Andrew Bakst1✉, Andrew Verbinnen1, Mitchell Graumenz3, Rutu Jagtap1, Lamia Tahsin1, Sneha Gowda1, Aarzoo Parikh1, Zachary Schrier1, Aonia Traxler1, Blake Bloxham1,2
(2026)
Low-dose oral minoxidil is increasingly prescribed off-label for androgenetic alopecia but is administered using an immediate-release (IR) tablet formulation originally developed for severe hypertension. The IR profile produces a sharp early serum peak that approaches the cardiac-effect anchor concentration, whereas the follicular pharmacodynamics rely on sustained SULT1A1-mediated sulfation of minoxidil. We characterize MINX, a lipid-matrix smooth-release oral minoxidil compounded formulation. In vitro dissolution at pH 1.2 and pH 6.8 showed progressive release over 12 h, contrasting with near-complete IR release within 5 min. In a two-subject pilot pharmacokinetic study (one subject each for fed and fasted dosing), fed dosing produced a flat, sustained serum profile still ascending at 8 h. Measured AUC₀–₈ was 29.0 ng·h/mL. The post-8 h tail was modeled and projected to AUC₀–∞ ≈ 60 ng·h/mL with a modeled C_max ≈ 6.5 ng/mL near 9–10 h, of the same order as the Fleishaker IR 5 mg cohort-mean AUC of 55.1 ng·h/mL. Fasted dosing produced an early washout (AUC₀–∞ ≈ 12.9 ng·h/mL; relative bioavailability ≈23%), consistent with gastric transit truncating matrix release. At comparable total exposure, MINX produced a modeled fed C_max approximately 6-fold lower than the IR reference and well below the published cardiac-effect anchor concentration. These pilot findings, bounded by the two-subject design and the modeled post-8 h tail, support advancing MINX to a powered within-subject crossover with concurrent cardiovascular monitoring as the appropriate confirmatory study.
Keywords: minoxidil; prolonged-release; pharmacokinetics; food effect; androgenetic alopecia; lipid matrix; SULT1A1; dissolution.
Androgenetic alopecia is the most common form of progressive hair loss in adults and remains incompletely addressed by topical minoxidil, whose efficacy is constrained by variable scalp penetration and adherence over chronic daily use. Over the past decade, low-dose oral minoxidil (LDOM) has emerged as an off-label standard-of-care in many dermatology practices, with multiple case series and retrospective cohorts supporting its use across male and female patterns at doses substantially below those historically used for hypertension [1,2,3,5,6]. Prescribing volumes have surged following lay-press coverage, expanding real-world exposure well beyond the originally reported referral populations [7]. International Delphi consensus has subsequently codified pragmatic dosing ranges, screening considerations, and monitoring practices for routine clinical use [8]. Despite these developments, immediate-release (IR) oral minoxidil was not designed for the follicle. It was developed and approved as a potent peripheral vasodilator for severe, refractory hypertension [14,16], and its labeled cardiovascular liabilities, including reflex tachycardia, sodium and fluid retention, and pericardial effusion, remain biologically continuous with the dose-limiting adverse events observed at the upper end of contemporary LDOM practice, alongside hypertrichosis [3,15,16].
At the 5 mg oral dose used widely in dermatology, IR minoxidil produces a sharp plasma peak of approximately 37 ng/mL within roughly 30 minutes of administration [13]. This peak approaches and exceeds the 21.7 ng/mL plasma concentration identified in the topical product monograph as the threshold at which heart-rate effects become clearly distinguishable from placebo [17]. The acute cardiovascular liabilities of minoxidil are widely understood to be peak-driven rather than total-exposure-driven, reflecting the kinetics of arteriolar smooth-muscle relaxation and reflex sympathetic activation [14,15]. The follicular mechanism is mechanistically distinct. Minoxidil itself is pharmacologically inert at the hair follicle; the active species is minoxidil sulfate, generated locally by SULT1A1 expressed in the outer root sheath [9,10]. SULT1A1 activity varies between individuals on a genetic and enzymatic basis and has been shown to predict clinical response to both topical and oral minoxidil [11,12]. Follicular pharmacodynamics are therefore believed to be sustained-substrate-dependent rather than peak-dependent. Together, these observations describe a pharmacological mismatch in which the IR oral profile maximizes the axis associated with acute cardiovascular liability while doing little to optimize the axis associated with follicular conversion. We hypothesized that a formulation decoupling these axes, by sustaining low-peak systemic exposure across the dosing interval, could preserve follicular substrate availability while attenuating the peak-driven component of the systemic dose-response.
