Phase 2a Dose Finding Study of Nu-3 Gel in Subjects With Mild iDFU

A Prospective, Randomized, Single-Blind Phase 2a Multi-Center Dose Comparative Study to Evaluated The Safety, Tolerability, and Antimicrobial Efficacy Of Topically Twice Daily Applied Bisphosphocin Nu-3 Gel At a 2%, 5%, And 10% Concentration To Infected Diabetic Foot Ulcers (iDFU)

The goal of this clinical trial is to learn if drug Nu-3 works to treat mildly infected diabetic foot ulcers in adults with Type 1 or Type 2 diabetes at 3 different dose levels. It will also learn about the safety of drug Nu-3. The main questions it aims to answer are:

Does drug Nu-3 lower the number of bacteria or viruses in the infected ulcer, or cure patients of the infection, at any or all of the dose levels being tested after 1 week or 2 weeks? What medical problems do participants have when taking the topical drug Nu-3? Researchers will compare drug Nu-3 at different dose levels to see if drug Nu-3 works to treat the infected ulcers at each of the dose levels.

Participants will:

Take drug ABC or a placebo every day for 4 months Visit the clinic once every 2 weeks for checkups and tests Keep a diary of their symptoms and the number of times they use a rescue inhaler

A prospective, randomized, single-blind Phase 2a multi-center dose comparative study to evaluate the safety, tolerability, and antimicrobial efficacy of topically twice daily applied Bisphosphocin® Nu-3 gel at a 2%, 5%, and 10% concentration to infected diabetic foot ulcers (iDFU) in adult subjects with type 1 and 2 diabetes. The study will be conducted in 3 phases: Screening, Treatment, and Follow-up.

Subjects will have the study explained to them and will be provided with the study specific informed consent form (ICF). For eligible subjects, the screening evaluations will be performed immediately after the subject provides a written informed consent. To further increase the probability of patients being enrolled who truly have a mildly infected diabetic foot ulcer (and not either no infection or a moderate or more severe infection) an additional external clinical specialist(s) will be consulted and provided with pictures obtained during the screening procedure. If the external specialist(s) confirms eligibility, the patient can be included. If the clinical specialist(s) has additional questions to determine the eligibility or disagrees with the study PI's assessment, a resolution call between the PI and the external specialist(s) takes place the same day. The specialist(s) may also join via appropriate telehealth technology to discuss other signs and symptoms which are difficult to be assessed via images with the site PI and the patient together. Only if consensus has been reached that the patient is eligible for the trial by wound status and all other inclusion and no exclusion criteria have been met, the baseline visit will be scheduled for the next morning. If consensus cannot be reached the patient will not be enrolled and recorded as screen failure as defined in the Safety Charter provided by CRO.

Enrollment and randomization need to take place as soon as screening lab-results from a local laboratory and possibly a plain foot X-ray have been obtained, because infection management will not permit a delay in enrollment and start of treatment for several days. If the patient does not meet eligibility criteria, the patient will not be randomized and will be considered a screening failure for the study. Subjects enrolled in the study at V2 will be followed by five scheduled visits plus an additional follow-up visit on day 28 (total = 7 visits from baseline until end of study).

Eligible subjects who enter the active treatment period will be randomized into one of the dose cohorts:

• Treatment/ Cohort 1: 2% Nu-3 gel + SOC (n = 8-10) twice daily
• Treatment/ Cohort 2: 5% Nu-3 gel + SOC (n = 8-10) twice daily
• Treatment/ Cohort 3:10% Nu-3 gel + SOC (n = 8-10) twice daily Study drug will be applied to the target iDFU for 14 days (± 2 days) twice daily.

All randomized subjects will continue to receive SOC throughout the study per Appendix 4 and other than any additional antibiotic treatment.

After completing the active treatment period, subjects will return after 2 weeks for a follow-up safety evaluation.

This study will permit an evaluation of a dose response among the three tested doses to establish the initial proof of concept. The study is a designed as a prospective, randomized, single-blind dose comparative study to evaluate the safety and tolerability of topically twice applied (2%, 5%, and 10% gel formulation) daily for 2 weeks, Bisphosphocin® Nu-3 Gel to infected diabetic foot ulcers (iDFU).

Previous study protocols for Nu-3 (protocol number LAI-Nu-3-CLIN002) and other developments of topicals for the treatment of infections in patients with iDFU typically focused either primarily on wound- healing or as a combined endpoint of infection control (clinically and anti-microbial response) and wound healing. For this phase 2a study we will evaluate primary and important secondary endpoints which measure predominantly the effect of study drug on the infection. This is based on the intended use as a stand-alone topical antimicrobial drug and reflects the purpose of the study to generate evidence and proof of concept that non-clinical and in-vitro findings about the antimicrobial effect also apply to a clinical situation in patients. Combining infection control with wound healing into a combined endpoint would confound the proof-of-concept approach. Wound healing information will therefore be collected as exploratory endpoints and to be potentially better addressed in confirmatory trials.

