AminoBODIPY Conjugates for Targeted Drug Delivery Systems and Real-Time Monitoring of Drug Release.

In this work, we report two concepts of drug delivery based on small-molecule drug conjugates with the ability of specific targeting and drug release monitoring via ratiometric fluorescence. The functionality of these concepts has been verified by two model systems consisting of three parts: (i) fluorescent aminoBODIPY for real-time detection of conjugate cleavage, (ii) a c(RGDfK) peptide specific for αvß3 integrin receptors targeting angiogenesis in most solid tumors or redBODIPY for conjugate cleavage monitoring via FRET, and (iii) pegylated-2-phenyl-3-hydroxy-4(1H)-quinolinone (3HQ) as a model drug. The model drug release is based on a self-immolative disulfide linker sensitive to environments containing thiols, especially glutathione, which is overexpressed in cancer cells. The results show effective thiol-mediated cleavage of the fluorescent reporter and the subsequent liberation of the drug in a tube. The conjugate with c(RGDfK) was confirmed to penetrate the cells via interaction with integrin receptors. Drug release from this conjugate is possible to monitor inside the cells. Further, the synthetic approach to the conjugates and the method of fluorescence monitoring of the drug release have also been described.

The effect of hydroxyquinoline-based gel on pessary-associated bacterial vaginosis: a multicenter randomized controlled trial.

OBJECTIVE: Pessaries are important options for women with pelvic floor disorders, but many pessary users experience bacterial vaginosis (BV). The aim of this study was to evaluate the effect of TrimoSan gel (Milex Pessaries, Cooper Surgical, Trumbull, CT) on BV prevalence among pessary users. STUDY DESIGN: Women presenting for a pessary fitting completed questionnaires on vaginal symptoms and hormone therapy use and underwent a BV BLUE test and slide collection for BV analysis by Nugent's criteria. Following pessary fitting, women were randomized to either standard pessary care with the use of TrimoSan placed vaginally twice weekly or to standard pessary care without TrimoSan gel. Women returned 2 weeks and 3 months later for a repeat slide collection for Gram stain, BV BLUE testing, and completion of questionnaires on vaginal symptoms and desire to continue the pessary. RESULTS: There were 184 women randomized after successful fitting (92 to the TrimoSan group), and 147 (79%) presented for 3-month follow up. Mean age was 56 ± 16 years; patients were mostly white (57%) or Hispanic (23%), and 36% were using hormone therapy. The groups did not differ in the prevalence of BV by Nugent's criteria at 2 weeks (20% TrimoSan vs 26% no gel, P = .46) or 3 months (24% TrimoSan vs 23% no gel, P = .82), nor did they differ in BV by BV BLUE testing at 2 weeks (0% TrimoSan vs 4% no gel, P = .12) or 3 months (3% TrimoSan vs 0% no gel, P = .15). The prevalence of at least one vaginal symptom did not differ between groups at 2 weeks (44% TrimoSan vs 45% no gel, P = .98) or 3 months (42% TrimoSan vs 32% no gel, P = .30). The TrimoSan group was equally likely to want to continue their pessary use compared with the standard care group at 2 weeks (90% vs 86%, P = .64) and 3 months (63% vs 60%, P = .76). CONCLUSION: TrimoSan gel in the first 3 months of pessary use does not decrease the prevalence of BV or vaginal symptoms and does not alter the likelihood of a woman desiring to continue pessary use.

Prediction of a potentially effective dose in humans for BAY 60-5521, a potent inhibitor of cholesteryl ester transfer protein (CETP) by allometric species scaling and combined pharmacodynamic and physiologically-based pharmacokinetic modelling.

