Affimer Tagged Cubosomes: Targeting of Carcinoembryonic Antigen Expressing Colorectal Cancer Cells Using In Vitro and In Vivo Models.

Nanomedicines, while having been approved for cancer therapy, present many challenges such as low stability, rapid clearance, and nonspecificity leading to off-target toxicity. Cubosomes are porous lyotropic liquid crystalline nanoparticles that have shown great premise as drug delivery vehicles; however, their behavior in vivo is largely underexplored, hindering clinical translation. Here, we have engineered cubosomes based on the space group Im3m that are loaded with copper acetylacetonate as a model drug, and their surfaces are functionalized for the first time with Affimer proteins via copper-free click chemistry to actively target overexpressed carcinoembryonic antigens on LS174T colorectal cancer cells. Unlike nontargeted cubosomes, Affimer tagged cubosomes showed preferential accumulation in cancer cells compared to normal cells not only in vitro (2D monolayer cell culture and 3D spheroid models) but also in vivo in colorectal cancer mouse xenografts, while exhibiting low nonspecific absorption and toxicity in other vital organs. Cancerous spheroids had maximum cell death compared to noncancerous cells upon targeted delivery. Xenografts subjected to targeted drug-loaded cubosomes showed a 5-7-fold higher drug accumulation in the tumor tissue compared to the liver, kidneys, and other vital organs, a significant decrease in tumor growth, and an increased survival rate compared to the nontargeted group. This work encompasses the first thorough preclinical investigation of Affimer targeted cubosomes as a cancer therapeutic.

Blood and urine analyses after radioembolization of liver malignancies with [166Ho]Ho-acetylacetonate-poly(l-lactic acid) microspheres.

BACKGROUND: [166Ho]Ho-acetylacetonate-poly(L-lactic acid) microspheres were used in radioembolization of liver malignancies by intra-arterial administration. The primary aim of this study was to assess the stability and biodistribution of these microspheres. MATERIALS AND METHODS: Peripheral blood and urine samples were obtained from two clinical studies. Patient and in vitro experiment samples were analyzed using inductively coupled plasma mass spectrometry (ICP-MS), gamma-ray spectroscopy, light microscopy, Coulter particle counting, and high performance liquid chromatography (HPLC). RESULTS: The median percentage holmium compared to the total amount injected into the hepatic artery was 0.19% (range 0.08-2.8%) and 0.32% (range 0.03-1.8%) in the 1 h blood plasma and 24 h urine, respectively. Both the blood plasma and urine were correlated with the neutron irradiation exposure required for [166Ho]Ho-AcAc-PLLA microsphere production (ρ = 0.616, p = 0.002). After a temporary interruption of the phase 2 clinical study, the resuspension medium was replaced to precipitate [166Ho]Ho3+ pre-administration using phosphate. The in vitro near-maximum neutron irradiation experiments showed significant [166Ho]Ho-AcAc-PLLA microsphere damage. CONCLUSION: The amount of holmium in the peripheral blood and urine samples after [166Ho]Ho-AcAc-PLLA microsphere intrahepatic infusion was low. A further decrease was observed after reformulation of the resuspension solution but minimization of production damage is necessary.

MicroRNA expression profiling of dibenzalacetone (DBA) treated intracellular amastigotes of Leishmania donovani.

BACKGROUND: Among different leishmanial infections, visceral leishmaniasis (VL) if not treated is the most severe form with high mortality rates. In India, it is caused by the protozoan parasite Leishmania donovani. The therapy of visceral leishmaniasis is limited due to high toxicity, resistance to existing drugs and increasing cases of Leishmania co-infections. Hence, there is a need to identify novel drug and targets to overcome these hindrances. MicroRNAs (miRNAs) are a class of small non-coding RNAs (∼22-24 nucleotide in length) that regulate gene expression in various biological processes. They play as intracellular mediators that are essential for different biological processes. OBJECTIVES: The aim of present study is to explore the leishmaniacidal role of trans-dibenzalacetone (DBA, a synthetic monoketone analog of curcumin) on the expression profile of miRNA in intracellular amastigotes of Leishmania donovani. METHODS: Small RNA libraries of samples (macrophages-infected with Leishmania amastigotes; and infected macrophages treated with DBA) were prepared by using Illumina Trueseq Small RNA kit. RESULTS: Using miRDIP database, we identified target gene of differentially expressed miRNAs (target miRNAs: hsa-mir-15b, hsa-mir-671, hsa-mir-151a and has-mir-30c) which was confirmed by real time stem-loop PCR. Ten KEGG pathways were significantly enriched with these target miRNA genes and they mainly relate to mitogen-activated protein kinases (MAPK) pathway. We have previously established the antiproliferative and apoptotic effect of trans-dibenzalacetone (DBA, a synthetic monoketone analog of curcumin) on the Leishmania donovani parasites. In the present study, using GFP-ATG8 gene as a marker for tracking putative autophagosomes, we confirmed that autophagic vacuolization may lead to autophagic cell death in the DBA-treated parasites. Our results demonstrated that curcumin analog DBA has a role to play in regulating the balance between autophagy and apoptosis. CONCLUSIONS: We conclude that curcumin analog DBA triggers imbalance between two known phenotypes of cell death viz apoptosis and autophagy.

