Phase 2 Trial of Iberdomide in Systemic Lupus Erythematosus.

BACKGROUND: Iberdomide, a cereblon modulator promoting degradation of the transcription factors Ikaros and Aiolos, which affect leukocyte development and autoimmunity, is being evaluated for the treatment of systemic lupus erythematosus (SLE). METHODS: In this phase 2 trial, we randomly assigned patients in a 2:2:1:2 ratio to receive oral iberdomide (at a dose of 0.45, 0.30, or 0.15 mg) or placebo once daily for 24 weeks, in addition to standard medications. The primary end point at week 24 was a response on the SLE Responder Index (SRI-4), which was defined as a reduction of at least 4 points in the Systemic Lupus Erythematosus Disease Activity Index 2000 score (a 24-item weighted score of lupus activity that ranges from 0 to 105, with higher scores indicating greater disease activity), no new disease activity as measured on the British Isles Lupus Assessment Group 2004 index, and no increase of 0.3 points or more in the Physician's Global Assessment score (on a visual-analogue scale ranging from 0 [no disease activity] to 3 [maximal disease]). RESULTS: A total of 288 patients received the assigned intervention: 81 received iberdomide at a dose of 0.45 mg, 82 received iberdomide at a dose of 0.30 mg, 42 received iberdomide at a dose of 0.15 mg, and 83 received placebo. At week 24, the percentages of patients with an SRI-4 response were 54% in the iberdomide 0.45-mg group, 40% in the iberdomide 0.30-mg group, 48% in the iberdomide 0.15-mg group, and 35% in the placebo group (adjusted difference between the iberdomide 0.45-mg group and the placebo group, 19.4 percentage points; 95% confidence interval, 4.1 to 33.4; P = 0.01), with no significant differences between the groups that received the lower doses of iberdomide and the group that received placebo. Iberdomide-associated adverse events included urinary tract and upper respiratory tract infections and neutropenia. CONCLUSIONS: In this 24-week, phase 2 trial involving patients with SLE, iberdomide at a dose of 0.45 mg resulted in a higher percentage of patients with an SRI-4 response than did placebo. Data from larger, longer trials are needed to determine the efficacy and safety of iberdomide in SLE. (Funded by Bristol Myers Squibb; ClinicalTrials.gov number, NCT03161483; EudraCT number, 2016-004574-17.).

First-in-Human, Single- and Multiple-Ascending-Dose Studies in Healthy Subjects to Assess Pharmacokinetics, Pharmacodynamics, and Safety/Tolerability of Iberdomide, a Novel Cereblon E3 Ligase Modulator.

Pharmacokinetics, pharmacodynamics, and safety/tolerability of iberdomide (CC-220), a highly potent oral cereblon E3 ligase modulator (CELMoD), were evaluated in escalating single-dose (0.03, 0.1, 0.3, 1, 2, 4, 6 mg) and multiple-dose (0.3 mg once daily for 14 days, 1 mg once daily for 28 days, 0.3 mg once daily for 28 days, or 1 mg once daily for 7 days with a 7-day washout, then once daily for 7 more days) studies in healthy subjects (n = 99). Iberdomide exposure increased in a dose-proportional manner. Terminal half-life was 9-13 hours after a single dose. Iberdomide decreased peripheral CD19+ B lymphocytes (Emax , 92.4%; EC50 , 0.718 ng/mL), with modest reductions in CD3+ T lymphocytes (Emax , 34.8%; EC50 , 0.932 ng/mL). Lipopolysaccharide-stimulated proinflammatory cytokines (IL-1α, IL-1ß) were reduced, but anti-CD3-stimulated IL-2 and interferon-γ were increased. Iberdomide 1 mg once daily partially decreased T-cell-independent antibody responses to PPV23 but did not change tetanus toxoid recall response. Pharmacodynamic data suggest dose-dependent, differential immunomodulatory effects on B and T lymphocytes. Iberdomide was tolerated up to 6 mg as a single dose and at 0.3 mg once daily for 4 weeks. Grade 3 asymptomatic neutropenia was observed following 1 mg once daily for 21 days; a 7-day drug holiday alleviated neutropenia. Further investigation of iberdomide in autoimmune and hematological diseases is warranted.

