Total Hip Arthroplasty in A Young Patient with Bernard-Soulier Syndrome.

INTRODUCTION: The management of patients with coagulopathic disorders undergoing orthopaedic surgery requires a dedicated, multi-disciplinary team with detailed perioperative planning. Bernard-Soulier Syndrome (BSS) is an extremely rare disorder, affecting 1 in 1 million individuals worldwide. It is caused by a deficiency in glycoprotein 1b-V-IX which is required for normal platelet-mediated clot formation. The deficiency results in prolonged bleeding time with high risk of spontaneous bleeds. Few reports exist in the clinical literature of BSS patients undergoing major surgery. CASE REPORT: A 40 year old, female with known BSS and developmental dysplasia of her left hip (DDH) was referred to us for consideration of left total hip arthroplasty (THA). Consultation with her Haematologist for pre-operative optimization of platelets and related clotting times together with detailed discussions of her intended anaesthesia protocol and surgery resulted in a successful operation with less than anticipated blood loss. She entered our rehabilitation program just one week after surgery. CONCLUSION: BSS is an extremely rare bleeding disorder that puts patients at very high risk of blood loss following surgery. This is the first report that we are aware of describing a BSS patient undergoing a THA. A cohesive, highly specialized, multi-disciplinary team is crucial to the success of these patients.

A survey of Canadian cancer patients' perspectives on the characteristics and treatment of breakthrough pain.

OBJECTIVE: Breakthrough pain is defined as a transient exacerbation of pain that occurs spontaneously or in response to a trigger despite stable and controlled background pain. The purpose of this study was to explore Canadian patients' awareness of and experience with breakthrough pain in cancer (BTPc). METHODS: Four Canadian cancer centers participated in a non-interventional survey recruiting cancer patients who experienced breakthrough pain. These patients were asked about their pain, its impact on functioning, current management and interest in new treatments of BTPc. RESULTS: Ninety-four Canadian cancer patients participated in this study, with 96% stating that cancer pain impacted their daily living with over half unable to go to work or shopping. Fifty percent of patients said that an episode of BTPc lasted greater than 60 minutes, with the pain score being on average 7.8/10, impacting normal work (7.2/10) and general activity (7.1/10). Only 35% of patients were very satisfied with the speed of relief of their medications. Those who did not take their breakthrough pain medication for every episode stated that was because the pain was not always severe (37%), or they were afraid of becoming tolerant (23%) or addicted (12%). Patients stated that the most important features of a new treatment for BTPc were the ability to relieve pain completely (47%), and quickly (43%). Patients expressed willingness to try transmucosal products (80%) or nasal products (59%). CONCLUSION: Breakthrough cancer pain in Canadian cancer patients greatly impacts their daily lives. There is room for improvement in the management of BTPc, and the majority of patients would be willing to try new treatments.

Antitumor actions of ruthenium(III)-based nitric oxide scavengers and nitric oxide synthase inhibitors.

The role of endogenous nitric oxide (NO) in the growth and vascularization of a rat carcinosarcoma (P22) has been investigated. Tumor-bearing animals were treated with (i) nitric oxide synthase (NOS) inhibitors, administered via the drinking water, including N(G)-nitro-l-arginine methyl ester (L-NAME), a nonisoform-selective inhibitor, and 2 others that target the inducible (NOS II) enzyme preferentially, namely 1-amino-2-hydroxyguanidine or N-[3-(aminomethyl)benzyl]acetamidine hydrochloride; or (ii) daily injections (intraperitoneally) of 2 Ru(III) polyaminocarboxylates, AMD6221 and AMD6245, both of which are effective NO scavengers. L-NAME, AMD6221, and AMD6245 reduced tumor growth by approximately 60% to 75% of control rates. Tumor sections stained with abs to CD-31/platelet endothelial cell adhesion molecule-1 or NOS III showed that this was associated with a marked reduction (60%-77%) of tumor microvascular densitiy (MVD). Tumors resumed growing promptly when treatment was discontinued, accompanied by partial or complete restoration of MVDs. In contrast, NOS-II selective inhibitors had no effect on tumor growth or vascularization, indicating that both responses require complete blockade of NO production. The results corroborate the view that endogenous NO facilitates tumor development. We suggest that NO deprivation causes tumor feeder vessels to constrict, reducing tumor blood flow. The delivery of oxygen and essential nutrients to the developing tumor is impaired as a consequence, hampering further growth. Normalizing NO levels by withholding treatment causes tumor feeder vessels to dilate, increasing tumor perfusion and reestablishing conditions that allow tumors to begin growing again.

