Molecular imaging of microbiota-gut-brain axis: searching for the right targeted probe for the right target and disease.

The highly bidirectional dialogue between the gut and the brain is markedly stimulated and influenced by the microbiome through integrated neuroendocrine, neurological and immunological processes. Gut microbiota itself communicate with the host producing hormonal intermediates, metabolites, proteins, and toxins responsible for a variety of biochemical and functional inputs, thereby shaping host homeostasis. Indeed, a dysregulated microbiota-gut-brain axis might be the origin of many neuroimmune-mediated disorders, e.g. autism, multiple sclerosis, depression, Alzheimer's and Parkinson's disease, which appear months or even years prior to a diagnosis, corroborating the theory that the pathological process is spread from the gut to the brain. A much deeper comprehension of how commensal microbe can be manipulated to interfere with disease progression is crucial for developing new strategies to diagnose and treat diseases. In recent years, the potential of positron-emission-tomography (PET) in the field of bacteria detection has gained attention. The uptake of several PET tracers has been evaluated to investigate infection pathophysiology, e.g. sterile or pathogen-mediated infection, monitoring of progression, or as a surrogate endpoint in clinical trials. In this minireview, we briefly describe the role of microbiome-gut-brain axis in health and disease and we discuss the imaging modalities and agents that could be applied to study the dynamic interactions between microbiome, gut and brain. These are key aspects in understanding the biochemical lexicon underpinning the microbiome-host crosstalk that would enable the development of diagnostics and therapeutics by targeting the human microbiota.

Detection of sites of infection in mice using 99mTc-labeled PN(2)S-PEG conjugated to UBI and 99mTc-UBI: a comparative biodistribution study.

UNLABELLED: The antimicrobial peptide ubiquicidin (UBI) directly labeled with technetium-99m ((99m)Tc) has recently been shown to be specifically taken up at sites of infection; however, its chemical structure is not well defined. To address this problem, the aim of the present study was to label UBI using poly(ethyleneglycol)-N-(N-(3-diphenylphosphinopropionyl)glycyl)-S-tritylcysteine ligand (PEG-PN(2)S) in order to compare its ability to detect infection sites with that of (99m)Tc-UBI. METHODS: The PN(2)S-PEG-UBI conjugate was prepared and labeled with (99m)Tc, and its radiochemical purity was subsequently assessed. The stability of the conjugate to cysteine challenge and dilution with both saline solution and phosphate buffer was determined and serum stability and protein binding were also assessed. In vivo studies were carried out in healthy mice to study the biodistribution of (99m)Tc-PN(2)S-PEG-UBI and its precursor (99m)Tc-PN(2)S-PEG and in infected mice to compare the uptakes of (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI at the site of infection using scintigraphic imaging and ex vivo tissue counting. RESULTS: (99m)Tc-PN(2)S-PEG-UBI was obtained with high radiochemical purity (98+/-1%) and high stability. The amphiphilic nature of the conjugate leads to a tendency to form micellar aggregates that explain the high protein binding values obtained. Biodistribution studies in mice showed low renal clearance followed by a predominant reticuloendothelial system clearance that limits its application in the abdominal area. Statistical analysis revealed no significant difference between (99m)Tc-UBI and (99m)Tc-PN(2)S-PEG-UBI uptake in infected mouse thigh, and the site of infection was clearly visualized using scintigraphic imaging. CONCLUSIONS: (99m)Tc-PN(2)S-PEG-UBI proved to be as effective as (99m)Tc-UBI in detecting sites of infection; however, the well-defined chemical structure of (99m)Tc-PN(2)S-PEG-UBI makes it a better candidate for clinical imaging of infection.

A paclitaxel-hyaluronan bioconjugate targeting ovarian cancer affords a potent in vivo therapeutic activity.