NHF-derived carbon dots: prevalidation approach in breast cancer treatment.

Metastatic breast cancer dominates the female cancer-related mortality. Tumour-associated molecules represents a crucial for early disease detection and identification of novel therapeutic targets. Nanomaterial technologies provide promising novel approaches to disease diagnostics and therapeutics. In the present study we extend the investigations of antitumoral properties of Carbon Dots prepared from N-hydroxyphthalimide (CD-NHF) precursor. We evaluate the effect of CD-NHF on tumour cell migration and invasion in vitro and their impact on tumour progression using an in vivo model. Furthermore, we investigate the molecular mechanisms involved in CD-NHF antitumour effects. In vivo mammary tumours were induced in Balb/c female mice by injecting 4T1 cells into the mammary fat pad. Conditional treatment with CD-NHF significantly impair both migration and invasion of metastatic breast cancer cells. The presence of CD-NHF within the 3D cell cultures strongly inhibited the malignant phenotype of MDA-MB-231, 4T1 and MCF-7 cells in 3D culture, resulting in culture colonies lacking invasive projections and reduction of mammospheres formation. Importantly, breast tumour growth and metastasis dissemination was significantly reduced upon CD-NHF treatments in a syngeneic mouse model and is associated with down-regulation of Ki67 and HSP90 expression. CD-NHF nanostructures provide exciting perspective for improving treatment outcome in breast cancer.

A novel insight into nitrogen and auxin signaling in lateral root formation in tea plant [Camellia sinensis (L.) O. Kuntze].

BACKGROUND: Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS: A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION: This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

A novel phenylphthalimide derivative, pegylated TC11, improves pharmacokinetic properties and induces apoptosis of high-risk myeloma cells via G2/M cell-cycle arrest.

Despite the development of new drugs for multiple myeloma (MM), the prognosis of MM patients with high-risk cytogenetic abnormalities such as t (4; 14) and del17p remains poor. We reported that a novel phenylphthalimide derivative, TC11, induced apoptosis of MM cells in vitro and in vivo, and TC11 directly bound to α-tubulin and nucleophosmin-1 (NPM1). However, TC11 showed low water solubility and poor pharmacokinetic properties. Here we synthesized a water-soluble TC11-derivative, PEG(E)-TC11, in which HOEtO-TC11 is pegylated with PEG through an ester bond, and we examined its anti-myeloma activity. We observed that PEG(E)-TC11 and its hydrolyzed product, HOEtO-TC11, induced G2/M arrest and the apoptosis of MM cells. Intraperitoneal administration of PEG(E)-TC11 to xenografted mice revealed improved pharmacokinetic properties and significantly delayed tumor growth. TC11 and its derivatives did not bind to cereblon (CRBN), which is a responsible molecule for thalidomide-induced teratogenicity. These results suggest that PEG(E)-TC11 is a good candidate drug for treating high-risk MM.

N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells.

