Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC₅₀ values and achieved picomolar DC₅₀ values and >99% of maximum degradation (D_max) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-MET^Y1230H and c-MET^D1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.     

Growth of human glioblastomas as xenografts in the brains of athymic rats

BACKGROUND: We have developed xenografts of human glioblastoma (GBM) and established the baseline growth parameters and histopathological features of these tumors. MATERIALS-METHODS: Cells from 4 different human GBM cell lines were injected into the right caudate-putamen of brain in athymic rats. We measured tumor weights and the estimated survival time of each rat. RESULTS-CONCLUSION: U-251 MG and U-87 MG cells produced solid intracerebral tumors with a 100% tumor take rate, while SF-767 and SF-126 cells did not grow in the brains of athymic rats. Under the conditions employed, U-87 MG tumors grew faster than U-251 MG tumors, but both types of tumors exhibited reproducible growth characteristics from animal to animal. There was heterogeneity in the growth characteristics and histologies between the 2 tumor types, indicating that these tumor models might be useful for simulating some of the heterogeneity that occurs between GBM in humans.     

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels – A Strategy for Brain Cancer Treatments

Purpose

The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression.

Methods

Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg^?1) or ethanolic lomustine (6.5 mg kg^?1) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg^?1) or ethanolic lomustine (daily 1.2 mg kg^?1 - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated.

Results

The MET formulation resulted in modest brain targeting (brain/ bone AUC_0-4h ratios for MET and ethanolic lomustine?=?0.90 and 0.53 respectively and brain/ liver AUC_0-4h ratios for MET and ethanolic lomustine?=?0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes.

Conclusions

Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity.

     

Yhhu3813 is a novel selective inhibitor of c-Met kinase that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells

Aim： c-Met kinase deregulation is strongly associated with the formation, progression and dissemination of human cancers. In this study we identified Yhhu3813 as a small-molecule inhibitor of c-Met kinase and characterized its antitumor properties both in vitro and in vivo.
Methods： The activities of different kinases were measured using ELISA assays and signaling proteins in the cells were detected with Western blotting. Cell proliferation was assessed using SRB or MTT assay in twenty human cell lines and cell cycle distribution was determined with flow cytometry. Transwell-based assay was used to evaluate cell migration and invasion. Cell invasive growth was detected by a morphogenesis assay. c-Met overactivated human NSCLC cell line EBC-1 xenografts were used to evaluate the in vivo anti-tumor efficacy.

Results： Yhhu3813 potently inhibited c-Met kinase activity in vitro with an IC50 value of 2.4±0.3 nmol/L, 〉400-fold higher than that for a panel of 15 different tyrosine kinases, suggesting a high selectivity of Yhhu3813. The compound （20, 100 and 500 nmol/L） dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and Erk signal cascades in multiple c-Met aberrant human cancer cell lines, regardless of the mechanistic complexity in c-Met activation across different cellular contexts. In 20 human cancer cell lines harboring different backgrounds of c-Met expression/activation, Yhhu3813 potently inhibited c-Met-driven cell proliferation via arresting cells at G1/S phase. Furthermore, Yhhu3813 substantially impaired c-Met-mediated cell migration, invasion, scattering, and invasive growth. Oral administration of EBC-1 xenograft mice with Yhhu3813 （50 or 100 mg·kg-1·d-1, qd, for 2 weeks） dose-dependently suppressed the tumor growth, which was correlated with a reduction in the intratumoral proliferation index and c-Met signaling.

Conclusion： Yhhu3813 is a potent selective inhibitor of c-Met that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.     

