Selective Neuropeptide Y Conjugates with Maximized Carborane Loading as Promising Boron Delivery Agents for Boron Neutron Capture Therapy.

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with the bound ligand. This mechanism was exploited to shuttle boron atoms into cancer cells for the application of boron neutron capture therapy (BNCT), a noninvasive approach to eliminate cancer cells. A maximized number of carboranes was introduced to the hY1R-preferring ligand [F7,P34]-NPY by solid phase peptide synthesis. Branched conjugates loaded with up to 80 boron atoms per peptide molecule exhibited a maintained receptor activation profile, and the selective uptake into hY1R-expressing cells was demonstrated by internalization studies. In order to ensure appropriate solubility in aqueous solution, we proved the need for eight hydroxyl groups per carborane. Thus, we suggest the utilization of bis-deoxygalactosyl-carborane building blocks in solid phase peptide synthesis to produce selective boron delivery agents for BNCT.

Polyborane-encapsulated PEGylated Liposomes Prepared Using Post-insertion Technique for Boron Neutron Capture Therapy.

PEGylated liposomes are one of the useful boron carriers for boron neutron capture therapy (BNCT). Recently, a method of adding PEG after liposome formation (post-insertion) was reported. In this study, we prepared polyborane-encapsulated PEGylated liposomes for BNCT with half the amount of DSPE-PEG of the conventional method using post-insertion technique (post-PEG liposomes), and their usefulness were evaluated in comparison with conventional PEGylated liposomes (pre-PEG liposomes). From the results of physicochemical property measurements, it was confirmed that particle size distributions, surface charge densities, and fixed aqueous layer thicknesses of these liposomes were equivalent. In vitro cytotoxicity and cell uptake tests were also carried out using B16 melanoma and RAW264.7 cells. Polyborane solution and bare liposomes were used for comparison. From the results of these tests, we confirmed that post-PEG liposomes and pre-PEG liposomes have the same influence of PEGylation. To evaluate biodistribution properties at 24 h post-administration, these liposomes and polyborane solution were injected into the tail veins of tumor-bearing mice. Boron concentration and tumor/blood ratios of PEGylated liposomes were 73.2-77.6 µg/g of tumor tissue and 5.5-5.8, respectively. From these results, it was found that by using post-insertion technique, liposomes for BNCT having same effect as the liposome prepared using the conventional method can be prepared with half amount of DSPE-PEG.

Metallacarboranes on the Road to Anticancer Therapies: Cellular Uptake, DNA Interaction, and Biological Evaluation of Cobaltabisdicarbollide [COSAN].

After uptake by U87 MG and A375 cancer cells, cobaltabisdicarbollide [COSAN]- distributes between membrane and nucleus and presents no relevant cytotoxicity against both cell lines even for long incubation times. The cytotoxicity of Na[COSAN] was also tested towards one normal cell line, the V79 fibroblasts, in order to ascertain the noncytotoxic profile of the compound. As the cell's nucleus contains DNA, the interaction between [COSAN]- and double-stranded calf thymus DNA (CT-dsDNA) has been investigated. There is a strong interaction between both molecules forming a nanohybrid CT-dsDNA-[COSAN] biomaterial, which was fully characterized. Moreover, Na[COSAN] shows characteristic redox peaks ascribed to the oxidation/reduction of Co3+/2+ at a formal potential of -1.444 V and it can be accumulated at a surface-immobilized DNA layer of glassy carbon electrodes. The equilibrium surface-binding constants (Kox /Kred ), which confirm that [COSAN]- interacts with DNA by an intercalative or electrostatic mode, depending on the ionic strength of the solution, were estimated. In addition, high binding affinity of Na[COSAN] to proteins was observed by 11 B{1 H} NMR and confirmed in vivo. Finally, biodistribution studies of [COSAN]- in normal mice were run. After administration, Na[COSAN] was distributed into many organs but mainly accumulated in the reticuloendothelial system (RES), including liver and spleen. After 1 h, the formation of aggregates by plasma protein interaction plays a role in the biodistribution profile; the aggregates accumulate mostly in the lungs. Na[COSAN], which displays low toxicity and high uptake by relevant cancer cells accumulating boron within the nucleus, could act as a suitable compound for further developments as boron neutron capture therapy (BNCT) agents.