One published low-peak alternative is sublingual minoxidil. In a phase 1B study, a 0.45 mg sublingual dose produced a mean peak plasma concentration of 1.62 ng/mL and was associated with hair-growth signals over four weeks [4]. Sublingual administration, however, presents adherence and practical limitations for chronic once-daily use. An oral prolonged-release formulation that achieves comparable peak suppression while delivering systemic exposure in the range of established LDOM regimens would be pharmacologically attractive on the basis of the hypothesis above. Here we characterize MINX, a lipid-matrix prolonged-release oral minoxidil compounded formulation. This pilot work has three aims: (i) to characterize in vitro dissolution behavior at two biorelevant pH values; (ii) to measure single-dose serum pharmacokinetics in two healthy adult males, one each under fed and fasted conditions; and (iii) to compare the in vivo profile against the Fleishaker IR reference [13] in order to quantify the degree of peak suppression and to estimate relative bioavailability. Efficacy and safety endpoints were not assessed in this pilot and are not claimed.
In vitro dissolution was used to confirm that the lipid-matrix capsule (MINX) released minoxidil progressively rather than as a bolus, and to compare its release kinetics against an immediate-release (IR) 2.5 mg oral tablet under non-compendial small-volume magnetic-stirred conditions at two physiologically relevant pH values [27,28]. Standard curves were linear over the working range in both media (pH 1.2: y = 0.04888x − 0.00328, R² = 0.9993; pH 6.8: y = 0.08031x − 0.00066, R² = 0.9999), supporting quantitative comparison of release profiles.
The IR comparator exhibited near-complete release within the first sampling window in both media, with 111.45% recovered at 5 min in pH 1.2 and 100.08% recovered at 5 min in pH 6.8, and recoveries remaining flat between approximately 100% and 113% across all subsequent time points through 12 h. This behavior is consistent with the rapid disintegration and high aqueous solubility expected of an IR minoxidil tablet [13].
MINX showed a markedly different profile (Fig. 1). In pH 1.2, both replicate vessels (R1, R2) released only 2.33% and 3.12% at 5 min, climbing to 11.06% and 20.46% at 60 min, 54.68% and 80.02% at 4 h, and 105.61% and 95.37% at 8 h, with values of 107.03% and 93.17% at 12 h. The contrast was more pronounced in pH 6.8, the medium considered most biorelevant for small-intestinal absorption [26]: no measurable release was detected at 5 min or 1 h in either vessel, with values of only 0.41% in both vessels at 2 h, 6.06% and 7.83% at 4 h, 16.00% and 20.04% at 6 h, 25.59% and 30.95% at 8 h, and 64.07% and 78.97% at 12 h. Inter-vessel variability was modest in pH 1.2 but larger in pH 6.8, where the spread between R1 and R2 reached approximately 15 percentage points at 12 h. Overall, the dissolution behavior observed for MINX is consistent with a prolonged-release lipid-matrix system, with both pH 1.2 and 6.8 profiles providing a clear prolonged-release signal.
To characterize systemic exposure, serum minoxidil was quantified by LC-MS/MS [21] in a single subject after a 5 mg MINX dose taken with food. Under fed conditions, serum minoxidil rose gradually across the 8-hour sampling window (Fig. 2). Concentrations were 0.072 ng/mL at 0.5 h (BLOQ; below 0.5 ng/mL LLOQ), 0.214 ng/mL at 1 h (BLOQ), 2.676 ng/mL at 2 h, 4.393 ng/mL at 4 h, 4.975 ng/mL at 6 h, and 6.026 ng/mL at 8 h. The curve was still ascending at the last measured time point, so an observed C_max and T_max were not captured within the protocol's sampling window. A modeled C_max of approximately 6.5 ng/mL near 9–10 h was projected based on physiological gastric emptying combined with small-intestinal transit (approximately 6 h plus an additional 3–4 h), recognizing that this value is extrapolated, not measured.
