Potential Clinical Benefits of D-ribose in Ischemic Cardiovascular Disease.

Cardiovascular disease still remains the leading cause of deaths worldwide. Atherosclerosis, the most common type of cardiovascular disease, has continued to progress due to many factors, genetics, and lifestyles. All cells require adequate adenosine triphosphate (ATP) levels to maintain their integrity and function. Myocardial ischemia commonly found in atherosclerosis can produce lower levels of ATP, which affects not only cellular energy, but also alters normal function. D-ribose, a naturally occurring pentose carbohydrate, has been shown to increase cellular energy levels and improve function following ischemia in pre-clinical studies and have demonstrated potential benefits in clinical evaluations. This review paper presents an overview of ischemic cardiovascular disease and the potential role that D-ribose could play in improving myocardial energy levels and function in the area of ischemic cardiovascular diseases.

The influence of D-ribose ingestion and fitness level on performance and recovery.

BACKGROUND: Skeletal muscle adenosine triphosphate (ATP) levels are severely depleted during and following prolonged high intensity exercise. Recovery from these lower ATP levels can take days, which can affect performance on subsequent days of exercise. Untrained individuals often suffer the stress and consequences of acute, repeated bouts of exercise by not having the ability to perform or recovery sufficiently to exercise on subsequent days. Conversely, trained individuals may be able to recover more quickly due to their enhanced metabolic systems. D-Ribose (DR) has been shown to enhance the recovery in ATP; however, it is not known if recovery and performance can be benefitted with DR ingestion. Therefore, this study was designed to determine what influence DR might have on muscular performance, recovery, and metabolism during and following a multi-day exercise regimen. METHODS: The study was a double blind, crossover study in 26 healthy subjects compared 10 g/day of DR to 10 g/day of dextrose (DEX, control). All subjects completed 2 days of loading with either DR or DEX, followed by 3 additional days of supplementation and during these 3 days of supplementation, each subject underwent 60 min of high intensity interval exercise in separate daily sessions, which involved cycling (8 min of exercise at 60% and 2 min at 80% VO2max), followed by a 2 min power output (PO) test. Subjects were divided into two groups based on peak VO2 results, lower VO2 (LVO2) and higher peak VO2 (HVO2). RESULTS: Mean and peak PO increased significantly from day 1 to day 3 for the DR trial compared to DEX in the LVO2 group. Rate of perceived exertion (RPE) and creatine kinase (CK) were significantly lower for DR than DEX in the LVO2 group. No differences in PO, RPE, heart rate, CK, blood urea nitrogen, or glucose were found between either supplement for the HVO2 group. CONCLUSION: DR supplementation in the lower VO2 max group resulted in maintenance in exercise performance, as well as lower levels of RPE and CK. Unlike no observed benefits with DEX supplementation.

Echocardiographic assessment of aortic valve annular and left ventricular outflow tract diameter in conscious and anesthetized adult sheep prior to prosthetic aortic valve implantation.

OBJECTIVE: Prosthetic heart valve implantation is commonly performed in patients that have valvular heart disease. Prior to clinical evaluation of newly developed prostheses, preclinical animal studies are performed for the assessment of both acute and chronic valvular function. Commonly, one size of valve is used in these preclinical studies, which can present difficulties with the implantation procedure and assessing valve function. Due to these potential problems, we developed a preoperative screening assessment in potential ovine candidates for prosthetic aortic valve implantation. By determining if there is a correlation between conscious and anesthetized echocardiographic examinations, an improvement in surgical confidence can predict that the animal is deemed a suitable candidate for a particular size of prosthetic valve for implantation prior to subjecting the animal to anesthesia and surgery. METHODS: A total of 53 crossbred sheep (Ovis aries), male and female, 10-37 months of age, weighing between 41 and 77 kg, underwent conscious echocardiography and a subset of 29 of these animals underwent echocardiographic assessment under anesthesia for a preoperative valve size comparison in these animals prior to surgical prosthetic aortic valve implantation. Using 2D echocardiographic assessment, left ventricular outflow tract (LVOT) dimensions were assessed. RESULTS: The mean paired difference between anesthetized and conscious LVOT diameter measurement was -0.87 mm (p = 0.0066, standard deviation 1.598, 95% confidence interval, -0.4796, -0.26378, n = 29). CONCLUSION: This pilot study evaluation revealed that conscious echocardiographic assessment can play a role preoperatively in selecting potential candidates for surgical prosthetic aortic valve implantation, thereby minimizing the potential in prosthetic-native annular mismatching, which can contribute to altered LVOT function.

