In vitro oxidation of [U-14C] palmitate, [1-14C] and [6-14C] glucose in newborn and adult rats and pigs

Studies have been made on the intensity of oxidation of [U-14C]-palmitate, [1-14C]- and [6-14C]-glucose by slices of the liver and skeletal muscles of new-born, 1-day, 5-day and adult Wistar rats and domestic pigs. It was found that the level of 14CO2 production from these substrates is higher in tissues of rats than in those of pigs. At early stages of ontogenesis, in tissues of both species intensive oxidation of glucose is observed together with oxidation of fatty acids. In the course of ontogenetic development, the intensity of glucose utilization significantly decreases, whereas the level of fatty acid catabolism remains relatively unaffected.     

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.     

In vivo biodistribution of two [18F]-labelled muscarinic cholinergic receptor ligands: 2-[18F]- and 4-[18F]-fluorodexetimide

Two [18F]-labelled analogues of the potent muscarinic cholinergic receptor (m-AChR) antagonist, dexetimide, were evaluated as potential ligands for imaging m-AChR by positron emission tomography (PET). Intravenous administration of both 2-[18F]- or 4-[18F]-fluorodexetimide resulted in high brain uptake of radioactivity in mice. High binding levels were observed in m-AChR rich areas, such as cortex and striatum, with low levels in the receptor-poor cerebellum. Uptake of radioactivity was saturable and could be blocked by pre-administration of dexetimide or atropine. Drugs with different sites of action were ineffective at blocking receptor binding. The results indicate that both radiotracers are promising candidates for use in PET studies.     

In vitro estimation of the rate of hexose phosphorylation, by sequential pulsing with [3H]- and [14C]2-deoxy-D-glucose

The rate of phosphorylation of 2-deoxy-D-glucose (2dGlc) was determined by incubating Schistosoma mansoni in vitro in [3H]2-deoxy-D-glucose; 60 sec after exposure to the [3H]dGlc, [14C]dGlc was added to the medium, and metabolic activity was arrested at 2 min by immersion of the tissue in ice-cold silicone oil. Column chromatographic separation of the neutral [3H]- and [14C]dGlc from the [3H]- and [14C]2-deoxy-D-glucose-6-phosphate permitted estimation of the quantity of [3H]dGlc phosphorylated in 2 min, and the proportion of [14C]dGlc phosphorylated in 1 min; thus a phosphorylation rate was determined from a single tissue sample. In male schistosomes derived from mouse infections 4.4 +/- 0.8% of the dGlc was phosphorylated each minute, and 4.2 +/- 0.9% in the females. Lower rates of phosphorylation were measured in schistosomes taken from hamsters where males phosphorylated 2.4 +/- 1.1% of the dGlc each minute, and in females 2.7 +/- 1.0%. These studies suggest the high rate of hexose utilization by schistosomes compares to the conscious rat brain, where 11% of the dGlc is phosphorylated each minute.     

In vitro and in vivo evaluation of 64Cu-labeled DOTA-linker-bombesin(7-14) analogues containing different amino acid linker moieties

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a variety of tumor types and has been targeted with radiolabeled peptides for detection and therapy of these cancers. Analogues of the 14 amino acid bombesin (BN) peptide have been radiolabeled with both gamma- and positron-emitting radionuclides for detection of GRPR-expressing tumors. We have previously evaluated BN analogues radiolabeled with the positron-emitter, copper-64 (64Cu), that contained various aliphatic linkers placed between the BN peptide and the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator. These studies showed that the analogues could be used for positron-emission tomographic (PET) imaging of GRPR-positive tumors in mice but clinical translation would be hindered by significant uptake in background tissues. Therefore, the purpose of this study was to determine if the use of amino acid linkers placed between the DOTA chelate and the BN peptide would reduce nontarget tissue uptake, while maintaining good prostate tumor uptake. The linkers studied utilized three amino acid combinations of glycine (G), serine (S), or glutamic acid (E). In vitro assays in PC-3 cells showed that the glutamic acid-containing linkers had poor binding and internalization, while the other analogues had IC50 values <100 nM and good internalization. In vivo, these same analogues demonstrated tumor-specific uptake and good imaging characteristics that were comparable to, or better than the previously reported 64Cu-labeled DOTA-BN analogues. Overall, this study shows that BN analogues containing amino acid linkers can be used for the PET imaging of GRPR-expressing prostate cancer and that these linkers lead to lower background tissue uptake.     