An oxygen-chelate complex, palladium bis-acetylacetonate, induces apoptosis in H460 cells via endoplasmic reticulum stress pathway rather than interacting with DNA.

Current precious-metal-containing anticancer agents are mostly chelated with N-containing ligands and function by interacting with DNA. In the present study, Pd(acac)2, a Pd(II) complex containing four O-donor ligands, has been evaluated as an active anticancer agent. Pd(acac)2 showed no interaction with N-ligand-containing DNA and the S-ligand-containing DMSO, probably because of the two six-member chelate rings that limit the release of the central Pd nuclei to bind to other ligands. Importantly, we found that Pd(acac)2 exhibited better growth inhibitory effects than cisplatin in several cancer cells. Treatment with Pd(acac)2 significantly induced apoptosis in H460 cells. Mechanistically, Pd(acac)2 induced the activation of a series of key components in ER stress-mediated apoptotic pathway, followed by caspase cleavage and activation, while cisplatin showed no similar effects. CHOP knockdown by specific siRNA significantly attenuated Pd(acac)2-induced cell apoptosis. Finally, Pd(acac)2 significantly inhibits H460 cell growth in xenograft mouse models. Taken together, these mechanistic insights on Pd(acac)2 provide us with a novel mechanism and strategy for the development of precious-metal-based anticancer drugs.

A new curcumin analogue exhibits enhanced antitumor activity in nasopharyngeal carcinoma.

The aim of the present study was to evaluate the antitumor effects of the curcumin analogue GL63 on radioresistant nasopharyngeal carcinoma (NPC) CNE2R cells and parental CNE2 cells. The cell viability and proliferation of NPC cells were detected by MTT assay and colony formation assay. The suppressive effect on tumor growth was examined using in vivo subcutaneously inoculated NPC tumor models using nude mice. The cell cycle distribution was detected using flow cytometry. Apoptosis was examined by Hoechst 33342 and Annexin V/PI staining assay. The protein expression of endoplasmic reticulum (ER) stress pathway markers, XBP-1, ATF-4 and CHOP, were examined by western blotting. A growth inhibitory effect was observed following treatment with GL63 in a dose-dependent manner and was more potent when compared to curcumin. GL63 at 5 µM induced significant G2/M arrest and apoptosis in NPC. The tumor-suppressive activity of GL63 in NPC xenograft models was more potent when compared to curcumin. Furthermore, GL63 induced an ER stress response, upregulation of CHOP, XBP-1 and ATF-4 expression, while the same concentration of curcumin had no effect on ER stress. These results suggest that GL63 has more potent antitumor activity than curcumin, which is associated with activation of ER stress, induction of G2/M arrest and apoptosis in NPC cells.

Radioactive holmium acetylacetonate microspheres for interstitial microbrachytherapy: an in vitro and in vivo stability study.

