Low Tissue Uptake Research Articles

306 Topical QR-313, an Antisense Oligonucleotide, in the Treatment of Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is caused by COL7A1 mutations encoding collagen VII (C7), comprised of 118 exons. In-frame exon 73 accounts for 9% of recessive (RDEB) and 55% of dominant (DDEB) cases. QR-313 is a 21-mer modified 2’O-methyl ribose phosphorothioate single strand antisense oligonucleotide which binds exon 73 and prevents its incorporation into mature mRNA. In vitro, QR-313 shows tissue uptake by fluorescent in situ hybridization (FISH) and generation of mRNAs lacking exon 73 by ddPCR. The shortened, exon-skipped protein effectively binds laminin-332 and collagen IV. Toxicology studies in mice and minipigs showed typical but reversible class effects in kidney and liver. We administered QR-313 1 mg/cm2 qod or vehicle for 8 weeks in a blinded fashion to 2 wounds in two patients (female, 13 years; male, 12 years) with RDEB due to mutations in exon 73 in at least one allele. There were no adverse events, no perturbations in renal or liver function or in coagulation studies. and both patients completed the study. Pharmacokinetics showed minimal systemic exposure. The wounds treated with QR-313 both showed a small amount of tissue uptake by FISH analysis and modest evidence of exon skipping in one of the patients on biopsies obtained at Week 2 or 4. Biopsies at Week 8 showed no difference between QR-313 and vehicle-treated wounds in C7 by indirect immunofluorescence staining or in anchoring fibrils by transmission electron microscopy, consistent with the limited tissue uptake of study drug. Both wounds in both patients waxed and waned in size during the study. In conclusion, QR-313 was safe and well-tolerated. Low tissue uptake at the study dose was likely responsible for the limited exon skipping observed and the lack of demonstrable C7 and anchoring fibrils on biopsy. Future studies will explore higher and/or more frequent dosing.

Clinical translation of 18F-fluoropivalate \u2013 a PET tracer for imaging short-chain fatty acid metabolism: safety, biodistribution, and dosimetry in fed and fasted healthy volunteers

BackgroundFatty acids derived de novo or taken up from the extracellular space are an essential source of nutrient for cell growth and proliferation. Radiopharmaceuticals including 11C-acetate, and 18F-FAC (2-18F-fluoroacetate), have previously been used to study short-chain fatty acid (SCFA) metabolism. We developed 18F-fluoropivalate (18F-FPIA; 3-18F-fluoro-2,2-dimethylpropionic acid) bearing a gem-dimethyl substituent to assert metabolic stability for studying SCFA metabolism. We report the safety, biodistribution, and internal radiation dosimetry profile of 18F-FPIA in 24 healthy volunteers and the effect of dietary conditions.Materials and methodsHealthy volunteer male and female subjects were enrolled (n = 24), and grouped into 12 fed and 12 fasted. Non-esterified fatty acids (NEFA) and carnitine blood measurements were assessed. Subjects received 159.48 MBq (range, 47.31–164.66 MBq) of 18F-FPIA. Radiochemical purity was > 99%. Safety data were obtained during and 24 h after radiotracer administration. Subjects underwent detailed multiple whole-body PET/CT scanning with sampling of venous bloods for radioactivity and radioactive metabolite quantification. Regions of interest were defined to derive individual and mean organ residence times; effective dose was calculated using OLINDA 1.1.ResultsAll subjects tolerated 18F-FPIA with no adverse events. Over 90% of radiotracer was present in plasma at 60 min post-injection. The organs receiving highest absorbed dose (in mGy/MBq) were the liver (0.070 ± 0.023), kidneys (0.043 ± 0.013), gallbladder wall (0.026 ± 0.003), and urinary bladder (0.021 ± 0.004); otherwise there was low tissue uptake. The calculated effective dose using mean organ residence times over all 24 subjects was 0.0154 mSv/MBq (SD ± 0.0010). No differences in biodistribution or dosimetry were seen in fed and fasted subjects, though systemic NEFA and carnitine levels reflected fasted and fed states.ConclusionThe favourable safety, imaging, and dosimetric profile makes 18F-FPIA a promising candidate radiotracer for tracing SCFA metabolism.

Accurate identification of myocardial viability after myocardial infarction with novel manganese chelate-based MR imaging.

