Enhanced tumor detection using a folate receptor-targeted near-infrared fluorochrome conjugate

Fluorescence optical imaging technologies are currently being developed to image specific molecular targets in vivo. Detection technologies range from those providing microscopic detail to whole body imaging systems with potential clinical use. A number of target-specific near-infrared imaging probes have recently been developed to image receptors, antigens, and enzymes. The goal of the current study was to evaluate a new near-infrared (NIR) folate receptor (FR)-targeted imaging probe for its ability to improve detection of FR-positive cancers. We hypothesized that modification of folate would retain receptor affinity in vivo, despite the bulkier NIR fluorochrome, NIR2 (em = 682 nm). Cellular uptake of the NIR conjugates was significantly higher in FR-positive nasopharyngeal epidermoid carcinoma, KB cells, compared to FR-negative human fibrosarcoma, HT1080 cells. When tumors were implanted in vivo, equal-sized KB tumors showed a 2.4-fold higher signal intensity compared to HT1080 tumors (24 h). The maximum signal-to-background ratio (3-fold) was observed at 24 h in KB tumor. Injection of the unmodified NIR2 fluorochrome did not result in persistent contrast increases under similar conditions. Furthermore, tumor enhancement with the NIR2-folate probe persisted over 48 h and was inhibitable in vivo by administration of unlabeled folate. These results indicate that folate-modified NIR fluorochrome conjugate can be used for improved detection of FR-positive tumors.     

Regioselective synthesis of folate receptor-targeted agents derived from epothilone analogs and folic acid

Efficient regioselective syntheses of conjugates of folic acid and cytotoxic agents derived from natural epothilones are described. These folate receptor (FR) targeting compounds are water soluble and incorporate a hydrophilic peptide-based spacer unit and a reducible self-immolative disulfide-based linker system between the FR-targeting ligand and the parent drug.     

Treatment of experimental adjuvant arthritis with the combination of methotrexate and lyophilized Enterococcus faecium enriched with organic selenium

The efficacy of combination therapy with methotrexate (MTX) and probiotic bacteriaEnterococcus faecium enriched with organic selenium (EFSe) in rats with adjuvant arthritis was determined. Rats with adjuvant arthritis were given MTX (0.3 mg/kg 2-times weekly, orally); lyophilizedE. faecium enriched with Se (15 mg/kg, 5 d per week, orally); and a combination of MTX plus EFSe for a period of 50 d from the immunization. Levels of serum albumin, serum nitrite/nitrate concentrations, changes in hind paw swellling, arthrogram score, bone erosions, whole body bone mineral density (BMD) and bone mineral content (BMC) were assayed in the rats as variables of inflammation and destructive arthritis-associated changes. Treatment with MTX and with the combination MTX+EFSe significantly inhibited markers of both inflammation and arthritis. Significant differences in favor of combination therapy with MTX+EFSe as compared to MTX alone were seen in serum albumin concentration, hind paw swelling and arthrogram score. Reductions in radiographic scores were also more pronounced in the combination therapy group. Combination therapy, but not MTX alone, inhibited the reduction of BMD and BMC; treatment with lyophilized EFSe alone had no significant effect on adjuvant arthritis in rats. The potent therapeutic effect of low dosage MTX therapy in combination with lyophilized EFSe on adjuvant arthritis in rats was shown.     

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.     

Efficient intracellular delivery of oligonucleotides formulated in folate receptor-targeted lipid vesicles

In this study, a novel lipid vector has been developed for targeted delivery of oligodeoxynucleotides (ODN) to tumors that overexpress folate receptor. This is based on a method developed by Semple et al. (1), which utilizes an ionizable aminolipid (1,2-dioleoyl-3-(dimethylammonio)propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. Folate is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) spacer. These vesicles are around 100-200 nm in diameter with an ODN entrapment efficiency of 60-80%. Folate mediated efficient delivery of ODN to KB cells that overexpress folate receptor. Uptake of folate-targeted lipidic ODN by KB cells is about 8-10-fold more efficient than that of lipidic ODN without a ligand or free ODN. This formulation is resistant to serum. Thus, targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress folate receptors.     

