Evaluation of Vamorolone Mineralocorticoid Receptor Antagonism in Healthy Subjects

The purpose of this study is to investigate the antagonistic effect of vamorolone on the mineralocorticoid receptor following mineralocorticoid challenge by fludrocortisone, compared to eplerenone as a positive control, and to a negative control with no study treatment. Furthermore, the safety and tolerability of vamorolone combined with fludrocortisone challenge will be assessed, and the Pharmacokinetic (PK) of a single dose of vamorolone and a single dose of eplerenone, combined with fludrocortisone challenge, will be evaluated.

• Vamorolone (Drug), also known as experimental drug: vamorolone 20 mg/kg single dose on Day 2
• Eplerenone (Drug), also known as positive control: Eplerenone 200 mg single dose on Day 2
• Fludrocortisone (Drug), also known as auxiliary medicinal product - mineralocorticoid agonist: Fludrocortisone challenge on Days 1 to 3 (for all subjects): Day 1: -Fludrocortisone 1 mg at 9 h predose vamorolone /eplerenone administration/corresponding timepoint for negative control arm Day 2: * Fludrocortisone 0.5 mg at the same time of vamorolone/eplerenone administration in the morning (0 h) /corresponding timepoint for negative control arm. * Fludrocortisone 0.1 mg at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 14 h vamorolone/eplerenone postdose administration/corresponding timepoint for negative control arm. * Fludrocortisone 0.5 mg at 16 h vamorolone/eplerenone postdose administration/corresponding timepoint for negative control arm. Day 3: -Fludrocortisone 0.1 mg at 24 h vamorolone/eplerenone postdose administration on Day 2/corresponding timepoint for negative control arm.

Primary outcomes

• Determination of the Ratio of Sodium to Potassium (Na/K) in Urine [Day 1, Day 2 and Day 3]: Amounts of Na and K were calculated by multiplying the respective concentration by the volume of urine for each collection interval. The considered timepoints were: Day 1: 24-9 h and 9-0 h predose of vamorolone/eplerenone; Day 2: 0-2 h, 2-4 h, 4-6 h, 6-8 h, 8-10 h, 10-12 h, 12-14 h, 14-16 h, 16-24 h postdose vamorolone/eplerenone or corresponding timepoint for negative control arm with fludrocortisone administrations (no treatment) Day 3: 24-32 h, 32-40 h, 40-48 h postdose vamorolone/eplerenone or corresponding timepoint for negative control arm with fludrocortisone administrations (no treatment) The individual Na/K ratio was then calculated for each urine collection interval using the amounts of Na and K. The corresponding logarithm of the Na/K ratio was determined. To avoid negative values, the ratio was multiplied by 10 before transformation, i.e., log10(10*Na/K).

Secondary outcomes

• Vamorolone PK Parameter AUC0-tlast [Day 2]: The area under the concentration-time curve (AUC) from time zero (= dosing time) to the time of the last quantifiable concentration (tlast). The collection points used to determine the curve were as follows: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Vamorolone PK Parameter AUC0-inf [Day 2]: The AUC from time zero (dosing time) extrapolated to infinity estimated. The collection points used to determine the curve were as follows: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Vamorolone PK Parameter Cmax [Day 2]: Maximum plasma drug concentration after administration of vamorolone. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Vamorolone PK Parameter Tmax [Day 2]: Time to reach maximum concentration following vamorolone administration. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Vamorolone PK Parameter t1/2 [Day 2]: Elimination half-life is the amount of time it takes for the concentration of a drug in the body to decrease by half. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Eplerenone PK Parameters AUC0-tlast [Day 2]: The area under the concentration-time curve (AUC) from time zero (= dosing time) to the time of the last quantifiable concentration (tlast).The collection points used to determine the curve were as follows: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Eplerenone PK Parameter AUC0-inf [Day 2]: The AUC from time zero (dosing time) extrapolated to infinity. The collection points used to determine the curve were as follows: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Eplerenone PK Parameter Cmax [Day 2]: Maximum plasma drug concentration after administration of eplerenone. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Eplerenone PK Parameter Tmax [Day 2]: Time to reach to the maximum concentration after eplerenone. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2
• Eplerenone PK Parameter t1/2 [Day 2]: Elimination half-life is the amount of time it takes for the concentration of a drug in the body to decrease by half. For this PK measure, the following timepoints were considered: predose; and 1, 2, 4, 8, 12, 24 h postdose on Day 2