We developed a novel manganese (Mn2+ ) chelate for magnetic resonance imaging (MRI) assessment of myocardial viability in acute and chronic myocardial infarct (MI) models, and compared it with Gadolinium-based delay enhancement MRI (Gd3+ -DEMRI) and histology. MI was induced in 14 rabbits by permanent occlusion of the left circumflex coronary artery. Gd3+ -DEMRI and Mn2+ chelate-based delayed enhancement MRI (Mn2+ chelate-DEMRI) were performed at 7days (acute MI, n=8) or 8weeks (chronic MI, n=6) after surgery with sequential injection of 0.15mmol/kg Gd3+ and Mn2+ chelate. The biodistribution of Mn2+ in tissues and blood was measured at 1.5 and 24h. Blood pressure, heart rate (HR), left ventricular (LV) function, and infarct fraction (IF) were analyzed, and IF was compared with the histology. The Mn2+ chelate group maintained a stable hemodynamic status during experiment. For acute and chronic MI, all rabbits survived without significant differences in HR or LV function before and after injection of Mn2+ chelate or Gd3+ (p>0.05). Mn2+ chelate mainly accumulated in the kidney, liver, spleen, and heart at 1.5h, with low tissue uptake and urine residue at 24h after injection. In the acute MI group, there was no significant difference in IF between Mn2+ chelate-DEMRI and histology (22.92±2.21% vs. 21.79±2.25%, respectively, p=0.87), while Gd3+ -DEMRI overestimated IF, as compared with histology (24.54±1.73%, p=0.04). In the chronic MI group, there was no significant difference in IF between the Mn2+ chelate-DEMRI, Gd3+ -DEMRI, and histology (29.50±11.39%, 29.95±9.40%, and 29.00±10.44%, respectively, p>0.05), and all three were well correlated (r=0.92-0.96, p<0.01). We conclude that the use of Mn2+ chelate-DEMRI is reliable for MI visualization and identifies acute MI more accurately than Gd3+ -DEMRI.

Water-soluble, deep-red fluorescent squaraine rotaxanes

Eight fluorescent squaraine rotaxanes with deep-red absorption/emission wavelengths were prepared and assessed for chemical stability and suitability as water-soluble, fluorescent tracers. The most stable squaraine rotaxanes have four large stopper groups attached to the ends of the encapsulated squaraine, and two members of this structural class have promise as highly fluorescent tracers with rapid renal clearance and very low tissue uptake in living mice.

Intravascular metabolism of different fatty acids during lipid infusion in man

The differential intravascular metabolism of individual fatty acids contained in triacylglycerol-rich particles was studied by infusing 6 normal subjects for 5h with a conventional soy-based emulsion and an experimental olive oil-based emulsion. Both emulsions contained similar amounts of palmitate (11%) and stearate (3–4%) but the former was quite rich in linoleate (54%) and alpha-linolenate (7%), while the latter was rich in oleate (69%). During hydrolysis of circulating triacylglycerols by endothelial lipases, the associated rise of non-esterified fatty acids (FFA) in plasma represents the balance between fatty acid release and tissue uptake. Plasma levels of triacylglycerols and FFA increased about 3 fold and total body fat oxidation was raised to similar values with both emulsions. Fatty acid pattern quickly changed in plasma triacylglycerols to resemble the composition of emulsion particles, with an exception for palmitate which increased markedly more, suggesting a high level of hepatic re-esterification and re-appearance in nascent very low density lipoprotein triglycerides (VLDL-TG) secreted into the circulation. In plasma FFA, stearate and palmitate increased more and alpha-linolenate much less than expected from their content in the emulsion, indicating probably low tissue uptake for the former ones but avid removal for the latter.

Effect of dietary Mo on Cu metabolism. Evidence for the involvement of Mo in abnormal binding of Cu to plasma proteins

The effects of feeding Mo at concentrations of 17μg/g to sheep and 100 μg/g to guinea pigs in the presence of adequate dietary Cu, on the distribution of Cu within plasma have been studied. The creation of a fraction of plasma Cu which is insoluble in trichloroacetic acid has been confirmed and it has been shown that this fraction also contains Mo. Complete removal of Cu and Mo from this protein cannot be achieved under acid conditions without at least partial hydrolysis of the protein. The estimated atomic ratio of Cu:Mo in the fraction was 1.7. It is suggested that the formation of such a stable Cu-Mo-protein compound may explain the observed low tissue uptake of Cu in the presence of high plasma total and “direct-reacting” Cu concentrations.