GnRH receptor-activating autoantibodies in polycystic ovary syndrome: identification of functional epitopes and development of epitope mimetic inhibitors.

PURPOSE: We have recently demonstrated that gonadotrophin-releasing hormone receptor-activating autoantibodies (GnRHR-AAb) are associated with polycystic ovary syndrome (PCOS). The aim of this study was to map the antigenic epitopes of GnRHR-AAb from PCOS patients, and develop retro-inverso peptide inhibitors that specifically target GnRHR-AAb. METHODS: Serum samples from ten GnRHR-AAb-positive PCOS patients and ten GnRHR-AAb-negative healthy controls were tested. Epitope mapping for GnRHR-AAb was performed using a set of 11 overlapping octapeptides spanning the second extracellular loop of GnRHR. Antibody-blocking effect of the designed retro-inverso peptide inhibitors was evaluated in a cell-based bioassay. RESULTS: Two peptide sequences, FSQCVTHC and HCSFSQWW, were found to react with all PCOS sera, but not with control sera. Two retro-inverso peptides that mimic the identified epitopes, d-CHTVCQSF and d-WWQSFSCH, significantly inhibited PCOS serum IgG-induced GnRHR activation. One of these two peptide inhibitors, d-CHTVCQSF, largely suppressed autoantibody-induced GnRHR activation, suggesting that the epitope sequence FSQCVTHC may be a major functional target of GnRHR-AAb. CONCLUSION: We have identified a dominant functional epitope for GnRHR-AAb associated with PCOS, and demonstrated effective blocking of GnRHR-AAb activity with epitope-mimicking retro-inverso peptide inhibitors. These proteolytically stable decoy peptides may have important therapeutic implications in subjects who harbor these autoantibodies.

Nanoparticles for Targeting of Prostate Cancer.

Prostate cancer (PCa) is the leading cause of death by cancer in men. Because of the drastic decline in the survival rate of PCa patients with advanced/metastatic disease, early diagnosis of disease and therapy without toxic side effects is crucial. Chemotherapy is widely used to control the progression of PCa at the later stages; however, it is associated with off-target toxicities and severe adverse effects due to the lack of specificity. Delivery of therapeutic or diagnostic agents by using targeted nanoparticles is a promising strategy to enhance accuracy and sensitivity of diagnosis of PCa and to increase efficacy and specificity of therapeutic agents. Numerous efforts have been made in past decades to create nanoparticles with different architectural bases for specific delivery payloads to prostate tumors. Major PCa associated cell membrane protein markers identified as targets for such purposes include folate receptor, sigma receptors, transferrin receptor, gastrin-releasing peptide receptor, urokinase plasminogen activator receptor, and prostate specific membrane antigen. Among these markers, prostate specific membrane antigen has emerged as an extremely specific and sensitive targetable marker for designing targeted nanoparticle-based delivery systems for PCa. In this article, we review contemporary advances in design, specificity, and efficacy of nanoparticles functionalized against PCa. Whenever feasible, both diagnostic as well as therapeutic applications are discussed.

Discovery and Development of a Novel mPGES-1/5-LOX Dual Inhibitor LFA-9 for Prevention and Treatment of Chronic Inflammatory Diseases.

BACKGROUND: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety. MATERIALS AND METHODS: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE2, LTB4, and PGI2 productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro. RESULTS: LFA-9 inhibited mPGES-1/5-LOX and their products PGE2 and LTB4, spared COX-1/2 and its product PGI2. LFA-9 bound strongly with human mPGES-1/5-LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE2 (82%) levels. CONCLUSION: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.

Preliminary Human Radiation Dose Estimates of PET Renal Agents, Para-18F-Fluorohippuric Acid and Ortho-124I-Iodohippuric Acid from Rat Biodistribution Data.

