Daily Treatment Of Mice Research Articles

Daily Treatment of Mice with Type 2 Diabetes with Adropin for Four Weeks Improves Glucolipid Profile, Reduces Hepatic Lipid Content and Restores Elevated Hepatic Enzymes in Serum.

Adropin is a peptide hormone encoded by Energy Homeostasis Associated gene. Adropin modulates energy homeostasis and metabolism of lipids and carbohydrates. There is growing evidence demonstrating that adropin enhances insulin sensitivity and lowers hyperlipidemia in obese mice. The aim of this study was to investigate the effects of daily administration of adropin for four weeks in mice with experimentally induced type 2 diabetes (T2D). Adropin improved glucose control without modulating insulin sensitivity. Adropin reduced body weight, size of adipocytes, blood levels of triacylglycerol and cholesterol in T2D mice. T2D mice treated with adropin had lower liver mass, reduced hepatic content of triacylglycerol and cholesterol. Furthermore, adropin attenuated elevated blood levels of hepatic enzymes (ALT, AST, GGT and ALP) in T2D mice. In T2D mice, adropin increased the circulating adiponectin level. Adropin had no effects on circulating insulin and glucagon levels and did not alter pancreatic islets morphology. These results suggest that adropin improves glucose control, lipid metabolism and liver functions in T2D. In conjunction with reduced lipid content in hepatocytes, these results render adropin as an interesting candidate in therapy of T2D.

Pharmacodynamic and Pharmacokinetic Properties of AFPep, a Novel Peptide for the Treatment of Breast Cancer

AFPep is a small synthetic cyclized peptide derived from alpha-fetoprotein that has been shown to have anti-estrogenic and anti-breast cancer activity. The purpose of this study was to establish blood levels of AFPep that are associated with biological activity. Blood levels of AFPep were measured by LC/MS/MS. Once daily treatment of mice with 4 mg/kg i.p. AFPep yielded a Cmax of 7 µg/ml and was sufficient to inhibit estrogen-stimulated growth of mouse uterus and of human breast cancer xenografts even though the half-life of this drug was only 11 min in mice, suggesting that its biological effects last much longer than its chemical half-life. In dose de-escalation studies, blood levels of 100 ng/ml of AFPep were still found to be biologically active. AFPep was effective by parenteral routes and with dose escalation also by the oral route. Blood levels of AFPep that were biologically effective in mice were readily achieved in dogs through parenteral as well as oral routes with no apparent evidence of host toxicity. In conclusion, AFPep blood levels can be measured by LC/MS/MS with accuracy into the ng/ml range. Blood levels in the 100 ng/ml range are associated with efficacious biological activity. This drug shows great promise for the treatment as well as prevention of breast cancer.

Abstract 5593: Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies

Abstract Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index <0.9) were observed in both anti-PD-1 native and refractory patients and correlated to patient responses. CD73 expression, CD8+ infiltration, TREG distribution, and gene expression signatures are currently being evaluated in serial tumor biopsy specimens and will be presented. In total, this shows that CPI-444 is well tolerated in cancer patients, exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of anti-tumor immunity and clinical activity. Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5593. doi:10.1158/1538-7445.AM2017-5593

Regulatory T-Cell Augmentation or Interleukin-17 Inhibition Prevents Calcineurin Inhibitor-Induced Hypertension in Mice.

The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17-producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4+/FoxP3+ regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17-producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor-treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17-neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A-treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice.

Aspalathin Protects the Heart against Hyperglycemia-Induced Oxidative Damage by Up-Regulating Nrf2 Expression.

