S. aureus colonization and clinical symptoms remain stable upon topical XZ.700 treatment: Results of a double‐blind randomized clinical trial in patients with mild to moderate atopic dermatitis

Abstract

Aim

Recovering dysbiosis may improve atopic dermatitis (AD) symptoms. XZ.700 is a recombinant chimeric endolysin that specifically targets Staphylococcus aureus and could be a new treatment option for patients with AD. The aim of this first‐in‐human study was to evaluate the safety, tolerability and efficacy of topical XZ.700 and explore the pharmacodynamic effects in patients with mild to moderate AD.

Method and Materials

This study consisted of Part A and Part B. In Part A, subjects were randomized and received XZ.700 10 μg/g, XZ.700 30 μg/g, XZ.700 100 μg/g or vehicle twice daily for 7 days on nonlesional skin and on all lesions (1% ≤ BSA ≤ 10%). In Part B, subjects received XZ.700 100 μg/g or vehicle on all lesions twice daily for 14 days (1% ≤ BSA ≤ 15%). Clinical scores and patient‐reported outcomes were recorded. Pharmacodynamic measurements were taken.

Results

In total, 35 patients completed the study. Tolerability of XZ.700 was acceptable. XZ.700 100 μg/g showed no evidence of effect on cultured S. aureus (estimated difference −52.9% CFU/mL; 95% CI −88.4% to 90.8%), oSCORAD (1.03; 95% CI −5.20 to 7.26) or EASI (−0.534; 95% CI −2.48 to 1.41). Furthermore, XZ.700 treatment did not result in a significant reduction in the relative abundance of S. aureus via metagenomics or other pharmacodynamic outcomes.

Conclusion

Tolerability and safety of short‐term topical administration of XZ.700 100 μg/g for 14 days were acceptable in most participants; however, some local application‐site events occurred, and one hypersensitivity reaction led to discontinuation. XZ.700 did not demonstrate target engagement or clinical benefit vs. vehicle under the tested conditions.

What is already known about this subject

• Atopic dermatitis (AD) is a chronic immune‐mediated inflammatory skin disease, with dysbiosis of the skin and an overgrowth of S. aureus.

• There is a need for novel topical treatments.

• Targeting S. aureus might be a novel treatment strategy.

What this study adds

• In a controlled first‐in‐human AD study, topical XZ.700 showed no measurable target engagement (no reduction of S. aureus by culture or metagenomics) and no clinical benefit vs. vehicle over 14 days.

• Local application‐site events were frequent in both arms; longer‐term tolerability and formulation optimization require further study. Future development should prioritize PK/PD bridging for topical protein therapeutics (skin deposition/penetration studies, predefined target engagement endpoints, formulation/delivery optimization) and consider enriched populations once engagement is demonstrated.

1. INTRODUCTION

In the clinical management of mild to moderate AD, topical medication is often considered the first‐line treatment. This is typically a topical corticosteroid (TCS) or topical calcineurin inhibitor (TCI) combined with emollients. Both TCS and TCI are considered effective in treating AD. However, misuse of TCS is associated with side effects, and TCIs are often less potent than TCS and not capable of inducing clinical remission. ¹ There is a need to develop a safe‐to‐use, effective topical treatment for atopic dermatitis (AD) patients.

Currently, multiple new therapies are being developed targeting different aspects of the pathophysiology of AD, which is complex and still not fully understood. ² , ³ Next to dysbiosis of the microbiome, genetic susceptibility, environmental factors, epidermal‐barrier abnormalities and immunological dysfunction contribute to disease manifestation. All the pathogenic drivers are interconnected and can continuously influence and reinforce each other. ¹ , ⁴ The variety of these drivers may explain the heterogeneous character of AD. ¹

Colonization with Staphylococcus aureus (S. aureus) is considered to play an important role in the pathogenesis of AD. ⁵ , ⁶ Colonization with S. aureus combined with a lower general microbial diversity is apparent in approximately 70%–90% of the AD patients. ⁷ Several factors contribute to enhanced S. aureus adhesion to AD skin. After adhesion, S. aureus may cause or exacerbate inflammation by binding its superantigens (SAgs) to MHCII molecules, which induces an excessive production of T cell cytokines. ⁸ In addition, S. aureus produces alpha toxin, which causes lysis of keratinocytes, and aerolysin, which activates antimicrobial peptides. ⁵