Targeted near-IR hybrid magnetic nanoparticles for in vivo cancer therapy and imaging

We report the use of immuno-targeted gold–iron oxide hybrid nanoparticles for laser-assisted therapy and for MRI-based imaging as demonstrated in xenograft colorectal cancer tumor model. Immuno-targeted gold–iron oxide nanoparticles selectively accumulate in SW1222 xenograft tumors as compared to the accumulation in non-antigen-expressing tumor xenografts. Effective photothermal treatment using near-IR laser irradiation (808 nm, 5 W cm^? 2) application is shown where > 65% of the antigen-expressing tumor cells presented corrupt extracellular matrix and cytoplasmic acidophilia suggesting effectiveness of nanoparticle-assisted thermal therapy. Cell killing was confirmed by hematoxylin and eosin (H&E) histological staining where scar-like structure containing collagen bundles was observed in the treatment group. Further, systemically injected HNPs were shown to be effective T₂ magnetic resonance (MR) imaging contrast agents, localized and detected at the antigen-expressing xenograft tumors. These findings suggest that the new class of bio-conjugated HNPs exhibits great potential for dual-therapy and diagnostics (theranostics) applications.From the Clinical EditorThis team reports the successful use of immuno-targeted gold-iron oxide hybrid nanoparticles for both laser-assisted therapy and MRI-based imaging in a xenograft colorectal cancer tumor model, demonstrating strong potentials for dual applications in cancer diagnosis and therapy.     

Effect of the catecholamine-depleting agent 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624) on drug metabolism in the rat

Male rats injected with 1-phenyl-3-(2-thiazolyl)-2-thiourea (U-14,624) (25 mg/kg/day i.p.) for 3 days prior to induction of anesthesia with pentobarbital (40 mg/kg, i.p.) slept significantly (P < 0.05) longer than control animals. Plasma and brain half-lives of pentobarbital were also prolonged in the treated animals, but both control and treated groups awakened with similar brain levels of pentobarbital. In addition, the plasma half-life of antipyrine in treated animals was also prolonged significantly. Subacute administration of U-14,624 (50 mg/kg/day i.p.) to male rats for 5–7 days suppressed the activities of aminopyrine N-demethylase, aniline hydroxylase and p-nitroanisole O-demethylase enzymes in vitro; this effect could not be demonstrated at lower doses. Single doses of U-14,624 (100–200 mg/kg, i.p.) also suppressed the activities of the three oxidative enzymes. The suppression was positively correlated with reduced levels of hepatic microsomal cytochrome P-450. Levels of cytochrome b₅ and NADPH-cytochrome c reductase activity were not affected consistently by acute dosage with U-14,624. The inhibitory effects of single doses (100–400 mg/kg, i.p.) on all enzymatic systems were reversible, and recovery was complete within 48 hr. Whereas all three oxidative drug-metabolizing enzymes were inhibited in a mixed manner by in vitro exposure to U-14,624 (10^?5–10^?2 M), neotetrazolium diaphorase was not inhibited by U-14,624 at concentrations as high as 5 mM. Inhibition of oxidative drug metabolism by U-14,624 is mechanistically related to depletion of cytochrome P-450, but inhibition of these enzymes in vitro indicates that a second inhibitory mechanism may also be operative.     

Dual T1 and T2 weighted magnetic resonance imaging based on Gd3+ loaded bioinspired melanin dots

In this report, a novel T₁/T₂ dual modal nanoprobe based on highly efficient and bioinspired melanin dots (M-dots) with directly loading gadolinium (Gd-M-dots) for magnetic resonance imaging (MRI) is described. In vitro and in vivo investigations have revealed that Gd-M-dots showed nontoxicity and good biocompatibilitity. Gd-M-dots relaxivity values on 3 T were determined to be r₁?=?23.4 and r₂?=?123.3 mM^?1 s^?1, which were much higher than both Gd-DTPA (r₁?=?5.1, r₂?=?6.2 mM^?1 s^?1) and Fe-M-dots (r₁?=?1.2, r₂?=?2.1 mM^?1 s^?1). For in vivo MRI, after injection of Gd-M-dots, simultaneous T₁ and T₂ contrast enhancement have been observed in the MRI of mice abdomen and mice bearing U87MG tumors. Furthermore, all the veins showed high signal intensity on T₁-weighted MRI and remained for 2 h. Overall, in vitro and in vivo studies indicate that Gd-M-dot with high r₁ relaxivity and r₂ relaxivity has high potential to be a promising nanoprobe for MR venography and molecular imaging.     