PURPOSE: This study was designed to evaluate the pharmacologic and biological properties of a paclitaxel-hyaluronan bioconjugate (ONCOFID-P) against IGROV-1 and OVCAR-3 human ovarian cancer xenografts following i.p. administration. EXPERIMENTAL DESIGN: In vitro tumor sensitivity to ONCOFID-P was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, whereas bioconjugate interaction with cells was studied cytofluorimetrically and by confocal microscopy. In vivo toxicity was assessed by a single-dose maximum-tolerated dose, peripheral blood cell count determination and by histologic analysis. Biodistribution of the compound was evaluated with a small animal-dedicated scintigraphy gamma camera following injection of 99mTc-labeled ONCOFID-P. Pharmacokinetic analysis was also carried out. Female severe combined immunodeficiency mice implanted with ovarian cancer cells underwent treatment with ONCOFID-P or free paclitaxel starting from day 7 or 14 after tumor injection, and survivals were compared. RESULTS: ONCOFID-P interacted with CD44, entered cells through a receptor-mediated mechanism, and exerted a concentration-dependent inhibitory effect against tumor cell growth. After i.p. administration, the bioconjugate distributed quite uniformly within the peritoneal cavity, was well-tolerated, and was not associated with local histologic toxicity. Pharmacokinetic studies revealed that blood levels of bioconjugate-derived paclitaxel were much higher and persisted longer than those obtained with the unconjugated free drug. Intraperitoneal treatment of tumor-bearing mice with the bioconjugate revealed that ONCOFID-P exerted a relevant increase in therapeutic activity compared with free drug. CONCLUSIONS: ONCOFID-P significantly improved results obtained with conventional paclitaxel, in terms of in vivo tolerability and therapeutic efficacy; these data strongly support its development for locoregional treatment of ovarian cancer.

Inhibition of hepatocellular carcinomas in vitro and hepatic metastases in vivo in mice by the histone deacetylase inhibitor HA-But.

PURPOSE: The purpose is to evaluate the CD44-mediated cellular targeting of HA-But, a hyaluronic acid esterified with butyric acid (But) residues, to hepatocellular carcinoma cell lines in vitro and to hepatic tumor metastases in vivo. EXPERIMENTAL DESIGN: In vitro, the CD44-dependent cytotoxicity in two human hepatocellular carcinoma cell lines (HepB3 and HepG2) with high and low CD44 expression was investigated; in vivo, the effect on liver metastases originating from intrasplenic implants of Lewis lung carcinoma (LL3) or B16-F10 melanoma in mice was compared with the pharmacokinetics of organ and tissue distribution using different routes of administration. RESULTS: HepB3 and HepG2 cell lines showed different expression of CD44 (78 and 18%, respectively), which resulted in a CD44-dependent HA-But inhibitory effect as demonstrated also by the uptake analysis performed using radiolabeled HA-But ((99m)Tc-HA-But). Pharmacokinetic studies showed different rates of (99m)Tc-HA-But distribution according to the route of administration (i.v., i.p., or s.c.): very fast (a few minutes) after i.v. treatment, with substantial accumulation in the liver and spleen; relatively slow after i.p. or s.c. treatment, with marked persistence of the drug at the site of injection. The effect of s.c. and i.p. treatment with HA-But on liver metastases originating from intrasplenic implants of LL3 carcinoma or B16-F10 melanoma (both CD44-positive: 68 and 87%, respectively), resulted in 87 and 100% metastases-free animals, respectively (regardless of the route of administration), and a significant prolongation of the life expectancy compared with control groups. CONCLUSIONS: HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions.

Technetium-99m labeling of N-[N-(3-diphenylphosphino propionyl)glycyl]cysteine (PN2S-OH) and its methyl ester derivative (PN2S-OMe).