This pilot characterization establishes one robust pharmacokinetic finding: at total exposure within approximately 10% of a published immediate-release (IR) 5 mg cohort mean (fed AUC ≈ 60 ng·h/mL vs Fleishaker IR 55.1 ng·h/mL [13]), MINX produced a peak serum concentration approximately six-fold lower than the IR reference (modeled fed C_max ≈6.5 ng/mL vs IR ≈37 ng/mL [13]). The in vitro and in vivo data are internally consistent. Prolonged release was observed in biorelevant dissolution at both pH 1.2 and pH 6.8, with more pronounced sustained release at pH 6.8, the pH most relevant to small-intestinal absorption. This dissolution behavior maps directly onto the flat, sustained serum profile observed under fed conditions, which was still rising at the final 8 h sampling timepoint. We frame this as a descriptive pharmaceutical characterization of the formulation. It is not a demonstration of clinical benefit, and is not proposed as such.
The dominant pharmaceutics observation is the approximately four- to five-fold difference between fed and fasted exposure (fed AUC ≈ 60 ng·h/mL; fasted AUC ≈ 12.9 ng·h/mL; F_rel,fasted ≈23%). Gastric residence appears to be the rate-limiting factor. Under fasted conditions, the matrix is swept distally before release completes; Wagner-Nelson reconstruction [18] indicated ongoing release into the decay phase with plateaus incomplete, consistent with the dosage form reaching the colon, where minoxidil absorptive surface is low, before substrate liberation is finished. Approximately three-quarters of the administered dose appears unaccounted for in systemic exposure under fasting. Under fed conditions, prolonged gastric residence combined with the small-intestinal transit window of approximately three to four hours aligns the matrix release profile with the absorptive window. The release behavior is consistent with the wax-matrix erosion-diffusion kinetics described for Compritol-based systems [26-30]. The clinical implication is unambiguous within the limits of this pilot: administration with food is not a preference for this formulation, it is a pharmaceutical requirement.
A mechanistic rationale for considering whether sustained substrate delivery is desirable at the follicle is worth articulating, with explicit caveats. Minoxidil itself is inert at the hair follicle; the pharmacologically active species is minoxidil sulfate, generated in situ by SULT1A1 . SULT1A1 activity in scalp follicles is genetically variable, and enzyme activity at baseline has been associated with the magnitude of clinical response to topical minoxidil . The enzyme operates over time and is believed to depend on the availability of substrate during the period it is active, rather than on transient peak substrate concentration. An IR oral dose presents a sharp substrate pulse followed by many hours of declining and low substrate availability, whereas a flat, sustained serum profile presents continuous substrate availability across many enzyme-hours. A first-principles argument can therefore be made that a sustained profile is not merely equivalent to an IR pulse on a total-exposure basis, but may be more aligned with the temporal structure of the follicular pharmacodynamics. This is a hypothesis. The present pilot does not measure hair growth, follicular sulfate generation, or any pharmacodynamic endpoint; it characterizes the systemic profile of the formulation and does not test the efficacy hypothesis.
MINX is a lipid-matrix prolonged-release oral capsule formulation of minoxidil intended for once-daily administration, containing 5 mg of minoxidil USP per capsule as the sole active pharmaceutical ingredient. The matrix-forming excipient is glyceryl behenate (Compritol® 888 ATO), a non-ionic lipid with documented utility as a rate-controlling matrix former in hot-melt and melt-granulation prolonged-release systems [26-30]. Manufacturing details, including granulation method, excipient ratios, and process parameters, are described in priority U.S. provisional application No. 64/078,367 (filed 29 May 2026, attorney docket HAIR-010) and are not reproduced in this manuscript. The finished product was compounded by a licensed 503A compounding pharmacy under valid individual prescription. MINX is not an FDA-approved drug product.
For in vitro dissolution, the comparator was a single commercial lot of Loniten® 2.5 mg immediate-release (IR) oral minoxidil tablets (Pharmacia & Upjohn/Pfizer), originally indicated for severe hypertension. The same lot was used throughout the dissolution work to minimize lot-to-lot variability. For in vivo pharmacokinetic comparison, no contemporaneous IR arm was dosed. Instead, cohort-mean parameters were drawn from the single-dose 5 mg IR oral minoxidil study of Fleishaker et al. (n = 29 completers; Latin-square crossover; HPLC bioanalytics) [13]: C_max 37.2 ng/mL at T_max 0.39 h, AUC 55.1 ng·h/mL, apparent oral clearance Cl₀ 95.2 L/h, terminal t½ 1.27 h, and apparent volume of distribution V_d/F 184 L.