Dynamic changes in 18F-borono-L-phenylalanine uptake in unresectable, advanced, or recurrent squamous cell carcinoma of the head and neck and malignant melanoma during boron neutron capture therapy patient selection.

BACKGROUND: We evaluated dynamic changes in 18F-borono-L-phenylalanine (18F-BPA) uptake in unresectable, advanced, or recurrent squamous cell carcinoma of the head and neck (SCC) and malignant melanoma (MM) during boron neutron capture therapy (BNCT) patient selection. METHODS: Dynamic changes in the maximum standardized uptake value (SUVmax), tumor-to-normal tissue ratio (TNR), and tumor-to-blood pool ratio (TBR) for 18F-BPA were evaluated in 20 patients with SCC and 8 patients with MM. RESULTS: SUVmax in SCC tumors decreased significantly from 30 to 120 min. There was a non-statistically significant decrease in SUVmax for SCC tumors from 30 to 60 min and from 60 to 120 min. Patients with MM had nonsignificant SUVmax changes in 18F-BPA uptake on delayed imaging. Nonsignificant 18F-BPA TNR and TBR changes were seen in patients with SCC and MM. CONCLUSIONS: Dynamic changes in SUVmax for 18F-BPA uptake had a washout pattern in SCC and a persistent pattern in MM. Dynamic 18F-BPA -PET studies should be performed to investigate the pharmacokinetics of 18F-BPA in humans and select appropriate candidates who may benefit from BNCT.

Polyester-based microdisc systems for sustained release of neuroprotective phosphine-borane complexes.

Phosphine-borane complexes are recently developed redox-active drugs that are neuroprotective in models of optic nerve injury and radioprotective in endothelial cells. However, a single dose of these compounds is short-lived, necessitating the development of sustained-release formulations of these novel molecules. We screened a library of biodegradable co- and non-block polyester polymer systems for release of incorporated phosphine-borane complexes to evaluate them as drug delivery systems for use in chronic disease. Bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) was combined with biodegradable polymers based on poly(D,L-lactide) (PDLLA), poly(L-lactide) (PLLA), poly(caprolactone) (PCL), poly(lactide-co-glycide) (PLGA), or poly(dioxanone-co-caprolactone) (PDOCL) to make polymer microdiscs, and release over time quantified. Of 22 polymer-PB1 formulations tested, 17 formed rigid polymers. Rates of release differed significantly based on the chemical structure of the polymer. PB1 released from PLGA microdiscs released most slowly, with the most linear release in polymers of 60:40 LA:GA, acid endcap, Mn 15 000-25 000 and 75:25 LA:GA, acid endcap, Mn 45 000-55 000. Biodegradable polymer systems can, therefore, be used to produce sustained-release formulations for redox-active phosphine-borane complexes, with PLGA-based systems most suitable for very slow release. The sustained release could enable translation to a clinical neuroprotective strategy for chronic diseases such as glaucoma.

Pyridinium Boranephosphonate Modified DNA Oligonucleotides.

The synthesis of previously unknown derivatives of boranephosphonate that contain amine substitutions at boron and the incorporation of these derivatives into the backbone of DNA oligonucleotides is described. These derivatives result from iodine-mediated replacement of one BH3 hydride of a boranephosphonate linkage by pyridine, various substituted pyridines, other aromatic amines, and certain unsaturated amines. Oligonucleotides containing these backbone modifications show enhanced uptake, relative to unmodified DNA, in mammalian cells. The redox behavior of the boranephosphonate and pyridinium boranephosphonate conjugated linkages has also been studied.

BA321, a novel carborane analog that binds to androgen and estrogen receptors, acts as a new selective androgen receptor modulator of bone in male mice.