AIMS: The purpose of this work was to support the prediction of a potentially effective dose for the CETP-inhibitor, BAY 60-5521, in humans. METHODS: A combination of allometric scaling of the pharmacokinetics of the CETP-inhibitor BAY 60-5521 with pharmacodynamic studies in CETP-transgenic mice and in human plasma with physiologically-based pharmacokinetic (PBPK) modelling was used to support the selection of the first-in-man dose. RESULTS: The PBPK approach predicts a greater extent of distribution for BAY 60-5521 in humans compared with the allometric scaling method as reflected by a larger predicted volume of distribution and longer elimination half-life. The combined approach led to an estimate of a potentially effective dose for BAY 60-5521 of 51 mg in humans. CONCLUSION: The approach described in this paper supported the prediction of a potentially effective dose for the CETP-inhibitor BAY 60-5521 in humans. Confirmation of the dose estimate was obtained in a first-in-man study.

[Novel controllers of airway obstruction in COPD patients]. / Nowe leki kontrolujace obturacje dróg oddechowych u chorych na POChP.

For improving effectiveness of chronic obstructive pulmonary disease (COPD) therapy is necessary to influence on pharmacologic receptors in the complementary way and to reduce the dose frequency. The once-daily dose administration is an important step which may allow to enhance of patients compliance. Novel long-acting bronchodilators--beta2-agonists or ultra long-acting beta2-agonists (LABAs) such as indacaterol and carmoterol--are under clinical application for the treatment of COPD patients. Moreover, some new long-acting antimuscarinic agents (LAMA) (aclidinium, glycopyrrolate) and dual-action ultra LABA+LAMA combination products are under development. The main target of therapeutic research is to produce a dimmer molecule known as M3 antagonist-beta2 agonist (MABA) bronchodilators which will open the door for a new range of combination products.

A Phase 2a randomized, single-center, double-blind, placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model.

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study. Trial registration: This study is registered with ClinicalTrials.gov as NCT04150250.

Co-delivery of IOX1 and doxorubicin for antibody-independent cancer chemo-immunotherapy.

Anti-programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) antibodies are currently used in the clinic to interupt the PD-1/PD-L1 immune checkpoint, which reverses T cell dysfunction/exhaustion and shows success in treating cancer. Here, we report a histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), which inhibits tumour histone demethylase Jumonji domain-containing 1A (JMJD1A) and thus downregulates its downstream ß-catenin and subsequent PD-L1, providing an antibody-independent paradigm interrupting the PD-1/PD-L1 checkpoint. Synergistically, IOX1 inhibits cancer cells' P-glycoproteins (P-gp) through the JMJD1A/ß-catenin/P-gp pathway and greatly enhances doxorubicin (DOX)-induced immune-stimulatory immunogenic cell death. As a result, the IOX1 and DOX combination greatly promotes T cell infiltration and activity and significantly reduces tumour immunosuppressive factors. Their liposomal combination reduces the growth of various murine tumours, including subcutaneous, orthotopic, and lung metastasis tumours, and offers a long-term immunological memory function against tumour rechallenging. This work provides a small molecule-based potent cancer chemo-immunotherapy.

Prophylactic P-selectin inhibition with PSI-421 promotes resolution of venous thrombosis without anticoagulation.

P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.

Changes in the Vaginal Microenvironment as Related to Frequency of Pessary Removal.

OBJECTIVE: The aim of the study was to describe the effect of frequency of pessary removal on the vaginal microenvironment. METHODS: We performed a secondary analysis of a multicenter randomized trial of hydroxyquinoline gel in women presenting for pessary fitting. Patients had vaginal secretions analyzed at baseline, 2 weeks, and 3 months. Patients were stratified by frequency of pessary removal at least once daily, at least once weekly, and less often than once weekly. These groups were compared for prevalence of Lactobacillus predominance (primary outcome), anaerobic predominance, Mobiluncus prominence, vaginal symptoms, and bacterial vaginosis by Nugent criteria, and correction for confounding variables was performed. RESULTS: One hundred thirty-seven women were included in this analysis: 34 (25%) removed the pessary daily, 54 (39%) at least weekly, and 49 (36%) less often than once weekly. Women who removed the pessary less often than weekly were older (P < 0.01), using more hormone therapy (P = 0.03), and more likely to have bacterial vaginosis at baseline (P < 0.01). At 2 weeks, the predominance of Lactobacillus in the group removing pessary daily was higher (41% daily vs 24% weekly vs 9% longer, P = 0.03) and this persisted after confounder correction (P < 0.01). Women who removed their pessary less than weekly were more likely to have anaerobic predominance at 3 months (P = 0.04). CONCLUSIONS: Women who remove their pessaries less often than once weekly have an increased prevalence of anaerobes at 3 months, but no difference in vaginal symptoms or pessary satisfaction.

