CN117110220A

CN117110220A - Method for stabilizing colorimetric assays for use with pulled-out human hair

Info

Publication number: CN117110220A
Application number: CN202210789982.1A
Authority: CN
Inventors: O·A·格伦; J·麦考伊
Original assignee: Fulia International Co
Current assignee: Fulia International Co
Priority date: 2022-05-17
Filing date: 2022-07-05
Publication date: 2023-11-24

Abstract

An apparatus for colorimetric analysis with pulled-out human hair using a device suitable for this purpose is disclosed. Also disclosed are methods for stabilizing a colorimetric chemical analysis such that it can be transported and used as a home device for performing the described colorimetric chemical analysis.

Description

Method for stabilizing colorimetric assays for use with pulled-out human hair

Cross Reference to Related Applications

The present patent application relates to and claims the benefit of priority from U.S. provisional patent application No. 63/364,837 filed on 5/17 of 2022, the entire contents of which are incorporated herein by reference.

Technical Field

The application relates to a device for colorimetric analysis with pulled-out human hair using a device suitable for this purpose. Also disclosed are methods for stabilizing a colorimetric chemical analysis such that it can be transported and used as a home device for colorimetric chemical analysis using extracted human hair.

Background

Point-of-care (POC) diagnostic devices are commonly used to analyze biomarkers of different human health or disease states. For example, pregnancy tests are lateral flow POC reporting the presence of the β subunit of human chorionic gonadotrophin (hCG) in urine. hCG is a biomarker produced by trophoblast cells of fertilized eggs. Thus, the presence of hCG in urine may be reported to the user of the device by a chemical reaction that produces a visible colour or change in the device, indicating to the user that pregnancy is occurring. Many different POC devices are currently available, typically using body fluids as substrates (e.g., blood, urine, or saliva). Each device has similar features: they are typically portable, hand-held and made of plastic. Most POCs can be used only once and are considered disposable.

Currently, there are laboratory-based tests for measuring the biomarker sulfotransferase 1A1 (SULT 1 A1) in extracted human hair follicles (see us patent 10,633,688 and 8,691,518). The method involves the use of chemical compositions that will spontaneously develop color over time (typically days or weeks). The spontaneous reaction can be slowed down by refrigerating the test solution. In the presence of SULT1A1 enzyme, the reaction rate increases (catalyzes), and color is produced in a much shorter time (several hours). The test is set up in such a way that the amount of color produced in a given amount of time (e.g., 24 hours) is indicative of the amount of SULT1A1 enzyme present in the hair follicle.

SULT1A1 is an enzyme that catalyzes the transfer of a sulfo group from a donor molecule to an acceptor alcohol. The enzyme has a broad distribution of human tissue, including the Outer Root Sheath (ORS) of the liver and hair follicles. Inter-individual changes in sulfonation capability are important in determining the metabolism of certain xenobiotics by an individual. Minoxidil is a drug that requires sulfonation to be effective. The sulfonation ability of an individual's ORS can be used to exclude non-responders to 5% minoxidil for treating androgenic alopecia.

Disclosure of Invention

Described herein are embodiments including an instant care device for measuring SULT1A1 biomarkers contained in one or more extracted human hairs. The chemical substance of the device is arranged in two parts in such a way that it does not react spontaneously. Thus, the described device has increased stability and can be stored at room temperature. The described device can be used to measure sulfotransferase activity in one or more pulled-out human hairs to predict the effectiveness of the drug minoxidil for treating androgenic alopecia.

In one embodiment of the application, the two-part chemical composition is prepared such that the reactants are sequestered and stabilized. In one embodiment, the reactant is potassium p-nitrophenyl sulfate (PNPS). In one embodiment, one part of the two-part chemical composition (part a) is prepared by adding PNPS to the stabilizing solution. In one embodiment, an aqueous solution containing a pH 9 to pH 11 buffer is used to stabilize PNPS. In one embodiment, the buffer is a carbonate buffer (pH 10) containing sodium bicarbonate and sodium carbonate. In another embodiment of the application, aprotic polar solvents are used to stabilize PNPS solutions. In yet another embodiment, the aprotic polar solvent is acetonitrile. In another embodiment, a protic nonaqueous solvent is used to stabilize the PNPS. In another embodiment, the protic non-aqueous solvent is isopropanol or ethanol.

