Dual-active hyaluronidase D016 reduces established atherosclerotic lesion area in LDLR⁻/⁻ mice

Dual-active hyaluronidase D016 reduces established atherosclerotic lesion area in LDLR⁻/⁻ mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dual-active hyaluronidase D016 reduces established atherosclerotic lesion area in LDLR⁻/⁻ mice Gunther Burgard This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9247777/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pathological accumulation of extracellular-matrix components, including hyaluronan and sulfated glycosaminoglycans (sGAGs), has been suggested to sustain advanced atherosclerotic plaques by altering tissue architecture and transport. D016 is a recombinant dual-active hyaluronidase designed to cleave both substrates, aiming to modulate matrix-associated components in the plaque microenvironment rather than lipid metabolism. Male LDLR−/− mice with established diet-induced atherosclerosis received intravenous vehicle or D016 once daily for 10 days (7,500 or 750,000 IU kg − 1), followed by a 19-day post-treatment interval. Aortic-root lesion area was quantified histologically on day 29. High-dose D016 was associated with an ~ 19% reduction in aortic-root plaque area versus vehicle (P < 0.05, one-way ANOVA with Dunnett’s test; n = 12 per group); the low dose showed no detectable effect. Protease-inhibitor co-treatment did not further reduce lesion area. This study did not quantify plaque ECM composition and therefore does not establish demonstrable ECM remodelling. An effect on lesion progression cannot be excluded under continued Western diet. Clinical condition, body weight, and hepatic and renal serum parameters were similar between groups. These results support further mechanistic evaluation of enzymatic modulation of matrix-associated pathways. Health sciences/Cardiology Health sciences/Cardiology/Cardiovascular biology Figures Figure 1 Introduction Atherosclerosis is characterised by progressive lipid accumulation, inflammatory cell infiltration and a pathologically altered extracellular matrix (ECM) within the arterial wall¹. The ECM is not merely a structural scaffold: it organises biomechanical cues and signalling landscapes that influence leukocyte recruitment, resident cell phenotype and tissue architecture, and disease-associated ECM remodelling can contribute to lesion persistence². Despite major advances in lipid lowering, reversal of established plaque burden is frequently limited, suggesting that matrix-dependent structure and function may constrain durable improvement of advanced disease. Hyaluronan (HA) is a major ECM glycosaminoglycan that forms highly hydrated networks with collagens and proteoglycans and thereby modulates tissue hydration, porosity and cell–matrix interactions³. In healthy tissues, HA is continuously synthesised and cleared; homeostasis therefore depends on a tightly regulated balance between production and enzymatic degradation⁴. When this balance is disrupted, HA-rich matrices can become compositionally and mechanically abnormal and may alter the transport of soluble mediators as well as the physical accessibility of receptor–ligand interactions in the pericellular space. Within the arterial wall, proteoglycans and their glycosaminoglycan chains are key determinants of matrix organisation and lipoprotein retention, linking ECM composition to atherogenesis and plaque composition⁵. Experimental studies further connect HA accumulation to maladaptive vascular remodelling, including increased arterial stiffness and altered vessel-wall mechanics⁶. Beyond bulk structural effects, HA processing can also shape inflammatory tone: hyaluronan fragments have been shown to function as endogenous danger signals capable of activating innate immune pathways⁷. These converging mechanisms support a model in which HA- and sulfated glycosaminoglycan-rich ECM domains are biologically active components of the plaque microenvironment rather than inert structural deposits. Clinical data are consistent with altered HA metabolism in human atherosclerotic disease. Plasma hyaluronidase activity has been reported to associate with atherosclerosis in patients with coronary artery disease⁸. In addition, plasma hyaluronan levels have been linked to plaque phenotypes in patients with ST-segment-elevation myocardial infarction⁹. Together, these observations suggest that a pathological HA/glycosaminoglycan axis is coupled to plaque characteristics and may represent a lipid-independent entry point for therapeutic investigation. Here, we investigated whether systemic enzymatic targeting of pathological HA- and sulfated chondroitin sulfate-rich ECM domains is associated with changes in established atherosclerotic lesion burden. D016 is a recombinant hyaluronidase candidate developed by Pharmact AG and reported to have activity against HA and sulfated chondroitin sulfate (Pharmact AG, internal data, 2021). Using LDLR⁻/⁻ mice maintained on a Western diet, we tested whether short-term D016 administration would be followed by a measurable reduction in aortic-root lesion area at a post-treatment endpoint. The study was designed to assess lesion area rather than to establish direct ECM remodelling, and it does not by itself distinguish unequivocal lesion regression from altered progression under continued Western-diet conditions. Results Dual-active hyaluronidase D016 reduces established aortic-root atherosclerotic lesion area in LDLR⁻/⁻ mice To determine whether transient enzymatic remodelling of hyaluronan- and sulfated glycosaminoglycan–rich extracellular matrix domains modifies established atherosclerotic burden, male LDLR⁻/⁻ mice were maintained on a Western-type diet for 12 weeks prior to intervention. Animals then received once-daily intravenous administration of vehicle or D016 for 10 consecutive days (7,500 or 750,000 U kg⁻¹ day⁻¹), followed by a 19-day post-treatment observation period. Aortic-root lesion area was quantified histologically at day 29 (Fig. 1 a, b). At necropsy, vehicle-treated animals (n = 12) exhibited a mean aortic-root lesion area of 2,514,408 ± 119,703 µm² (mean ± SEM). Low-dose D016 (7,500 U kg⁻¹ day⁻¹; n = 12) did not significantly alter lesion burden (2,502,474 ± 138,252 µm²). In contrast, high-dose D016 (750,000 U kg⁻¹ day⁻¹; n = 12) resulted in a reduction of mean lesion area to 2,042,619 ± 126,841 µm², corresponding to an 18.8% decrease relative to vehicle. Group comparisons were performed using one-way analysis of variance (ANOVA; α = 0.05, two-sided), followed by Dunnett’s multiple comparisons test versus vehicle. One-way ANOVA across vehicle, low-dose and high-dose groups showed a significant overall difference (F(2,33) = 4.38, P = 0.0205). Dunnett’s multiple comparisons test versus vehicle showed no effect at low dose (adjusted P = 0.9968) and a significant reduction at high dose (adjusted P = 0.0261). Compared with vehicle, high-dose D016 significantly reduced aortic-root lesion area (mean difference − 471,790 µm²; 95% confidence interval − 891,727 to − 51,852 µm²; adjusted P = 0.0261). Low-dose D016 did not differ from vehicle (mean difference − 11,934 µm²; 95% confidence interval − 431,872 to 408,004 µm²; adjusted P = 0.9968). To explore whether co-administration of protease-inhibitor systems altered the lesion-area response to high-dose D016, high-dose D016 was administered with three distinct protease-inhibitor systems (sys1–3; n = 4 per group). Mean lesion areas were 2,487,133 ± 360,626 µm² (sys1), 2,123,220 ± 149,032 µm² (sys2), and 2,174,085 ± 84,650 µm² (sys3). Given the small group sizes, these exploratory arms were evaluated descriptively. The point estimates did not indicate a clear or consistent reduction beyond that observed with high-dose D016 monotherapy; however, no inferential conclusions should be drawn from these underpowered comparisons. Across all treatment groups, clinical condition, body-weight trajectories, and serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, urea nitrogen and creatinine, did not differ from vehicle-treated controls. Collectively, these findings show that short-term systemic administration of D016 at 750,000 U kg⁻¹ day⁻¹ was associated with a significant reduction in aortic-root lesion area at the day-29 post-treatment endpoint in LDLR⁻/⁻ mice under continued Western-diet conditions. Figure 1 | Dual-active hyaluronidase D016 reduces established aortic-root atherosclerotic lesion area in LDLR⁻/⁻ mice. a , Quantification of aortic-root lesion area. Male LDLR⁻/⁻ mice were maintained on a cholesterol-containing Western-type diet for approximately 12 weeks and then received once-daily intravenous administration of vehicle or D016 (7,500 or 750,000 U kg⁻¹ day⁻¹) for 10 consecutive days (days 1–10). Animals were euthanised on day 29 and lesion area in the aortic root was quantified (µm²) on histological sections (H&E) by image analysis. Data are mean ± SEM with individual animals shown (n = 12 per group). One-way ANOVA followed by Dunnett’s multiple comparisons test versus vehicle (two-sided, α = 0.05). High-dose D016 significantly reduced lesion area compared with vehicle (adjusted P = 0.0261); the low-dose group did not differ from vehicle (adjusted P = 0.9968). b , Representative H&E-stained cross-sections of the aortic root from each treatment group at day 29. Scale bars, 200 µm. Discussion Short-term systemic administration of the dual-active hyaluronidase D016 reduced aortic-root lesion area in male LDLR⁻/⁻ mice with established diet-induced atherosclerosis. In animals maintained on a Western-type diet, a 10-day intravenous dosing period followed by 19 days without treatment yielded a significant reduction in lesion area at the high dose (750,000 U kg⁻¹ day⁻¹), whereas the low dose (7,500 U kg⁻¹ day⁻¹) produced no detectable change. The persistence of the effect beyond the dosing window indicates that the intervention altered lesion dynamics over time rather than exerting a purely acute pharmacological effect. We cannot exclude that the observed reduction reflects altered lesion progression rather than unequivocal structural regression. The present design does not include serial in vivo measurements or matched baseline histology within the same animals, and therefore the term “regression” must be interpreted with caution. The working hypothesis underlying this study is that enzymatic targeting of hyaluronan- and sulfated glycosaminoglycan–rich domains may modify the biophysical and signalling milieu of the plaque microenvironment. However, we did not directly quantify plaque hyaluronan content, sulfated glycosaminoglycan abundance, or extracellular matrix architecture in this dataset. Accordingly, the data do not demonstrate extracellular matrix remodelling per se; rather, they are consistent with the possibility that enzymatic modulation of matrix-associated components may have contributed to the observed change in lesion burden. This distinction is important. Without compositional or structural matrix readouts, any mechanistic attribution remains inferential. Direct assessment of hyaluronan distribution (for example by HABP staining), sulfated glycosaminoglycan content, collagen organisation, or inflammatory cell composition would be required to establish whether matrix structure was measurably altered and whether such alterations preceded or accompanied changes in plaque area. The clear dose separation suggests a threshold requirement for effective intralesional exposure or substrate engagement. Yet the relationship between administered enzyme dose and plaque-level biochemical activity is not directly characterised here. We did not measure circulating hyaluronan species, intraplaque enzyme activity, or pharmacodynamic biomarkers that could bridge systemic dosing to local target engagement. This gap limits mechanistic resolution. Future studies incorporating plasma hyaluronan kinetics, tissue-level substrate mapping, and spatially resolved plaque phenotyping would substantially strengthen causal interpretation. Co-administration of D016 with protease-inhibitor systems did not further reduce lesion area beyond high-dose monotherapy. Given the small group sizes (n = 4 per inhibitor arm), these comparisons were exploratory and underpowered to detect modest additive effects. The point estimates do not indicate a clear enhancement, but absence of statistical significance in this context should not be overinterpreted. No differences were observed in body weight trajectories or routine hepatic and renal serum parameters between vehicle and D016-treated groups. These findings provide preliminary reassurance for short-term dosing; they do not address longer-term safety, immunogenicity, or cumulative effects of repeated administration. Importantly, reduction in plaque area alone does not inform on plaque stability. Fibrous cap thickness, necrotic core size, and inflammatory burden were not assessed, and it remains possible that structural remodelling - if present - could have heterogeneous effects on plaque vulnerability. Clinical reports linking circulating hyaluronidase or hyaluronan measures to coronary phenotypes provide contextual support for a pathological HA axis in vascular disease¹⁰. Small, uncontrolled case observations of intravenous hyaluronidase use in advanced vascular disease have been published 11 , 12 , 13 ; however, these reports are anecdotal and cannot establish causality. They should be regarded as hypothesis-generating rather than confirmatory. Taken together, the present findings show that short-term administration of a dual-active hyaluronidase was associated with a measurable reduction in aortic-root lesion area in LDLR⁻/⁻ mice under continued Western diet conditions. Whether this effect reflects altered extracellular matrix organisation, modified lipoprotein retention, shifts in inflammatory signalling, or a combination of these processes cannot be determined from the current dataset. The