Resolution of venous thrombosis using a novel oral small-molecule inhibitor of P-selectin (PSI-697) without anticoagulation.

P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; 2) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma D-dimer levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.

Additive Neuroprotective Effects of the Multifunctional Iron Chelator M30 with Enriched Diet in a Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common degenerative disease of the motoneuron system, involving various abnormalities, such as mitochondrial dysfunction, oxidative stress, transitional metal accumulation, neuroinflammation, glutamate excitotoxicity, apoptosis, decreased supply of trophic factors, cytoskeletal abnormalities, and extracellular superoxide dismutase (SOD)-1 toxicity. These multiple disease etiologies implicated in ALS gave rise to the perception that future therapeutic approaches for the disease should be aimed at targeting multiple pathological pathways. In line with this view, we have evaluated in the current study the therapeutic effects of low doses of the novel multifunctional monoamine oxidase (MAO) inhibitor/iron-chelating compound, M30 in combination with high Calorie Energy supplemented Diet (CED) in the SOD1-G93A transgenic mouse model of ALS. Our results demonstrated that the combined administration of M30 with CED produced additive neuroprotective effects on motor performance and increased survival of SOD1-G93A mice. We also found that both M30 and M30/CED regimens caused a significant inhibition of MAO-A and -B activities and decreased the turnover of dopamine in the brain of SOD1-G93A mice. In addition, M30/CED combined treatment resulted in a significant increase in mRNA expression levels of various mitochondrial biogenesis and metabolism regulators, such as peroxisome proliferator-activated receptor-γ (PPARγ)-co activator 1 alpha (PGC-1α), PPARγ, uncoupling protein 1, and insulin receptor in the gastrocnemius muscle of SOD1-G93A mice. These results suggest that a combination of drug/agents with different, but complementary mechanisms may be beneficial in the treatment of ALS.

Effect of Linomide on adhesion molecules, TNF-alpha, nitrogen oxide, and cell adhesion.

Linomide (quinoline-3-carboxamide) is an immunomodulator with anti-inflammatory effects in rodents with autoimmune diseases. Its mode of action still remains to be elucidated. We hypothesized that an investigation of T cell interactions with the extracellular matrix (ECM), composed of glycoproteins such as fibronectin (FN) and laminin (LN), might provide better understanding of their in vivo mode of action in extravascular inflammatory sites. We examined the effect of Linomide on T cell adhesion to intact ECM, and separately to LN, and FN, and on the release and production of tumor necrosis factor (TNFalpha) and nitrogen oxide (NO) in relation to adhesive molecules in non-obese diabetic (NOD) female spleen cells, focusing on intracellular adhesion molecule-1 (ICAM-1) and CD44. NOD female mice that developed spontaneous autoimmune insulitis, which destroys pancreatic islets and subsequently leads to insulin-deficient diabetes mellitus, were studied. Linomide, given in the drinking water or added to tissue cultures in vitro, inhibited the beta1 integrin-mediated adhesion of T cells to ECM, FN and LN, as well as the production and release of TNFalpha and NO, which play a major role in the induction and propagation of T cell-mediated insulitis. In addition, exposure of T cells to Linomide resulted in increased expression of CD44 and ICAM-1 molecules on spleen cells of Linomide-treated mice; such an increase in adhesion molecule expression may lead to more effective arrest of T cell migration in vivo. The regulation of T-cell adhesion, adhesion receptor expression, and inhibition of TNFalpha and NO secretion by Linomide may explain its beneficial role and provide a new tool for suppressing self-reactive T cell-dependent autoimmune diseases.

The antiangiogenic agent linomide inhibits the growth rate of von Hippel-Lindau paraganglioma xenografts to mice.

