Differential MRGPRX2-dependent activation of human mast cells by polymyxins and octapeptins.

The emergence of multi-drug resistant Gram-negative bacteria has led to renewed interest in the antimicrobial activity of polymyxins and novel polymyxin analogues (e.g. nonapeptides and octapeptin). In some individuals, clinically used polymyxins can cause acute hypersensitivity reactions through mast cell activation, with a recent study attributing this effect to activation of the MAS-related G protein-coupled receptor X2 (MRGPRX2). In the present study, HEK293 cells expressing human MRGPRX2 and the human mast cell line LAD2 were used to characterize the activity of the broader family of polymyxins. Octapeptin C4, polymyxin B and colistin produced concentration-dependent calcium mobilization, degranulation, and CCL-2 (MCP-1) release in LAD2 mast cells, with the former being highly potent. CRISPR-Cas9 knockdown of MRGPRX2 in LAD2 cells and a MRGPRX2 inverse agonist caused a significant reduction in calcium mobilization, degranulation, and CCL-2 release, demonstrating dependency on MRGPRX2 expression. In contrast, polymyxin nonapeptides were far less potent calcium mobilisers and failed to induce functional degranulation in LAD2 cells. Our results confirm that activation of mast cells induced by polymyxin-related antibiotics is MRGPRX2-dependent and reveal that octapeptin C4 might be more liable, whilst nonapeptides are less liable, to trigger immediate hypersensitivity reactions clinically. The mechanism underpinning the difference in MRGPRX2 activation between polymyxin-related antibiotics is important to better understand as it may help design new, safer polymyxins and guide the optimal clinical use of existing polymyxin drugs.

Semisynthesis of A6-A11 lactam insulin.

Insulin replacement therapy is essential for the management of diabetes. However, despite the relative success of this therapeutic strategy, there is still a need to improve glycaemic control and the overall quality of life of patients. This need has driven research into orally available, glucose-responsive and rapid-acting insulins. A key consideration during analogue development is formulation stability, which can be improved via the replacement of insulin's A6-A11 disulfide bond with stable mimetics. Unfortunately, analogues such as these require extensive chemical synthesis to incorporate the nonnative cross-links, which is not a scalable synthetic approach. To address this issue, we demonstrate proof of principle for the semisynthesis of insulin analogues bearing nonnative A6-A11 cystine isosteres. The key feature of our synthetic strategy involves the use of several biosynthetically derived peptide precursors which can be produced at scale cost-effectively and a small, chemically synthesised A6-A11 macrocyclic lactam fragment. Although the assembled A6-A11 lactam insulin possesses poor biological activity in vitro, our synthetic strategy can be applied to other disulfide mimetics that have been shown to improve thermal stability without significantly affecting activity and structure. Moreover, we envisage that this new semisynthetic approach will underpin a new generation of hyperstable proteomimetics.

An Engineered Nanosugar Enables Rapid and Sustained Glucose-Responsive Insulin Delivery in Diabetic Mice.

Glucose-responsive insulin-delivery platforms that are sensitive to dynamic glucose concentration fluctuations and provide both rapid and prolonged insulin release have great potential to control hyperglycemia and avoid hypoglycemia diabetes. Here, biodegradable and charge-switchable phytoglycogen nanoparticles capable of glucose-stimulated insulin release are engineered. The nanoparticles are "nanosugars" bearing glucose-sensitive phenylboronic acid groups and amine moieties that allow effective complexation with insulin (≈95% loading capacity) to form nanocomplexes. A single subcutaneous injection of nanocomplexes shows a rapid and efficient response to a glucose challenge in two distinct diabetic mouse models, resulting in optimal blood glucose levels (below 200 mg dL-1 ) for up to 13 h. The morphology of the nanocomplexes is found to be key to controlling rapid and extended glucose-regulated insulin delivery in vivo. These studies reveal that the injected nanocomplexes enabled efficient insulin release in the mouse, with optimal bioavailability, pharmacokinetics, and safety profiles. These results highlight a promising strategy for the development of a glucose-responsive insulin delivery system based on a natural and biodegradable nanosugar.

The effect of substance P and its common in vivo-formed metabolites on MRGPRX2 and human mast cell activation.

