Granzyme B PET Imaging of Immune Checkpoint Inhibitor Combinations in Colon Cancer Phenotypes.

PURPOSE: Immune checkpoint inhibitor (ICI) monotherapy and combination regimens are being actively pursued as strategies to improve durable response rates in cancer patients. However, the biology surrounding combination therapies is not well understood and may increase the likelihood of immune-mediated adverse events. Accurate stratification of ICI response by non-invasive PET imaging may help ensure safe therapy management across a wide number of cancer phenotypes. PROCEDURES: We have assessed the ability of a fluorine-labelled peptide, [18F]AlF-mNOTA-GZP, targeting granzyme B, to stratify ICI response in two syngeneic models of colon cancer, CT26 and MC38. In vivo tumour uptake of [18F]AlF-mNOTA-GZP following ICI monotherapy, or in combination with PD-1 was characterised and correlated with changes in tumour-associated immune cell populations. RESULTS: [18F]AlF-mNOTA-GZP showed good predictive ability and correlated well with changes in tumour-associated T cells, especially CD8+ T cells; however, overall uptake and response to monotherapy or combination therapies was very different in the CT26 and MC38 tumours, likely due to the immunostimulatory environment imbued by the MSI-high phenotype in MC38 tumours. CONCLUSIONS: [18F]AlF-mNOTA-GZP uptake correlates well with changes in CD8+ T cell populations and is able to stratify tumour response to a range of ICIs administered as monotherapies or in combination. However, tracer uptake can be significantly affected by preexisting phenotypic abnormalities potentially confusing data interpretation.

Imaging Fibrogenesis in a Diet-Induced Model of Nonalcoholic Steatohepatitis (NASH).

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v ß 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v ß 3 imaging agent [18F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging. Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [18F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v ß 3, smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis. Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [18F]FtRGD correlated well with integrin α v ß 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [18F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49. Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v ß 3 and extracellular matrix protein expression. [18F]FtRGD showed very good correlation between liver uptake and integrin α v ß 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis.

Imaging adipose tissue browning using the TSPO-18kDa tracer [18F]FEPPA.

OBJECTIVES: The browning of white adipose tissue (WAT) into beige has been proposed as a strategy to enhance energy expenditure to combat the growing epidemic of obesity. Research into browning strategies are hampered by the lack of sensitive, translatable, imaging tools capable of detecting beige fat mass non-invasively. [18F]FDG is able to detect activated beige fat but provides little information on unstimulated beige fat mass. We have assessed the use of [18F]FEPPA, a tracer for the TSPO-18KDa found on the outer mitochondrial membrane, as an alternative imaging agent capable of detecting unstimulated brown fat (BAT) and beige fat. METHODS: Female Balb/c mice (n = 5) were treated for 7 days with the ß3 adrenergic agonist CL-316,243 to induce the browning of inguinal WAT (beige fat). Animals were imaged longitudinally with [18F]FDG and [18F]FEPPA and uptake in interscapular BAT and inguinal WAT assessed. The browning of inguinal WAT was confirmed using H&E and immunohistochemical detection of UCP-1 and TSPO. RESULTS: Repeated dosing with ß3-adrenergic agonist CL-316,243 caused a significant increase in [18F]FDG uptake in both interscapular BAT and inguinal WAT associated with the increased metabolic activity of brown and beige adipocytes respectively. [18F]FEPPA uptake was likewise increased in inguinal WAT but showed no increase in BAT uptake due to stimulation over the same time course. Furthermore, inguinal WAT uptake was unaffected by pharmacological blockade, indicating that [18F]FEPPA uptake is associated with the expression of mitochondria in BAT and beige adipocytes and independent of activation. CONCLUSION: These data show that [18F]FEPPA can detect BAT and newly formed beige fat under non-stimulated, thermoneutral conditions and that uptake after stimulation is linked to mitochondrial expression as opposed to activation.

Imaging the Proangiogenic Effects of Cardiovascular Drugs in a Diabetic Model of Limb Ischemia.

Purpose: Peripheral artery disease (PAD) causes narrowing of arteries in the limbs, leading to tissue ischemia, gangrene, and eventually limb amputation. The presence of diabetes greatly exacerbates the course of PAD, accounting for the majority of lower limb amputations. Therapeutic strategies focussing on macrovascular repair are less effective in diabetic patients where smaller vessels are affected, and proangiogenic therapies offer a viable adjunct to improve vascularisation in these at risk individuals. The purpose of the current study was to assess the proangiogenic effects of drugs routinely used to treat cardiovascular disease in a diabetic murine model of hind limb ischemia longitudinally using multimodal imaging. Procedures: Diabetic mice underwent surgical intervention to induce hind limb ischemia and were treated with simvastatin, metformin, or a combination orally for 28 days and compared to diabetic and nondiabetic mice. Neovascularisation was assessed using [18F]FtRGD PET imaging, and macrovascular volume was assessed by quantitative time of flight MRI. At each imaging time point, VEGF expression and capillary vessel density were quantified using immunohistochemical analysis, and functional recovery and disease progression were assessed. Results: Combined use of simvastatin and metformin significantly increased neovascularisation above levels measured with either treatment alone. Early angiogenic events were accurately assessed using PET [18F]FtRGD, showing maximal retention in the ischemic hind limb by day 8, which translated to a sustained increase in vascular volume at later time points. Immunohistochemical analysis shows that combined therapy significantly increased VEGF expression and capillary density (CD31+) in a similar time course and also slowed disease progression while simultaneously improving functional foot use. Conclusions: Combined treatment with simvastatin and metformin led to a significant improvement in limb angiogenesis, vascular volume, and sustained functional recovery in a diabetic murine model of HLI. PET imaging with [18F]FtRGD provides a robust method for early detection of these proangiogenic effects preclinically and may be useful for the assessment of proangiogenic therapies used clinically to treat diabetic PAD patients.