Effects of photodynamic therapy on the structural integrity of vertebral bone.

STUDY DESIGN: This study investigates the effects of photodynamic therapy (PDT) on the structural integrity of vertebral bone in healthy rats. OBJECTIVE: To determine the short-term (1 week) and intermediate term (6 weeks) effects of a single PDT treatment on the mechanical and structural properties of vertebral bone. SUMMARY OF BACKGROUND DATA: Spinal metastasis develops in up to one-third of all cancer patients, compromising the mechanical integrity of the spine and thereby increasing the risk of pathologic fractures and spinal cord damage. PDT has recently been adapted to ablate metastatic tumors in the spine in preclinical animal models. However, little is known about the effects of PDT on the structural integrity of vertebral bone. METHODS: A single PDT treatment was administered to healthy Wistar rats at photosensitizer and light doses known to be effective in athymic rats bearing human breast cancer metastases. At both 1 and 6 weeks posttreatment, changes in trabecular architecture, global stiffness and strength of vertebrae were quantified using micro-CT stereological analysis and axial compression testing. RESULTS: At 6 weeks, there was a significant increase in bone volume fraction (to 55.7 +/- 11.1% vs. 38.5 +/- 6.4%, P < 0.001) and decrease in bone surface area-to-volume ratio (16.9 +/- 5.0/mm vs. 22.8 +/- 4.5/mm, P = 0.001), attributed to trabecular thickening (130 +/- 40 microm vs. 90 +/- 20 microm, P < 0.001). Similar trends were found at 1 week after PDT. There was a significant increase in stiffness from control (306 +/- 123 N/mm) at 1 week (399 +/- 150 N/mm, P = 0.04) and 6 weeks (410 +/- 113 N/mm, P = 0.05) post PDT. There was a positive trend toward increased ultimate stress at 1 week, which became statistically significant at 6 weeks compared with control (39.3 +/- 11.3 MPa vs. 27.5 +/- 9.5 MPa control, P = 0.002). CONCLUSION: Not only may PDT be successful in ablating metastatic tumor tissue in the spine, but the positive effects of PDT on bone found in this study suggest that PDT may also improve vertebral mechanical stability.

Phototoxic aptamers selectively enter and kill epithelial cancer cells.

The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5' end with the photodynamic therapy agent chlorin e(6) and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells.

Photodynamic therapy of vertebral metastases: evaluating tumor-to-neural tissue uptake of BPD-MA and ALA-PpIX in a murine model of metastatic human breast carcinoma.

Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin-derivative monoacid ring A (BPD-MA) and by 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor-bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD-MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD-MA, ALA-PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor-bearing vertebrae compared to spinal cord. The highest ratio for BPD-MA concentration was found 15 min after injection, which can be recommended for therapy in this model.

A new technique for physiodesis using photodynamic therapy.

Vascularization of developing growth plates is integral to the process of endochondral ossification. We hypothesized photodynamic therapy could be used to initiate premature vascularization and calcification of growth plates in mice on the induction of vascular endothelial growth factor. Three-week-old transgenic mice that emit bioluminescence on activation of the vascular endothelial growth factor gene were treated with different regimens of benzoporphyrin derivative mono-acid-mediated photodynamic therapy in the right, proximal tibial growth plate. We quantified changes in vascular endothelial growth factor-related bioluminescence at times after photodynamic therapy in vivo. The expression of vascular endothelial growth factor protein and CD31-labeled vasculature in growth plates also were examined with growth plate histology. Measurements of limb length were assessed in vivo using conventional radiography and confirmed on harvesting. Mice exposed to repeat treatments of 10 J x2 displayed enhanced bioluminescence 2 weeks after photodynamic therapy. Histology confirmed increased vasculature immediately adjacent to the growth plates with evidence of physeal closure. At 4 weeks posttreatment, limbs were shortened by an average of 9.5% +/- 4.4% without complication, confirming the potential application of photodynamic therapy for physiodesis.