Light induced drug release from a folic acid-drug conjugate

A major area of cancer research focuses on improving the specificity of therapeutic agents by engineering drug-delivery vehicles that target overexpressed receptors on tumor cells. One of the most commonly used approaches involves targeting of folate receptors using folic acid conjugated to a drug-containing macromolecular cargo. Once internalized via endocytosis, the drugs must be released from these constructs in order to avoid being trapped in the endosomes. Here, we describe the synthesis of a small-molecule conjugate that couples folic acid to doxorubicin via a photocleavable linker. Using HPLC we show that the doxorubicin can be released with light rapidly and with high efficiency. This approach has advantages over macromolecular systems due to its simplicity and efficiency.     

Synthesis and activity of a folate targeted monodisperse PEG camptothecin conjugate

A folate targeted camptothecin small molecule drug conjugate (SMDC) was synthesized using a monodisperse PEG spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity in human KB cells exhibited an IC₅₀ of 6 nM. Importantly, activity of the prodrug was blocked by excess folate, demonstrating receptor-mediated celluar uptake of the PEG conjugate.     

A novel DAD type and folic acid conjugated fluorescent monomer as a targeting probe for imaging of folate receptor overexpressed cells

We describe a modification and post‐functionalization technique for a donor–acceptor–donor type monomer; 6‐(4,7‐bis(2,3‐dihydrothieno[3,4‐b][1,4]dioxin‐5‐yl)‐2H‐benzo[d][1,2, 3]triazol‐2‐yl)hexan‐1‐amine. Folic acid was attached to the fluorescent structure. The conjugation was confirmed via NMR and Fourier transform infrared analyses. Cytotoxicity was investigated and the comparison of association of targeted monomeric structures in tumor cells was monitored via fluorescence microscopy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:952–959, 2014     

Genetic control of sensitivity to experimental adjuvant arthritis in mice of inbred lines

Genetic control over sensitivity to experimental adjuvant arthritis was studied in different strains of inbred, recombinant and congeneic mice. The development of adjuvant arthritis is genetically regulated, with sensitivity to adjuvant arthritis controlled by H62-complex. The genes controlling sensitivity to adjuvant arthritis were shown to be located in (S-region) H-2 system.     

Treatment of adjuvant arthritis with granulocyte-colony stimulating factor and peptide derived from heat shock protein 65

Adjuvant arthritis in Lewis rats is induced by the subcutaneous injection of Mycobacterium tuberculosis in mineral oil, and the predominant T cell immune reactivity is against the heat shock protein 65 derived peptide 176-190. We treated Lewis rats with human recombinant G-CSF followed by (i.v) administration of peptide 176-190 after induction of adjuvant arthritis (AA), and observed decreased disease severity, joint destruction, new bone formation and joint ankylosis. Treatment with G-CSF alone was also effective, but to a lesser extent. In addition, we found that splenocytes from rats treated with G-CSF had reduced antigen presenting capacity compared with splenocytes from vehicle treated rats. Primed lymph node cells from G-CSF plus peptide treated rats showed a marked reduction in proliferation and secretion of IFN-gamma after stimulation with the heat shock protein peptide in vitro as compared to controls.     

Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.     

18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.     

Further evidence validating adjuvant arthritis as an experimental model of chronic pain in the rat

The experiments described here were aimed at further validating adjuvant arthritis as an animal model of chronic pain. It was found that the relative oral intake of a 0.008 mg/ml solution of fentanyl was higher in arthritic than in normal control rats; this difference was predicted by the notion that the analgesic effect of a substance may reinforce its intake in animals exposed to pain, more so than in normal pain-free animals. It was also found that body weight decreases and that vocalizations of aggregated rats increase as a result of the challenge; these effects suggest that the vegetative signs and the behavioral irritability which are characteristic of chronic pain in humans, also occur in arthritic animals. The pain which thus seems to be associated with adjuvant arthritis was estimated to have its onset on days 10–11, to peak on days 18–21, and to terminate on days 35–40 after inoculation with $\text{Mycobacterium butyricum}$.     