Styryl sulfonyl compounds inhibit translation of cyclin D1 in mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is characterized by the uncontrolled overexpression of cyclin D1. Styryl sulfonyl compounds have shown potent antitumor activity against MCL by inducing cell-cycle arrest and apoptosis. However, the exact molecular mechanism by which these compounds function is yet to be elucidated. Here, we show that the prototypical styryl sulfonyl compound ON 01910.Na decreased cyclin D1 and c-Myc protein levels in MCL cells, whereas mRNA levels of cyclin D1 were minimally affected. Notably, ON 01910.Na suppressed eukaryotic translation initiation factor 4E (eIF4E)-mediated cyclin D1 mRNA translation, decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR) and eIF4E-binding protein (eIF4E-BP), lowered the cap site binding activity of eIF4E and directly inhibited activity of phosphatidylinositol-3 kinase (PI-3K). Analysis of apoptotic signaling pathways revealed that ON 01910.Na induced the release of cytochrome c from mitochondria, altered expression of Bcl-2 family of proteins and stimulated activation of caspases. Taken together, styryl sulfonyls can cause a rapid decrease of cyclin D1 by blocking cyclin D1 mRNA translation through inhibition of the PI-3K/Akt/mTOR/eIF4E-BP signaling pathway and triggering a cytochrome c-dependent apoptotic pathway in MCL cells.

Analgesic activity of methanol extract of Eupatorium adenophorum Spreng. leaves.

The methanol extract of the leaves of E. adenophorum (100, 200 and 300 mg/kg, po) showed significant analgesic activity, as compared to standard drugs diclofenac sodium and pentazocine, employing acetic acid-induced writhing test, tail immersion test and tail flick test models.

ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent.

Elevated expression of polo-like kinase1 (Plk1) has been reported in many human tumors, and inhibition of Plk1 activity results in their mitotic arrest and apoptosis. Here we describe the profile of ON01910, a small molecule inhibitor of Plk1 activity, which induces mitotic arrest of tumor cells characterized by spindle abnormalities leading to their apoptosis. This compound was not ATP-competitive, but competed for the substrate binding site of the enzyme. In vivo, this compound did not exhibit hematotoxicity, liver damage, or neurotoxicity, and was a potent inhibitor of tumor growth in a variety of xenograft nude mouse models. ON01910 showed strong synergy with several chemotherapeutic agents, often inducing complete regression of tumors.

Pharmamacgnostical evaluation of ionldium suffrlitlcosam ging (family: violaceae).

The macroscopic character of the whole plant, physical constant values, extractive values, behaviour on treatment with different chemical reagents. Fluorescence characters under uv light after treatment with different chemical reagents of the powdered whole plant of lonidium suffruticosam Ging (Family; violaceae) were studied to characterize some pharmacognostical parameters. Preliminary photochemical study on different extracts of the whole plant were also performed.

Evaluation of antitussive activity of Lagerstroemia parviflora leaf extract.

The methanol extract of the leaves of Lagerstroemia parviflora Roxb was investigated for its effect on a cough model induced by sulfur dioxide gas in mice. It exhibited significant antitussive activity when compared with the control in a dose-dependent manner. The L. parviflora extract (100, 200, 300 mg/kg) showed maximum inhibition of cough reflex at 90 min after drug administration and the antitussive activity was comparable to that of codeine phosphate, a standard antitussive agent.

Evaluation of antipyretic potential of Clitoria ternatea L. extract in rats.

The methanol extract of Clitoria ternatea L. root (MECTR) blue flowered variety (Family: Fabaceae), was evaluated for its anti-pyretic potential on normal body temperature and yeast-induced pyrexia in albino rats. Yeast suspension (10 ml/kg body wt.) increased rectal temperature after 19 hours of subcutaneous injection. The extract, at doses of 200, 300 and 400 mg/kg body wt., p.o., produced significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner. The effect extended up to 5 hours after the drug administration. The anti-pyretic effect of the extract was comparable to that of paracetamol (150 mg/kg body wt., p.o.), a standard anti-pyretic agent.

Anti-inflammatory evaluation of Ionidium suffruticosam Ging. in rats.