BACKGROUND: Para-18F-fluorohippuric acid (18F-PFH) and ortho-124I-iodohippuric acid (124IOIH) were recently identified as potential radiotracers suitable for conducting renography using positron emission tomography (PET). The aim of this work was to estimate preliminary human-equivalent internal radiation dose of 18F-PFH and 124I-OIH using the biodistribution data reported in healthy rats. The results were compared with the absorbed dose data of technetium-99m-mercaptoacetyltriglycine (99mTc- MAG3) as documented in the International Commission on Radiological Protection (ICRP) publication 80. METHODS: The medical internal radiation dose (MIRD) formula was applied to extrapolate data from rats to human and to project the absorbed radiation dose for various organs in humans. S factor was calculated by Monte-Carlo N-particle (MCNP) simulation. RESULTS: Our dose prediction shows that an injection of 18F-PFH or 124I-OIH in humans would result in an estimated effective absorbed dose of 0.09 or 0.17 µSv/MBq respectively for whole body, which is about 135 or 73 times respectively lower than that obtained with an injection of 99mTc-MAG3. All organs except kidneys would receive an estimated effective absorbed dose of <0.1 µSv/MBq for 18F-PFH or 124I-OIH. Kidneys would receive a dose of 0.83 or 0.77 µSv/MBq respectively for 18F-PFH or 124I-OIH. CONCLUSIONS: Our results indicate that 18F-PFH and 124I-OIH would deliver much safer levels and lower radiation doses to the patients compared to 99mTc-MAG3 and warrants a clinical trial to estimate the radiation doses more accurately.

Lack of chemopreventive effects of P2X7R inhibitors against pancreatic cancer.

Pancreatic cancer (PC) is an almost uniformly lethal disease with inflammation playing an important role in its progression. Sustained stimulation of purinergic receptor P2X7 drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome, we performed transcriptomic analysis of p48Cre/+-LSL-KrasG12D/+ mice pancreatic tumors by next generation sequencing. Results showed that P2X7R's key inflammasome components, IL-1ß and caspase-1 are highly expressed (p < 0.05) in pancreatic tumors. Hence, to target P2X7R, we tested effects of two P2X7R antagonists, A438079 and AZ10606120, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PC in p48Cre/+-LSL-KrasG12D/+ mice. Following dose optimization studies, for chemoprevention efficacy, six-week-old p48Cre/+-LSL-KrasG12D/+ mice (24-36/group) were fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC incidence. A marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug-treated mice. Importantly, the carcinoma spread in untreated mice was 24% compared to 43-53% in treatment groups. Reduced survival rates were observed in mice exposed to P2X7R inhibitors. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of P2X7R, NLRP3, and IL-33, whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show chemopreventive effects against PC and slightly enhanced PanIN progression to PDAC. Hence, caution is needed while treating high-risk individuals with P2X7R inhibitors.

Primary radiation dosimetry of a novel PET radiopharmaceutical 68Ga-NODAGA-glycine in comparison with 99mTc-DTPA in renal studies.

OBJECTIVE: In this study, we tried to estimate human absorbed dose of 68Ga-NODAGA-glycine as a new potential positron emission tomography (PET) renal agent based on the biodistribution data reported in healthy rats, and compare our estimation with the available absorbed dose data from technetium-99m-diethylenetriaminepentaacetic acid (99mTc-DTPA). SUBJECTS AND METHODS: The medical internal radiation dose (MIRD) formulation was applied to extrapolate from rats to human and to project the absorbed radiation dose for various organs in humans. S factor calculated by Monte-Carlo N-particle (MCNP) simulation and also this factor has been taken from the tables presented in MIRD pamphlet No.11. Hence, two radiation absorbed dose were calculated for organs. RESULTS: Our dose prediction shows that an 185MBq injection of gallium-68-1,4,7-triazacyclononane-1-γ-glutamylglycine-4,7-diacetic acid (68Ga-NODAGA-glycine) in humans might result in an estimated absorbed dose of 0.063mGy in the whole body when S factor calculated by MCNP simulation. The highest absorbed doses are observed in kidneys, lungs, spleen, liver, and red marrow with 3.510, 0.453, 0.335, 0.268, and 0.239mGy, respectively. In addition to, the estimated absorbed dose for total body after injection of 185MBq of 68Ga-NODAGA-glycine is 0.053mGy when S factor has been taken from MIRD pamphlet No.11. The highest absorbed doses are observed in kidneys, lungs, liver, spleen, and red marrow with 3.110, 0.438, 0.209, 0.203, and 0.203mGy, respectively. Comparison between human absorbed dose estimation for 68Ga-NODAGA-glycine and 99mTc-DTPA indicated that the absorbed dose of the most organs after injection of 99mTc-DTPA is higher than the amount after 68Ga-NODAGA-glycine. CONCLUSION: The results showed that 68Ga-NODAGA-glycine delivers lower dose to the patients. Also due to its application in PET (which offers higher sensitivity and spatial resolution compared to planar or SPET), 68Ga-NODAGA-glycine would be a superior choice than 99mTc-DTPA for renography and impose less radiation doses to patients.