In one embodiment of the application, one part of the two-part chemical composition (part a) is deposited in a reaction vessel. In another embodiment, the solvent is removed, leaving only part a of the stabilized solute. In another embodiment, the solvent is removed by air drying and vacuum. In yet another embodiment, a bench top concentrator (e.g., speedvac) is used to remove solvent from the stabilized part a solution. In yet another embodiment, the solvent is removed using a lyophilization process.

In one embodiment of the application, a 50mM PNPS solution is stabilized in 10mM carbonate buffer (pH 10). In yet another embodiment, 10 μl of 50mM PNPS solution stabilized in 10mM carbonate buffer (pH 10) is added to a reaction vessel (e.g., 500 μl tube) and the water is evaporated under vacuum. In another embodiment, a bench top concentrator (speedvac) is used to remove water from the stabilized PNPS solution.

In one embodiment of the application, the second part (part B) of the two-part chemical composition is prepared by adding all non-reactive ingredients to the solution. In one embodiment, the second part of the two-part chemical composition contains 50mM phosphorusAcid salt buffer (pH 8), 100. Mu.M minoxidil and 5mM MgCl ₂ 。

In one embodiment of the application, a kit of parts a and B is prepared containing a two-part chemical composition. In another embodiment, part a and part B components are combined to make a functional device. In yet another embodiment, a functional device produced by combining part a and part B components is used to test for the presence of an enzyme. In yet another embodiment, the enzyme is SULT1A1.

In one embodiment, the device as described above may be used to predictively evaluate the likelihood that an individual will respond favorably to the drug minoxidil. Minoxidil requires biochemical activation by minoxidil sulfotransferase to form an active minoxidil sulfate metabolite. The exact mechanism of action of minoxidil-based treatment of androgenic alopecia is not fully understood. However, in vitro studies have demonstrated that minoxidil sulfate is an active metabolite of minoxidil. The response to minoxidil treatment of androgenic alopecia is related to differences in scalp sulfotransferase activity, in particular sulfotransferase 1A1 (SULT 1 A1). Thus, subjects with high levels of SULT1A1 activity will produce more minoxidil sulfate, and thus will likely have a good response to minoxidil treatment of androgenic alopecia. On the other hand, subjects with low levels of SULT1A1 activity will not produce too much minoxidil sulfate and will likely have a poor response to minoxidil treatment of androgenic alopecia.

According to one method described herein, a hair follicle sample of a subject can be obtained. Preferably, at least two hair follicles can be obtained such that if only one hair follicle is analyzed, there will be at least one alternate hair follicle if desired. In one embodiment of the application, one hair is added to a functional device created by combining the part a and part B components of a two-part chemical. In yet another embodiment, two hair follicles are added. In yet another embodiment, at least 3 hair follicles are added to the functional device.

In one embodiment, the reaction may be carried out in a vessel other than those specifically described herein. For example, it may be a transparent container with a lid or other opening into which the hair follicle sample may be inserted. In one non-limiting example, the total amount of liquid in the analysis vessel may be about 0.1ml.

As part of the above reaction, it is understood that p-nitrophenyl sulfate is converted to colorimetric p-nitrophenol in the presence of minoxidil sulfotransferase activity.

In one embodiment, the reactions may be mixed and then incubated at room temperature for about 4 to 24 hours, depending on the number of hair follicles used in the assay. Mixing may be performed by any mixing means known in the art, including shaking the container. In one embodiment, a greater number of hair follicles may require a shorter incubation time. In one embodiment, the assay using one hair follicle can be incubated for about 24 hours. In another embodiment, the assay using two hair follicles can be incubated for about 16 hours.

After incubation, the absorbance of p-nitrophenol was read with a spectrophotometer at about 405 nm. The absorbance can be explained as follows: (a) An absorbance of less than 0.4 Absorbance Units (AU) means that minoxidil will be less likely to function; (b) Absorbance equal to 0.4AU means an ambiguous test; or (c) absorbance greater than 0.4AU means minoxidil can function. Alternatively, the color intensity of p-nitrophenol may be compared to a reference color chart having an intensity range corresponding to SULT1A1 activity. Subjects with relatively high levels of sulfotransferase activity will have relatively strong colorimetric readings, resulting in relatively significant color changes. In contrast, a patient with a relatively low level of sulfotransferase activity will have a relatively weak colorimetric reading and, correspondingly, relatively minimal color change. Patients with a strong colorimetric response are expected to respond to minoxidil for hair regrowth or maintenance. However, patients with weak colorimetric responses are expected to have poor responses to minoxidil.