HPMA copolymer-cyclic RGD conjugates for tumor targeting

This review describes the design and development of N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-cyclic RGD conjugates for targeting tumor angiogenesis. Relative to non-targetable systems, HPMA copolymer-RGD4C and -RGDfK conjugates have shown increased tumor accumulation in a variety of solid tumors including prostate, lung, and breast tumor xenografts. Compared to free peptides, copolymers had increased tumor accumulation and decreased uptake in non-target organs such as the liver and spleen. Clinically relevant imaging agents such as ^99mTc, ¹¹¹In, and Gd enabled in vivo imaging of the constructs by scintigraphy and magnetic resonance techniques. Targeted delivery of ⁹⁰Y, a radiotherapeutic agent by HPMA copolymer-RGD4C conjugates resulted in tumor size reduction in mice bearing prostate tumor xenografts. Delivery of the geldanamycin derivative 17-(6-aminohexylamino)-17-demethoxygeldanamycin by HPMA copolymer-RGDfK conjugates resulted in increased tumor concentration of the free drug in a prostate xenograft model. These constructs show promise for targeted delivery of therapeutics and imaging agents to solid tumors.     

The in vitro effect of chlordecone and mirex on Ca2+-activated ATPase in the teleost Saccobranchus fossilis

The effects of the insecticides chlordecone (Kepone^®) and mirex on Ca²⁺-ATPase were studied in vitro using mitochondrial fraction prepared from gill, skeletal muscle, heart, liver, kidney and brain of the fresh-water fish Saccobranchus fossilis. The enzyme from brain and heart was most sensitive to chlordecone [IC₅₀ = (50% inhibitory concentration of pesticides for the enzyme), 6 and 13 μM, respectively] as compared to that from muscle, liver, gill and kidney (IC₅₀, 17, 19, 24 and 27 μM, respectively). Mirex, a close structural analog of chlordecone, on the other hand was much less toxic. The IC₅₀ values for brain and heart were 300 and 250 μM, respectively, while for other tissues, due to <50% inhibition, the values were not obtained. The results suggest that chlordecone is a potent neurotoxic inhibitor which may alter the calcium regulatory activity of cells resulting in the impairment of calcium dependent metabolism and its transport.     

Genetic engineering and characterisation of chlorotoxin-fused gelonin for enhanced glioblastoma therapy

Abstract

Despite substantial advances in its treatment, brain cancer remains a life-threatening disease with a poor survival rate. The main challenges for the conventional chemotherapy include an insufficient efficacy of drugs and toxicity caused by their nonselective mode of action. Recently, great attention has been paid to highly potent macromolecules such as gelonin, a type 1 ribosome-inactivating protein that inhibits protein translation. However, gelonin is poorly internalised into tumour cells and cannot distinguish between cancer and normal cells. To overcome these challenges, we engineered in this study a recombinant gelonin fusion protein with chlorotoxin, known as a brain cancer-homing peptide. The gelonin–chlorotoxin (Gel-CLTX) fusion chimera, produced in Escherichia coli, possessed an equipotent N-glycosidase activity with that of unmodified gelonin and, furthermore, could be selectively internalised into U-87?MG glioma cells over noncancerous glial cells. Consequently, Gel-CLTX displayed substantial inhibition of protein translation in U-87?MG cells, which eventually led to significantly augmented tumouricidal effects. When tested against xenograft tumour-bearing mice, Gel-CLTX showed higher tumour accumulation and inhibition of tumour growth than did gelonin, with a low systemic toxicity. Taken together, our results demonstrate the feasibility of using a fusion strategy for enhanced chemotherapy of brain tumours.     