Chemodrug delivery using integrin-targeted PLGA-Chitosan nanoparticle for lung cancer therapy.

In this study, we report the efficacy of RGD (arginine-glycine-aspartic acid) peptide-modified polylactic acid-co-glycolic acid (PLGA)-Chitosan nanoparticle (CSNP) for integrin αvß3 receptor targeted paclitaxel (PTX) delivery in lung cancer cells and its impact on normal cells. RGD peptide-modified chitosan was synthesized and then coated onto PTX-PLGA nanoparticles prepared by emulsion-solvent evaporation. PTX-PLGA-CSNP-RGD displayed favorable physicochemical properties for a targeted drug delivery system. The PTX-PLGA-CSNP-RGD system showed increased uptake via integrin receptor mediated endocytosis, triggered enhanced apoptosis, and induced G2/M cell cycle arrest and more overall cytotoxicity than its non-targeted counterpart in cancer cells. PTX-PLGA-CSNP-RGD showed less toxicity in lung fibroblasts than in cancer cells, may be attributed to low drug sensitivity, nevertheless the study invited close attention to their transient overexpression of integrin αvß3 and cautioned against corresponding uptake of toxic drugs, if any at all. Whereas, normal human bronchial epithelial (NHBE) cells with poor integrin αvß3 expression showed negligible toxicity to PTX-PLGA-CSNP-RGD, at equivalent drug concentrations used in cancer cells. Further, the nanoparticle demonstrated its capacity in targeted delivery of Cisplatin (CDDP), a drug having physicochemical properties different to PTX. Taken together, our study demonstrates that PLGA-CSNP-RGD is a promising nanoplatform for integrin targeted chemotherapeutic delivery to lung cancer.

An Open-Source Automated Peptide Synthesizer Based on Arduino and Python.

The development of the first open-source automated peptide synthesizer, PepSy, using Arduino UNO and readily available components is reported. PepSy was primarily designed to synthesize small peptides in a relatively small scale (<100 µmol). Scripts to operate PepSy in a fully automatic or manual mode were written in Python. Fully automatic script includes functions to carry out resin swelling, resin washing, single coupling, double coupling, Fmoc deprotection, ivDde deprotection, on-resin oxidation, end capping, and amino acid/reagent line cleaning. Several small peptides and peptide conjugates were successfully synthesized on PepSy with reasonably good yields and purity depending on the complexity of the peptide.

Small-Molecule Inhibition of GCNT3 Disrupts Mucin Biosynthesis and Malignant Cellular Behaviors in Pancreatic Cancer.

Pancreatic cancer is an aggressive neoplasm with almost uniform lethality and a 5-year survival rate of 7%. Several overexpressed mucins that impede drug delivery to pancreatic tumors have been therapeutically targeted, but enzymes involved in mucin biosynthesis have yet to be preclinically evaluated as potential targets. We used survival data from human patients with pancreatic cancer, next-generation sequencing of genetically engineered Kras-driven mouse pancreatic tumors and human pancreatic cancer cells to identify the novel core mucin-synthesizing enzyme GCNT3 (core 2 ß-1,6 N-acetylglucosaminyltransferase). In mouse pancreatic cancer tumors, GCNT3 upregulation (103-fold; P < 0.0001) was correlated with increased expression of mucins (5 to 87-fold; P < 0.04-0.0003). Aberrant GCNT3 expression was also associated with increased mucin production, aggressive tumorigenesis, and reduced patient survival, and CRISPR-mediated knockout of GCNT3 in pancreatic cancer cells reduced proliferation and spheroid formation. Using in silico small molecular docking simulation approaches, we identified talniflumate as a novel inhibitor that selectively binds to GCNT3. In particular, docking predictions suggested that three notable hydrogen bonds between talniflumate and GCNT3 contribute to a docking affinity of -8.3 kcal/mol. Furthermore, talniflumate alone and in combination with low-dose gefitinib reduced GCNT3 expression, leading to the disrupted production of mucins in vivo and in vitro Collectively, our findings suggest that targeting mucin biosynthesis through GCNT3 may improve drug responsiveness, warranting further development and investigation in preclinical models of pancreatic tumorigenesis. Cancer Res; 76(7); 1965-74. ©2016 AACR.

