Method for Stabilizing Colorimetric Assay for Use with Plucked Human Hair

Abstract

Apparatuses are disclosed for performing colorimetric assays with plucked human hair using a device adapted for that purpose. Also disclosed are methods for stabilizing a colorimetric chemical assay so that it can be transported and used as an at-home device for conducting a colorimetric chemical assay are described.

Claims

1. A kit for measuring sulfotransferase enzyme activity in a hair follicle comprising: a container configured to hold a two-part chemistry composition, the two-part chemistry composition having a reactive portion and non-reactive portion; and a reaction vessel configured for combining or mixing contents of the reactive portion and contents of the non-reactive portion; wherein contents of the reactive portion include potassium p-nitrophenyl sulfate; wherein contents of the non-reactive portion include minoxidil, magnesium chloride, and phosphate buffer.

2. The kit of claim 1, wherein the container is configured to hold the contents of the reactive portion in physical and/or chemical isolation from the contents of the non-reactive portion.

3. The kit of claim 1, wherein contents of the reactive portion include a stabilizing solution.

4. The kit of claim 1, wherein the stabilizing solution stabilizes potassium p-nitrophenyl sulfate to form stabilized potassium p-nitrophenyl sulfate.

5. The kit of claim 4, wherein the stabilizing solution is a solvent comprising: a water solution and a buffering agent; an aprotic polar solvent; and/or a protic non-aqueous solvent.

6. The kit of claim 5, wherein the stabilizing solution has a pH within a range from about 9 to about 11.

7. The kit of claim 5, wherein the buffering agent is a carbonate buffer comprising sodium bicarbonate and sodium carbonate.

8. The kit of claim 5, wherein the aprotic polar solvent comprises acetonitrile.

9. The kit of claim 5, wherein the protic non-aqueous solvent comprises isopropanol or ethanol.

10. The kit of claim 1, wherein the phosphate buffer has a pH of about 8.

11. The kit of claim 1, wherein the container is in selective fluid communication, via an actuator, with the reaction vessel such that the contents of the reactive portion and the contents of the non-reactive portion are introduced into the reaction vessel upon actuation of the actuator.

12. The kit of claim 1, wherein the reaction vessel includes an opening configured to receive a hair follicle.

13. A method for determining whether a minoxidil formulation will be effective in a subject in need of hair-regrowth or maintenance, the method comprising: combining or mixing contents of a reactive portion and contents of a non-reactive portion of a two-part chemistry composition to form a mixture or combination, wherein: contents of the reactive portion include potassium p-nitrophenyl sulfate; and contents of the non-reactive portion include minoxidil, magnesium chloride, and phosphate buffer; and placing a hair follicle from the subject in contact with the mixture or combination.

14. The method of claim 13, further comprising: measuring presence of a p-nitrophenol product, wherein the presence of the p-nitrophenol product is positively correlated with sulfotransferase enzyme activity of the hair follicle.

15. The method of claim 14, further comprising: comparing the measured presence of the p-nitrophenol product to a predetermined level; and using the comparison as an indicator that the subject will have a good response to minoxidil treatment.

16. The method of claim 15, wherein: a presence of the p-nitrophenol product greater than the predetermined level indicates a level of sulfotransferase enzyme activity signifying that the subject will have a good response to minoxidil treatment.

17. The method of claim 13, further comprising: mixing and/or incubating the hair follicle with the mixture or combination.

19. The method of claim 17, wherein: mixing and/or incubating the hair follicle with the mixture or combination involves mixing and/or incubating for a period of time that depends on a number of hair follicles placed into contact with the mixture or combination.

20. The method of claim 14, wherein: measuring presence of a p-nitrophenol product involves measuring absorbance of the p-nitrophenol product at about 405 nm using a spectrophotometer; and/or measuring presence of a p-nitrophenol product involves involves comparing a color intensity of the p-nitrophenol product to a color intensity reference card with a range of intensities corresponding to sulfotransferase enzyme activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other objects, aspects, features, advantages and possible applications of the present innovation will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings.

[0019] FIG. 1 shows an exemplary kit that may be used for performing colorimetric assays with plucked human hair.

[0020] FIG. 2 is an exemplary representation of the PART A and PART B components of an exemplary two-part chemistry composition.