Multimodality imaging for vertebral metastases in a rat osteolytic model.

Imaging modalities facilitate the detection of early bony metastases. Few studies specifically address vertebral metastases in animal models for preclinical (early, asymptomatic) disease. We performed intracardiac injection of human breast cancer (MT-1) cells in 35 athymic nude rats. We evaluated potential temporal differences in appendicular versus axial metastases as detectable by longitudinal in vivo conventional radiography (ie, fine detail radiography and two-dimensional fluoroscopy). We compared bioluminescent reporter imaging with conventional radiographs in the detection of vertebral metastasis, and compared bioluminescent imaging with subsequent ex vivo microcomputed tomography analysis of osteolysis. The mean survival was 25 days in the animals that had metastases develop. Conventional radiographs identified appendicular osteolysis by 14 days; however, vertebral osteolysis was identified late in the metastatic spread (Days 25-28). Bioluminescence imaging was more sensitive in earlier detection of vertebral lesions in all imaged animals at Day 21, which corresponded to microcomputed tomography evaluation of osteolysis. Conventional radiographs do not appear useful for early detection of vertebral metastasis. Early identification of metastasis is important when considering the use of this model to evaluate therapeutic outcomes directed toward vertebral metastasis.

High-frequency ultrasound assessment of antimicrobial photodynamic therapy in vitro.

Ultrasound imaging is proving to be an important tool for medical diagnosis of dermatological disease. Backscatter spectral profiles using high-frequency ultrasound (HFUS, 10-100 MHz) are sensitive to subtle changes in eukaryotic cellular morphology and mechanical properties that are indicative of early apoptosis, the main type of cell death induced following photodynamic therapy (PDT). We performed experiments to study whether HFUS could also be used to discern changes in bacteria following PDT treatment. Pellets of planktonic Staphylococcus aureus were treated with different PDT protocols and subsequently interrogated with HFUS. Changes in ultrasound backscatter response were found to correlate with antimicrobial effect. Despite their small size, distinct changes in bacterial morphology that are indicative of cell damage or death are detectable by altered backscatter spectra from bacterial ensembles using HFUS. This highlights the potential for HFUS in rapidly and non-invasively assessing the structural changes related to antimicrobial response.

Pre-clinical in vitro and in vivo studies to examine the potential use of photodynamic therapy in the treatment of osteomyelitis.

Osteomyelitis can lead to severe morbidity and even death resulting from an acute or chronic inflammation of the bone and contiguous structures due to fungal or bacterial infection. Incidence approximates 1 in 1000 neonates and 1 in 5000 children in the United States annually and increases up to 0.36% and 16% in adults with diabetes or sickle cell anaemia, respectively. Current regimens of treatment include antibiotics and/or surgery. However, the increasing number of antibiotic resistant pathogens suggests that alternate strategies are required. We are investigating photodynamic therapy (PDT) as one such alternate treatment for osteomyelitis using a bioluminescent strain of biofilm-producing staphylococcus aureus (S. aureus) grown onto kirschner wires (K-wire). S. aureus-coated K-wires were exposed to methylene blue (MB) or 5-aminolevulinic acid (ALA)-mediated PDT either in vitro or following implant into the tibial medullary cavity of Sprague-Dawley rats. The progression of S. aureus biofilm was monitored non-invasively using bioluminescence and expressed as a percentage of the signal for each sample immediately prior to treatment. S. aureus infections were subject to PDT 10 days post inoculation. Treatment comprised administration of ALA (300 mg kg(-1)) intraperitoneally followed 4 h later by light (635 +/- 10 nm; 75 J cm(-2)) delivered transcutaneously via an optical fiber placed onto the tibia and resulted in significant delay in bacterial growth. In vitro, MB and ALA displayed similar cell kill with > or =4 log(10) cell kill. In vivo, ALA-mediated PDT inhibited biofilm implants in bone. These results confirm that MB or ALA-mediated PDT have potential to treat S. aureus cultures grown in vitro or in vivo using an animal model of osteomyelitis.