Drawings

The above and other objects, aspects, features, advantages and possible applications of the present application will become more apparent from the following more particular description, which is presented in conjunction with the following drawings.

FIG. 1 illustrates an exemplary kit that may be used for colorimetric analysis with pulled-out human hair.

FIG. 2 is an exemplary schematic of the part A and part B components of an exemplary two-part chemical composition.

FIG. 3 is an exemplary reaction scheme for analysis.

Detailed Description

Embodiments are described herein in the context of a point-of-care diagnostic device for measuring biomarkers from extracted human hair. The following description is illustrative only and is not intended to be limiting in any way. 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 exemplary embodiments.

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 embodiment, 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 these specific goals will vary from one implementation to another and from one developer to another. Moreover, while such a development effort might be complex and time-consuming, it would nevertheless be a routine undertaking of engineering for those of ordinary skill in the art having the benefit of this disclosure.

The term "exemplary" is used 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.

Referring to fig. 1-3, embodiments relate to a kit 100 for measuring sulfotransferase activity in hair follicles. The kit 100 may include a container 102 and a reaction container 104. The kit 100 may include a container 102 and a reaction container 104 as separate components. For example, the kit 100 may include a container 102 as one unit and a reaction container 104 as a separate and distinct unit. In the alternative, the kit 100 may include the container 102 and the reaction container 104 as part of a single component. For example, the kit 100 may include a housing or other structure that includes the container 102 and the reaction container 104 as a single unit. The kit 100 may include any number of containers 102 and/or reaction containers 104. There may be one vessel 102 for one or more reaction vessels 104, separate vessels 102 for each separate reaction vessel 104, etc. As will be explained herein, the contents of vessel 102 will be introduced into reaction vessel 104. This may be accomplished by pouring the contents of vessel 102 into reaction vessel 104, allowing (e.g., gravity feed, osmotic motion, capillary motion, etc.) the contents to flow from vessel 102 to reaction vessel 104 via conduit 103, forcing (e.g., via a pump, unbalanced kinetic motion, etc.) the contents to flow from vessel 102 to reaction vessel 104, etc. via conduit 103. Conduit 103 may be, for example, a flow tube or flow path comprising vessel 102 and reaction vessel 104 (if vessel 103 and reaction vessel 104 are a single unit, they may be formed in a housing or other structure). Conduit 103 may be configured to place vessel 102 in fluid communication with reaction vessel 104. Some embodiments may include an actuator 106 (e.g., valve, solenoid valve, etc.) to control the flow of contents from the vessel 102 to the reaction vessel 104. The actuator 106 may include a processor, control module, switch, etc. configured to control aspects of the pump, valve, etc. The actuator 106 may be mechanically operated, electromechanically operated, etc. Other aspects of the kit 100 may include membranes, filters, gaskets, switches, timers, etc. to facilitate proper control and flow of the contents, control of flow rates and amounts, control of flow start and stop times, etc. Thus, the kit 100 may be configured such that the container 102 is in selective fluid communication with the reaction container 104 via the actuator 106 such that the contents of the reactive portion 108 and the contents of the non-reactive portion 110 are introduced into the reaction container 104 upon actuation of the actuator 106. It should be appreciated that there may be one or more actuators 106 for a given kit 100.

The container 102 may be configured to hold a two-part chemical composition. The two-part chemical composition may have a reactive part 108 and a non-reactive part 110. For example, the container 102 may be a container (e.g., bottle, vessel, tank, flask, etc.) having a bifurcated structure 109 forming a reaction chamber and a non-reaction chamber, each configured to contain and retain contents (e.g., chemical composition). The contents of each section 108, 110 are expected to be fluid. The furcation structure 109 may 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 may physically and/or chemically isolate the contents of the reactive portion 108 from the non-reactive portion 110 and the environment outside the container 102-e.g., prevent permeable movement, prevent light entry, prevent mixing of the respective contents, etc. The material of the container 102 may physically and/or chemically isolate the contents of the non-reactive portion 110 from the reactive portion 108 and the environment outside the container 102-e.g., prevent permeable movement, prevent light entry, prevent mixing of the respective contents, etc.

