Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT)

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.     

Spontaneous nucleophilic addition of hydroxide ions to the meso-position of high-valent antimony-oxo porphyrin complexes

Bimolecular reactions of the antimony(V) porphyrin complexes SbO(tpp)OH, 1 and SbO(oep)OH, 2 with tetra-n-butylammonium hydroxide were investigated at 298 K in acetonitrile solution (tpp, dianion of 5,10,15,20-tetraphenylporphyrin and oep, dianion of 2,3,7,8,12,13,17,18-octaethylporphyrin). Spontaneous nucleophilic addition of hydroxide ions to the non-oxidized porphyrin macrocycle leads to novel hydroxyphlorin derivatives, which contain a saturated meso-carbon bridge. While this process is a reversible equilibrium reaction for the TPP derivative, the OEP complex undergoes subsequent demetallation and oxidative ring cleavage in the presence of dioxygen. Possible implications for the competitive inhibition of heme-oxygenase by high-valent metalloporphyrin therapeutics are discussed.     

Boronated derivatives of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e ₆ and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e ₆ by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec⁻¹, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.     

Comparative molecular field analysis and comparative molecular similarity indices analysis of boron-containing human thymidine kinase 1 substrates

Three-dimensional quantitative structure-activity relationship (3D-QSAR) using CoMFA and CoMSIA techniques was applied to evaluate 56 pyrimidine nucleosides as substrates of human thymidine kinase 1 (hTK1), 27 of them containing a carborane substituent either at the 3-, 5-, or 3'-position of the 2'-deoxyuridine scaffold. This is the first report describing 3D-QSAR studies of compounds containing boron atoms. Both CoMFA and CoMSIA models were derived from a training set of 47 molecules and the predictive capacity of the CoMSIA model was successfully validated by accurately calculating known phosphorylation rates of both boronated and non-boron hTK1 substrates that were not included in the training set. The optimal CoMSIA model provided the following values: q(2) 0.622, r(2) 0.983, s 0.165, and F 187.5. Contour maps obtained from the CoMSIA model were in agreement with the experimentally determined biological data.     

Association of sugar-based carboranes with cationic liposomes: an electron spin resonance and light scattering study

The possibility of cationic (di-oleoyltrimethylammonium propane, DOTAP)/(L-alpha-dioleoylphosphatidyl-ethanolamine, DOPE) liposomes to act as carriers of boronated compounds such as 1,2-dicarba-closo-dodecaboran(12)-1-ylmethyl](beta-D-galactopyranosyl)-(1-->4)-beta-D-glucopyranoside and 1,2-di-(beta-D-gluco-pyranosyl-ox)methyl-1,2-dicarba-closo-dodeca-borane(12) has been investigated by Electron Spin Resonance (ESR) of n-doxyl stearic acids (n-DSA) and Quasi-Elastic Light Scattering (QELS). Both these carboranes have potential use in Boron Neutron Capture Therapy (BNCT), which is a targeted therapy for the treatment of radiation resistant tumors. They were shown to give aggregation both in plain water and in saline solution. Carborane aggregates were, however, disrupted when DOTAP/DOPE liposome solutions were used as dispersing agents. The computer analysis of the ESR spectra from carborane-loaded liposomes allowed to establish an increase of the order degree in the liposome bilayer with increasing carborane concentration, together with a decreased mobility. The same discontinuities of both correlation time and order parameter with respect to temperature variations were observed in carborane-containing and carborane-free liposomes. This suggested that a homogeneous dispersion of nitroxides and carboranes occurred in the liposome bilayer. The ESR line shape analysis proved that no dramatic changes were induced in the liposome environment by carborane insertion. QELS data showed that the overall liposome structure was preserved, with a slight decrease in the mean hydrodynamic radius and increase in polydispersity caused by the guest molecules.     

Beta-lactam congeners of orlistat as inhibitors of fatty acid synthase

Beta-lactam derivatives of orlistat were prepared and their inhibitory activities toward the thioesterase domain of fatty acid synthase (FAS-TE) were evaluated using a recombinant form of the enzyme. While in general these derivatives showed lower potency compared to beta-lactones, a reasonably potent, lead compound (-)-9 (IC(50)=8.6microM) was discovered that suggests that this class of compounds should be evaluated further.     

Design, synthesis, and antitumor evaluation of novel acenaphtho[1,2-b]pyrrole-carboxylic acid esters with amino chain substitution

8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid esters and derivatives were prepared and evaluated for cytotoxicity against A549 and P388 cell lines. Based on a novel chromophore precursor 8-oxo-8H-acenaphtho[1,2-b]pyrrol-9-carbonitrile 1, the very insoluble 1 was converted to more soluble esters 5 and a series of 3-amino derivatives from 5 were obtained by mild S(N)Ar(H) reaction between 5 and various amines. The biological evaluation indicated that methyl esters 5a are the most cytotoxic with IC(50) values of 0.45 and 0.80 microM (against A549 and P388, respectively) among the parent esters 5a-5f, but 3-amino derivatives 4b and 4c of 5f with bromine showed the highest activity (with IC(50) values of 0.019-0.60 microM) among the 3-amino derivatives.     