In vitro dissolution testing was performed using a non-compendial small-volume magnetic-stirred method (not USP Apparatus II or I) to provide preliminary release-rate characterization of the lipid-matrix capsule under two physiologically relevant pH conditions. Each dissolution vessel was a 500 mL borosilicate glass conical (Erlenmeyer) flask filled with 250 mL of pre-warmed dissolution medium, stirred by a magnetic stir bar of uniform size on a Corning PC-420D digital hotplate-stirrer (Corning Inc., CLS6795420D). The stirrer dial was set between positions 6 and 7 and maintained consistently across vessels to avoid surface vortexing. Per the Corning PC-420D manufacturer manual, dial 6 corresponds to approximately 380 rpm and dial 7 to approximately 550 rpm; the operating stirring speed in this run is therefore estimated at approximately 380–550 rpm. A digital tachometer reading was not available; the value reported here is derived from manufacturer-specified dial-to-rpm calibration rather than directly measured. Medium temperature was maintained at 37 ± 0.5 °C throughout each run. Two dissolution media were evaluated in accordance with USP-recommended pH conditions for orally administered solid dosage forms: pH 1.2 acidic medium (NaCl 4.0 g, concentrated HCl ≈14 mL adjusted to final pH, Milli-Q water q.s. to 2 L; acceptable range 1.1–1.3) simulating gastric conditions, and pH 6.8 phosphate buffer (KH₂PO₄ 13.6 g, NaOH for pH adjustment, Milli-Q water q.s. to 2 L; acceptable range 6.7–6.9) simulating small-intestinal conditions. Both media were pre-warmed prior to use. The 2.5 mg IR comparator was run in a single vessel (n = 1) in each medium; MINX 5 mg was run in duplicate vessels (n = 2; R1 and R2) in each medium. Dosage forms were added intact (not crushed or opened) at t = 0. Because comparator dissolution was conducted at n = 1, comparator vessel-to-vessel variability cannot be assessed from this run.
All authors are affiliated with Anagen and/or HairDAO, which develops the MINX formulation characterized in this work. A U.S. provisional patent application covering aspects of the MINX formulation has been filed (USPTO application 64/078,367; attorney docket HAIR-010; filed 29 May 2026; first-named inventor A. Bakst). The authors declare no other competing interests.
All quantitative data underlying the analyses presented in this manuscript are tabulated in the Results section, the Methods section, and the figure legends. The raw bioanalytical worksheets (LC-MS/MS serum minoxidil concentrations and in vitro dissolution UV absorbance values) are available from the corresponding author upon reasonable request. Python source code for the non-compartmental analysis, Wagner-Nelson reconstruction, and Bateman reference simulation is available upon reasonable request from the corresponding author.
The authors thank the staff of the licensed compounding pharmacy that prepared MINX under valid prescription. We acknowledge the broader HairDAO community for ongoing scientific discussion. This work was supported by Anagen / HairDAO internal research funds. No external funding was received.
The modeled T_max is materially later than the 0.5–6 h T_max range previously reported for IR oral minoxidil formulations [13,14]. Measured exposure over the sampled interval was AUC₀–₈ = 29.0 ng·h/mL (single subject, descriptive only); extrapolation of the release-limited tail yielded a modeled AUC₀–∞ of approximately 60 ng·h/mL. Against the cohort-mean AUC of 55.1 ng·h/mL reported by Fleishaker and colleagues for a 5 mg IR oral dose in 29 healthy volunteers [13], this corresponds to an estimated relative bioavailability of approximately 100%.
We note the point estimate exceeds 100% by approximately 9%, which reflects two known artefacts: tail extrapolation beyond the last measured concentration, and the use of cohort-mean clearance borrowed from a separate study to convert single-subject data. Relative bioavailability cannot, in principle, exceed administered dose; the honest interpretation is that the fed exposure was of the same order as the immediate-release cohort mean of the 5 mg dose, with the caveat that this estimate derives from a single subject and is descriptive only. No formal bioequivalence inference is implied. The fed-state profile is qualitatively that of a flat, sustained absorption phase rather than a sharp peak-and-decay curve, consistent with release-limited (flip-flop) kinetics.