Sex: Male, ages: 18 Years to 55 Years, accepts healthy volunteers

Inclusion Criteria:

1. Age of 18 to 55 years inclusive, at the time of signing the informed consent.
2. Male is overtly healthy as determined by medical evaluation including medical history, physical examination, vital signs, laboratory tests and ECG.
3. Body weight ≥ 50 kg and a BMI ≥ 18 kg/m2 and ≤ 29.9 kg/m2 at screening
4. Contraceptive use by men and women should be consistent with local regulations regarding the methods of contraception for those participating in clinical studies.

   If the subject is a sexually active man and not surgically sterilized, he must be willing to:
   * Abstain from sexual intercourse or
   * Use a condom plus another form of contraception (e.g., spermicide, Intrauterine device (IUD), birth control pills taken by female partner, diaphragm with spermicide) if engaging in sexual intercourse with a woman who could become pregnant.
   * Use a condom during sexual intercourse with pregnant or lactating women.
   * Must not father a child and must refrain from donating sperm from administration of the first dose and up to 3 months after the last dose of study treatment.
5. Subject is a non-smoker for at least 3 months prior to exposure to the study treatments.

   Subjects must also have abstained from use of other nicotine containing products (e.g., nicotine patch, chewing gum or e-cigarettes) for at least 3 months before exposure to the study treatment.
6. Capable of giving signed informed consent, which includes compliance with the requirements and restrictions listed in the Informed consent form (ICF) and in this protocol prior to any clinical study specific procedure.
7. Supine systolic blood pressure of ≥ 90 mmHg and ≤ 140 mmHg, diastolic blood pressure of ≥ 50 mmHg and ≤ 90 mmHg, pulse rate of ≥ 45 bpm and ≤ 90 bpm, and tympanic body temperature of ≥ 35.0 and ≤ 37.5°C at screening.
8. Subjects must be able to communicate well with the Investigator and comply with the protocol requirements, instructions, and protocol related restrictions (e.g., dietary, fluid and lifestyle restrictions from screening to study completion)
9. Subjects must be able to swallow the study treatments as per protocol

Exclusion Criteria:

1. A past medical history of clinically significant abnormalities or a history/family history of long QT interval syndrome.
2. An abnormal ECG, defined as:

   * Pulse rate (PR) > 215 msec and < 120 msec, QRS (Part of electrocardiographic wave representing ventricular depolarization) complex > 120 msec; QTcF > 440 msec by automated reading
   * Any clinically significant cardiac conduction abnormalities
   * Any atrial or ventricular arrhythmias
3. A past medical history of myocardial infarction, angina pectoris, atherosclerosis, or other clinically significant heart disease (e.g.congestive heart failure, uncontrolled hypertension, history of labile hypertension)
4. A past medical history of peptic ulcers, diverticulitis, and non-specific ulcerative colitis.
5. History of complaints of frequent dizziness and /or vomiting spells or lightheadedness.
6. Any history or evidence of any clinically relevant, gastrointestinal, respiratory, hepatic, renal, endocrinologic, hematologic, immunologic, metabolic, genitourinary, pulmonary, neurologic, dermatologic, musculoskeletal, and/or other major disease or malignancy as determined by medical evaluation (including physical examination) capable of significantly altering the absorption, metabolism, or elimination of drugs; constituting a risk when taking the study treatments; or interfering with the interpretation of data.
7. Known Gilbert's syndrome.
8. Any clinically relevant history of allergic conditions requiring hospitalization or prolonged systemic treatment (including drug allergies, allergic asthma, eczema, allergies requiring therapy with corticosteroids or anaphylactic reactions); but excluding untreated, asymptomatic, seasonal allergies at time of dosing or allergic contact sensitizations (e.g., nickel allergy).
9. Known or suspected hypersensitivity or contraindications to the study treatments or any components of the formulation used, e.g., vamorolone, eplerenone and fludrocortisone.
10. Relevant current acute or chronic/recurrent viral, bacterial, fungal or parasitic infections (e.g., pulmonary/upper respiratory, gastrointestinal, urinary, skin, or ear, nose and throat (ENT) infections) at screening or within 28 days prior to administration of the study treatments.
11. Use of any concomitant medication or any drugs / medicines (including dietary supplements, natural and herbal remedies, and hormone replacement therapy) within 2 weeks or 5 times the half-life of the respective drug, whichever is longer, prior to the first administration of the study treatments.