Aspalathin (ASP) can protect H9c2 cardiomyocytes against high glucose (HG)-induced shifts in myocardial substrate preference, oxidative stress, and apoptosis. The protective mechanism of ASP remains unknown. However, as one of possible, it is well known that phytochemical flavonoids reduce oxidative stress via nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation resulting in up-regulation of antioxidant genes and enzymes. Therefore, we hypothesized that ASP protects the myocardium against HG- and hyperglycemia-induced oxidative damage by up-regulating Nrf2 expression in H9c2 cardiomyocytes and diabetic (db/db) mice, respectively. Using an oxidative stress RT2 Profiler PCR array, ASP at a dose of 1 µM was demonstrated to protect H9c2 cardiomyocytes against HG-induced oxidative stress, but silencing of Nrf2 abolished this protective response of ASP and exacerbated cardiomyocyte apoptosis. Db/db mice and their non-diabetic (db/+) littermate controls were subsequently treated daily for six weeks with either a low (13 mg/kg) or high (130 mg/kg) ASP dose. Compared to nondiabetic mice the db/db mice presented increased cardiac remodeling and enlarged left ventricular wall that occurred concomitant to enhanced oxidative stress. Daily treatment of mice with ASP at a dose of 130 mg/kg for six weeks was more effective at reversing complications than both a low dose ASP or metformin, eliciting enhanced expression of Nrf2 and its downstream antioxidant genes. These results indicate that ASP maintains cellular homeostasis and protects the myocardium against hyperglycemia-induced oxidative stress through activation of Nrf2 and its downstream target genes.

Inhibition of Leukotriene B4 Action Mitigates Intracerebral Hemorrhage-Associated Pathological Events in Mice.

Infiltration of neutrophils has been suggested to play an important role in the pathogenesis of intracerebral hemorrhage (ICH) for which effective therapeutic interventions remain unavailable. In the present study we focused on leukotriene B4 (LTB4) as a potent chemotactic factor for neutrophils in order to address its contribution to the pathologic events associated with ICH. ICH with hematoma expansion into the internal capsule that resulted in severe sensorimotor dysfunction was induced by injection of collagenase in mouse striatum. We found that LTB4 as well as mRNAs of 5-lipoxygenase (5-LOX) and 5-LOX-activating protein were increased in the brain after ICH. Daily treatment with a 5-LOX inhibitor zileuton (3 or 10 mg/kg, i.v.) prevented ICH-induced increase in LTB4, attenuated neutrophil infiltration into the hematoma, and ameliorated sensorimotor dysfunction. In addition, mice deficient in LTB4 receptor BLT1 exhibited a lower number of infiltrating neutrophils in the hematoma and lower levels of sensorimotor dysfunction after ICH than did wild-type mice. Similarly, daily treatment of mice with BLT antagonist ONO-4057 (30 or 100 mg/kg, by mouth) from 3 hours after induction of ICH inhibited neutrophil infiltration and ameliorated sensorimotor dysfunction. ONO-4057 also attenuated inflammatory responses of microglia/macrophages in the perihematoma region and axon injury in the internal capsule. These results identify LTB4 as a critical factor that plays a major role in the pathogenic events in ICH, and BLT1 is proposed as a promising target for ICH therapy.

Abstract 2337: The adenosine A2A receptor antagonist CPI-444 blocks adenosine-mediated T-cell suppression and exhibits antitumor activity alone and in combination with anti-PD-1 and anti-PD-L1

Abstract Elevated levels of extracellular adenosine within the tumor microenvironment create an immunosuppressive niche that promotes tumor growth and metastasis. Adenosine signaling via the A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapies such as anti-PD-L1 or anti-PD-1 monoclonal antibodies (mAbs). CPI-444 is an oral A2AR antagonist that has been evaluated previously in phase 1 clinical trials for non-oncology indications. CPI-444 demonstrates binding to A2AR with a Ki of 3.5 nM and > 50-fold selectivity over other adenosine receptor subtypes. We examined the immune activating and anti-tumor properties of CPI-444 alone and in combination with anti-PD-1/PDL-1 mAbs. In vitro studies using activated primary human T cells demonstrated that CPI-444 fully inhibited the production of intracellular cAMP following incubation of the cells with 5’-N-ethylcarboxamidoadenosine (NECA), a stable analog of adenosine. A2AR agonists suppressed rapid TCR-mediated ERK phosphorylation and, subsequently, production of IL-2 and IFNγ by activated T cells; blockade of A2AR with CPI-444 restored T cell signaling and function. The efficacy of CPI-444 was evaluated in MC38 and CT26 syngeneic mouse tumor models. In the MC38 model, daily treatment of mice with CPI-444 (1, 10, 100 mg/kg) led to a dose-dependent reduction in tumor growth, leading to full tumor elimination in 9/30 treated mice. New tumors failed to establish when the cured mice were re-challenged with MC38 cells, indicating that CPI-444 induced systemic anti-tumor immune memory. Combining CPI-444 with anti-PD-L1 mAb treatment in the MC38 model synergistically inhibited tumor growth and led to elimination of tumors in 9/10 treated mice. In the CT26 model, CPI-444 alone or anti-PD-1 alone led to non-significant reductions in tumor growth; however the combination of CPI-444 and anti-PD-1 led to a synergistic inhibition of tumor growth and prolonged survival compared to either agent alone. These results, and others, suggest that adenosine signaling may be an important resistance mechanism in tumors that incompletely respond to anti-PD-1/PD-L1 mAb therapy. Based on these results and others, we plan to initiate a Phase 1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with anti-PD-L1 (atezolizumab) in patients with solid tumors. Citation Format: Stephen Willingham, Po Ho, Robert Leone, Emily Piccione, Carmen Choy, Andrew Hotson, Joseph Buggy, Jonathan Powell, Richard Miller. The adenosine A2A receptor antagonist CPI-444 blocks adenosine-mediated T-cell suppression and exhibits antitumor activity alone and in combination with anti-PD-1 and anti-PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2337.