Cellular protection during oxidative stress: a potential role for D-ribose and antioxidants.

A healthy cellular system involves the maintenance of an intracellular metabolic balance. Reactive oxygen species (ROS) are constantly produced as a normal product of cellular metabolism; however, during situations of cellular stress, these levels can increase dramatically with the potential to cause deleterious cellular structural and/or functional consequences. There is a significant elevation in these ROS following stressful situations, such as ischemia, hypoxia, high-intensity exercise, and in many diseases. To combat these ROS, neutralizing endogenous enzymes, as well as exogenous antioxidants, can aid in minimizing their potential untoward cellular effects. Exogenous reducing antioxidant agents, such as vitamin C and/or E, play a role in addressing these formed species; however, recent research has suggested that fruit seed extracts may provide additional cellular benefits beyond their antioxidant features. Furthermore, supplemental D-ribose enhances the recovery of high-energy phosphates following stress and appears to potentially offer additional benefits by reducing radical formation. Specifically, during periods of hypoxia/ischemia, supplemental D-ribose may play an inhibitory role in the breakdown of adenine nucleotides, influencing the subsequent formation of xanthine and uric acid compounds; and thereby affecting the release of superoxide anion radicals. The combination of D-ribose with reducing antioxidants may provide a more optimal state of cellular protection during and following times of oxidative stress.

GLUT 5 is not over-expressed in breast cancer cells and patient breast cancer tissues.

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

The patented uses of D-ribose in cardiovascular diseases.

Cardiovascular diseases account for more deaths worldwide than any other illness. Myocardial ischemia, a common finding in cardiovascular diseases, lowers cellular energy levels, which affects a cell's integrity and function. Pre-clinical animal studies have reported lower cellular energy levels with an associated decreased function following myocardial ischemia. Recently, scientists have reported that the failing heart is energy starved and yet no pharmaceuticals have been able to address this issue with satisfactory results. Over decades, researchers have explored the use of various metabolites to replenish deficient cellular energy levels following induced ischemia with mixed results. However, D-ribose, a natural occurring carbohydrate, has demonstrated significant enhancing abilities in replenishing deficient cellular energy levels following myocardial ischemia, as well as improving depressed function in numerous animal investigations. Subsequent clinical trials have further substantiated these benefits of D-ribose in patients afflicted with ischemic cardiovascular disease and those carrying the diagnosis of congestive heart failure. The future of effective therapies for ischemic heart disease and congestive heart failure must strongly consider novel pharmaceuticals directed at replenishing cellular energy levels. Intellectual property and the represented patents in this paper emphasize the use of D-ribose for its cellular energy enhancing potential, reflected in both objective and subjective clinical improvements; therefore, substantiating its value in patients with ischemic cardiovascular diseases.

Metabolic supplementation with enhanced external counterpulsation improves myocardial function in acquired cardiomyopathy: a case report.

BACKGROUND: Cardiomyopathy is reflected in a deterioration of heart function, increased risk of developing arrhythmias, and the potential for sudden cardiac death. The use of enhanced external counterpulsation has been recommended for treating chronic stable angina in high-risk surgical patients. Furthermore, cells require adequate levels of adenosine triphosphate for the maintenance of integrity and function. Lower myocardial levels of adenosine triphosphate are commonly found with ischemia and heart failure. d-Ribose, a natural occurring carbohydrate, enhances the regeneration of adenosine triphosphate levels and improves diastolic function following ischemia. SUBJECT: We present a patient with cardiomyopathy and marked reduced cardiac function. CONCLUSIONS: This patient underwent enhanced external counterpulsation and metabolic supplementation, including d-ribose, and achieved a significant functional improvement.

The role of ribose on oxidative stress during hypoxic exercise: a pilot study.

