Late Development of a Large Arteriovenous Fistula with Aneurysm in an In Situ Vein Bypass Graft.

We report a case of saphenous vein bypass aneurysm and arteriovenous fistula in a 65-year-old man, 20 years after an in situ vein bypass for occlusive disease. He was found to have patent venous branches which kept the bypass open despite distal anastomotic occlusion. The saphenous vein was successfully excised without distal revascularization due to sufficient native arterial flow. This is the first reported case of aneurysmal degeneration of an in situ vein conduit with occluded distal anastomosis and patent venous side branches. Ultrasound surveillance is warranted for all bypass procedures, and early endovascular or open revisions can prevent late complications.

Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Orally Bioavailable and Brain Penetrant Mutant IDH1 Inhibitors.

Mutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma, and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This Letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker 2-HG.

CSF1R-dependent myeloid cells are required for NK­mediated control of metastasis.

Myeloid leukocytes are essentially involved in both tumor progression and control. We show that neo-adjuvant treatment of mice with an inhibitor of CSF1 receptor (CSF1R), a drug that is used to deplete tumor-associated macrophages, unexpectedly promoted metastasis. CSF1R blockade indirectly diminished the number of NK cells due to a paucity of myeloid cells that provide the survival factor IL-15 to NK cells. Reduction of the number of NK cells resulted in increased seeding of metastatic tumor cells to the lungs but did not impact on progression of established metastases. Supplementation of mice treated with CSF1R-inhibitor with IL-15 restored numbers of NK cells and diminished metastasis. Our data suggest that CSF1R blockade should be combined with administration of IL-15 to reduce the risk of metastasis.

Discovery and Evaluation of Clinical Candidate IDH305, a Brain Penetrant Mutant IDH1 Inhibitor.

Inhibition of mutant IDH1 is being evaluated clinically as a promising treatment option for various cancers with hotspot mutation at Arg132. Having identified an allosteric, induced pocket of IDH1R132H, we have explored 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors for in vivo modulation of 2-HG production and potential brain penetration. We report here optimization efforts toward the identification of clinical candidate IDH305 (13), a potent and selective mutant IDH1 inhibitor that has demonstrated brain exposure in rodents. Preclinical characterization of this compound exhibited in vivo correlation of 2-HG reduction and efficacy in a patient-derived IDH1 mutant xenograft tumor model. IDH305 (13) has progressed into human clinical trials for the treatment of cancers with IDH1 mutation.

Structure-Guided Design of EED Binders Allosterically Inhibiting the Epigenetic Polycomb Repressive Complex 2 (PRC2) Methyltransferase.

PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.

The tumor microenvironment underlies acquired resistance to CSF-1R inhibition in gliomas.

Macrophages accumulate with glioblastoma multiforme (GBM) progression and can be targeted via inhibition of colony-stimulating factor-1 receptor (CSF-1R) to regress high-grade tumors in animal models of this cancer. However, whether and how resistance emerges in response to sustained CSF-1R blockade is unknown. We show that although overall survival is significantly prolonged, tumors recur in >50% of mice. Gliomas reestablish sensitivity to CSF-1R inhibition upon transplantation, indicating that resistance is tumor microenvironment-driven. Phosphatidylinositol 3-kinase (PI3K) pathway activity was elevated in recurrent GBM, driven by macrophage-derived insulin-like growth factor-1 (IGF-1) and tumor cell IGF-1 receptor (IGF-1R). Combining IGF-1R or PI3K blockade with CSF-1R inhibition in recurrent tumors significantly prolonged overall survival. Our findings thus reveal a potential therapeutic approach for treating resistance to CSF-1R inhibitors.

CDK9 inhibitors selectively target estrogen receptor-positive breast cancer cells through combined inhibition of MYB and MCL-1 expression.

Our previous studies showed that MYB is required for proliferation of, and confers protection against apoptosis on, estrogen receptor-positive (ER(+ve)) breast cancer cells, which are almost invariably also MYB(+ve). We have also shown that MYB expression in ER(+ve) breast cancer cells is regulated at the level of transcriptional elongation and as such, is suppressed by CDK9i. Here we examined the effects of CDK9i on breast cancer cells and the involvement of MYB in these effects. ER(+ve) breast cancer cell lines including MCF-7 were much more sensitive (> 10 times) to killing by CDK9i than ER(-ve)/MYB(-ve) cells. Moreover, surviving cells showed a block at the G2/M phase of the cell cycle. Importantly, ectopic MYB expression conferred resistance to apoptosis induction, cell killing and G2/M accumulation. Expression of relevant MYB target genes including BCL2 and CCNB1 was suppressed by CDK9 inhibition, and this too was reversed by ectopic MYB expression. Nevertheless, inhibition of BCL2 alone either by MYB knockdown or by ABT-199 treatment was insufficient for significant induction of apoptosis. Further studies implied that suppression of MCL-1, a well-documented target of CDK9 inhibition, was additionally required for apoptosis induction, while maximal levels of apoptosis induced by CDK9i are likely to also involve inhibition of BCL2L1 expression. Taken together these data suggest that MYB regulation of BCL2 underlies the heightened sensitivity of ER(+ve) compared to ER(-ve) breast cancer cells to CDK9 inhibition, and that these compounds represent a potential therapeutic for ER(+ve) breast cancers and possibly other MYB-dependent cancers.

Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism.

CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

Phenotypic and metabolic investigation of a CSF-1R kinase receptor inhibitor (BLZ945) and its pharmacologically active metabolite.

1. 4-[2((1R,2R)-2-Hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide (BLZ945) is a small molecule inhibitor of CSF-1R kinase activity within osteoclasts designed to prevent skeletal related events in metastatic disease. Key metabolites were enzymatically and structurally characterized to understand the metabolic fate of BLZ945 and pharmacological implications. The relative intrinsic clearances for metabolites were derived from in vitro studies using human hepatocytes, microsomes and phenotyped with recombinant P450 enzymes. 2. Formation of a pharmacologically active metabolite (M9) was observed in human hepatocytes. The M9 metabolite is a structural isomer (diastereomer) of BLZ945 and is about 4-fold less potent. This isomer was enzymatically formed via P450 oxidation of the BLZ945 hydroxyl group, followed by aldo-keto reduction to the alcohol (M9). 3. Two reaction phenotyping approaches based on fractional clearances were applied to BLZ945 using hepatocytes and liver microsomes. The fraction metabolized (fm) or contribution ratio was determined for each metabolic reaction type (oxidation, glucuronidation or isomerization) as well as for each metabolite. The results quantitatively illustrate contribution ratios of the involved enzymes and pathways, e.g. the isomerization to metabolite M9 accounted for 24% intrinsic clearance in human hepatocytes. In summary, contribution ratios for the Phase I and Phase II pathways can be determined in hepatocytes.

Rapamycin drives selection against a pathogenic heteroplasmic mitochondrial DNA mutation.

Mitochondrial DNA (mtDNA) mutations cause a variety of mitochondrial disorders for which effective treatments are lacking. Emerging data indicate that selective mitochondrial degradation through autophagy (mitophagy) plays a critical role in mitochondrial quality control. Inhibition of mammalian target of rapamycin (mTOR) kinase activity can activate mitophagy. To test the hypothesis that enhancing mitophagy would drive selection against dysfunctional mitochondria harboring higher levels of mutations, thereby decreasing mutation levels over time, we examined the impact of rapamycin on mutation levels in a human cytoplasmic hybrid (cybrid) cell line expressing a heteroplasmic mtDNA G11778A mutation, the most common cause of Leber's hereditary optic neuropathy. Inhibition of mTORC1/S6 kinase signaling by rapamycin induced colocalization of mitochondria with autophagosomes, and resulted in a striking progressive decrease in levels of the G11778A mutation and partial restoration of ATP levels. Rapamycin-induced upregulation of mitophagy was confirmed by electron microscopic evidence of increased autophagic vacuoles containing mitochondria-like organelles. The decreased mutational burden was not due to rapamycin-induced cell death or mtDNA depletion, as there was no significant difference in cytotoxicity/apoptosis or mtDNA copy number between rapamycin and vehicle-treated cells. These data demonstrate the potential for pharmacological inhibition of mTOR kinase activity to activate mitophagy as a strategy to drive selection against a heteroplasmic mtDNA G11778A mutation and raise the exciting possibility that rapamycin may have therapeutic potential for the treatment of mitochondrial disorders associated with heteroplasmic mtDNA mutations, although further studies are needed to determine if a similar strategy will be effective for other mutations and other cell types.

CSF-1R inhibition alters macrophage polarization and blocks glioma progression.

Glioblastoma multiforme (GBM) comprises several molecular subtypes, including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment, such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model, which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly, TAMs were not depleted in treated mice. Instead, glioma-secreted factors, including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ), facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs, which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM.

2-Aminothiazole based P2Y(1) antagonists as novel antiplatelet agents.

ADP receptors, P2Y1 and P2Y12 have been recognized as potential targets for antithrombotic drugs. A series of P2Y1 antagonists that contain 2-aminothiazoles as urea surrogates were discovered. Extensive SAR of the thiazole ring is described. The most potent compound 7j showed good P2Y1 binding (Ki=12nM), moderate antagonism of platelet aggregation (PA IC50=5.2µM) and acceptable PK in rats.

Liraglutide-induced acute kidney injury.