Evaluation of the novel folate receptor ligand [18F]fluoro-PEG-folate for macrophage targeting in a rat model of arthritis

Introduction

Detection of (subclinical) synovitis is relevant for both early diagnosis and monitoring of therapy of rheumatoid arthritis (RA). Previously, the potential of imaging (sub)clinical arthritis was demonstrated by targeting the translocator protein in activated macrophages using (R)-[¹¹C]PK11195 and positron emission tomography (PET). Images, however, also showed significant peri-articular background activity. The folate receptor (FR)-β is a potential alternative target for imaging activated macrophages. Therefore, the PET tracer [¹⁸F]fluoro-PEG-folate was synthesized and evaluated in both in vitro and ex vivo studies using a methylated BSA induced arthritis model.

Methods

[¹⁸F]fluoro-PEG-folate was synthesized in a two-step procedure. Relative binding affinities of non-radioactive fluoro-PEG-folate, folic acid and naturally circulating 5-methyltetrahydrofolate (5-Me-THF) to FR were determined using KB cells with high expression of FR. Both in vivo [¹⁸F]fluoro-PEG-folate PET and ex vivo tissue distribution studies were performed in arthritic and normal rats and results were compared with those of the established macrophage tracer (R)-[¹¹C]PK11195.

Results

[¹⁸F]fluoro-PEG-folate was synthesized with a purity >97%, a yield of 300 to 1,700 MBq and a specific activity between 40 and 70 GBq/µmol. Relative in vitro binding affinity for FR of F-PEG-folate was 1.8-fold lower than that of folic acid, but 3-fold higher than that of 5-Me-THF. In the rat model, [¹⁸F]fluoro-PEG-folate uptake in arthritic knees was increased compared with both contralateral knees and knees of normal rats. Uptake in arthritic knees could be blocked by an excess of glucosamine-folate, consistent with [¹⁸F]fluoro-PEG-folate being specifically bound to FR. Arthritic knee-to-bone and arthritic knee-to-blood ratios of [¹⁸F]fluoro-PEG-folate were increased compared with those of (R)-[¹¹C]PK11195. Reduction of 5-Me-THF levels in rat plasma to those mimicking human levels increased absolute [¹⁸F]fluoro-PEG-folate uptake in arthritic joints, but without improving target-to-background ratios.

Conclusions

The novel PET tracer [¹⁸F]fluoro-PEG-folate, designed to target FR on activated macrophages provided improved contrast in a rat model of arthritis compared with the accepted macrophage tracer (R)-[¹¹C]PK11195. These results warrant further exploration of [¹⁸F]fluoro-PEG-folate as a putative PET tracer for imaging (sub)clinical arthritis in RA patients.     

Bone selective effect of an estradiol conjugate with a novel tetracycline-derived bone-targeting agent

In this study a novel bone-targeting agent containing elements of the tricarbonylmethane system of ring A of tetracycline was developed and was shown to bind to the mineral constituent of bone, hydroxyapatite. Conjugation of this bone-targeting agent to estradiol resulted in a bone-targeted estrogen (BTE₂-A1) with an enhanced ability to bind to hydroxyapatite. In an ovariectomized rat model of osteoporosis a partial separation of the skeletal effects of estradiol from the uterine effects was observed following subcutaneous administration of BTE₂-A1. This novel bone-targeting estradiol delivery system has the potential to improve the safety profile of estradiol in the treatment of osteoporosis.     

