The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36.

Fatty acid translocase (FAT)/CD36 has been associated with diverse normal and pathologic processes. These include scavenger receptor functions (uptake of apoptotic cells and modified lipid), lipid metabolism and fatty acid transport, adhesion, angiogenesis, modulation of inflammation, transforming growth factor-beta activation, atherosclerosis, diabetes and cardiomyopathy. Although CD36 was identified more than 25 years ago, it is only with the advent of recent genetic technology that in vivo evidence has emerged for its physiologic and pathologic relevance. As these in vivo studies are expanded, we will gain further insight into the mechanism(s) by which CD36 transmits a cellular signal, and this will allow the design of specific therapeutics that impact on a particular function of CD36.

Stereoselective synthesis, in vitro, and initial in vivo evaluation of 1-methylpiperidin-4-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IPIP): a novel radioiodinated molecular probe with high affinity for the muscarinic receptor.

1-Methylpiperidin-4-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IPIP, Fig. 1) was investigated as a potential radioiodinated molecular probe targeted to the muscarinic receptor complex. The IPIP stereoisomers were synthesized via a chiral intermediate in >95% enantiomeric excess. The R-isomers demonstrated a M(1) to M(2) subtype selectivity of approximately 3 to 1 and the S-isomers demonstrated non-subtype selective binding in vitro. IPIP was radiolabeled with iodide-125 with an average radiochemical yield of 74.4% (+/-14.8, n = 5), specific activities >800 mCi/micromol, and radiochemical purities >97%. In vivo the Z-isomers demonstrated high uniform cerebral uptake suggesting non-subtype selective binding. In contrast, E-R-IPIP, after allowing a low uptake in M(2) rich areas to clear, demonstrated a retention of activity in M(1) and M(4) rich cerebral regions. In addition, the cerebral uptake of E-R-IPIP and Z-S-IPIP were inhibited by 70-90% via pretreatment with R-QNB, an established muscarinic antagonist. An ex vivo metabolism study demonstrated Z-S-IPIP was stable at the receptor site with an absence of radiolabeled metabolites.

Simple new method for effective concentration of 188Re solutions from alumina-based 188W-188Re generator.

UNLABELLED: (188)Re is a useful generator-produced radioisotope currently under evaluation for a variety of therapeutic applications, including bone pain palliation and intravascular radiation therapy. Because the (188)W parent is available only in a relatively low specific activity (<0.15-0.19 GBq/mg) from reactor irradiation of enriched (186)W, relatively large volumes of 0.9% saline (>15 mL) are required for elution of the (188)Re daughter from traditional alumina-based (188)W-(188)Re generators. Because these large bolus volumes result in solutions with a relatively low specific volume activity of (188)Re (<1 GBq/mL for the 18.5-GBq generator), the availability of effective methods for eluent concentration is important. Our new approach is based on the use of 0.3 mol/L ammonium acetate as a representative salt of a weak acid instead of saline for generator elution. METHODS: After generator elution, the ammonium acetate generator eluent (15-20 mL) is passed through a tandem IC-H Plus cation (Dowex-H)-anion (QMA Light) column system. Exchange of ammonium cations with hydrogen ions on the cation column forms an acetic acid solution containing perrhenate anions from which the macroscopic levels of the acetate anion of the eluent have been effectively removed. Because perrhenic acid is fully dissociated at this pH, the QMA Light column specifically traps the (188)Re-perrhenate, which is subsequently eluted with a low volume (<1 mL) of saline. Concentration ratios greater than 20:1 are readily achieved with this method. RESULTS: A typical clinical-scale generator loaded with 19.2 GBq (188)W was used to validate the approach. Saline elution provided (188)Re in a 75%-80% yield. Although elution with 0.15 mol/L NH4OAc gave lower yields (55%-60%), use of 0.3 mol/L NH4OAc provided yields comparable with those of saline (70%-75%). (188)W parent breakthrough was not detected after passage of the bolus through the tandem concentration system. Bolus volumes of 15-20 mL, which initially contained as much as 11.1-14.8 GBq (188)Re, were readily concentrated to less than 1 mL saline using QMA Light cartridges. The generator was evaluated for more than 3 mo with no decrease in performance. CONCLUSION: This approach represents a simple, rapid, and effective method using inexpensive disposable components of concentrating solutions of (188)Re for preparation of therapeutic agents.

Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice.

The transmembrane protein CD36 has been identified in isolated cell studies as a putative transporter of long chain fatty acids. In humans, an association between CD36 deficiency and defective myocardial uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid (BMIPP) has been reported. To determine whether this association represents a causal link and to assess the physiological role of CD36, we compared tissue uptake and metabolism of two iodinated fatty acid analogs BMIPP and 15-(p-iodophenyl) pentadecanoic acid (IPPA) in CD36 null and wild type mice. We also investigated the uptake and lipid incorporation of palmitate by adipocytes isolated from both groups. Compared with wild type, uptake of BMIPP and IPPA was reduced in heart (50-80%), skeletal muscle (40-75%), and adipose tissues (60-70%) of null mice. The reduction was associated with a 50-68% decrease in label incorporation into triglycerides and in 2-3-fold accumulation of label in diglycerides. Identical results were obtained from studies of [(3)H]palmitate uptake in isolated adipocytes. The block in diglyceride to triglyceride conversion could not be explained by changes in specific activities of the key enzymes long chain acyl-CoA synthetase and diacylglycerol acyltransferase, which were similar in tissues from wild type and null mice. It is concluded that CD36 facilitates a large fraction of fatty acid uptake by heart, skeletal muscle, and adipose tissues and that CD36 deficiency in humans is the cause of the reported defect in myocardial BMIPP uptake. In CD36-expressing tissues, uptake regulates fatty acid esterification at the level of diacylglycerol acyltransferase by determining fatty acyl-CoA supply. The membrane transport step may represent an important control site for fatty acid metabolism in vivo.

Evaluation of Z-(R,R)-IQNP for the potential imaging of m2 mAChR rich regions of the brain and heart.

Alterations in the function or density of the m2 muscarinic (mAChR) subtype have been postulated to play an important role in various dementias such as Alzheimer's disease. The ability to image and quantify the m2 mAChR subtype is of importance for a better understanding of the m2 subtype function in various dementias. Z-(R)-1-Azabicyclo[2.2.2]oct-3-y (R)-alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z-(R,R)-IQNP) has demonstrated significant uptake in cerebral regions that contain a high concentration of m2 mAChR subtype in addition to heart tissue. The present study was undertaken to determine if the uptake of Z-(R,R)-IQNP in these regions is a receptor mediated process and to identify the radiospecies responsible for binding at the receptor site. A blocking study demonstrated cerebral and cardiac levels of activity were significantly reduced by pretreatment (2-3 mg/kg) of (R)-3-quinuclidinyl benzilate, dexetimide and scopolamine, established muscarinic antagonists. A direct comparison of the cerebral and cardiac uptake of [I-125]-Z-(R,R)-IQNP and [I-131]-E-(R,R)-IQNP (high uptake in ml, m4 rich mAChR cerebral regions) demonstrated Z-(R,R)-IQNP localized to a higher degree in cerebral and cardiac regions containing a high concentration of the m2 mAChR subtype as directly compared to E-(R,R)-IQNP. In addition, a study utilizing [I-123]-Z-(R,R)-IQNP, [I-131]-iododexetimide and [I-125]-R-3-quinuclidinyl S-4-iodobenzilate, Z-(R,R)-IQNP demonstrated significantly higher uptake and longer residence time in those regions which contain a high concentration of the m2 receptor subtype. Folch extraction of global brain and heart tissue at various times post injection of [I-125]-Z-(R,R)-IQNP demonstrated that approximately 80% of the activity was extracted in the lipid soluble fraction and identified as the parent ligand by TLC and HPLC analysis. These results demonstrate Z-(R,R)-IQNP has significant uptake, long residence time and high stability in cerebral and cardiac tissues containing high levels of the m2 mAChR subtype. These combined results strongly suggest that Z-(R,R)-IQNP is an attractive ligand for the in vivo imaging and evaluation of m2 rich cerebral and cardiac regions by SPECT.