Chloroquine has tumor-inhibitory and tumor-promoting effects in triple-negative breast cancer

Toll-like receptor-9 (TLR9) is an intracellular DNA receptor that is widely expressed in breast and other cancers. We previously demonstrated that low tumor TLR9 expression upon diagnosis is associated with significantly shortened disease-specific survival times in patients with triple-negative breast cancer (TNBC). There are no targeted therapies for this subgroup of patients whose prognosis is among the worst in breast cancer. Due to the previously detected in vitro anti-invasive effects of chloroquine in these cell lines, the present study aimed to investigate the in vivo effects of chloroquine against two clinical subtypes of TNBC that differ in TLR9 expression. Chloroquine suppressed matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression and protein activity, whereas MMP-13 mRNA expression and proteolytic activity were increased. Despite enhancing TLR9 mRNA expression, chloroquine suppressed TLR9 protein expression in vitro. Daily treatment of mice with intraperitoneal (i.p.) chloroquine (80 mg/kg/day) for 22 days, did not inhibit the growth of control siRNA or TLR9 siRNA MDA-MB-231 breast cancer cells. In conclusion, despite the favorable in vitro effects on TNBC invasion and viability, particularly in hypoxic conditions, chloroquine does not prevent the growth of the triple-negative MDA-MB-231 cells with high or low TLR9 expression levels in vivo. This may be explained by the activating effects of chloroquine on MMP-13 expression or by the fact that chloroquine, by suppressing TLR9 expression, permits the activation of currently unknown molecular pathways, which allow the aggressive behavior of TNBC cells with low TLR9 expression in hypoxia.

High Mobility Group Box 1 Contributes to the Pathogenesis of Experimental Pulmonary Hypertension via Activation of Toll-like Receptor 4

Survival rates for patients with pulmonary hypertension (PH) remain low, and our understanding of the mechanisms involved are incomplete. Here we show in a mouse model of chronic hypoxia (CH)-induced PH that the nuclear protein and damage-associate molecular pattern molecule (DAMP) high mobility group box 1 (HMGB1) contributes to PH via a Toll-like receptor 4 (TLR4)-dependent mechanism. We demonstrate extranuclear HMGB1 in pulmonary vascular lesions and increased serum HMGB1 in patients with idiopathic pulmonary arterial hypertension. The increase in circulating HMGB1 correlated with mean pulmonary artery pressure. In mice, we similarly detected the translocation and release of HMGB1 after exposure to CH. HMGB1-neutralizing antibody attenuated the development of CH-induced PH, as assessed by measurement of right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and endothelial activation and inflammation. Genetic deletion of the pattern recognition receptor TLR4, but not the receptor for advanced glycation end products, likewise attenuated CH-induced PH. Finally, daily treatment of mice with recombinant human HMGB1 exacerbated CH-induced PH in wild-type (WT) but not Tlr4(-/-) mice. These data demonstrate that HMGB1-mediated activation of TLR4 promotes experimental PH and identify HMGB1 and/or TLR4 as potential therapeutic targets for the treatment of PH.

