BIS-QUATERNARY PYRIDINIUM-ALDOXIME SALTS AND TREATMENT OF EXPOSURE TO CHOLINESTERASE INHIBITORS

Government Rights Clause

This invention was made with United States Government support under Contract No. W9113M-05-C-0199 awarded by the United States Army. The Government has certain rights in this invention.

Cross Reference To Related Applications

The present application claims the benefit of the filing date of U.S. Patent Application Serial No. 12/047,988 filed March 13, 2008, the teachings of which are incorporated herein by reference.

Field Of The Invention

The present invention relates to bis-quaternary pyridinium-aldoxime salts, their associated methods of preparation including their use for treatment of exposure to cholinesterase inhibitors, such as a phosphorous containing cholinesterase inhibitor type compounds.

Background

Stimulating signals are typically carried by acetylcholine within a nervous system synapse. Such signals may be discontinued by a specific type of cholinesterase enzymes, acetylcholinesterase, which breaks down acetylcholine. If cholinesterase inhibiting chemicals are present, they may then prevent the breakdown of acetylcholine thereby disrupting normal nervous system activity. For example, certain chemical classes of pesticides, such as organophosphates and carbamates, may result in toxic cholinesterase inhibition. Accordingly, if an individual is regularly exposed to such inhibitors, there remains a need to prophylactically or therapeutically treat such toxicity. Among other things,

individuals or animals who may have been exposed to a carbamate type cholinesterase inhibitor may currently be treated with atropine, and those exposed to organophosphates may beneficially be treated with a pralidoxime antidote.

Summary

In a first exemplary embodiment, the present disclosure relates to a bis-quaternary pyridinium-2-aldoxime salt of the formula:

wherein R is a halogen and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-35 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 35 degrees. In another exemplary embodiment, the present disclosure is directed at a bis- quaternary pyridinium-2-aldoxime salt of the formula:

wherein R is a halogen and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-45 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 45 degrees.

In yet another exemplary embodiment, the present disclosure is directed at a bis- quaternary pyridinium-2-aldoxime salt of the formula:

wherein Rl is a methyl and/or ethyl group and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-30 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 30 degrees and said salt has cubic rectangular crystal structure.

In another exemplary embodiment, the present disclosure is directed at a bis- quaternary pyridinium-2-aldoxime salt of the formula:

wherein Rl is a methyl and/or ethyl group and said salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-30 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 30 degrees and said salt has hexagonal crystal structure.

In yet another exemplary embodiment, the present disclosure is directed at a method for preparing a bis-quaternary pyridinium-2-aldoxime salt comprising supplying pyridine-4- aldoximine of the structure:

treating the pyridine-4-aldoximine with diodomethane to form l,l'-methylenebis[4- [(hydroxyimino)methyl]-pyridinium] diodide of the following formula:

converting the l,r-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] diodide to the following formula via ion exchange of the iodine to provide the following bis-quaternary pyridinium-2-aldoxime salt:

wherein R is a chlorine atom or an alkyl sulfonate group and where the alkyl sulfonate is of the general structure:

O

Il R1-S-0

O where Rl is a methyl or ethyl group.

In yet another exemplary embodiment, the present disclosure is directed at a prophylactic or therapeutic method of treating a person or animal for intoxication with a phosphorous containing cholinesterase inhibitor, comprising administering to a person or animal one or more of the following compounds:

(i) a bis-quaternary pyridinium-2-aldoxime salt of the formula:

wherein R is a halogen and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-35 degrees as compared to non-distinguishing peaks at 2 Theta angles greater than 35 degrees; or

(ii) a bis-quaternary pyridinium-2-aldoxime salt of the formula:

wherein R is a halogen and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-45 degrees as compared to non-distinguishing peaks at 2 Theta angles greater than 45 degrees; or

(iii) a bis-quaternary pyridinium-2-aldoxime salt of the formula:

wherein Rl is a methyl and/or ethyl group and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-30 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 30 degrees and said salt has cubic rectangular crystal structure; or

(iv) a bis-quaternary pyridinium-2-aldoxime salt of the formula:

wherein Rl is a methyl and/or ethyl group and the salt indicates one or more distinguishing x-ray diffraction peaks at 2 Theta angles of 10-30 degrees as compared to non-distinguishing x-ray diffraction peaks at 2 Theta angles greater than 30 degrees and said salt has hexagonal crystal structure.

