In vivo multimodal imaging of hyaluronan-mediated inflammatory response in articular cartilage.

OBJECTIVE: One driving factor in the progression to posttraumatic osteoarthritis (PTOA) is the perpetuation of the inflammatory response to injury into chronic inflammation. Molecular imaging offers many opportunities to complement the sensitivity of current imaging modalities with molecular specificity. The goal of this study was to develop and characterize agents to image hyaluronan (HA)-mediated inflammatory signaling. DESIGN: We developed optical (Cy5.5-P15-1) and magnetic resonance contrast agents (Gd-DOTA-P15-1) based in a hyaluronan-binding peptide (P15-1) that has shown anti-inflammatory effects on human chondrocytes, and validated them in vitro and in vivo in two animal models of PTOA. RESULTS: In vitro studies with a near infrared (NIR) Cy5.5-P15-1 imaging agent showed a fast and stable localization of Cy5.5-P15-1 on chondrocytes, but not in synovial cells. In vivo NIR showed significantly higher retention of imaging agent in PTOA knees between 12 and 72 h (n = 8, Cohen's d > 2 after 24 h). NIR fluorescence accumulation correlated with histologic severity in cartilage and meniscus (ρ between 0.37 and 0.57, P < 0.001). By using in vivo magnetic resonance imaging with a Gd-DOTA-P15-1 contrast agent in 12 rats, we detected a significant decrease of T1 on injured knees in all cartilage plates at 48 h (-15%, 95%-confidence interval (CI) = [-18%,-11%]) while no change was observed in the controls (-2%, 95%-CI = [-5%,+1%]). CONCLUSIONS: This study provides the first in vivo evidence that hyaluronan-related inflammatory response in cartilage after injury is a common finding. Beyond P15-1, we have demonstrated that molecular imaging can provide a versatile technology to investigate and phenotype PTOA pathogenesis, as well as study therapeutic interventions.

The development of a near infrared inulin optical probe for measuring glomerular filtration rate.

The polysaccharide, inulin, is considered the clinical gold standard for measuring glomerular filtration rate (GFR), an assessment of kidney filtering capacity and renal function, and therefore, is a prognostic indicator of chronic kidney disease (CKD). The classic method of measuring GFR is laborious, tedious and invasive. Therefore, estimated GFR (eGFR) has become the favoured measurement, but unfortunately suffers in its accuracy. Here, we describe the development of a near infrared dye-labeled inulin, Cy7.5-inulin conjugate, for use as an optical probe to accurately and non-invasively measure GFR in patients by transcutaneous pulse dye densitometer (TPDD). We have characterized the modifications made to inulin and the dye-polysaccharide conjugate by a number of analytical techniques and demonstrated that it is stable under experimental in vivo conditions. To this end, the probe has been successfully used in a pig model to accurately measure GFR non-invasively.

Development of a [68Ga]-ghrelin analogue for PET imaging of the ghrelin receptor (GHS-R1a).

The ghrelin receptor is a member of the growth hormone secretagogue receptor (GHS-R) family and is present at low concentrations in tissues such as the brain, kidney, cardiovascular system, and prostate. The ghrelin receptor plays an important role in cellular proliferation, apoptosis, invasion, and migration associated with the progression of many cancers, including prostate, breast, ovarian, testicular, and intestinal carcinomas. Ghrelin, the endogenous ligand, is a 28 amino acid peptide (IC50 = 3.1 nM) known to have poor in vivo stability. Herein, we report the synthesis and evaluation of [Dpr3(octanoyl),Lys19(Ga-DOTA)]ghrelin(1-19). This new ghrelin analogue has a binding affinity (IC50 = 5.9 nM) comparable to that of natural ghrelin. Preliminary in vivo evaluation shows higher uptake of [Dpr3(octanoyl),Lys19(68Ga-DOTA)]ghrelin(1-19) in HT1080/GHSR-1a xenografts than the non-transfected HT1080 xenografts in NOD-SCID mice, although considerable uptake is observed in the kidneys. This is the first example of ghrelin receptor PET imaging in a xenograft model using a peptide derived directly from the endogenous ligand and serves as motivation for developing more effective ghrelin-based radiopeptides.

Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p = 0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

Molecular imaging probes derived from natural peptides.

Covering: up to the end of 2015.Peptides are naturally occurring compounds that play an important role in all living systems and are responsible for a range of essential functions. Peptide receptors have been implicated in disease states such as oncology, metabolic disorders and cardiovascular disease. Therefore, natural peptides have been exploited as diagnostic and therapeutic agents due to the unique target specificity for their endogenous receptors. This review discusses a variety of natural peptides highlighting their discovery, endogenous receptors, as well as their derivatization to create molecular imaging agents, with an emphasis on the design of radiolabelled peptides. This review also highlights methods for discovering new and novel peptides when knowledge of specific targets and endogenous ligands are not available.

Single isomer technetium-99m tamoxifen conjugates.

To produce an imaging agent for breast cancer using a technetium-99m-labeled agent specific for estrogen receptors, an N(2)S(2) bifunctional chelator was conjugated to Z- and E-aminotamoxifens through an amide linker. These bioconjugates have been chelated with both technetium-99m and rhenium. For the Z-isomer, chelation with rhenium in the presence of sodium acetate yields a mixture of two isomers, anti and syn, in a 1:1 ratio and in the presence of hydroxide results in only the anti isomer. Both the Z- and E-tamoxifen conjugates have been chelated with technetium-99m at the tracer level yielding a single isomer product, which is assigned as anti based on chromatographic comparison to the rhenium complexes. Radiochemical yields were consistently greater than 80%, with Sep-Pak column purification yielding a final product with >99% radiochemical purity and no residual starting material. Both in vitro and in vivo biological evaluation of the tamoxifen chelates indicated very limited estrogen receptor binding.

An N2S2 bifunctional chelator for technetium-99m and rhenium: complexation, conjugation, and epimerization to a single isomer.

A bifunctional chelator 6 was prepared bearing an N2S2 core for binding rhenium or technetium and a carboxylic acid group for conjugation to amino groups of biomolecules. Complexation of 6 with rhenium(V) resulted in two kinetic isomers, anti-7 and syn-7, being formed in approximately equal amounts. Epimerization with 0.5 M NaOH yields a single isomer anti-7, as determined by NMR spectroscopy and single-crystal X-ray analysis. The 99mTc complex was prepared at the tracer level by reaction of the ligand with 99mTcO4-, tin(II) chloride and sodium gluconate giving a mixture of two isomers, but showing a preference for the anti isomer. Chelation in the presence of 1 M NaOH results in anti-8 being formed as the sole product. The bifunctional ability of the ligand was explored by amide formation with (S)-alpha-phenethylamine, either by direct DCC coupling or through the N-hydroxy succinimidyl ester 9 intermediate. The deprotected bioconjugate 11 was complexed with rhenium, yielding similar amounts of two isomeric rhenium complexes, anti-12 and syn-12, which were isolated and characterized by NMR spectroscopy. Treatment of the kinetic mixture of anti-12 and syn-12 with 1 M NaOH resulted in quantitative conversion to a single rhenium complex anti-12. With technetium-99m in 0.1 M sodium acetate, bioconjugate 11 yielded both technetium-99m complexes anti-13 and syn-13, in a 2:1 ratio, respectively. In contrast, complexation in the presence of 1 M NaOH gave only one technetium-99m complex, assigned the structure anti-13.