Sparing the microbiome, notably Staphylococcus epidermidis, is important to keep S. aureus in check, as low abundances of S. epidermidis are associated with more severe disease. ⁹ Additionally, previous research in mice has shown that recovering this dysbiosis and targeting S. aureus colonization may improve AD symptoms. ¹⁰ Targeting the microbiome in AD is considered an interesting novel treatment target. ¹¹ , ¹² , ¹³ XZ.700 is a recombinant chimeric endolysin that specifically targets S. aureus, sparing the remaining microbiome. Endolysins are phage‐derived enzymes that are produced at the end of the reproduction cycle of bacteriophages in the bacteria to lyse the peptidoglycan cell wall and to allow the newly assembled phages to leave the host cell. XZ.700 is produced via recombinant technology. Preclinical studies have shown that XZ.700 selectively inhibits the growth of S. aureus in vivo and in vitro, due to its bacteriolytic activity. ¹⁴ , ¹⁵ In addition, in vivo data have shown that XZ.700 dose‐dependently restores microbial diversity. ¹⁴ Furthermore, it effectively treats MRSA biofilms in two biofilm models. ¹⁶ Together, these findings provide a strong rationale for an exploratory evaluation of XZ.700. However, the combination of (i) high molecular weight, (ii) protein stability constraints (e.g., pH) and (iii) formulation requirements in AD (the need for an emollient‐like vehicle) may influence delivery from the vehicle to target micro‐niches and/or biofilms, underscoring the importance of exploratory clinical investigation.

We hypothesized that the topical use of XZ.700 in patients with AD could reduce the S. aureus abundance and restore the microbiome, thus leading to improvement of the AD symptoms. Given the exploratory nature of both the clinical and pharmacodynamic (PD) endpoints, we designed a first‐in‐human study to evaluate the safety and tolerability of topical XZ.700, while exploring its potential PD effects in patients with mild‐to‐moderate AD colonized with S. aureus. Moreover, we considered the compound's molecular properties and biofilm‐related barriers using exploratory endpoints within a translational context.

2. MATERIALS AND METHODS

In this study, we included 36 patients with mild to moderate AD. The study was conducted from 17 September 2020 to 17 March 2022 at the Centre for Human Drug Research, Leiden, the Netherlands. Patients were recruited via media advertisements, the database of the Centre for Human Drug Research (CHDR) (Leiden, Netherlands), the Clinical Network for Trials in Dermatology (CONNECTED) and the website of CHDR. Patients were included if AD was assessed as mild to moderate (IGA 2 or 3 and 1%–15% body surface area [BSA]). In addition, patients had to have a suitable target lesion within a range of 0.5% ≤ BSA ≤ 5% and were required to test positive on two consecutive occasions for S. aureus on the target lesion. We included patients with mild‐to‐moderate AD to ensure a favourable safety profile for first‐in‐human clinical exposure while still enrolling an S. aureus–colonized population amenable to repeated sampling and non‐invasive imaging. This disease‐severity range was considered optimal to balance feasibility and participant safety with a sufficient likelihood of detecting PD effects.

Patients who had other clinically significant skin conditions and clinically significant diseases or were not able to comply with lifestyle restrictions were excluded. The health status was verified by a detailed medical history assessment, a complete physical examination, vital sign assessment, 12‐lead electrocardiography (ECG) and laboratory tests (including hepatic and renal panels, complete blood count, chemistry panel, virology and urinalysis). Participants were requested to follow the following lifestyle restrictions: Refrain from excessive sun exposure or the tanning booth, refrain from washing the target lesion 12 h before every visit and use a microbiome‐friendly bodywash (La Roche‐Posay, Lipikar Syndet AP+), which was provided by the sponsor. A protocol‐defined washout period prior to baseline was required for prohibited AD treatments (14 days for topical therapies and systemic antibiotics, 7 days for emollients [target lesion only], 3 weeks for phototherapy, 4 weeks for systemic immunosuppressants and 5 half‐lives for biologics); topical emollients were permitted until baseline. All design details, inclusion and exclusion criteria are provided in the Supporting Information.

The Declaration of Helsinki was the guiding principle for this trial execution. The study was approved by the independent medical ethics committee ‘Medisch Ethische Toetsingscommissie van de Stichting Beoordeling Ethiek Biomedisch Onderzoek’ (Assen, the Netherlands). Before any study activity, all patients provided written informed consent. The study is registered under EudraCT number 2020‐002767‐56.