results therefore suggest that enzymatic modulation of matrix-associated components warrants further mechanistic evaluation rather than providing definitive evidence of extracellular matrix remodelling. Methods Animals and study design Male low-density lipoprotein receptor–deficient mice (LDLR⁻/⁻; JAX 002207) were used. Animals were received at 9–11 weeks of age and maintained on a cholesterol-containing Western-type diet (Research Diets D12079B) ad libitum for an ~ 12-week pretreatment period; the same diet was continued throughout the dosing phase and the subsequent post-treatment observation period. Animals were single-housed in polycarbonate cages with appropriate bedding and provided environmental enrichment (nesting material) except during designated procedures. Health status was monitored at least twice daily, with additional cage-side observations as warranted. Body weight was recorded three times per week throughout the in-life phase. Animals were allocated to treatment groups based on body weight and pre-dose serum lipid measurements (total cholesterol and triglycerides) to generate groups with no significant differences in these parameters. Allocation was performed before dosing using objective baseline measures (body weight and serum lipids) to reduce the risk of baseline imbalance. Treatment initiation was staggered across cohorts to ensure balanced representation of groups across dosing days. Ethics approval and regulatory compliance All animal procedures were approved by the Institutional Animal Care and Use Committee of Charles River Laboratories Massachusetts (CRMA IACUC; Study No. 20238304) and conducted in accordance with Directive 2010/63/EU of the European Parliament, the U.S. Animal Welfare Act (9 CFR), the Public Health Service Policy, and the NIH-Guide for the Care and Use of Laboratory Animals (8th edition). Humane endpoints were predefined. No unexpected mortality occurred. The study complied with ARRIVE guidelines. Test article, formulations and dosing D016 (test article designation at the testing facility: Hyphilase Groningen / HyPGSP) was supplied by the sponsor. We characterised D016, a recombinant dual‑acting hyaluronidase produced in Escherichia coli . The enzyme merges PH20‑like β1→4 exolytic activity with HYAL4‑like β1→3 endolytic activity, enabling cleavage of sulfated glycosaminoglycan-rich chondroitin sulfate A/C meshes. D016 exhibits a specific activity of 1.3 × 10⁶ IU mg⁻¹--approximately 2 000‑fold higher than bovine testicular hyaluronidase. Vehicle consisted of Tris–HCl/NaCl buffer (pH 7.4). Dose formulations were prepared according to protocol specifications and stored at 4°C until use; dosing solutions were allowed to equilibrate to room temperature before administration. Mice received once-daily intravenous administration of vehicle or D016 for 10 consecutive days. D016 was administered at 7,500 U kg⁻¹ day⁻¹ (low dose) or 750,000 U kg⁻¹ day⁻¹ (high dose). The dose volume was 5.0 mL kg⁻¹. In exploratory arms, high-dose D016 was co-administered with one of three protease-inhibitor systems (sys1–sys3; n = 4 per system arm), prepared as specified in the study protocol. Blood sampling and serum biochemistry For group allocation, blood was collected pre-dose to measure serum total cholesterol and triglycerides. At study termination, terminal blood was collected by intracardiac puncture into serum separator tubes, processed to serum by centrifugation (2,200 g, 10 min, room temperature), and stored at − 20°C until analysis. Clinical chemistry parameters were measured on terminal serum and included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, total protein, albumin, calculated globulin and albumin/globulin ratio, glucose, sodium, potassium and chloride. Necropsy, tissue collection and lesion quantification Animals were euthanised by carbon dioxide asphyxiation. For vascular tissue collection, the thoracic cavity was opened and animals were perfused with phosphate-buffered saline, followed by 10% neutral buffered formalin. The aorta (to the bifurcation) was collected as a single specimen in 10% neutral buffered formalin, taking care not to disrupt luminal lesions. The apex of the heart was removed; the remaining heart with the attached ascending aorta segment was embedded in OCT and frozen for aortic-root sectioning. Aortic-root lesion area was quantified histologically on haematoxylin and eosin (H&E)-stained cryosections. Serial 10-µm sections were collected through the aortic sinus; images were acquired at predefined step levels spanning the sinus, and total lesion area was quantified using computer-assisted image analysis. Histological lesion quantification was performed by personnel blinded to treatment allocation. The primary endpoint was total aortic-root lesion area at day 29 (post-treatment time point) in vehicle, low-dose and high-dose groups (n = 12 per group). Exploratory sys1–sys3 groups were quantified similarly but were not used as primary inferential comparisons because of limited group size. Statistical analysis Statistical tests were two-sided with a prespecified α = 0.05. For comparisons of aortic-root lesion area across the primary groups (vehicle vs low-dose vs high-dose), a one-way analysis of variance (ANOVA) was performed, followed by Dunnett’s multiple comparisons test comparing each active dose to vehicle. Data are presented as mean ± SEM with individual animals shown. Exploratory protease-inhibitor arms were evaluated descriptively and are reported without over-interpretation given the small sample size. Data availability The data that support the findings of this study are available in the Zenodo repository at https://doi.org/10.5281/zenodo.16785010 . The repository includes raw aortic-root histology images, region-of-interest (ROI) masks, per-animal plaque-area measurements (CSV files), statistical analysis scripts reproducing the one-way ANOVA with Dunnett’s multiple comparisons test (CRL Study No. 20238304), and de-identified clinical summary tables. Source data are provided with this paper. Code availability The analysis scripts used to reproduce the statistical analyses reported in this study (one-way ANOVA with Dunnett’s multiple comparisons test; CRL Study No. 20238304) are available in the Zenodo repository at https://doi.org/10.5281/zenodo.16785010 . No custom code beyond the scripts deposited in the repository was used. Declarations Acknowledgements This work was funded by Pharmact AG, which sponsored the preclinical mouse study. The in vivo study was performed at Charles River Laboratories (CRL Study No. 20238304), and histological lesion quantification was performed by Vascular Strategies LLC. G.M.B. performed the formal data analysis, interpreted the results, prepared the figures, wrote the manuscript and decided to submit it for publication. The author thanks Charles River Laboratories and Vascular Strategies (Fort Washington, PA, USA) for independent conduct of the in vivo study and