BACKGROUND: N-Hydroxyphthalimide (NHPI), an important chemical raw material, was found to have potent and selective anti-proliferative effect on human breast carcinoma BT-20 cells, human colon adenocarcinoma LoVo and HT-29 cells during our screening for anticancer compounds. The purpose of this study is to assess the antitumor efficacy of NHPI in vitro and in vivo and to explore the underlying antitumor mechanism. METHODS: Cell cytotoxicity of NHPI was evaluated using MTS assay and cell morphological analysis. After NHPI treatment, cell cycle, apoptosis and mitochondrial membrane potential were analyzed using flow cytometer. The subcellular localization of eukaryotic initiation factor 4E (eIF4E) was analyzed by immunofluorescence assay. The antitumor efficacy of NHPI in vivo was tested in BT-20 xenografts. The underlying antitumor mechanisms of NHPI in vitro and in vivo were investigated with western blot analysis in NHPI-treated cancer cells and tumor tissues. Statistical significance was determined using Student's t-test. RESULTS: In vitro, NHPI selectively inhibited the proliferation and induced G2/M phase arrest in BT-20 and LoVo cells, which was attributed to the inhibition of cyclin B1 and cdc2 expressions. Furthermore, NHPI induced apoptosis via mitochondrial pathway. Of note, NHPI effectively inhibited mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling, and overcame the feedback activation of Akt and extracellular signal-regulated kinase (ERK) caused by mTORC1 inhibition in BT-20 and LoVo cells. In vivo, NHPI inhibited tumor growth and suppressed mTORC1 and mTORC2 signaling in BT-20 xenografts with no obvious toxicity. CONCLUSIONS: We found for the first time that NHPI displayed antitumor activity which is associated with the inhibition of mTOR signaling pathway. Our findings suggest that NHPI may be developed as a promising candidate for cancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

An open-label, dose-escalation study to evaluate the safety and pharmacokinetics of CEP-9722 (a PARP-1 and PARP-2 inhibitor) in combination with gemcitabine and cisplatin in patients with advanced solid tumors.

Poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors may potentiate chemotherapy by hindering DNA damage repair pathways. CEP-9722 is the prodrug of CEP-8983, a selective inhibitor of PARP-1 and PARP-2. Preclinical studies and a prior phase 1 study suggested that CEP-9722 may cause less myelosuppression than has been observed with other oral PARP inhibitors. The primary objective of this study was to determine the maximum-tolerated dose of CEP-9722 in combination with gemcitabine and cisplatin in patients with advanced solid tumors. All patients received cisplatin 75 mg/m(2) on day 1 and gemcitabine 1250 mg/m(2) on days 1 and 8 of a 21-day cycle. Patients who completed one cycle of chemotherapy alone continued chemotherapy in combination with CEP-9722 150, 200, 300, or 400 mg orally twice daily on days 2-7, with dose-limiting toxicity assessed in cycle 2. Patients experiencing clinical benefit could continue treatment until disease progression or unacceptable toxicity. Thirty-two patients enrolled; 18 patients completed cycle 1 and received chemotherapy plus CEP-9722. The median (range) treatment administration with CEP-9722 was five (1-12) cycles. No patient experienced dose-limiting toxicity with CEP-9722 treatment. Grade 3/4 hematologic adverse events included neutropenia (28%) and leukopenia (11%); adverse events led to discontinuation in 33% of patients. One patient achieved complete response, three had partial responses, and 11 had stable disease; however, the relative contribution of CEP-9722 and/or the chemotherapeutic agents cannot be determined from this single-arm design. This study was discontinued before determination of the maximum-tolerated dose because of highly variable CEP-8983 exposure in all cohorts and toxicity, particularly chemotherapy-induced myelosuppression.

Pharmacokinetics of the Experimental Non-Nucleosidic DNA Methyl Transferase Inhibitor N-Phthalyl-L-Tryptophan (RG 108) in Rats.

DNA methyl transferase (DNMT) inhibitors can re-establish the expression of tumour suppressor genes in malignant diseases, but might also be useful in other diseases. Inhibitors in clinical use are nucleosidic cytotoxic agents that need to be integrated into the DNA of dividing cells. Here, we assessed the in vivo kinetics of a non-nucleosidic inhibitor that is potentially free of cytotoxic effects and does not require cell division. The non-specific DNMT inhibitor N-phthalyl-L-tryptophan (RG 108) was injected subcutaneously in rats. Blood was drawn 0, 0.5, 1, 2, 4, 6, 8 and 24 hr after injection and RG 108 in plasma was measured by high-performance liquid chromatography coupled to mass spectrometry. Trough levels and area under the curve (AUC) were significantly higher with multiple-dose administration and cytochrome inhibition. In this group, time to maximal plasma concentration (tmax , mean ± S.D.) was 37.5 ± 15 min., terminal plasma half-life was approximately 3.7 h (60% CI: 2.1-15.6 h), maximal plasma concentration (Cmax) was 61.3 ± 7.6 µM, and AUC was 200 ± 54 µmol·h/l. RG 108 peak levels were not influenced by cytochrome inhibition or multiple-dose administration regimens. Maximal tissue levels (Cmax in µmol/kg) were 6.9 ± 6.7, 1.6 ± 0.4 and 3.4 ± 1.1 in liver, skeletal and heart muscle, respectively. We conclude that despite its high lipophilicity, RG 108 can be used for in vivo experiments, appears safe and yields plasma and tissue levels in the range of the described 50% inhibitory concentration of around 1 to 5 µM. RG 108 can therefore be a useful tool for in vivo DNMT inhibition.