In vitro and in vivo activity of AS101 against West Nile virus (WNV)

There are currently no effective drugs to treat serious complications caused by WNV infection. The inhibition of WNV by the pluripotent immunomodulator AS101 [ammonium trichloro(dioxyethylene-0-0′)tellurate] was evaluated in vitro and in vivo, and its mechanism was explored. Adding AS101 to Vero cells 1h or 5min before infection increased cell survival from 21% to 84% and decreased plaque formation by 87% and virus yield by 2 logs. Following infection, high titer of WNV remained in the culture supernatants indicating interference with virus cell attachment. The binding of αVβ3 integrin to WNV and of Vero cells to anti-αVβ3 antibody were inhibited by AS101, suggesting that AS101 may block this cellular WNV receptor. Daily treatment of mice with AS101 starting 1 day before lethal infection with WNV resulted in 48% survival. However, treatment beginning 3 days post infection resulted only in 16% survival. Similarly, a single dose of anti-WNV IVIG three days post infection resulted in 16% survival compared to 100% if IVIG was given on the same day of infection or 1 day later. However, when mice received combined treatment with AS101 and IVIG starting 3 days post infection, an additive effect of 33% survival was observed. Our study suggests that AS101 has a potential preventive and therapeutic effect against WNV infection.

Metformin inhibits renal cell carcinoma in vitro and in vivo xenograft

ObjectiveTo evaluate the effects of metformin on renal cell carcinoma (RCC) and its underlying mechanisms. Materials and methodsWe used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and colony formation assays to investigate the effects of metformin on RCC cell growth. Flow cytometry was used to evaluate the cell cycle changes after metformin treatment. We further determined the possible signaling molecules involved in this process by immunoblot analysis of various proteins. Furthermore, a xenograft model was used to study the effects of metformin on RCC tumor growth. ResultsWe demonstrated that metformin effectively inhibits cell proliferation in 786-O and OS-RC-2 RCC cell lines. Moreover, metformin down-regulated cyclin D1 expression and induced G0/G1 cell cycle arrest in these cells. Further study revealed metformin induced the activation of AMP-activated protein kinase (AMPK), and inhibited mammalian target of rapamycin (mTOR), which is a central regulator of protein synthesis and cell growth, and negatively regulated by AMPK. Most importantly, daily treatment of mice with metformin prevented RCC tumor growth in a xenograft model. ConclusionsMetformin was able to induce G0/G1 cell cycle arrest and inhibit RCC growth in vitro and in vivo. These results suggest that metformin may be a potential therapeutic agent for the treatment of RCC.

Cardioprotective effect of NO‐metoprolol in murine coxsackievirus B3‐induced myocarditis

The effect of NO-metoprolol, that is, 3-nitrooxypivaloyl metoprolol-amide, a novel NO-releasing derivative of the β1-blocking drug metoprolol was investigated in A.CA/SnJ mice infected with coxsackievirus B3 (CVB3) and compared to metoprolol and placebo. Daily treatment of mice with the respective drug started immediately (experiment A) or 3 days after virus infection (experiment B) and was continued until day 13 post-infection (p.i.). Two doses of NO-metoprolol were administered. Body mass differences, viral load, and histopathological signs of myocarditis were compared between the several groups. As a result, NO-metoprolol diminished significantly the body weight loss, the viral load and the histopathology, whereas metoprolol treatment led solely to a significant attenuation of myocardial damage. In experiment A, low dose NO-metoprolol decreased significantly enteroviral copy numbers. Both doses of NO-metoprolol had a significant effect on reduction of myocardial infiltrates and fibrosis. The data suggest that delayed drug administration might more advantageous. Both doses of NO-metoprolol reduced significantly the scores of four tested parameters compared to placebo. Body weight loss, virus titers, plus-strand as well as minus-strand enteroviral RNA levels, infiltration and fibrosis scores were diminished significantly when NO-metoprolol was given 3 days p.i. In addition, a significant difference regarding the enteroviral copy numbers was observed between low dose NO-metoprolol- and metoprolol-treated mice. Treatment with metoprolol reduced insignificantly the viral load and body weight loss (experiment A and B) but led to a significant reduction of myocardial histopathology in experiment A. The results indicate that NO-metoprolol treatment has a greater therapeutic benefit than metoprolol.