Dual-label studies with [125I]-3(R)/[131I]-3(S)-BMIPP show similar metabolism in rat tissues.

UNLABELLED: Biodistribution studies with the radioiodinated 3(R)- and 3(S)-isomers of 15-(p-iodophenyl)-3-methylpentadecanoic acid (BMIPP) in rats have shown that 3(R)-BMIPP has 20%-25% higher heart uptake than 3(S)-BMIPP (15-180 min). In contrast, the 3(S)-isomer has slightly higher liver uptake, and uptake in other tissues examined is similar. METHODS: To evaluate the possible differences in metabolic fate of the two isomers, a mixture of [125I]-3(R)/[131I]-3(S)-BMIPP was administered to fasted female Fisher rats. Groups of rats (3 per group) were killed 15, 60 and 180 min after administration. Urine and feces were collected from a fourth group (n = 3) over 7 d. Samples of blood, heart, liver, lungs, kidney and urine were Folch extracted. The distributions of 125I and 131I in the organic (lipid), aqueous and pellet samples were determined. The lipid samples as well as the organic fractions from base-hydrolyzed triglyceride (TG) fractions and acid-hydrolyzed urine samples were then analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). RESULTS: The relative distributions of 125I and 131I in the lipid, aqueous and pellet samples were similar for both isomers. Distribution of 125I and 131I in the various components of the lipid extracts observed by TLC (hexane:ether:HOAc, 70:30:1) was also similar, with principal incorporation into the free fatty acid (FFA) and TG pools. HPLC analyses (C18) of the FFA fraction showed similar 125I and 131I profiles, corresponding to BMIPP, and the alpha-methyl-C14 (14-(p-iodophenyl)-3-(R,S)-methyltetradecanoic acid) and C12, C10 and C6 carbon chain-length catabolites. By TLC, radioactive components of 125I and 131I in the urine had the same TLC mobility as hippuric acid. HPLC analyses (C18) of acid-hydrolyzed urine gave a single 125I/131I component with the same relative retention time as 2-(p-iodophenyl)acetic acid, which is the final alpha/beta-oxidative BMIPP catabolite. Unexpectedly, HPLC of lipids from base-hydrolyzed TG from the heart tissue showed 125I/131I components with the same retention times as shorter-chain fatty acids, similar to the FFA fraction, with only low levels of activity detected in BMIPP. CONCLUSION: These results show that 3(R)-BMIPP and 3(S)-BMIPP are metabolized similarly in rat tissues and that higher myocardial extraction observed for 3(R)-BMIPP may reflect differences in the relative membrane transport of the two isomers.

Endovascular beta irradiation for prevention of restenosis using solution radioisotopes: pharmacologic and dosimetric properties of rhenium-188 compounds.

