Selective Depletion of Staphylococcus aureus Restores the Skin Microbiome and Accelerates Tissue Repair after Injury.

Our skin is home to a diverse community of commensal microorganisms integral to cutaneous function. However, microbial dysbiosis and barrier perturbation increase the risk of local and systemic infection. Staphylococcus aureus is a particularly problematic bacterial pathogen, with high levels of antimicrobial resistance and direct association with poor healing outcome. Innovative approaches are needed to selectively kill skin pathogens, such as S aureus, without harming the resident microbiota. In this study, we provide important data on the selectivity and efficacy of an S aureus-targeted endolysin (XZ.700) within the complex living skin/wound microbiome. Initial cross-species comparison using Nanopore long-read sequencing identified the translational potential of porcine rather than murine skin for human-relevant microbiome studies. We therefore performed an interventional study in pigs to assess the impact of endolysin administration on the microbiome. XZ.700 selectively inhibited endogenous porcine S aureus in vivo, restoring microbial diversity and promoting multiple aspects of wound repair. Subsequent mechanistic studies confirmed the importance of this microbiome modulation for effective healing in human skin. Taken together, these findings strongly support further development of S aureus-targeted endolysins for future clinical management of skin and wound infections.

Endolysin Inhibits Skin Colonization by Patient-Derived Staphylococcus Aureus and Malignant T-Cell Activation in Cutaneous T-Cell Lymphoma.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Linker-Improved Chimeric Endolysin Selectively Kills Staphylococcus aureus In Vitro, on Reconstituted Human Epidermis, and in a Murine Model of Skin Infection.

Staphylococcus aureus causes a broad spectrum of diseases in humans and animals. It is frequently associated with inflammatory skin disorders such as atopic dermatitis, where it aggravates symptoms. Treatment of S. aureus-associated skin infections with antibiotics is discouraged due to their broad-range deleterious effect on healthy skin microbiota and their ability to promote the development of resistance. Thus, novel S. aureus-specific antibacterial agents are desirable. We constructed two chimeric cell wall-lytic enzymes, Staphefekt SA.100 and XZ.700, which are composed of functional domains from the bacteriophage endolysin Ply2638 and the bacteriocin lysostaphin. Both enzymes specifically killed S. aureus and were inactive against commensal skin bacteria such as Staphylococcus epidermidis, with XZ.700 proving more active than SA.100 in multiple in vitro activity assays. When surface-attached mixed staphylococcal cultures were exposed to XZ.700 in a simplified microbiome model, the enzyme selectively removed S. aureus and retained S. epidermidis. Furthermore, XZ.700 did not induce resistance in S. aureus during repeated rounds of exposure to sublethal concentrations. Finally, we demonstrated that XZ.700 formulated as a cream is effective at killing S. aureus on reconstituted human epidermis and that an XZ.700-containing gel significantly reduces bacterial numbers compared to an untreated control in a mouse model of S. aureus-induced skin infection.

Topical S. aureus - Targeting Endolysin Significantly Improves Symptoms and QoL in Individuals With Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic skin condition affecting an increasing number of children and adults whose quality of life is impacted by chronic itch and pain. It is characterized by an altered epidermal barrier, skin inflammation, and skin microbiome dysbiosis particularly over-colonization of Staphylococcus aureus. The efficacy and tolerance of a cream containing a S. aureus-targeting technology (endolysin) was assessed in an open-label, two-week study in children and adults with mild-to-moderate atopic dermatitis. A total of 43 patients ranging from 7 months to 57 years old were included and all patients finished the study without any tolerance problem. Disease severity, measured with SCORAD, quickly reduced by 43% in 7 days and by 68 % in 14 days. The benefit was perceived by the whole panel with a marked improvement in overall QoL. This study shows the efficacy of a highly specific S. aureus-targeted technology in alleviating symptoms and improving QoL in children and adults with atopic dermatitis. It could also be beneficial in reducing and preventing flares in subjects with S. aureus load due to its good tolerance and specific action. J Drugs Dermatol. 2021;20(12):1323-1328. doi:10.36849/JDD.6363.