Vessel 102 and reaction vessel 104 may each include an inlet 112 and an outlet 114 to facilitate ingress and egress of contents. The vessel 102 may have a separate inlet 112 for each section 108, 110, a separate outlet 114 for each section 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 an inlet and an outlet 114. Either the inlet 112 or the outlet 114 of the vessel 102 or the reaction vessel 104 may have a lid 109 or plug to selectively cover the inlet 112 or the outlet 114. For embodiments in which the vessel 102 is separate from the reaction vessel 104, a user may manipulate the vessel 102 with their hand and pour the contents from each portion 108, 110 into the inlet 112 of the reaction vessel 104 via the outlet 114. Additionally or in the alternative, a transfer device, such as a liquid drop pipette (pipette), may be used to transfer the contents-for example, a user may introduce a pipette into the outlet 114 of the vessel 102 to restore the volume of the contents and dispense the contents to the inlet 112 of the reaction vessel 104. In addition, a pump having conduit 103 lines (extending to and from inlet 112 and outlet 114) may be used to transfer the contents from vessel 102 to reaction vessel 104. For embodiments in which the vessel 102 is part of the same unit as the reaction vessel 104, the actuator 106 may be actuated to cause/allow the contents to flow from the outlet 114 of the vessel 102 to the inlet 112 of the reaction vessel 104.

The contents of reactive portion 108 are not expected to mix or combine with the contents of non-reactive portion 110 until the respective contents are introduced into reaction vessel 104. Thus, the arrangement and configuration of the conduits 103, transfer devices, valves, actuators 106, etc. of the method of transferring the contents may be such that no mixing or combination of the contents occurs prior to introducing the contents into the reaction vessel 104.

The reaction vessel 104 may be configured to combine or mix the contents of the reactive portion 108 and the contents of the non-reactive portion 110. For example, the reaction vessel 104 may be a vessel having a cavity that receives the contents of the vessel 102 as the contents are allowed/forced to flow from the vessel 102 to the reaction vessel 104. The cavity allows for combining or mixing the contents of the reactive portion 108 with the contents of the non-reactive portion 110. This may be via free mixing, forced mixing, other forms of agitation, etc. as the contents enter the reaction vessel 104.

The contents of the reactive moiety 108 are contemplated to include potassium p-nitrophenyl sulfate. The contents of the reactive portion 108 may also include a stabilizing solution. The stabilizing solution stabilizes the potassium p-nitrophenyl sulfate to form stabilized potassium p-nitrophenyl sulfate. Hydrogen is required to convert potassium p-nitrophenyl sulfate (PNPS) to potassium p-nitrophenyl (PNP) and SO ₃ . Having an alkaline pH minimizes the amount of protons available and has a stabilized PNPS. The stabilizing solution may have a pH in the range of about 9 to about 11. In some embodiments, the stabilizing solution may be a solvent comprising an aqueous solution and a buffer. The buffer may be a carbonate buffer comprising sodium bicarbonate and sodium carbonate. In some embodiments, the stabilizing solution may be a solvent comprising an aprotic polar solvent. Non-massThe sub-polar solvent may comprise acetonitrile. In some embodiments, the stabilizing solution may be a protic nonaqueous solvent. The protic nonaqueous solvent may comprise isopropanol or ethanol.

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

The reaction vessel 104 may include an opening 116 configured to receive at least one hair follicle. In some cases, the opening 116 may be the same as the inlet of the reaction vessel 104. The reaction vessel 104 may be configured to receive at least a portion of a hair follicle (e.g., only a portion of a hair follicle is inserted into the reaction vessel 104) or an entire hair follicle (e.g., an entire hair follicle is inserted into the reaction vessel 104). It is contemplated that the hair follicle is placed in contact with a mixture or combination of contents (the mixture or combination of contents of the reactive portion 108 and the contents of the non-reactive portion). This may include contacting with the mixture or combination, immersing in a bath of the mixture or combination, and the like. The reaction vessel 104 may be configured to receive more than one hair follicle via one opening 116 or via multiple openings 116.