The tachykinin neuropeptide substance P (SP) is the canonical agonist peptide for the neurokinin 1 receptor (NK1 R). More recently, it has also been shown to activate the Mas-related G protein-coupled receptor X2 (MRGPRX2) receptor on mast cells (MCs), triggering degranulation and release of inflammatory mediators. SP undergoes rapid C-terminal truncation in vivo by a number of proteases to generate the metabolites SP(1-9)-COOH and in particular SP(1-7)-COOH. While the C terminus of SP is critical for NK1 R activation, studies have shown that the peptide polycationic N terminus is key for MRGPRX2 and mast cell activation. The study thus aimed to determine if the C-terminally truncated metabolites of SP, SP(1-9)-COOH, and SP(1-7)-COOH retained stimulatory activity at MRGPRX2. SP, SP(1-9)-COOH, and SP(1-7)-COOH were synthesized and tested on HEK293 cells expressing NK1 R or MRGPRX2, and LAD2 human mast cells, to determine the activity of SP and its metabolites in Ca2+ mobilization, degranulation, and cytokine assays. As expected from prior studies, both C-terminally truncated SP metabolites had essentially no activity at NK1 R, even at very high concentrations. In contrast, the in vivo metabolite of SP, SP(1-9)-COOH retained ability to activate MRGPRX2 across all parameters tested, albeit with reduced potency compared to intact SP. SP(1-7)-COOH did not produce any significant MRGRPX2 activation. Our results suggest that the SP metabolite, SP(1-9)-COOH, may play a regulatory role through the activation of MRGPRX2. However, given the relatively low potency of both SP and SP(1-9)-COOH at MRGPRX2, additional work is needed to better understand the biological importance of this expanded SP/MRGPRX2 pathway.

The Chemical Synthesis of Insulin: An Enduring Challenge.

The pancreatic peptide hormone insulin, first discovered exactly 100 years ago, is essential for glycemic control and is used as a therapeutic for the treatment of type 1 and, increasingly, type 2 diabetes. With a worsening global diabetes epidemic and its significant health budget imposition, there is a great demand for new analogues possessing improved physical and functional properties. However, the chemical synthesis of insulin's intricate 51-amino acid, two-chain, three-disulfide bond structure, together with the poor physicochemical properties of both the individual chains and the hormone itself, has long represented a major challenge to organic chemists. This review provides a timely overview of the past efforts to chemically assemble this fascinating hormone using an array of strategies to enable both correct folding of the two chains and selective formation of disulfide bonds. These methods not only have contributed to general peptide synthesis chemistry and enabled access to the greatly growing numbers of insulin-like and cystine-rich peptides but also, today, enable the production of insulin at the synthetic efficiency levels of recombinant DNA expression methods. They have led to the production of a myriad of novel analogues with optimized structural and functional features and of the feasibility for their industrial manufacture.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133-150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133-150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.

Endosomal escape cell-penetrating peptides significantly enhance pharmacological effectiveness and CNS activity of systemically administered antisense oligonucleotides.

Antisense oligonucleotides (ASOs) are an emerging class of gene-specific therapeutics for diseases associated with the central nervous system (CNS). However, ASO delivery across the blood-brain barrier (BBB) to their CNS target cells remains a major challenge. Since ASOs are mainly taken up into the brain capillary endothelial cells interface through endosomal routes, entrapment in the endosomal compartment is a major obstacle for efficient CNS delivery of ASOs. Therefore, we evaluated the effectiveness of a panel of cell-penetrating peptides (CPPs) bearing several endosomal escape domains for the intracellular delivery, endosomal release and antisense activity of FDA-approved Spinraza (Nusinersen), an ASO used to treat spinal muscular atrophy (SMA). We identified a CPP, HA2-ApoE(131-150), which, when conjugated to Nusinersen, showed efficient endosomal escape capability and significantly increased the level of full-length functional mRNA of the survival motor neuron 2 (SMN2) gene in SMA patient-derived fibroblasts. Treatment of SMN2 transgenic adult mice with this CPP-PMO conjugate resulted in a significant increase in the level of full-length SMN2 in the brain and spinal cord. This work provides proof-of-principle that integration of endosomal escape domains with CPPs enables higher cytosolic delivery of ASOs, and more importantly enhances the efficiency of BBB-permeability and CNS activity of systemically administered ASOs.

Structure-Activity Relationships of Daptomycin Lipopeptides.

