG-132, were also shown to reduce tumour growth and vascularity of squamous cell carcinoma and pancreatic cancer xenograft probably through inhibition of NF–κB-dependent release of pro-angiogenic gene products, VEGF and IL-8 (Sunwoo et al., 2001; Nawrocki et al., 2002; Matsuo et al., 2009). Similarly, inhibition of B-cell lymphoma 2 (Bcl-2), a prosurvival protein that regulates apoptosis by preventing the mitochondrial release of pro-apoptogenic factors, was shown to prevent NF-κB-mediated release of the pro-angiogenic factors IL-8 and CXC chemokine ligand 1 (CXCL1) as well as VEGF in tumor-associated endothelial cells and pancreatic cell lines respectively (Karl et al., 2005; Wang et al., 2008). Moreover, (−)-gossypol, a natural BH3 mimetic that inhibits BH3 domain of Bcl-2 as well as related prosurvival proteins (Bcl-xL and Mcl-1), was shown to remarkably decrease microvessel density in human prostate tumour PC-3 xenografts through decrease of VEGF and IL-8 release as well as blocking multiple steps in VEGF-activated biological events (Karaca et al., 2008; Pang et al., 2011).

Matrix degrading and remodelling-targeted therapy

Matrix degrading and remodelling are activated by tumors to modify local micro-environment, which in turn promote their angiogenic potential (Bergers et al., 2000; Vlodavsky and Friedmann, 2001). Up-regulation of expression and activity of several endogenous MMPs including MMP-2, MMP-9 as well as MMP-3 and MMP-7 have been identified in invasive tumors (for a review, see Bourboulia and Stetler-Stevenson, 2010). Consequently, inhibitors of MMPs were extensively pursued as a therapeutic strategy for treating cancer. Unfortunately, MMPs intervention strategies had met with limited clinical success because of severe toxicities and associated metastasis-promoting effect (Coussens et al., 2002; Devy et al., 2009). Furthermore, the paradoxical roles of tissue inhibitors of metalloproteinases (TIMPs) may contribute to such failure depending on the net balance of TIMPs and MMPs in tumour stroma (Jiang et al., 2002). As a result, efforts were directed at therapies exploiting endogenous MMP inhibitors, TIMPs or monoclonal antibodies against individual MMPs (Martens et al., 2007; Jarvelainen et al., 2009). For example, DX-2400, a highly selective fully human MMP-14 inhibitory antibody, was found to block pro-MMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions (Devy et al., 2009). Alternatively, in order to reduce toxicity and enhance drug delivery, polymeric nanoparticulate delivery systems could be used to target individual components of ECM. For example, targeted delivery of antisense inhibitors of laminin-8, a vascular basement membrane component, by conjugation to the natural drug carrier β-poly(L-malic acid) significantly reduced tumour microvessel density and increased animal survival in an experimental model of glioblastoma (Fujita et al., 2006). Similarly, a nano delivery system that incorporate peptides against proteolytically processed type IV collagen significantly accumulated in tumors and blocked angiogenesis in experimental models (Mueller et al., 2009). However, the highly sulfated oligosaccharides, Heparan (HS) mimetics highly sulfated oligosaccharides, were shown to have a heparanase-inhibiting effect sequestering, in turn, many heparan sulfate proteoglycan (HSPG)-binding factors (Johnstone et al., 2010; Dredge et al., 2011). In preclinical studies, HS mimetics have effectively targeted multiple HSPG-dependent functions and have resulted in decreased in vivo tumor growth, tumor invasion, tumor metastasis and angiogenesis (Johnstone et al., 2010; Dredge et al., 2011; Zhou et al., 2011). Clinically, the heparanase inhibitor PI-88 showed preliminary efficacy as an adjunct therapy for post-operative hepatocellular carcinoma (Liu et al., 2009).