Carboranes are a class of carbon-containing polyhedral boron cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors such as estrogen receptor (ER) and androgen receptor (AR). We have synthesized BA321, a novel carborane compound, which binds to AR. We found here that it also binds to ERs, ERα and ERß. In orchidectomized (ORX) mice, femoral bone mass was markedly reduced due to androgen deficiency and BA321 restored bone loss in the male, whilst the decreased weight of seminal vesicle in ORX mice was not recovered by administration of BA321. In female mice, BA321 acts as a pure estrogen agonist, and restored both the loss of bone mass and uterine atrophy due to estrogen deficiency in ovariectomized (OVX) mice. In bone tissues, the trabecular bone loss occurred in both ORX and OVX mice, and BA321 completely restored the trabecular bone loss in both sexes. Cortical bone loss occurred in ORX mice but not in OVX mice, and BA321 clearly restored cortical bone loss due to androgen deficiency in ORX mice. Therefore, BA321 is a novel selective androgen receptor modulator (SARM) that may offer a new therapy option for osteoporosis in the male.

Metabolism and disposition of [(14)C]dimethylamine borane in male Harlan Sprague Dawley rats following gavage administration, intravenous administration and dermal application.

1. Dimethylamine borane (DMAB) is used as a reducing agent in the manufacturing of a variety of products and in chemical synthesis. National Toxicology Program is evaluating the toxicity of DMAB in rodents following dermal application. The objective of this study was to evaluate the metabolism and disposition of DMAB in male Harlan Sprague Dawley (HSD) rats. 2. Disposition of radioactivity was similar between gavage and intravenous administration of 1.5 mg/kg [(14)C] DMAB, with nearly 84%-89% of the administered radioactivity recovered in urine 24 h post dosing. At 72 h, only 1% or less was recovered in feces, 0.3% as CO2, and 0.5%-1.4% as volatiles and 0.3%-0.4 % in tissues. 3. The absorption of [(14)C]DMAB following dermal application was moderate; percent dose absorbed increased with the dose, with 23%, 32% and 46% of dose absorbed at 0.15, 1.5 and 15 mg/kg, respectively. Urinary and fecal excretion ranged from 18%-37% and 2%-4% of dose, respectively, and 0.1%-0.2% as CO2, and 1%-3% as volatiles. Tissue retention of the radiolabel was low ∼1%, but was higher than following the gavage or intravenous administration. 4. Following co-adminsitration of DMAB and sodium nitrite by gavage, N-nitrosodimethylamine was not detected in blood or urine above the limit of quantitation of the analytical method of 10 ng/mL. 5. Absorption of DMAB in fresh human skin in vitro was ∼41% of the applied dose: the analysis of the receptor fluid shows that the intact DMAB complex can be absorbed through the skin.

Biological evaluation of dodecaborate-containing L-amino acids for boron neutron capture therapy.

To develop a boron carrier for practical purposes, new boron-containing amino acids with an undecahydro-closo-dodecaboranylthio ([(10)B(12)H(11)S](2-)-) unit in the side chain of the α-amino acid have already been designed and synthesized. In the present paper, cytotoxicity, the incorporation amounts into tumor cells, and the tumor cell killing effects of these compounds were elucidated to evaluate their usefulness as boron carriers. Furthermore, the microdistribution of the amino acids in tumor cells was established.

Synthesis, characterisation, and biodistribution of radioiodinated C-hydroxy-carboranes.

The synthesis, radiolabelling and biodistribution of iodinated C-hydroxy-nido-carborane ligands is described. Microwave heating by using NaF in aqueous ethanol was used to prepare {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate], nido-carboranol} and {sodium [7-hydroxy-7,8-dicarba-nido-undecaborate-8-carboxylic acid], nido-salborin} in 97 and 90 % yield, respectively. Radioiodination of these nido-carboranes was completed by using both (125)I and (123)I, and the products were obtained in high radiochemical purity (>99 %) and yield (72 to 87 %). The structures of the radiolabelled products were validated through comparison to authentic standards. Biodistribution studies in BALB/c mice showed low accumulation of the labelled compounds in the liver and intestines, which are sites where labelled carboranes typically localise. The labelled cluster bearing hydroxy and carboxylic acid groups on the two carbon vertices demonstrated preferential clearance through the kidneys and low thyroid uptake. This compound had substantially reduced non-specific binding than the deshydroxy analogue making it an attractive bifunctional ligand for preparing targeted molecular imaging and therapy agents.