Brief Description Of The Drawings

FIG. 1 is an X-ray diffraction pattern for MMB4-Dichloride Polymorph A.

FIG. 2 is a scanning electron micrograph of MMB4 Dichloride Polymorph A. FIG. 3 is an X-ray diffraction pattern for MMB4 Dichloride Polymorph B.

FIG. 4 is a scanning electron micrograph of MMB4 Dichloride Polymorph B/

FIG. 5 is an X-ray diffraction pattern for MMB4 DMS Polymorph A.

FIG. 6A is a scanning electron micrograph of MMB4 DMS Polymorph A.

FIG. 6B is an illustration of the crystalline structure of MMB4 DMS Polymorph A identified in FIG. 6A.

FIG. 7 is an X-ray diffraction pattern for MMB4 DMS Polymorph B.

FIG. 8A is a scanning electron micrograph of MMB4 DMS Polymorph B.

FIG. 8B is an illustration of the crystalline structure of MMB4 DMS Polymorph B identified in FIG. 8A.

Detailed Description

It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

As alluded to above, the present disclosure is directed at the formation of certain bis- quaternary pyridinium aldoxime salts and their associated use to prophylactically and/or therapeutically treat intoxication in a person or animal due to the presence of a cholinesterase inhibitor, such as a phosphorous containing cholinesterase inhibitor. It is therefore worth pointing out that organophosphates (OPs) may act as hemi-substrates of cholinesterase by specifically phosphorylating the active site serine. As the rate of hydrolysis of the phosphoryl or phosphonyl enzyme may be relatively slower than deacylation of

acytylcholine, OPs are effectively irreversible cholinesterase inhibitors. OPs have also been developed as chemical weapon systems, and relatively potent insecticides, due to their inhibition of the insects' flight muscle cholinesterase, with resulting paralysis and death. It may therefore be appreciated that intoxication by anti-cholinesterase compounds may develop following accidental exposure to organophosphorus insecticides and/or other associated chemical agents. Furthermore, the overall pharmacologic effect of anticholinesterases may be due to the inhibition of cholinesterase enzymes throughout the body.

In a first exemplary embodiment, the present disclosure relates to the preparation of a l,l'-methylenebis[4-(hydroxyimino)methyl]-pyridinium salt, which may be represented by the following general formula:

where R may be a halide counteranion such as a halogen (e.g. Cl ^" or Br ^" or I ^" ) in which case the compound may be referred to as "MMB4 Dihalide.". More generally, R may be derived from a salt of an inorganic or organic acid. For example, the anion may be derived from hydrogen sulfate (H ₂ SO ₄ ), nitrate, fumarate, lactate, tartate, citrate, and/or acetate. In addition, R may be a counteranion such as an alkyl sulfonate group. In such a case, the l,l'-methylenebis[4-(hydroxyimino)methyl]-pyridinium salt would assume the following general formula:

wherein Rl may be selected such that it does not interfere (e.g. steric interference) with the formation of the particular polymorphic pyridinium salts noted below. Accordingly, Rl may be a methyl (-CH ₃ ) group, and it is contemplated herein that it may also include ethyl type group functionality (-CH ₂ CH ₃ ). One particularly useful and convenient synthetic procedure for the formation of the pyridinium salts of the present disclosure may involve the preparation of 1,1'- methylenebis [4- [(hydroxyimino)methyl] -pyridinium] diodide hereinafter referred to as "MMB4 DI", which may then be converted to l,l'-methylenebis[4-[(hydroxyimino)methyl]- pyridinium] dimethanesulfonate "MMB4 DMS." This synthetic procedure is outlined in the general reaction scheme illustrated below:

Py

MMB4 DMS

In addition, it may be appreciated that the MMB4 DI may be converted, again by the convenient procedure of ion exchange, to a particular dihalide salt, such as the dichloride salt, as illustrated below:

MMB4 Dl

Ion Exchange Conversion

MMB4 Dichloride

It has been determined that the MMB4 dichloride and/or the MMB4 DMS compounds noted above may be isolated in one of two polymorphic forms, as disclosed herein, by control of, e.g., the solvents that may be employed for the pyridinium salt recrystallization. In addition, such polymorphic forms, as also noted above, provided the ability to offer improved prophylactic or therapeutic treatment of a person or animal intoxicated with a cholinesterase inhibitor. Accordingly, attention is therefore next directed to FIG. 1, which provides the x-ray diffraction pattern [intensity (counts) versus 2- Theta(degrees)] for the MMB4 dichloride compound in the form of what may now be termed MMB4-dichloride Polymorph A. The diffraction patterns (as well as the other diffraction patterns reported herein) were made on a Siemens Kristalloflex 805 with a model D500 goniometer, serial number WM80030464X. The diffraction patterns were then processed using JADE v3.1 from Materials Data, Inc (program serial number MDI-R95704. In general, a representative portion of the sample for analysis was ground to a grain size of less than 25

microns and then spread on a polycarbonate specimen holder. The x-ray tube was run at 4OkV and 3OmA with a 2-theta range of 10-60 degrees. The instrument may be calibrated at regular intervals using appropriate standards.

As can be seen from FIG. 1, the MMB4 dichloride compound in the form of polymorph A herein indicates one or more x-ray diffraction peaks with relative intensity counts (artificial units) between 500-1500 at the 2 Theta angles of between 10-35 degrees, which relatively intensity counts for the peaks drop to a level of less than 500 counts at 2 Theta angles of greater than about 35 degrees. That is, no peaks are present with relative intensity counts of more than 250 at 2 Theta angles between 35-60 degrees. Accordingly, it may be understood herein that the MMB4 dichloride compound in the form of polymorph A may be characterized as having an x-ray diffraction pattern with distinguishing peaks at the 2 Theta angles of between 10-35 degrees as compared to the non-distinguishing x-ray diffraction peaks at the 2 Theta angles of greater than 35 degrees. By reference to distinguishing peaks, it may be understood (upon consideration of FIG. 1) as those peaks and/or collection of peaks within the 2 Theta angles of 10-35 degrees which then may be employed to provide identifiable d-spacing (Braggs Law) for the MMB4 dichloride polymorph A. Accordingly, reference to a collection of peaks herein may include, e.g. information sourced from 2-100 peaks, including all values and increments within the range of 2-100. Attention is therefore next directed to FIG. 2, which provides a scanning electron micrograph of MMB4 dichloride Polymorph A. As can be seen, MMB4 dichloride Polymorph A may also be characterized as having a needle-like particulate structure, with an aspect ratio (AR) or length divided by largest diameter of greater than 2:1. More particularly, the aspect ratio may be in the range of 2:1 to 16: 1, including all values and increments therein.

Attention is next directed to FIG. 3, which provides the x-ray diffraction pattern of MMB4 dichloride Polymorph B. As can be seen, MMB4 dichloride Polymorph B indicates one or more x-ray diffraction peaks having relative intensity counts (artificial units) between 500-1500 at the 2 Theta angles of between 10-45 degrees, which relatively intensity counts for the peaks drop to a level of less than 500 counts at 2 Theta angles greater than about 45 degrees. That is, no peaks are present with relative intensity counts of more than 250 at 2 Theta angles between 45-60 degrees. Accordingly, it may be understood herein that the

MMB4 dichloride compound in the form of polymorph B may be characterized as having an x-ray diffraction pattern with distinguishing peaks at the 2 Theta angles of between 10-45 degrees as compared to the non-distinguishing x-ray diffraction peaks at the 2 Theta angles of greater than 45 degrees. By reference to distinguishing peaks, it may again be understood (upon consideration of FIG. 3) as those peaks and/or collection of peaks within the 2 Theta angles of 10-45 degrees which then may be employed to provide identifiable d-spacing (Braggs Law) for the MMB4 dichloride polymorph B.

Attention is therefore next directed to FIG. 4 which provides a scanning electron micrograph of MMB4 dichloride Polymorph B. As can be seen, MMB4 dichloride Polymorph B may also be characterized as having either a particulate structure that is of a square, rectangular, rhomboid (i.e. a parallelogram in which adjacent sides are of unequal lengths) and/or rhombus (a rhomboid with right angled corners) type geometry.