Daptomycin is a calcium-dependent cyclic lipodepsipeptide derived from the soil saprotroph Streptomyces roseosporus, and its antibiotic properties make it a key agent for treatment of drug-resistant Gram-positive infections. It is most commonly used clinically for the treatment of Gram-positive skin and skin structure infections (SSSI), Staphylococcus aureus bacteremia, and right-sided endocarditis infections associated with S. aureus, including methicillin resistant S. aureus (MRSA). It has also been used "off-label" for Enterococcal infections. There has been a tremendous amount of research investigating its mode of action, resistance mechanisms, and biosynthesis of this clinically important antimicrobial agent. Although we cover the latter aspects in detail, the primary focus of this review is to provide the most comprehensive and up-to-date reference for the medicinal chemist on the structure-activity-toxicity of this important class of lipopeptide antibiotics.

The Antimicrobial Activity of Cannabinoids.

A post-antibiotic world is fast becoming a reality, given the rapid emergence of pathogens that are resistant to current drugs. Therefore, there is an urgent need to discover new classes of potent antimicrobial agents with novel modes of action. Cannabis sativa is an herbaceous plant that has been used for millennia for medicinal and recreational purposes. Its bioactivity is largely due to a class of compounds known as cannabinoids. Recently, these natural products and their analogs have been screened for their antimicrobial properties, in the quest to discover new anti-infective agents. This paper seeks to review the research to date on cannabinoids in this context, including an analysis of structure-activity relationships. It is hoped that it will stimulate further interest in this important issue.

The Killing Mechanism of Teixobactin against Methicillin-Resistant Staphylococcus aureus: an Untargeted Metabolomics Study.

Antibiotics have served humankind through their use in modern medicine as effective treatments for otherwise fatal bacterial infections. Teixobactin is a first member of newly discovered natural antibiotics that was recently identified from a hitherto-unculturable soil bacterium, Eleftheria terrae, and recognized as a potent antibacterial agent against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. The most distinctive characteristic of teixobactin as an effective antibiotic is that teixobactin resistance could not be evolved in a laboratory setting. It is purported that teixobactin's "resistance-resistant" mechanism of action includes binding to the essential bacterial cell wall synthesis building blocks lipid II and lipid III. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of the synthetic teixobactin analogue Leu10-teixobactin against a MRSA strain, S. aureus ATCC 700699. The metabolomes of S. aureus ATCC 700699 cells 1, 3, and 6 h following treatment with Leu10-teixobactin (0.5 µg/ml, i.e., 0.5× MIC) were compared to those of the untreated controls. Leu10-teixobactin significantly perturbed bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan (lipid I and II) metabolism, and cell wall teichoic acid (lipid III) biosynthesis as early as after 1 h of treatment, reflecting an initial activity on the cell envelope. Concordant with its time-dependent antibacterial killing action, Leu10-teixobactin caused more perturbations in the levels of key intermediates in pathways of amino-sugar and nucleotide-sugar metabolism and their downstream peptidoglycan and teichoic acid biosynthesis at 3 and 6 h. Significant perturbations in arginine metabolism and the interrelated tricarboxylic acid cycle, histidine metabolism, pantothenate, and coenzyme A biosynthesis were also observed at 3 and 6 h. To conclude, this is the first study to provide novel metabolomics mechanistic information, which lends support to the development of teixobactin as an antibacterial drug for the treatment of multidrug-resistant Gram-positive infections.IMPORTANCE Antimicrobial resistance is one of the greatest threats to the global health system. It is imperative that new anti-infective therapeutics be developed against problematic "superbugs." The cyclic depsipeptide teixobactin holds much promise as a new class of antibiotics for highly resistant Gram-positive pathogens (e.g., methicillin-resistant Staphylococcus aureus [MRSA]). Understanding its molecular mechanism(s) of action could lead to the design of new compounds with a broader activity spectrum. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of teixobactin against MRSA. Our findings revealed that teixobactin significantly disorganized the bacterial cell envelope, as reflected by a profound perturbation in the bacterial membrane lipids and cell wall biosynthesis (peptidoglycan and teichoic acid). Importantly, teixobactin significantly suppressed the main intermediate d-alanyl-d-lactate involved in the mechanism of vancomycin resistance in S. aureus These novel results help explain the unique mechanism of action of teixobactin and its lack of cross-resistance with vancomycin.

1,3-Dichloroacetone: A Robust Reagent for Preparing Bicyclic Peptides.