histological analyses. Author contributions G.M.B. conceived the study, developed the therapeutic concept, designed the experimental framework, oversaw study execution, performed the formal data analysis, interpreted the results, prepared the figures, and wrote and revised the manuscript. Competing interests G.M.B. is the founder of the Pharmact group and serves as Chairman of the Board and Chief Medical Officer of Pharmact Holding AG (Switzerland), the parent company of Pharmact AG, which sponsored the preclinical mouse study reported in this manuscript. G.M.B. holds equity in Pharmact Holding AG and is an inventor on issued and pending patents covering the therapeutic use of hyaluronidase for extracellular-matrix-related diseases, including D016. As the sole author and a sponsor-affiliated investigator, G.M.B. performed the formal data analysis, interpreted the data, prepared the figures, wrote the manuscript and decided to submit it for publication. No other competing interests are declared. References Hynes, R. O. The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219 (2009). Bonnans, C., Chou, J. & Werb, Z. Remodelling the extracellular matrix in development and disease. Nat. Rev. Mol. Cell Biol. 15, 786–801 (2014). Lierova, A. et al. Hyaluronic acid: known for almost a century, but still in vogue. Pharmaceutics 14, 838 (2022). Laurent, U. B. G. & Reed, R. K. Turnover of hyaluronan in the tissues. Adv. Drug Deliv. Rev. 7, 237–256 (1991). Wight, T. N. Cell biology of arterial proteoglycans. Arterioscler. Thromb. 12, 114–128 (1992). Lorentzen, K. A. et al. Mechanisms involved in extracellular matrix remodeling and arterial stiffness induced by hyaluronan accumulation. Atherosclerosis 244, 195–203 (2016). Scheibner, K. A. et al. Hyaluronan fragments act as an endogenous danger signal by engaging TLR2. J. Immunol. 177, 1272–1281 (2006). Karadağ, B. et al. Association of plasma hyaluronidase activity with atherosclerosis in patients with coronary artery disease. Atherosclerosis Suppl. 9, 68–69 (2008). Li, J. et al. The association between plasma hyaluronan level and plaque types in ST-segment-elevation myocardial infarction patients. Front. Cardiovasc. Med. 8, 628529 (2021). Afify, A. M., Stern, M., Guntenhöner, M. W. & Yamaguchi, Y. Purification and characterization of human plasma hyaluronidase. Biochem. Biophys. Res. Commun. 261, 340–345 (1999). Burgard, G. M. & Pfützner, A. Intravenöse Hyaluronidase-Infusionstherapie bei austherapierter finaler Arteriosklerose – Fallbericht. Diabetes Stoffwechsel Herz 24, 171–174 (2015). Pfützner, A., Sachsenheimer, D. & Burgard, G. Possible role of intravenous hyaluronidase treatment in coronary lesion and hypertension. J. Case Rep. 11, 160–163 (2021). Pfützner, A., Segiet, T., Hanna, M., Kalasauske, D. & Sachsenheimer, D. Treatment of diabetic foot syndrome by means of hyaluronidase infusion therapy – single patient case report. Med. Clin. Case Rep. 3, 1–4 (2023). Additional Declarations Yes there is potential Competing Interest. G.M.B. is the founder of the Pharmact group and serves as Chairman of the Board and Chief Medical Officer of Pharmact Holding AG (Switzerland), the parent company of Pharmact AG, which sponsored the preclinical mouse study reported in this manuscript. G.M.B. holds equity in Pharmact Holding AG and is an inventor on issued and pending patents covering the therapeutic use of hyaluronidase for extracellular-matrix-related diseases, including D016. As the sole author and a sponsor-affiliated investigator, G.M.B. performed the formal data analysis, interpreted the data, prepared the figures, wrote the manuscript and decided to submit it for publication. No other competing interests are declared. Supplementary Files NaturePortfolioReportingSummaryLifeSciences.docx Reporting Summary Figure1rawdataaorticrootlesionarea.csv Dataset 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9247777","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":616870778,"identity":"1d3898ae-da8c-4362-9e0a-5303d4988080","order_by":0,"name":"Gunther Burgard","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDACCQbGB4wNUA6IwcfAQ1ALswFUC2MDiMHGRlgLmwRpWuSje8yqC3fY5eu2H37+gHGHjTybfO8Bhh8V23BqMbxzxuz2zDPJltvOpBk2MAKJNja+BMaeM7dxa5mRY3abt43ZwOxADmPz37bDjG1sPAbMjG34tRTzttUbmJ1/A/RL2397glrkJXLMmHnbDhuY3cgBaTmQSFCLgcyxYumZbceBWp4ZzmBsS05uY8sxOIjPL/Kzmzd+LmyrBjos+cEHxjY7237mM4YPflTgseUAAwMzhugBnOpBtjRg0zIKRsEoGAWjABkAAOR/VB6C2Uy3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0002-0451-8002","institution":"Pharmact Holding AG","correspondingAuthor":true,"prefix":"","firstName":"Gunther","middleName":"","lastName":"Burgard","suffix":""}],"badges":[],"createdAt":"2026-03-27 19:05:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9247777/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9247777/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106094680,"identity":"c7681111-d778-49db-80d7-1a2a93a96ae1","added_by":"auto","created_at":"2026-04-03 11:43:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11532082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual-active hyaluronidase D016 reduces established aortic-root atherosclerotic lesion area in LDLR⁻/⁻ mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea,\u003c/strong\u003e Quantification of aortic-root lesion area. Male LDLR⁻/⁻ mice were maintained on a cholesterol-containing Western-type diet for approximately 12 weeks and then received once-daily intravenous administration of vehicle or D016 (7,500 or 750,000 U kg⁻¹ day⁻¹) for 10 consecutive days (days 1–10). Animals were euthanised on day 29 and lesion area in the aortic root was quantified (µm²) on histological sections (H\u0026amp;E) by image analysis. Data are mean ±SEM with individual animals shown (n = 12 per group). One-way ANOVA followed by Dunnett’s multiple comparisons test versus vehicle (two-sided, α = 0.05). High-dose D016 significantly reduced lesion area compared with vehicle (adjusted P = 0.0261); the low-dose group did not differ from vehicle (adjusted P = 0.9968).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb,\u003c/strong\u003e Representative H\u0026amp;E-stained cross-sections of the aortic root from each treatment group at day 29. Scale bars, 200 µm.