COMPARISON OF SOME PROPERTIES OF PRONETHALOL AND PROPRANOLOL

Much evidence has accumulated to justify the classification of pronethalol (Alderlin; I.C.I.) as a sympathetic β-receptor antagonist described in terms of Ahlquist's (1948) dual-receptor hypothesis. There has now been confirmation in animal experiments of antagonism to adrenergically induced vasodilatation (Ahlquist, 1963; Abboud, Eckstein & Zimmerman, 1963), cardiac stimulation (Meester, Hardman & Barboriak, 1963; Kako, Krayenbuhl, Luthy & Hegglin, 1964; Donald, Kvale & Shepherd, 1964), bronchodilatation (Nagasaka, Schaepdryver & Heymans, 1964), uterine relaxation (Levy & Tozzi, 1963) and gut relaxation after block or loss of α-receptors (Lum & Kermani, 1963). In human pharmacological studies antagonism to catecholamine-induced vasodilatation and cardiac stimulation (Dornhorst & Robinson, 1962; Lowe & Robinson, 1964) has been reported. Reduction of exercise tachycardia in man has been described by Bishop & Segel (1963), Chamberlain & Howard (1964), Harrison, Braunwald, Glick, Mason, Chidsey & Ross (1964) and Schroder & Werko (1964). The effects of pronethalol in experimental cardiac arrhythmias in dogs and guinea-pigs have been investigated. Antagonism of arrhythmias induced by cardiac glycosides has been reported by Erlij & Mendes (1964), Vaughan-Williams & Sekiya (1963) and Somani, Hardman & Lum (1963). Prevention of arrhythmias induced by adrenaline during anaesthesia with a hydrocarbon has been found by Murray, McKnight & Davis (1963) and by Hess & Hampton (1964). Somani et al. (1963) have shown that pronethalol will block ectopic ventricular tachycardia produced by large doses of adrenaline, and Ehringer & Gogel (1963) claim that it reduces the apical myocardial necrosis produced in rats by large doses of isoprenaline. This interest in the cardiovascular actions of pronethalol is also shown in various clinical reports. Success in the treatment of some cardiac arrhythmias, including digitalis intoxication, has been reported by Stock & Dale (1963) and Grandjean & Rivier (1963). Encouraging results in the treatment of angina have been found in a multicentre double-blind trial (Alleyne, Dickinson, Dornhorst, Fulton, Green, Hill, Hurst, Laurence, Pilkington, Prichard, Robinson & Rosenheim, 1963) and in a small, placebo-controlled, trial of effort tolerance (Apthorp, Chamberlain & Hayward, 1964). Potential therapeutic value of pronethalol in the management of phaeochromocytoma (Dornhorst & Laurence, 1963) and hypertrophic obstructive cardiomyopathy (Harrison et al., 1964; Cohen, Effat, Goodwin, Oakley & Steiner, 1964) has been described. Effective treatment of ventricular arrhythmias occurring during halothane anaesthesia has been reported by Johnstone (1964) and by Payne & Senfield (1964). These reports confirm the suggestions by Black & Stephenson (1962) that sympathetic β-receptor antagonists might be valuable in the study and treatment of certain cardiac disorders. The overt toxic effects of pronethalol described by Black & Stephenson (1962) were tremors and convulsions. These signs were correlated with the evidence that the brain concentrations of pronethalol were of the order of 100-times the blood concentration. The acute toxic signs appeared at doses seven- to ten-times greater than those needed for effective β-receptor blockade. While tremors and convulsions have not been described in man, effective doses are often associated with unpleasant subjective side-effects. Mild disorientation, slight inco-ordination, nausea and vomiting have been described (Alleyne et al., 1963). Since the toxic signs in animals are seen with the inactive (+)-isomer as as well as with the active (–)-isomers (Howe, 1963), it has been assumed that the effects of pronethalol on the central nervous system are probably not due to β-receptor blockade. A more serious toxic manifestation was found in mice. Studies showed that pronethalol was a moderately potent carcinogen in this species. Malignant tumours, mainly thymic lymphosarcomata, began to appear after 10 weeks continuous dosing (Paget, 1963; Alcock & Bond, 1964). This combination of nonspecific central actions and carcinogenic potential in a compound of clinical interest put a premium on finding a less toxic compound. A preliminary communication (Black, Crowther, Shanks, Smith & Dornhorst, 1964) on such a compound has been published; this compound is propranolol (Inderal; LCI.). The relationship of propranolol to pronethalol is shown by the structural formulae: This paper presents details of the comparative activity of these compounds. Detailed studies of their toxicity by our colleagues have shown that daily treatment of mice with propranolol over a period of 18 months did not produce thymic tumours (Alcock, Baker & Tucker, 1965).