In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide E-[c(RGDfK)2] that targets integrin alphavbeta3

Integrins, especially integrin alpha vbeta3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK)2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK)2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK)2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK)2]-DOXO-1 and E-[c(RGDfK)2]-DOXO-2. In E-[c(RGDfK)2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK)2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK)2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK)2], E-[c(RGDfK)2]-DOXO-1, E-[c(RGDfK)2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg.     

Type and location of fluorescent probes incorporated into the potent mu-opioid peptide [Dmt]DALDA affect potency, receptor selectivity and intrinsic efficacy.

The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.     

Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.     

Biosynthesis of thyroglobulin. Incorporation of [1-14C]galactose, [1-14C]mannose and [4,5-3H2]leucine into soluble proteins by rat thyroids in vitro

1. Rat thyroid lobes were incubated for various periods of time in Krebs–Ringer bicarbonate containing [³H]leucine and either [1-¹⁴C]galactose or [1-¹⁴C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [¹⁴C]-mannose into soluble thyroid proteins was parallel to that observed for [³H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17–18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3–8s). Radioactivity from double-labelled fractions 12s and 3–8s was transferred to protein 17–18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [¹⁴C]galactose was immediately incorporated into protein 17–18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [³H]leucine and [¹⁴C]mannose into all protein fractions, but had little effect on the incorporation of [¹⁴C]galactose into protein 17–18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17–18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17–18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17–18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.     

In vivo evaluation of [18F]FECIMBI-36, an agonist 5-HT2A/2C receptor PET radioligand in nonhuman primate

We recently reported the radiosynthesis and in vitro evaluation of [¹⁸F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([¹⁸F]FECIMBI-36) or ([¹⁸F]1), an agonist radioligand for 5HT_2A/2C receptors in postmortem samples of human brain. Herein we describe the in vivo evaluation of [¹⁸F]FECIMBI-36 in vervet/African green monkeys by PET imaging. PET images show that [¹⁸F]FECIMBI-36 penetrates the blood-brain barrier and a low retention of radioactivity is observed in monkey brain. Although the time activity curves indicate a somehow heterogeneous distribution of the radioligand in the brain, the low level of [¹⁸F]FECIMBI-36 in brain may limit the use of this tracer for quantification of 5-HT_2A/2C receptors by PET.     

In vivo evaluation of 7 alpha-[11-(4-[125I]iodophenoxy)undecyl]-17 beta-estradiol: a potential vector for therapy of adrenal and estrogen receptor-positive cancers

7 alpha-[11-(4-[125I]Iodophenoxy)undecyl]-17 beta-estradiol ([125I]IPUE2) was synthesized and its tissue distribution studied in immature female Fischer rats. Upon intravenous administration, [125I]IPUE2 was shown to accumulate in the adrenals and, to some extent, in the uterus and the ovaries. Coinjection with estrogen receptor (ER)-saturating quantities of unlabeled 17 beta-estradiol did not significantly reduce the uptake of [125I]IPUE2 in these tissues. The high adrenal uptake of [125I]IPUE2 is most likely associated with the lipophilic nature of the 7 alpha-substituted estradiol. The potential to use the 7 alpha-undecylestradiol as a vector to direct therapeutic groups to adrenal and ER-positive cancers is discussed.     

In vivo incorporation of [U-14C]glycerol and [2-3H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals

A double label design was used to study the in vivo incorporation of [U-¹⁴C] and [2-³H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected.The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.     