PURPOSE: Irradiation of the arterial wall with beta particles has been shown to be effective in inhibiting neointimal hyperplasia following percutaneous transluminal coronary angioplasty (PTCA). In this study, we describe the use of 188W/188Re generators to obtain 188Re (half-life 16.9 h, maximal beta energy of 2.12 MeV) as a new candidate radioisotope for endovascular irradiation. We have evaluated two [188Re]-compounds as candidates for use as solution-based radiation sources that would allow conventional liquid-filled balloon inflation for delivery of radiation to the vessel wall. While balloon rupture at nominal inflation pressures is a very rare event, (<1 per 10,000 at high pressure), radioisotope release could potentially result in significant dose to radiation-sensitive organs. We have thus evaluated the biodistribution, dosimetry, and kinetics of excretion in rats of two 188Re-labeled compounds that are proposed for intravascular therapy. MATERIALS AND METHODS: Rhenium-188 was obtained as [188Re]-sodium perrhenate by saline elution of an alumina-based 188W/188Re generator system (>500 mCi). High specific volume solutions of the [188Re]-sodium perrhenate (>50 mCi/ml) were obtained by post-elution concentration of the generator bolus by passage through a tandem silver cation/anion column system. Rhenium-188-labeled benzoylthioacetyltriglycine (MAG3) was prepared by stannous ion reduction of [188Re]-perrhenate in the presence of the benzyl-MAG3 substrate, and was characterized as a single radioactive component. Rhenium-188-perrhenate and [188Re]-MAG3 were administered to separate groups of Fischer rats, which were sacrificed at various times and the tissue distribution of 88Re determined in the major organs. Excretory products were also collected daily from separate groups of rats for each agent over 7 days. The effects of perchlorate and iodide preblocking and postdisplacement of thyroid uptake of [188Re]-perrhenate were also evaluated. RESULTS: Organ uptake values were modest for both agents [<0.25 % injected dose(ID)/gram of tissue at 6 h] for all organs evaluated except for the thyroid, with the intestines and intestinal contents showing the highest uptake values (0.72-1.97 %ID/gram). Whereas thyroid uptake of 188Re after injection of [188Re]-MAG3 was low (0.16 %ID/gram), uptake after injection of [188Re]-perrhenate was higher and could be blocked by pretreatment with perchlorate (intravenous [IV]) or displaced by perchlorate posttreatment. Also, oral or IV iodide pre- or postadministration could also significantly block or displace thyroid uptake of [188Re]-perrhenate. Both [188Re] agents were excreted primarily via the urinary bladder. The excretion half-life of [188Re]-perrhenate was about 7 h; in contrast, the [188Re]-MAG3 complex showed 50% excretion in less than 2 h. The large intestines received the most significant adsorbed dose, with values of 2.0 cGy/ mCi for [188Re]-perrhenate and 4.6 x 10(-3) cGy/mCi for [188Re]-MAG3. CONCLUSIONS: Rhenium-188-MAG3 shows more rapid urinary bladder excretion in rats than perrhenate and both agents show low organ uptake. Thyroid uptake of free [188Re]-perrhenate can be blocked or displaced with oral perchlorate administration. For the projected use of [188Re]-MAG3 for balloon inflation required for irradiation of the arterial wall, calculated organ dose values are within acceptable limits in the unlikely event of low pressure balloon rupture. Rhenium-188-MAG3 in solution is thus a new candidate for balloon dilation providing uniform endovascular irradiation following PTCA for restenosis therapy.

Reactor-produced radioisotopes from ORNL for bone pain palliation.

The treatment of painful skeletal metastases is a common clinical problem, and the use of therapeutic radionuclides which localize at metastatic sites has been found to be an effective method for treatment of pain, especially for multiple sites for which the use of external beam irradiation is impractical. There are currently several metastatic-targeted agents radiolabeled with various therapeutic radionuclides which are in various stages of clinical investigation. Since neutron-rich radionuclides are produced in research reactors and often decay by emission of beta- particles, most radionuclides used for bone pain palliation are reactor-produced. Key examples of radionuclides produced by single neutron capture of enriched targets include rhenium-186 and samarium-153. In addition, generator systems are also of interest which provide therapeutic daughter radionuclides from the decay of reactor-produced parent radionuclides. One important example is rhenium-188, available from generators via decay of reactor-produced tungsten-188. Tin-117m is an example of a reactor-produced radionuclide which decays with the emission of low-energy conversion electrons rather than by beta- decay. Each of these agents and/or radionuclides has specific advantages and disadvantages, however, the ideal agent for bone pain palliation has not yet been identified. The goal of this paper is to briefly review the production and use of several reactor-produced radionuclides for bone pain palliation, and to discuss the role of the ORNL High Flux Isotope Reactor (HFIR) for the production of many of these radionuclides.

Effects of configuration on the myocardial uptake of radioiodinated 3(R)-BMIPP and 3(S)-BMIPP in rats.

