First example of well-characterized Re and Tc tricarbonyl complexes of ciprofloxacin and norfloxacin in the development of infection-specific imaging agents

Aiming at the development of ^99mTc-based infection-specific imaging agents, the synthesis and characterization of rhenium and technetium-99m tricarbonyl complexes with derivatized ciprofloxacin and norfloxacin is hereby reported. The ligands were prepared by coupling the tridentate chelator picolylamino-N,N-diacetic acid (PADA) with the piperazinyl (NH) nitrogen of ciprofloxacin or norfloxacin, through the employment of the PADA anhydride. The corresponding rhenium complexes were synthesized using the fac-[NEt₄]₂[ReBr₃(CO)₃] precursor and were fully characterized by elemental analysis and NMR spectroscopy. X-ray crystallography of the ciprofloxacin complex showed that the geometry about rhenium is distorted octahedral defined by the NNO donor atom set of the tridentate chelator and the three carbonyl groups. The analogous technetium-99m complexes were prepared quantitatively through the use of the fac-[^99mTc(H₂O)₃(CO)₃]⁺ precursor and their structure was established by comparative HPLC studies using the well-characterized rhenium complexes as reference. Preliminary studies with the technetium-99m complexes showed high bacterial uptake in vitro.     

Benzimidazole derivatives as NSO ligands for the fac-[M(CO)3] (M = Re, Tc)

Two rhenium(I) tricarbonyl complexes with the tridentate monoanionic NSO ligands, 4-(benzimidazol-2-yl)-3-thiabutanoic acid (complex 3) and [1-(11-carboxyundecanyl)-4-(benzimidazol-2-yl)]-3-thiabutanoic acid (complex 4) were synthesized and characterized by spectroscopic methods and elemental analysis. X-ray crystallographic analysis of complex 3 revealed a distorted octahedral geometry around rhenium defined by the three facially bound CO groups and the NSO donor atom set of the tridentate ligand. The analogous technetium-99m complexes (complexes 5 and 6) were also prepared quantitatively by reaction of the NSO ligands with the fac-[^99mTc(H₂O)₃(CO)₃]⁺ synthon and their identity was established by chromatographic comparison to their rhenium congeners. Biodistribution in mice of complex 6 bearing the fatty acid chain showed significant heart uptake (6.26 ± 0.79% ID/g p.i.) at 1 min accompanied, however, with a heart:blood ratio below 1.     

Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

Different glycosides were grafted on the surface of liposomes containing ¹²⁵I-labelled γ-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of $\text{p-}\text{aminophenyl-}\text{d}\text{-glycosides}$ to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ¹²⁵I-labelled γ-globulin was determined in mouse tissues from 5–60 min after a single injection of these liposomes. The liver uptake of encapsulated ¹²⁵I-labelled γ-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ¹²⁵I-labelled γ-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of ¹²⁵I-labelled γ-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of ¹²⁵I-labelled γ-globulin from liposomes containing asialogangliosides depends upon the phospholipid/ glycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.     

Reactions of the fac-[Re(CO)3] and [ReO] moieties with substituted benzothiazoles

The reaction of 2-(2-aminophenyl)benzothiazole (Habt) with [Re(CO)₅Br] led to the isolation of the rhenium(I) complex fac-[Re(Habt)(CO)₃Br] (1). With trans-[ReOCl₃(PPh₃)₂], the ligand Habt decomposed to form the oxofree rhenium(V) complex [Re(itp)₂Cl(PPh₃)] (2) (itp = 2-amidophenylthiolate). From the reaction of trans-[ReOBr₃(PPh₃)₂] with 2-(2-hydroxyphenyl)benzothiazole (Hhpd) the complex [Re^VOBr₂(hpd)(PPh₃)] (3) was obtained. Complexes 1-3 are stable and lipophilic. ¹H NMR and infrared assignments, as well as the X-ray crystal structures, of the complexes are reported.     