The aim of this study was to ascertain the potential usefulness of the antiangiogenic compound linomide for treatment of von Hippel-Lindau (VHL)-related tumors. Paraganglioma tissue fragments obtained at surgery from a VHL type 2a patient were transplanted s.c. to male BALB/c nu/nu (nude) mice: (a) 2-3-mm fragments for "prevention" experiments; and (b) 2-3-mm fragments allowed to grow to 1 cm for "intervention" studies. Both groups received either 0.5 mg/ml linomide in drinking water or acidified water and were followed until tumor diameter reached 3 cm or for 4 weeks. In both the prevention and intervention experiments, a significant diminution of tumor size and weight was observed in the drug-treated animals. In vivo nuclear magnetic resonance analysis of tumor blood flow in linomide-treated animals showed localization of blood vessels almost exclusively to the periphery of the poorly vascularized tumors with a significant reduction of both vascular functionality and vasodilation. Histological examination of tumors from linomide-treated animals revealed marked avascularity. Treated animals also displayed a 2.4-fold reduction of tumor vascular endothelial growth factor mRNA levels. Taken together, our data indicate that in VHL disease, therapy directed at inhibition of constitutively expressed VEGF induction of angiogenesis by VHL tumors may constitute an effective medical treatment.

Amelioration of diabetes in nonobese diabetic mice with advanced disease by linomide-induced immunoregulation combined with Reg protein treatment.

Oral linomide, (quinoline-3-carboxamide), has been shown to prevent autoimmune insulitis, islet destruction, and diabetes in NOD mice treated at an early stage of the disease, but confers only partial protection in animals with advanced disease. Reg protein, the gene product of a complementary DNA isolated from a regenerating rat islet library, has been previously shown to induce expansion of beta-cell mass in pancreatectomized rats. To determine the effect of treatment combining immunomodulation and Reg protein on advanced autoimmune diabetes, we treated female NOD mice with oral linomide and i.p. Reg protein injections. In 14-week-old animals with less severe disease (glucose tolerant), treatment with each agent alone resulted in amelioration of diabetes, as did treatment with Reg alone in 5-week-old prediabetic mice. In 14-week-old animals with more severe disease (glucose intolerant), only treatment with the combination of both agents, but not that with each separately, resulted in amelioration of diabetes. Our study suggests that treatment aimed at abrogation of autoimmunity combined with expansion of beta-cell mass constitutes a potential therapeutic approach for treatment of insulin-dependent diabetes mellitus.

Linomide in relapsing and secondary progressive MS: part I: trial design and clinical results. North American Linomide Investigators.

OBJECTIVE: To determine whether linomide (roquinimex) is better than placebo in slowing the time to confirmed clinical worsening in patients with relapsing-remitting (RR) and secondary progressive (SP) MS. METHODS: In this 27-center, randomized, double-blind, placebo-controlled, multiple-dose, phase III trial, 715 patients with active RRMS (n = 90) or SPMS (n = 625) were randomized to receive either linomide (1.0, 2.5, or 7.5 mg orally daily) or placebo. Patients were evaluated at 3-month intervals clinically and with MRI. The planned primary outcome was the time to the development of "confirmed" clinical worsening (increase of >/= 1.0 Expanded Disability Status Scale [EDSS] score for an enrollment EDSS score /= 0.5 point for an enrollment EDSS score of >/= 5.5) not associated with an acute relapse. RESULTS: The trial was terminated 1 month after it became fully enrolled due to unanticipated serious cardiopulmonary toxicities (pericarditis, pleural effusion, myocardial infarction, and possible pulmonary embolism), pancreatitis, and death. Notable arthralgia, myalgia, bursitis, and facial and peripheral edema were common adverse events. The high dose of linomide (7.5 mg) was not well tolerated. The trial was too brief to determine unequivocal clinical benefits. Trends suggested an unconfirmed early effect on change in EDSS score at 6 months for the medium dose (2.5 mg daily). CONCLUSION: MS patients may be more prone to develop important linomide treatment-related adverse events than other previously studied patients. However, linomide may be a potentially more toxic drug than was suspected from observations made in smaller studies for other indications. Phase III trials may identify infrequent and important toxicities that may not be anticipated by phase I and II trials.