Attention is next directed to FIG. 5 which provides the x-ray diffraction pattern of MMB4 DMS Polymorph A. As can be seen, MMB4 DMS Polymorph A indicates one or more x-ray diffraction peaks with relative intensity counts (artificial units) between 500-1500 at the 2 Theta angles of between 10-30 degrees, which relatively intensity counts for the peaks drop to a level of less than 500 counts at 2 Theta angles greater than about 30 degrees. That is, no peaks are present with relative intensity counts of more than 250 at 2 Theta angles between 30-60 degrees. Accordingly, it may be understood herein that the MMB4 DMS compound in the form of Polymorph A may be characterized as having an x-ray diffraction pattern with distinguishing peaks at the 2 Theta angles of between 10-30 degrees as compared to the non-distinguishing x-ray peaks at the 2 Theta angles in the range of greater than 30 degrees, e.g. in the range of greater than 30 degrees to about 60 degrees. By reference to distinguishing peaks, it may again be understood (upon consideration of FIG. 5) as those peaks and/or collection of peaks within the 2 Theta angles of 10-30 degrees which then may be employed to provide identifiable d-spacing (Braggs Law) for the MMB4 DMS Polymorph A.

FIG. 6A next provides a scanning electron micrograph of MMB4 DMS Polymorph A. As can be seen, MMB4 DMS Polymorph A may be described as having cubic rectangular type crystal structure or geometry. A cubic rectangular geometry may be understood as a cubic configuration that may be stretched along its (c) axis to provide a rectangular configuration, consisting of three substantially equal or equatorial (a, b and c) axes at 90° (+/-

5°) and the c axis is longer than the horizontal axis. See FIG. 6B and angles α, β, and γ which are at 90° (+/- 5°).

Attention is next directed to FIG. 7 which provides the x-ray diffraction pattern of MMB4 DMS polymorph B. As can be seen, MMB4 DMS Polymorph B indicates one or more x-ray diffraction peaks with relative intensity counts (artificial units) between 1000- 4500 at the 2 Theta angles of between 10-30 degrees, which relatively intensity counts for the peaks drop to a level of less than 500 counts at 2 Theta angles greater than about 30 degrees. That is, no peaks are present with relative intensity counts of more than 500 at 2 Theta angles between 30-60 degrees. Accordingly, it may be understood herein that the MMB4 DMS compound in the form of polymorph B may be characterized as having an x-ray diffraction pattern with distinguishing peaks at the 2 Theta angles of between 10-30 degrees as compared to the non-distinguishing x-ray diffraction peaks at the 2 Theta angles of greater than 30 degrees. By reference to distinguishing peaks, it may again be understood (upon consideration of FIG. 7) as those peaks and/or collection of peaks within the 2 Theta angles of 10-30 degrees which then may be employed to provide identifiable d-spacing (Braggs Law) for the MMB4 DMS polymorph B.

FIG. 8A next provides a scanning electron micrograph of MMB4 DMS Polymorph B. As can be seen, MMB4 DMS Polymorph B may be described as having primarily hexagonal structure. A hexagonal crystal structure may be understood as having four crystallographic axes consisting of three substantially equal or equatorial (a, b, and d) axes at 120° (+/- 5°) and one vertical (c) axis that is 90° (+/- 5°) to the other three. See, e.g., FIG. 8B, wherein angle α is shown being equal to 120° (+/- 5°) and angle β being equal to 90° (+/- 5°). The (c) axis may be shorter or longer than the horizontal axis.

Once prepared, the l,l'-methylenebis-quaternary pyridinium-4-aldoximine compounds, either in the form of polymorph A and/or polymorph B, may be readily incorporated into a pharmaceutically acceptable carrier. As noted, such compounds may then be administered in an antidotal amount to therapeutically treat exposure to a phosphorous containing cholesterase inhibitor. A pharmaceutically acceptable carrier may therefore be understood herein as an aqueous formulation containing the above l,l'-methylenebis- quaternary pyridinium-4-aldoximine salt compounds in the form of what has been identified herein as polymorph A and/or polymorph B. Such formulations may therefore amount to aqueous solutions, suspensions and/or emulsions and may be adjusted to have a pH of 1.0 to

5.0, including all values and increments therein. Furthermore, it may be appreciated that an emulsion may be understood as an aqueous mixture containing the subject compound in the presence of suitable hydrophobic/hydrophilic emulsifying agents.