γ-Aminobutyric acid-stimulated chloride permeability in crayfish muscle

γ-Aminobutyric acid selectively increased Cl^? permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in VAn Harreveld's solution at room temperature took up ³⁶Cl^? to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-s incubations, the control ³⁶Cl^? uptake space was 131 ± 4 ml/kg (n = 60) and this was significantly increased by γ-aminobutyric acid at 200 μM or higher concentrations to 177 ± 4 ml/kg (n = 48, P < 0.05). This effect was specific for chloride since γ-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. γ-Aminobutyric acid stimulation of ³⁶Cl^? uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The γ-aminobutyric acid stimulation of Cl^? permeability is dose dependent with 50% of the maximal effect at 40 μM γ-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The γ-aminobutyric acid agonist muscimol causes the same maximal effect on Cl^? uptake as γ-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist γ-amino, β-hydroxybutyric acid produced little or no stimulation of ³⁶Cl^? flux. The response to γ-aminobutyric acid was blocked by 2 mM β-guanidinopropionate or γ-guanidinobutyrate, 0.5 mM bicuculline, and 10 μM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 μM. Antagonists did not affect control ³⁶Cl^? uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter γ-aminobutyric acid involves a rapid increase in membrane permeability to Cl^?     

Assessment of the potential uses of liposomes for lymphoscintigraphy and lymphatic drug delivery failure of 99m-technetium marker to represent intact liposomes in lymph nodes

The in vivo fate of subcutaneously injected neutral SUV liposomes in rats was examined using a membrane marker, ^99mTc, and an aqueous marker, ¹²⁵I-labelled poly(vinyl pyrrolidone). Liposomes with entrapped ¹²⁵I-labelled poly(vinyl pyrrolidone) were labelled with ^99mTc by the SnCl₂ method [2]. ^99mTc-radioactivity was localized several-fold more in the primary and secondary regional lymph nodes than ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity. Similarly, ^99mTc-radioactivity appeared and was subsequently cleared from the circulation much more rapidly than ¹²⁵I-labelled poly(vinyl pyrrolidone). The gel chromatography of the lymph node homogenate revealed that 60–70% of ¹²⁵I-labelled poly(vinyl pyrrolidone)-radioactivity was in the liposome fractions, whereas only 3% of ^99mTc-radioactivity was co-eluted with liposomes. Thus, the two markers have different fates in the lymphatics, and the presence of all ^99mTc-radioactivity does not represent the 60–70% of intact liposomes present in lymph nodes. Using the aqueous marker ¹²⁵I-labelled poly(vinyl pyrrolidone), the lymphnode localization of positive, negative and neutral small unilamellar vesicles was studied, and it was found that ¹²⁵I-radioactivity was more localized from negative liposomes than from positive liposomes, which in turn was more localized than that from neutral liposomes. Thus, these findings differ from those reported earlier [2], where the authors used ^99mTc as a liposomal marker. In vitro studies showed that liposomes of preparations containing 20 mol% cholesterol became ‘leaky’ to low-molecular-weight drugs, for example, methotrexate (M_r 454) to a much greater extent than with a large-molecular-weight substance, ¹²⁵I-labelled poly(vinyl pyrrolidone) (M_r 30 000–40 000), when incubated with rat lymph at 37°C. Using the two markers ^99mTc and ¹²⁵I-labelled poly(vinyl pyrrolidone) it was found that the localization of both radioactivities was reduced in lymph nodes draining λ-carrageenan-treated footpads. In conclusion, it is suggested that liposomes can be used for the delivery of drugs to diseased lymph nodes, and it would be worthwhile examining the possibilities of using alternative methods of labelling liposomes with ^99mTc rather than using the SnCl₂ technique [2], or using other radionuclides as markers for γ-scan imaging.     