Under fasted conditions, the same 5 mg MINX dose produced a quantitatively and qualitatively different exposure profile. Serum minoxidil was below the limit of quantification (BLOQ) at baseline, rose to an early peak of 3.037 ng/mL at 2 h, and then declined through 1.588 ng/mL at 4 h, 0.784 ng/mL at 6 h, and 0.483 ng/mL at 8 h (BLOQ), returning to BLOQ by 12 h. Measured AUC₀–₁₂ was 12.3 ng·h/mL (single subject, descriptive only), and extrapolated AUC₀–∞ approximately 12.9 ng·h/mL, yielding an estimated relative bioavailability of approximately 23% relative to the Fleishaker IR 5 mg reference [13].
The apparent terminal slope of the fasted curve was k = 0.31 h⁻¹, corresponding to an apparent half-life of approximately 2.3 h. This value should not be interpreted as an intrinsic elimination half-life of minoxidil. The measured terminal half-life of minoxidil after IR oral administration in the Fleishaker cohort is 1.07–1.34 h [13], well below the 2.3 h observed here. The most parsimonious interpretation is that fasted MINX exhibits release-limited (flip-flop) kinetics, in which the apparent decline rate reflects the rate of dissolution from the matrix rather than elimination from plasma.
Wagner-Nelson absorption reconstruction [18] supported this interpretation (Fig. 3). The cumulative amount absorbed, A(t) = C(t) + k_el·AUC(0→t), was computed under the assumption of dose-linear, single-compartment behavior, justified by the dose-linearity established in the Fleishaker dataset [13]. Although serum concentration was decaying from 2 h onward (3.04 → 1.59 → 0.78 → 0.48 ng/mL), A(t) continued to climb monotonically, indicating that the matrix was still releasing as it transited the gastrointestinal tract. The fasted A(t) curve plateaued incomplete near a normalized index of approximately 7, consistent with the formulation being swept into the colon, where minoxidil absorption is minimal, before release was complete. By contrast, the fed A(t) curve was still climbing steeply at 8 h. This mechanism provides a parsimonious explanation for the approximately 4–5-fold fed/fasted difference in relative bioavailability: food prolongs gastric residence and slows transit, aligning the slow-release window of the matrix with the small-intestinal absorption window.
To place the fed-state MINX profile in context, the measured concentrations were compared with a reconstructed IR 5 mg reference curve (Fig. 4). The reference was simulated using a one-compartment Bateman model parameterized to the Fleishaker cohort-mean values: C_max = 37.2 ng/mL, T_max = 0.39 h, and k = 0.546 h⁻¹ [13]. We emphasize that the IR curve is a literature-derived cohort-mean simulation, not a within-subject comparator.
The two profiles differed qualitatively. The IR reference rose sharply to 37 ng/mL within approximately 25 min and then declined exponentially. The MINX fed profile rose gradually to a modeled C_max of approximately 6.5 ng/mL near 9–10 h with no observed spike. The C_max ratio is therefore approximately 6-fold lower for MINX fed (6.5 vs 37.2 ng/mL), the modeled T_max is approximately 25-fold delayed (≈9–10 h vs 0.39 h), and total exposure (AUC₀–∞) is comparable (≈60 vs 55.1 ng·h/mL, a difference of approximately 9%).
Two literature-derived horizontal references are overlaid on Fig. 4 for orientation. The first, at 21.7 ng/mL, corresponds to the serum concentration anchor associated with cardiovascular effects of minoxidil derived from a topical minoxidil monograph [17]. The IR reference approaches this concentration at T_max, whereas the modeled MINX fed curve remains 3–4-fold below it throughout. The second, at 1.62 ng/mL, corresponds to the mean peak serum concentration achieved after a 0.45 mg sublingual minoxidil dose associated with hair regrowth at 4 weeks in a phase 1B study [4]. Both profiles cross this line for several hours. We explicitly caution against framing time-above-1.62-ng/mL as a clean MINX advantage. This duration is sensitive to the assumed terminal half-life. Using Fleishaker's measured terminal half-life of 1.27 h [13], the IR curve clears the 1.62 ng/mL line for approximately 6 h; using the 4.2 h Loniten® label terminal half-life [16], it clears for approximately 21 h. The modeled MINX fed curve clears it for approximately 11–17 h depending on assumed release-limited slope. The robust, descriptively defensible finding in this pilot dataset is therefore peak suppression at comparable total exposure, not a difference in duration above an exploratory threshold.