Embodiments may relate to methods for determining whether minoxidil formulations are effective in a subject in need of hair regrowth or maintenance. The method may include combining or mixing the contents of the reactive portion 108 and the contents of the non-reactive portion 110 of the two-part chemical composition to form a mixture or combination. The contents of the reactive moiety may include potassium p-nitrophenyl sulfate. The contents of the reactive portion 108 may also include a stabilizing solution. The stabilizing solution stabilizes the potassium p-nitrophenyl sulfate to form stabilized potassium p-nitrophenyl sulfate. The stabilizing solution may be a solvent comprising an aqueous solution and a buffer, an aprotic polar solvent or a protic nonaqueous solvent. It is contemplated to use pure solvents for aprotic polar or protic nonaqueous solvents. For aqueous based solutions, 50mM carbonate buffer is contemplated. The range may be 10 to 100mM and 100mM to 1M buffer; however, the lowest amount that would stabilize PNPS is preferred. The contents may include stabilized potassium p-nitrophenyl sulfate, wherein the solvent is removed to leave only the stabilized solute. The solvent may be removed (e.g., 90% -100% removed) via air drying, vacuum (e.g., using a bench concentrator, lyophilization, etc.). For example, the contents may be subjected to air drying under vacuum (e.g., 20 to 0.1 torr) for 20 minutes.

The contents of the non-reactive moiety may include minoxidil, magnesium chloride, and phosphate buffer.

The method may include placing hair follicles from the subject in contact with the mixture or combination. The method may include mixing and/or incubating the hair follicle with the mixture or combination. Mixing may include stirring, using a magnetic mixer, shaking, other forms of agitation, and the like. Mixing and/or incubating may include mixing and/or incubating for a period of time depending on the number of hair follicles placed in contact with the mixture or combination. For example, the more hair follicles that are contemplated for a given embodiment, the less mixing and/or incubation is required. For example, the incubation time may be 24 hours for 2 hair follicles, 12 hours for 4 hair follicles, etc. The volume of the contents used in the reaction vessel 104 may also depend on the number of hair follicles placed in contact with the mixture or combination. For example, an incubation time with a reaction volume of 100uL would be twice that if a reaction volume of 50uL were used.

The presence of the p-nitrophenol product is positively correlated with the sulfotransferase enzymatic activity of the hair follicle, and thus the method may include measuring the presence of the p-nitrophenol product within the mixture or combination. The method may include comparing the measured presence of the p-nitrophenol product to a predetermined level. The predetermined level may be a threshold level that is used as an indication that the subject will have a good response to minoxidil treatment. For example, the presence of a p-nitrophenol product greater than a predetermined level may indicate a level of sulfotransferase enzyme activity that indicates that the subject will have a good response to minoxidil treatment. The presence of a p-nitrophenol product less than a predetermined level may indicate a level of sulfotransferase enzymatic activity that indicates that the subject will not have a good response to minoxidil treatment.

In the presence of minoxidil sulfotransferase activity, p-nitrophenyl sulfate is converted to colorimetric p-nitrophenol. Thus, measuring the presence of the p-nitrophenol product may comprise measuring the absorbance of the p-nitrophenol product at about 400nm-410nm (preferably 405 nm) using a spectrophotometer 118 (e.g., a spectrometer having a sensor for collecting electromagnetic emissions of light passing through the solution and a processor for resolving or quantifying the collected electromagnetic emissions). In an exemplary embodiment, the absorbance may be explained as follows: (a) An absorbance of less than 0.4AU may indicate that minoxidil will be less likely to function; (b) Absorbance equal to 0.4AU may indicate an ambiguous test; or (c) an absorbance greater than 0.4AU may indicate minoxidil may be functional.