Radiolabeled biomolecules with 186Re: Potential for radioimmunotherapy

Rhenium-186 has been theoretically determined to be among the best therapy radiolabels due to its unique half-life, paniculate and γ emissions and chelation properties. Traditionally, rhenium chelates have been synthesized by the tin reduction method at low pH which frequently produces denaturation of acid labile proteins. A comparative study has been carried out to assess three techniques of reducing ¹⁸⁶ReO⁻₄ in order to label biologically active macromolecules (i.e. human serum albumin (HSA), anti-human serum albumin antibody (HSA-Ab) and a monoclonal antibody to T-cells [T-101]). These experiments showed that stannous and dithionite reduction methods provide for an overall labeling yield of between 5 and 18% with an associated immunoreactivity of 12–40%. The hypophosphorous acid (H₃PO₂) reduction method, however, yielded no usable radiolabeled product. The preparation of ¹⁸⁶Re-DTPA-HSA produced an 18.2% radiochemical yield (7.4 × 10⁶ Bq/mg) and 40% retention of binding affinity. Using the stannous or dithionite reduction methods for the radiolabeling of HSA-Ab and T-101 resulted in a relatively low yield (9%), but the labeled product retained binding affinity of 12–25% with Protein A.     

Ortho-metalation, rotational isomerization, and hydride-hydride coupling at rhenium (V) polyhydride complexes stabilized by aromatic amine ligands

An ortho-metalated rhenium (V) polyhydride complex has been prepared through the reaction of ReH₇(PPh₃)₂ with 2-phenylpyridine. Additionally, a small series of neutral rhenium (V) pentahydride complexes, each of which is stabilized by an aromatic amine ligand, has been prepared. E and Z rotational isomers of the ReH₅(PPh₃)₂(aromatic amine) complexes have been observed at low temperatures by NMR spectroscopy. The E and Z rotational isomers arise from a combination of the lack of a mirror plane symmetry element orthogonal to the aromatic ring in the aromatic amine ligands and the restricted rotation about the Re-N bond in such complexes. Restricted rotation about the Re-N bond in the related complex, ReH₅(PPh₃)₂(Py) has previously been observed by Crabtree et al. The restricted rotation about the Re-N bond seems to result from π-donation of the lone electron pair on the rhenium (V) center to the π∗ system of the aromatic amine ligands. Different populations of the E and Z rotational isomers arise from interactions of substituents on the aromatic ring with the other ligands bound to rhenium. The values of ΔG^‡ for the restricted rotation about the Re-N bonds, for the complexes containing 4-phenylpyrimidine, 2-aminopyrimidine, or 2-aminopyridine, range from 9.9 to 11.3 kcal/mole. One of the new compounds reported herein, ReH₅(PPh₃)₂[1-(2-NH₂Pyr)] is the first rhenium (V) polyhydride complex to display hydride-hydride coupling in its ¹H NMR spectrum.     

Dodecaborate lipid liposomes as new vehicles for boron delivery system of neutron capture therapy

closo-Dodecaborate lipid liposomes were developed as new vehicles for boron delivery system (BDS) of neutron capture therapy. The current approach is unique because the liposome shell itself possesses cytocidal potential in combination with neutron irradiation. The liposomes composed of closo-dodecaborate lipids DSBL and DPBL displayed high cytotoxicity with thermal neutron irradiation. The closo-dodecaborate lipid liposomes were taken up into the cytoplasm by endocytosis without degradation of the liposomes. Boron concentration of 22.7 ppm in tumor was achieved by injection with DSBL-25% PEG liposomes at 20 mg B/kg. Promising BNCT effects were observed in the mice injected with DSBL-25% PEG liposomes: the tumor growth was significantly suppressed after thermal neutron irradiation (1.8 × 10¹² neutrons/cm²).     