[0021] FIG. 3 is an exemplary reaction scheme of the assay.

DETAILED DESCRIPTION

[0022] Examples are described herein in the context of a point-of-care diagnostic device for measuring biomarkers from plucked human hair. The following description is illustrative only and is not intended to be in any way limiting. Other embodiments will readily suggest themselves to such skilled persons having the benefit of this disclosure. Reference will now be made in detail to implementations of the example embodiments.

[0023] In the interest of clarity, not all of the routine features of the implementations described herein are shown and described. In the development of any such actual implementation, numerous implementation-specific decisions must be made in order to achieve the developer's specific goals, such as compliance with application- and business-related constraints, and that these specific goals will vary from one implementation to another and from one developer to another. Moreover, such a development effort might be complex and time-consuming but would nevertheless be a routine undertaking of engineering for those of ordinary skill in the art having the benefit of this disclosure.

[0024] The term “exemplary” is used exclusively herein to mean “serving as an example, instance or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0025] Referring to FIGS. 1-3, embodiments relate to a kit 100 for measuring sulfotransferase enzyme activity in a hair follicle. The kit 100 can include a container 102 and a reaction vessel 104. The kit 100 can include the container 102 and the reaction vessel 104 as separate components. For instance, the kit 100 can include the container 102 as one unit and the reaction vessel 104 as a separate and distinct unit. In the alternative, the kit 100 can include the container 102 and the reaction vessel 104 as part of a single component. For instance, the kit 100 can include a housing or other structure that includes the container 102 and the reaction vessel 104 as a single unit. The kit 100 can include any number of containers 102 and/or reaction vessels 104. There can be one container 102 for one or more reaction vessels 104, an individual container 102 for each individual reaction vessel 104, etc. As will be explained herein, contents of the container 102 will be introduced into the reaction vessel 104. This can be done by pouring the contents of the container 102 into the reaction vessel 104, allowing (e.g., gravity fed, osmosis movement, capillary action movement, etc.) the contents to flow via conduit 103 from the container 102 to the reaction vessel 104, forcing (e.g., via a pump, non-equilibrium kinetics movement, etc.) the contents to flow via conduit 103 from the container 102 to the reaction vessel 104, etc. The conduit 103 can be flow tubes or flow paths (which may be formed in the housing or other structure if the container 103 and reaction vessel 104 are a single unit) that includes the container 102 and the reaction vessel 104, for example. The conduit 103 can be configured to place the container 102 in fluid communication with the reaction vessel 104. Some embodiments can include an actuator 106 (e.g., a valve, solenoid valve, etc.) to control the flow of contents from the container 102 to the reaction vessel 104. The actuator 106 can include a processor, control module, switch, etc. configured to control aspects of the pump, valve, etc. The actuator 106 can be mechanically operated, electro-mechanically operated, etc. Other aspects of the kit 100 can include membranes, filters, gaskets, switches, timers, etc. to facilitate proper control and flow of contents, control of flow rate and amounts, control of flow start and stop times, etc. Thus, the kit 100 can be configured such that the container 102 is in selective fluid communication, via the actuator 106, with the reaction vessel 104 such that the contents of the reactive portion 108 and the contents of the non-reactive portion 110 are introduced into the reaction vessel 104 upon actuation of the actuator 106. It is understood that there may be one or more actuators 106 for a given kit 100.

[0026] The container 102 can be configured to hold a two-part chemistry composition. The two-part chemistry composition can have a reactive portion 108 and non-reactive portion 110. For instance, the container 102 can be a container (e.g., bottle, vessel, tank, flask, etc.) with a bifurcated structure 109 forming a reactive cavity and a non-reactive cavity, each cavity configured to hold and retain contents (e.g., chemical composition). It is contemplated for the contents of each portion 108, 110 to be fluids. The bifurcated structure 109 can be a wall configured to physically and/or chemically separate the contents of the reactive portion 108 and the contents of the non-reactive portion 110. The material of the container 102 can physically and/or chemically isolate the contents of the reactive portion 108 from the non-reactive portion 110 and from the environment outside the container 102—e.g., prevent permeable movement, prevent light from entering, prevent contents of each from mixing, etc. The material of the container 102 can physically and/or chemically isolate the contents of the non-reactive portion 110 from the reactive portion 108 and from the environment outside the container 102—e.g., prevent permeable movement, prevent light from entering, prevent contents of each from mixing, etc.