N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine and its methyl ester derivative were labeled with (99m)Tc at neutral pH, leading to a single species in high yield (>95%) in 30 min. RP-HPLC comparison with the analogue Re(V)-oxo complexes identified the labeled compounds as the anti isomers of pentacoordinated (99m)TcO[PN(2)S]-OH and (99m)TcO[PN(2)S]-OMe complexes. The compounds are stable from pH 7 to pH 9 when (99m)TcO[PN(2)S]-OH interconverts to related syn isomer, while (99m)TcO[PN(2)S]-OMe undergoes both saponification and interconversion. They exhibit high stability in human plasma and in vivo (mice), and a biodistribution characterized by hepatobiliary excretion.

Radiochemical and biological characteristics of 99mTc-UBI 29-41 for imaging of bacterial infections.

A technetium-99m-labeled peptide derived from ubiquicidine, further referred to as 99mTc-UBI 29-41, targets bacterial and fungal infections, but not sterile inflammatory processes, in experimental animals. This paper reports on the radiochemical and biological features of this radioactive agent and the importance of the amino acid sequence of UBI 29-41 for imaging of infections. Radiochemical analyses of 99mTc-UBI 29-41 and a radiolabeled scrambled version of this peptide, i.e. 99mTc-Sc-UBI 29-41, revealed that both peptides were labeled rapidly (within 10 min) and effectively with little colloid formation (less than 5% of the total radioactivity) and very little free pertechnetate (or radioactive intermediates) in the preparations containing radiolabeled peptide. Furthermore, association of the peptides with bacteria could be competed with excess unlabeled peptide and this association proved to be temperature-dependent. Based on this in vitro data we concluded that labeling of peptides with 99mTc by this direct method is rapid, efficient, and safe. Scintigraphy demonstrated that radioactivity is rapidly removed from the circulation (half-lifes of UBI 29-41 and Sc-UBI 29-41 were 16 and 21 min, respectively) mainly by renal clearance. Analysis of murine blood revealed that only a small proportion of the intravenously injected 99mTc-peptides is associated with blood cells. Although both radiolabeled peptides accumulated rapidly at sites of infection, the values for 99mTc-UBI 29-41 were higher (P < 0.05) than for 99mTc-Sc-UBI 29-41. Moreover, injection of excess unlabeled UBI 29-41, but not Sc-UBI 29-41, into Staphylococcus aureus-infected mice prior to injection of 99mTc-UBI 29-41 significantly (P < 0.05) reduced the accumulation of this radiopharmaceutical at the site of infection. In addition, we observed significantly (P < 0.01) higher amounts of 99mTc-UBI 29-41 at the site of infection in mice using a carrier-free radiolabeled UBI 29-41 as compared with unpurified preparations containing radiolabeled UBI 29-41. This in vivo data indicates that the amino acid sequence of 99mTc-UBI 29-41 contributes to its accumulation at the site of infection.

Biokinetic and dosimetric studies of 188Re-hyaluronic acid: a new radiopharmaceutical for treatment of hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and has very limited therapeutic options. Recently, it has been found that hyaluronic acid (HA) shows selective binding to CD44 receptors expressed in most cancer histotypes. Since the trend in cancer treatment is the use of targeted radionuclide therapy, the aim of this research was to label HA with rhenium-188 and to evaluate its potential use as a hepatocarcinoma therapeutic radiopharmaceutical. METHODS: (188)Re-HA was prepared by a direct labelling method to produce a ReO(O-COO)(2)-type coordination complex. (188)Re-HA protein binding and its stability in saline, phosphate buffer, human serum and cysteine solutions were determined. Biokinetic and dosimetric data were estimated in healthy mice (n=60) using the Medical Internal Radiation Dose methodology and mouse model beta-absorbed fractions. To evaluate liver toxicity, alanine aminotranferase (AST) and aspartate aminotranferase (ALT) levels in mice were assessed and the liver maximum tolerated dose (MTD) of (188)Re-HA was determined. RESULTS: A stable complex of (188)Re-HA was obtained with high radiochemical purity (>90%) and low serum protein binding (2%). Biokinetic studies showed a rapid blood clearance (T(1/2)alpha=21 min). Four hours after administration, (188)Re-HA was almost totally removed from the blood by the liver due to the selective uptake via HA-specific receptors (73.47+/-5.11% of the injected dose). The liver MTD in mice was approximately 40 Gy after 7.4 MBq of (188)Re-HA injection. CONCLUSIONS: (188)Re-HA complex showed good stability, pharmacokinetic and dosimetric characteristics that confirm its potential as a new agent for HCC radiation therapy.

