Janus kinase inhibitors are potential therapeutics for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a poorly treated multifactorial neurodegenerative disease associated with multiple cell types and subcellular organelles. As with other multifactorial diseases, it is likely that drugs will need to target multiple disease processes and cell types to be effective. We review here the role of Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) signalling in ALS, confirm the association of this signalling with fundamental ALS disease processes using the BenevolentAI Knowledge Graph, and demonstrate that inhibitors of this pathway could reduce the ALS pathophysiology in neurons, glia, muscle fibres, and blood cells. Specifically, we suggest that inhibition of the JAK enzymes by approved inhibitors known as Jakinibs could reduce STAT3 activation and modify the progress of this disease. Analysis of the Jakinibs highlights baricitinib as a suitable candidate due to its ability to penetrate the central nervous system and exert beneficial effects on the immune system. Therefore, we recommend that this drug be tested in appropriately designed clinical trials for ALS.

The AI-Assisted Identification and Clinical Efficacy of Baricitinib in the Treatment of COVID-19.

During the current pandemic, the vast majority of COVID-19 patients experienced mild symptoms, but some had a potentially fatal aberrant hyperinflammatory immune reaction characterized by high levels of IL-6 and other cytokines. Modulation of this immune reaction has proven to be the only method of reducing mortality in severe and critical COVID-19. The anti-inflammatory drug baricitinib (Olumiant) has recently been strongly recommended by the WHO for use in COVID-19 patients because it reduces the risk of progressive disease and death. It is a Janus Kinase (JAK) 1/2 inhibitor approved for rheumatoid arthritis which was suggested in early 2020 as a treatment for COVID-19. In this review the AI-assisted identification of baricitinib, its antiviral and anti-inflammatory properties, and efficacy in clinical trials are discussed and compared with those of other immune modulators including glucocorticoids, IL-6 and IL-1 receptor blockers and other JAK inhibitors. Baricitinib inhibits both virus infection and cytokine signalling and is not only important for COVID-19 management but is "non-immunological", and so should remain effective if new SARS-CoV-2 variants escape immune control. The repurposing of baricitinib is an example of how advanced artificial intelligence (AI) can quickly identify new drug candidates that have clinical benefit in previously unsuspected therapeutic areas.

Repurposing Vandetanib plus Everolimus for the Treatment of ACVR1-Mutant Diffuse Intrinsic Pontine Glioma.

Somatic mutations in ACVR1 are found in a quarter of children with diffuse intrinsic pontine glioma (DIPG), but there are no ACVR1 inhibitors licensed for the disease. Using an artificial intelligence-based platform to search for approved compounds for ACVR1-mutant DIPG, the combination of vandetanib and everolimus was identified as a possible therapeutic approach. Vandetanib, an inhibitor of VEGFR/RET/EGFR, was found to target ACVR1 (K d = 150 nmol/L) and reduce DIPG cell viability in vitro but has limited ability to cross the blood-brain barrier. In addition to mTOR, everolimus inhibited ABCG2 (BCRP) and ABCB1 (P-gp) transporters and was synergistic in DIPG cells when combined with vandetanib in vitro. This combination was well tolerated in vivo and significantly extended survival and reduced tumor burden in an orthotopic ACVR1-mutant patient-derived DIPG xenograft model. Four patients with ACVR1-mutant DIPG were treated with vandetanib plus an mTOR inhibitor, informing the dosing and toxicity profile of this combination for future clinical studies. SIGNIFICANCE: Twenty-five percent of patients with the incurable brainstem tumor DIPG harbor somatic activating mutations in ACVR1, but there are no approved drugs targeting the receptor. Using artificial intelligence, we identify and validate, both experimentally and clinically, the novel combination of vandetanib and everolimus in these children based on both signaling and pharmacokinetic synergies.This article is highlighted in the In This Issue feature, p. 275.

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.

Variation in postacute care utilization after complex surgery.