[0027] The container 102 and the reaction vessel 104 can each include an inlet 112 and an outlet 114 to facilitate ingress and egress of contents. The container 102 may have an individual inlet 112 for each portion 108, 110, a separate individual outlet 114 for each portion 108, 110, etc. In some embodiments, the inlet 112 serves as both the inlet 112 and the outlet 114. The reaction vessel 104 may have an inlet 112 and an outlet 114, or its inlet 112 may serve as both the inlet and outlet 114. Any of the inlets 112 or outlets 114 of the container 102 or the reaction vessel 104 can have a lid 109 or plug to selectively cover the inlet 112 or outlet 114. With embodiments in which the container 102 is separate from the reaction vessel 104, a user can handle the container 102 in their hand and pour to contents from each portion 108, 110, via the outlet 114, into the reaction vessel 104 inlet 112. In addition, or in the alternative, a transfer device, such as a pipette droplet, can be used to transfer the contents—e.g., a user can introduce the pipette into the outlet 114 of the container 102 to retrieve a volume of the contents and dispense the contents into the reaction vessel 104 inlet 112. Also, a pump with conduit 103 lines (extending to and from the inlets 112 and outlets 114) can be used to transfer the contents from the container 102 to the reaction vessel 104. With embodiments in which the container 102 is part of the same unit as the reaction vessel 104, actuator 106 can be actuated to cause/allow flow of contents from the container 102 outlet 114 to the reaction vessel 104 inlet 112.

[0028] It is contemplated for the contents of the reactive portion 108 to not mix or combine with the contents of the non-reactive portion 110 until the contents of each are introduced into the reaction vessel 104. Thus, the methods of content transfer, the arrangement and configuration of conduit 103, transfer devices, valves, actuators 106, etc. can be such that no mixing or combining of the contents occurs until they are introduced into the reaction vessel 104.

[0029] The reaction vessel 104 can be configured for combining or mixing contents of the reactive portion 108 and contents of the non-reactive portion 110. For instance, the reaction vessel 104 can be a vessel having a cavity that receives the contents of the container 102 when the contents are allowed/forced to flow from the container 102 to the reaction vessel 104. The cavity allows for combining or mixing of the reactive portion 108 contents and the non-reactive portion 110 contents. This can be via free mixing as the contents enter the reaction vessel 104, forced mixing, other forms of agitation, etc.

[0030] It is contemplated for the contents of the reactive portion 108 to include potassium p-nitrophenyl sulfate. The contents of the reactive portion 108 can also include a stabilizing solution. The stabilizing solution stabilizes potassium p-nitrophenyl sulfate to form stabilized potassium p-nitrophenyl sulfate. A hydrogen is required to convert potassium p-nitrophenyl sulfate (PNPS) to potassium p-nitrophenyl (PNP) and SO.sub.3. Having a basic pH minimizes the amount of available protons and stabilized PNPS. The stabilizing solution can have a pH within a range from about 9 to about 11. In some embodiments, the stabilizing solution can be a solvent comprising a water solution and a buffering agent. The buffering agent can be a carbonate buffer comprising sodium bicarbonate and sodium carbonate. In some embodiments, the stabilizing solution can be a solvent comprising aprotic polar solvent. The aprotic polar solvent can comprise acetonitrile. In some embodiments, the stabilizing solution can be a protic non-aqueous solvent. The protic non-aqueous solvent can comprise isopropanol or ethanol.

[0031] It is contemplated for the contents of the non-reactive portion 110 to include minoxidil, magnesium chloride, and phosphate buffer. The SULT1A1 enzymatic reaction is optimized at about pH 8, and thus the phosphate buffer can have a pH of about 8.

[0032] The reaction vessel 104 can include an opening 116 configured to receive at least one hair follicle. This opening 116 can be the same as the reaction vessel 104 inlet in some instances. The reaction vessel 104 can be configured to receive at least a portion of the hair follicle (e.g., only a portion of the hair follicle is inserted within the reaction vessel 104) or the entire hair follicle (e.g., the entire hair follicle is inserted within the reaction vessel 104). It is contemplated for the hair follicle to be placed into contact with the mixture or combination of contents (the mixture or combination of reactive portion 108 contents and non-reactive portion contents). This can include making contact with the mixture or combination, being submerged within a bath of the mixture or combination, etc. The reaction vessel 104 can be configured to receive more than one hair follicle, which can be via one opening 116 or via multiple openings 116.