PURPOSE: The clinical application of holmium acetylacetonate microspheres (HoAcAcMS) for the intratumoral radionuclide treatment of solid malignancies requires a thorough understanding of their stability. Therefore, an in vitro and an in vivo stability study with HoAcAcMS was conducted. METHODS: HoAcAcMS, before and after neutron irradiation, were incubated in a phosphate buffer at 37°C for 6 months. The in vitro release of holmium in this buffer after 6 months was 0.5%. Elemental analysis, scanning electron microscopy, infrared spectroscopy and time of flight secondary ion mass spectrometry were performed on the HoAcAcMS. RESULTS: After 4 days in buffer the acetylacetonate ligands were replaced by phosphate, without altering the particle size and surface morphology. HoAcAcMS before and after neutron irradiation were administered intratumorally in VX2 tumor-bearing rabbits. No holmium was detected in the faeces, urine, femur and blood. Histological examination of the tumor revealed clusters of intact microspheres amidst necrotic tissue after 30 days. CONCLUSION: HoAcAcMS are stable both in vitro and in vivo and are suitable for intratumoral radionuclide treatment.

Nitecapone reduces development and symptoms of neuropathic pain after spinal nerve ligation in rats.

Neuropathic pain is caused by damage or malfunctioning of the nervous system. It is fairly common and more resistant to treatment than other types of pain. Since nitecapone, an inhibitor of catechol-O-methyl-transferase (COMT), has decreased neuropathic symptoms in diabetic rats, we studied its effects in another model of neuropathic pain, the spinal nerve ligation (SNL) model. Spinal nerves L5-6 were ligated in male Wistar rats under anaesthesia to produce the SNL model of neuropathic pain. Nitecapone (30 mg/kg, i.p.) or vehicle was administered once daily starting either 1h before or 2 days after surgery and continued for 14-19 days. Threshold for mechanical allodynia was measured with the digital von Frey test and responses to cold stimuli with the acetone test, before surgery and every other day after it 1h before drug administration. Mechanical and cold allodynia developed in all study groups. Both nitecapone treatments significantly reduced mechanical allodynia and withdrawal thresholds were 80-95% higher compared with the control group. In the acetone test, both nitecapone groups also showed less signs of cold allodynia than the control groups. In nitecapone-naïve animals a single dose of nitecapone also reduced mechanical allodynia on the 14th day after the surgery. Nitecapone reduced the symptoms of neuropathic pain after the SNL, which is in line with the earlier study. Our results suggest that nitecapone and other COMT inhibitors should be studied further in the treatment of neuropathic pain.

Anticonvulsant effects of methyl ethyl ketone and diethyl ketone in several types of mouse seizure models.

The anticonvulsant effects of acetone have been reported in various animal models of epilepsy. We recently demonstrated that other ketone bodies, methyl ethyl ketone (MEK) and diethyl ketone (DEK), suppressed status epilepticus that was induced by lithium-pilocarpine in rat. In the present study, the anticonvulsant effects of MEK and DEK were evaluated by using four different types of mouse seizure models, which were induced by pentylenetetrazole, kainic acid, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), and electroshock. The effects of clonazepam, a typical anticonvulsant, and acetone were also evaluated and compared with MEK and DEK. In this study, MEK (5 and 10 mmol/kg, i.p.) and DEK (2.5 and 5 mmol/kg, i.p.) produced anticonvulsant activity against all types of seizure models. Furthermore, MEK and DEK showed almost the same potencies against four different seizure models used, while clonazepam showed significant higher ED(50) values against kainic acid-induced and electroshock-induced seizure models as compared with the pentylenetetrazole- or DMCM-induced seizure model. In each study, the highest doses of clonazepam (1 or 3mg/kg) did not show clear anticonvulsant effects against the kainic acid- or electroshock-induced seizures. In conclusion, MEK and DEK showed broad-spectra anticonvulsant effects in both chemically- and electroshock-induced experimental seizure models.

Methyl ethyl ketone blocks status epilepticus induced by lithium-pilocarpine in rats.