Recombinant Human Antithrombin in Pregnant Patients with Hereditary Antithrombin Deficiency: Integrated Analysis of Clinical Data.

OBJECTIVES: The purpose of this analysis was to evaluate the use of recombinant human antithrombin (rhAT) in preventing venous thromboembolism (VTE) in pregnant patients with hereditary AT deficiency (HATD). STUDY DESIGN: Data from two clinical trials were pooled. Dosing of rhAT was based on body weight and baseline AT activity, started up to 24 hours before scheduled induction or cesarean delivery, or at the onset of labor. RESULTS: A total of 21 pregnant HATD patients were enrolled. Mean rhAT therapy duration was 4.3 days and dose was 245.1 IU/kg/day. All patients achieved target mean AT activity (80-120% of normal) during rhAT therapy. There were no confirmed VTEs during rhAT treatment or within 7 ( ± 1) days after dosing. Two VTE events (one deep vein thrombosis and one pulmonary embolism) occurred 11 and 14 days after discontinuation of rhAT, in patients managed with prophylactic doses of heparin or low-molecular-weight heparin following delivery. CONCLUSION: rhAT was safe and effective in pregnant HATD patients when administered during the peripartum period, the period of highest VTE risk and a time when anticoagulation therapy is normally withheld. Pregnant HATD patients may benefit from therapeutic, rather than prophylactic, doses of anticoagulation after delivery to protect against postpartum VTE.

Perioperative and peripartum prevention of venous thromboembolism in patients with hereditary antithrombin deficiency using recombinant antithrombin therapy.

Recombinant human antithrombin (rhAT; ATryn), isolated from the milk of transgenic goats, provides an alternative to human plasma-derived antithrombin (AT) concentrate for perioperative and peripartum prophylaxis of venous thromboembolism (VTE) in patients with hereditary AT deficiency. Optimized rhAT dosing algorithms and improved plasma AT monitoring protocol were used in an open-label, single-arm, multinational, pivotal safety and efficacy study that was conducted in patients with hereditary AT deficiency in perioperative and peripartum settings. Loading and maintenance doses were calculated on the basis of pretreatment AT activity levels. Specific dosing regimens were used for pregnant and surgical patients; rhAT was to be given for at least 3 days and for 14 days or less. The primary efficacy end point was the incidence of any thromboembolic event during rhAT therapy or within 7 days of rhAT discontinuation. Safety and AT activity levels were secondary end points. Six surgical and 12 pregnant patients were treated for a median of 3.2 days (range 0.9-14 days). With the optimized dosing regimens, a median of 1 dose adjustment (range 0-6 dose adjustments) was needed to maintain AT activity levels within 80-120% of normal. No confirmed VTEs occurred during treatment or in the subsequent 7 days. Overall, rhAT was well tolerated, but some bleeding complications occurred after rhAT discontinuation and anticoagulation reinstitution. No antibodies to rhAT or goat milk proteins were detected. Perioperative and peripartum prophylactic rhAT therapy in patients with hereditary AT deficiency is well tolerated and effective in preventing VTE.

Clinical syndromes associated with acquired antithrombin deficiency via microvascular leakage and the related risk of thrombosis.

Antithrombin (AT) is a 65kDa glycoprotein belonging to a group of inhibitory factors known as serpins (serine protease inhibitors). It plays a critical role in the inhibition of coagulation and inflammation processes within the environment of the vascular endothelium. Inadequate levels of functional AT in plasma results in an increased risk of thrombotic events, both venous and arterial. AT deficiency can be inherited or acquired. Congenital AT deficiency is the most severe inherited thrombophilic condition with an odds ratio of 20 for the increased risk of venous thrombosis. Acquired AT deficiency occurs in a variety of physiologic and pathologic medical conditions with similar risks of increased thrombosis. In this article, we review clinical settings characterized by an acquired AT deficiency largely or partly subsequent to protein microvascular leakage. Other different mechanisms of AT depletion are implied in some clinical conditions together with endothelial loss, and, therefore, outlined. In addition, we provide a description of the current knowledge on the specific mechanisms underlying endothelial AT leakage and on the consequences of this protein decrease, specifically looking at thrombosis. We identify potential directions of research that might prove useful in patients with acquired AT deficiency.