2.1. Design

This was a randomized, double‐blind, vehicle controlled, study in 36 AD patients with mild to moderate AD to assess the safety, tolerability and efficacy, as well as exploring the PD effects, of topical XZ.700. Part A of the study comprised of 12 patients and had a safety run‐in period of 7 days, during which patients received XZ.700 10 μg/g, XZ.700 30 μg/g, XZ.700 100 μg/g or vehicle BID on 20 × 20 cm nonlesional skin and simultaneously on all lesions, with a maximum of 10% BSA. Dosing rationale was based on the preclinical antibacterial activity data including MIC‐based potency estimates. Across the S. aureus panel, XZ.700 showed a modal MIC of approximately 0.125 μg/mL, with most isolates inhibited within the range of 0.06–0.5 μg/mL. In addition, formulation constraints for a topical in AD, i.e., patient preference for an emollient vehicle and established clinical recommendations, ¹⁷ and a stepwise first‐in‐human approach with a 10‐fold dose‐range across three cohorts were included in the dosing rationale strategy. After 7 days, nonlesional skin treatment was stopped, and patients continued lesional treatment for seven additional days. After an unblinded interim analysis of Part A, the protocol was updated with the removal of the dose groups of 10 and 30 μg/g of XZ.700, due to lack of activity. In Part B, 24 AD patients were included using 14 days of treatment on all lesions with a maximum of 15% BSA (Figure 1B,C). Treatment compliance was for both parts recorded with a mobile e‐diary app including a notification and photo capture function to enable documentation. Patients were stratified by baseline S. aureus load on the target lesion (high vs. low based on semi‐quantitative culture) and randomized within strata using block randomization (block size 4 in Part A). This approach was used to distribute patients as evenly as possible over the treatment groups. The randomization code was generated by a study‐independent CHDR statistician and was done in blocks of four patients for Part A. During the study, topical triamcinolone 0.1% was available as rescue medication at the discretion of the study physician.

FIGURE 1.

(A) CONSORT flowchart. (B) Part A study design. Schematic representation of the study design of Part A. AD, atopic dermatitis; EOS, end of study; EOT, end of treatment; NL, nonlesional. Created with BioRender.com. © 2025 BioRender. (C) Part B study design. Schematic representation of the study design of Part B. AD, atopic dermatitis; EOS, end of study; EOT, end of treatment. Created with BioRender.com. © 2025 BioRender.

2.2. Methods of measurement

During the study, safety and tolerability endpoints were evaluated by means of adverse event monitoring, physical examination, vital signs measurements, 12‐lead ECG and laboratory tests. Furthermore, in Part A of the study, skin irritation of nonlesional skin treated with XZ.700 was assessed using a local irritation grading scale (LIGS).

Treatment efficacy was assessed with multiple clinical outcome measurements and patient‐reported outcomes. Disease activity was evaluated with the Eczema Area and Severity Index (EASI) score, the objective SCORing Atopic Dermatitis (oSCORAD) score and Investigator Global Assessment (IGA). The target lesion was assessed with the local oSCORAD score, total signs and symptoms (TSS) and the size of the lesion (cm²). Patient‐reported outcomes consisted of weekly Patient‐Oriented Eczema Measure and Dermatology Life Quality Index questionnaires and twice‐daily numeric rating scale itch scores via the mobile diary app.

To assess S. aureus cultures and the full microbiome composition of the skin with metagenomic sequencing, swabs were collected from lesional and nonlesional skin during every study visit (same location each visit). For the S. aureus cultures, nylon flocked swabs (490CE eSwab, Copan, Italy) were used. After collection, the skin swab was placed in an eSwab tube containing 1‐mL Amies medium. The swabs were cultured under aerobic conditions for 48 h on SA‐ID agar plates (Biomérieux, France), and the determination of grown colonies was assessed using MALDI‐TOF (MALDI Biotyper System, Bruker, Germany). The grown colonies were assessed semi‐quantitatively (0 to 4+) and quantitatively (CFU/mL). The samples for microbiome analysis were taken with sterile noncotton FLOQ swabs (COPAN Diagnostics, 50U002DS01/50U006DS02). After collection, the skin swab was placed in a DNA‐free empty Eppendorf tube and stored in the freezer at −80°C. Microbial DNA was extracted from the samples using the Macherey–Nagel NucleoSpin DNA extraction kit. DNA was sequenced on the Illumina platform (150PE). The metagenomic profiling of the samples was done by mapping the data to the skin metagenomic species database built by Clinical Microbiomics to create a bacterial abundance table of the samples (compositional data).

PD effects of XZ.700 over time on the target lesion were assessed using multiple imaging tools. Skin erythema and roughness were recorded using a Miravex Antera 3D camera (Dublin, Ireland). In addition, blood flow was assessed with the laser speckle contrast imaging (LSCI) device (PeriCam PSI imager [Perimed Jäfälla, Sweden]). The barrier status of the skin was assessed during the study by measuring the trans epidermal water loss (TEWL) using an AquaFlux AF200 system (Biox, London, UK). The target lesion and nonlesional skin were photo documented with standardized photography.