    Occasional use of paracetamol up to 2 g/day (medicinal product in its original packaging, approved and marketed in Germany) is permitted.

    Oral, injectable, and implantable contraceptives as outlined in protocol section 5.1 are permitted.
12. Previous exposure to vamorolone.
13. Any use of corticoids within 6 months prior to the first administration of the study treatments.
14. Administration of live, attenuated, replication-competent vaccines within 6 weeks prior to the first administration of study treatment until 2 weeks after the follow-up visit. Administration of inactivated vaccines or vector-based or messenger ribonucleic acid (mRNA ) COVID-19 vaccines within 2 weeks prior to the first administration of the study treatments until 2 weeks after the follow-up visit.
15. Treatment with biologic agents (such as monoclonal antibodies including marketed drugs) within 3 months or 5 half-lives (whichever is longer) prior to the first administration of the study treatments.
16. Use of any investigational drug or participation in any clinical study within 30 days or 5 half-lives (whichever is longer) prior to the expected date of first administration of study treatments or planning to take other investigational drugs during the study.
17. Positive results for HBsAg, anti-HCV (Hepatitis C virus), anti-HIV 1 and 2, and HIV 1-p24 antigen at screening.
18. Positive screen for alcohol, drugs of abuse and cotinine at screening.
19. Elevations in Alanine aminotransferase (ALT) ≥ 1.1 x ULN (Upper limit of normal) , AST (Aspartate aminotransferase) ≥ 1.1 x ULN, serum bilirubin ≥ 1.0 x ULN, creatinine ≥1.0 x ULN and HbA1c > ULN at screening. A case-by-case decision for any abnormality must be discussed with the Sponsor before inclusion.
20. Sodium, potassium, magnesium, chloride blood concentration below the lower limit of normal at screening.
21. Thyroid-stimulating hormone (TSH) and coagulation outside of normal ranges.
22. eGFR (Estimated glomerular filtration rate) based on the Chronic kidney disease epidemiology collaboration (CKD-EPI) of < 90 mL/min at screening.
23. Subjects who are unwilling to adhere to contraceptive requirements.
24. Higher than low-risk alcohol consumption i.e., consumption of an average weekly alcohol intake of > 21 units/week for men. 1 unit (12 g) corresponds to 0.3 L of beer/day or 0.12 L of wine/day or 1 glass (at 2 cL) of spirits/day.
25. Excessive consumption of caffeine- or xanthine-containing food or beverages (> 5 cups of coffee a day or equivalent) or inability to stop consuming from 48 hours prior to first planned administration of study treatments.
26. Consumption of alcohol from 48 hours prior to admission
27. Consumption of high-dose resveratrol-containing products or products with enzyme-inducing or enzyme-inhibiting properties 14 days prior to first administration of study treatments.
28. Regular consumption of poppy seed containing food prior to first administration of study treatments.
29. No sweets/teas containing liquorice are allowed within 2 weeks prior to first administration of study treatments up to the follow-up examination.
30. Any use of drugs-of-abuse or alcohol abuse within 1 month prior to dosing.
31. Subject with vegetarian, vegan, or restricted diet (e.g., gluten-free) or not willing or able to eat the complete standard meals.
32. Donation or loss of more than 400 mL of blood or received a transfusion of any blood or blood products within 30 days, or donated plasma within 30 days prior to first administration of study treatments.
33. Strenuous physical activity within 72 h to admission.
34. Employee of the Sponsor, the Nuvisan Group, or other Contract Research Organization involved in the clinical study.
35. Legal incapacity or limited legal capacity, or incarceration and vulnerable subjects.
36. Inability to understand or communicate reliably with the Investigator or considered by the Investigator to be unable to or unlikely to co-operate with the protocol requirements, instructions, and study-related restrictions
37. History of non-compliance to medical regimens and subjects who are considered potentially unreliable (e.g., refuse to comply with study regulations).
38. Any other conditions or factors which in the opinion of the Investigator may interfere with study conduct.
39. Changes in medical conditions compared to screening, as judged by the Investigator.
40. Body weight < 50 kg.
41. Changes in prior/concomitant therapy compared to screening, as judged by the Investigator.
42. Positive for an acute common cold/respiratory or other infection upon admission.
43. Positive screen for alcohol, drugs of abuse and cotinine test upon admission.
44. Changes in other exclusion criteria compared to screening, as judged by the Investigator.