A second hypothesis, separable from the first, concerns cardiovascular safety. The dose-limiting acute cardiovascular liabilities of oral minoxidil (reflex tachycardia, fluid retention, and rarely pericardial effusion) have long been hypothesized to be peak-concentration-driven [14,15]. The topical product monograph anchors this empirically, reporting that a 6.86 mg oral dose yielding a mean serum concentration of approximately 21.7 ng/mL was the lowest dose clearly distinguishable from placebo on heart rate [17]. The MINX fed C_max of approximately 6.5 ng/mL sits three- to four-fold below that anchor. If the peak-concentration hypothesis is correct, a formulation that delivers full systemic exposure while capping C_max at roughly one-sixth of the IR value may warrant prospective testing of whether the cardiovascular liability profile differs from that of IR dosing. This pilot, however, did not measure heart rate, blood pressure, body weight, peripheral edema, or any cardiovascular endpoint, and provides no direct evidence on this point. The hypothesis requires prospective testing in a powered cohort with cardiac monitoring.
Within the broader oral minoxidil literature, low-dose oral minoxidil (LDOM) has seen rapid uptake for androgenetic alopecia, with the largest safety series describing favorable tolerability in 1,404 patients [3] and consensus-based prescribing guidance now emerging [1,2,7,8]. LDOM is, however, an off-label use of an IR cardiovascular formulation never engineered for chronic dermatologic dosing. Sublingual minoxidil [4] represents one published low-peak alternative; an oral PR formulation is mechanistically related but practically distinct, since chronic daily compliance generally favors conventional oral dosing. The present work contributes a two-subject pilot characterization of one such oral PR candidate.
Aliquots of 1.2–1.5 mL were withdrawn from the middle of each vessel without contacting the dosage form at 5, 10, 15, 30, and 45 min and at 1, 2, 4, 6, 8, and 12 h post-immersion. Each aliquot was filtered immediately through a 0.45 µm syringe filter and 1 mL of filtered sample was retained for analysis; 1 mL of pre-warmed matching medium was replenished after each draw. A cumulative-correction formula was applied to account for analyte mass removed in prior aliquots; cumulative amount released was computed as M_n = C_n · 250 + Σ_{i=1}^{n-1} C_i µg, and percent release was referenced to nominal dose (2,500 µg for comparator; 5,000 µg for MINX). Minoxidil was quantified by ultraviolet absorbance at 230 nm using a Thermo Scientific Varioskan LUX multimode plate reader (Cat. No. N16045) controlled by SkanIt Software 7.0 Research Edition (Thermo Fisher Scientific). Each filtered dissolution sample (200 µL) was loaded into duplicate wells of a UV-compatible 96-well plate alongside the matching medium blank in duplicate; duplicate absorbance values were averaged and blank-corrected before concentration calculation. Calibration standards were prepared in matching dissolution medium from a 1,000 µg/mL minoxidil stock in methanol, diluted to 0, 2.5, 5, 10, 15, 20, and 25 µg/mL, and loaded in triplicate on each plate. Standard-curve fitting used ordinary linear regression; the pH 1.2 curve was y = 0.04888x − 0.00328 (R² = 0.9993) and the pH 6.8 curve was y = 0.08031x − 0.00066 (R² = 0.9999); both met the acceptance criterion R² > 0.99. Values exceeding 100% were interpreted as complete release with superimposed analytical and method variability and were not truncated.
The in vivo phase comprised two single-dose, single-period pharmacokinetic studies conducted in two healthy adult male subjects, one each for fed and fasted conditions, as a physician-supervised self-experimentation protocol. Subject 1 (fed period) was a 30-year-old male, height 185 cm (6 ft 1 in), body weight 82 kg (180 lb). Subject 2 (fasted period) was a 30-year-old male, height 193 cm (6 ft 4 in), body weight 93 kg (205 lb). This was not a within-subject crossover; fed and fasted exposure estimates therefore reflect two different individuals with broadly comparable demographics but a body-weight difference of approximately 13% that may contribute to inter-period variability independent of food effect.