UNLABELLED: Radioiodinated 3(R)-(+)- and 3(S)-(-)-15-(p-iodophenyl)-3-(R,S)-methylpentadecanoic acid (BMIPP) were prepared and evaluated in rats to investigate the effects of absolute configuration of the 3(beta)-methyl group on myocardial uptake and release kinetics. METHODS: The 3(R)-(+)-BMIPP analog was synthesized by initial acylation of a thiophene template with the acid chloride of ethyl 3(R)-methylglutarate. 3(S)-(-)-BMIPP was obtained by separation from the mixture of diastereomeric amides prepared from reaction of the acid chloride of racemic BMIPP with the S-(-)-alpha-methylbenzylamine. The amide of synthetic 3(R)-BMIPP prepared from S-(-)-alpha-methylbenzylamine was identical to the chromatographically more polar isomer. Free acids were obtained by acid hydrolysis of the amides, fully characterized and then converted to the radioiodinated BMIPP isomers. RESULTS: Biodistribution studies in rats with the dual-labeled [(131)I]-3(S)-BMIPP/[(125)I]-3(R)-BMIPP mixture demonstrated greater myocardial uptake of 3(R)-BMIPP compared with the 3(S)-BMIPP isomer [60 min: 3(R)-BMIPP = 4.37 %ID/g; 3(S)-BMIPP = 3.44; p < 0.05; 180 min, 2.31 and 1.78 %ID/G, respectively, p < 0.01], although both isomers had similar myocardial washout curves (5-180 min). Percent ID/g values for other tissues which were examined (blood, lungs, thyroid) were similar. CONCLUSION: Higher myocardial uptake of the 3(R)-BMIPP isomer observed in these animal studies may suggest differences in carrier-mediated myocyte uptake of the two isomers. These studies suggest that [(123)I]-3(R)-BMIPP is a candidate for clinical evaluation and may show greater myocardial uptake than the 3(S)-BMIPP isomer and may thus require reduced injected dose.

Availability of rhenium-188 from the alumina-based tungsten-188/rhenium-188 generator for preparation of rhenium-188-labeled radiopharmaceuticals for cancer treatment.

Rhenium-188 (beta- = 2.2 MeV; gamma = 155 keV; T1/2 16.9 hours) is an attractive therapeutic radioisotope which is produced from decay of the reactor-produced tungsten-188 parent (T1/2 69 days) and thus conveniently obtained on demand by elution from the alumina-based tungsten-188 /rhenium-188 generator system. The rhenium-188 is obtained as sodium perrhenate by elution of the generator with 0.9% saline. The post elution use of disposable tandem, ion-exchange columns is a simple method for the concentration of rhenium-188 saline solutions with specific volumes > 500 mCi/ml. This method can also extend the useful shelf-life of the generator, which can be as long as one year. The long useful shelf-life of the generator is expected to provide rhenium-188 at very reasonable costs for routine preparation of a variety of radiopharmaceuticals for the treatment of a variety of cancers including breast cancer. We are evaluating two types of Re-188-labeled agents under investigation which have potential for the treatment of breast cancer. Rhenium-188-labeled hydroxyethylidenediphosphonate (HEDP) and Re-188-dimercaptosuccinic acid (DMSA) are being applied for palliative treatment of pain associated with skeletal metastases, and the Re-188-RC-160 somatostatin analogue [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Lys-Val-Cys-Trp-NH2] for somatostatin-receptor-positive tumors. The results of initial clinical studies with the two bone pain agents demonstrate good targeting to skeletal metastases, and use of Re-188-HEDP has resulted in pain palliation with minimal bone marrow suppression in the initial patient studies. While these initial studies have been conducted in patients with prostate cancer, similar results are expected in planned studies in breast cancer patients. In animal studies, Re-188-RC-160 has been successfully used for the local/regional treatment of experimental breast cancer and other cancers. Re-188-RC-160 binds to somatostatin-receptor-positive cells both in vitro and in vivo, including breast cancer cells (ZR-75-1 breast carcinoma and NCI-H69 human small cell ling carcinoma), but not to binding-negative cells (Raji, Burkitt's lymphoma). A structurally similar Re-188-cyclic peptide with different binding specificity (CTOP [cyclic NH2-(D)-Phe-Cys-Try-(D)-Trp-Orn-Thr-Pen-Thr-ol]; an opiate-receptor antagonist) did not bind to target cells. Both gentisic acid and ascorbic acid are present in the Re-188-HEDP and Re-188-RC-160 formulations, and have been found to also significantly reduce radiolytic degradation of the somatostatin peptide analogues, and may have general application in the stabilization of Re-188-labeled radio-pharmaceuticals.