Overcoming the stromal barrier for targeted delivery of HPMA copolymers to pancreatic tumors

Delivery of macromolecules to pancreatic cancer is inhibited by a dense extracellular matrix composed of hyaluronic acid, smooth muscle actin and collagen fibers. Hyaluronic acid causes a high intratumoral fluidic pressure which prevents diffusion and penetration into the pancreatic tumor. This study involves the breaking down of hyaluronic acid by treating CAPAN-1 xenograft tumors in athymic nu/nu mice with targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers radiolabeled with ¹¹¹In for single photon emission computerized tomography (SPECT) imaging. Two targeting strategies were investigated including α_vβ₃ integrin and HER2 receptors. HPMA copolymers were targeted to these receptors by conjugating short peptide ligands cRGDfK and KCCYSL to the side chains of the copolymer. Results demonstrate that tumor targeting can be achieved in vivo after treatment with hyaluronidase. This approach shows promise for enhanced delivery of polymer–peptide conjugates to solid tumors.     

Influence of Ammonium Chloride on the Tissue Distribution of Anticholinergic Drugs in Rats

Ammonium chloride (NH₄***C1) increases lysosomal pH and thereby abolishes intralysosomal accumulation of drugs. Its effect on the tissue distribution of biperiden and trihexyphenidyl in rats has been investigated. The tissue-plasma concentration ratios (K_p) of these drugs in various tissues were determined by infusion studies at steady-state in the presence or absence of NH₄***C1. Treatment with NH₄***C1 reduced the K_p values for both drugs, causing the largest reduction in K_p in the lung (52.1 to 11.8 for biperiden and 59.5 to 18.9 for trihexyphenidyl; ratios of decrease 0.77 and 0.68, respectively), followed by the heart and kidneys, with relatively small reductions in the brain, gut, muscle and fat. Subcellular fractionation studies in the lung indicated that the subcellular fraction-plasma concentration ratio of each drug at the steady state (K_p,sf) was reduced by treatment with NH₄***C1, with the largest decrease in the post-nuclear fraction (ratio of decrease 0.82 for biperiden and 0.74 for trihexyphenidyl), followed by the nucleus, microsomes and supernatant, in that order. A strong correlation was found between the ratio of decrease in K_p,sf after NH₄***Cl treatment and the specific activity of acid phosphatases, a marker of lysosomes, in each fraction (biperiden, r = 0.948; trihexyphenidyl, r = 0.945). These results suggest that acidic organelles contribute significantly to the distribution kinetics of anticholinergic drugs.     

Synergistic suppression of noscapine and conventional chemotherapeutics on human glioblastoma cell growth

Aim:

Noscapine (NOS) is a non-narcotic opium alkaloid with anti-tumor activity. The aim of this study was to investigate the effects of the combination of NOS with conventional chemotherapeutics temozolamide (TMZ), bis-chloroethylnitrosourea (BCNU), or cisplatin (CIS)on human glioblastoma cells.

Methods:

U87MG human glioblastoma cells were examined. Cell proliferation was quantified using MTT assay. Western blotting and flow cytometry were used to examine apoptosis and the expression of active caspase-3 and cleaved PARP. Mouse tumor xenograft model bearing U87MG cells was treated with TMZ (2 mg·kg⁻¹·d⁻¹, ip) or CIS (2 mg/kg, ip 3 times a week) alone or in combination with NOS (200 mg·kg⁻¹·d⁻¹, ig) for 3 weeks. Immunohistochemistry was used to investigate the expression of active caspase-3 and Ki67 following treatment in vivo. The safety of the combined treatments was evaluated based on the body weight and histological studies of the animal''s organs.

Results:

NOS (10 or 20 mol/L) markedly increased the anti-proliferation effects of TMZ, BCNU, and CIS on U87MG cells in vitro. The calculated combination index (CI) values of NOS-CIS, NOS-TMZ, and NOS-BCNU (20 μmol/L) were 0.45, 0.51, and 0.57, respectively, demonstrating synergistic inhibition of the drug combinations. In tumor xenograft models, combined treatment with NOS robustly augmented the anti-cancer actions of TMZ and CIS, and showed no detectable toxicity. The combined treatments significantly enhanced the apoptosis, the activated caspase-3 and PARP levels in U87MG cells in vitro, and reduced Ki67 staining and increased the activated caspase-3 level in the shrinking xenografts in vivo.