BACKGROUND: Variation in use of postacute care (PAC), including skilled nursing facilities and inpatient rehabilitation, accounts for 73% of regional variation in Medicare spending. Studies of hospital variation in PAC use have typically focused on nonsurgical patients or have been limited to Medicare data. Consequently, there is no nationally representative data on how rates of postoperative discharge to PAC differ between hospitals. The purpose of this study was to explore hospital-level variation in PAC utilization after cardiovascular and abdominal surgery. MATERIALS AND METHODS: We evaluated 3,487,365 patients from the Nationwide Inpatient Sample and 60,666 from the Veterans Affairs health system, who had colorectal surgery, hepatectomy, pancreatectomy, coronary bypass, aortic aneurysm repair, and peripheral vascular bypass from 2008 to 2011. For each hospital, we calculated unadjusted and risk-adjusted observed-to-expected ratios for discharge to PAC facilities (skilled nursing or inpatient rehabilitation). RESULTS: A total of 631,199 (18%) non-veterans and 4744 (8%) veterans were discharged to PAC facilities. For veterans, 32% were ≥70 y old, and 98% were men. For non-veterans, 39% were ≥70, and 60% were men. Hospital rates of discharge to PAC facilities varied from 1% to 36% for veterans hospitals and from 1% to 59% for non-veteran hospitals. Risk-adjusted observed-to-expected ratios ranged from 0.10 to 4.15 for veterans and from 0.11 to 4.3 for non-veteran hospitals. CONCLUSIONS: There is substantial variation in PAC utilization and rates of home discharge after abdominal and cardiovascular surgery. To reduce variation, further research is needed to understand health systems factors that influence PAC utilization.

The novel CXCR4 antagonist, PRX177561, reduces tumor cell proliferation and accelerates cancer stem cell differentiation in glioblastoma preclinical models.

Glioblastoma is the most frequent and the most lethal primary brain tumor among adults. Standard of care is the association of radiotherapy with concomitant or adjuvant temozolomide. However, to date, recurrence is inevitable. The CXCL12/CXCR4 pathway is upregulated in the glioblastoma tumor microenvironment regulating tumor cell proliferation, local invasion, angiogenesis, and the efficacy of radio-chemotherapy. In this study, we evaluated the effects of the novel CXCR4 antagonist, PRX177561, in preclinical models of glioblastoma. CXCR4 expression and PRX177561 effects were assessed on a panel of 12 human glioblastoma cells lines and 5 patient-derived glioblastoma stem cell cultures. Next, the effect of PRX177561 was tested in vivo, using subcutaneous injection of U87MG, U251, and T98G cells as well as orthotopic intrabrain inoculation of luciferase-transfected U87MG cells. Here we found that PRX177561 impairs the proliferation of human glioblastoma cell lines, increases apoptosis, and reduces CXCR4 expression and cell migration in response to stromal cell-derived factor 1alpha in vitro. PRX177561 reduced the expression of stem cell markers and increased that of E-cadherin and glial fibrillary acidic protein in U87MG cells consistent with a reduction in cancer stem cells. In vivo, PRX177561 reduced the weight and increased the time to progression of glioblastoma subcutaneous tumors while increasing disease-free survival and overall survival of mice bearing orthotopic tumors. Our findings suggest that targeting stromal cell-derived factor 1 alpha/CXCR4 axis by PRX177561 might represent a novel therapeutic approach against glioblastoma and support further investigation of this compound in more complex preclinical settings in order to determine its therapeutic potential.

The brain-penetrating CXCR4 antagonist, PRX177561, increases the antitumor effects of bevacizumab and sunitinib in preclinical models of human glioblastoma.

