James R. Ballinger

Combination of dopamine transporter and D2 receptor SPECT in the diagnostic evaluation of PD, MSA, and PSP

It is often difficult to differentiate clinically between Parkinsons disease (PD), multiple system atrophy (MSA), and progressive supranuclear palsy (PSP).The objective of this work was to investigate whether combined pre‐ and postsynaptic dopaminergic single photon emission computed tomography (SPECT) scanning can reliably demonstrate changes in the nigrostriatal dopaminergic system and help differentiate between normal controls, PD, MSA, and PSP patients. We performed SPECT evaluation of the dopamine transporter (DAT) and dopamine D2 receptors (D2). SPECT scans using [123I]β‐CIT (for DAT) and [123I]IBF (for D2) were performed in 18 patients with PD (12 dopa‐naïve and 6 on levodopa and/or dopamine agonists), 7 with MSA of the striatonigral degeneration type, 6 with PSP, and 29 normal controls. Antiparkinsonian drugs were withheld for at least 12 hours before the scans. DAT and D2 binding potentials (Rv = V3/V2) were measured for caudate, anterior, and posterior putamen on the sides ipsilateral and contralateral to the worst motor symptoms. DAT binding in the posterior putamen was markedly reduced in all patients. However, D2 binding in posterior putamen was significantly increased in dopa‐untreated PD, being greater than the normal range in 4 of 12 (33%), and it was significantly reduced in MSA, being below the normal range in 5 of 7 (71%). None of the patients with PD showed reduced D2 binding below the normal range in posterior putamen. The degree of DAT binding could not discriminate between the patient groups. The ratio of posterior putamen to caudate percentage D2 Rv compared with the controls showed an opposite pattern between PD or PSP and MSA; the caudate was greater in 16 of 18 with PD and 6 of 6 with PSP, whereas caudate was less in 5 of 7 with MSA. These findings suggest that DAT SPECT may be useful in differentiating parkinsonism from controls and D2 SPECT in further differentiating MSA from Parkinsons disease and possibly PSP.

A study of doxorubicin loading onto and release from sulfopropyl dextran ion-exchange microspheres

The objective of this study was to investigate various factors that influence doxorubicin (Dox) loading onto and release from sulfopropyl dextran ion-exchange microspheres (MS), and to evaluate the anticancer activity of the released drug in vitro. Dox was incorporated into the MS by incubating the MS with aqueous solutions of Dox at room temperature. The drug release was carried out at 37 degrees C in aqueous solutions containing NaCl with or without CaCl2. The kinetics of drug absorption and release, the amount of Dox released, and the stability of Dox after loading, freeze-drying, and release were determined by spectrophotometry. The cytotoxicity of Dox (the original drug or that released from MS) against murine EMT6 breast cancer cells was assessed using a clonogenic assay. An increase in the MS to drug ratio resulted in a higher absorption rate and a higher fraction of the drug extracted from the solution. The release rate and the equilibrium fraction of Dox released increased with a decrease in the initial amount of Dox loaded or an increase in the salt concentration. The addition of divalent ions (Ca2+) promoted drug release compared to NaCl alone. The percent loss of colony forming ability of the cells, a measure of cytotoxicity of the released Dox, was the same as parent Dox solutions, indicating that the drug bioactivity was fully preserved after the drug loading and release cycle. This work demonstrated that various drug release rates were achieved by varying the drug loading and that the MS-delivered Dox was effective against the cancer cells in vitro.

SPECT imaging of pre- and postsynaptic dopaminergic alterations in l-dopa–untreated PD