Additionally or in the alternative, measuring the presence of the p-nitrophenol product may include comparing the color intensity of the p-nitrophenol product to a color intensity reference card having an intensity range corresponding to the enzymatic activity of the sulfotransferase. For example, the color intensity of p-nitrophenol may be compared to a reference color chart having an intensity range corresponding to SULT1A1 activity. Subjects with relatively high levels of sulfotransferase activity will have relatively strong colorimetric readings, resulting in relatively significant color changes (color changes in the mixture due to the conversion of any p-nitrophenyl sulfate to colorimetric p-nitrophenol). In contrast, a subject with a relatively low level of sulfotransferase activity will have a relatively weak colorimetric reading (color change of the mixture due to the conversion of any p-nitrophenyl sulfate to p-nitrophenol) and correspondingly a relatively minimal color change. It is expected that subjects with a strong colorimetric response will respond to minoxidil for hair regrowth or maintenance. However, it is expected that subjects with a weak colorimetric response will have a poor response to minoxidil. The color intensity reference card may be a physical card or a virtual card (e.g., a computer graphical user interface) that is pre-made based on data from previous tests. The comparison of the colorimetric reading with the color intensity reference card may be performed by a human visual inspection, by a computer algorithm inspection, or the like.

Thus, if the hair follicle contains SULT1A1 (or a sufficient amount of SULT1 A1), then the p-nitrophenyl sulfate is converted to colorimetric p-nitrophenol (or a sufficient amount of p-nitrophenyl sulfate is converted to colorimetric p-nitrophenol) in order 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.

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 application, therefore, is not to be restricted except in the spirit of the appended claims.

Example 1: minoxidil Response Test (MRT), two-part chemical formulation

Example procedure the following reagents were used: sodium phosphate buffer (500 mM), sodium carbonate, sodium bicarbonate, potassium p-nitrophenyl sulfate, minoxidil, mgCl ₂ Solution (1M), water (0.1 μm filtration) and methanol.

The procedure was implemented to generate 1000 units of MRT reaction. PNPS deposited sample tubes were prepared (part a), and solutions were reconstituted (solution B). A single MRT reaction unit was created by adding 100 μl of solution B to PNPS deposited sample tubes (part a).

Preparation procedure

1.0 solution 1: PNPS buffer 10mM (pH 10)

TABLE 1 preparation of PNPS buffer 10mM (pH 10)

Component (A)	quantity/1L	Calculated amount	Actual amount of
				Sodium bicarbonate	0.388g	g	g
Sodium carbonate	0.571g	g	g
				Pure water	1.0L	L	L

Solutions were formulated according to the calculated amounts indicated in the table as follows: pure water was added to a suitably sized vessel, sodium bicarbonate and sodium carbonate were added and mixed on a magnetic stir plate until dissolved.

The pH of the final solution was measured (pH 10).

2.0 solution 2: potassium p-nitrophenyl sulfate (PNPS) 50mM (pH 10)

TABLE 2 preparation of potassium p-nitrophenyl sulfate (PNPS) 50mM (pH 10)

Component (A)	Amount/5 mL	Calculated amount	Actual amount of
				P-nitrophenyl potassium sulfate	0.128g	g	g
PNPS buffer	10.0mL	mL	mL

Solutions were formulated according to the calculated amounts indicated in the table as follows: PNPS buffer was added to a properly sized container, potassium p-nitrophenyl sulfate was added and mixed on a magnetic stir plate until dissolved.

3.0 solution 3-100mM minoxidil stock solution

TABLE 3 preparation of 100mM minoxidil stock solution

Component (A)	Amount/5 mL	Calculated amount	Actual amount of
				Minoxidil	0.105g	g	g
Methanol	5.0mL	mL	mL

Solutions were formulated according to the calculated amounts indicated in the table as follows: methanol was added to a suitably sized vessel, minoxidil was added and mixed on a magnetic stir plate until dissolved.

The stock solution was stored at about-20 ℃ for up to 1 year.

4.0PNP deposited tube (section A)

TABLE 4 solution 2 calculation

Material	Quantity-	Calculated amount
			Solution 2	10uL	mL

The materials in step 4.0 include: solution 2 (PNPS 50mM, pH 10) and sample tube (500. Mu.L).

The following steps are carried out: (1) 10 μl of solution 2 was added to the MRT sample tube, (2) the open tube was placed in a speedvac and air dried under vacuum while spinning until all the liquid had evaporated (about 20 min), and (3) stored at ambient temperature.