For the circulating cytokine assessment, blood was collected before treatment and after 15 days of treatment. Blood was collected in a 6‐mL sodium heparin tube and centrifuged within 30 min of collection for 15 min at 2000G at 4°C. The following biomarkers were investigated: TARC, IL31 and IL8.

2.3. Analysis

For the efficacy and PD analysis, the 10 and 30 μg/g groups were excluded because of the low number of patients. For the safety endpoints, all treatment groups were included in the analysis. All calculations were conducted with SAS 9.4 for Windows or newer (SAS Institute Inc., Cary, NC, USA). Power calculations were performed for the parameter TSS and oSCORAD based on an interim analysis of a previous study in AD patients at CHDR. ¹⁸ The group size was based on empirical considerations for a first‐in‐human study with the main objective of evaluating safety and tolerability but also having sizable treatment arms to investigate signals of activity in PD and efficacy. No formal power analysis was performed. At each time point, absolute values and change from baseline of the variables were summarized with n, mean, SD, SEM, median, min and max values. The number of available observations was presented. Efficacy and PD endpoints were analysed with a mixed effects model using treatment, time and treatment by time as fixed factors; subject as the random factor; and pretreatment value as the covariate. In case of log‐normal distribution of the data, the data are log‐transformed before analysis, and the results are back‐transformed to percentages. Before log transformation, zeroes in the S. aureus data are dealt with by adding 1 to every S. aureus value. The following contrast is estimated within the model: XZ.700 100 μg/g − vehicle within Day 1 to Day 15.

2.4. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2025/26. ¹⁹

3. RESULTS

During the study, a total of 39 patients were enrolled, of whom 35 (89.7%) completed the study. See Figure 1A for the flow chart of patient enrolment. The demographic characteristics at baseline were comparable between treatment groups (Table 1). The eczema severity parameters (EASI, oSCORAD and %BSA) and the colony‐forming units of S. aureus per millilitre on the target lesion were higher in the XZ.700 100 μg/g treatment group compared with the other treatment groups due to the increase in allowed maximum % BSA in Part B.

TABLE 1.

Baseline characteristics.

Characteristics	All patients (n = 39)	XZ.700 10 μg/g (n = 4)	XZ.700 30 μg/g (n = 3)	XZ.700 100 μg/g (n = 19)	Vehicle (n = 13)
Patients completed, n a (%)	35 (89.7%)
Age, years
Mean (SD)	22.6 (3.8)	23.8 (5.7)	20.3 (2.3)	22.0 (3.4)	23.5 (4.1)
Median	22	22	19	21	22
Min, max	18, 34	19, 32	19, 23	18, 31	19, 34
Sex
Female, n (%)	23 (59.0%)	2 (50.0%)	0 (0.0%)	13 (68.4%)	8 (61.5%)
Male, n (%)	16 (41.0%)	2 (50.0%)	3 (100.0%)	6 (31.6%)	5 (38.5%)
oSCORAD
Mean (SD)	24.77 (7.60)	19.88 (2.12)	21.80 (5.73)	27.39 (8.13)	23.14 (7.29)
Min, max	14.6, 44.2	18.0, 22.0	18.1, 28.4	14.6, 44.2	14.8, 39.9
oSCORAD (local)
Mean (SD)	23.73 (7.95)	18.55 (3.33)	21.20 (9.26)	27.29 (8.40)	20.69 (6.06)
Min, max	10.7, 45.7	14.2, 21.2	10.7, 28.2	17.7, 45.7	10.7, 31.8
EASI
Mean (SD)	4.33 (3.87)	4.61 (2.20)	2.50 (1.18)	5.13 (5.21)	3.49 (1.63)
Min, max	0.80, 18.50	2.95, 7.80	1.20, 3.50	0.80, 18.50	1.50, 7.20
BSA
Mean (SD)	5.85 (3.62)	3.13 (1.31)	4.00 (2.65)	7.08 (4.31)	5.31 (2.47)
Min, max	2.0, 15.0	2.0, 5.0	2.0, 7.0	(2.0, 15.0)	3.0, 12.0
Fitzpatrick skin type, n (%)
1	5 (12.8%)	0 (0%)	0 (0%)	3 (15.8%)	2 (15.4%)
2	20 (51.3%)	1 (25.0%)	0 (0%)	11 (57.9%)	8 (61.5%)
3	10 (25.6%)	2 (50.0%)	2 (66.7%)	4 (21.1%)	2 (15.4%)
4	3 (7.7%)	0 (0%)	1 (33.3%)	1 (5.3%)	1 (7.7%)
5	1 (2.6%)	1 (25.0%)	0 (0%)	0 (0%)	0 (0%)
6	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Staphylococcus aureus colonization
CFU/mL, mean (SD)		191.0 (3)	164.3 (3)	29 009.3 (18)	229 994.3 (12)

Abbreviations: BSA, body surface area; CFU, colony‐forming units; EASI, Eczema Area and Severity Index; oSCORAD, objective Scoring Atopic Dermatitis; SD, standard deviation.