Synthesis and biological evaluation of folate receptor-targeted boronated PAMAM dendrimers as potential agents for neutron capture therapy

Successful treatment of cancer by boron neutron capture therapy (BNCT) requires the selective delivery of (10)B to constituent cells within a tumor. The expression of the folate receptor is amplified in a variety of human tumors and potentially might serve as a molecular target for BNCT. In the present study we have investigated the possibility of targeting the folate receptor on cancer cells using folic acid conjugates of boronated poly(ethylene glycol) (PEG) containing 3rd generation polyamidoamine dendrimers to obtain (10)B concentrations necessary for BNCT by reducing the uptake of these conjugates by the reticuloendothelial system. First we covalently attached 12-15 decaborate clusters to 3rd generation polyamidoamine dendrimers. Varying quantities of PEG units with varying chain lengths were then linked to these boronated dendrimers to reduce hepatic uptake. Among all prepared combinations, boronated dendrimers with 1-1.5 PEG(2000) units exhibited the lowest hepatic uptake in C57BL/6 mice (7.2-7.7% injected dose (ID)/g liver). Thus, two folate receptor-targeted boronated 3rd generation polyamidoamine dendrimers were prepared, one containing approximately 15 decaborate clusters and approximately 1 PEG(2000) unit with folic acid attached to the distal end, the other containing approximately 13 decaborate clusters, approximately 1 PEG(2000) unit, and approximately 1 PEG(800) unit with folic acid attached to the distal end. In vitro studies using folate receptor (+) KB cells demonstrated receptor-dependent uptake of the latter conjugate. Biodistribution studies with this conjugate in C57BL/6 mice bearing folate receptor (+) murine 24JK-FBP sarcomas resulted in selective tumor uptake (6.0% ID/g tumor), but also high hepatic (38.8% ID/g) and renal (62.8% ID/g) uptake, indicating that attachment of a second PEG unit and/or folic acid may adversely affect the pharmacodynamics of this conjugate.     

Liposomal encapsulation enhances and prolongs the anti-inflammatory effects of water-soluble dexamethasone phosphate in experimental adjuvant arthritis

Introduction

The objective of this study was to evaluate the efficacy of intravenous (i.v.) injection of liposomally encapsulated dexamethasone phosphate (DxM-P) in comparison to free DxM-P in rats with established adjuvant arthritis (AA). This study focused on polyethylene glycol (PEG)-free liposomes, to minimize known allergic reactions caused by neutral PEG-modified (PEG-ylated) liposomes.

Methods

Efficacy was assessed clinically and histologically using standard scores. Non-specific and specific immune parameters were monitored. Activation of peritoneal macrophages was analyzed via cytokine profiling. Pharmacokinetics/biodistribution of DxM in plasma, synovial membrane, spleen and liver were assessed via mass spectrometry.

Results

Liposomal DxM-P (3 × 1 mg/kg body weight; administered intravenously (i.v.) on Days 14, 15 and 16 of AA) suppressed established AA, including histological signs, erythrocyte sedimentation rate, white blood cell count, circulating anti-mycobacterial IgG, and production of interleukin-1beta (IL-1β) and IL-6 by peritoneal macrophages. The suppression was strong and long-lasting. The clinical effects of liposomal DxM-P were dose-dependent for dosages between 0.01 and 1.0 mg/kg. Single administration of 1 mg/kg liposomal DxM-P and 3 × 1 mg/kg of free DxM-P showed comparable effects consisting of a partial and transient suppression. Moreover, the effects of medium-dose liposomal DxM-P (3 × 0.1 mg/kg) were equal (in the short term) or superior (in the long term) to those of high-dose free DxM-P (3 × 1 mg/kg), suggesting a potential dose reduction by a factor between 3 and 10 by liposomal encapsulation. For at least 48 hours after the last injection, the liposomal drug achieved significantly higher levels in plasma, synovial membrane, spleen and liver than the free drug.

Conclusions

This new PEG-free formulation of macrophage-targeting liposomal DxM-P considerably reduces the dose and/or frequency required to treat AA, with a potential to enhance or prolong therapeutic efficacy and limit side-effects also in the therapy of rheumatoid arthritis. Depot and/or recirculation effects in plasma, inflamed joint, liver, and spleen may contribute to this superiority of liposomally encapsulated DxM-P.