All volumes should be within 5% of the specification. The minimum time required for each instrument (speedvac) used to evaporate the liquid should be determined. Excessive drying may decrease the stability of part a.

5.0 solution B

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

TABLE 5 preparation of solution B

The solutions were formulated according to the calculated amounts indicated in the table and then mixed on a magnetic stirring plate until homogeneous.

All volumes should be within 5% of the specification. All weights should be within 5% of the specification. The pH should be 8.+ -. 0.5.

Example 2: accelerated stability scheme

Test component

Two (2) validated batches (lot number) of p-nitrophenyl potassium sulfate (PNPS) deposited tubes were prepared and stored at elevated temperature. Each lot contains twenty four (24) PNPS deposited tubes in total. During the course of the accelerated stability study, 3 samples of each lot number will be taken from the elevated temperature and reconstituted with the second part of the two-part chemistry (solution B). After 24h of reconstitution, the samples were visually inspected for yellow color.

Rationality description of model

Subjecting the test device to extreme temperature conditions aims to simulate degradation of the product over time, but at an accelerated rate. Using the arrhenius equation, the shelf life data can be extrapolated as a predicted measure of product performance. The arrhenius equation relates the chemical reaction rate (k) to the absolute temperature (T):

d(Ink)/dT＝ΔE _a /RT ²

wherein E is _a Is the activation energy and R is the universal gas constant.

TABLE 6 Arrhenius model for minoxidil sensitivity analysis

Using the Arrhenius model at 20 kcal, the test agent would theoretically have satisfactory performance for 1 year if it met the specification after 56.2 days at 50℃or 112.3 days at 40 ℃.

Annotation: in the case that the 50 ℃ conditions are too extreme for the test sample, the 40 ℃ sample is operated as an emergency measure. The 40 ℃ experiment was terminated with the test sample remaining stable at 50 ℃ for 56.2 days.

Assuming storage conditions

The performance of PNPS deposited tubes was evaluated when stored at 50 ℃ and 40 ℃. All storage locations were in compliance with the application biology QSR to ensure that the appropriate temperature range was met during the study.

Targets and purposes of testing

The purpose of the stability test at 50 ℃ and 40 ℃ was to generate data to determine the shelf life of the product. The data are used as predictors of the stability of the final product form.

Storage conditions of test kit

The kit (reagents) were stored at 50℃and 40 ℃ (2 ℃ for all temperatures).

Interval between analyses

TABLE 7 Minodl sensitivity analysis accelerated stability study

The stability of minoxidil sensitivity assay reagents stored at 50 ℃ ± 2 ℃ and 40 ℃ ± 2 ℃ was accelerated as described in table 7. Reagents were tested in triplicate. If the test day falls on a weekend or holiday, the test point is cancelled (depending on the test interval) or tested on the first weekday after the scheduled time.

Stability criterion

Accelerated stability studies must initially pass the accepted targets detailed in table 8.

TABLE 8 minoxidil sensitivity assay specific Release test and stability Specification

Description of the application	Result specification (visual)	Result determination
			PNPS deposited tube+100 uL solution B	Clarified	By passing through
PNPS deposited tube+100 uL solution B	Pale yellow	By passing through
			PNPS deposited tube+100 uL solution B	Yellow colour	Failed to pass

Annotation:

(1) The results were evaluated 24 hours after reconstitution with solution B.

(2) The reagent must meet the following specifications (# 4) for 56.2 days at 50 ℃ or 112.3 days at 40 ℃ to obtain the expected shelf life of 12 months at 23 ℃.

(3) Failure at a particular temperature may not be an indication of shelf life, as higher temperatures may adversely affect solution chemistry. Thus, a shelf life of 1 year may not be expected when treated at 50 ℃, whereas a shelf life of 1 year may be expected when treated at 40 ℃.

(4) When a failure is observed at two (2) consecutive test time points, the time points are considered to be failure. The expiration time may be deferred from the time of expiration at the first point in time. If invalid test results exist, the test may be retested to confirm the results. A failure may also be retested to confirm the results. If the failure is not confirmed at the time of retesting, additional tests may be run to evaluate the status of the time point.

Appendix A

	0	6	14	28	42	56	70	84	98	126	154	182	Total number of bottles
														50	√	√	√	√	√	√	√	√	24
40		√	√	√			√		√	√	√	√	24

Annotation:

samples should be evaluated in triplicate.