BACKGROUND: Glioblastoma recurrence after treatment with the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. It has been demonstrated that anti-VEGF/VEGFR therapies control the invasive phenotype and that relapse occurs through the increased activity of CXCR4. We therefore hypothesized that combining bevacizumab or sunitinib with the novel CXCR4 antagonist, PRX177561, would have superior antitumor activity. METHODS: The effects of bevacizumab, sunitinib, and PRX177561 were tested alone or in combination in subcutaneous xenografts of U87MG, U251, and T98G cells as well as on intracranial xenografts of luciferase tagged U87MG cells injected in CD1-nu/nu mice. Animals were randomized to receive vehicle, bevacizumab (4 mg/kg iv every 4 days), sunitinib (40 mg/kg po qd), or PRX177561 (50 mg/kg po qd). RESULTS: The in vivo experiments demonstrated that bevacizumab and sunitinib increase the in vivo expression of CXCR4, SDF-1α, and TGFß1. In addition, we demonstrate that the co-administration of the novel brain-penetrating CXCR4 antagonist, PRX177561, with bevacizumab or sunitinib inhibited tumor growth and reduced the inflammation. The combination of PRX177561 with bevacizumab resulted in a synergistic reduction of tumor growth with an increase of disease-free survival (DSF) and overall survival (OS), whereas the combination of PRX177561 with sunitinib showed a mild additive effect. CONCLUSIONS: The CXC4 antagonist PRX177561 may be a valid therapeutic complement to anti-angiogenic therapy, particularly when used in combination with VEGF/VEGFR inhibitors. Therefore, this compound deserves to be considered for future clinical evaluation.

CXCR4 and Glioblastoma.

This article focuses on the possible application of antagonists of the G-protein coupled chemokine receptor, CXCR4, for the treatment of glioblastoma and summarises the evidence for CXCR4 antagonism being a viable therapeutic approach. Particular attention is paid to the role of this receptor in cancer stem cell biology, and the maintenance of CXCR4 expression by the glioblastoma key driver mutations. The expression of the CXCR4 receptor, and of its ligand stromal derived factor 1 (SDF-1, CXCL12), is maintained by intracellular pathways via positive feedback loops, and is associated with the epithelial mesenchymal transition (EMT) and the generation and self-renewal of cancer stem cells. SDF-1 and CXCR4 also play a role in the generation and maintenance of the perivascular stem cell niche which contains these cancer stem cells. The available data suggest that most, if not all, glioblastoma cancer stem cells rely on CXCR4 mediated signalling to maintain their phenotype. SDF-1 and CXCR4 are alsoinvolved in many other aspects of brain tumour biology including resistance to radio- and chemotherapy, the migration of cancer cells through the brain, the generation of the tumour blood supply and the recruitment of vascular progenitor cells. These properties suggest that a CXCR4 antagonist would help in the control of this disease.

Leptin-mediated inhibition of the insulin-stimulated increase in fatty acid uptake in differentiated 3T3-L1 adipocytes.

The effects of insulin and leptin on fatty acid uptake in differentiated (adipocytes) and undifferentiated 3T3-L1 cells were investigated. It was demonstrated that in undifferentiated 3T3-L1 cells, insulin and leptin have no effect on fatty acid uptake. In differentiated 3T3-L1 adipocytes, insulin had a concentration-dependent stimulatory effect on fatty acid uptake, whereas leptin on its own had no effect. Leptin, when coincubated with 10 nmol/L insulin, resulted in a concentration-dependent inhibition of the insulin-stimulated fatty acid uptake in differentiated 3T3-L1 cells. These results indicate that leptin has a direct inhibitory effect on the stimulation of fatty acid uptake by insulin in differentiated murine adipocytes.

Dual presynaptic control by ATP of glutamate release via facilitatory P2X1, P2X2/3, and P2X3 and inhibitory P2Y1, P2Y2, and/or P2Y4 receptors in the rat hippocampus.