Conclusion:

NOS synergistically potentiated the efficacy of FDA-approved anti-cancer drugs against human glioblastoma cells, thereby allowing them to be used at lower doses and hence minimizing their toxic side effects.     

Studies on L-640,035: a novel antagonist of contractile prostanoids in the lung

1 The effects of L-640,035 (3-hydroxymethyl-dibenzo [b,f] thiepin-5,5-dioxide) have been studied on pulmonary smooth muscle contraction in vitro and in vivo.

2 When studied in vitro on guinea-pig tracheal chains, L-640,035 produced significant shifts in the dose-response curves to a prostaglandin (PG) endoperoxide analogue (U-44069) (pA₂ 7.0), PGF_2α (pA₂ 5.9) and PGD₂ (pA₂ 6.5). L-640,035 produced no significant shift in the dose-response curves to leukotriene D₄ or histamine and produced a small but statistically significant shift in the dose-response curve to 5-hydroxytryptamine (5-HT) (pA₂ 5.2). With the exception of PGF_2α, Schild analysis did not in general indicate competitive inhibition. The main metabolite of L-640,035, L-636,499, also produced significant parallel shifts in the dose-response curves to U-44069 (pA₂ 6.0) and PGF_2α (pA₂ 6.0), but with some reduction in the maximal contraction.

3 When L-640,035 was administered intravenously to guinea-pigs, significant inhibition of increases in pulmonary resistance or insufflation pressure induced by U-44069 (ED₅₀ 0.16 mg kg^-1), leukotriene D₄ (ED₅₀ 0.25 mg kg^-1) and 5-HT (ED₅₀ 3.4 mg kg^-1) but not histamine (ED₅₀ > 10 mg kg^-1) was observed.

4 When L-640,035 was administered intravenously to dogs a significant inhibition of increases in pulmonary resistance induced by U-44069 (ED₅₀ 0.85 mg kg^-1) but not histamine (ED₅₀ > 30 mg kg^-1) was observed.

5 When L-640,035 was administered by the intraduodenal route to dogs at doses of 3 and 10 mg kg^-1 significant inhibition of increases in pulmonary resistance induced by sodium arachidonate (3 mg kg^-1 i.v.) was observed with a duration of action of > 255 min.

6 It is concluded that L-640,035 is a novel, relatively selective, and orally active antagonist of the actions of contractile prostanoids on pulmonary smooth muscle.

     

Eigenschaften der Cholinesterasen in Geweben des Dorsches(Gadus callarias) und ihre Inaktivierung durchin vivo Einwirkung von Paraoxon und Tabun

The investigations presented in this paper were carried out in connection with possible contamination of the Baltic Sea by dumped tabun ammunition. By means of different substrates it was possible to demonstrate that most tissues of Gadus callarias (cod), (brain > myocardium ≧ skeletal muscle ? gills) contain only acetyleholinesterase (AChE, E.C.3.1.1.7.), whereas the liver also contains some butyrylcholinesterase (E.C.3.1.1.8.). Neither type of cholinesterase can be detected in erythrocytes and serum. The properties of the AChE of brain were characterized by determination of the substrate optimum (1 · 10^?3M ACh), the substrate inhibition (I₅₀ at 2·10^?2M ACh), the Michaelis constant (Km = 1 · 10^?4M ACh), and the dependence on pH and temperature (maximal activity at pH 8.5 and 37°C). The specific activity of AChE of brain or skeletal muscle is practically independent of the body weight. Exposure of living fish to concentrations of paraoxon or tabun between 1 · 10^?8 M and 1 · 10^?6M in sea water for 0.5 to 24 h yielded the following results: Both organophosphates were absorbed rapidly. Paraoxon inactivates the AChE of brain and of skeletal muscle with an exponential activity course at similar rates. The bimolecular rate constant (k ₂) hardly differs from the one measured in vitro (k ₂ in vivo 1.15 · 10⁴M^?1min^?1,in vitro 1.6 · 10⁴M^?1min^?1). Thein vitro reaction of tabun was probably falsified by its hydrolysis in sea water (k ₂ for AChE of brain is approximately 8 · 10³M^?1min^?1). The AChE of muscle was inactivated to a lesser degree by tabun than was cerebral AChE. The fish died when the activity of the AChE of the brain was lowered to a 5 % level by paraoxon or tabun. In spite of the extremely slow rate of reaction of cerebral AChE with either organophosphate it was possible to detect concentrations of 3 · 10^?8 - 1· 10^?7M ( 0.005 to 0.015 ppm. of paraoxon and tabun) by means of the AChE inactivation after 3 to 6 h of exposure.     