If the test day falls on a weekend or holiday, the test point is cancelled (depending on the test interval) or tested on the first weekday after the scheduled time.

Stability is expected for 1 year: 50 ℃ =56.2 days, 40 ℃ =112.3 days

Results

Days (days)

0

6

14

28

42

56

70

84

98

126

154

182

50C

By passing through

Failed to pass

NA

40C

By passing through

Failed to pass

NA

Accelerated stability reporting for Table X-formulations (pH 7.5-9.5)

Formulation G2 (pH 7.5-9.5)

Days (days)

0

6

14

28

42

56

70

84

98

126

154

182

50C

By passing through

40C

By passing through

Accelerated stability reporting of Table Y-formulations (pH 9.5-10.5)

Example 3: functional testing of reconstructed part A and part B

Samples from example 2 that were considered stable, i.e., those that remained clear after 24h of reconstitution, were reacted with two human hair follicles to test whether the reconstituted chemicals were functional.

Specification of specification

Description of the application	Result specification (visual)	Result determination
			PNPSDeposited tube +100uL solution B + hair	Clarified	Failed to pass
PNPS deposited tube+100 uL solution B+hair	Yellow colour	By passing through

Results

Days (days)

0

6

14

28

42

56

70

84

98

126

154

182

50C

By passing through

NA

40C

By passing through

NA

Accelerated stability reporting for Table X-formulations (pH 7.5-9.5)

Formulation G2 (pH 7.5-9.5)

Days (days)

0

6

14

28

42

56

70

84

98

126

154

182

50C

By passing through

40C

By passing through

Accelerated stability reporting of Table Y-formulations (pH 9.5-10.5)

Claims

1. A kit for measuring sulfotransferase activity in hair follicles comprising:

a container configured to hold a two-part chemical composition having a reactive part and a non-reactive part; and

a reaction vessel configured for combining or mixing the contents of the reactive portion and the contents of the non-reactive portion;

wherein the contents of the reactive moiety comprise potassium p-nitrophenyl sulfate;

wherein the contents of the non-reactive moiety comprise minoxidil, magnesium chloride, and phosphate buffer.

2. The kit of claim 1, wherein the container is configured to maintain the contents of the reactive portion physically and/or chemically isolated from the contents of the non-reactive portion.

3. The kit of claim 1, wherein the contents of the reactive moiety comprise 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:

an aqueous solution and a buffer;

aprotic polar solvents; and/or

A protic nonaqueous solvent.

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

7. The kit of claim 5, wherein the buffer 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 with the reaction container via an actuator such that the contents of the reactive portion and the contents of the non-reactive portion are introduced into the reaction container upon actuation of the actuator.

12. The kit of claim 1, wherein the reaction vessel comprises 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 the contents of the reactive portion and the contents of the non-reactive portion of the two-part chemical composition to form a mixture or combination, wherein:

the contents of the reactive moiety comprise potassium p-nitrophenyl sulfate; and

the contents of the non-reactive portion include minoxidil, magnesium chloride, and phosphate buffer; and

hair follicles from the subject are placed in contact with the mixture or combination.

14. The method of claim 13, further comprising:

measuring the presence of a p-nitrophenol product, wherein the presence of the p-nitrophenol product is positively correlated with sulfotransferase enzymatic 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

the comparison is used as an indication that the subject will have a good response to minoxidil treatment.

16. The method of claim 15, wherein:

the presence of the p-nitrophenol product of greater than the predetermined level is indicative of a level of sulfotransferase enzyme activity, the level indicating 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.

18. The method of claim 17, wherein:

mixing and/or incubating the hair follicle with the mixture or combination includes mixing and/or incubating for a period of time that depends on the number of hair follicles placed in contact with the mixture or combination.

19. The method of claim 14, wherein:

measuring the presence of a p-nitrophenol product comprises measuring an absorbance of the p-nitrophenol product at about 405nm using a spectrophotometer; and/or

Measuring the presence of a p-nitrophenol product comprises comparing the color intensity of the p-nitrophenol product to a color intensity reference card having an intensity range corresponding to the enzymatic activity of the sulfotransferase.