The study was conducted as physician-supervised self-experimentation by Dr. Blake Bloxham (Bloxham Medical, Long Island, NY), outside the jurisdiction of a formal Institutional Review Board (IRB) or Independent Ethics Committee (IEC). Both participants provided written informed consent prior to dosing. This design is consistent with the published methodological and ethical framework for N-of-1 trials [23] and is offered as descriptive pilot characterization, not as a controlled clinical investigation.
Subject 1 (fed period) consumed a standard high-fat meal totaling approximately 900 kcal with 50 g of fat (450 kcal from fat, ≈50% of total caloric content), consistent with the FDA Food-Effect Bioavailability and Fed Bioequivalence Studies Guidance specification for a high-fat / high-calorie test meal, within 30 min before dosing. Subject 2 (fasted period) observed an overnight fast of at least 10 h before dosing and abstained from food for 4 h post-dose. In both cases a single 5 mg MINX capsule was administered orally with water. No concomitant medications were taken in the 24 h preceding or following dosing.
Venous blood samples were collected by peripheral venipuncture, 8 mL per timepoint, into BD Vacutainer Plus Plastic Serum Tubes (BD #367820; 10 mL, 16 × 100 mm, red conventional stopper, silicone-coated with clot activator, no separator gel). Samples were allowed to clot at room temperature for 30 min before centrifugation in a Thermo Scientific Sorvall X Pro Series centrifuge at 2,800 rpm for 10 min at 4 °C, with acceleration and deceleration ramps both set to 9 (maximum). The supernatant serum fraction was aliquoted into three cryovials of 500 µL each per timepoint, stored overnight at −80 °C, and transferred next morning on dry ice to the contracted bioanalytical laboratory for LC-MS/MS analysis. Subject 1 (fed) sampling times were 0 (pre-dose), 0.5, 1, 2, 4, 6, and 8 h post-dose; Subject 2 (fasted) sampling times were 0 (pre-dose), 2, 4, 6, 8, and 12 h post-dose. The collection protocol yields serum rather than plasma; the reference Fleishaker cohort used plasma [13], a methodological difference noted in the Limitations.
Chemicals and reagents.
Minoxidil reference standard and deuterated internal standard minoxidil-d10 were used for preparation of calibration standards and quality-control (QC) samples. Reference standard purity ≥ 99% and vendor/lot details are maintained in the bioanalytical laboratory records.
Sample preparation.
Serum aliquots were processed by protein precipitation with methanol. Following protein precipitation, supernatant was transferred to an autosampler vial for injection.
Liquid chromatography.
Chromatographic separation was performed on a Phenomenex Kinetex XB-C18 column (50 × 2.1 mm, 2.6 µm particle size) using a Sciex ExionLC HPLC system. The mobile phase consisted of (A) 0.1% (v/v) formic acid in water and (B) 0.1% (v/v) formic acid in methanol, delivered as a gradient over a 4-minute run at a flow rate of 0.5 mL/min; the chromatographic configuration is consistent with published LC-MS approaches for minoxidil quantification [22].
Mass spectrometry.
Detection was performed on a Sciex API 5500 triple-quadrupole tandem mass spectrometer with electrospray ionization (ESI) interface operated in positive ion mode using multiple reaction monitoring (MRM). The quantifier transition for minoxidil was m/z 210.2 → 164.0, and for the deuterated internal standard minoxidil-d10 was m/z 220.2 → 169.1, both at collision energy 32 V.
Calibration and quality control.
Calibration standards were prepared at 8 non-zero levels spanning 0.5–50 ng/mL, encompassing the 0.5–10 ng/mL concentration range observed in this pilot. The calibration curve was fitted by 1/x² weighted linear regression, with an observed coefficient of determination R² = 0.9958. Quality control samples were prepared at three concentrations (LQC 1.5 ng/mL, MQC 15 ng/mL, HQC 37.5 ng/mL) with two replicates per level. All QC concentrations fell within ±8% of nominal target value, well within the FDA 2018 Bioanalytical Method Validation Guidance for Industry [19] acceptance criterion of ±15%.
Lower limit of quantification (LLOQ).