Oxygen free radicals are produced during stress, are unstable, and potentially interact with other cellular components or molecules. This reactivity can influence cellular function, including a prolongation in tissue recovery following exercise. We tested the effect of ribose (d-ribose), a pentose carbohydrate, in a double-blinded, crossover study on markers of free radical production during hypoxic exercise. Seven healthy volunteers cycled at their lactate threshold for 25 minutes while inhaling 16% O(2) with a subsequent 60-minute resting period at room air. Subjects ingested either placebo or 7 g of ribose in 250 mL of water before and after the exercise session. Urinary malondialdehyde (MDA) and plasma reduced glutathione levels increased significantly during placebo ingestion (0.2 +/- 0.03 nM/mg and 0.26 +/- 0.29 microM, respectively) but were lower with ribose supplementation (0.04 +/- 0.03 nM/mg and 0.38 +/- 0.29 microM, respectively; P < .05). Uric acid levels were similar between groups (ribose vs. placebo, 4.55 +/- 0.06 mg/dL vs. 4.67 +/- 0.06 mg/dL). Ribose demonstrated a beneficial trend in lower MDA and reduced glutathione levels during hypoxic stress.

D-ribose, a metabolic substrate for congestive heart failure.

The incidence of congestive heart failure continues to escalate worldwide, taxing health care systems. Current therapies focus on clinical management. Current accepted regimens have provided some success; however, most patients show progression of their disease. Because of this failure, research continues to explore therapies directed at stabilization of their disease and hopefully to improve the downward spiral. Publications have asserted that the failing heart is energy starved. D-ribose, a naturally occurring pentose carbohydrate and a key component in the adenosine triphosphate (ATP) molecule, has demonstrated an ability to replenish ATP levels and improve diastolic dysfunction following myocardial ischemia, which has been shown to improve the clinical state of patients afflicted with congestive heart failure. D-ribose may provide the necessary metabolic substrate to benefit this energy-deficient state found in heart failure.

Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine.

UNLABELLED: The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. METHODS: A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. RESULTS: (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. CONCLUSION: The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

Dermal benefits of topical D-ribose.

Our aging skin undergoes changes with reductions in collagenous and elastic fibers, fibroblasts, mast cells, and macrophages with free radical production, which can result in reduced skin tone and wrinkle formation. Fibroblasts are important for dermal integrity and function with a decrease in function producing less skin tone, thinning, and wrinkle formation. Dermal levels of adenosine triphosphate (ATP) decline with aging, potentially altering dermal function. Supplemental D-ribose, a natural occurring carbohydrate, enhances ATP regeneration. D-ribose-based studies demonstrated benefits in both cell culture fibroblastic activities and a subsequent clinical study in women with decreased skin tone with wrinkles. Supplemental D-ribose may offer this needed cellular benefit.

Ribose enhances retinoic acid-induced differentiation of HL-60 cells.

Ribose, a critical building block for nucleotides, plays an important role in energy metabolism, transcription, translation, and second messenger systems. This 5-carbon sugar, synthesized from glucose via the pentose phosphate pathway, has a rate-limiting step at glucose-6-phosphate dehydrogenase. Therefore, we hypothesized that when cells are required to proliferate or differentiate, as in an immune response, the requirement for D-ribose may be greater than what could be supplied by the synthetic pathway. We hypothesized that providing an exogenous source of D-ribose during cell differentiation will enhance the process of differentiation. We used a retinoic acid-induced HL-60 cell differentiation culture as a model of neutrophil maturation. The addition of 10 to 25 mmol/L D-ribose was shown to reduce cell proliferation and move the cell population toward apoptosis in a dose-dependent manner. The expression of a cell surface marker representing maturity (CD11b) significantly increased and a cell surface marker indicative of immaturity (CD117) significantly decreased. Functionally, the cells had a greater oxidative burst function dependent on time and dose. The mechanism by which ribose enhances HL-60 cell differentiation is not known; however, as adenosine triphosphate levels did not change, adenosine triphosphate is not thought to be involved. We conclude that in this cell culture model, ribose supplementation enhanced cellular differentiation and function. Thus, ribose might be conditionally essential during time of higher need as in an immune response.

Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks.

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

Ribose in the heart.