Photodynamic therapy of diseased bone.

OBJECTIVE: Photodynamic therapy (PDT) defines the light activation of a photosensitizing compound, leading to the generation of cytotoxic, reactive oxygen species. We are investigating the unique application of PDT for diseased bone. METHODS: Bioluminescent, human breast cancer cells (MT-1) were injected intracardially into athymic rats. At 2-3 weeks following injection rats developed diffuse bone metastases that were visible using an in vivo bioluminescence imaging system. Metastatic lesions within vertebrae and long bones were treated with differing regimens of 690nm laser light (25-150J) following injection of benzoporphyrin-derivative monoacid (BPD-MA) and the tumour response assayed histologically upon sacrifice. Subsequent large animal studies involved light propagation studies in porcine vertebrae and BPD-MA-PDT treatment of large primary osteosarcomas in canine. Canine were subject to high dose PDT (500J/cm). A second rat model involved bacterial infection in bone. Bioluminescent Staphylococcus aureus (S. aureus) were grown as biofilms onto wires, implanted into rat tibia and exposed to 5-aminolevulinic acid (ALA)-PDT delivered percutaneously with 635nm laser light (75J/cm(2)). Response to therapy was monitored as changes in bioluminescence signal and colony forming assay upon sacrifice. RESULTS: PDT (150J) proved effective in ablating metastatic lesions within rat femur and lumbar vertebrae. Porcine studies confirmed the average depth of light penetration into trabecular bone as 0.16±0.04cm with an average incident fluence rate of 4.3mW/cm(2), however, the necrotic/non-necrotic interface extended 0.6cm out from the treatment fiber. In canine a single, high dose treatment created well-circumscribed margins of effect encompassing the entire 3-4cm osteosracoma. Finally, ALA-PDT proved effective against S. aureus biofilms in bone, although recurrent infection did occur in some. CONCLUSIONS: Results support the application of PDT to the treatment of primary or metastatic lesions within bone. Secondly, ALA-PDT may be useful as a treatment for osteomyelitis with repeat or chronic treatment regimens.

Fluorescence image-guided brain tumour resection with adjuvant metronomic photodynamic therapy: pre-clinical model and technology development.

Fluorescence-guided resection (FGR) and photodynamic therapy (PDT) have previously been investigated separately with the objectives, respectively, of increasing the extent of brain tumour resection and of selectively destroying residual tumour post-resection. Both techniques have demonstrated trends towards improved survival, pre-clinically and clinically. We hypothesize that combining these techniques will further delay tumour re-growth. In order to demonstrate technical feasibility, we here evaluate fluorescence imaging and PDT treatment techniques in a specific intracranial tumour model. The model was the VX2 carcinoma grown by injection of tumour cells into the normal rabbit brain. An operating microscope was used for white light imaging and a custom-built fluorescence imaging system with co-axial excitation and detection was used for FGR. PDT treatment light was applied by intracranially-implanted light emitting diodes (LED). The fluorescent photosensitizer used for both FGR and PDT was ALA-induced PpIX. For PDT, ALA (100 mg kg(-1)) and low light doses (15 and 30 J) were administered over extended periods, which we refer to as metronomic PDT (mPDT). Eighteen tumour bearing rabbits were divided equally into three groups: controls (no resection); FGR; and FGR followed by mPDT. Histological whole brain sections (H&E stain) showed primary and recurrent tumours. No bacteriological infections were found by Gram staining. Selective tumour cell death through mPDT-induced apoptosis was demonstrated by TUNEL stain. These results demonstrate that the combined treatment is technically feasible and this model is a candidate to evaluate it. Further optimization of mPDT treatment parameters (drug/light dose rates) is required to improve survival.

Assessment of cutaneous photosensitivity of TOOKAD (WST09) in preclinical animal models and in patients.