Role of (drug) transporters in imaging in health and disease.

This report is the summary of presentations at the symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics, April 26-30, at Experimental Biology 2014 in San Diego, CA. The presentations focused on the role of transporters in imaging in health and disease and on assessing transporter function in vivo. Imaging is an important diagnostic tool in clinics and is a novel tool for in vivo visualization of transporter function. Many imaging substrates and endogenous markers for organ function are organic anions. In this symposium, the bile salt transporter sodium taurocholate cotransporting polypeptide and the liver organic anion transporting polypeptides (OATPs) as well as the renal organic anion transporters (OATs) were addressed in detail; e.g., OATPs mediate transport of contrast agents used for magnetic resonance imaging of the liver or transport agents used for hepatobiliary scintigraphy, and OATs transport substances used in renography. In addition, the symposium also focused on the multidrug-resistance transporter 1 (MDR1 or P-gp), which is the most important gatekeeper in epithelial or endothelial barriers for preventing entry of potentially harmful substances into organs. Novel substrates suitable for positron emission tomography (PET) allow the study of such transporters at the blood-brain barrier or while they are mediating uptake of drugs into hepatocytes, and, importantly, PET tracers also now allow renography. Finally, quantitative data on transporter expression in human organs allow the development of improved physiologically based pharmacokinetic (PBPK) models for drug disposition. Hence, the combined efforts using novel substrates for in vivo visualization of transporters and quantification of transporters will lead to a deeper understanding of transporter function in disease and allow development of novel PBPK models for disease states.

Evaluation of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney aminopeptidase N expression.

Aminopeptidase N (APN; CD13; EC 3.4.11.2) is a zinc-dependent membrane-bound exopeptidase that catalyzes the removal of N-terminal amino acids from peptides. APN is known to be highly expressed on renal cortical proximal tubules. APN expression levels are markedly decreased under the influence of nephrotoxins and in the tumor regions of renal cancers. Thus, molecular imaging of kidney APN expression could provide pathophysiological information about kidneys noninvasively. Probestin is a potent APN inhibitor and binds to APN. Abdominal SPECT imaging was conducted at 1 h postinjection of (99m)Tc-probestin in a group of 12 UPII-SV40T transgenic and wild-type mice. UPII-SV40T mice spontaneously develop urothelial carcinoma in situ and invasive transitional cell carcinoma (TCC) that invade kidneys. Histopathology and immunohistochemistry analysis were used to confirm the presence of tumor and to evaluate APN expression in kidney. Radioactivity in normal tissue regions of renal cortex was clearly visible in SPECT images, whereas tumor regions of renal cortex displayed significantly lower or no radioactivity uptake. Histopathological analysis of kidney sections showed normal morphology for both renal pelvic and cortical regions in wild-type mice and abnormal morphology in some transgenic mice. Proliferating cell nuclear antigen staining confirmed the presence of tumor in those abnormal regions. Immunohistochemical analysis of kidney sections using anti-CD13 antibody showed significantly lower APN expression in tumor regions compared to normal regions. Results obtained in this study demonstrate the potential use of (99m)Tc-probestin SPECT as a novel technique for noninvasive imaging of kidney APN expression.

Synthesis and in vivo evaluation of N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide for inhibition of intestinal tumorigenesis in APC(Min/+) mice.