Background: In PD, presynaptic dopamine transporters are known to be decreased, whereas postsynaptic striatal D2 receptors are proposed to be upregulated. However, the relationship between these alterations is not clear. Objective: To evaluate the ability of SPECT to detect both the pre- and postsynaptic dopaminergic alterations of the striatum in patients with l-dopa–untreated PD. Methods: We studied 10 l-dopa–untreated patients with clinically mild PD and 21 age-matched normal controls. Individuals had both presynaptic [123I]β-CIT dopamine transporter and postsynaptic [123I]IBF D2 SPECT studies 1 week apart. Results: In PD patients, the dopamine transporter binding potential Rv ipsilateral/contralateral to the most affected limbs was 30%/41%, 41%/50%, and 59%/68% lower than controls for caudate, anterior putamen, and posterior putamen, respectively. These bilateral Rv decreases showed a lateralized difference more reduced in the contralateral striatum as well as intrastriatal differences most reduced in the posterior putamen. In contrast, in PD patients the D2 binding potential Rv ipsilateral/contralateral was 15%/16% higher for caudate, 18%/14% higher for anterior putamen, and 28%/31% higher for posterior putamen. These bilateral Rv increases showed no lateralized differences and less marked intrastriatal differences. The motor Unified Parkinson’s Disease Rating Scale scores negatively correlated with dopamine transporter Rv but not with D2 Rv. Conclusions: SPECT imaging can detect characteristic dopaminergic alterations in the striatum of dopa-untreated PD patients including the upregulation of postsynaptic D2 receptors (denervation supersensitivity). SPECT is widely available and is a promising clinical tool to evaluate PD patients.

Delivery of an anticancer drug and a chemosensitizer to murine breast sarcoma by intratumoral injection of sulfopropyl dextran microspheres.

Intratumoral injection of controlled‐release microsphere formulations of anticancer compounds has the potential to selectively increase tumour exposure to drugs. This work aimed to evaluate the therapeutic effect and toxicity of microsphere formulations containing the anticancer drug, doxorubicin, in a murine tumour model. The effect of co‐administration of verapamil, a P‐glycoprotein modulator or chemosensitizer, was investigated. Initial in‐vitro studies confirmed the ability of verapamil to enhance the accumulation of both doxorubicin and [99mTc]sestamibi, also a P‐glycoprotein substrate, in EMT6 murine breast sarcoma cells and a doxorubicin‐selected multidrug‐resistant variant, EMT6/AR1.0. Ex‐vivo studies using confocal microscopy demonstrated release of doxorubicin from microspheres and diffusion of the drug through tissue. For in‐vivo studies, EMT6 and EMT6/AR1.0 cells were grown in BALB/c mice. Following intratumoral injection of doxorubicin‐loaded microspheres, alone or in combination with verapamil‐loaded microspheres, the tumour diameter was measured serially as an indication of therapeutic effect, while the weight, appearance, and behaviour of the mice were monitored as an indication of general toxicity. Intratumoral injections of doxorubicin‐loaded microspheres were tolerated much better than systemic administration of equivalent drug concentrations. There was a modest (up to 34%) delay of tumour growth compared with groups receiving no treatment or blank microspheres. Co‐injection of verapamil microspheres with doxorubicin microspheres produced a moderate increase in toxicity but no further delay in tumour growth. Controlled‐release microsphere formulations of anticancer agents administered intratumorally were an efficient way to deliver high drug doses to the tumour with little systemic toxicity.

Electrochemical monofluorination of pyridine: synthesis of 2-fluoropyridine at a platinum anode☆

2-Fluoropyridine has been synthesized in 22% yield by electrochemical fluorination of pyridine at a platinum anode at 2.5 V vs Ag/Ag+ (0.1 M) and with 0.5 M tetramethylammonium dihydrogen trifluoride (Me4NF·2HF) in dry acetonitrile as supporting electrolyte and fluoride source. At lower ME4NF·2HF concentrations and at lower applied potentials both yield and reaction rate are decreased whereas at higher potentials the supporting electrolyte/solvent system decomposes. The choice and purity of supporting electrolyte is an important factor in determining the product distribution and yield. The relatively low fluoride concentration allows use of a glass cell.

Synthesis and Characterization of Surface‐Hydrophobic Ion‐Exchange Microspheres and the Effect of Coating on Drug Release Rate

Biodegradable, dextran-based ion-exchange microspheres (IE-MS) have been used for localized delivery of anticancer drugs and chemosensitizers. Because of their hydrophilic nature, the IE-MS release their payload quickly. The purpose of this work was to develop an IE-MS system that could provide a broad range of release rates for in vitro and in vivo applications. Sulfopropylated dextran microspheres (SP C25 MS) were surface-modified by acylation. These hydrophobically modified sulfopropylated dextran microspheres (HM-MS) were further coated with the cationic acrylic polymer Eudragit RL100 (EU-MS). The changes in chemical composition after the surface modification and coating were characterized by X-ray photoelectron spectroscopy. The effects of the modification and coating on the surface hydrophobicity, equilibrium swelling, surface morphology, and drug loading capacity were investigated. The HM-MS showed little change in swelling and functionality, despite significantly increased affinity to oil and carbon content on the surface. The coated microspheres (EU-MS) exhibited a profoundly decreased swelling ratio, an even higher affinity to oil, a higher loading capacity, and a lower drug release rate. Through further coating of the EU-MS with different amounts of corn oil, the rate of drug release could be tailored to cover a relatively wide range. These results suggest that a versatile delivery system with various release profiles can be prepared by a combination of hydrophobic modification, polymer coating, and oil coating.