Tumour-associated stromal cell-targeted therapy

Tumour-associated stromal cells crosstalk is a perquisite for the formation of a tumour vasculature, an essential step for tumour progression (Lorusso and Ruegg, 2008). Interference with those crosstalk circuits through intervention of cellular adhesion (highlighted in next paragraph) or tumor-induced recruitment of different stromal cells may be considered as an indirect way of anti-angiogenic therapy (Ferrara and Kerbel, 2005). The latter can be supported by studies in which inhibition of macrophage infiltration, for example, by either genetic ablation of the macrophage CSF-1 or liposomal clodronate-induced macrophage depletion, was shown to delay the angiogenic switch and malignant transition (Giraudo et al., 2004; Lin et al., 2006). Furthermore, CSF-1R kinase inhibitors were found to reduce tumor-associated vascularity in two different tumor mouse models (Kubota et al., 2009; Mantheyet al., 2009). In addition, clodronate and other related bisphosphonates, originally used to treat skeletal complications in patients with tumour-induced osteolysis, were shown to exert potent anti-tumour and anti-angiogenic effects in many other studies (Fournier et al., 2002; Santini et al., 2003; Stathopoulos et al., 2008). Zoledronic acid, a third-generation bisphosphonate, was also found to reduce a number of tumour-associated macrophages and shift their phenotype from M2 to M1, resulting in a reduction in TAM-associated production of VEGF in murine models of spontaneous mammary carcinogenesis and mesothelioma (Coscia et al., 2010; Veltman et al., 2010). Clinically, repeated low-dose therapy with zoledronic acid, which maintains active drug plasma concentration, was able to induce an early remarkable and long-lasting decrease of VEGF levels in patients with cancer (Santini et al., 2007). In another example, inhibition of mobilization of neutrophils, from bone marrow and their infiltration into tumour, using neutralizing anti–prokineticin-2, an antibody against a secreted protein known also as BV8, was shown to impair the initial angiogenic switch in a multistage pancreatic beta cell tumorigenesis model (Shojaei et al., 2008). Furthermore, the neutralizing anti-BV8 was found to prevent myeloid cell-dependent tumour angiogenesis in several xenograft models (Shojaei et al., 2007). Cancer-associated fibroblasts (CAF) can also be targeted with thapsigargin analogue coupled with peptides specific for fibroblast activation protein (FAP), a CAF membrane-bound protease whose catalytic site has access to the peritumoural fluid of the tumor micro-environment. This extracellular activation results in the death of CAFs as well as pericytes and endothelial cells within milieu of different human tumor xenografts (Brennen et al., 2012).

Cell adhesion molecules (CAMs)-targeted therapy

CAMs are cell surface proteins known to be involved in binding with other counter-receptors on adjacent cells or surrounding ECM macromolecules (Aplin et al., 1998). Many CAMs, such as αv-integrins, E-selectin, N-cadherin and VE-cadherin, have been implicated in tumour angiogenesis (Bischoff, 1997; Tei et al., 2002; Nakashima et al., 2003; Weis and Cheresh, 2011). For example, αv-integrins are expressed on surface of endothelial cells and can determine whether cells can adhere to and survive in a particular micro-environment. A number of matrix-derived fragments have the ability to act as endogenous angiogenesis inhibitors through binding to integrins on endothelial cells, disrupting physical connections and suppressing signalling events associated with cell survival, migration and proliferation (Nyberg et al., 2005). Consequently, integrins antagonism using peptidomimetics (e.g. cilengitide), monoclonal antibodies (e.g. volociximab) or oral small-molecule compounds have been investigated in a wide range of malignancies (Huveneers et al., 2007). Cilengitide is a cyclized pentapeptide peptidomimetic designed to compete for the arginine-glycine-aspartic acid (RGD) peptide sequence, thereby blocking the ligation of the αvβ3 and αvβ5 integrins to matrix proteins (Hariharan et al., 2007). Cilengitide is mainly under clinical development for glioblastoma; however, clinical trials of other malignancies such as head and neck cancer as well as lung cancer were also initiated (Reardon and Cheresh, 2011; Vermorken et al., 2012; Manegold et al., 2013). Alternatively, cyclic peptides containing RGD motif could guide nanoparticulate delivery system, which incorporates anti-angiogenic cytotoxic agents such as doxorubicin, paclitaxel or combretastatin A4, to accumulate specifically in tumor vasculature with no overt systemic toxicity (Murphy et al., 2008; Ruoslahti et al., 2010; Wang et al., 2011). Volociximab, a chimeric humanized monoclonal antibody that selectively inhibits the αvβ1 integrin interaction with fibronectin, has been evaluated also in clinical trials for solid tumours such as renal cell carcinoma, recurrent ovarian cancer, advanced non–small-cell lung cancer and metastatic pancreatic cancer (Figlin et al., 2006; Evans et al., 2007; Jarvelainen et al., 2009; Vergote et al., 2009; Besse et al., 2013). Cadherins constitute a superfamily of molecules that mediate calcium-dependent cell–cell adhesions. The intracellular domains of cadherins directly bind to β-catenin and link with cytoskeletal components, providing the molecular basis for stable cell–cell adhesion (Zhang et al., 2010). Targeting cadherin signalling may also represent another way for tumor angiogenesis intervention. For example, ADH-1, a cyclic pentapeptide containing the cell adhesion recognition site (His-Ala-Val) required for N-cadherin adhesion, was shown to possess anti-angiogenic and anti-tumour activity (Blaschuk et al., 2005; Blaschuk, 2012). Similarly, monoclonal antibody directed against specific region of VE-cadherin was able to inhibit tumor angiogenesis and growth with no side effects on normal vasculature (Corada et al., 2002; May et al., 2005).