Biodistribution imaging of a paclitaxel-hyaluronan bioconjugate.

INTRODUCTION: Gamma-ray detectors represent sensitive and noninvasive instruments to evaluate in vivo the metabolic trapping of radiopharmaceuticals. This study aimed to assess the imaging biodistribution of a [(99m)Tc]-radiolabelled new prototype bioconjugate composed of paclitaxel linked to hyaluronan (ONCOFID-P). METHODS: A small gamma camera providing high-resolution images was employed. Imaging of biodistribution following intravenous, intraperitoneal, intravesical and oral administration was carried out for a 2-h period in anesthetized mice receiving [(99m)Tc]ONCOFID-P. At the end of the observation time, radioactivity in organs was directly measured. As a control, groups of mice were treated with free [(3)H]paclitaxel given according to the same administration routes, and organ biodistribution of the drug was assessed after 2 h. RESULTS: Intravenous inoculation of [(99m)Tc]ONCOFID-P was followed by a rapid and strong liver uptake. In fact, almost 80% of the imaging signal was detected in this organ 10 min after injection and such value remained constant thereafter, thus indicating that the bioconjugate given through the intravenous route could be well suited to targeting primary or metastatic liver neoplasias. Imaging of the bladder, abdomen and gastrointestinal tract after local administration disclosed that the radiolabelled compound remained confined to the cavities, suggesting a potential regional application for transitional bladder cell carcinomas, ovarian cancers and gastric tumors, respectively. Free [(3)H]paclitaxel biodistribution profoundly differed from that of [(99m)Tc]ONCOFID-P. CONCLUSIONS: Conjugation of drugs with polymers results in new chemical entities characterized by a modified biodistribution pattern. Therefore, preclinical studies based on imaging analysis of such new compounds can suggest novel therapeutic applications.

Tris(1,2-dimethyl-3-hydroxy-4(1H)-pyridone)oxotantalum(v): a new water-soluble tantalum complex containing the [Ta=O]3+ core.

The synthesis, the characterization and the X-ray crystal structure of a novel tantalum(v) complex tris(1,2-dimethyl-3-hydroxy-4(1H)-pyridone)oxotantalum(v) [TaO(C7H8O2N)3] 1 is reported. Starting from the tantalum pentaethoxide a two-step reaction was carried out observing rigorous anhydrous conditions in methanol in the first step, while the insertion of the oxo group was achieved using water as oxygen donor in the second step. X-Ray diffraction analysis of a crystal of 1 obtained upon evaporation of an aqueous solution shows a seven-coordinate monomeric complex containing the [Ta=O]3+ core in the triclinic space group P1, a = 7.282(3), b = 14.055(5), c = 16.987(6) A, alpha = 65.704(5), beta = 89.155(6), gamma = 75.270(6) degrees, V = 1525(1)A3, Z = 2. Spectroscopic investigation reveals that the complex is highly soluble and stable in water at physiological pH values; as a consequence the complex may represent a potential candidate for the development of new studies on aqueous tantalum(v) chemistry for radiopharmaceutical applications.

Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol).

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate.

Technetium-99m labelled fluconazole and antimicrobial peptides for imaging of Candida albicans and Aspergillus fumigatus infections.