Complexes with the fac-{M(CO)3} (M = Tc, Re) moiety and long alkyl chain ligands as Lipiodol surrogates

Accumulation of radiopharmaceuticals in the liver is frequently observed and represents in general a limiting factor when developing novel labeled compounds for any purpose in nuclear medicine. Aiming at the treatment of liver cancer with radiopharmaceuticals, such accumulation is desired but the compounds have to remain in the liver over an extended time period rather than being washed out or redistributed over time in the whole body. Lipiodol is known to remain in the liver and we present here a study for the preparation of ¹⁸⁶Re and ^99mTc labeled Lipiodol surrogates expected to behave similarly. We have synthesized two bidentate and two tridentate ligands conjugated to a pendant C18 chain as well as their corresponding fac-[Re(CO)₃]⁺ and fac-[Tc(CO)₃]⁺ complexes. Three of the rhenium complexes have been structurally characterized. Labelling with [¹⁸⁶Re(OH₂)₃(CO)₃]⁺ and [^99mTc(OH₂)₃(CO)₃]⁺, respectively, gave yields in the range of 90%. The complexes could be extracted into Lipiodol due to their high lipophilicity and close structural relationship with the major components of Lipiodol. The complexes are stable in water and in Lipiodol for more than 24 h. These Lipiodol surrogates present new low-valent technetium and rhenium complexes for applications in liver cancer imaging and therapy.     

Energy transfer pathways in pyridylporphyrin Re(I) adducts

Dimeric and pentameric adducts between a meso-4′ pyridylporphyrin core and either one or four rhenium(I) bipyridine tricarbonyl units, fac-[Re(CO)₃(bipy)(4′MPyP)][CF₃SO₃] and fac-[{Re(CO)₃(bipy)}₄(4′TPyP)][CF₃SO₃]₄(4′MPyP = 4′-monopyridylporphyrin, 4′TPyP = 4′-tetrapyridylporphyrin), respectively, were synthesized and their photophysical behaviors were investigated by emission and absorption time resolved experiments. The adducts exhibit distinctive supramolecular features, different from those of the molecular components. Upon excitation of the core, the typical porphyrin fluorescence is quenched. This effect is attributed to enhanced intersystem crossing in the porphyrin unit, owing to the heavy atom effect provided by the attached rhenium unit(s). Following excitation of rhenium fragments the typical rhenium MLCT emission is not observed while partial sensitization of the porphyrin fluorescence occurs, indicating that fast intercomponent energy and/or electron transfer processes take place in competition with the intersystem crossing in the rhenium unit.     

The [Re(N)(PN)] building-block

The formation of the uncommon molecular ion fragment [Re(N)(P^∩N)] is ESI-MS conditions is confirmed by the synthesis and characterization of the representative [Re(N)Cl₂(P^∩NMe₂)PPh₃] complex. This species contains the unprecedented [Re(N)(P^∩NMe₂)] moiety which constitutes an additional example of metal-based building-block potentially useful in the design of rhenium radiopharmaceuticals.     

Purification of an ammonium-inducible glutamate dehydrogenase and the use of its antigen affinity column-purified antibody in specific immunoprecipitation and immunoadsorption procedures

A modified five-step purification procedure was developed which gave an 80–87% yield of pure NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella. The enzyme was shown to be composed of six identical subunits with alanine as the C-terminal amino acid. The purified enzyme was covalently coupled to CNBr-activated Sepharose-4B, and then the subunits were linked together with dimethyl suberimidate to make a stable antigen affinity column for purification of anti-NADP-GDH IgG from rabbit antiserum. When the subunits of the column-bound holoenzyme were not linked together, elution of the anti-NADP-GDH IgG resulted in a 50% loss of enzyme subunits from the column. This loss of subunits inactivated the column. The monospecific, affinity-purified anti-NADP-GDH IgG was used in an indirect immunoprecipitation procedure with purified sheep anti-rabbit IgG or in an indirect procedure with Staphylococcus Protein A Sepharose 4B to obtain a 95–98% recovery (by either procedure) of ³⁵S-labeled NADP-GDH from radioactive Chlorella cell homogenates. As shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the [³⁵S]NADP-GDH recovered by these procedures was free of contaminating radioactive cellular proteins. When direct precipitation was used with the purified antibody, only an 85–90% recovery of the radioactive enzyme was obtained. Thus, the indirect procedures would be the ones of choice for measurements of the in vivo rates of synthesis and degradation of the NADP-GDH which comprises approximately 0.2% of the total soluble protein of Chlorella.     