^a Four subjects dropped out during the study. Two were replaced (vehicle and XZ.700 10 μg/g group), and two were not replaced (XZ.700 100 μg/g group).

3.1. Safety/tolerability/compliance

In total, 40 treatment‐emergent AEs (TEAEs) were recorded in 26 subjects. Eight were considered probably related, 11 were considered possibly related, and all other AEs were considered either unlikely related or unrelated. Most AEs were rated as mild; three AEs were rated moderate, and one was rated as severe. The severe rated AE consisted of an application site hypersensitivity reaction after the first dosing, and the patient was excluded from the study. Regardless of their severity, all AEs were self‐limiting. The most frequent TEAE in both the vehicle and XZ.700 groups was application site irritation. No serious adverse events occurred during the study. See Table 2 for all AEs. Rescue triamcinolone 0.1% was not used by participants in the XZ.700 100 μg/g group and vehicle group; however, one subject did drop out due to severe disease progression (see CONSORT chart). During the study, no notable changes were seen in physical assessments, vital signs measurements, 12‐lead ECGs and laboratory tests. The treatment compliance for 10 μg/g XZ.700 and 30 μg/g XZ.700 was 100.0%. For the 100 μg/g XZ.700 treatment group, the compliance ranged from 60.7% to 100.0% (mean 94.8%, SD: 11.37) and for the vehicle group from 89.3% to 100.0% (mean 97.0%, SD: 3.67). The relatively low minimal range in the XZ.700 is due to the dropout of one subject on Day 10 due to significant disease progression.

TABLE 2.

Summary of treatment‐emergent adverse events.

	XZ.700 10 μg/g (n = 4)		XZ.700 30 μg/g (n = 3)		XZ.700 100 μg/g (n = 19)		Vehicle (n = 13)
	Events N	Subjects N	Events N	Subjects N	Events N	Subjects N	Events N	Subjects N
Treatment‐emergent AEs	7	4	3	2	21	13	9	7
Mild	6	4	3	2	18	12	9	7
Moderate	1	1	0	0	2	2	0	0
Severe	0	0	0	0	1 a	1	0	0
Treatment related b	3	1	3	2	10	7	3	3
Events that occurred in more than 1 patient	Occurrence of AE		Occurrence of AE		Occurrence of AE		Occurrence of AE
Application site pruritus	1		‐		‐		1
Application site irritation	1		1		2		1
Disease progression c	‐		2		2		‐
Coronavirus infection	‐		‐		1		2
Headache	1		‐		2		‐
Presyncope	‐		‐		1		1
Procedural haemorrhage	‐		‐		1		1

Abbreviations: AE, adverse event; SAE, severe adverse event; TEAE, treatment‐emergent adverse event.

^a Hypersensitivity reaction after application of XZ.700, subject dropped out of the study.

^b Probable or possible related.

^c One subject dropped out due to significant disease progression.

3.2. Treatment efficacy

The treatment efficacy scores (EASI, SCORAD and IGA) showed no significant changes or trends in the active treatment group (XZ.700 100 μg/g) compared with vehicle. Patient‐reported outcomes (POEM, DLQI and NRS itch) showed no significant changes in the active treatment group compared with the vehicle. Both groups (vehicle and XZ.700 100 μg/g showed a small numerical improvement when looking at SCORAD, EASI, IGA, DLQI and POEM; however, the change was not statistically significant, and no significant difference was observed between the treatment and vehicle groups. The Harmonizing Outcome Measures for Eczema (HOME) outcomes are presented in Figure 2, and the full analysis results are presented in Table 3.

FIGURE 2.

Staphylococcus aureus cultures and HOME outcome measures (EASI, POEM, Itch). Mild to moderate AD. Red indicates XZ.700 100 mg, and blue indicates vehicle. D1 indicates before treatment measurement. D15 indicates EOT, and D22 is the follow‐up visit, 7 days post last treatment with XZ.700 or vehicle. (A) Mean POEM total score on different timepoints. (B) Mean EASI total score on different time points during treatment period. (C) Mean NRS itch in the evening measured via e‐diary on mobile app. (D) Quantitative S. aureus cultures on lesional AD skin. Error bars represent SD; lower limits are truncated for visual clarity. Y‐axis is presented on a logarithmic scale for improved clarity of data visualization. Graph generated using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

TABLE 3.