Synthesis,characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives

In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropagation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring, were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the corresponding benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melting point determinations, CI+ MS and ¹H NMR spectroscopy. The cytokinin activity of all the prepared derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3 in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the parent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N⁹ position decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective, the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal cytotoxicity compared to the corresponding N⁹-ribosides. Finally, the pH stability of the two model compounds was assessed in acidic and neutral water solutions (pH 3–7) by high-performance liquid chromatography (HPLC).     

Synthesis and SAR studies of a novel series of T-type calcium channel blockers

For the novel, potent, and selective T-type Ca2+ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinazoline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca2+ channels. Among them, 9c (KYS05064, IC50 = 0.96 +/- 0.22 microM) was found to be as potent as Mibefradil and also showed the highest selectivity for T-type Ca2+ channel with no effect on N-type Ca2+ channel.     

Benzofuran- and furan-2-yl-(phenyl)-3-pyridylmethanols: synthesis and inhibition of P450 aromatase

A series of benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives were prepared using an efficient 1-step procedure in good yields. In addition furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were also prepared to determine the effect of the benzene ring in benzofuran with respect to inhibitory activity. The pyridylmethanol derivatives were all evaluated in vitro for inhibitory activity against aromatase (P450(AROM), CYP19), using human placental microsomes. The benzofuran-2-yl-(phenyl)-3-pyridylmethanol derivatives showed good to moderate activity (IC50 = 1.3-25.1 microM), which was either better than or comparable with aminoglutethimide (IC50 = 18.5 microM) but lower than arimidex (IC50 = 0.6 microM), with the 4-methoxyphenyl substituted derivative displaying optimum activity. Molecular modelling of the benzofuran-2-yl-(4-fluorophenyl)-3-pyridylmethanol derivative suggested activity to reside with the (S)-enantiomer. The furan-2-yl-(phenyl)-3-pyridylmethanol derivatives were devoid of activity indicating the essential role of the benzene ring of the benzofuran component for enzyme binding.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy

Boron neutron capture therapy (BNCT) depends on the selective delivery of a sufficient number of (10)B atoms to tumor cells to sustain a lethal (10)B(n,alpha)(7)Li reaction. Expression of FR frequently is amplified among human tumors. The goal of the present study was to investigate folate receptor (FR)-targeted liposomes as potential carriers for a series of boron-containing agents. Two highly ionized boron compounds, Na(2)[B(12)H(11)SH] and Na(3) (B(20)H(17)NH(3)), were incorporated into liposomes by passive loading with encapsulation efficiencies of 6% and 15%, respectively. In addition, five weakly basic boronated polyamines were investigated. Two were the spermidine derivatives: N(5)-(4-carboranylbutyl)spermidine.3HCl (SPD-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermidine.4HCl (ASPD-5). Three were the spermine derivatives: N(5)-(4-carboranylbutyl)spermine.4HCl (SPM-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermine.5HCl (ASPM-5), and N(5),N(10)-bis(4-carboranylbutyl)spermine.4 HCl (SPM-5,10). These were incorporated into liposomes by a pH-gradient-driven remote-loading method with varying loading efficiencies, which were influenced by the specific trapping agent and the structure of the boron compound. Greater loading efficiencies were obtained with lower molecular weight boron derivatives, using ammonium sulfate as the trapping agent, compared to those obtained with sodium citrate. The in vitro uptake of folate-derivatized, boronated liposomes was investigated using human KB squamous epithelial cancer cells, which have amplified FR expression. Higher cellular boron uptake (up to 1584 microg per 10(9) cells) was observed with FR-targeted liposomes than with nontargeted control liposomes (up to 154 microg per 10(9) cells), irrespective of the chemical form of the boron and the method used for liposomal preparation. KB cell binding of the FR-targeted liposomes was saturable and could be blocked by 1 mM free folic acid. Our findings suggest that further evaluation of FR-targeted liposomes is warranted to assess their potential as boron carriers for neutron capture therapy.     