The anti-inflammatory activity of Ionidium suffruticosam (Violaceae) methanol extract was evaluated on carrageenin, histamine and serotonin-induced rat hind paw oedema acute models. The extract at doses of 200 and 400 mg/kg has been found to possess significant anti-inflammatory activity on the tested experimental models. The extract at the dose level of 400 mg/kg exhibited maximum anti-inflammatory activity in all the animal models. In a chronic test, the extract (400 mg/kg) showed 42.78% reduction in granuloma weight. The effect produced by the extract was comparable to that of phenylbutazone, a proto type of a non-steroidal anti-inflammatory agent.

Studies on psychopharmacological effects of Cleome viscosa Linn. extract in rats and mice.

Methanol extract of the entire plant Cleome viscosa Linn. (CVME) was evaluated for different psychopharmacological actions such as general behaviour, exploratory behaviour, muscle relaxant activity and phenobarbitone induced sleeping time and effects on normal body temperature in rats and mice. The extract was found to cause reduction in spontaneous activity, decrease in exploratory behavioural pattern by the head dip and Y-maze test, reduction in the muscle relaxant by rotarod, 30 degrees inclined screen and traction tests and caused significant lowering of body temperature. In addition, CVME significantly potentiated the phenobarbitone-induced sleeping time. Preliminary tests indicate that the methanol extract of Cleome viscosa Linn. in doses of 200-400 mg/kg has significant psychopharmacological activity.

Evaluation of anti-inflammatory potential of Pavetta indica Linn. leaf extract (family: Rubiaceae) in rats.

The anti-inflammatory potential of methanol extract of Pavetta indica Linn. leaves (Family: Rubiaceae) was evaluated against several models of inflammation such as carragenin, histamine and dextran induced pedal inflammation in rats. The extract showed 48.41%, 41.10% and 24.22% inhibition respectively; when compared to that of control animals. The effect was comparable with that of the standard drug indomethacin, a standard non-steroidal anti-inflammatory drug. Simultaneous subplantar administration of the extract and carrageenin in a mixture helps in differentiating true anti-inflammatory action from an apparent anti-inflammatory effect due to counter-irritant activity. The methanol extract also effectively and significantly reduced cotton pellet induced granuloma. The percentage of inhibition was 62.78 at the dose 500 mg/kg, thereby suggesting its activity in the proliferative phase of the inflammatory process.

Evaluation of antitussive potential of Ionidium suffruticosam Ging. (Violaceae) extract in albino mice.

The present study was carried out to evaluate the antitussive potential of methanol extract of Ionidium suffruticosam Ging. (Violaceae) which was investigated for its effect on a cough model induced by sulfur dioxide gas in albino mice. It exhibited significant dose-dependent antitussive activity when compared with the control, 250 and 500 mg/kg (p.o.) of the extract showing 28.37% and 54.16% inhibition of the cough with respect to control group. The antitussive activity of the extract was comparable to that of codeine phosphate, a prototype antitussive agent.

Evaluation of antipyretic potential of Cleome viscosa Linn. (Capparidaceae) extract in rats.

The antipyretic activity of a methanol extract of Cleome viscosa Linn. (CVME) was investigated for its potential on normal body temperature and yeast-induced pyrexia in albino rats. The CVME, at doses of 200, 300, and 400mg/kg BW p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner. The effect also extended upto 5h after the drug administration. The anti-pyretic effect of CVME was comparable to that of paracetamol (150mg/kg p.o.), a standard anti-pyretic agent.

Anti-inflammatory, analgesic and antipyretic properties of Clitoria ternatea root.

Clitoria ternatea roots methanol extract when given by oral route to rats was found to inhibit both the rat paw oedema caused by carrageenin and vascular permeability induced by acetic acid in rats. Moreover, the extract exhibited a significant inhibition in yeast-induced pyrexia in rats. In the acetic acid-induced writhing response, the extract markedly reduced the number of writhings at doses of 200 and 400 mg/kg (p.o.) in mice.

Studies on analgesic activity of Cleome viscosa in mice.

The analgesic activity of methanol extract of Cleome viscosa, given orally at the doses of 100, 200, 400 mg/kg was evaluated for its analgesic activity in mice using the acetic acid-induced writhing and the tail flick, tail clip, tail immersion methods. The extract showed promising activity in all the tests.

Studies of pharmacognostical profiles of cleome viscosa L (family:capparidaceae).

The macroscopic characters of the whole plant, physical constant values, extractive values, behavior on treatment with different chemical reagents, fluorescence characters under ultra violet light after treatment with different reagents of the powered entire plant of Cleome viscose Linn.(Capparidaceae) were studied to fix some pharmacognostical parameters. Preliminary phytochemical screening on the methanol extract of the plant also performed. These studies will help in identification of this plant for further research.

Evaluation of anti-diarrheal activity of Cleome viscosa L. extract in rats.

A study was undertaken to evaluate the effect of a methanol extract of the entire plant Cleome viscosa L. (CVME) (Family; Capparidaceae) for its anti-diarrheal potential against some of the experimental models of diarrhea in rats. CVME showed significant inhibitory activity against castor-oil-induced diarrhea and PGE2 induced enteropooling in rats. The extract also showed a significant reduction in gastrointestinal motility in the charcoal meal test in rats. The results obtained establish the efficacy and substantiate the folklore claim as an anti-diarrheal agent.