Clinical outcomes table (summary of all results).

	XZ.700 100 μg/g vs. vehicle up to end of treatment
Outcome	Estimated difference	95% CI	p‐value
S. aureus in culture (quantitative) (CFU/mL)	−52.9%	−88.4% to 90.8%	0.2804
oSCORAD overall	1.03	−5.20 to 7.26	0.7390
oSCORAD target lesion	0.82	−5.34 to 6.97	0.7879
IGA	0.0	−0.4 to 0.4	0.8998
EASI	−0.534	−2.479 to 1.411	0.5820
NRS (0–100) morning itch	−9.8	−24.4 to 4.9	0.1810
NRS (0–100) evening itch	−9.3	−25.1 to 6.6	0.2389
NRS (0–100) sleeplessness	−2.6	−13.2 to 8.0	0.6131
DLQI total score	0.271	−2.869 to 3.411	0.8623
POEM total score	3.130	−1.011 to 7.270	0.1337
Erythema target lesion multispectral	−0.4420	−3.4606 to 2.5766	0.7667
Roughness target lesion multispectral	0.1265	−1.5091 to 1.7622	0.8755
LSCI target lesion perfusion	6.442	−10.803 to 23.686	0.4532
TEWL target lesion a : Steady state flux (g/m2/h)	1.991	−10.302 to 14.284	0.7437
IL31 expression in blood pg/mL, [%]	−1.518	−38.094 to 35.058	0.9312
IL8 in blood pg/mL, [%]	0.770	−0.586 to 2.127	0.2532
TARC expression in blood, pg/mL, [%]	21.996	−15.292 to 59.283	0.2357

Abbreviations: 3D, 3‐dimensional; CFU, colony forming units; CI, confidence interval; DLQI, Dermatology Life Quality Index; EASI, Eczema Area and Severity Index; IL, interleukin; LSCI, Laser Speckly Contrast Imaging; NRS, numeric rating scale; oSCORAD, objective Scoring Atopic Dermatitis; POEM, Patient Oriented Eczema Measure; TARC, thymus and activation‐regulated chemokine; TEWL, trans epidermal water loss assessment; TSS, total signs and symptoms.

^a Nonlesion corrected.

3.3. Microbiome (metagenomics)

At baseline, there were significant differences between target‐lesion and nonlesion sites despite interparticipant variation including (i) higher percentage of host reads, (ii) higher S. aureus relative abundance and (iii) lower alpha diversity at target‐lesion sites than nonlesion sites. The results of the microbiome swabs did not differ significantly between participants treated with vehicle or XZ.700 100 μg/g before (Day 1) and after treatment (Day 15) in alpha diversity (Figure 3) and beta diversity. Importantly, we did not detect a reduction in S. aureus relative abundance by the treatment with XZ.700 100 μg/g in comparison with vehicle treatment of the target‐lesion sites (Figures 4 and 5). Untargeted tests uncovered very few taxa with a significant change (using an FDR of 10%) in relative abundance during XZ.700 (100 μg/g) treatment in comparison to the vehicle treatment.

FIGURE 3.

The plot shows alpha diversity values calculated from metagenomic species (MGS) relative abundances at Day 1 and Day 15 separately for the two lesion types and treatment groups. Samples from the same participant are connected by a grey line. Richness values of the target‐lesion samples decreased from Day 1 to Day 15 for eight out of 10 participants treated with XZ.700 with both samples present.

FIGURE 4.

The plot shows S. aureus relative abundance [%] over time separately for the target lesion (TL) and nonlesional skin (NL) for vehicle and XZ.700 100 μg/g group. Samples from the same participant are connected by a grey line. No consistent decrease in S. aureus relative abundance over time is seen neither for the vehicle nor the XZ.700 100 μg/g treatment group. There is a strong spread in these relative abundances ranging from 0% to 100%; we therefore showed the plot on a pseudo‐log scale.

FIGURE 5.

The plot shows S. aureus relative abundance at Day 1 and Day 15 separately for the nonlesional (NL) and target lesion (TL) per treatment group (vehicle or XZ.700 100 μg/g). Samples from the same participant are connected by a grey line. In none of the four cases shown, there was a significant difference between Day 1 and Day 15 values in paired Mann–Whitney U tests.

3.4. Microbiology (cultures)

In general, the S. aureus cultures had a high individual variability. Treatment with XZ.700 did not lead to a reduction of S. aureus in the cultures (both semi‐quantitative and quantitative). See Figure 2.