A rapid screening system evaluates novel inhibitors of DNA methylation and suggests F-box proteins as potential therapeutic targets for high-risk neuroblastoma.

After extensive research on radiochemotherapy, 5-year survival rates of children with high risk neuroblastoma still do not exceed 50%, owing to adverse side-effects exemplified by doxorubicin-induced cardiomyopathy. A promising new approach is the combination of conventional therapies with specific modulation of cell signaling pathways promoting therapeutic resistance, such as inhibition of aberrant kinase activity or re-expression of silenced tumor suppressor genes by means of chromatin remodeling. In this regard, we established a system that allows to identify potential drug targets as well as to validate respective candidate inhibitors in high-risk neuroblastoma model cell lines. Cell culture, drug exposure, shRNA-mediated knockdown and phenotype analysis are integrated into an efficient and versatile single well-based protocol. By utilizing this system, we assessed RG108, SGI-1027 and nanaomycin A, three novel DNA methyltransferase inhibitors that have not been tested in neuroblastoma cell lines so far, for their potential of synergistic anti-tumor activity in combination with doxorubicin. We found that, similarly to azacytidine, SGI-1027 and nanaomycin A mediate synergistic growth inhibition with doxorubicin independently of N-Myc status. However, they display high cytotoxicity but lack global DNA demethylation activity. Secondly, we conducted a lentiviral shRNA screen of F-box proteins, key regulators of protein stability, and identified Fbxw11/ß-TrCP2 as well as Fbxo5/Emi1 as potential therapeutic targets in neuroblastoma. These results complement existing studies and underline the reliability and versatility of our single well-based protocol.

A combination treatment with DNA methyltransferase inhibitors and suramin decreases invasiveness of breast cancer cells.

The treatment of patients with invasive breast cancer remains a major issue because of the acquisition of drug resistance to conventional chemotherapy. Here we propose a new therapeutic strategy by combining DNA methyltransferase inhibitors (DMTIs) with suramin. Cytotoxic effects of suramin or combination treatment with DMTIs were determined in highly invasive breast cancer cell lines MDA-MB-231, BT-20 and HCC1954, or control cells. In addition, effects on cell invasion were determined in 3-dimensional cell culture assays. DMTI-mediated upregulation of Protein Kinase D1 (PKD1) expression was shown by Western blotting. Effects of suramin on PKD1 activity was determined in vitro and in cells. The importance of PKD1 in mediating the effects of such combination treatment in cell invasion was demonstrated using 3D cell culture assays. A proof of principal animal experiment was performed showing that PKD1 is critical for breast cancer growth. We show that when used in combination, suramin and DMTIs impair the invasive phenotype of breast cancer cells. We show that PKD1, a kinase that previously has been described as a suppressor of tumor cell invasion, is an interface for both FDA-approved drugs, since the additive effects observed are due to DMTI-mediated re-expression and suramin-induced activation of PKD1. Our data reveal a mechanism of how a combination treatment with non-toxic doses of suramin and DMTIs may be of therapeutic benefit for patients with aggressive, multi-drug resistant breast cancer.

Anti-tumoral effect of the non-nucleoside DNMT inhibitor RG108 in human prostate cancer cells.