In vivo study of effects of artesunate nanoliposomes on human hepatocellular carcinoma xenografts in nude mice

Abstract

To investigate the effect of artesunate nanoliposomes on cultured cells in vitro and hepatocellular carcinoma xenografts in BALB/c-nu mice. Fluorescence polarization was applied for measurement of mitochondrial membrane fluidities; inhibition test of tumor cell proliferation in vitro was performed and nude mice xenograft model from human hepatocellular carcinoma (HCC) was established. Cytotoxicity of these compounds was evaluated by MTT assay on hepatocellular carcinoma xenografts in nude mice. Anisotropy (r-value) of blank nanoliposomes didn’t change, it had no statistically significance between the blank nanoliposomes group and the control group, it indicated that artesunate had no obvious effect on L-O2 human normal liver cells. IC₅₀ values of artesunate nanoliposomes and artesunate API (active pharmaceutical ingredient) against HepG-2 cells were 15.997 and 19.706?μg/ml; IC₅₀ values of the same drugs against L-O2 normal human liver cells were 100.23 and 105.54?μg/ml, respectively. Tumor growth inhibitory effect of artesunate nanoliposomes was 32.7%, and artesunate API was 20.5%, respectively. HepG-2 cells treated with artesunate nanoliposomes showed dose-dependent apoptosis. The antitumor effect of artesunate nanoliposomes on human hepatoma HepG2 cells were stronger than that of artesunate API at the same concentration.     

Neuropharmacology: Effects on Rat Brain K1- and K2-Opioid Receptors after Chronic Treatment with Non-peptide K-Agonists

Injection of K-agonist dynorphins and non-peptide K-agonists into the hippocampus induces a reduction in blood pressure. It has been postulated that K-opioid agonists and K-receptors are important in one mechanism of antihypertension and might have clinical potential for the treatment of hypertension. We have investigated whether chronic treatment with U-50488H and U-62066E, two non-peptide K-agonists, effects brain K₁- or K₂-receptor numbers or affinities in areas that might correlate with changes in blood pressure. K₁- and K₂-Opioid receptor affinities and densities were determined in cortex, hippocampus, hypothalamus, midbrain and pons after 14 days subcutaneous infusion of two non-peptide K-agonists, U-50488H and U-62066E, 9.6 mg kg day^?1, by means of osmotic minipumps, to spontaneously hypertensive rats (SHR) and to Wistar-Kyoto (WKY) rats. This infusion significantly reduced blood pressure. Brains were removed within 48 h of the end of drug infusion and K-receptor binding studies were performed on homogenates from each brain area using [³H]U-69593 to assay K₁-receptors and [³H]bremazocine to assay K₂-receptors. U-62066E treatment seemed to cause a slight decrease in the number of [³H]bremazocine binding sites (K₂-receptors) from 98.2 ± 9 to 74.9 ± 8 fmol (mg protein)^?1 in the hippocampus when compared with SHR controls. A small decrease in K₂-receptor density in the pons of WKY rats was also observed after U-50488H treatment (control, 51.2±5; U-50488H-treated, 24.3±9 fmol (mg protein)^?1). Although SHR blood pressure values were consistently reduced by treatment with K-agonists, there was little if any significant change in apparent numbers of K₁- or K₂-receptors or their affinities in any of the brain regions examined. These data indicate that although chronic treatment with K-agonists reduces blood pressure in SHR, the treatment does not elicit major changes in brain K-receptors either in SHR or in WKY rats. The potential use of K-agonists for treating hypertension might not cause receptor changes in the brain and might, therefore, result in fewer side effects or negligible rebound hypertension.     