Every cell needs energy, i.e., adenosine triphosphate (ATP), to carry out its function. Decreased oxygen levels, decreased blood flow, and other stressful conditions can drastically effect the intracellular concentrations of these energy compounds. Skeletal muscle, unlike the heart, can address this drop in ATP by employing the myokinase reaction, ultimately producing ATP with a subsequent elevation in adenosine monophosphate (AMP). Ribose, a naturally occurring 5-carbon monosaccharide, is a key component of RNA, DNA (which has deoxyribose), acetyl coenzyme A, and ATP. Each cell produces its own ribose, involved in the pentose phosphate pathway (PPP), to aid in ATP production. States of ischemia and/or hypoxia can severely lower levels of cellular energy compounds in the heart, with an associated compromise in cellular processes, ultimately reflected in altered function. Ribose appears to provide a solution to the problem in replenishing the depressed ATP levels and improving functional status of patients afflicted with cardiovascular diseases.

Isolation, characterization, and biological evaluation of syn and anti diastereomers of [(99m)Tc]technetium depreotide: a somatostatin receptor binding tumor imaging agent.

The early and later eluting [(99m)TcO]depreotide products on RP-HPLC were confirmed to be the anti and syn diastereomers, respectively, based on proton NMR and circular dichroism spectroscopy. NMR provided evidence of a folded, conformationally constrained structure for the syn diastereomer. The syn diastereomer is predominant (anti/syn approximately 10:90) in the [(99m)TcO]depreotide preparation and shows a slightly higher affinity (IC50 = 0.15 nM) for the somatostatin receptor than the anti diastereomer (IC50 = 0.89 nM). Both diastereomers showed higher binding affinities than the free peptide (IC(50) = 7.4 nM). Biodistribution studies in AR42J tumor xenograft nude mice also showed higher tumor uptake for syn [(99m)TcO]depreotide (6.58% ID/g) than for the anti [(99m)TcO]depreotide (3.38% ID/g). Despite the differences in biological efficacy, the favorable binding affinity, tumor uptake, and tumor-to-background ratio results for both diastereomeric species predict that both are effective for imaging somatostatin receptor-positive tumors in vivo.

MO tripeptide diastereomers (M=99/99mTc, Re): models to identify the structure of 99mTc peptide targeted radiopharmaceuticals.

Biologically active molecules, such as many peptides, serve as targeting vectors for radiopharmaceuticals based on 99mTc. Tripeptides can be suitable chelates and are easily and conveniently synthesized and linked to peptide targeting vectors through solid-phase peptide synthesis and form stable TcVO complexes. Upon complexation with [TcO]3+, two products form; these are syn and anti diastereomers, and they often have different biological behavior. This is the case with the approved radiopharmaceutical [99mTcO]depreotide ([99mTcO]P829, NeoTect) that is used to image lung cancer. [99mTcO]depreotide indeed exhibits two product peaks in its HPLC profile, but assignment of the product peaks to the diastereomers has proven to be difficult because the metal peptide complex is difficult to crystallize for structural analysis. In this study, we isolated diastereomers of [99TcO] and [ReO] complexes of several tripeptide ligands that model the metal chelator region of [99mTcO]depreotide. Using X-ray crystallography, we observed that the early eluting peak (A) corresponds to the anti diastereomer, where the Tc=O group is on the opposite side of the plane formed by the ligand backbone relative to the pendant groups of the tripeptide ligand, and the later eluting peak (B) corresponds to the syn diastereomer, where the Tc=O group is on the same side of the plane as the residues of the tripeptide. 1H NMR and circular dichroism (CD) spectroscopy report on the metal environment and prove to be diagnostic for syn or anti diastereomers, and we identified characteristic features from these techniques that can be used to assign the diastereomer profile in 99mTc peptide radiopharmaceuticals like [99mTcO]depreotide and in 188Re peptide radiotherapeutic agents. Crystallography, potentiometric titration, and NMR results presented insights into the chemistry occurring under physiological conditions. The tripeptide complexes where lysine is the second amino acid crystallized in a deprotonated metallo-amide form, possessing a short N1-M bond. The pKa measurements of the N1 amine (pKa approximately 5.6) suggested that this amine is rendered more acidic by both metal complexation and the presence of the lysine residue. Furthermore, peptide chelators incorporating a lysine (like the chelator of [TcO]depreotide) likely exist in the deprotonated form in vivo, comprising a neutral metal center. Deprotonation possibly mediates the interconversion process between the syn and anti diastereomers. The N1 amine group on non-lysine-containing metallopeptides is not as acidic (pKa approximately 6.8) and does not deprotonate and crystallize as do the metallo-amide species. Three of the tripeptide ligands (FGC, FSC, and FKC) were radiolabeled with 99mTc, and the individual syn and anti isomers were isolated for biodistribution studies in normal female nude mice. The main organs of uptake were the liver, intestines, and kidneys, with the FGC compounds exhibiting the highest liver uptake. In comparing the diastereomers, the syn compounds had substantially higher organ uptake and slower blood clearance than the anti compounds.