ATP is released in a vesicular manner from nerve terminals mainly at higher stimulation frequencies. There is a robust expression of ATP (P2) receptors in the brain, but their role is primarily unknown. We report that ATP analogs biphasically modulate the evoked release of glutamate from purified nerve terminals of the rat hippocampus, the facilitation being mediated by P2X1, P2X2/3, and P2X3 [antagonized by 8-(benzamido)naphthalene-1,3,5-trisulfonate and 2',3'-O-(2,4,6-trinitrophenyl)-ATP] and the inhibition by P2Y1, P2Y2, and/or P2Y4 [antagonized by reactive blue 2 and 2'deoxy-N6-methyladenosine-3',5'-bisphosphate and mimicked by P1-(urinine 5'-),P4-(inosine 5'-) tetraphosphate and 2-methylthio-ADP] receptors. The combination of single-cell PCR analysis of rat hippocampal pyramidal neurons, Western blot analysis of purified presynaptic active zone fraction, and immunocytochemical analysis of hippocampal glutamatergic terminals revealed that the P2 receptors expressed in glutamatergic neurons, located in the active zone and in glutamatergic terminals, were precisely P2X1, P2X2, and P2X3 subunits and P2Y1, P2Y2, and P2Y4 receptors. This provides coincident functional and molecular evidence that P2 receptors are present and act presynaptically as a modulatory system controlling hippocampal glutamate release.

Detection of enterovirus capsid protein VP1 in myocardium from cases of myocarditis or dilated cardiomyopathy by immunohistochemistry: further evidence of enterovirus persistence in myocytes.

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected chi(2)=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.

Adenosine modulates the striatal GABAergic inputs to the globus pallidus via adenosine A2A receptors in rats.

Previous studies have shown presynaptic modulation of adenosine A(2A) receptors for GABAergic synaptic transmission in the globus pallidus (GP). The pallidal A(2A) receptor-mediated modulation is caused by an action on the terminals of striatopallidal medium spiny neurons (MSNs) and/or axon collaterals of GP neurons. Herein, we examined the precise target neurons of the A(2A) receptor-mediated modulation. Activation of A(2A) receptors enhanced striatopallidal GABAergic transmission onto GP neurons, accompanied by a reduction in the paired-pulse facilitation, indicating the presynaptic contribution of A(2A) receptors at terminals of striatopallidal MSNs in the GP. Besides, no A(2A) receptor mRNA was detected in GP neurons by single-cell reverse transcription-polymerase chain reaction analysis, implying no contribution of axon collaterals of GP neurons to the A(2A) receptor regulation. These results demonstrate that the target neurons of adenosinergic modulation via A(2A) receptors in the GP are the striatopallidal MSNs.

Adenosine A3 receptors are located in neurons of the rat hippocampus.

Adenosine is a neuromodulator acting mainly via inhibitory A1 and facilitatory A2A receptors. Whole tissue PCR also identified adenosine A3 receptors in the brain and A3 receptor agonists affect CNS neuronal responses and viability. However, recent reports failed to detect A3 receptor expression in CNS neurons and showed that A3 receptor agonists can bind and activate A1 receptors. We now present evidence for the presence of A3 receptor mRNA in CNS neurons using single cell PCR analysis of laser dissected hippocampal neurons. Western blot analysis showed that A3 receptors are present in rat hippocampal nerve terminal membranes. This indicates that A3 receptors are present in CNS neurons in the hippocampus.

Orexins induce increased excitability and synchronisation of rat sympathetic preganglionic neurones.

The neuropeptides orexin A and B are synthesised by perifornical and lateral hypothalamic (LH) neurones and exert a profound influence on autonomic sympathetic processes. LH neurones project to spinal areas containing sympathetic preganglionic neurones (SPNs) and therefore may directly modulate sympathetic output. In the present study we examined the possibility that orexinergic inputs from the LH influence SPN activity. Orexin-positive neurones in the LH were labelled with pseudorabies virus injected into the liver of parasympathetically denervated animals and orexin fibres were found adjacent to the soma and dendrites of SPNs. Orexin A or B (10-1000 nM) directly and reversibly depolarised SPNs in spinal cord slices. The response to orexin A was significantly reduced in the presence of the orexin receptor 1 (OX1R) antagonist SB334867A at concentrations of 1-10 micro M. Single cell reverse transcriptase-polymerase chain reaction revealed expression of mRNA for both OX1R and OX2R in the majority of orexin-sensitive SPNs. The orexin-induced depolarisation involved activation of pertussis toxin-sensitive G-proteins and closure of a K+ conductance via a protein kinase A (PKA)-dependent pathway that did not require an increase in intracellular Ca2+. Orexins also induced biphasic subthreshold membrane potential oscillations and synchronised activity between pairs of electrically coupled SPNs. Coupling coefficients and estimated junctional conductances between SPNs were not altered indicating synchronisation is due to activation of previously silent coupled neurones rather than modulation of gap junctions. These findings are consistent with a direct excitation and synchronisation of SPNs by orexinergic neurones that in vivo could increase the frequency and coherence of sympathetic nerve discharges and mediate LH effects on sympathetic components of energy homeostasis and cardiovascular control.