Conjugation of phenyl isothiocyanate derivatives of carborane to antitumor antibody and in vivo localization of conjugates in nude mice

A systematic study of the conjugation of 1-(p-isothiocyanatophenyl)-1,2-dicarba-closo-[2-3H]dodeca borane(12), 3H-1, and 7-(p-isothiocyanatophenyl)dodecahydro-7,8-dicarba-nido -[8-3H] undecaborate(1-)ion, 3H-2, to the murine monoclonal anti-CSAp antibody, Mu-9, was carried out to compare charged and uncharged boron cages in their effect upon antibody loading. Approximately one neutral cage and four of the anionic cages were successfully linked to antibody in two separate conjugates which were subsequently radioiodinated and evaluated in vivo. No significant loss of the antibody or its immunoreactivity was observed in either case. In nude mice bearing GW-39 tumor xenografts the conjugate containing the anionic carborane derivative showed a reduced tumor uptake although the tumor:non-tumor ratio was similar to that of the native antibody. The carborane cage in 2 was radiolabeled with 125I followed by attempts to purify and conjugate product 4 to a model goat IgG protein. This exploratory conjugation study was undertaken as a prelude to linking new conjugation reagents, which contain multiple anionic boron cages, to antitumor antibodies. The latter conjugates are required to maximize boron loading for the purpose of neutron-capture therapy.     

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: a new fluorescence displacement titration technique for characterizing the nucleotide binding site on the catalytic subunit

We determined the dissociation constant (Kd) of a series of nucleotides for the bovine skeletal muscle type II catalytic subunit by displacing lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) with increasing concentrations of competing nucleotide. The Kd of each nucleotide was calculated from the decreases in the fluorescence polarization of lin-benzo-ADP that accompany its displacement from the catalytic subunit. We found that modifications of the adenine moiety reduce nucleotide affinity for the enzyme. The effect was most pronounced with modifications at position 6 of the base. Replacement of the 3'-hydroxyl group of ribose with a hydrogen increased the affinity of the nucleotide; addition of phosphate to the 2'- or 3'-hydroxyl groups, on the other hand, decreased nucleotide affinity. MgATP and MgADP exhibited Kd's of about 10 microM. AMP, which contains a negatively charged alpha-phosphate, bound with reduced affinity (643 microM). Adenosine, which lacks a charged alpha-phosphate, bound with a higher affinity (32 microM). To learn more about the nature of the alpha-phosphate binding site, a series of uncharged and positively charged derivatives of the 5'-position on the ribose moiety was prepared. The uncharged derivatives bound with much greater affinity than the negatively charged AMP. The Kd's for 5'-tosyladenosine and 5'-iodo-5'-deoxyadenosine were 30 and 32 microM, respectively. Like the negatively charged AMP, positively charged derivatives also bound less tenaciously than the neutral species. The positively charged 5'-amino-5'deoxyadenosine, for example, exhibited a 15-fold higher Kd (506 microM) than the neutral congenors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic activities of novel hexahydroindolizino[8,7-b]indole derivatives prepared by 1,3-dipolar cycloaddition reactions of 3,4-dihydro-beta-carboline ylides

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-beta-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-beta-carboline were found to be the most active, with IC(50)'s in the 2-50 microM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c, respectively, were found by cytometric flux analysis to stop cancer cell growth at the G(2)M and 8N (>G(2)M) stage of the cell cycle. These two compounds also showed no loss of cytotoxic activity in K562R cancer cells resistant to doxorubicin.     

Synthesis and biological evaluation of disubstituted N6-cyclopentyladenine analogues: the search for a neutral antagonist with high affinity for the adenosine A1 receptor

Novel 3,8- and 8,9-disubstituted N(6)-cyclopentyladenine derivatives were synthesised in moderate overall yield from 6-chloropurine. The derivatives were made in an attempt to find a new neutral antagonist with high affinity for adenosine A(1) receptors. N(6)-Cyclopentyl-9-methyladenine (N-0840) was used as a lead compound. Binding affinities of the new analogues were determined for human adenosine A(1) and A(3) receptors. Their intrinsic activity was assessed in [35S]GTPgammaS binding experiments. Elongation of the 9-methyl of N-0840 to a 9-propyl substituent was very well tolerated. A 9-benzyl group, on the other hand, caused a decrease in adenosine A(1) receptor affinity. Next, the 8-position was examined in detail, and affinity was increased with appropriate substitution. Most derivatives were A(1)-selective and 20 of the new compounds (6-9, 15-21, 23-26, 28, 31, 33, 35, and 36) had higher adenosine A(1) receptor affinity than the reference substance, N-0840. Compound 31 (N(6)-cyclopentyl-8-(N-methylisopropylamino)-9-methyladenine, LUF 5608) had the highest adenosine A(1) receptor affinity, 7.7 nM. In the [35S]GTPgammaS binding experiments, derivatives 5, 14, 22, 23, 25, 26, 33 and 34 did not significantly change basal [35S]GTPgammaS binding, thus behaving as neutral antagonists. Moreover, four of these compounds (23, 25, 26, and 33) displayed a 4- to 10-fold increased adenosine A(1) receptor affinity (75-206 nM) compared to N-0840 (852 nM). In summary, we synthesised a range of N-0840 analogues with higher affinity for adenosine A(1) receptors. In addition, four new derivatives, LUF 5666 (23), LUF 5668 (25), LUF 5669 (26) and LUF 5674 (33), behaved as neutral antagonists when tested in [35S]GTPgammaS binding studies. Thus, these compounds have improved characteristics as neutral adenosine A(1) receptor antagonists.     