Preservation of diastolic function in monocrotaline-induced right ventricular hypertrophy in rats.

During ischemic heart diseases and when heart failure progresses depletion of myocardial energy stores occurs. D-Ribose (R) has been shown to improve cardiac function and energy status after ischemia. Folic acid (FA) is an essential cofactor in the formation of adenine nucleotides. Therefore, we assessed whether chronic R-FA administration during the development of hypertrophy resulted in an improved cardiac function and energy status. In Wistar rats (n = 40) compensatory right ventricular (RV) hypertrophy was induced by monocrotaline (30 mg/kg; MCT), whereas saline served as control. Both groups received a daily oral dose of either 150 mg.kg(-1).day(-1) dextrose (placebo) or R-FA (150 and 40 mg.kg(-1).day(-1), respectively). In Langendorff-perfused hearts, RV and left ventricular (LV) pressure development and collagen content as well as total RV adenine nucleotides (TAN), creatine content, and RV and LV collagen content were determined. In the control group R-FA had no effect. In the MCT-placebo group, TAN and creatine content were reduced, RV and LV diastolic pressure-volume relations were steeper, RV systolic pressures were elevated, RV and LV collagen content was increased, and RV-LV diastolic interaction was altered compared with controls. In the MCT-R-FA group, TAN, RV and LV diastolic stiffness, RV and LV collagen content, and RV-LV diastolic interaction were normalized to the values in the control group while creatine content remained depressed and RV systolic function remained elevated. In conclusion, the depression of energy status in compensated hypertrophic myocardium observed was partly prevented by chronic R-FA administration and accompanied by a preservation of diastolic function and collagen deposition.

Orthotopic total aortic root replacement model in adult sheep.

Prosthetic heart valves undergo mandatory preclinical animal testing prior to human clinical trials. Historically, a non-site-specific placement of a valve prosthesis has been commonly performed; however, recently site-specific placement continues to attract interest. Various animal models have been used for preclinical evaluation of both aortic and mitral valve prostheses; however, a universally accepted animal model for orthotopic total aortic root replacement with acceptable early and late mortality for long-term evaluation has been lacking. This article reports a successful orthotopic model for placement of tissue valve conduit prosthesis for total aortic root replacement in adult sheep. This model utilized preoperative echocardiographic assessment, specific intraoperative surgical techniques, and both early and late postoperative management therapies. The combination of all of these components resulted in a successful model for orthotopic placement of a tissue valve prosthesis for total aortic root replacement in adult sheep for potential long-term assessment.

Somatostatin receptor-binding peptides suitable for tumor radiotherapy with Re-188 or Re-186. Chemistry and initial biological studies.

Somatostatin derivative peptides previously designed for radiodiagnostic purposes (99mTc P829 or 99mTc depreotide) were reoptimized for radiotherapy of tumors with rhenium radioisotopes. An optimized pharmacophore peptide P1839 was derived by in vitro binding affinity assay to AR42J rat pancreatic tumor cell membranes. Peptides with chelating domains and their oxorhenium(V) complexes were tested in vitro for binding to NCI H69 human SCLC tumor membranes. Further optimization entailed radiolabeling with 99mTc and biodistribution in an AR42J xenograft mouse model. Kidney uptake was decreased substantially by removing positively charged residues. Neutral N3S diamide amine thiol chelators with no adjacent positive charges had the best overall properties. Substituting an aromatic amino acid into the chelator approximately doubled the tumor uptake. The final optimized peptide P2045 (39) radiolabeled with 99mTc exhibited increased tumor uptake ( approximately 25 %ID/g at 1.5 h), lower kidney uptake ( approximately 4.8 %ID/g at 1.5 h), and extensive urinary excretion (59 %ID at 1.5 h). Finally, comparison biodistribution studies between 99mTc and 188Re (39) showed a good correlation between the two metal complexes and demonstrated prolonged tumor retention (> or =24 h).