[0033] Embodiments can relate to a method for determining whether a minoxidil formulation will be effective in a subject in need of hair-regrowth or maintenance. The method can involve combining or mixing contents of a reactive portion 108 and contents of a non-reactive portion 110 of a two-part chemistry composition to form a mixture or combination. Contents of the reactive portion can include potassium p-nitrophenyl sulfate. The contents of the reactive portion 108 can also include a stabilizing solution. The stabilizing solution stabilizes potassium p-nitrophenyl sulfate to form stabilized potassium p-nitrophenyl sulfate. The stabilizing solution can be a solvent comprising a water solution and a buffering agent, an aprotic polar solvent, or a protic non-aqueous solvent. It is contemplated using pure solvent for the aprotic polar or protic non-aqueous solvents. For the water based solution, it is contemplated using 50 mM carbonate buffer. The range can be from 10 to 100 mM and from 100 mM to 1M buffer; however, the lowest amount possible to stabilize PNPS is preferred. The contents can include stabilized potassium p-nitrophenyl sulfate in which the solvent is removed so as to leave only stabilized solutes. The solvent can be removed (e.g., 90-100% is removed) via air drying, vacuum, e.g., using a benchtop concentrator, lyophilizaton, etc. For instance, the contents can be subjected to 20 minutes of air drying under vacuum (e.g., 20 to 0.1 Torr).

[0034] Contents of the non-reactive portion can include minoxidil, magnesium chloride, and phosphate buffer.

[0035] The method can involve placing a hair follicle from the subject in contact with the mixture or combination. The method can involve mixing and/or incubating the hair follicle with the mixture or combination. Mixing can involve stirring, use of a magnetic mixer, shaking, other forms of agitation, etc. Mixing and/or incubating can involve mixing and/or incubating for a period of time that depends on a number of hair follicles placed into contact with the mixture or combination. For instance, it is contemplated that the more hair follicles used for given implementation, the less mixing and/or incubation is needed. For instance, the incubation time can be 24 hrs. for 2 hair follicles, 12 hrs. for 4 hair follicles, etc. The volume of contents used in the reaction vessel 104 can also depend on the number of hair follicles placed into contact with the mixture or combination. For instance, the incubation time using a reaction volume of 100 uL would be twice the incubation time if reaction volume of 50 uL is used.

[0036] Presence of the p-nitrophenol product is positively correlated with sulfotransferase enzyme activity of the hair follicle, and thus the method can involve measuring presence of a p-nitrophenol product within the mixture or combination. The method can involve comparing the measured presence of the p-nitrophenol product to a predetermined level. The predetermined level can be a threshold level used as an indicator that the subject will have a good response to minoxidil treatment. For instance, a presence of the p-nitrophenol product greater than the predetermined level can indicate a level of sulfotransferase enzyme activity signifying that the subject will have a good response to minoxidil treatment. A presence of the p-nitrophenol product less than the predetermined level can indicate a level of sulfotransferase enzyme activity signifying that the subject will not have a good response to minoxidil treatment.

[0037] In the presence of minoxidil sulfotransferase activity, p-nitrophenyl sulfate is converted to the colorimetric p-nitrophenol. Thus, measuring presence of a p-nitrophenol product can involve measuring absorbance of the p-nitrophenol product at about 400-410 nm (preferably 405 nm) using a spectrophotometer 118 (e.g., spectroscopy instrument having a sensor to collect electromagnetic emission of light passing through a solution and a processor for interpreting or quantifying the collected electromagnetic emissions). In an exemplary embodiment, absorbance may be interpreted as follows: (a) absorbance less than 0.4 AU can indicate minoxidil will not likely work; (b) absorbance equal to 0.4 AU can indicate an equivocal test; or (c) absorbance greater than 0.4 AU can indicate minoxidil may work.