The study includes 3 different dose levels which will be compared. No placebo cohort nor an oral broad-spectrum antibiotic therapy as active comparator is included in this first proof of concept study. A placebo control in a double-blind design is planned for the subsequent phase 2b study. Guidelines recommend for the treatment of mildly infected foot ulcers to initiate a broad-spectrum oral antibiotic therapy when signs of infection are present. We opted against the inclusion of an active comparator in the early phase of clinical development for the following reasons.

1. An active comparator (oral antibiotic drug) should have proven and documented efficacy over standardized physical wound care alone (e.g., debridement, dressing).

   • The evidence that oral antibiotic therapy has significant additional benefit over appropriate wound care alone is weak and highly uncertain. No studies are published which demonstrate this outcome and show an outcome benefit. The IDSA (2012) and the IWGDF (2019) guideline highlight the limited available evidence.

2. It must be feasible to incorporate the active comparator into a clinical trial and would require a double-blinded design.

   • Switching the comparator, based on wound microbiology cultures, is not viable in a double blinded clinical trial.

3. Inclusion of close "infection stewardship" is required for all cohorts as the trial will be blinded to the subjects the test product may or may not be efficacious.

   Based on the above information and in agreement with external experts it is concluded that for this particular study and its intended use an active comparator would not be appropriate. Potentially, a non- inferiority outcome vs. an active comparator may be misleading towards further development if such outcome is due to the SOC wound care and not an oral antibiotic therapy (Pexiganan, for example). Only a placebo comparison would allow a robust assessment of an added antimicrobial efficacy benefit of Nu3 compared to SOC wound care, which therefore is our comparator of choice for the planned subsequent phase 2b study.

   Risk reduction and mitigation for the patient is the most important objective in the study design and conduct with the goal to identify any clinical signs of worsening of the infection early, to then put the patient on oral antibiotic treatment and to terminate the study for this patient as a treatment failure. Key measures for risk mitigation as defined in the study protocol are:

   • External Specialist(s) will discuss the eligibility of a patient with the PI during or after the screening examination based on images taken and if needed via telehealth tools to reach consensus.

   • Frequent physician office visits for wound inspection, especially during the first few days as the most vulnerable time period.

   • Appropriate training for PI and other study personnel at the site.

   • Training for home-care providers and caregivers and patients.

     o Education for the patients, caregivers and home-care providers with specific focus of recognizing any signs of worsening of the infection condition.

   • Periodic review of any safety findings (e.g. treatment failures) by the It should be mentioned that this study introduces potentially substantial bias in favor of the Standard of Care. In this study the rigor and frequency in the physical wound care clearly exceeds the real-world reality and hence maximizes the possible benefit from wound care alone which subsequently increases the hurdles to demonstrate any additional pharmacological treatment effect on the infection.

   Standard of Care recommends starting with an active treatment when clinical signs of infection exist. For this study this means that patients need to be enrolled and treated quickly. A several-day screening period is not acceptable from a patient safety perspective. Timely enrollment, randomization, and treatment as well as the thorough clinical monitoring of the infection condition by the PI as defined in this protocol, immediately after the patient has provided written consent, the lab samples have been collected and were sent to a local laboratory and the plain X-ray of the foot is obtained at Visit 1, the results are available and all eligibility criteria are met, the patient can be randomized the following morning (Visit 2).

   Once the patient is enrolled and assigned to one of the cohorts, close monitoring of the clinical situation continues, specifically during the first week as signs of clinical worsening will lead to discontinuation from the study and the immediate treatment with an oral antibiotic will be initiated. If that happens before results from cultures are available, the therapy may be modified accordingly when the results become available and suggest a different choice of antibiotic treatment.

   Among the options to obtain material for bacterial cultures at several timepoints to monitor treatment the following is proposed for the study: A total of 3 punch biopsies per patient, which are considered as the gold standard, during the 14 days of active treatment. Any higher number is seen as an unacceptable burden to the patient due to the more invasive nature of a punch biopsy. To allow for some data generation on the microbiological environment and wound infection during the intervals between biopsies we will obtain material from swabs during all physician office visit biopsies. While swabs are considered to be less accurate than biopsies, some studies have been published in recent years which suggest that the accuracy of the swab-based results depends on the swab technique. Using the Levine technique is expected to get results of similar quality as biopsy results. The study will also provide additional comparative data between biopsies and Levine Swabs.