\u003c/p\u003e","description":"","filename":"Figure1NatCom180mm600dpi1.png","url":"https://assets-eu.researchsquare.com/files/rs-9247777/v1/7644a3b1b153e02cdc6e3267.png"},{"id":106724586,"identity":"e5bd9611-2a8f-4627-a2ed-cad7acd4dc40","added_by":"auto","created_at":"2026-04-12 18:28:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9528585,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9247777/v1/1e4dd086-d31a-4c19-ad22-cb4592e25164.pdf"},{"id":106074214,"identity":"a10f9964-ef27-47f3-9f40-d0bbe6727270","added_by":"auto","created_at":"2026-04-03 07:11:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19834,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"NaturePortfolioReportingSummaryLifeSciences.docx","url":"https://assets-eu.researchsquare.com/files/rs-9247777/v1/d7963c876735e3172a43311a.docx"},{"id":106074216,"identity":"1fb66af6-11a6-47ad-a050-62c10273c332","added_by":"auto","created_at":"2026-04-03 07:11:59","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":331,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"Figure1rawdataaorticrootlesionarea.csv","url":"https://assets-eu.researchsquare.com/files/rs-9247777/v1/5df23cd56a913a6a1f5f07c9.csv"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nG.M.B. is the founder of the Pharmact group and serves as Chairman of the Board and Chief Medical Officer of Pharmact Holding AG (Switzerland), the parent company of Pharmact AG, which sponsored the preclinical mouse study reported in this manuscript. G.M.B. holds equity in Pharmact Holding AG and is an inventor on issued and pending patents covering the therapeutic use of hyaluronidase for extracellular-matrix-related diseases, including D016. As the sole author and a sponsor-affiliated investigator, G.M.B. performed the formal data analysis, interpreted the data, prepared the figures, wrote the manuscript and decided to submit it for publication. No other competing interests are declared.","formattedTitle":"Dual-active hyaluronidase D016 reduces established atherosclerotic lesion area in LDLR⁻/⁻ mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerosis is characterised by progressive lipid accumulation, inflammatory cell infiltration and a pathologically altered extracellular matrix (ECM) within the arterial wall\u0026sup1;. The ECM is not merely a structural scaffold: it organises biomechanical cues and signalling landscapes that influence leukocyte recruitment, resident cell phenotype and tissue architecture, and disease-associated ECM remodelling can contribute to lesion persistence\u0026sup2;. Despite major advances in lipid lowering, reversal of established plaque burden is frequently limited, suggesting that matrix-dependent structure and function may constrain durable improvement of advanced disease.\u003c/p\u003e \u003cp\u003eHyaluronan (HA) is a major ECM glycosaminoglycan that forms highly hydrated networks with collagens and proteoglycans and thereby modulates tissue hydration, porosity and cell\u0026ndash;matrix interactions\u0026sup3;. In healthy tissues, HA is continuously synthesised and cleared; homeostasis therefore depends on a tightly regulated balance between production and enzymatic degradation⁴. When this balance is disrupted, HA-rich matrices can become compositionally and mechanically abnormal and may alter the transport of soluble mediators as well as the physical accessibility of receptor\u0026ndash;ligand interactions in the pericellular space.\u003c/p\u003e \u003cp\u003eWithin the arterial wall, proteoglycans and their glycosaminoglycan chains are key determinants of matrix organisation and lipoprotein retention, linking ECM composition to atherogenesis and plaque composition⁵. Experimental studies further connect HA accumulation to maladaptive vascular remodelling, including increased arterial stiffness and altered vessel-wall mechanics⁶. Beyond bulk structural effects, HA processing can also shape inflammatory tone: hyaluronan fragments have been shown to function as endogenous danger signals capable of activating innate immune pathways⁷. These converging mechanisms support a model in which HA- and sulfated glycosaminoglycan-rich ECM domains are biologically active components of the plaque microenvironment rather than inert structural deposits.\u003c/p\u003e \u003cp\u003eClinical data are consistent with altered HA metabolism in human atherosclerotic disease. Plasma hyaluronidase activity has been reported to associate with atherosclerosis in patients with coronary artery disease⁸. In addition, plasma hyaluronan levels have been linked to plaque phenotypes in patients with ST-segment-elevation myocardial infarction⁹. Together, these observations suggest that a pathological HA/glycosaminoglycan axis is coupled to plaque characteristics and may represent a lipid-independent entry point for therapeutic investigation.\u003c/p\u003e \u003cp\u003eHere, we investigated whether systemic enzymatic targeting of pathological HA- and sulfated chondroitin sulfate-rich ECM domains is associated with changes in established atherosclerotic lesion burden. D016 is a recombinant hyaluronidase candidate developed by Pharmact AG and reported to have activity against HA and sulfated chondroitin sulfate (Pharmact AG, internal data, 2021). Using LDLR⁻/⁻ mice maintained on a Western diet, we tested whether short-term D016 administration would be followed by a measurable reduction in aortic-root lesion area at a post-treatment endpoint. The study was designed to assess lesion area rather than to establish direct ECM remodelling, and it does not by itself distinguish unequivocal lesion regression from altered progression under continued Western-diet conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDual-active hyaluronidase D016 reduces established aortic-root atherosclerotic lesion area in LDLR⁻/⁻ mice\u003c/p\u003e \u003cp\u003eTo determine whether transient enzymatic remodelling of hyaluronan- and sulfated glycosaminoglycan\u0026ndash;rich extracellular matrix domains modifies established atherosclerotic burden, male LDLR⁻/⁻ mice were maintained on a Western-type diet for 12 weeks prior to intervention. Animals then received once-daily intravenous administration of vehicle or D016 for 10 consecutive days (7,500 or 750,000 U kg⁻\u0026sup1; day⁻\u0026sup1;), followed by a 19-day post-treatment observation period. Aortic-root lesion area was quantified histologically at day 29 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt necropsy, vehicle-treated animals (n\u0026thinsp;=\u0026thinsp;12) exhibited a mean aortic-root lesion area of 2,514,408\u0026thinsp;\u0026plusmn;\u0026thinsp;119,703 \u0026micro;m\u0026sup2; (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). Low-dose D016 (7,500 U kg⁻\u0026sup1; day⁻\u0026sup1;; n\u0026thinsp;=\u0026thinsp;12) did not significantly alter lesion burden (2,502,474\u0026thinsp;\u0026plusmn;\u0026thinsp;138,252 \u0026micro;m\u0026sup2;). In contrast, high-dose D016 (750,000 U kg⁻\u0026sup1; day⁻\u0026sup1;; n\u0026thinsp;=\u0026thinsp;12) resulted in a reduction of mean lesion area to 2,042,619\u0026thinsp;\u0026plusmn;\u0026thinsp;126,841 \u0026micro;m\u0026sup2;, corresponding to an 18.8% decrease relative to vehicle.