TOOKAD (WST09) is a new, long-wavelength palladium bacteriopheophorbide photosensitizer that targets tissue vasculature. The cutaneous phototoxicity of TOOKAD was assessed in normal rat and pig animal models and in patients in a Phase-I trial of TOOKAD-mediated photodynamic therapy (PDT) for recurrent prostate cancer. Controlled skin exposures were administered using solar-simulated light at various times after drug administration. Two different spectral ranges were used. In the first, the UV portion of the spectrum was removed (UV(-)) because UV irradiation in nondrugged control animals produced an erythema response at incident energy densities (J/cm(2)) lower than those required to induce a PDT response. In the second, the full solar spectrum (UV(+)) was used, and the potentiation by the photosensitizer of the UV-mediated minimum erythema dose was assessed. Results showed that the PDT skin response was negligible at clinical drug doses of 2 mg/kg for any period after administration at light doses of 128 J/cm(2) in the animal models. In patients, there was no observed UV(-) skin response at doses of up to 2 mg/kg, drug-light intervals of 1-3 h or greater and light exposures up to 128 J/cm(2). At higher drug doses in the rat and pig models, the duration of skin phototoxicity was found to be approximately 3 h and less than 1 h, respectively. Using the full spectrum of solar-simulated light, the presence of TOOKAD did not measurably enhance the UV(+)-induced erythema in the rats, pigs or patients.

Bioluminescence imaging of the response of rat gliosarcoma to ALA-PpIX-mediated photodynamic therapy.

Photodynamic therapy (PDT) is a promising modality for the treatment of solid tumors that combines a photosensitizing agent and light to produce cytotoxic reactive oxygen species that lead to tumor cell death. The recent introduction of bioluminescence imaging (BLI), involving the use of the luciferase gene (luc) transferred into target tumor cells, followed by systemic administration of luciferin and detection of the emitted visible chemiluminescence photons, offers the potential for longitudinal imaging of tumor growth and therapeutic response in single animals. We demonstrate in this study the first results of the use of BLI to assess the response of an intracranial brain tumor model (9L rat gliosarcoma) to aminolevulinic acid (ALA)-mediated PDT. Complementary in vitro experiments with the luciferase-transfected 9L cells show that the decrease in the luminescent signal after PDT correlates with cell kill. In vivo imaging shows a decrease in the BLI signal from the tumor after ALA-PDT treatment, followed by tumor regrowth. Furthermore, preliminary studies using cells transfected with a hypoxia-responsive vector show an increase in bioluminescence within 4 h after Photofrin-mediated PDT, demonstrating the ability to observe stress-gene responses. These results suggest that BLI can be used to provide spatiotemporal information of intracranial brain tumor responses after PDT and may serve as a valuable response-endpoint measure.

Metronomic photodynamic therapy as a new paradigm for photodynamic therapy: rationale and preclinical evaluation of technical feasibility for treating malignant brain tumors.

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low-dose PDT using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA-mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate, sodium salt nick-end labeling positive) cells as compared with an acute, high-dose regimen (ALA-aPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectrofluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3-4 fold increase in the tumor-to-brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light-emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.

Photodynamic actinometry using microspheres: concept, development and responsivity.

Photodynamic therapy (PDT) relies on three main ingredients, oxygen, light and photoactivating compounds, although the PDT response is definitively contingent on the site and level of reactive oxygen species (ROS) generation. This study describes the development of a novel, fluorescent-based actinometer microsphere system as a means of discerning spatially resolved dosimetry of total fluence and ROS production. Providing a high resolution, localized, in situ measurement of fluence and ROS generation is critical for developing in vivo PDT protocols. Alginate-poly-L-lysine-alginate microspheres were produced using ionotropic gelation of sodium alginate droplets, ranging from 80 to 200 microm in diameter, incorporating two dyes, ADS680WS (ADS) and Rhodophyta-phycoerythrin (RPE), attached to the spheres' inside and outside layers, respectively. To test the responsivity and dynamic range of RPE for ROS detection, the production of ROS was initiated either chemically using increasing concentrations of potassium perchromate or photochemically using aluminum tetrasulphonated phthalocyanine. The generation of singlet oxygen was confirmed by phosphorescence at 1270 nm. The resulting photodegradation and decrease in fluorescence of RPE was found to correlate with increased perchromate or PDT treatment fluence, respectively. This effect was independent of pH (6.5-8) and could be inhibited using sodium azide. RPE was not susceptible to photobleaching with light alone (670 nm; 150 Jcm(-2)). ADS, which absorbs light between 600 and 750 nm, showed a direct correlation between radiant exposure (670 nm; 0-100 Jcm(-2)) and diminished fluorescence. Photobleaching was independent of irradiance (10-40 mW cm(-2)). We propose that actinometer microspheres may provide a means for obtaining high spatial resolution information regarding delivered PDT dose within model systems during investigational PDT development and dosimetric information for clinical extracorporeal PDT as in the case of ex vivo bone marrow purging.