In vitro characterization of radioiodinated (+)-2-[4-(4-iodophenyl) piperidino]cyclohexanol [(+)-pIV] as a sigma-1 receptor ligand

We investigated the binding characteristics of a (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[125I]pIV], radioiodinated at the para-position of the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (+)-pIV (Ki=1.30 nM) had more than 10 times higher affinity to the sigma-1 (sigma-1) receptor than (+)-pentazocine (Ki=19.9 nM) or haloperidol (Ki=13.5 nM) known as sigma ligands. Also, the binding affinity of (+)-pIV for the sigma-1 receptor (Ki=1.30 nM), was about 16 times higher than the sigma-2 (sigma-2) receptor (Ki=20.4 nM). (+)-pIV (Ki=1260 nM) had a much lower affinity for VAChT than (-)-vesamicol (Ki=13.0 nM) or (-)-pIV (Ki=412 nM). (+)-[125I]pIV had low affinity for the dopamine, serotonin, adrenaline, and acetylcholine receptors. Furthermore, in a saturation binding study, (+)-[125I]pIV exhibited a K) of 6.96 nM with a Bmax of 799 fmol/mg of protein. These results showed that (+)-pIV binds to the sigma-1 receptor with greater affinity than sigma receptor ligands such as (+)-pentazocine or haloperidol, and that radioiodinated (+)-pIV is suitable as radiotracer for sigma-1 receptor studies in vitro.     

Synthesis, in vitro and in vivo evaluation of [O-methyl-11C] 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]-triazine-3,5-dione: a novel agonist 5-HT1A receptor PET ligand

Synthesis and in vivo evaluation of 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (5 or MMT), a high affinity and selective serotonin 5-HT1AR agonist PET tracer, are described. GTPgammaS assay shows that MMT is an agonist with an EC50 comparable to 5-HT. Radiolabeling of 5 was achieved in 30% yield (EOS) from desmethyl-MMT (4) with >99% chemical and radiochemical purities and a specific activity >1000 Ci/mmol. PET studies in baboon show that [11C]5 penetrates the blood-brain barrier but, because of low specific binding and fast clearance of radioactivity it is not a suitable PET tracer for the in vivo quantification of 5-HT1AR.     

Synthesis,radiolabeling and preliminary in vivo evaluation of [18F]FE-PE2I,a new probe for the dopamine transporter

A new dopamine transporter (DAT) ligand, (E)-N-(3-iodoprop-2-enyl)-2β-carbofluoroethoxy-3β-(4′-methyl-phenyl) nortropane (FE-PE2I, 6), derived from PE2I (1), was prepared and found to be a potent inhibitor of rodent DAT in vitro. Compound 6 was radiolabelled with fluorine-18 (t_1/2 = 109.8 min) for PET studies in monkeys. In vivo PET measurements showed a regional distribution in brain that corresponds to the known distribution of DAT. This binding was specific, reversible and the kinetics of [¹⁸F]6 binding in brain were faster than for its lead compound, [¹¹C]1. The possible presence of a hydroxymethyl-radiometabolite formed by oxidation in the 3β-benzylic position of [¹⁸F]6 warrants further detailed evaluation of the metabolism of [¹⁸F]6. [¹⁸F]6 is a potential radioligand for imaging DATs in the human brain with PET.     

Synthesis and evaluation of 1-[2-(4-[11C]methoxyphenyl)phenyl]piperazine for imaging of the serotonin 5-HT7 receptor in the rat brain