The LLOQ was 0.5 ng/mL, corresponding to the lowest non-zero calibrator. Most measured concentrations fell above the LLOQ. Three timepoints (Subject 1, 0.5 h and 1 h; Subject 2, 8 h) yielded values below the LLOQ of 0.5 ng/mL and are reported as below-quantitation-limit (BLOQ) signals rather than fully quantitative concentrations. These BLOQ values were retained in the trapezoidal AUC calculation; their inclusion changes AUC₀–₈ by less than 1%.
Pilot bioanalytical validation status.
The bioanalytical run used stable-isotope dilution with minoxidil-d10 as the deuterated internal standard, providing isotope-dilution quantitation rather than external-standard semi-quantitation. The QC replication structure (n = 2 replicates per level across three concentration levels) is fewer than the n ≥ 5 per concentration recommended by FDA 2018 [19] and ICH M10 [20] guidance for full bioanalytical method validation, so the present pilot should be considered a pilot bioanalytical assay rather than a fully validated method. Two unused 500 µL serum aliquots per collection timepoint remain in storage at −80 °C and are available for a confirmatory rerun against a fully validated calibration and QC structure if subsequent work warrants.
Non-compartmental analysis (NCA) was performed in Python 3 using numpy and scipy. AUC₀–t was computed by the linear trapezoidal rule on measured serum minoxidil concentrations, with pre-dose (t = 0) and below-quantifiable-limit (BLOQ) values set to zero. AUC₀–∞ was computed as AUC₀–last + C_last / k_el, where k_el denotes the apparent terminal elimination rate constant. In the fasted state, k_el was estimated by log-linear regression of the terminal four measured timepoints (2–8 h), giving an apparent terminal half-life t½ = ln(2)/k_el = 2.3 h.
For a prolonged-release product whose in vivo release rate is slower than the intrinsic elimination rate of the parent drug, the apparent terminal slope reflects absorption (release) rate rather than elimination rate, a condition known as 'flip-flop' kinetics. The intrinsic terminal t½ of minoxidil reported in the Fleishaker IR cohort is 1.07–1.34 h [13]. The 2.3 h apparent t½ observed in the fasted MINX state therefore reflects release-limited rather than elimination-limited kinetics. For the fed state, serum concentrations had not yet peaked at the final (8 h) sample, so the post-8 h tail was modeled rather than measured. Three terminal-slope assumptions were applied to bound the projected AUC: 1.2 h (intrinsic elimination; a non-physical floor for a PR product), 2.3 h (the observed fasted release-limited slope; central estimate), and 4.2 h (slower-release / fed-prolonged; upper bound). The projected tail followed C(t) = 6.5 · exp(−k(t − 10)) for k ∈ {0.58, 0.31, 0.165} h⁻¹, respectively.
Wagner-Nelson cumulative absorption reconstruction was applied to both fed and fasted profiles using A(t) = C(t) + k_el · AUC(0 → t), consistent with the approximate dose-proportionality of minoxidil pharmacokinetics reported by Fleishaker et al. across 2.5–10 mg single oral doses [13,18]. Relative oral bioavailability was computed as F_rel = AUC₀–∞(MINX) / AUC₀–∞(Fleishaker 5 mg IR = 55.1 ng·h/mL) and capped at 100%. For Fig. 4, a one-compartment first-order oral absorption (Bateman) model was scaled to the Fleishaker cohort-mean C_max (37.2 ng/mL) and T_max (0.39 h), yielding k_a = 7.14 h⁻¹ and k_el = 0.546 h⁻¹, and used as the IR reference overlay.
Two horizontal reference lines appear on the serum concentration–time figures and are presented as literature-derived context, not as MINX-validated thresholds. The 21.7 ng/mL line is the minoxidil-monograph-derived serum concentration described as 'clearly distinguishable from placebo, based on heart rate data' [17]; it is a published anchor for the cardiac-effect concentration and is not a safety threshold qualified for MINX. The 1.62 ng/mL line is the mean peak serum minoxidil concentration observed after a single 0.45 mg sublingual dose in the phase 1B trial of Bokhari et al. [4], a concentration compatible with hair-growth efficacy after 4 weeks of dosing rather than a derived minimum efficacious concentration. Topical minoxidil studies have not demonstrated a correlation between systemic minoxidil concentration and hair-growth response [24]. Both lines are therefore interpreted as literature reference values, not as MINX-qualified efficacy or safety thresholds.