Linomide in relapsing and secondary progressive MS: part II: MRI results. MRI Analysis Center of the University of Texas-Houston, Health Science Center, and the North American Linomide Investigators.

OBJECTIVE: To determine the safety and efficacy of roquinimex (linomide) in the management of relapsing-remitting and secondary progressive MS as monitored by MRI. BACKGROUND: Preclinical studies and several short term randomized trials of linomide suggested clinical and MRI-measured benefits with acceptable risk for closely followed MS patients. METHODS: The North American Linomide Trial formally screened 853 individuals for relapsing or secondary progressive, clinically definite MS; recent disease activity or progression; and an Expanded Disability Status Scale score at entry of 3.0 to 6.5 inclusive. MRI was obtained on 811 subjects at pre-enrollment, 718 cases at enrollment, and then at three monthly intervals until the trial was prematurely terminated for unacceptable toxicity. RESULTS: Enhancement was found on 40.2% of 718 entry scans. Statistically robust correlations were found between clinical demographic data and several entry MRI measures including CSF volume, a reflection of brain atrophy. Assessment of the effect of treatment on MRI-measured disease was limited by early trial termination. However, active treatment for 3 months reduced the proportion of patients with one or more enhancements. An exploratory analysis suggested that 2.5 mg was the most active of three doses tested in limiting the total volume of enhanced tissue, the proportion of MRI-defined lesions designated as "black holes," and by a novel MRI composite disease measure. CONCLUSIONS: The short term signature of the effect of linomide on MRI-measured aspects of the disease suggests that safer drugs of this class might be useful in the management of MS. The use of a composite index of the heterogeneous nature of the pathology of MS as captured by MRI may have merit as an outcome measure in clinical trials.

Abolition of the effect of cyclosporine on rat cardiac allograft rejection by the new immunomodulator LS-2616 (Linomide).

The effect of the quinoline-3-carboxamide LS-2616 (Linomide), given alone or together with cyclosporine, was studied in the first set cardiac allograft transplantation model in the rat. PVG rat hearts were transplanted heterotopically to Wistar/Kyoto rat recipients on day 0. The recipients were given LS-2616 orally on day -1 to rejection and/or CsA orally on days 0-9. In untreated animals rejection occurred on days 8-9, as judged by the absence of palpable pulsations. Treatment with CsA (5 or 10 mg/kg) resulted in prolongation of graft survival to days 17-21, i.e., the rejection occurred 8-10 days after cessation of treatment. LS-2616 in a dose of 160 mg/kg did not in itself have any impact on graft survival, but when given in doses of 40 or 160 mg/kg simultaneously with CsA (10 mg/kg), the effect of CsA was totally abolished. Animals treated with LS-2616 together with CsA had slightly lower trough blood levels than those treated with CsA alone. This interaction with CsA pharmacokinetics does not explain the results, as doubling of the CsA dose to 20 mg/kg, which well compensated for the difference in blood levels, was not sufficient to reverse the effect of LS-2616. To our knowledge this is the first compound known to abolish the effect of CsA. The mechanism is unknown, but is is possible that studies on the interaction between these two drugs will shed further light on the molecular basis of their modes of action.

Liposomes in silicosis investigations.

The effects of quartz and sodium metasilicate on liposomes were studied in order to understand the mechanism of silicosis. 8-Hydroxyquinoline-5-sulfonic acid was tested for its in situ silicosis-prevention capacity. Two types of liposomes--(A) those incorporating cholesterol and (B) those without cholesterol--were used. The tests consisted of measuring permeability changes caused by the above-mentioned chemicals. Permeabilities were found to depend on membrane composition. Tests on quartz action led us to the conclusion that liposomes of this composition did not simulate the erythrocytes very well. It was also observed that absence or presence of cholesterol and the mode of contact altered the effect of quartz. Silicate destabilized type A liposomes, but this was less than that caused by quartz. This was explained by the concentration of monosilicic acid that dissolves out from quartz and silicate. When quartz was pretreated with the preventive, the type A liposomes were stabilized, but a slight destabilizing effect was observed on type B. 8-Hydroxyquinoline-5-sulfonic acid augmented the destabilizing effect of silicate, whereas it decreased the hemolytic activity of uncoated quartz, indicating a preventive potential in in vivo.