Functional and molecular characterization of metabotropic glutamate receptors expressed in rat striatal cholinergic interneurones.

In the present study we have used single-cell RT-PCR in conjunction with electrophysiology to examine the expression and functional properties of metabotropic glutamate receptors (mGluRs) expressed within biochemically identified cholinergic interneurones in the rat striatum. Using single-cell RT-PCR, it was possible to demonstrate the presence of mGluR1, mGluR2, mGluR3, mGluR5 and mGluR7 mRNAs within single cholinergic interneurones. Bath application of the non-selective mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 S,3R-ACPD) or the group-I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) depolarized all cholinergic neurones tested by activation of an inward current at -60 mV. The effects of DHPG were partially inhibited by the mGluR5 selective antagonist 6-methyl-2-(pherazo)-3-pyridinol and by the non-selective group-I antagonist alpha-methyl-4-carboxyphenylglycine but were not mimicked by the group-II and group-III selective mGluR agonists 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and L-2-amino-4-phosphonobutanoate (L-AP4), respectively. Intrastriatal stimulation evoked an excitatory postsynaptic current within cholinergic neurones that was reversibly inhibited by bath application of the group-II and group-III selective mGluR agonists DCG-IV and L-AP4, respectively, via presynaptic actions. In summary, we have identified the mGluRs expressed by striatal cholinergic interneurones and demonstrated that their activation produces modulatory effects via both pre- and postsynaptic mechanisms.

Presynaptic adenosine A2A receptors enhance GABAergic synaptic transmission via a cyclic AMP dependent mechanism in the rat globus pallidus.

1. We previously reported a presynaptic facilitatory action of A(2A) receptors on GABAergic synaptic transmission in the rat globus pallidus (GP). In the present study we identify the intracellular signalling mechanisms responsible for this facilitatory action of A(2A) receptors, using biochemical and patch-clamp methods in rat GP slices. 2. The adenosine A(2A) receptor selective agonist CGS21680 (1, 10 microM) and the adenylyl cyclase activator forskolin (1, 10 microM) both significantly increased cyclic AMP accumulation in GP slices. The CGS21680 (1 microM)-mediated increase in cyclic AMP was inhibited by the A(2A) receptor selective antagonist KF17837 (10 microM). 3. In an analysis of miniature inhibitory postsynaptic currents (mIPSCs), forskolin (10 microM) increased the mIPSC frequency without affecting their amplitude distribution, a result similar to that previously reported with CGS21680. 4. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536, 300 microM) abolished the CGS21680-induced enhancement in the frequency of mIPSCs. 5. H-89 (10 microM), a selective inhibitor for cyclic AMP-dependent protein kinase (PKA), blocked the CGS21680-induced enhancement of the mIPSC frequency. 6. The calcium channel blocker CdCl(2) (100 microM) did not prevent CGS21680 from increasing the frequency of mIPSCs. 7. These results indicate that A(2A) receptor-mediated potentiation of mIPSCs in the GP involves the sequential activation of the A(2A) receptor, adenylyl cyclase, and then PKA, and that this facilitatory modulation could occur independently of presynaptic Ca(2+) influx.