Cross-sensitization between caffeine- and l-dopa-induced behaviors in hemiparkinsonian mice

Adenosine A 2A receptor (A 2AR) antagonists, including the non-specific adenosine antagonist caffeine, have been proposed as a novel, non-dopaminergic treatment strategy for Parkinson's disease (PD). However, the long-term interaction between caffeine and l-dopa treatment in PD models has not been characterized. We examined the interaction between caffeine and l-dopa following a repeated treatment paradigm in hemiparkinsonian mice. In contrast to the progressively sensitized rotational behavior induced by daily l-dopa (2.0 mg/kg) treatment, tolerance for the rotational response to daily caffeine (2.5 or 10 mg/kg) treatment tended to develop over several weeks. However, after a subsequent two-week washout, challenge with same drug demonstrated an extinction of the sensitized l-dopa-induced rotation, but a sensitization of the caffeine-induced rotation. In a cross-challenge paradigm, daily treatment of mice with l-dopa (compared to daily saline) produced a three-fold enhancement in the rotational response to a subsequent re-challenge with caffeine. Similarly, daily treatment of mice with caffeine produced a six-fold enhancement in the rotational response to a subsequent re-challenge with l-dopa. Furthermore, daily co-administration of caffeine plus l-dopa produced enhanced rotational behavior, compared to caffeine or l-dopa alone, indicating an additive or synergistic interaction between caffeine and l-dopa during repeated treatment. Cross-sensitization between caffeine and l-dopa following repeated treatment and their positive interaction during chronic co-adminstration in hemiparkinsonian mice suggest that repeated exposure to caffeine may alter l-dopa responses in PD.

Neurobehavioral effects of tetramisole in mice

Acute toxicity and neurobehavioral effects of the veterinary anthelmintic tetramisole were examined in male albino mice. The 24-h median lethal doses of tetramisole were determined by the up-and-down method in mice after oral, subcutaneous and intraperitoneal administrations, and they were 110, 57 and 34 mg/kg, respectively. The intoxicated mice manifested nervousness, crouching, piloerection and tremor. Subcutaneous injection of tetramisole at 0.5 and 1 mg/kg did not significantly affect general locomotor activity of the mice in 5-min open-field activity test or negative geotaxis performance and landing foot splay. However, the 1 mg/kg dose of tetramisole significantly increased the stereotyped behavior (grooming, sniffing, biting and licking and head bobbing) of the mice when compared to the control group. Daily treatment of mice with tetramisole at 0.5 and 1 mg/kg for six consecutive days did not significantly affect their general locomotor activity in the open-field activity test. Both doses of tetramisole significantly increased stereotyped behavior of the mice on days 3, 5 and 8 in comparison with respective control values. Repeated tetramisole treatments (0.5 and 1 mg/kg) also significantly decreased the time needed to complete the negative geotaxis task and reduced landing foot splay. All tetramisole-treated mice showed crouching behavior and were nervous and difficult to handle. The results suggest nervous involvement in the acute toxicity of tetramisole. Stereotyped behavior and changes in the negative geotaxis and landing foot splay are reported for the first time in mice treated with tetramisole.

Metabotropic glutamate receptors and neuroadaptation to antidepressants: imipramine‐induced down‐regulation of β‐adrenergic receptors in mice treated with metabotropic glutamate 2/3 receptor ligands

Antidepressant drugs have a clinical latency that correlates with the development of neuroadaptive changes, including down-regulation of beta-adrenergic receptors in different brain regions. The identification of drugs that shorten this latency will have a great impact on the treatment of major depressive disorders. We report that the time required for the antidepressant imipramine to reduce the expression of beta-adrenergic receptors in the hippocampus is reduced by a co-administration with centrally active ligands of type 2/3 metabotropic glutamate (mGlu2/3) receptors. Daily treatment of mice with imipramine alone (10 mg/kg, i.p.) reduced the expression of beta-adrenergic receptors in the hippocampus after 21 days, but not at shorter times, as assessed by western blot analysis of beta1-adrenergic receptors and by the amount of specifically bound [3H]CGP-12177, a selective beta-adrenergic receptor ligand. Down-regulation of beta-adrenergic receptors occurred at shorter times (i.e. after 14 days) when imipramine was combined with low doses (0.5 mg/kg, i.p.) of the selective mGlu2/3 receptor agonist LY379268, or with the preferential mGlu2/3 receptor antagonist LY341495 (1 mg/kg, i.p.). Higher doses of LY379268 (2 mg/kg, i.p.) were inactive. This intriguing finding suggests that neuroadaptation to imipramine--at least as assessed by changes in the expression of beta1-adrenergic receptors--is influenced by drugs that interact with mGlu2/3 receptors and stimulates further research aimed at establishing whether any of these drugs can shorten the clinical latency of classical antidepressants.

Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris , Candida albicans , and Candida dubliniensis

Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC50) of 30 microM in 2 or 4 mM Mg2+, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s ranged from 1 to 9 microM for strains with group I introns and were 12 and 32 microM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.

Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13.

1. Exposure to midrange ultraviolet radiation (UVB) is known to produce skin inflammation similar to sunburn. The aim of this study was to characterize the hyperalgesia and cytokine upregulation induced by UVB and their modulation by antiinflammatory cytokines. 2. Acute exposure of the dorsal skin of mice to UVB (200, 250 and 300 mJ cm(2)) resulted in a dose-dependent decrease in the latencies of the hot plate and tail flick tests, without evident signs of skin lesions. 3. The observed hyperalgesia displayed a biphasic temporal evolution with an acute phase (3 - 6 h) and a late (48 - 96 h) phase. 4. Exposure to UVB (300 mJ cm(2)) elicited significant upregulation of interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha and nerve growth factor (NGF), determined by ELISA in the exposed skin. This upregulation was more important during the acute phase of hyperalgesia. 5. Daily treatment of mice, with i.p. injections of either IL-10 or IL-13 (1.5, 7.5 and 15 ng in 100 microl saline) produced a dose-dependent attenuation of the UVB-induced hyperalgesia. 6. Treatment with the highest doses of either IL-10 or IL-13, produced significant attenuation of the levels of the cytokines and NGF by UVB, with relatively more pronounced effects by IL-13. 7. Acute exposure to moderate amounts of UVB results in a systemic hyperalgesia related to the upregulation of cytokine and NGF levels, since both were prevented by treatment with antiinflammatory cytokines.

Induction of dendritic cells (DC) by Flt3 Ligand (FL) promotes the generation of tumor-specific immune responses in vivo.

Daily treatment of mice with human Flt3 ligand (FL) induces the production of large numbers of MHC-class II+, CD11c+, DEC205+ dendritic cells (DC) in both lymphoid and nonlymphoid tissues. Because DC play a pivotal role in the induction of immune responses, we evaluated the effects of FL in the augmentation of anti-tumor immune responses in vivo. Treatment with daily subcutaneous injections of FL not only caused complete regression of tumors in a significant proportion of mice challenged with a syngeneic methylcholanthrene-induced fibrosarcoma, but also caused a significant decrease in the tumor growth rate in the remaining mice. The effects of FL were both dose and schedule dependent, induced regression of tumors established as much as 7 days prior to beginning cytokine treatment, and was mediated by both T-cell mediated and non-T cell-mediated mechanisms. Histopathologic evaluation of tumors during and after systemic treatment with FL revealed a dense infiltration of mononuclear and myeloid precursor cells into both the tumor and tissue surrounding the tumor that generally correlated with the degree of regression and resolved after rejection of the tumor. Collectively, the data suggest that FL may be an important cytokine in the generation of effective anti-tumor immune responses in vivo and the treatment of cancer in situ.

Flt3 ligand induces tumor regression and antitumor immune responses in vivo.

Daily treatment of mice with recombinant human Flt3 ligand (huFlt3L) results in a dramatic numerical increase in the number of dendritic cells (DCs) in vivo. Since DCs are pivotal in the induction of immune responses, we tested whether Flt3L treatment of mice challenged with a syngeneic methylcholanthrene (MCA)-induced fibrosarcoma would augment the generation of effective antitumor immune responses in vivo. Flt3L treatment not only induced complete tumor regression in a significant proportion of mice, but also decreased tumor growth rate in the remaining mice. A preliminary characterization of the cellular mechanisms involved suggests that Flt3L may be important in the treatment of cancer in situ through the generation of specific antitumor immune responses.