\u003c/p\u003e \u003cp\u003eGroup comparisons were performed using one-way analysis of variance (ANOVA; α\u0026thinsp;=\u0026thinsp;0.05, two-sided), followed by Dunnett\u0026rsquo;s multiple comparisons test versus vehicle. One-way ANOVA across vehicle, low-dose and high-dose groups showed a significant overall difference (F(2,33)\u0026thinsp;=\u0026thinsp;4.38, P\u0026thinsp;=\u0026thinsp;0.0205). Dunnett\u0026rsquo;s multiple comparisons test versus vehicle showed no effect at low dose (adjusted P\u0026thinsp;=\u0026thinsp;0.9968) and a significant reduction at high dose (adjusted P\u0026thinsp;=\u0026thinsp;0.0261). Compared with vehicle, high-dose D016 significantly reduced aortic-root lesion area (mean difference\u0026thinsp;\u0026minus;\u0026thinsp;471,790 \u0026micro;m\u0026sup2;; 95% confidence interval\u0026thinsp;\u0026minus;\u0026thinsp;891,727 to \u0026minus;\u0026thinsp;51,852 \u0026micro;m\u0026sup2;; adjusted P\u0026thinsp;=\u0026thinsp;0.0261). Low-dose D016 did not differ from vehicle (mean difference\u0026thinsp;\u0026minus;\u0026thinsp;11,934 \u0026micro;m\u0026sup2;; 95% confidence interval\u0026thinsp;\u0026minus;\u0026thinsp;431,872 to 408,004 \u0026micro;m\u0026sup2;; adjusted P\u0026thinsp;=\u0026thinsp;0.9968).\u003c/p\u003e \u003cp\u003eTo explore whether co-administration of protease-inhibitor systems altered the lesion-area response to high-dose D016, high-dose D016 was administered with three distinct protease-inhibitor systems (sys1\u0026ndash;3; n\u0026thinsp;=\u0026thinsp;4 per group). Mean lesion areas were 2,487,133\u0026thinsp;\u0026plusmn;\u0026thinsp;360,626 \u0026micro;m\u0026sup2; (sys1), 2,123,220\u0026thinsp;\u0026plusmn;\u0026thinsp;149,032 \u0026micro;m\u0026sup2; (sys2), and 2,174,085\u0026thinsp;\u0026plusmn;\u0026thinsp;84,650 \u0026micro;m\u0026sup2; (sys3). Given the small group sizes, these exploratory arms were evaluated descriptively. The point estimates did not indicate a clear or consistent reduction beyond that observed with high-dose D016 monotherapy; however, no inferential conclusions should be drawn from these underpowered comparisons.\u003c/p\u003e \u003cp\u003eAcross all treatment groups, clinical condition, body-weight trajectories, and serum biochemical parameters, including alanine aminotransferase, aspartate aminotransferase, urea nitrogen and creatinine, did not differ from vehicle-treated controls.\u003c/p\u003e \u003cp\u003eCollectively, these findings show that short-term systemic administration of D016 at 750,000 U kg⁻\u0026sup1; day⁻\u0026sup1; was associated with a significant reduction in aortic-root lesion area at the day-29 post-treatment endpoint in LDLR⁻/⁻ mice under continued Western-diet conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003e| Dual-active hyaluronidase D016 reduces established aortic-root atherosclerotic lesion area in LDLR⁻/⁻ mice.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ea\u003c/b\u003e, Quantification of aortic-root lesion area. Male LDLR⁻/⁻ mice were maintained on a cholesterol-containing Western-type diet for approximately 12 weeks and then received once-daily intravenous administration of vehicle or D016 (7,500 or 750,000 U kg⁻\u0026sup1; day⁻\u0026sup1;) for 10 consecutive days (days 1\u0026ndash;10). Animals were euthanised on day 29 and lesion area in the aortic root was quantified (\u0026micro;m\u0026sup2;) on histological sections (H\u0026amp;E) by image analysis. Data are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM with individual animals shown (n\u0026thinsp;=\u0026thinsp;12 per group). One-way ANOVA followed by Dunnett\u0026rsquo;s multiple comparisons test versus vehicle (two-sided, α\u0026thinsp;=\u0026thinsp;0.05). High-dose D016 significantly reduced lesion area compared with vehicle (adjusted P\u0026thinsp;=\u0026thinsp;0.0261); the low-dose group did not differ from vehicle (adjusted P\u0026thinsp;=\u0026thinsp;0.9968).\u003c/p\u003e \u003cp\u003e \u003cb\u003eb\u003c/b\u003e, Representative H\u0026amp;E-stained cross-sections of the aortic root from each treatment group at day 29. Scale bars, 200 \u0026micro;m.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eShort-term systemic administration of the dual-active hyaluronidase D016 reduced aortic-root lesion area in male LDLR⁻/⁻ mice with established diet-induced atherosclerosis. In animals maintained on a Western-type diet, a 10-day intravenous dosing period followed by 19 days without treatment yielded a significant reduction in lesion area at the high dose (750,000 U kg⁻\u0026sup1; day⁻\u0026sup1;), whereas the low dose (7,500 U kg⁻\u0026sup1; day⁻\u0026sup1;) produced no detectable change. The persistence of the effect beyond the dosing window indicates that the intervention altered lesion dynamics over time rather than exerting a purely acute pharmacological effect.\u003c/p\u003e \u003cp\u003eWe cannot exclude that the observed reduction reflects altered lesion progression rather than unequivocal structural regression. The present design does not include serial in vivo measurements or matched baseline histology within the same animals, and therefore the term \u0026ldquo;regression\u0026rdquo; must be interpreted with caution.\u003c/p\u003e \u003cp\u003eThe working hypothesis underlying this study is that enzymatic targeting of hyaluronan- and sulfated glycosaminoglycan\u0026ndash;rich domains may modify the biophysical and signalling milieu of the plaque microenvironment. However, we did not directly quantify plaque hyaluronan content, sulfated glycosaminoglycan abundance, or extracellular matrix architecture in this dataset. Accordingly, the data do not demonstrate extracellular matrix remodelling per se; rather, they are consistent with the possibility that enzymatic modulation of matrix-associated components may have contributed to the observed change in lesion burden.\u003c/p\u003e \u003cp\u003eThis distinction is important. Without compositional or structural matrix readouts, any mechanistic attribution remains inferential. Direct assessment of hyaluronan distribution (for example by HABP staining), sulfated glycosaminoglycan content, collagen organisation, or inflammatory cell composition would be required to establish whether matrix structure was measurably altered and whether such alterations preceded or accompanied changes in plaque area.\u003c/p\u003e \u003cp\u003eThe clear dose separation suggests a threshold requirement for effective intralesional exposure or substrate engagement. Yet the relationship between administered enzyme dose and plaque-level biochemical activity is not directly characterised here. We did not measure circulating hyaluronan species, intraplaque enzyme activity, or pharmacodynamic biomarkers that could bridge systemic dosing to local target engagement. This gap limits mechanistic resolution. Future studies incorporating plasma hyaluronan kinetics, tissue-level substrate mapping, and spatially resolved plaque phenotyping would substantially strengthen causal interpretation.\u003c/p\u003e \u003cp\u003eCo-administration of D016 with protease-inhibitor systems did not further reduce lesion area beyond high-dose monotherapy. Given the small group sizes (n\u0026thinsp;=\u0026thinsp;4 per inhibitor arm), these comparisons were exploratory and underpowered to detect modest additive effects. The point estimates do not indicate a clear enhancement, but absence of statistical significance in this context should not be overinterpreted.