   The target iDFU will be established and characterized using information obtained from the subject's medical history and physical examination as well as data from the following tests and measurements: Doppler Sonography, Transcutaneous oxygen tension (TcPO2), or AB index. In addition, qualitative and semiquantitative measurement of ulcer depth and measurement of maximal width/length, depth, and shape, respectively, will be obtained. Final classification will employ the Wagner Scale. Other scales have been considered but rejected because most of them are an aggregate of wound and infection parameters which would partially conflict with the established infection scores.

   The Wagner system (Wagner 1979) assesses ulcer depth and presence of osteomyelitis or gangrene by using the grades listed below. The Wagner Scale outlined in this section will be used for grading ulcers for eligibility.

   1. Pre- or post-ulcerative lesion completely epithelialized.
   2. Superficial, full-thickness ulcer limited to the dermis, not extending to the subcutis.
   3. Ulcer of the skin extending through the subcutis with exposed tendon or bone and without osteomyelitis or abscess formation.
   4. Deep ulcers with osteomyelitis or abscess formation.
   5. Localized gangrene of the toes or the forefoot.
   6. Foot with extensive gangrene.

   The infection grading of the target ulcer will be assessed during screening and will involve at least one external specialist to be consulted for consensus and then at baseline, day 7 and day 14 as defined by the IDSA infection severity criteria. The following criteria are considered:

   Primary clinical signs of infection

   The presence of purulent drainage* or at least two of the following criteria determines a mild infection:

   I. erythema*, II. warmth*, III. pain or tenderness*, IV. edema*, or V. induration*

   *Definitions and exact symptom description are in the protocol appendix. The infection must only involve the skin and subcutaneous tissue (without involvement of deeper tissues and without systemic signs of infections as described below). If erythema is present, it must be > 0.5 cm to ≤ 2cm around the ulcer.

   Other cases of inflammatory response of the skin (e.g., trauma, gout, acute Charcot, neuro- osteoarthropathy, fracture, thrombosis, and venous stasis) must be excluded.

   The above-mentioned clinical signs will be evaluated by the PI as absent, present, and if present as mild, moderate, or severe.

   Cave: the assessment of the infection grade involves symptoms such as erythema and edema which may also occur or worsen as a result of tissue irritation caused by the gel treatment. Special consideration needs to be given to this option in case symptoms occur or worsen under treatment and additional clinical signs must be evaluated to increase the likelihood of a correct causal interpretation of the findings.

   Secondary/ additional indicators of infection:

   The presence or absence of non-purulent secretion, friable or discolored granulation and of a foul odor will be recorded, all being indicative of an infection as well.

   Clinical signs and symptoms for moderate/ severe infection to be excluded.

   The absence of any symptoms which were indicative of moderate and /or severe infection must be documented for eligibility. These symptoms are based on the IDSA guideline (Lipsky 2012):

   • Signs of local infections with erythema > 2 cm or involving structures deeper than skin and subcutaneous tissues (e.g., abscess, Osteomyelitis, septic arthritis, fasciitis).

   • Local infection with signs of systemic infection with 2 or more of

   o Temperature >38° C or <36° C

   • Heart Rate > 90 beats/min
   • Respiratory rate > 20 breath/min or PaCO2, 32mm HG
   • White Blood Cell count > 12000 or < 4000 cells/ µL or ≥10% immature (band) forms This diagnosis of mild infection must be confirmed immediately following debridement at Baseline.

   There is no fully validated semi-quantitative scoring system for infection, wound or combined assessment. However, for exploratory purposes a semi-quantitative scoring as proposed by Lipsky (2009) will be used but slightly modified to better reflect the study objective and purpose of this trial. Lipsky proposed/used in several studies an infection score which also included parameters related to wound healing. The score will be split into two separate scores: one for infection assessment and one for wound healing assessment. The information obtained in this study can be compared to the clinical assessment as well as to the microbiological results to potentially guide assessments in a larger confirmatory study.

Inclusion Criteria:

1. Male and female subjects ≥18 years of age.

2. Voluntary written informed consent, including information about the provisions of the Health Insurance Portability and accountability act (HIPAA) as applicable.

3. Non-hospitalized ambulatory subjects diagnosed with diabetes mellitus, Type I or II per ADA criteria with signs of a localized mild foot infection as defined by the IDSA infection severity criteria (Lipsky,2012). The presence of purulent drainage or at least two of the following criteria:

   i. erythema, ii. warmth, iii. pain or tenderness, iv. edema, or v. induration (The diagnosis of mild infection must be confirmed immediately following debridement at Baseline).