[0038] In addition, or in the alternative, measuring presence of a p-nitrophenol product can involve comparing a color intensity of the p-nitrophenol product to a color intensity reference card with a range of intensities corresponding to sulfotransferase enzyme activity. For instance, the color intensity of p-nitrophenol may be compared to a reference color card with a range of intensities corresponding to SULT1A1 activity. Subjects with a relatively high level of sulfotransferase activity will have a relatively strong colorimetric readout, resulting in a relatively significant color change (change in color of the mixture due to any of the p-nitrophenyl sulfate converting to colorimetric p-nitrophenol). In comparison, subjects with a relatively low level of sulfotransferase activity will have a relatively weak colorimetric readout (change in color of the mixture due to any of the p-nitrophenyl sulfate converting to colorimetric p-nitrophenol), and correspondingly, a relatively minimal color change. Subjects with a strong colorimetric assay response would be expected to respond to minoxidil for hair re-growth or retention. Whereas, subjects with a weak colorimetric assay response would be expected to have a poor response to minoxidil. The color intensity reference card can be a physical card or virtual card (e.g., computer graphical user interface) that is pre-made based data from previous tests. The comparison of the colorimetric readout to the color intensity reference card can be by visual human inspection, by computer algorithmic inspection, etc.

[0039] Hence, if the hair follicle contains SULT1A1 (or a sufficient amount of SULT1A1), p-nitrophenyl sulfate is converted to the colorimetric p-nitrophenol (or a sufficient amount of p-nitrophenyl sulfate is converted to the colorimetric p-nitrophenol) so as to indicate a sufficient level of sulfotransferase enzyme activity, which can be used as a predictor that the subject will have a good response to minoxidil treatment.

[0040] While embodiments and applications have been shown and described, it would be apparent to those skilled in the art having the benefit of this disclosure that many more modifications than mentioned above are possible without departing from the inventive concepts disclosed herein. The invention, therefore, is not to be restricted except in the spirit of the appended claims.

Example 1: Minoxidil Response Test (MRT), Two Part Chemistry Formulation

[0041] The example procedure uses the following reagents: sodium phosphate buffer (500 mM), sodium carbonate, sodium bicarbonate, potassium p-nitrophenyl sulfate, minoxidil, MgCl.sub.2 solution (1 M), water (0.1 μm filtered) and methanol.

[0042] The example procedure is for production of 1000 units of MRT reactions. PNPS deposited sample tubes are prepared (PART A), as well as the reconstituting solution (SOLUTION B). A single MRT reaction unit is created by adding 100 μL of SOLUTION B to a PNPS deposited sample tube (PART A).

Manufacturing Procedure

1.0 Solution 1: PNPS Buffer 10 mM (pH 10)

[0043]

TABLE-US-00001 TABLE 1 PNPS Buffer 10 mM (pH 10) Preparation Calculated Actual Component Amount per 1 L Amount Amount Sodium Bicarbonate 0.388 g g g Sodium Carbonate 0.571 g g g Pure Water  1.0 L L L

[0044] Compound the solution according to the calculated amounts indicated in the table as follows: add the pure water to an appropriately sized container, add sodium bicarbonate and sodium carbonate and mix on a magnetic stir plate until dissolved.

[0045] Measure the pH of the final solution (pH 10).

2.0 Solution 2: Potassium p-Nitrophenyl Sulfate (PNPS) 50 mM (pH 10)

TABLE-US-00002 TABLE 2 Potassium p-nitrophenyl sulfate (PNPS) 50 mM (pH 10) Preparation Amount per 5 Calculated Actual Component mL T Amount Amount Potassium p-Nitrophenyl Sulfate 0.128 g g g PNPS Buffer 10.0 mL mL mL

[0046] Compound the solution according to the calculated amounts indicated in the table as follows: add PNPS buffer to an appropriately sized container, add potassium p-nitrophenyl sulfate and mix on a magnetic stir plate until dissolved.

3.0 Solution 3-100 mM Minoxidil Stock Solution

[0047]

TABLE-US-00003 TABLE 3 100 mM Minoxidil Stock Solution Preparation Amount Calculated Actual Component per 5 mL Amount Amount Minoxidil 0.105 g g g Methanol 5.0 mL mL mL

[0048] Compound the solution according to the calculated amounts indicated in the table as follows: add the methanol to an appropriately sized container, add the minoxidil and mix on a magnetic stir plate until dissolved.

[0049] Store stock solution at about −20° C. for up to 1 year.