Biosynthesis of γ-aminobutyric acid from spermine in maize seedlings

The administration of labelled spermine [tetramethylene-1,4-¹⁴C] to Zea mays shoots resulted in the formation of radioactive γ-aminobutyric acid (GABA). A chemical degradation of radioactive GABA suggested that its radioactivity was located on C-1 and C-4, indicating that GABA is a product of spermine metabolism in maize seedlings.     

A new tricarbonyl fac-[M(acac)(isc)(CO)3] complex (M = Re, Tc) with acetylacetonate (acac) and isocyanide (isc) in a 2+1 combination

The synthesis and characterization of the neutral 2+1 mixed ligand complex fac-Re(CO)₃(acac)(isc) (4) with acetylacetonate (acac) as the bidentate ligand and an isocyanide (the isocyanocyclohexane, isc) as the monodentate ligand is described. The synthesis of 4 proceeds through the intermediate formation of the fac-Re(acac)(H₂O)(CO)₃ precursor complex 2. Complex 4 was characterized by elemental analysis, spectroscopic methods, and X-ray crystallography showing a distorted octahedral arrangement of the ligands around Re. At technetium-99m level, the corresponding fac-^99mTc(acac)(isc)(CO)₃ complex 5 was obtained in high yield by reacting the fac-^99mTc(acac)(H₂O)(CO)₃ precursor complex 3 with isocyanocyclohexane and its structure was established by chromatographic comparison with the prototypic rhenium complex using high performance liquid chromatography.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn²⁺) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn²⁺ complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO₄ or MnCl₂. In response to a pH gradient or a Mn²⁺ ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn²⁺ suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn²⁺ complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn²⁺ complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

γ-Terpinene synthetase: A key enzyme in the biosynthesis of aromatic monoterpenes

Previous studies with thyme (Thymus vulgaris L.) leaf slices indicated that γ-terpinene (1,4-p-menthadiene) is the precursor of the aromatic monoterpenes p-cymene (4-isopropyl toluene) and thymol (5-methyl-2-isopropyl phenol) (Poulose, A. and Croteau, R. (1978) Arch. Biochem. Biophys.187, 307–314). A 105,000g supernatant obtained from an extract of young thyme leaves catalyzed the cyclization of both [1-³H]neryl pyrophosphate and [1-³H]geranyl pyrophosphate to γ-[3-³H]terpinene. No evidence for the interconversion of the acyclic precursors was obtained, and isotopic dilution experiments suggested that γ-terpinene was synthesized directly from these acyclic precursors without the involvement of any free intermediates. Competing phosphatase activity in the soluble preparation was removed by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150. In these fractionation steps, γ-terpinene synthetase activity co-purified with small amounts of α-thujene (1-isopropyl-4-methylbicyclo[3.1.0]-hex-3-ene) and α-terpineol (p-menth-1-en-8-ol) synthetase activities, and these three activities could not be resolved by subsequent hydroxylapatite chromatography, anion exchange chromatography on QAE-Sephadex, or affinity chromatography on neroic acid-substituted agarose. All the enzymatic products were identified by radio chromatography and by the synthesis of derivatives followed by radio chromatography or crystallization to constant specific activity. γ-Terpinene synthetase has an apparent molecular weight of 96,000, shows a pH optimum at about 6.8, and requires Mg²⁺ for catalytic activity. Mn²⁺ can partially substitute for Mg²⁺, but other divalent cations are ineffective. Estimated values of V and K_m are 3.5 nmol/h/mg and 9 μm, respectively, for neryl pyrophosphate, and 3.0 nmol/h/mg and 14 μm, respectively, for geranyl pyrophosphate. Enzymic activity is inhibited by sulfhydryl-directed reagents and inorganic pyrophosphate, but not by γ-terpinene, p-cymene, or thymol. Based on the specific location of tritium in the product, a mechanism is proposed which involves the cyclization of the acyclic precursor, loss of a proton from C5 to form the Δ⁴ double bond, and a 1,2-hydride shift from C4 to C8 to give γ-terpinene. A similar mechanism, but with loss of the proton from C6 and the formation of a cyclopropane ring, would yield α-thujene.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">In vivo</Emphasis> Studies on a Novel Bioadhesive Colloidal System: Cationic Liposomes of Ibuprofen