4. The target ulcer is classified as a grade 1 ulcer according to the Wagner Scale (Wagner 1979). The ulcer is a superficial, full-thickness ulcer limited to the dermis, not extending to the subcutis. Target ulcer is >1 cm2 and <12 cm2 post debridement at baseline and must be no higher than the ankle, on or below the malleolus (ankle bone) with ≥50% below the malleolus.

5. Adequate vascular perfusion as evidenced by one of the following:

   1. Dorsal transcutaneous oxygen measurement (TCOM) or a skin perfusion pressure (SPP) measurement of ≥ 40 mmHg
   2. Ankle Branchial Index (ABI) between 0.9 and 1.3 within 3 months of Screening using the extremity with the target ulcer.
   3. Arterial Doppler ultrasound evaluating for biphasic or triphasic dorsalis pedis and posterior tibial vessels at the level of the ankle or a TBI (Toe Brachial Index) of >0.75.

6. Subject has a caregiver who will attend the Baseline visit (V2) and/or watch the dosing and dressing demonstration video and apply wound treatment along with study dressings for the study duration.

7. Must meet one of the following criteria:

   a. Female subjects of Non-Child-Bearing Potential i. Postmenopausal for at least 1 year ii. Surgically sterilized (i.e., hysterectomy or bilateral oophorectomy more than 3 months prior to Screening) iii. Bilateral tube ligation > 6 months prior to screening iv. A negative serum β-hCG pregnancy test at screening and no breastfeeding after the administration of the study drug.

   b. Male subjects of Non-Childbearing Potential defined as: i. Vasectomized subjects for > 6 months prior to Screening ii. Those diagnosed as sterile by a physician. c. Females and Males of Childbearing Potential who practice an acceptable method of contraception defined as the i. Use of any form of hormonal contraceptive ii. Use of a barrier method with spermicide, condoms, intrauterine device, iii. Abstinence from sexual intercourse starting at least 60 days prior to Screening and continuing at least 14 days following the last treatment.

8. Subjects must be willing to undergo all clinical investigation-related procedures, attend all required visits, and cooperate fully with the investigator and site personnel.

9. Subject must be willing to wear offloading RCW, if necessary (defined as ulcer at plantar site of the foot, see Appendix 6), throughout the duration of the clinical treatment.

10. Subject must have plain radiograph taken at screening and prior to randomization showing no evidence of bony abnormalities consistent with osteomyelitis, or gas compatible with tissue crepitus, in the affected foot.

Exclusion Criteria:

1. Ulceration with exposed tendon, capsule, or bone
2. IDSA-defined moderate or severe DFU infection.
3. Infected diabetic foot ulcer that is associated with local wound complications such as prosthetic materials or protruding surgical hardware.
4. > 1 infected foot ulcer
5. Subject is currently receiving topical antimicrobial treatment for a localized infection of the study ulcer or received such topical antimicrobial treatment < 72 hours prior to study enrollment.
6. Subject has received systemic treatment with a long-lasting antibiotic such as azathioprine (within less than 10 days prior to Screening) or any other systemic antibiotic within 48 hours prior to Screening.
7. Concurrent or expected to require systemic antimicrobials during the active treatment study period for any infection including diabetic foot ulcer.
8. Any subject that has active viral hepatitis (A, B, C) and/or untreated HIV/AIDS.
9. Any subject that has vascular compromise requiring surgical intervention or has undergone vascular reconstruction or angioplasty less than 1 month prior to randomization. Any planned surgical procedures during the study participation
10. eGFR <60 and/or subject on hemodialysis within 3 months prior to randomization.
11. Hemoglobin A1c (HbA1c) >12% within 3 months prior to randomization.
12. Aspartate Aminotransferase (AST, GOT) and/or Alanine Aminotransferase (ALT, GPT) >3.0 x the upper limit of normal and/or bilirubin >1.5 x the upper limit of normal within 3 months prior to randomization.
13. Acute active Charcot foot
14. Any subject that would be unable to safely monitor the infection status at home and return for scheduled visits.
15. History of immunosuppression within 3 months prior to randomization, or taking immunosuppressive agents including systemic corticosteroids, except stable daily doses of 5 mg/day or less for chronic conditions
16. Any subject with a life expectancy ≤ 6 months
17. Use of investigational drugs within 28 days prior to screening
18. Use of Aspirin® or acetylsalicylic acid containing medication (except low-dose aspirin) < 7 days before baseline,
19. Use of oral anticoagulants (e.g., warfarin, Xarelto® or comparable products).
20. History of concurrent condition that, in the Investigator's opinion, would jeopardize the safety of the subject or compliance with the protocol including known or suspected active abuse of alcohol, narcotics, or non-prescription drugs.
21. Prior randomization in this clinical trial, or a previous Bisphosphocin study