BACKGROUND: Current therapeutic strategies for advanced prostate cancer (PCa) are largely ineffective. Because aberrant DNA methylation associated with inappropriate gene-silencing is a common feature of PCa, DNA methylation inhibitors might constitute an alternative therapy. In this study we aimed to evaluate the anti-cancer properties of RG108, a novel non-nucleoside inhibitor of DNA methyltransferases (DNMT), in PCa cell lines. METHODS: The anti-tumoral impact of RG108 in LNCaP, 22Rv1, DU145 and PC-3 cell lines was assessed through standard cell viability, apoptosis and cell cycle assays. Likewise, DNMT activity, DNMT1 expression and global levels of DNA methylation were evaluated in the same cell lines. The effectiveness of DNA demethylation was further assessed through the determination of promoter methylation and transcript levels of GSTP1, APC and RAR-ß2, by quantitative methylation-specific PCR and RT-PCR, respectively. RESULTS: RG108 led to a significant dose and time dependent growth inhibition and apoptosis induction in LNCaP, 22Rv1 and DU145. LNCaP and 22Rv1 also displayed decreased DNMT activity, DNMT1 expression and global DNA methylation. Interestingly, chronic treatment with RG108 significantly decreased GSTP1, APC and RAR-ß2 promoter hypermethylation levels, although mRNA reexpression was only attained for GSTP1 and APC. CONCLUSIONS: RG108 is an effective tumor growth suppressor in most PCa cell lines tested. This effect is likely mediated by reversion of aberrant DNA methylation affecting cancer related-genes epigenetically silenced in PCa. However, additional mechanism might underlie the anti-tumor effects of RG108. In vivo studies are now mandatory to confirm these promising results and evaluate the potential of this compound for PCa therapy.

Synthesis and pharmacological evaluation of novel phenyl sulfonamide derivatives designed as modulators of pulmonary inflammatory response.

In this paper we report the design, synthesis and pharmacological evaluation of a new series of phenyl sulfonamide derivatives 2a-h and 3-8 planned by structural modification on the anti-inflammatory prototype LASSBio-468 (1). Among the synthesized analogues, the tetrafluorophthalimide LASSBio-1439 (2e) stands out showing an in vitro anti-TNF-α effect similar to the standard thalidomide. The relevance of tetrafluorination of the phthalimide nucleus was also confirmed by the anti-inflammatory profile of 2e, through oral administration, in a murine model of pulmonary inflammation. The corresponding tetrafluorocarboxyamide metabolite LASSBio-1454 (15), generated from partial hydrolysis of the derivative 2e, presented a significant in vitro effect and a pronounced anti-inflammatory activity in vivo.

Potential inhibitory effect of LASSBio-596, a new thalidomide hybrid, on inflammatory corneal angiogenesis in rabbits.

AIMS: Evaluate the effect of LASSBio-596, structurally designed as a new hybrid of thalidomide, on inflammatory corneal angiogenesis. METHODS: Eighteen rabbits were submitted to an alkaline cauterization in the right cornea. The animals were randomly allocated to three groups: vehicle, dexamethasone and LASSBio-596. Drugs were administered by eyedrops 3 times a day for 21 days. Evaluations were performed on days 3, 6, 9, 12, 15, 18 and 21 after cauterization. At these time points, digital images of the cornea were captured in a standard fashion. The angiogenic response was measured using software that was developed specifically for this purpose. It calculated the following parameters: neovascularization area (NA), total vascular length (TVL) and blood vessel number (BVN). RESULTS: It was observed that dexamethasone significantly decreased NA, TVL and BVN during all assessments. From the NA the angiogenesis rate (AR) was calculated in each group. Therefore, dexamethasone completely inhibited the inflammatory corneal angiogenesis with an AR of -0.001 ± 0.006 mm(2)/day, which was significantly lower (p < 0.001) than that observed after treatment with vehicle (0.078 ± 0.024 mm(2)/day) and LASSBio-596 (0.054 ± 0.012 mm(2)/day). Although LASSBio-596 reduced angiogenesis in relation to vehicle, according to NA, TVL and BVN values, this difference was not statistically significant. However, it was found that the AR as measured in the LASSBio-596 group was significantly lower (p < 0.05) than that seen in control animals, indicating a potential antiangiogenic effect. CONCLUSION: We conclude that topical application of LASSBio-596 at 1.0% has a potential inhibitory effect on inflammatory corneal angiogenesis in rabbits.