BACKGROUND AND PURPOSE: A ketogenic diet has been used successfully to treat patients with intractable epilepsy, although the mechanism is unknown. Acetone has been shown to have an anticonvulsive effect in various animal models. The main purpose of this study was to determine whether other ketones, 2-butanone (methyl ethyl ketone: MEK) and 3-pentanone (diethyl ketone: DEK), also show anticonvulsive effects in lithium-pilocarpine (Li-pilocarpine)-induced status epilepticus (SE) in rats. EXPERIMENTAL APPROACH: Anticonvulsive effects of MEK and DEK in Li-pilocarpine SE rats were measured by behavioural scoring. Anti-seizure effects of MEK were also evaluated using electroencephalography (EEG). Neuroprotective effect of MEK was investigated by haematoxylin and eosin staining 4 weeks after the treatment with pilocarpine. KEY RESULTS: Acetone, MEK and DEK showed anticonvulsant effects in Li-pilocarpine-induced SE rats. Treatment with MEK twice (8 mmol.kg(-1) and 5 mmol.kg(-1)) almost completely blocked spontaneous recurrent cortical seizure EEG up to 4 weeks after the administration of pilocarpine. MEK also showed strong neuroprotective effects in Li-pilocarpine-treated rats 4 weeks following the administration of pilocarpine. Significant neural cell death occurred in the hippocampus of Li-pilocarpine SE rats, especially in the CA1 and CA3 subfields. In contrast, normal histological characteristics were observed in these regions in the MEK-pretreated rats. CONCLUSIONS AND IMPLICATIONS: Both MEK and DEK showed strong anticonvulsive effects in Li-pilocarpine-induced SE rats. They also inhibited continuous recurrent seizure and neural damage in hippocampal region for 4 weeks after the treatment with pilocarpine. These findings appear to be of value in the investigation of epilepsy.

Nitecapone and selegiline as effective adjuncts to L-DOPA in reserpine-induced catatonia in mice.

Reserpine-induced catatonia is a widely accepted animal model of Parkinson's disease. In the present study, reserpine (5 mg/kg i.p.) and alpha-methylpara-tyrosine (AMPT) (200 mg/kg i.p.) induced catatonia in mice 20 h and 1 h before the experiment, respectively, as assessed using the rota-rod and bar tests after reserpine treatment. There was a significant decrease in fall-off time in the rota-rod test and a significant increase in time spent on the bar in the bar test as compared to the untreated control mice. Combination therapy with L-DOPA (100 mg/kg i.p.) and carbidopa (10 mg/kg i.p.) was less effective in reversing catatonia as compared to higher doses of L-DOPA (200 mg/kg i.p.) and carbidopa (20 mg/kg i.p.), which showed intense hyperactivity in reserpinized mice. Pretreatment with nitecapone (30 mg/kg i.p.), a COMT inhibitor, or selegiline (10 mg/kg i.p.), a MAO-B inhibitor potentiated the motor stimulant actions of subthreshold doses of the L-DOPA (100 mg/kg i.p.) and carbidopa (10 mg/kg i.p.) combination. Amantadine (40 mg/kg i.p.), but not bromocriptine, potentiated the effects of L-DOPA treatment. The NMDA antagonistic action of amantadine may have beneficial effects. It is concluded that COMT and MAO-B enzymes play an important role in the metabolism of dopamine and administration of a COMT or MAO-B inhibitor may prove to be a better adjunct to L-DOPA therapy than a dopamine receptor agonist in Parkinson's disease.

Combined antioxidant and COMT inhibitor treatment reverses renal abnormalities in diabetic rats.

The development and progression of diabetic nephropathy is dependent on glucose homeostasis and many other contributing factors. In the present study, we examined the effect of nitecapone, an inhibitor of the dopamine-metabolizing enzyme catechol-O-methyl transferase (COMT) and a potent antioxidant, on functional and cellular determinants of renal function in rats with streptozotocin-induced diabetes. Administration of nitecapone to diabetic rats normalized urinary sodium excretion in a manner consistent with the dopamine-dependent inhibition of proximal tubule Na,K-ATPase activity. Hyperfiltration, focal glomerulosclerosis, and albuminuria were also reversed by nitecapone, but in a manner that is more readily attributed to the antioxidant potential of the agent. A pattern of elevated oxidative stress, measured as CuZn superoxide dismutase gene expression and thiobarbituric acid-reactive substance content, was noted in diabetic rats, and both parameters were normalized by nitecapone treatment. In diabetic rats, activation of glomerular protein kinase C (PKC) was confirmed by isoform-specific translocation and Ser23 phosphorylation of the PKC substrate Na,K-ATPase. PKC-dependent changes in Na,K-ATPase phosphorylation were associated with decreased glomerular Na,K-ATPase activity. Nitecapone-treated diabetic rats were protected from these intracellular modifications. The combined results suggest that the COMT-inhibitory and antioxidant properties of nitecapone provide a protective therapy against the development of diabetic nephropathy.

The Helicobacter felis mouse model in assessing anti-Helicobacter therapies and gastric mucosal prostaglandin E2 levels.