Antitumor activity of cytokine-induced killer cells in nude mouse xenograft model

Malignant glioma is the most common primary brain tumor in adults and the median survival for patients is less than a year. Despite aggressive treatments including surgical resection, radiotherapy, and chemotherapy, only modest improvement has been achieved in the survival of patients with glioma. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against human glioma cancer was evaluated in vitro and in vivo. Human peripheral blood mononuclear cells were cultured with IL-2-containing medium in anti-CD3 antibody-coated flasks for 5 days, followed by incubation in IL-2-containing medium for 9 days. The number of cells increased more than 200-fold and the viability was >90%. The resulting populations were consisted of 96% CD3⁺, 2% CD3⁻CD56⁺, 68% CD3⁺CD56⁺, 2% CD4⁺, <1% CD4⁺CD56⁺, 80% CD8⁺, and 49% CD8⁺CD56⁺. This heterogeneous cell population was called as CIK cells. At an effector-target cell ratio of 30:1, CIK cells destroyed 43% of U-87 MG human glioma cells, as measured by the ⁵¹Cr-release assay. In addition, CIK cells at doses of 0.3, 1, and 3 million cells per mouse inhibited 23%, 40%, and 50% of U-87 MG tumor growth in nude mouse xenograft assays, respectively. This study suggests that CIK cells may be used as an adoptive immunotherapy for glioma cancer patients. These authors contributed equally to this work.     

Irgm1 knockout indirectly inhibits regeneration after skeletal muscle injury in mice

Immunity-related GTPase family M1 protein (lRGM1) plays an important role in host resistance to infection, immune inflammation, and tumors, and it is expressed in various tissues and cells, including the central nervous system, cardiovascular system, bone marrow-derived cells, glioma, and melanoma. However, the effect of IRGM1 in the muscles has not been reported to date. In this study, Irgm1^−/− mice were used to evaluate the effect of lrgm1 on regeneration after skeletal muscle injury. The tibialis anterior muscle in Irgm1^−/− mice was poorly repaired after BaCl₂-induced injury, whereas lrgm1 knockout itself had no significant effect on the differentiation of myoblasts. However, the microenvironment of Irgm1^−/− mice with a high interferon-gamma level inhibited the differentiation of myoblasts in vivo. These results suggest that lrgm1 knockout indirectly inhibits skeletal muscle regeneration after injury, providing new insights into the biological function of IRGM1.     

Prediction of drug distribution in vivo on the basis of in vitro binding data

For 11 drugs it was investigated whether tissue distribution in vivo can be predicted by use of binding data obtained in vitro. The selection of drugs represented a broad spectrum of physicochemical and pharmacokinetic properties thought to be important for distribution of drugs in vivo. The extent of binding to plasma and to tissue-homogenates of rabbits was determined in vitro. The drug concentrations in plasma, liver, lungs, kidneys, and skeletal muscle of rabbits were determined in vivo after i.v. administration of the drug. The tissue-plasma partition ratios measured in vivo were compared with the theoretical tissue-plasma partition ratios calculated from the in vitro binding data. For all drugs investigated the muscle-plasma partition ratio could be reasonably well predicted by the in vitro binding data. In liver, lungs, and kidneys good agreement was found between measured and predicted tissue-plasma ratio for anionic drugs; marked differences, however, were observed between measured and predicted tissue-plasma ratios of lipophilic cationic drugs. A significant correlation was found between binding of drugs to muscle tissue in vitro and the volume of distribution of the unbound drug (Vf), opening the possibility to approximate Vf from in vitro binding studies with rabbit muscle tissue.