Toxicokinetics of captan and folpet biomarkers in orally exposed volunteers.

The time courses of key biomarkers of exposure to captan and folpet was assessed in accessible biological matrices of orally exposed volunteers. Ten volunteers ingested 1 mg kg(-1) body weight of captan or folpet. Blood samples were withdrawn at fixed time periods over the 72 h following ingestion and complete urine voids were collected over 96 h post-dosing. The tetrahydrophthalimide (THPI) metabolite of captan along with the phthalimide (PI) and phthalic acid metabolites of folpet were then quantified in these samples. Plasma levels of THPI and PI increased progressively after ingestion, reaching peak values ~10 and 6 h post-dosing, respectively; subsequent elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 15.7 and 31.5 h, respectively. In urine, elimination rate time courses of PI and phthalic acid evolved in parallel, with respective t(½) of 27.3 and 27.6 h; relatively faster elimination was found for THPI, with mean t(½) of 11.7 h. However, phthalic acid was present in urine in 1000-fold higher amounts than PI. In the 96 h period post-treatment, on average 25% of folpet dose was excreted in urine as phthalic acid as compared with only 0.02% as PI. The corresponding value for THPI was 3.5%. Overall, THPI and PI appear as interesting biomarkers of recent exposure, with relatively short half-lives; their sensitivity to assess exposure in field studies should be further verified. Although not a metabolite specific to folpet, the concomitant use of phthalic acid as a major biomarker of exposure to folpet should also be considered.

Post-intoxication inhibition of botulinum neurotoxin serotype A within neurons by small-molecule, non-peptidic inhibitors.

Botulinum neurotoxins (BoNTs) comprise seven distinct serotypes that inhibit the release of neurotransmitter across neuromuscular junctions, resulting in potentially fatal flaccid paralysis. BoNT serotype A (BoNT/A), which targets synaptosomal-associated protein of 25kDa (SNAP-25), is particularly long-lived within neurons and requires a longer time for recovery of neuromuscular function. There are currently no treatments available to counteract BoNT/A after it has entered the neuronal cytosol. In this study, we examined the ability of small molecule non-peptidic inhibitors (SMNPIs) to prevent SNAP-25 cleavage post-intoxication of neurons. The progressive cleavage of SNAP-25 observed over 5 h following 1 h BoNT/A intoxication was prevented by addition of SMNPIs. In contrast, anti-BoNT/A neutralizing antibodies that strongly inhibited SNAP-25 cleavage when added during intoxication were completely ineffective when added post-intoxication. Although Bafilomycin A1, which blocks entry of BoNT/A into the cytosol by preventing endosomal acidification, inhibited SNAP-25 cleavage post-intoxication, the degree of inhibition was significantly reduced versus addition both during and after intoxication. Post-intoxication application of SMNPIs, on the other hand, was nearly as effective as application both during and after intoxication. Taken together, the results indicate that competitive SMNPIs of BoNT/A light chain can be effective within neurons post-intoxication.

LASSBio 596 per os avoids pulmonary and hepatic inflammation induced by microcystin-LR.