The chemical synthesis of cyclic peptides is a well-established area of research. This has been further expanded by development of bio-orthogonal reactions that enable access to peptides of greater structural complexity. One approach utilizes 1,3-dichloroacetone to selectively link free cysteine side-chains with an acetone-like bridge via an SN2 reaction. Here, we have used this reaction to dimerize cyclic peptide monomers to create novel bicyclic dimeric peptides. We investigated a range of reaction parameters to identify the optimal dimerization conditions for our model systems. One of the acetone-linked dimeric peptides was analyzed for proteolytic stability in human serum and was observed to still be fully intact after 48 h. This study provides valuable insights into the application of 1,3-dichloroacetone as a tool in the synthesis of complex, multicyclic peptides.

Structure of micelle bound cationic peptides by NMR spectroscopy using a lanthanide shift reagent.

[Tm(DPA)3]3- was used to generate multiple, paramagnetic nuclear Overhauser effect NMR spectra of cationic peptides when weakly bound to a lipopolysaccharide micelle. Increased spectral resolution combined with a marked increase in the number of distance restraints yielded high resolution structures of polymyxin and MSI-594 in the liposaccharide bound state.

Total Chemical Synthesis of a Nonfibrillating Human Glycoinsulin.

Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B in vitro and high glucose-lowering effects in vivo, irrespective of the route of administration (s.c. vs i.p.). The glycoinsulin retained insulin-like helical structure and exhibited improved stability in human serum. Importantly, our disialo-glycoinsulin analogue does not form fibrils at both high concentration and temperature. Therefore, it is an excellent candidate for clinical use in insulin pumps.

Synthesis and structure-activity relationships of teixobactin.

The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium and found to possess potent antibacterial activity against several Gram positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting. This is proposed to be a result of a mechanism of action that involves binding to essential cell wall synthesis building blocks, lipid II and lipid III. Since the initial isolation report in 2015, significant efforts have been made to understand its unique mechanism of action, develop efficient synthetic routes for its production, and thus enable the generation of analogues for structure-activity relationship studies and optimization of its pharmacological properties. Our review provides a comprehensive treatise on the progress in understanding teixobactin chemistry, structure-activity relationships, and mechanisms of antibacterial activity. Teixobactin represents an exciting starting point for the development of new antibiotics that can be used to combat multidrug-resistant bacterial ("superbug") infections.

The impact of backbone N-methylation on the structure-activity relationship of Leu10 -teixobactin.

Antimicrobial resistance is a serious threat to global human health; therefore, new anti-infective therapeutics are required. The cyclic depsi-peptide teixobactin exhibits potent antimicrobial activity against several Gram-positive pathogens. To study the natural product's mechanism of action and improve its pharmacological properties, efficient chemical methods for preparing teixobactin analogues are required to expedite structure-activity relationship studies. Described herein is a synthetic route that enables rapid access to analogues. Furthermore, our new N-methylated analogues highlight that hydrogen bonding along the N-terminal tail is likely to be important for antimicrobial activity.

Rapid Photolysis-Mediated Folding of Disulfide-Rich Peptides.

Structure-activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated "one-pot" strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin.

Thiol-Cyanobenzothiazole Ligation for the Efficient Preparation of Peptide-PNA Conjugates.

Antisense oligonucleotide (ASO)-based drugs are emerging with great potential as therapeutic compounds for diseases with unmet medical needs. However, for ASOs to be effective as clinical entities, they should reach their intracellular RNA and DNA targets at pharmacologically relevant concentrations. Over the past decades, various covalently attached delivery vehicles have been utilized for intracellular delivery of ASOs. One such approach is the use of biocompatible cell-penetrating peptides (CPPs) covalently conjugated to ASOs. The stability of the linkage is of paramount importance for maximal intracellular delivery to achieve the desired therapeutic effect. In this study, we have investigated the efficiency and stability of four different bioorthogonal and nonreductive linkages including triazole, thioether, thiosuccinimide thioether and thiazole moieties. Here we have shown that thiazole and thiosuccinimide are the two most efficient and facile approaches for the preparation of peptide-ASO conjugates. The thiazole linkage had a higher stability compared to the thiosuccinimide thioether at physiological conditions (pH 7.4, 37 °C) in the presence of a biologically relevant concentration of glutathione. We have also shown that the peptide-ASO conjugate with a thiosuccinimide linkage has a significantly lower antisense activity compared to the peptide-ASO with the thiazole linkage, which maintains its antisense activity after 24 h of exposure to glutathione. In summary, we have demonstrated that the bioorthogonal thiazole linkage offers the benefits of mild reaction conditions, fast reaction kinetics, absence of any byproducts, and higher stability compared to other conjugation approaches. This facile ligation can be used for the synthesis of a variety of bioconjugates where a stable linkage is required.