Linomide does not prevent spontaneous autoimmune thyroiditis in NOD mice.

Linomide is a potent immunomodulator and has been reported to prevent type 1 diabetes mellitus in non-obese diabetic (NOD) mice and to reduce the incidence of other autoimmune diseases in animal models. The mechanisms of action seem to involve antigen expression by down regulation of macrophage activity and to antagonise the activation of Th1 cells during the cellular immune response. With the purpose to investigate the effect of Linomide on the incidence of spontaneous autoimmune thyroiditis (AIT) in female NOD mice we administered Linomide in drinking water (100 mg/kg/day) to NOD mice from 5th to 19th week of age. The mice were sacrificed at the end of week 19. None of the mice developed diabetes during the study period. The incidence of thyroiditis was evaluated on paraffin HE-stained sections and graduated on a scale from 0 to 4. Thirty-two percent of 37 mice treated with Linomide developed thyroiditis compared to 45% of 22 controls (p=0.31, chi2 =1.00). Among the mice who developed thyroiditis no difference in the degree of thyroiditis was found. Therefore no beneficial effect of Linomide on the incidence of spontaneous AIT in NOD mice could be demonstrated.

Effects of the immunomodulator linomide on macrophage migration and myelin phagocytic activity in peripheral nerve trauma: an experimental study.

Wallerian degeneration after peripheral nerve transection leads to the phagocytosis of degenerated myelin and axon components by macrophages. These phagocytes are recruited from the systemic circulation and Wallerian degeneration may therefore be used as a model for myelin removal by hematogenous macrophages, a feature that is also a hallmark of demyelinating diseases of the central and peripheral nervous system. The immunomodulator linomide has been shown to be effective in the treatment of experimental demyelinating diseases although the exact mode of its action is not yet defined. The present study investigated the effect of linomide on monocyte invasion and myelin phagocytosis after sciatic nerve transection. Linomide had a dual effect in Wallerian degeneration. Monocyte migration from the circulation to the damaged nervous system was significantly reduced. Additionally, the myelin phagocytic capacity of macrophages was impaired, finally resulting in a significant delay in the removal of myelin. The present experiments may provide an explanation for the effects of linomide during the course of demyelinating diseases of the nervous system.

The immunomodulator LS-2616 (linomide) more effectively than sensitization of the donor enhances graft-versus-host reaction after small bowel transplantation.

The effects of the immunomodulating substance LS-2616 (linomide) on graft-versus-host reaction (GVHR) were investigated in a semi-syngeneic small bowel transplantation model. The entire bowel of Lewis donors were transplanted heterotopically into (Lewis x BN) F1 hybrids. Both untreated animals and animals treated with LS-2616, in a daily dose of 160 mg/kg, developed a lethal GVHR. The median survival time in untreated animals was 14.5 days while in LS-2616 treated animals it was just eight days (p < 0.01). LS-2616 in combination with cyclosporin A (CyA), 15 mg/kg given orally on days 0-20, did not seem to alter the survival times compared with CyA treatment alone; 56% of the animals treated with CyA survived for more than 100 days and after combined treatment with CyA/LS-2616 there were 50% permanent survivors. Also the effect of earlier sensitization of the donor on the course of GVHR was investigated. Hearts from BN rats were transplanted heterotopically to the neck vessels of Lewis rats. The hearts were rejected on about day six; five days later the bowels were harvested and transplanted into (Lewis x BN) F1 hybrids. The median survival time in this group was 12.5 days. Taken together our results, in combination with earlier findings, suggest that, at the level of effector mechanisms, GVHR is not an exact mirror image of rejection. Also, LS-2616 appears to be a useful tool for further studies of the mechanisms of action of GVHR.