Inflammatory angiogenesis-targeted therapy

Targeting inflammatory angiogenesis, responsible for a substantial part of tumour vascularization initiated by infiltrating leukocytes, may be considered as another indirect anti-angiogenic strategy (Albini et al., 2005). Moreover, as mentioned before, tumour-infiltrating leukocytes contribute into malignant progression through production of many pro-inflammatory cytokines, chemokines and enzymes that can mostly induce angiogenic cascade (Balkwill et al., 2005). Such vital roles have been supported by the early observation that nonsteroidal anti-inflammatory drugs can inhibit tumour angiogenesis and, in turn, tumor progression (Albini et al., 2005). For example, ibuprofen was found to decrease tumor growth and metastatic potential in mice models through modulation of angiogenesis (Yao et al., 2005). Moreover, selective inhibitors of COX-2, an inducible enzyme that catalyses the production of prostanoids from arachidonic acid, were also shown to inhibit angiogenesis (Tsujii et al., 1998; Wei et al., 2004). The anti-angiogenic effect of COX-2 inhibitors may be contributed, in part, by decreasing the COX-2 metabolic product PGE2, the predominant PG in solid tumors known to stimulate cancer cells to produce pro-angiogenic factors such as VEGF and bFGF as well as many other factors belonging to CXC chemokines family (Strieter et al., 2004; Wang et al., 2006; Wang and Dubois, 2010). Members of the CXC chemokine family are heparin-binding proteins that possess disparate regulative roles in angiogenesis. For example, the ELR+ CXC chemokines, characterized by highly conserved three amino acid motifs (Glu-Leu-Arg; ‘ELR’ motif), are potent promoters of angiogenesis, whereas the IFN-inducible (ELR−) CXC chemokines are inhibitors of angiogenesis (Strieter et al., 2004). The use of repertaxin, originally designed to target the ELR+ CXC chemokine receptors CXCR1 and CXCR2 on neutrophils to prevent their migration to sites of inflammation, was found to inhibit tumor angiogenesis, thereby suppressing tumour progression in a genetic model of pancreatic ductal adenocarcinoma (Ijichi et al., 2011). It would be beneficial to explore other small-molecule CXCR2 antagonists that have already been developed for the treatment of inflammatory diseases in different preclinical models of cancer, especially inflammation-associated cancers (refer to Chapman et al., 2009 for a list of newly developed CXCR2 antagonists used in the treatment of inflammatory diseases of the lung).

Mechanisms of enhanced therapeutic efficacy

  • Dual targeting of tumor vasculature
  • Targeting different cell types of tumor micro-environment
  • Normalization of tumor vasculature
  • Chemosensitization of tumor cells
  • Interference with the repair of cytotoxic drug-induced damage and resistance mechanisms

Consequences of anti-angiogenic therapy with other anticancer therapy

  • Contrary to initial expectations, treatment with angiogenesis inhibitors was associated with unexpected toxicities. The toxicity profiles of those inhibitors reflect the systemic disturbance of growth factor signalling pathways that mediate their anti-angiogenic activity (Elice and Rodeghiero, 20102012). In this context, disturbance of the tight endothelial cell-platelet interaction that maintains vascular integrity results in bleeding complications, gastrointestinal perforations, and disturbed wound and ulcer healing (Verheul and Pinedo, 2007). In general, the incidence of those adverse effects increases when anti-angiogenic agent is combined with chemotherapy. For example, bleeding complications have been observed in patients with colorectal cancer treated with chemotherapy in combination with bevacizumab (Kabbinavar et al., 2003; Giantonio et al., 2006). In non–small-cell lung cancer, some patients treated with bevacizumab in combination with carboplatin and paclitaxel experienced severe or fatal pulmonary haemorrhage (Johnson et al., 2004). Furthermore, a higher incidence of gastrointestinal perforation was observed in patients with colorectal cancer given bevacizumab in combination with chemotherapy compared with chemotherapy alone (Hurwitz et al., 2004). Similarly, thrombotic events have been observed in patients treated with angiogenesis inhibitors, especially when these agents are given in combination with chemotherapy (Verheul and Pinedo, 2007). Treatment of patients with cancer with angiogenesis inhibitors is frequently associated with hypertension, which may require the addition of regular anti-hypertensive agent (Izzedine et al., 2009).