Radiofluorination with reactor-produced Cesium [18F]fluoride: No-carrier-added [18F]2-fluoronicotine and [18F]6-fluoronicotine

Abstract We have synthesised no-carrier-added [18F]2-fluoronicotine by the halogen-exchange reaction of reactor-produced Cs18F upon 2-bromonicotine in refluxing DMSO. No special drying of the Cs18F was required. [18F]2-Fluoronicotine was isolated by reversed-phase HPLC and production of 220 MBq (6 mCi) ready for injection represented a decay-corrected radiochemical yield of 23% and required 3.5 h from end-of-bombardment. Similarly, [18F]6-fluoronicotine was prepared from 6-bromonicotine in amounts of 80 MBq (2.2 mCi) or 8% yield. These quantities are sufficient for studies in humans.

A solid-phase technique for preparation of no-carrier-added technetium-99m radiopharmaceuticals : Application to the streptavidin/biotin system

A high effective specific activity (HESA) formulation of a biotin-containing (99m)Tc ligand [RP488: dimethyl-Gly-Ser-Cys(Acm)-Lys(Biotin)-Gly] conveniently prepared from solid phase was compared to a typical low effective specific activity (LESA) solution formulation to demonstrate improved targeting to streptavidin in an in vitro assay and in an in vivo rat model. RP488 was coupled to a maleimide-functionalized polyethylene glycol resin via a thiol ether linkage and labeled with (99m)Tc-gluconate at room temperature, followed by elution of the HESA (99m)Tc-RP488 in saline (minimum specific activity approximately 1000 TBq/mmol by amino acid analysis). Both HESA and LESA (99m)Tc-RP488 labeled at > 90% purity. In vitro, HESA (99m)Tc-RP488 incubated with streptavidin-agarose was bound quantitatively, but there was competition from addition of increasing amounts of cold RP488. In rats, radiotracer uptake was evident at the site of implantation of streptavidin-agarose beads for the HESA dose, less uptake of low effective specific activity (LESA) material, and no appreciable uptake in the control rats of the LESA or HESA dose. The target-to-background ratio for HESA (99m)Tc-RP488 was 5.4 times that of the control. The solid-phase technology offers a convenient way to prepare high specific activity receptor-targeting (99m)Tc radiopharmaceuticals.

Accumulation of Sestamibi in Lymphoma Cell Lines In Vitro

Although some authors have suggested that sestamibi imaging is useful in evaluation of patients with lymphoma, others have obtained equivocal results. This discrepancy has been further investigated in vitro using two patient-derived non-Hodgkins lymphoma cell lines, OCI-Ly3 and OCI-Ly18. Sestamibi (0.2 MBq/ml) was added to a suspension of OCI-Ly3 or OCI-Ly18 cells and aliquots were removed over 1 h and centrifuged to determine cell-associated radioactivity. Further experiments studied the effect of addition of a P-glycoprotein (Pgp) modulator or alteration in plasma and/or mitochondrial membrane potentials. Accumulation of sestamibi reached plateau values within 30 min, but these values were 6-fold higher in OCI-Ly3 than in OCI-Ly18. Inhibition of Pgp function with GG918 or PSC833 did not affect OCI-Ly3 cells but increased accumulation in OCI-Ly18 cells 3-fold, indicating a moderate level of Pgp. However, both cell lines responded similarly to membrane potential alterations: hyperpolarization of the mitochondrial membrane with nigericin had little effect on accumulation; in contrast, depolarization of the plasma membrane with an isotonic high potassium buffer reduced accumulation of sestamibi to 52% of control and additional depolarization of the mitochondrial membrane with valinomycin further reduced accumulation to 12% of control levels. These studies suggest that there can be wide differences in accumulation between cell lines, in part due to Pgp-mediated efflux, but that both of these cell lines have highly polarized mitochondria with little further capacity for hyperpolarization.