1-[2-(4-Methoxyphenyl)phenyl]piperazine (4) is a potent serotonin 5-HT₇ receptor antagonist (K_i = 2.6 nM) with a low binding affinity for the 5-HT_1A receptor (K_i = 476 nM). As a potential positron emission tomography (PET) radiotracer for the 5-HT₇ receptor, [¹¹C]4 was synthesized at high radiochemical yield and specific activity, by O-[¹¹C]methylation of 2′-(piperazin-1-yl)-[1,1′-biphenyl]-4-ol (6) with [¹¹C]methyl iodide. Autoradiography revealed that [¹¹C]4 showed in vitro specific binding with 5-HT₇ in the rat brain regions, such as the thalamus which is a region with high 5-HT₇ expression. Metabolite analysis indicated that intact [¹¹C]4 in the brain exceeded 90% of the radioactive components at 15 min after the radiotracer injection, although two radiolabeled metabolites were found in the rat plasma. The PET study of rats showed moderated uptake of [¹¹C]4 in the brain (1.2 SUV), but no significant regional difference in radioactivity in the brain. Pretreatment with 5-HT₇-selective antagonist SB269970 (3) did not decrease the uptake of [¹¹C]4 in the rat brain. Further studies are warranted that focus on the development of PET ligand candidates with higher binding affinity for 5-HT₇ and higher in vivo stability in brain than 4.     

Synthesis and in vivo evaluation of [18F]-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide as a PET imaging probe for COX-2 expression

Synthesis of [18F]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ([18F]celecoxib), a selective COX-2 inhibitor, is achieved via a bromide to [18F]F- exchange reaction. Synthesis of the precursor for radiolabeling was achieved from 4'-methylacetophenone in four steps with 22% overall yield. Under non-radioactive conditions, fluorination was achieved using TBAF in DMSO at 135 degrees C in 80% yield. Synthesis of [18F]celecoxib was achieved using [18F]TBAF in DMSO at 135 degrees C in 10+/-2% yield (EOS) with >99% chemical and radiochemical purities. The specific activity was 120+/-40 mCi/micromol (EOB). [18F]celecoxib was found to be stable in ethanol, however, de[18F]fluorination (6.5%) was observed after 4 h in 10% ethanol-saline solution. Rodent PET studies show bone labeling indicating in vivo de[18F]fluorination of [18F]celecoxib. PET studies in baboon indicated a lower rate of de[18F]fluorination than rat and retention of radioactivity in brain regions consistent with the known distribution of COX-2. A radiolabeling method that can generate consistent high specific activity is needed for routine human use.     

Design, synthesis, and evaluation of 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate (8MDP-fluor), as a novel equilibrative nucleoside transporter probe

Nucleoside transporters are integral membrane glycoproteins that play critical roles in physiological nucleoside and nucleobase fluxes, and influence the efficacy of many nucleoside chemotherapy drugs. Fluorescent reporter ligands/substrates have been shown to be useful in the analysis of nucleoside transporter (NT) protein expression and discovery of new NT inhibitors. In this study, we have developed a novel dipyridamole (DP)-based equilibrative nucleoside transporter 1 (ENT1) fluorescent probe. The potent ENT1 and ENT2 inhibitor analogue of dipyridamole, 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (4, 8MDP), was modified to replace one β-hydroxyethyl group of the amino substituent at the 2-position with a β-aminoethyl group and then conjugated through the amino group to 6-(fluorescein-5-carboxamido)hexanoyl moiety to obtain a new fluorescent molecule, 2-diethanolamino-4,8-diheptamethyleneimino-2-(N-aminoethyl-N-ethanolamino)-6-(N,N-diethanolamino)pyrimido[5,4-d]pyrimidine-fluorescein conjugate, designated 8MDP-fluorescein (8MDP-fluor, 6). The binding affinities of 8MDP-fluor at ENT1 and ENT2 are reflected by the uridine uptake inhibitory K(i) values of 52.1 nM and 285 nM, respectively. 8MDP-fluor was successfully demonstrated to be a flow cytometric probe for ENT1 comparable to the nitrobenzylmercaptopurine riboside (NBMPR) analogue ENT1 fluorescent probe SAENTA-X8-fluorescein (SAENTA-fluor, 1). This is the first reported dipyridamole-based ENT1 fluorescent probe, which adds a novel tool for probing ENT1, and possibly ENT2.