4.0 PNP Deposited Tube (PART A)

[0050]

TABLE-US-00004 TABLE 4 Solution 2 Calculation Materials Amount per Calculated Amount Solution 2 10 uL mL

[0051] The materials in step 4.0 include: Solution 2 (PNPS 50 mM, pH 10) and a sample tube (500 μL).

[0052] Perform the following: (1) add 10 μL of Solution 2 to MRT sample tube, (2) place open tube in speedvac and air dry under vacuum with spinning until all liquid has evaporated (approximately 20 min) and (3) store at ambient temperature.

[0053] All volumes shall be within 5% of the specification. The minimum time required to evaporate liquid should be determined for each instrument (speedvac) used. Overdrying may reduce stability of PART A.

5.0 Solution B

[0054] The materials in step 5.0 include: 500 mM phosphate buffer, pH 8.0; 1 M magnesium chloride solution; 100 mM minoxidil stock solution and water.

TABLE-US-00005 TABLE 5 Solution B Preparation Amount per Calculated Actual Component 100 mL Amount Amount 500 mM Phosphate Buffer, pH    10 mL mL mL 8.0 1M Magnesium Chloride  0.100 mL mL mL Solution 100 mM Minoxidil Solution  0.500 mL mL mL Pure Water 89.400 mL mL mL

[0055] Compound the solution according to the calculated amounts indicated in the table, then mix on a magnetic stir plate until uniform.

[0056] All volumes shall be within 5% of specification. All weights shall be within 5% of specification. The pH should be 8±0.5.

Example 2: Accelerated Stability Protocol

Test Components

[0057] Two (2) verification batches (lots) of potassium p-nitrophenyl sulfate (PNPS) deposited tubes were created and stored at elevated temperature. Each lot contains a total of twenty-four (24) PNPS deposited tubes. During the course of the accelerated stability study, 3 samples of each lot will be removed from the elevated temperature and reconstituted with the second part of the two-part chemistry (Solution B). After 24 h of reconstitution, samples are visually inspected for yellow color.

justification FOR MODEL

[0058] Subjecting test devices to extreme temperature conditions is intended to simulate product degradation over time, but at an accelerated rate. Using the Arrhenius equation, shelf-life data can be extrapolated as a predictive measurement for product performance. The Arrhenius equation relates chemical reaction rate (k) to absolute temperature (T):

d(Ink)/dT=ΔE.sub.a/RT.sup.2

[0059] Where E.sub.a is the activation energy and R is the universal gas constant.

TABLE-US-00006 TABLE 6 Arrhenius Model for MINOXIDIL SENSITIVITY ASSAY Treatment Days Required for Days Required for Days Required for Temperature 1 year stability @ 1.5 year stability @ 2 year stability @ (° C.) 23° C. 23° C. 23° C. 50  56.2 days  84.3 days 112.3 days 40 112.3 days 168.6 days 224.6 days

[0060] Using the Arrhenius model at 20 kcal, the test reagent would have satisfactory performance theoretically for 1 years if specifications were met after 56.2 days at 50° C., or 112.3 days at 40° C.

[0061] NOTE: The 40° C. samples are run as a contingency in the event that the 50° C. conditions are too extreme for the test samples. In the event that test samples remain stable for 56.2 days at 50° C., the 40° C. experiment is terminated.

Presumed Storage Conditions

[0062] The performance of the PNPS deposited tubes is evaluated when stored at 50° C. and 40° C. All storage locations conform to Applied Biology QSR to assure appropriate temperature ranges are met during the study.

Objective and Purpose of Testing

[0063] The purpose of stability testing at 50° C. and 40° C. is to generate data to determine the shelf life of the product. The data is used as a predictor of stability for the final product form.

Test Kit Storage Conditions

[0064] Kits (reagent) is stored at 50° C. and 40° C. (f 2° C. for all temperatures).

Interval Between Analyses

[0065]

TABLE-US-00007 TABLE 7 MINOXIDIL SENSITIVITY ASSAY Accelerated Stability Study Storage Temp Duration 50° C. Day 6, 14, 28, 42, 56, 70, 84, 98 40° C. Day 6, 14, 28, 70, 98, 126, 154, 182

[0066] An accelerated stability is carried out on the MINOXIDIL SENSITIVITY ASSAY reagent stored at 50° C.±2° C. and 40° C.±2° C. as described in Table 7. Reagent is tested in triplicate. If a testing day falls on a weekend or holiday, the test point is either eliminated (depending on the testing interval) or tested on the first workday following the scheduled time.