\u003c/p\u003e \u003cp\u003eNo differences were observed in body weight trajectories or routine hepatic and renal serum parameters between vehicle and D016-treated groups. These findings provide preliminary reassurance for short-term dosing; they do not address longer-term safety, immunogenicity, or cumulative effects of repeated administration. Importantly, reduction in plaque area alone does not inform on plaque stability. Fibrous cap thickness, necrotic core size, and inflammatory burden were not assessed, and it remains possible that structural remodelling - if present - could have heterogeneous effects on plaque vulnerability.\u003c/p\u003e \u003cp\u003eClinical reports linking circulating hyaluronidase or hyaluronan measures to coronary phenotypes provide contextual support for a pathological HA axis in vascular disease\u0026sup1;⁰. Small, uncontrolled case observations of intravenous hyaluronidase use in advanced vascular disease have been published\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e; however, these reports are anecdotal and cannot establish causality. They should be regarded as hypothesis-generating rather than confirmatory.\u003c/p\u003e \u003cp\u003eTaken together, the present findings show that short-term administration of a dual-active hyaluronidase was associated with a measurable reduction in aortic-root lesion area in LDLR⁻/⁻ mice under continued Western diet conditions. Whether this effect reflects altered extracellular matrix organisation, modified lipoprotein retention, shifts in inflammatory signalling, or a combination of these processes cannot be determined from the current dataset. The results therefore suggest that enzymatic modulation of matrix-associated components warrants further mechanistic evaluation rather than providing definitive evidence of extracellular matrix remodelling.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and study design\u003c/h2\u003e \u003cp\u003eMale low-density lipoprotein receptor\u0026ndash;deficient mice (LDLR⁻/⁻; JAX 002207) were used. Animals were received at 9\u0026ndash;11 weeks of age and maintained on a cholesterol-containing Western-type diet (Research Diets D12079B) ad libitum for an ~\u0026thinsp;12-week pretreatment period; the same diet was continued throughout the dosing phase and the subsequent post-treatment observation period. Animals were single-housed in polycarbonate cages with appropriate bedding and provided environmental enrichment (nesting material) except during designated procedures. Health status was monitored at least twice daily, with additional cage-side observations as warranted. Body weight was recorded three times per week throughout the in-life phase.\u003c/p\u003e \u003cp\u003eAnimals were allocated to treatment groups based on body weight and pre-dose serum lipid measurements (total cholesterol and triglycerides) to generate groups with no significant differences in these parameters. Allocation was performed before dosing using objective baseline measures (body weight and serum lipids) to reduce the risk of baseline imbalance. Treatment initiation was staggered across cohorts to ensure balanced representation of groups across dosing days.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthics approval and regulatory compliance\u003c/h3\u003e\n\u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee of Charles River Laboratories Massachusetts (CRMA IACUC; Study No. 20238304) and conducted in accordance with Directive 2010/63/EU of the European Parliament, the U.S. Animal Welfare Act (9 CFR), the Public Health Service Policy, and the NIH-Guide for the Care and Use of Laboratory Animals (8th edition). Humane endpoints were predefined. No unexpected mortality occurred. The study complied with ARRIVE guidelines.\u003c/p\u003e\n\u003ch3\u003eTest article, formulations and dosing\u003c/h3\u003e\n\u003cp\u003eD016 (test article designation at the testing facility: Hyphilase Groningen / HyPGSP) was supplied by the sponsor. We characterised D016, a recombinant dual‑acting hyaluronidase produced in \u003cem\u003eEscherichia coli\u003c/em\u003e. The enzyme merges PH20‑like β1\u0026rarr;4 exolytic activity with HYAL4‑like β1\u0026rarr;3 endolytic activity, enabling cleavage of sulfated glycosaminoglycan-rich chondroitin sulfate A/C meshes. D016 exhibits a specific activity of 1.3 \u0026times; 10⁶ IU mg⁻\u0026sup1;--approximately 2 000‑fold higher than bovine testicular hyaluronidase.\u003c/p\u003e \u003cp\u003eVehicle consisted of Tris\u0026ndash;HCl/NaCl buffer (pH 7.4). Dose formulations were prepared according to protocol specifications and stored at 4\u0026deg;C until use; dosing solutions were allowed to equilibrate to room temperature before administration.\u003c/p\u003e \u003cp\u003eMice received once-daily intravenous administration of vehicle or D016 for 10 consecutive days. D016 was administered at 7,500 U kg⁻\u0026sup1; day⁻\u0026sup1; (low dose) or 750,000 U kg⁻\u0026sup1; day⁻\u0026sup1; (high dose). The dose volume was 5.0 mL kg⁻\u0026sup1;. In exploratory arms, high-dose D016 was co-administered with one of three protease-inhibitor systems (sys1\u0026ndash;sys3; n\u0026thinsp;=\u0026thinsp;4 per system arm), prepared as specified in the study protocol.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBlood sampling and serum biochemistry\u003c/h2\u003e \u003cp\u003eFor group allocation, blood was collected pre-dose to measure serum total cholesterol and triglycerides. At study termination, terminal blood was collected by intracardiac puncture into serum separator tubes, processed to serum by centrifugation (2,200 g, 10 min, room temperature), and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until analysis. Clinical chemistry parameters were measured on terminal serum and included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, total bilirubin, urea nitrogen, creatinine, calcium, phosphorus, total protein, albumin, calculated globulin and albumin/globulin ratio, glucose, sodium, potassium and chloride.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNecropsy, tissue collection and lesion quantification\u003c/h3\u003e\n\u003cp\u003eAnimals were euthanised by carbon dioxide asphyxiation. For vascular tissue collection, the thoracic cavity was opened and animals were perfused with phosphate-buffered saline, followed by 10% neutral buffered formalin. The aorta (to the bifurcation) was collected as a single specimen in 10% neutral buffered formalin, taking care not to disrupt luminal lesions. The apex of the heart was removed; the remaining heart with the attached ascending aorta segment was embedded in OCT and frozen for aortic-root sectioning.