Selective uptake of boronophenylalanine by glioma stem/progenitor cells.

The success of boron neutron capture therapy (BNCT) depends on the amount of boron in cells and the tumor/blood and tumor/(normal tissue) boron concentration ratios. For the first time, measurements of boron uptake in both stem/progenitor and differentiated glioma cells were performed along with measurements of boron biodistribution in suitable animal models. In glioma stem/progenitor cells, the selective accumulation of boronophenylalanine (BPA) was lower, and retention of boron after BPA removal was longer than in differentiated glioma cells in vitro. However, boron biodistribution was not statistically significantly different in mice with xenografts.

The formulation of polyhedral boranes for the boron neutron capture therapy of cancer.

The early promise of boron neutron capture therapy as a method for the treatment of cancer has been inhibited by the inherent toxicity associated with therapeutically useful doses of ¹°B-containing pharmacophores, the need for target-tissue specificity and the challenges imposed by biological barriers. Although developments in the synthetic chemistry of polyhedral boranes have addressed issues of toxicity to a considerable extent, the optimisation of the transport and the delivery of boronated agents to the site of action--the subject of this review--is a challenge that is addressed by the development of innovative formulation strategies.

A cell-permeable phosphine-borane complex delays retinal ganglion cell death after axonal injury through activation of the pro-survival extracellular signal-regulated kinases 1/2 pathway.

The reactive oxygen species (ROS) superoxide has been recognized as a critical signal triggering retinal ganglion cell (RGC) death after axonal injury. Although the downstream targets of superoxide are unknown, chemical reduction of oxidized sulfhydryls has been shown to be neuroprotective for injured RGCs. On the basis of this, we developed novel phosphine-borane complex compounds that are cell permeable and highly stable. Here, we report that our lead compound, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1) promotes RGC survival in rat models of optic nerve axotomy and in experimental glaucoma. PB1-mediated RGC neuroprotection did not correlate with inhibition of stress-activated protein kinase signaling, including apoptosis stimulating kinase 1 (ASK1), c-jun NH2-terminal kinase (JNK) or p38. Instead, PB1 led to a striking increase in retinal BDNF levels and downstream activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. Pharmacological inhibition of ERK1/2 entirely blocked RGC neuroprotection induced by PB1. We conclude that PB1 protects damaged RGCs through activation of pro-survival signals. These data support a potential cross-talk between redox homeostasis and neurotrophin-related pathways leading to RGC survival after axonal injury.

Prevention of clinical and histological signs of proteolipid protein (PLP)-induced experimental allergic encephalomyelitis (EAE) in mice by the water-soluble carbon monoxide-releasing molecule (CORM)-A1.

We have evaluated the effects of the carbon monoxide-releasing molecule CORM-A1 [Na(2) (BH(3) CO(2) ); ALF421] on the development of relapsing-remitting experimental allergic encephalomyelitis (EAE) in SJL mice, an established model of multiple sclerosis (MS). The data show that the prolonged prophylactic administration of CORM-A1 improves the clinical and histopathological signs of EAE, as shown by a reduced cumulative score, shorter duration and a lower cumulative incidence of the disease as well as milder inflammatory infiltrations of the spinal cords. This study suggests that the use of CORM-A1 might represent a novel therapeutic strategy for the treatment of multiple sclerosis.

Acidic heterocycles as novel hydrophilic pharmacophore of androgen receptor ligands with a carborane core structure.

A novel series of androgen receptor (AR) ligands bearing an acidic heterocycle with hydrogen-bonding ability as the terminal polar group was developed. Since most non-steroidal AR ligands so far known are structurally limited to nitro- or cyanobenzanilide as the polar pharmacophore, development of alternative hydrogen-bonding components is required to obtain novel AR ligands. Various acidic heterocycles were introduced into a hydrophobic phenylcarborane (1-phenyl-1,12-dicarba-closo-dodecaborane) core structure to provide a moiety that could interact effectively with the critical basic arginine residue of the AR ligand binding domain. The most potent compounds, 1,2,4-oxadiazole-5-thione derivatives 21a and 21b, exhibited higher affinity for hAR than did the well-known anti-androgen hydroxyflutamide. The results suggest that this heterocyclic functionality is potential bioisoster of the nitro and cyano groups forming the polar pharmacophores of known non-steroidal AR ligands.