High speed, wide velocity dynamic range Doppler optical coherence tomography (Part III): in vivo endoscopic imaging of blood flow in the rat and human gastrointestinal tracts.

We previously described a fiber based Doppler optical coherence tomography system [1] capable of imaging embryo cardiac blood flow at 4~16 frames per second with wide velocity dynamic range [2]. Coupling this system to a linear scanning fiber optical catheter design that minimizes friction and vibrations, we report here the initial results of in vivo endoscopic Doppler optical coherence tomography (EDOCT) imaging in normal rat and human esophagus. Microvascular flow in blood vessels less than 100 microm diameter was detected using a combination of color-Doppler and velocity variance imaging modes, during clinical endoscopy using a mobile EDOCT system.

A simple two-step approach for introducing a protected diaminedithiol chelator during solid-phase assembly of peptides.

A simple synthetic strategy is described to incorporate a protected diaminedithiol (N(2)S(2)) chelator during Fmoc solid-phase synthesis of short peptides. The resulting constructs could be efficiently labeled with technetium-99m (99mTc). The chelator was assembled at the N-terminus of peptides in a two-step procedure where the deprotected terminal amino group was first reacted with di-Fmoc-diaminopropionic acid (Fmoc-DAP-[Fmoc]-OH). The two protected amino groups were then simultaneously deprotected and subsequently reacted with S-benzoylthiolglycolic acid (TGA) to generate a protected N(2)S(2) chelator. This metal binding site was introduced into di- and tripeptides. Each peptide construct was composed of a C-terminal lysine residue and an N-terminal diaminopropionic moiety modified to create the chelator site. The epsilon-amino group at the C-terminal lysine was further derivatized with a nitroimidazole group to facilitate cellular retention. The resulting constructs were then cleaved from the resin support, purified, and labeled with [99mTc]pertechnetate. Six constructs were prepared differing by a single amino acid inserted between the diaminopropionic acid and lysine residues. Optimal labeling yields of >70% were achieved around neutral pH and heating at 75 degrees C for 10 min. Purified 99mTc-labeled constructs were found to accumulate in Chinese hamster ovary (CHO) cells in vitro as a function of charge and hydrophobicity.

Designing peptide-based scaffolds as drug delivery vehicles.

Methods for delivering drugs into cells remain an important part of the process of designing drugs. One promising approach is the concept of loligomers, synthetic peptides composed of a branched polylysine core harboring identical arms. Loligomers are typically synthesized with eight arms, each carrying peptide signals guiding their import and localization into cells. The most important advantage of loligomers is the multivalent presentation of targeting signals resulting from a tentacular arrangement. Multivalency increases the efficiency of import and intracellular routing signals as compared to similar linear peptides. Secondly, it reduces and delays the impact of peptide degradation in terms of cellular processing and compartmentalization. The vectorial delivery of nucleus-directed loligomers into cells has recently been confirmed by microscopy and flow cytometry studies. Practical uses of loligomers as intracellular vehicles include the import of plasmid DNA into cells, the conjugation of chemical groups, such as photosensitizers for use in photodynamic therapy, and the incorporation of cytotoxic T-lymphocyte (CTL) epitopes with a view to creating synthetic vaccines. Branched peptides such as loligomers represent simple and versatile molecular vehicles with potential applications in a wide variety of drug design approaches.