The Assembly of Fluorescently Labeled Peptide-Oligonucleotide Conjugates via Orthogonal Ligation Strategies.

Efficient intracellular delivery is critical to the successful application of synthetic antisense oligonucleotides (ASOs) to modulate gene expression. The conjugation of cell-penetrating peptides (CPPs) to ASOs has been shown to significantly improve their intracellular delivery. It is important, however, that formation of the covalent linkage between the peptide and oligonucleotide is efficient and orthogonal, to ensure high yields and a homogeneous product. Described herein are efficient and facile methodologies for the conjugation of peptides to ASOs, and their subsequent labeling with various moieties such as fluorescent dyes for intracellular tracking studies.

Sputum Active Polymyxin Lipopeptides: Activity against Cystic Fibrosis Pseudomonas aeruginosa Isolates and Their Interactions with Sputum Biomolecules.

The mucoid biofilm mode of growth of Pseudomonas aeruginosa ( P. aeruginosa) in the lungs of cystic fibrosis patients makes eradication of infections with antibiotic therapy very difficult. The lipopeptide antibiotics polymyxin B and colistin are currently the last-resort therapies for infections caused by multidrug-resistant P. aeruginosa. In the present study, we investigated the antibacterial activity of a series of polymyxin lipopeptides (polymyxin B, colistin, FADDI-003, octapeptin A3, and polymyxin A2) against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa cystic fibrosis isolates grown under planktonic or biofilm conditions in artificial sputum and their interactions with sputum component biomolecules. In sputum media under planktonic conditions, the lipopeptides FADDI-003 and octapeptin A3 displayed very promising activity against the polymyxin-resistant isolate FADDI-PA066 (polymyxin B minimum inhibitory concentration (MIC) = 32 mg/L), while retaining their activity against the polymyxin-sensitive strains FADDI-PA021 (polymyxin B MIC = 1 mg/L) and FADDI-PA020 (polymyxin B MIC = 2 mg/L). Polymyxin A2 was only effective against the polymyxin-sensitive isolates. However, under biofilm growth conditions, the hydrophobic lipopeptide FADDI-003 was inactive compared to the more hydrophilic lipopeptides, octapeptin A3, polymyxin A2, polymyxin B, and colistin. Transmission electron micrographs revealed octapeptin A3 caused reduction in the cell numbers in biofilm as well as biofilm disruption/"antibiofilm" activity. We therefore assessed the interactions of the lipopeptides with the component sputum biomolecules, mucin, deoxyribonucleic acid (DNA), surfactant, F-actin, lipopolysaccharide, and phospholipids. We observed the general trend that sputum biomolecules reduce lipopeptide antibacterial activity. Collectively, our data suggests that, in the airways, lipopeptide binding to component sputum biomolecules may reduce antibacterial efficacy and is dependent on the physicochemical properties of the lipopeptide.

Rhenium and Technetium-oxo Complexes with Thioamide Derivatives of Pyridylhydrazine Bifunctional Chelators Conjugated to the Tumour Targeting Peptides Octreotate and Cyclic-RGDfK.

This research aimed to develop new tumor targeted theranostic agents taking advantage of the similarities in coordination chemistry between technetium and rhenium. A γ-emitting radioactive isotope of technetium is commonly used in diagnostic imaging, and there are two ß- emitting radioactive isotopes of rhenium that have the potential to be of use in radiotherapy. Variants of the 6-hydrazinonicotinamide (HYNIC) bifunctional ligands have been prepared by appending thioamide functional groups to 6-hydrazinonicotinamide to form pyridylthiosemicarbazide ligands (SHYNIC). The new bidentate ligands were conjugated to the tumor targeting peptides Tyr3-octreotate and cyclic-RGD. The new ligands and conjugates were used to prepare well-defined {M═O}3+ complexes (where M = 99mTc or natRe or 188Re) that feature two targeting peptides attached to the single metal ion. These new SHYNIC ligands are capable of forming well-defined rhenium and technetium complexes and offer the possibility of using the 99mTc imaging and 188/186Re therapeutic matched pairs.