BACKGROUND: The aims of the present study were to assess the usefulness of the Helicobacter felis mouse model in the evaluation of antimicrobial therapies and the effect of Helicobacter infection on gastric mucosal prostaglandin E2 release. METHODS: Barrier-maintained BALB/c mice were infected with H. felis and treated with different antibacterial therapies. H. felis status was determined by bacterial culture, urease test, and bacterial and histologic stainings. Release of prostaglandin E2 from the gastric mucosa was measured by radioimmunoassay. RESULTS: All triple-treated mice were cleared of bacteria both 24 h and 1 month after treatment. However, tinidazole alone also resulted in 100% eradication. Monotherapies with erythromycin acistrate, tetracycline, colloidal bismuth subcitrate, and nitecapone failed to eradicate the bacteria. The release of gastric prostaglandin E2 was doubled in the infected mice (554 +/- 39, mean +/- SE) compared with the noninfected mice (270 +/- 35) (p < 0.01). CONCLUSIONS: The H. felis mouse model proved satisfactory for assessing both anti-Helicobacter therapies and the prostaglandin E2 release. The reliability of this method was improved when several methods to assess the H. felis status were used in parallel.

Nitecapone protects the Langendorff perfused heart against ischemia-reperfusion injury.

We assessed the protection afforded against myocardial ischemia/reperfusion by nitecapone in the Langendorff heart model using male Sprague-Dawley rats. We found that when present in the perfusate 10 microM nitecapone improved the mechanical function of the heart and lowered the enzyme leakage of lactate dehydrogenase after 40 minutes of global ischemia. In nitecapone treated hearts the content of oxidized proteins and lipids (carbonyl groups and endogenous lipid fluorescent products) decreased. Nitecapone partially prevented the loss of total sulfhydryl groups and vitamin E after ischemia and reperfusion. We suggest that the mechanism of nitecapone protection most likely involves direct antioxidant action and enhancing the activity of other antioxidants.

[18F]-6-fluorodopa PET scanning in Parkinson's disease after selective COMT inhibition with nitecapone (OR-462).

PET studies were performed to investigate the effects of a new cathechol-O-methyltransferase (COMT) inhibitor, nitecapone (OR-462 [3-(3,4-dihydroxy-5-nitrobenzylidene)- 2,4-pentadione]), on the accumulation of dopamine in the striatum and whether it is able to improve [18F]6-fluorodopa imaging of the brain. Altogether, three patients with Parkinson's disease (PD) and three normal volunteers were examined, first without nitecapone and then with an oral dose of 100 mg of nitecapone 1 hour before the IV injection of 3 mCi of [18F]6-fluorodopa. High-pressure liquid chromatography analysis of arterial plasma samples showed a significant reduction in the metabolic conversion rate from [18F]6-fluorodopa to [18F]3-O-methylfluorodopa after the administration of nitecapone. PET studies showed that nitecapone significantly (p less than 0.05) increased the [18F]6-fluorodopa accumulation in the striatum both in PD patients and normal controls; the magnitude of this increase was 20.0 +/- 5.5% (mean +/- SEM). The ratio of radioactivity in the striatum and arterial plasma was increased 39.0 +/- 5.0% (mean +/- SEM) after the administration of nitecapone. Consequently, the quality of PET images after OR-462 was better, which has implications for future [18F]6-fluorodopa studies. In addition, COMT inhibition may have clinical advantages by improving levodopa treatment in PD.

Effect of some 4-alkyl-5-dimethylamino-1-phenyl-1-penten-3-one hydrochlorides and related compounds on respiration in mouse liver mitochondria.

5-Dimethylamino-1-phenyl-1-penten-3-one hydrochloride (1a) has high inhibiting properties in mitochondria isolated from mice liver. Substitution at the 4-methylene group of 1a by different alkyl substituents lead to compounds with wide variation in respiration-inhibiting properties. No correlations were found between either the Charton steric parameter (v), Taft inductive value (f*) or fragmental constant (f) of the substituents at position 4 of the Mannich bases with inhibition of respiration. However the biological results suggested two receptor sites were present in the mitochondria which may interact with the Mannich bases namely a common receptor for all compounds and in addition a narrow hydrophobic binding area which can accommodate a n-butyl or higher alkyl groups which are present in some of the compounds.