A selective KGFR tyrosine kinase inhibitor, N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide, was synthesized and its possible inhibitory effects on the development of colon polyps and colorectal tumors was examined in APC(Min/+) mice, a mouse model of human intestinal familial adenomatous polyposis. The present study shows for the first time that a dietary administration of a selective KGFR tyrosine kinase inhibitor lacks the overt-toxicities and significantly reduced the growth of small intestinal polyps in both male and female APC(Min/+) mice. This inhibition of polyp growth appears to occur at a greater extent in female mice.

Raloxifene and antiestrogenic gonadorelin inhibits intestinal tumorigenesis by modulating immune cells and decreasing stem-like cells.

Studies suggest that estrogen plays a contributing role in colorectal cancer. This project examined the preventive effects of raloxifene, a selective estrogen receptor modulator (SERM), and gonadorelin, an antiestrogenic drug, in female Apc(Min/+) mouse intestinal tumorigenesis. Six-week-old Apc(Min/+)mice were fed diet containing 1 ppm raloxifene or control diet. Gonadorelin (150 ng/mouse) was injected subcutaneously into one treatment group. Intestinal tumors were evaluated for tumor multiplicity and size. Mice treated with raloxifene and gonadorelin showed colon tumor inhibition of 80% and 75%, respectively. Both drugs significantly inhibited small intestinal tumor multiplicity and size (75%-65%, P < 0.0001). Raloxifene and gonadorelin showed significant tumor inhibition with 98% and 94% inhibition of polyps >2 mm in size. In mice fed with raloxifene or injected with gonadorelin, tumors showed significantly reduced proliferating cell nuclear antigen expression (58%-65%, P < 0.0001). Raloxifene treatment decreased ß-catenin, cyclin D1, laminin 1ß, Ccl6, and stem-like cells (Lgr 5, EpCAM, CD44/CD24), as well as suppressed inflammatory genes (COX-2, mPGES-1, 5-LOX,). Gonadorelin showed significant decrease in COX-2, mPGES-1, iNOS, and stem-like cells or increased NK cells and chemokines required for NK cells. Both drugs were effective in suppressing tumor growth albeit with different mechanisms. These observations show that either suppression of estrogen levels or modulation of estrogen receptor dramatically suppresses small intestinal and colonic tumor formation in female Apc(Min/+) mice. These results support the concept of chemoprevention by these agents in reducing endogenous levels of estrogen or modulating ER signaling.

Evaluation of 99mTc-probestin for imaging APN expressing tumors by SPECT.

Aminopeptidase N (APN) is known to play important roles in tumor angiogenesis, tumor cell invasion, and metastasis. Thus, APN is an attractive biomarker for imaging tumor angiogenesis. Here we report results obtained from biodistribution and single photon emission computed tomography (SPECT) imaging studies of a technetium-99m labeled probestin (a potent APN inhibitor) conjugate containing a tripeptide, Asp-DAP-Cys (DAP=2,3-diaminopropionic acid), chelator and a 8-amino-3,6-dioxaoctanoic acid (PEG2) linker conducted in nude mice xenografted with HT-1080 human fibrosarcoma tumors (APN-positive tumors). These results collectively demonstrate that (99m)Tc-probestin uptake by tumors and other APN expressing tissues in vivo is specific and validate the use of probestin as a vector for targeting APN in vivo.

Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor.

Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth.

Synthesis and evaluation of novel Tc-99m labeled probestin conjugates for imaging APN/CD13 expression in vivo.

The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here, we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide (N(3)S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and (99m)Tc(V) by transmetalation using corresponding Re(V) or (99m)Tc(V) gluconate synthon. The mass spectral analyses of ReO-N(3)S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N(3)S complexes. Initial biological activity of ReO-N(3)S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of (99m)TcO-N(3)S-PEG(2)-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64%ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3, respectively, at 1 h postinjection (p.i.). Tumors were clearly visible in whole body planar image obtained at 1 h p.i., but not when the APN was competitively blocked with a coinjection of excess nonradioactive ReO-N(3)S-PEG(2)-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN.

Design and synthesis of a bombesin peptide-conjugated tripodal phosphino dithioether ligand topology for the stabilization of the fac-[M(CO)3]+ core (M=(99 m)Tc or Re).