3.5. Imaging

Multiple imaging tools did not show any significant changes in the active treatment group (XZ.700 100 μg/g) compared with vehicle. Multispectral 3D imaging showed no improvement in erythema or roughness. Skin perfusion as measured by Laser speckle contrast imaging did not show any change. In addition, the TEWL did not change significantly. The full analysis results are presented in Table 3.

3.6. Blood‐based biomarkers

At baseline, slightly increased measurements were observed for IL31. The blood‐based biomarkers (TARC, IL8 and IL31) did not show any significant changes in the active treatment group (XZ.700 100 μg/g) compared with vehicle. The full analysis results are presented in Table 3.

4. DISCUSSION

The aim of this exploratory study was to evaluate the safety, tolerability and efficacy of XZ.700 as well as to explore the PD effects with a PD test battery in patients with mild to moderate AD. Treatment with XZ.700 100 μg/g for 14 days was acceptable in most patients; however, local site application events were common, and one hypersensitivity reaction led to discontinuation. Treatment with XZ.700 did not result in a significant reduction in oSCORAD, IGA, EASI or any of the PD endpoints vs. vehicle, consistent with the exploratory nature of this trial. A key finding of this trial is the absence of measurable target engagement: XZ.700 did not reduce S. aureus by culture or metagenomics, nor did it meaningfully modulate microbiome composition. Accordingly, efficacy conclusions are limited to the tested formulation and regimen. These results underscore the importance of demonstrating skin exposure/target engagement for large protein antimicrobials in AD, where vehicle constraints, pH, molecular size and biofilm accessibility may critically limit delivery to microbial niches.

4.1. Comparison with SA.100

Similar results were reported for a comparable product, Staphefekt SA.100. SA.100 is a chimeric peptidoglycan hydrolase, which was also developed to target the skin microbiome and more specifically S. aureus. In a randomized double‐blind vehicle‐controlled study in patients with mild to moderate AD, no target engagement of SA.100 was found on any of the outcome measures including S. aureus. ¹⁸ SA.100 and XZ.700 feature the same domain architecture but differ in the linker region and amidase domains connecting M23. XZ.700 is a linker‐improved endolysin with higher antibacterial activity in in vitro studies. ¹⁵ However, we could not prove the clinical relevance of this in the present study. Despite the enhanced in vitro activity and expanded PD readouts, no measurable engagement was observed under a clinically acceptable cream formulation and 14‐day dosing. This highlights a key translational lesson for topical protein antimicrobials in AD: Formulation‐ and penetration‐driven PK/PD bridging is essential before progressing to larger efficacy trials. In the in vitro study, however, the endolysins were diluted in phosphate‐buffered saline and not used in a lipophilic base such as a cream. Possibly, improvement and innovation into more novel drug delivery systems could be beneficial for novel endolysins and phages in topical management. ²⁰

4.2. Potential reasons for the lack of effect on S. aureus

Regarding lack of or too low activity, it might be contemplated that the endolysin XZ.700 is unable to diffuse from the vehicle substance to the skin, due to the high lipophilic conditions of the cream. Therefore, it might not be able to reduce S. aureus on the skin. Potentially, the efficacy of XZ.700 could be improved with the change of vehicle to a less lipophilic compound, as a too lipophilic solution could counteract drug delivery to the skin. ²¹ Nonetheless, the use of a less lipophilic compound raises another challenge in AD patients since hydrophilic‐based compounds could induce an AD exacerbation. In addition, endolysins are proteinaceous and therefore can quickly be affected by changing pH levels. ²² As pH levels on the skin are known to be higher in AD patients, this may be a reason for the lack of effect. ²³ Moreover, the drug's molecular weight is far above 500 Da, hindering skin penetration of exogenous compounds. ²⁴ Most commonly used and effective drugs for dermatological therapies have a molecular weight under or around 500 Da. For example, fusidic acid has a weight of 517 Da and is used (mainly in Europe and Australia) for the treatment of acute skin infections or MRSA/S. aureus colonization. ²⁴ , ²⁵ As the molecular weight of XZ.700 is 100‐fold higher, 51.828 Da, this impedes efficient drug delivery into the skin and potentially explains the lack of effect, when assuming that some degree of skin and biofilm penetration is necessary for a relevant effect.

