A new member of the human cystatin superfamily, called cystatin E, has been found by expressed sequence tag (EST) sequencing in amniotic cell and fetal skin epithelial cell cDNA libraries. The sequence of a fulllength amniotic cell cDNA clone contained an open reading frame encoding a putative 28-residue signal peptide and a mature protein of 121 amino acids, including four cysteine residues and motifs of importance for the inhibitory activity of Family 2 cystatins like cystatin C. Recombinant cystatin E was produced in a baculovirus expression system and isolated. An antiserum against the recombinant protein could be used for affinity purification of cystatin E from human urine, as confirmed by N-terminal sequencing. The mature recombinant protein processed by insect cells started at amino acid 4 (cystatin C numbering), and displayed reversible inhibition of papain and cathepsin B (K i values of 0.39 and 32 nM, respectively), in competition with substrate. Cystatin E is thus a functional cysteine proteinase inhibitor despite relatively low amino acid sequence similarities with human cystatins (26 -34% identity with sequences for the Family 2 cystatins C, D, S, SN, and SA; <30% with the Family 1 cystatins, A and B, and domains 2 and 3 of the Family 3 cystatin, kininogen). Unlike other human low M r cystatins, cystatin E is a glycoprotein, carrying an N-linked carbohydrate chain at position 108. Northern blot analysis revealed that the cystatin E gene is expressed in most human tissues, with the highest mRNA amounts found in uterus and liver. A strikingly high incidence of cystatin E clones in cDNA libraries from fetal skin epithelium and amniotic membrane cells (>0.5% of clones sequenced) indicates a protective role of cystatin E during fetal development.

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Human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of papain-like mammalian proteases, has been produced in its full-length form by recombinant techniques and crystallized in two polymorphic forms: cubic and tetragonal. A selenomethionyl derivative of the protein, obtained by Escherichia coli expression and with complete Met-->Se-Met substitution confirmed by mass spectrometry, amino-acid analysis and X-ray absorption spectra, was crystallized in the cubic form. A truncated variant of the protein, lacking ten N-terminal residues, has also been crystallized. The crystals of this variant are tetragonal and, like the two polymorphs of the full-length protein, contain multiple copies of the molecule in the asymmetric unit, suggesting oligomerization of the protein.

Background/Aims Prostacyclin analogues, although effective treatment for systemic sclerosis (SSc)-related Raynaud’s phenomenon (RP), have the disadvantage of requiring intravenous or subcutaneous (SC) infusion. Treprostinil subcutaneous depot (CAM2043) is a novel extended-release SC formulation of treprostinil. The aim of this trial was to explore the effect of a single SC dose of CAM2043 on skin temperature (indirect measure of perfusion), as evaluated by thermography following cold challenge. Methods This was an exploratory, open-label, single-dose Phase 2 trial. Ten female patients (mean age 54.9 years) with mean SSc duration of 11.5 years (all anticentromere antibody positive) and mean RP duration of 14.5 years, were recruited. To be included, patients had to experience ≥5 attacks of RP/week. Patients attended on 6 occasions: screening, baseline (Day 1), and on Days 2, 3, 8, and 15. At baseline, each patient received a single SC injection of 2.5 mg CAM2043 (except the first patient who received 5mg CAM2043). A standard cold challenge test of the hands (15 °C for one minute) was performed pre-dose and at 3-, 6-, 24-, 72-, 168- and 336-hours post-dose, with temperature responses over the subsequent 15 minutes measured by infrared thermography. The primary endpoint was the mean change from baseline to 6 hours post-dose in the area under the curve (AUCtherm) for rewarming (8 fingers). Secondary endpoints included: change from baseline in AUCtherm, maximum temperature after rewarming (MAX) and Raynaud’s Condition Score (RCS). Safety and tolerability were assessed throughout the trial. Results AUCtherm increased 6 hours post-dose, but not statistically significantly: mean increase 192.7 °C×sec (95% CI: -727.1, 1112.6). AUCtherm was significantly greater at 24 hours than at baseline (mean increase 1175.8 °C×sec (95% CI: 127.3, 2224.3)), with a trend towards mean AUCtherm values higher than baseline up to 168 hours post-dose (Day 8) and returning to baseline values at 336 hours (Day 15). A statistically significant increase from baseline was also seen for MAX at 24 hours: mean increase 1.4 °C (95% CI: 0.1, 2.7). At baseline, mean (SD) RCS was 3.7 (1.3) units (10-point scale), and was significantly improved post dosing, including Day 8 (mean reduction 1.6 units (95% CI: -2.68, -0.52)). This positive treatment effect persisted at Day 15 (mean reduction 1.6 units (95% CI: -3.33, 0.19)). Adverse events were reported by all 10 patents: all reported erythema and pain at the injection site. Conclusion Finger skin temperature rose after CAM2043 treatment (significantly at 24 hours post-dose), with significant improvements in RP symptoms (RCS) over the 15-day period following dosing indicating a positive treatment effect. Overall, the safety profile for CAM2043 was consistent with known safety profile for treprostinil, including injection site reactions. The results indicate that CAM2043 could be further investigated in clinical trials of RP. Disclosure A.L. Herrick: Consultancies; Camurus AB. A. Murray: None. G. Dinsdale: None. J. Manning: None. C. Chukwu: None. M. Alvarez Fernandez: Corporate appointments; Camurus AB (employee). J.R. Seibold: Corporate appointments; Medical Lead, Prometheus Biosciences. Consultancies; Camurus AB, Alexion, GlaxoSmithKline, Mitenyi. Shareholder/stock ownership; Prometheus Biosciences. Other; Advisory Committee/Boards: CSL Behring, Prometheus Biosciences, Mitenyi. F. Tiberg: Corporate appointments; President & CEO Camurus AB. Shareholder/stock ownership; Camurus AB. Other; Board Member Camurus AB and Amniotics AB.