Synthesis and biological activity of olomoucine II

Based on our previous experiences with synthesis of purines, novel 2,6,9-trisubstituted purine derivatives were prepared and assayed for the ability to inhibit CDK1/cyclin B kinase. One of newly synthesized compounds designated as olomoucine II, 6-[(2-hydroxybenzyl)amino]-2-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine, displays 10 times higher inhibitory activity than roscovitine, potent and specific CDK1 inhibitor. Olomoucine II in vitro cytotoxic activity exceeds purvalanol A, the most potent CDK inhibitor, as it kills the CEM cells with IC(50) value of 3.0 microM.     

Synthesis of feruloyl-myo-insitol derivatives and their inhibitory effects on phorbol ester-induced superoxide generation and Esptein-Barr virus activation

We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O(2)(-)) generation. And further, their suppressive effect on the TPA-induced Epstein-Barr virus (EBV) activation was examined in order to estimate their anti-carcinogenic potentials. Among the derivatives tested, 1,6-O-bis[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol (6b) showed an excellent suppressive activity on the O(2)(-) generation at a concentration of 20 microM. For the suppressive effects on the EBV activation, 2,4,6-O-tris[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate (9b) showed the highest activity at a concentration of 100 microM among the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules.     

Synthesis and biological evaluation of folate receptor-targeted boronated PAMAM dendrimers as potential agents for neutron capture therapy

Successful treatment of cancer by boron neutron capture therapy (BNCT) requires the selective delivery of (10)B to constituent cells within a tumor. The expression of the folate receptor is amplified in a variety of human tumors and potentially might serve as a molecular target for BNCT. In the present study we have investigated the possibility of targeting the folate receptor on cancer cells using folic acid conjugates of boronated poly(ethylene glycol) (PEG) containing 3rd generation polyamidoamine dendrimers to obtain (10)B concentrations necessary for BNCT by reducing the uptake of these conjugates by the reticuloendothelial system. First we covalently attached 12-15 decaborate clusters to 3rd generation polyamidoamine dendrimers. Varying quantities of PEG units with varying chain lengths were then linked to these boronated dendrimers to reduce hepatic uptake. Among all prepared combinations, boronated dendrimers with 1-1.5 PEG(2000) units exhibited the lowest hepatic uptake in C57BL/6 mice (7.2-7.7% injected dose (ID)/g liver). Thus, two folate receptor-targeted boronated 3rd generation polyamidoamine dendrimers were prepared, one containing approximately 15 decaborate clusters and approximately 1 PEG(2000) unit with folic acid attached to the distal end, the other containing approximately 13 decaborate clusters, approximately 1 PEG(2000) unit, and approximately 1 PEG(800) unit with folic acid attached to the distal end. In vitro studies using folate receptor (+) KB cells demonstrated receptor-dependent uptake of the latter conjugate. Biodistribution studies with this conjugate in C57BL/6 mice bearing folate receptor (+) murine 24JK-FBP sarcomas resulted in selective tumor uptake (6.0% ID/g tumor), but also high hepatic (38.8% ID/g) and renal (62.8% ID/g) uptake, indicating that attachment of a second PEG unit and/or folic acid may adversely affect the pharmacodynamics of this conjugate.     

Polyamines and the NMDA receptor: modifying intrinsic activities with aromatic substituents

Thirty-four spermidine (SPD) and spermine (SPM) derivatives with aromatic substituents were synthesized and tested as inhibitors of specific binding of the NMDA channel blocker [3H]MK-801 to membranes prepared from rat hippocampus and cerebral cortex. SPD and SPM derivatives with aromatic substituents at the primary amino groups were the most potent inhibitors (IC50 3.9-4.7 microM). These compounds most likely act directly at the NMDA ion channel, since 30 microM SPM had no pronounced influence on their inhibiting activities. SPD derivatives with aromatic substituents at the secondary amino group were either inactive or highly SPM-sensitive inhibitors (IC50 10-82 microM), depending on the size of the substituent. Our results support the hypothesis that an aromatic interaction site near the center of polyamine derivatives contributes to polyamine inverse agonism.