Participant flow

Outcome results

• Determination of the Ratio of Sodium to Potassium (Na/K) in Urine (Primary), Geometric mean, log10(10*Na/K): Study Arm 1 (Test Arm)Vamorolone 1.12929 +/- 1.090602; Study Arm 2 (Positive Control Arm): Eplerenone 0.98207 +/- 1.290309; Study Arm 3 (Negative Control Arm): no Treatment 1.04238 +/- 1.407236
• Vamorolone PK Parameter AUC0-tlast (Secondary), Mean, h*ng/ml: Study Arm 1 (Test Arm): Vamorolone 19802.6 +/- 4422.56
• Vamorolone PK Parameter AUC0-inf (Secondary), Mean, h*ng/ml: Study Arm 1 (Test Arm): Vamorolone 19879.8 +/- 4419.77
• Vamorolone PK Parameter Cmax (Secondary), Mean, ng/mL: Study Arm 1 (Test Arm): Vamorolone 3942 +/- 1018.7
• Vamorolone PK Parameter Tmax (Secondary), Median, h: Study Arm 1 (Test Arm): Vamorolone 1.992 (1.00 to 2.00)
• Vamorolone PK Parameter t1/2 (Secondary), Mean, h: Study Arm 1 (Test Arm): Vamorolone 3.170 +/- 0.6253
• Eplerenone PK Parameters AUC0-tlast (Secondary), Mean, h*ng/ml: Study Arm 2 (Positive Control Arm): Eplerenone 18631.4 +/- 9242.04
• Eplerenone PK Parameter AUC0-inf (Secondary), Mean, h*ng/ml: Study Arm 2 (Positive Control Arm): Eplerenone 19899.9 +/- 11361.57
• Eplerenone PK Parameter Cmax (Secondary), Mean, ng/ml: Study Arm 2 (Positive Control Arm): Eplerenone 2362 +/- 551.42
• Eplerenone PK Parameter Tmax (Secondary), Median, h: Study Arm 2 (Positive Control Arm): Eplerenone 2.000 (0.97 to 2.02)
• Eplerenone PK Parameter t1/2 (Secondary), Mean, h: Study Arm 2 (Positive Control Arm): Eplerenone 4.453 +/- 2.2996

Adverse events

Time frame: All adverse events AEs)and serious AE(SAEs) were collected from the signing of the informed consent until the safety follow-up phone call on Day11. All SAEs were to be recorded & reported to the sponsor within 24hours of awareness,filing in the safety form provided. Investigators were not obligated to actively seek AEs or SAEs after conclusion of the study participation. If the investigator learned of any SADR (SAE considered related to the IMP) at any time it had to be reported to the sponsor

Other adverse events by term

• Results publication: de Vera A, Clemens PR, Dang UJ, Dutreix C, Gresko E, Guglieri M, Hagerty L, Hasham S, Damsker J, Hathout Y, Linden A, Berglund A, Tobin R, Wahbi K, Hoffman EP. Mineralocorticoid receptor antagonism of vamorolone: Evidence from LIONHEART and VISION-DMD clinical trials. Steroids. 2025 Nov;223:109689. doi: 10.1016/j.steroids.2025.109689. Epub 2025 Sep 14. PMID 40957504

• Study Protocol (PDF, 2024-02-26)
• Statistical Analysis Plan (PDF, 2024-07-17)

• Steffen Haffner, MD, Principal investigator, Nuvisan GmbH