A new tumor-seeking tridentate topology consisting of a phosphino dithioether ((HOCH(2))(2)PCH(2)CH(2)S(CH(2))(n)CH(2)SR; PS(2)) ligand framework for the production of kinetically inert and in vivo stable facial [(99m)Tc(CO)(3)(PS(2))](+) or [Re(CO)(3)(PS(2))](+) is described. The X-ray crystal structure of fac-Re(CO)(3)(PS(2))PF(6) is reported. The bioconjugation strategies for incorporating bombesin (BBN) peptides on to the PS(2) tripodal framework and, thereby, de novo designing of GRP receptor-seeking Tc(PS(2)-BBN)(CO)(3) are developed.

Synthesis and in vivo evaluation of Tc-99m-labeled cyclic CisoDGRC peptide conjugates for targeting αvß3 integrin expression.

Two α(v)ß(3) integrin-binding peptide conjugates containing the cyclic CisoDGRC motif, a linker, and a chelator to enable Tc-99m labeling via the fac-[(99m)Tc(CO)(3)]+ core were synthesized. In vivo biodistribution studies in U87MG tumor-bear nude mice at 1h post-injection revealed a profound effect of the linker on the clearance of the radiotracer from the blood stream. In vivo blocking studies demonstrated the selective binding to the tumors expressing α(v)ß(3)-integrin and other tissues. The HPLC analysis of urine samples collected upon necropsy showed no degradation indicating their metabolic stability. These results suggest that cyclic CisoDGRC motif could be exploited as a new α(v)ß(3)-targeting vector by an appropriate selection of a linker between the peptide and the payload to obtain optimum pharmacokinetic properties.

Photodynamic therapy with fullerenes.

Fullerenes are a class of closed-cage nanomaterials made exclusively from carbon atoms. A great deal of attention has been focused on developing medical uses of these unique molecules especially when they are derivatized with functional groups to make them soluble and therefore able to interact with biological systems. Due to their extended pi-conjugation they absorb visible light, have a high triplet yield and can generate reactive oxygen species upon illumination, suggesting a possible role of fullerenes in photodynamic therapy. Depending on the functional groups introduced into the molecule, fullerenes can effectively photoinactivate either or both pathogenic microbial cells and malignant cancer cells. The mechanism appears to involve superoxide anion as well as singlet oxygen, and under the right conditions fullerenes may have advantages over clinically applied photosensitizers for mediating photodynamic therapy of certain diseases.

Functionalized fullerenes mediate photodynamic killing of cancer cells: Type I versus Type II photochemical mechanism.

Photodynamic therapy (PDT) employs the combination of nontoxic photosensitizers (PS) and harmless visible light to generate reactive oxygen species (ROS) and kill cells. Most clinically studied PS are based on the tetrapyrrole structure of porphyrins, chlorines, and related molecules, but new nontetrapyrrole PS are being sought. Fullerenes are soccer-ball shaped molecules composed of 60 or 70 carbon atoms and have attracted interest in connection with the search for biomedical applications of nanotechnology. Fullerenes are biologically inert unless derivatized with functional groups, whereupon they become soluble and can act as PS. We have compared the photodynamic activity of six functionalized fullerenes with 1, 2, or 3 hydrophilic or 1, 2, or 3 cationic groups. The octanol-water partition coefficients were determined and the relative contributions of Type I photochemistry (photogeneration of superoxide in the presence of NADH) and Type II photochemistry (photogeneration of singlet oxygen) were studied by measurement of oxygen consumption, 1270-nm luminescence and EPR spin trapping of the superoxide product. We studied three mouse cancer cell lines: (J774, LLC, and CT26) incubated for 24 h with fullerenes and illuminated with white light. The order of effectiveness as PS was inversely proportional to the degree of substitution of the fullerene nucleus for both the neutral and the cationic series. The monopyrrolidinium fullerene was the most active PS against all cell lines and induced apoptosis 4-6 h after illumination. It produced diffuse intracellular fluorescence when dichlorodihydrofluorescein was added as an ROS probe, suggesting a Type I mechanism for phototoxicity. We conclude that certain functionalized fullerenes have potential as novel PDT agents and phototoxicity may be mediated both by superoxide and by singlet oxygen.