The objective of this study was to develop an ocular drug delivery system built on the cationic liposomes, a novel bioadhesive colloidal system, which could enhance the precorneal residence time, ocular permeation, and bioavailability of ibuprofen. The optimal formulation of cationic liposomes prepared by ethanol injection method was ultimately confirmed by an orthogonal L₉ (3³) test design. In addition, γ-scintigraphic technology and the microdialysis technique were utilized in the assessment of in vivo precorneal retention capability and ocular bioavailability individually. In the end, we acquired the optimal formulation of ibuprofen cationic liposomes (Ibu-CL) by orthogonal test design, and the particle size and entrapment efficiency (EE%) were 121.0 ± 3.5 nm and 72.9 ± 3.4%, respectively. In comparison to ibuprofen eye drops (Ibu-ED), Ibu-CL could significantly prolong the T _max to 100 min and the AUC to 1.53-folds, which indicated that the Ibu-CL could improve the precorneal retention time and bioavailability of ibuprofen. Consequently, these outcomes designated that the ibuprofen cationic liposomes we researched probably are a promising application in ocular drug delivery system.     

Synthesis and evaluation of Re/99mTc(I) complexes bearing a somatostatin receptor-targeting antagonist and labeled via a novel [N,S,O] clickable bifunctional chelating agent

The somatostatin receptor subtype 2 (SSTR2) is often highly expressed on neuroendocrine tumors (NETs), making it a popular in vivo target for diagnostic and therapeutic approaches aimed toward management of NETs. In this work, an antagonist peptide (sst₂-ANT) with high affinity for SSTR2 was modified at the N-terminus with a novel [N,S,O] bifunctional chelator (2) designed for tridentate chelation of rhenium(I) and technetium(I) tricarbonyl cores, [Re(CO)₃]⁺ and [^99mTc][Tc(CO)₃]⁺. The chelator-peptide conjugation was performed via a Cu(I)-assisted click reaction of the alkyne-bearing chelator (2) with an azide-functionalized sst₂-ANT peptide (3), to yield NSO-sst₂-ANT (4). Two synthetic methods were used to prepare Re-4 at the macroscopic scale, which differed based on the relative timing of the click conjugation to the [Re(CO)₃]⁺ complexation by 2. The resulting products demonstrated the expected molecular mass and nanomolar in vitro SSTR2 affinity (IC₅₀ values under 30?nM, AR42J cells, [¹²⁵I]iodo-Tyr¹¹-somatostatin-14 radioligand standard). However, a difference in their HPLC retention times suggested a difference in metal coordination modes, which was attributed to a competing N-triazole donor ligand formed during click conjugation. Surprisingly, the radiotracer scale reaction of [^99mTc][Tc(OH₂)₃(CO)₃]⁺ (^99mTc; t_½?=?6?h, 141?keV γ) with 4 formed a third product, distinct from the Re analogues, making this one of the unusual cases in which Re and Tc chemistries are not well matched. Nevertheless, the [^99mTc]Tc-4 product demonstrated excellent in vitro stability to challenges by cysteine and histidine (≥98% intact through 24?h), along with 75% stability in mouse serum through 4?h. In vivo biodistribution and microSPECT/CT imaging studies performed in AR42J tumor-bearing mice revealed improved clearance of this radiotracer in comparison to a similar [^99mTc][Tc(CO)₃]-labeled sst₂-ANT derivative previously studied. Yet despite having adequate tumor uptake at 1?h (4.9% ID/g), tumor uptake was not blocked by co-administration of a receptor-saturating dose of SS-14. Aimed toward realignment of the Re and Tc product structures, future efforts should include distancing the alkyne group from the intended donor atoms of the chelator, to reduce the coordination options available to the [M(CO)₃]⁺ core (M?=?Re, ^99mTc) by disfavoring involvement of the N-triazole.