Polyhedral boron compounds as potential diagnostic and therapeutic antitumor agents.

The use of polyhedral boron hydrides for cancer treatment is traditionally connected with boron neutron capture therapy. More recently, polyhedral borate anions were proposed as carriers of radionuclide label for targeted radionuclide therapy and diagnostics of cancer. Some metal derivatives of carboranes were found to demonstrate significant antitumor activity themselves. This review is designed to highlight the recent work concerning various fields of potential application of polyhedral boron compounds in anticancer diagnostics and therapy.

Organic syntheses on an icosahedral borane surface: closomer structures with twelvefold functionality.

The syntheses of a series of novel ester-linked derivatives of the icosahedral [closo-B12(OH)12]2- boron cluster (closomer esters) are described using several synthetic methods. The reaction of bis(tetrabutylammonium)-closo-dodecahydroxy-dodecaborate, [NBu4]2 1, with carboxylic acid chlorides and anhydrides, vinyl esters with a Y5(OiPr)13O catalyst and 1,1'-carbonyldiimidazole-activated carboxylic acids yields the corresponding dianionic dodeca-ester closomers. The method using 1,1'-carbonyldiimidazole-activated carboxylic acids may be employed as a general synthetic strategy. The use of elevated reaction temperatures, achievable under pressure, to expedite syntheses is described. An attractive methodology using immobilized scavenger reagents for the expeditious purification of the closomer esters was employed. The developed methodology is compatible with a variety of peripheral functional groups attached to the termini of densely packed, carboxylate ester-linked radial arms bonded to the icosahedral borane surface. A closomer ester having twelve terminal amino groups was prepared, and without isolation, fully acetylated in good yield.

Synthesis and cellular studies of porphyrin-cobaltacarborane conjugates.

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.

Accumulation of boron in human malignant glioma cells in vitro is cell type dependent.

It has been shown that human malignant glioma tumours consist of several subpopulations of tumour cells. Due to heterogeneity and different degrees of vascularisation cell subpopulations possess varying resistance to chemo- or radiation therapy. Therefore, therapy is dependent on the ability to specifically target a tumour cell. Boron neutron capture therapy (BNCT) is a bimodal method, in radiation therapy, taking advantage of the ability of the stable isotope boron-10 to capture neutrons. It results in disintegration products depositing large amounts of energy within a short length, approximately one cell diameter. Thereby, selective irradiation of a target cell may be accomplished if a sufficient amount of boron has been accumulated and hence the cell-associated boron concentration is of critical importance. The accumulation of boron, boronophenylalanine (BPA), was investigated in two human glioma cell subpopulations and a human fibroblast cell line in vitro. The cells were incubated at low boron concentrations (0-5 microg B/ml). Oil filtration was then used for separation of extracellular and cell-associated boron. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for boron determination. Significant (P < 0.05) differences in accumulation ratio (relation between cell-associated and extracellular boron concentration) between human malignant glioma cell lines were found. Human fibroblasts, used to represent normal cells, showed a growth-dependent uptake and a lower accumulation ratio than the glioma cells. Our findings indicate that BPA concentration, incubation time and differences in boron uptake between cell subpopulations should be considered in BNCT.

Synthesis, radioiodination, and biodistribution of some nido- and closo-monocarbon carborane derivatives.

Iodination and radioiodination reactions of several anionic nido- and closo-monocarbon carboranes were conducted. Iodinations occurred more rapidly with nido-carboranes than with closo-carboranes. The most rapid iodination and radioiodination reactions occurred with unsubstituted carboranes. C-amino and C-ammonium derivatives did not iodinate under the conditions studied. Both nido- and closo-carboranes with C-NH-acetyl and C-NH-succinyl substituents iodinated, but the nido-carboranes iodinated under milder reaction conditions. Biodistributions of nido-1-succinylamido-[(131)I]carborane and closo-1-succinylamido-[(125)I]carborane were similar in mice, but blood clearance of the nido- compound was slower.