Cyanobacterial blooms that generate microcystins (MCYSTs) are increasingly recognized as an important health problem in aquatic ecosystems. We have previously reported the impairment of pulmonary structure and function by microcystin-LR (MCYST-LR) exposure as well as the pulmonary improvement by intraperitoneally injected (i.p.) LASSBio 596. In the present study, we aimed to evaluate the usefulness of LASSBio 596 per os on the treatment of pulmonary and hepatic injuries induced by MCYST-LR. Swiss mice received an intraperitoneal injection of 40 µl of saline (CTRL) or a sub-lethal dose of MCYST-LR (40 µg/kg). After 6 h the animals received either saline (TOX and CTRL groups) or LASSBio 596 (50 mg/kg, LASS group) by gavage. Eight hours after the first instillation, lung impedance (static elastance, elastic component of viscoelasticity and resistive, viscoelastic and total pressures) was determined by the end-inflation occlusion method. Left lung and liver were prepared for histology. In lung and hepatic homogenates MCYST-LR, TNF-α, IL-1ß and IL-6 were determined by ELISA. LASSBio 596 per os (LASS mice) kept all lung mechanical parameters, polymorphonuclear (PMN) cells, pro-inflammatory mediators, and alveolar collapse similar to control mice (CTRL), whereas in TOX these findings were higher than CTRL. Likewise, liver structural deterioration (hepatocytes inflammation, necrosis and steatosis) and inflammatory process (high levels of pro-inflammatory mediators) were less evident in the LASS than TOX group. LASS and CTRL did not differ in any parameters studied. In conclusion, orally administered LASSBio 596 prevented lung and hepatic inflammation and completely blocked pulmonary functional and morphological changes induced by MCYST-LR.

Enhanced therapeutic effect of LK-423 in treating experimentally induced colitis in rats when administered in colon delivery microcapsules.

LK-423 is a phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity. In the present study the therapeutic efficacy of a colon-specific drug delivery system–LK-423 microcapsules–was examined in the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced ulcerative colitis model in rats. The colon-specific delivery of the drug using microcapsules relies on the combination of pH (outer gastroresistant coating), time (inner retard coating of Eudragit® RS and RL) and enzyme (pectin core) controlled drug release mechanisms. The optimal in vitro dissolution profile for LK-423 delivery to the colon of rats was obtained after coating newly developed LK-423 loaded pectin cores with 20% w/w of retard coating with a Eudragit® RS/RL ratio of 8.5/1.5 and 30% w/w of enteric coating. Orally administered LK-423 microcapsules were therapeutically more beneficial in treating TNBS-induced ulcerative colitis in rats than orally or rectally administered LK-423 in the form of suspension. Clinical activity scores and colon weight to length ratio were insignificantly lower and the macroscopically estimated degree of healing was significantly greater. On the histological level, the administration of LK-423 microcapsules resulted in most physiological regeneration of intestinal mucosa, indicated by regular architecture of all mucosal tissue components, what is probably related to local drug delivery near the site of inflammation achieved using microcapsules. These results demonstrate that LK-23 colon delivery microcapsules enhance the therapeutic efficacy of the drug and therefore appear to be a useful approach for treating various inflammatory diseases in the large intestine.

Physicochemical and preclinical pharmacokinetic and toxicological evaluation of LK-423--a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity.

INTRODUCTION: LK-423 is a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity. As optimized delivery to the site of action appears crucial for further preclinical development of LK-423, the aim of this study was to perform a physicochemical and preclinical pharmacokinetic and toxicological evaluation. METHODS: The solubility, partition coefficient, permeability, and stability profile were determined. Pharmacokinetics were evaluated in rats following intravenous and oral application of LK-423, and in dogs after intravenous administration and oral administration of microcapsules, designed for colon-specific delivery of LK-423 based on pH-, time-, and enzyme-controlled release mechanisms. Additionally, the acute and subchronic toxicity was examined. RESULTS AND DISCUSSION: LK-423 is hydrophilic, sparingly to slightly soluble, and poorly permeable. Stability profile in aqueous solution is pH dependent. A pharmacokinetic study following intravenous application to rats and dogs revealed that LK-423 is rapidly eliminated with a short terminal phase half-life, and high plasma clearance, as well as a limited distribution to the peripheral tissue. Oral bioavailability of LK-423 is low, presumably due to low permeability. Debris of insoluble microcapsule coating in feces and obtained plasma concentration profiles confirm that LK-423 microcapsules are a promising approach for local treatment of inflammatory diseases of the large intestine. Acute and a subchronic toxicity results indicate that LK-423 is a safe and nontoxic drug under the applied experimental conditions.