Furthermore, possibly the penetration into the S. aureus biofilm was insufficient. Biofilm formation by S. aureus is complex, and in AD patients, it can contribute to the persistence of S. aureus and resistance to antimicrobial treatment. ²⁶ Biofilm‐associated effects of XZ.700 have so far been demonstrated only under in vitro conditions, where XZ.700 successfully treats biofilms. ¹⁶

In addition, the severity of the disease was potentially too low to explore activity. Patients were all of mild to moderate severity and colonized with S. aureus. Possibly, a more severe disease status, AD flare or acute S. aureus infection is better suited for additional XZ.700 treatment. Though the local tolerability issues may be more pertinent in the severe population, as this is generally associated with a larger BSA, which will increase exposure to the treatment. A case series with a similar treatment (SA.100), also developed to target the skin microbiome and more specifically S. aureus, showed efficacy in three patients with recurrent S. aureus infections. However, in a previous study with a similar compound but in another formulation (gel), in which the same techniques were used in the same mild to moderate AD population, a small but significant reduction in S. aureus culture and microbiome composition was seen. ¹¹ It shows that effects have been seen on this PD endpoint specifically. Additionally, the formulation did not lead to exacerbation of AD in this study, but the patients did experience increased dryness of the skin.

4.3. Limitations

Limitations that could possibly have influenced the outcome are both the small sample size and the treatment duration of 14 days, which could have been too short to observe stable microbiome shifts and downstream clinical improvement. In addition, the CFU levels in the XZ.700 group were 10‐fold lower at baseline compared with vehicle, potentially influencing the observed effects. Moreover, the inclusion of mild–moderate patients instead of a more severe population could have influenced the likelihood of detecting microbiome‐driven effects, given spontaneous fluctuations and vehicle responses in this population. However, we would expect an indication of improvement in the PD biomarkers such as S. aureus load or early signs of improvement with the sensitive imaging tools. Furthermore, there is a small discrepancy in AD severity between the treatment groups, with the treatment group for XZ.700 100 μg exhibiting higher clinical scores at baseline.

4.4. Bridge to the clinic

The question remains whether the elimination of S. aureus as monotherapy in colonized AD patients would be sufficient to reduce clinical symptoms. In a preclinical study by Kobayashi et al., it was shown that antibiotic treatment of mice suffering from S. aureus‐induced AD had an improvement of AD symptoms. ¹⁰ In contrast, the study of van der Kolk et al. shows that restoration of dysbiosis had no impact on clinical symptoms, which supports the hypothesis that dysbiosis is a consequence of AD and not the key driver. ¹¹

5. CONCLUSION

In conclusion, this exploratory study shows that the tolerability and safety of XZ.700 were acceptable in most patients; however, local application site events were common, and one hypersensitivity reaction led to discontinuation in the study. Furthermore, XZ.700 did not demonstrate target engagement or clinical benefit vs. vehicle. Future development should prioritize PK/PD bridging for topical protein therapeutics including skin deposition/penetration studies, predefined target engagement endpoints and formulation/delivery optimization and consider enriched populations once engagement is demonstrated.

AUTHOR CONTRIBUTIONS

Laura W. J. van der Meulen: Formal analysis; investigation; data curation; writing—original draft; writing—review and editing. Menthe E. Bergmans: Investigation; data curation; writing—review and editing. Salma Assil: Investigation; data curation; writing—review and editing. Naomi Klarenbeek: Supervision; writing—review and editing. Marieke L. de Kam: Formal analysis; data curation; writing—review and editing. Alexa J. Tibboel: Formal analysis; data curation; writing—review and editing. Thorsten Brach: Microbiome analysis; writing—review and editing. Bjorn L. Herpers: Conceptualization; writing—review and editing. Johan Frieling: Conceptualization; writing—review and editing. Vincent de Jong: Conceptualization; writing—review and editing. Benthe Freyee: Conceptualization; writing—review and editing. Martijn B. A. van Doorn: Supervision; conceptualization; writing—review and editing. Robert Rissmann: Supervision; conceptualization; writing—review and editing. Tessa Niemeyer‐van der Kolk: Supervision; conceptualization; writing—original draft; writing—review and editing.

CONFLICT OF INTEREST STATEMENT

L.v.d.M., M.B., S.A., T.v.d.K., M.K., A.T., N.K., and R.R. are employees of the Centre for Human Drug Research, which received funding from Micreos Human Health B.V. to conduct this study. B.H. is a consultant to Micreos Human Health B.V. V.J. is an employee of Micreos Human Health B.V. B.F. and J.F. were employees of Micreos Human Health B.V. at study conduct. J.F. and B.H. have stock option of Micreos Human Health B.V.

Supporting information

Data S1. Supporting information.

van der Meulen LWJ, Bergmans ME, Assil S, et al. S. aureus colonization and clinical symptoms remain stable upon topical XZ.700 treatment: Results of a double‐blind randomized clinical trial in patients with mild to moderate atopic dermatitis. Br J Clin Pharmacol. 2026;92(10):3448‐3459. doi: 10.1002/bcp.70630

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.