Summary and future directions

  • Angiogenesis is a critical process that occurs pathologically in many malignancies due to changing balance of endogenous angiogenesis inducers and inhibitors, leading to the activation of nearby endothelial cells to form new vasculature. Consequently, angiogenesis can be targeted to restrict initiation, growth and progression of most of angiogenesis-dependent malignancies. Numerous angiogenic inhibitors have been identified, some of which are currently being investigated in clinical trials and some others were even approved for cancer therapies. These angiogenesis inhibitors were classified based on their target into two main classes: direct and indirect inhibitors. Indirect angiogenesis inhibitors can be further subclassified based on their interference mechanisms with the angiogenic cascade. A list of major categories and molecular targets for angiogenesis inhibitors is shown in Table 2.
  • Most angiogenesis inhibitors conferred clinical benefits mainly when combined with other chemotherapeutic/targeted therapies rather than being used as monotherapy. Unfortunately, many anti-angiogenic agents were shown to be associated with overt systemic toxicity as well as resistance emergence and disease recurrence. Drug resistance in anti-angiogenic therapy may result from a plethora of pro-angiogenic factors released by inappropriately functioning host cells in the tumor micro-environment as a compensatory mechanism. Therefore, the strategy of targeting endothelial cells alone may not be enough as explained in the previous texts, requiring the proposal of different rationales in which other cellular compartments of tumor micro-environment are targeted to attain proper anti-angiogenic and anti-tumor response. That highlights the importance of considering tumor micro-environment as a dynamic system, as depicted in Figure 1 in which interference with any of its components may be an approach to interfere with cancer hallmarks, including angiogenesis.

9.5.5 LUCITANIB a VEGFR/FGFR dual kinase inhibitor in Phase 2 trials

Dr.  Anthony Melvin Crasto

source: http://medcheminternational.blogspot.com/2015/01/lucitanib-vegfrfgfr-dual-kinase.html

Lucitanib.png
LUCITANIB
6-[7-[(1-aminocyclopropyl)methoxy]-6-methoxyquinolin-4-yl]oxy-N-methylnaphthalene-1-carboxamide
6-(7-((l-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yloxy)- N-methyl- 1 -naphthamide
1058137-23-7 (E-3810 free base); 1058137-84-0  (E-3810 HCl salt)
E-3810, E-3810 amine, UNII-PP449XA4BH, E3810, Lucitanib [INN], AL3810
Molecular Formula:C26H25N3O4
Molecular Weight:443.4944 g/mol
PATENT SUBMITTED GRANTED
Spiro Substituted Compounds As Angiogenesis Inhibitors [US8163923] 2008-09-18 2012-04-24
A 4-(3-methoxypropoxy)-3-methylpyridinyl derivative of timoprazole that is used in the therapy of STOMACH ULCERS and ZOLLINGER-ELLISON SYNDROME. The drug inhibits H(+)-K(+)-EXCHANGING ATPASE which is found in GASTRIC PARIETAL CELLS.
For in advanced solid tumors.
Lucitanib (E-3810): Lucitanib, also known as E-3810,  is a novel dual inhibitor targeting human vascular endothelial growth factor receptors (VEGFRs) and fibroblast growth factor receptors (FGFRs) with antiangiogenic activity. VEGFR/FGFR dual kinase inhibitor E-3810 inhibits VEGFR-1, -2, -3 and FGFR-1, -2 kinases in the nM range, which may result in the inhibition of tumor angiogenesis and tumor cell proliferation, and the induction of tumor cell death. Both VEGFRs and FGFRs belong to the family of receptor tyrosine kinases that may be upregulated in various tumor cell type
Lucitanib (E-3810) Structure

Overview

Lucitanib is an oral, potent inhibitor of the tyrosine kinase activity of fibroblast growth factor receptors 1 through 3 (FGFR1-3), vascular endothelial growth factor receptors 1 through 3 (VEGFR1-3) and platelet-derived growth factor receptors alpha and beta (PDGFR α-ß). We own exclusive development and commercial rights to lucitanib on a global basis, excluding China. Lucitanib rights to markets outside of the U.S. and Japan have been sublicensed to Les Laboratoires Servier (Servier). We are collaborating with Servier on the global clinical development of lucitanib.

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