Stability Criteria

[0067] The accelerated stability studies must initially pass the acceptance targets detailed in Table 8.

TABLE-US-00008 TABLE 8 MINOXIDIL SENSITIVITY ASSAY Specificity Release Testing and Stability Specifications Result Specification Result Description (visual) Determination PNPS deposited tubes + 100 uL Solution B Clear Pass PNPS deposited tubes + 100 uL Solution B Slight Yellow Pass PNPS deposited tubes + 100 uL Solution B Yellow Fail

Notes:

[0068] (1) Results are evaluated 24 hours after reconstitution with Solution B. [0069] (2) Reagent must meet the specifications below (#4) for 56.2 days at 50° C. or 112.3 days at 40° C. to yield a projected 12-month shelf life at 23° C. [0070] (3) A failure at a specific temperature may not be an indicator of the shelf life since higher temperatures may adversely affect the solution chemistry. Thus, a 1 year shelf life may not be projected at the 50° C. treatment, where it may be projected at the 40° C. treatment. [0071] (4) A time point is deemed a failure when fail is observed on two (2) consecutive testing time points. The failure time may defer from that of the first time point failure. If there is an invalid test result, the test may be retested once to confirm the result. A failure may also be retested once to confirm the result. If upon retest, the failure is not confirmed, an additional test may be run to assess the status of the time point.

Attachment A

[0072]

TABLE-US-00009 Total 0 6 14 28 42 56 70 84 98 126 154 182 Vials 50 0 0 0 0 0 0 0 0 24 40 0 0 0 0 0 0 0 0 24

Notes:

[0073] Samples shall be evaluated in triplicate.

[0074] If a testing day falls on a weekend or holiday, the test point is eliminated (depending on the testing interval) or tested on the first work day following the scheduled time.

[0075] 1 Year Estimated stability: 50° C.=56.2 days, 40° C.=112.3 days

Results

[0076]

TABLE-US-00010 TABLE X Days 0 6 14 28 42 56 70 50C Pass Pass Fail NA NA NA NA 40C Pass Pass Pass Pass Pass Pass Pass Days 84 98 126 154 182 50C NA NA NA NA NA 40C Fail Fail NA NA NA Accelerated Stability Report for Formula (pH 7.5-9.5)

TABLE-US-00011 TABLE Y Formula G2 (pH 7.5-9.5) Days 0 6 14 28 42 56 70 50C Pass Pass Pass Pass Pass Pass Pass 40C Pass Pass Pass Pass Pass Pass Pass Days 84 98 126 154 182 50C Pass Pass Pass Pass Pass 40C Pass Pass Pass Pass Pass Accelerated Stability Report for Formula (pH 9.5-10.5)

Example 3: Functional Testing of Reconstituted PART A and PART B

[0077] Samples deemed stable from Example 2, i.e., those that remained clear after 24 h of reconstitution, were reacted with two human hair follicles to test whether the reconstituted chemistries were functional.

[0078] Specifications

TABLE-US-00012 Result Specification Result Description (visual) Determination PNPS deposited tubes + 100 uL Solution Clear Fail B + Hair PNPS deposited tubes + 100 uL Solution Yellow Pass B + Hair

Results

[0079]

TABLE-US-00013 TABLE X Days 0 6 14 28 42 56 70 50C Pass Pass NA NA NA NA NA 40C Pass Pass Pass Pass Pass Pass Pass Days 84 98 126 154 182 50C NA NA NA NA NA 40C NA NA NA NA NA Accelerated Stability Report for Formula (pH 7.5-9.5)

TABLE-US-00014 TABLE Y Formula G2 (pH 7.5-9.5) Days 0 6 14 28 42 56 70 50C Pass Pass Pass Pass Pass Pass Pass 40C Pass Pass Pass Pass Pass Pass Pass Days 84 98 126 154 182 50C Pass Pass Pass Pass Pass 40C Pass Pass Pass Pass Pass Accelerated Stability Report for Formula (pH 9.5-10.5)