Pharmacokinetics of recombinant human antithrombin in delivery and surgery patients with hereditary antithrombin deficiency.

Population pharmacokinetic (PK) analyses were conducted to refine dosing recommendations for recombinant human anti-thrombin therapy in surgery and delivery patients with hereditary antithrombin deficiency (HD). Single-dose PK data from patients with HD and nonlinear mixed-effects modeling were used to devise a dosing regimen to target antithrombin (AT) activity levels between 80% and 120% of normal. External validation with data from a phase 3 trial confirmed the correctness of a covariate-free model for surgery patients, but dosing adjustment was necessary for delivery patients. After different covariates were tested, the model was updated to incorporate the influential covariate, delivery. Simulations were used to develop a therapeutic drug-monitoring scenario that results in steady state AT activity levels within the target range as quickly as practically feasible. Data from a second clinical trial provided additional external validation and confirmed the accuracy of the dosing model for both groups of patients.

Exploring the role of antithrombin replacement for the treatment of preeclampsia: a prospective randomized evaluation of the safety and efficacy of recombinant antithrombin in very preterm preeclampsia (PRESERVE-1).

PROBLEM: Antithrombin (AT) replacement has been described in patients with hereditary AT deficiency undergoing delivery; however, the kinetics of AT replacement in preeclampsia is not adequately understood. Therefore, the Prospective Randomized Evaluation of the Safety and Efficacy of Recombinant Antithrombin in Very Preterm Preeclampsia (PRESERVE-1) study has been proposed. METHODS: Sixty women aged≥18 years at 24 0/7-28 0/7 weeks' gestation and with hypertension and proteinuria will be enrolled and randomly assigned to receive recombinant human AT or placebo until fetal and/or maternal indications cause cessation of expectant management or until 34 0/7 weeks' gestation. The primary endpoint is the increase in gestational age from randomization to delivery. Safety assessments and laboratory assays will also be performed. RESULTS: PRESERVE-1 study enrollment will begin during the second half of 2013. CONCLUSION: The PRESERVE-1 study will provide further insight into the pharmacokinetic activity and safety of AT therapy in preeclampsia.

Antithrombin alfa in hereditary antithrombin deficient patients: A phase 3 study of prophylactic intravenous administration in high risk situations.

During surgery and childbirth, patients with hereditary antithrombin (AT) deficiency are at high risk for thrombosis, and heparin prophylaxis may not be sufficiently efficacious. In these patients, exogenous AT may be used in association with heparin. A recombinant human AT (generic name: antithrombin alfa) has been developed. This multi-center study assessed the efficacy and safety of prophylactic intravenous administration of antithrombin alfa to hereditary AT deficient patients in high risk situations, including elective surgery, childbirth, or cesarean section. Antithrombin alfa was administered prior to and during the high risk period for restoration and maintenance of AT activity at 100% of normal. Heparin, low-molecular-weight heparin, and/or vitamin K antagonists were used according to standard of care. The primary efficacy endpoint was the incidence of acute deep vein thrombosis (DVT) from baseline up to day 30 post dosing as assessed by independent central review of duplex ultrasonograms and/or venograms. Safety was assessed based on adverse events (AEs) and laboratory evaluations. Five surgical and nine obstetrical hereditary AT deficiency patients received antithrombin alfa for a mean period of seven days. No clinically overt DVT occurred. Central review of ultrasonograms identified signs of acute DVT in two out of 13 evaluable patients. No antithrombin alfa-related AEs were reported. No patient developed anti-antithrombin alfa antibodies. In conclusion, this study suggests that antithrombin alfa is a safe and effective alternative to human plasma-derived AT for treating hereditary AT deficiency patients at high risk for thromboembolic events.