\u003c/p\u003e \u003cp\u003eAortic-root lesion area was quantified histologically on haematoxylin and eosin (H\u0026amp;E)-stained cryosections. Serial 10-\u0026micro;m sections were collected through the aortic sinus; images were acquired at predefined step levels spanning the sinus, and total lesion area was quantified using computer-assisted image analysis. Histological lesion quantification was performed by personnel blinded to treatment allocation. The primary endpoint was total aortic-root lesion area at day 29 (post-treatment time point) in vehicle, low-dose and high-dose groups (n\u0026thinsp;=\u0026thinsp;12 per group). Exploratory sys1\u0026ndash;sys3 groups were quantified similarly but were not used as primary inferential comparisons because of limited group size.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical tests were two-sided with a prespecified α\u0026thinsp;=\u0026thinsp;0.05. For comparisons of aortic-root lesion area across the primary groups (vehicle vs low-dose vs high-dose), a one-way analysis of variance (ANOVA) was performed, followed by Dunnett\u0026rsquo;s multiple comparisons test comparing each active dose to vehicle. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM with individual animals shown. Exploratory protease-inhibitor arms were evaluated descriptively and are reported without over-interpretation given the small sample size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available in the Zenodo repository at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.16785010\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.16785010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe repository includes raw aortic-root histology images, region-of-interest (ROI) masks, per-animal plaque-area measurements (CSV files), statistical analysis scripts reproducing the one-way ANOVA with Dunnett\u0026rsquo;s multiple comparisons test (CRL Study No. 20238304), and de-identified clinical summary tables.\u003c/p\u003e \u003cp\u003eSource data are provided with this paper.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCode availability\u003c/h2\u003e \u003cp\u003eThe analysis scripts used to reproduce the statistical analyses reported in this study (one-way ANOVA with Dunnett\u0026rsquo;s multiple comparisons test; CRL Study No. 20238304) are available in the Zenodo repository at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5281/zenodo.16785010\u003c/span\u003e\u003cspan address=\"10.5281/zenodo.16785010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eNo custom code beyond the scripts deposited in the repository was used.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by Pharmact AG, which sponsored the preclinical mouse study. The in vivo study was performed at Charles River Laboratories (CRL Study No. 20238304), and histological lesion quantification was performed by Vascular Strategies LLC. G.M.B. performed the formal data analysis, interpreted the results, prepared the figures, wrote the manuscript and decided to submit it for publication.\u003c/p\u003e\n\u003cp\u003eThe author thanks Charles River Laboratories and Vascular Strategies (Fort Washington, PA, USA) for independent conduct of the in vivo study and histological analyses.\u003c/p\u003e\n\n\n\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.M.B. conceived the study, developed the therapeutic concept, designed the experimental framework, oversaw study execution, performed the formal data analysis, interpreted the results, prepared the figures, and wrote and revised the manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.M.B. is the founder of the Pharmact group and serves as Chairman of the Board and Chief Medical Officer of Pharmact Holding AG (Switzerland), the parent company of Pharmact AG, which sponsored the preclinical mouse study reported in this manuscript. G.M.B. holds equity in Pharmact Holding AG and is an inventor on issued and pending patents covering the therapeutic use of hyaluronidase for extracellular-matrix-related diseases, including D016. As the sole author and a sponsor-affiliated investigator, G.M.B. performed the formal data analysis, interpreted the data, prepared the figures, wrote the manuscript and decided to submit it for publication. No other competing interests are declared.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHynes, R. O. The extracellular matrix: not just pretty fibrils. \u003cem\u003eScience\u003c/em\u003e 326, 1216\u0026ndash;1219 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnans, C., Chou, J. \u0026amp; Werb, Z. Remodelling the extracellular matrix in development and disease. \u003cem\u003eNat. Rev. Mol. 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Case Rep.\u003c/em\u003e 3, 1\u0026ndash;4 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9247777/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9247777/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePathological accumulation of extracellular-matrix components, including hyaluronan and sulfated glycosaminoglycans (sGAGs), has been suggested to sustain advanced atherosclerotic plaques by altering tissue architecture and transport. D016 is a recombinant dual-active hyaluronidase designed to cleave both substrates, aiming to modulate matrix-associated components in the plaque microenvironment rather than lipid metabolism.\u003c/p\u003e \u003cp\u003eMale LDLR\u0026minus;/\u0026minus; mice with established diet-induced atherosclerosis received intravenous vehicle or D016 once daily for 10 days (7,500 or 750,000 IU kg\u0026thinsp;\u0026minus;\u0026thinsp;1), followed by a 19-day post-treatment interval. Aortic-root lesion area was quantified histologically on day 29. High-dose D016 was associated with an ~\u0026thinsp;19% reduction in aortic-root plaque area versus vehicle (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, one-way ANOVA with Dunnett\u0026rsquo;s test; n\u0026thinsp;=\u0026thinsp;12 per group); the low dose showed no detectable effect. Protease-inhibitor co-treatment did not further reduce lesion area.\u003c/p\u003e \u003cp\u003eThis study did not quantify plaque ECM composition and therefore does not establish demonstrable ECM remodelling. An effect on lesion progression cannot be excluded under continued Western diet. Clinical condition, body weight, and hepatic and renal serum parameters were similar between groups. These results support further mechanistic evaluation of enzymatic modulation of matrix-associated pathways.\u003c/p\u003e","manuscriptTitle":"Dual-active hyaluronidase D016 reduces established atherosclerotic lesion area in LDLR⁻/⁻ mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 07:11:54","doi":"10.21203/rs.3.rs-9247777/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"453a42b8-c915-4ad1-9e5b-43da1a8db4d2","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65646876,"name":"Health sciences/Cardiology"},{"id":65646877,"name":"Health sciences/Cardiology/Cardiovascular biology"}],"tags":[],"updatedAt":"2026-04-09T04:30:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 07:11:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9247777","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9247777","identity":"rs-9247777","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}