The aim of this study was to investigate whether technetium-99m labelled fluconazole can distinguish fungal from bacterial infections. Fluconazole was labelled with (99m)Tc and radiochemical analysis showed less than 5% impurities. The labelling solution was injected into animals with experimental infections. For comparison, we used two peptides for infection detection, i.e. UBI 29-41 and hLF 1-11, and human IgG, all labelled with (99m)Tc. Mice were infected with Candida albicans or injected with heat-killed C. albicans or lipopolysaccharides to induce sterile inflammation. Also, mice were infected with Staphylococcus aureus or Klebsiella pneumoniae. Next, accumulation of (99m)Tc-fluconazole and (99m)Tc-labelled peptides/IgG at affected sites was determined scintigraphically. (99m)Tc-fluconazole detected C. albicans infections (T/NT ratio=3.6+/-0.47) without visualising bacterial infections (T/NT ratio=1.3+/-0.04) or sterile inflammatory processes (heat-killed C. albicans: T/NT ratio=1.3+/-0.2; lipopolysaccharide: T/NT ratio=1.4+/-0.1). C. albicans infections were already seen within the first hour after injection of (99m)Tc-fluconazole (T/NT ratio=3.1+/-0.2). A good correlation (R(2)=0.864; P<0.05) between T/NT ratios for this tracer and the number of viable C. albicans was found. Although (99m)Tc-UBI 29-41 and (99m)Tc-hLF 1-11 were able to distinguish C. albicans infections from sterile inflammatory processes in mice, these (99m)Tc-labelled peptides did not distinguish these fungal infections from bacterial infections. It is concluded that (99m)Tc-fluconazole distinguishes infections with C. albicans from bacterial infections and sterile inflammations.

Synthesis and characterization of rhenium(V) oxo complexes with N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine methyl ester. X-ray crystal structure of (ReO[Ph(2)P(CH(2))(2)C(O)-Gly-Cys-OMe(P,N,N,S)]).

The PN(2)S chelate N-[N-(3-diphenylphosphinopropionyl)glycyl]-S-tritylcysteine methyl ester [PN(2)S(Trt)-OMe] was synthesized and reacted with ReOCl(3)(PPh(3))(2) and Ph(4)P[ReOCl(4)]. The reactions of both tritylated and detritylated ligands with Re(V)O precursors gave two diastereomers, 9a and 9b, of the ReO(PN(2)S-OMe) complex. The two isomers, produced in a 1:1 molar ratio, are stable and do not interconvert. They were separated by reverse-phase HPLC and characterized by NMR, FT-IR, and UV-visible spectroscopy and electrospray mass spectrometry. X-ray analysis established for 9a the presence in the solid of the syn isomer. Compound 9a, C(21)H(23)N(2)O(5)PSRe, crystallized from warm acetonitrile in the triclinic space group Ponemacr;, a = 9.828(2) A, b = 11.163(2) A, c = 11.641(2) A, alpha = 106.48(3) degrees, beta = 109.06(3) degrees, gamma = 102.81(3) degrees, V = 1085.7(4) A(3), Z = 2. The PN(2)S coordination set is in the equatorial plane, and the complex shows a distorted square pyramidal coordination. The anti configuration assigned to 9b is consistent with all the available physicochemical data. Follow-up of the reaction of the detritylated ligand with Ph(4)P[ReOCl(4)] in ethanol or acetonitrile indicated that the phosphorus atom of the chelate binds first to the metal and that this bond acts as the driving force for coordination.

CCK8 peptide derivatized with diphenylphosphine for rhenium labelling: synthesis and molecular mechanics calculations.

A novel CCK8 derivative bearing a chelating agent at its N- end and its oxo-rhenium(V) complex have been synthesized and characterized. The chelating agent N-[N-13-(diphenylphosphino)propionyl]glycyl]cysteine (PN2S) ligand, the coordination set of which is made by the phosphorus atom of phosphine, the nitrogen atoms of the two amido groups and the sulphur atom of cysteine, has been used due to its high affinity towards the oxo-rhenium(V) moiety. Molecular modelling studies indicate that the CCK8 peptide adopts the right conformation for cholecystokinin receptor binding, and that modifications on the N-terminal side of CCK8 obtained by introducing chelating agents and its metal complexes should not affect the interaction with CCK(A) receptor.