Published case reports have documented the relationship between exenatide use and acute kidney injury. However, to our knowledge, no cases of this adverse effect with liraglutide, another glucagon-like peptide-1 receptor agonist approved for the treatment of type 2 diabetes mellitus, have been reported. We describe a 53-year-old Caucasian woman who came to a community hospital with severe and progressively worsening gastrointestinal symptoms for several weeks, leading to dehydration and development of acute kidney injury. Laboratory results showed an increase in her serum creatinine concentration to 22.8 mg/dl and blood urea nitrogen to 150 mg/dl; she also had lost 8.9 kg in the previous month. One month earlier, the patient had begun liraglutide 1.8 mg/day subcutaneously for uncontrolled type 2 diabetes. Use of the Naranjo adverse drug reaction probability scale indicated a possible relationship (score of 3) between the patient's development of acute kidney injury and liraglutide. Renal biopsy and laboratory testing were helpful in ruling out other potential causes of renal failure and adverse drug reactions due to other drugs such as ciprofloxacin and quinapril. After the reports of the renal biopsy were obtained, liraglutide was determined to be a likely cause of acute tubular necrosis. The patient was successfully treated with discontinuation of liraglutide, volume repletion, and hemodialysis. To our knowledge, this is the first documented case of liraglutide-induced acute tubular necrosis. Clinicians should be aware of this possible complication and closely follow liraglutide's dosage titration recommendations in the package insert. Patients should also be educated about the need to report unusual or prolonged gastrointestinal symptoms.

Increased melanoma risk in Parkinson disease: a prospective clinicopathological study.

OBJECTIVE: To evaluate the possible association of Parkinson disease (PD) and melanoma in North America. DESIGN, SETTING, AND PATIENTS: Thirty-one centers enrolled patients with idiopathic PD. At visit 1, a neurologist obtained a medical history. At visit 2, a dermatologist recorded melanoma risk factors, performed a whole-body examination, and performed a biopsy of lesions suggestive of melanoma for evaluation by a central dermatopathology laboratory. We compared overall prevalence of melanoma with prevalence calculated from the US Surveillance Epidemiology and End Results (SEER) cancer database and the American Academy of Dermatology skin cancer screening programs. RESULTS: A total of 2106 patients (mean [SD] age, 68.6 [10.6] years; duration of PD, 7.1 [5.7] years) completed the study. Most (84.8%) had received levodopa. Dermatology examinations revealed 346 pigmented lesions; dermatopathological findings confirmed 20 in situ melanomas (0.9%) and 4 invasive melanomas (0.2%). In addition, histories revealed 68 prior melanomas (3.2%). Prevalence (5-year limited duration) of invasive malignant melanoma in the US cohort of patients with PD (n = 1692) was 2.24-fold higher (95% confidence interval, 1.21-4.17) than expected in age- and sex-matched populations in the US SEER database. Age- or sex-adjusted relative risk of any melanoma for US patients was more than 7 times that expected from confirmed cases in American Academy of Dermatology skin cancer screening programs. CONCLUSIONS: Melanoma prevalence appears to be higher in patients with PD than in the general population. Despite difficulties in comparing other databases with this study population, the study supports increased melanoma screening in patients with PD.

Terapia de reemplazo hormonal y riesgo de enfermedades neurodegenerativas / Hormone replacement therapy and risk for neurodegenerative diseases

En los últimos años se han dado controversias importantes acerca de los efectos de la estrogenoterapia sustitutiva y, en términos más generales, la terapia de reemplazo hormonal (TRH) sobre la enfermedad de Alzheimer (EA) y otras enfermedades neurodegenerativas. Esta revisión presenta algunos de los hallazgos y aportes más recientes en estudios de ciencias básicas, ensayos clínicos aleatorizados e investigación epidemiológica relacionada con los efectos neuroprotectores potenciales de la TRH en EA, demencia asociada con VIH y enfermedad de Parkinson (EP). Ha ido ganado más adeptos la convicción acerca de la capacidad de la TRH de reducir el riesgo de EA y mejorar el funcionamiento cognitivo de las mujeres posmenopáusicas, sobre todo cuando se consideran también las limitaciones del Women’s Health Initiative Memory Study de 2002. También se está prestando mayor atención a los efectos sintomáticos y neuroprotectores de la TRH en el tratamiento de la EP, así como al papel de la TRH como estrategia novedosa en la prevención y el tratamiento de la demencia asociada con VIH. Existen limitaciones importantes en la investigación actual, pero también razones convincentes para volver a examinar el modo en que algunas formas de TRH pueden ayudar a preservar las capacidades cognitivas en mujeres posmenopáusicas y evitar las enfermedades neurodegenerativas.

In the past several years, there has been a significant amount of controversy about the effects of estrogen replacement therapy (ERT) and, more generally, hormonereplacement therapy (HRT) on Alzheimer’s (AD) and otherneurodegenerative conditions. This review presents some of the more recent findings and developments in basicscience studies, randomized clinical trials, and epidemiological research regarding the potential neuroprotective effects of HRT in AD, HIV-associated dementia (HAD), and Parkinson’s disease (PD). Increased support iscontinuing to emerge for HRT’s ability to reduce the riskof AD and improve the cognitive functioning ofpostmenopausal women, particularly when consideredalongside the limitations of the 2002 Women’s HealthInitiative Memory Study. Greater attention is also beinggiven to the symptomatic and neuroprotective effects ofHRT in the management of PD, as well as the role of HRTas a novel strategy in the prevention and treatment of HAD. There are important limits to the existing research, but there are also persuasive reasons for reexamininghow some forms of HRT may help preserve cognitiveabilities in post-menopausal women and stave off neurodegenerative diseases.