The pharmaceutically acceptable carrier may also provide a pharmaceutically acceptable anion, which as noted above, may be sourced from an inorganic or organic acid, which acids may or may not include a preservative. Accordingly, the l,l'-methylenebis- quaternary pyridinium-4-aldoximine salt compounds, either in the form of polymorph A and/or polymorph B may be present herein in the aqueous formulation at a concentration of 0.1-50% by weight, including all values and increments therein. It may be appreciated, however, that the specific doses may depend on a variety of factors, for example, the age, body weight, general state of health and time of administration and the time and severity of exposure. It is worth noting that parenteral administration may be utilized herein, whether for prophylaxis or therapeutically (i.e., before exposure to a cholinesterase inhibitor).

In addition, the pharmaceutically acceptable aqueous carrier herein may include other diluents suitable for preparing oral pharmaceutical suspension. For example, an oral pharmaceutical suspension of the present invention may include, if necessary, pharmaceutically acceptable additives including auxiliary substances, stabilizing agents, suspending agents, surface tension modifiers, viscosity modifiers, colorants, preservatives, flavoring agents and other commonly used additives such as lactose, citric acid, tartaric acid, stearic acid, magnesium stearate, sucrose, and the like.

The present disclosure may therefore now be considered with respect to the following various non-limiting examples.

* * *

Example 1 - Preparation of MMB4 Dimethane Sulfonate (Laboratory Scale) (1) Production ofMMB4 Diiodide

To 21.53 g (0.176mol) of pyridine-4-aldoxime in 250 mL of acetonitrile was added 27.21 g (0.176 mol) of diiodomethane. The reaction mixture was refluxed under argon for 90 hours. The mixture was cooled, filtered and the filter cake washed with 100 ml of acetonitrile. The filter cake was air dried for 30 minutes to yield 41.52 g. The cake was dried under high vacuum to give 41.02 g (91% yield). (2) Dimethanesulfonate Resin Preparation

In a 250 mL beaker, 30 g of Dowex 550A (OH form), available from the Dow Chemical Company, was added to 84 mL of 10% (v/v) methanesulfonic acid in methanol. The resin was stirred at room temperature for 2 h then filtered through a 150 mL sintered funnel. The resin bed was washed with 2 x 84 mL portions of methanol and then air-dried for 30 minutes. Total resin weight: 17.6 g, divided into 2 x 8.8 g portions. (3) Conversion of MMB4 Diiodide to MMB4 Dimethanesulfonate

A sample of 2.0 g (3.9 mmol) of MMB4 diiodide was dissolved in 100 mL of methanol with stirring in a 50 ⁰ C water bath. The solution was cooled to room temperature, then 8.8 g of the mesylate form of Dowex 550A was added and stirred at room temperature for 2 hours. The mixture was filtered through a sintered funnel, washing the resin bed with 10 mL of methanol. An additional 8.8 g of the mesylate form of Dowex 550A was added to the filtrate and the mixture stirred for an additional 2 h. The mixture was filtered and the resin bed washed with 10 mL of methanol.

The filtrate was concentrated to 10 mL, then 35 mL of denatured ethanol (denatured with 5% isopropanol and 5% methanol) was added. The mixture was heated to 50 ⁰ C with stirring until complete dissolution (30 min). The solution was allowed to stand for 16 hours at ambient temperature with slow stirring. The mother liquor was decanted and the solids rinsed with 2 x 5 mL of cold (5 ⁰ C) denatured ethanol. The solid was dried at 23 mm Hg and room temperature to yield 1.35 g (77%) of a tan-amber solid (Polymorph A). * * *

Example 2 -Preparation Of MMB4 Dimethanesulfonate (Production Scale)

(1) Production ofMMB4 Diiodide

A 100-gallon (380L) reactor is charged with 21.9 kg (179 moles) of pyridine-4- aldoxime and 170 kg of acetonitrile, followed by 48.3 kg (180 moles) of diiodomethane and 37.5 kg of acetonitrile. The mixture is brought to a gentle reflux (approximately 84 ⁰ C) with vigorous mechanical stirring under an inert atmosphere (nitrogen). After 72 hours, the mixture is cooled to 40-45 ⁰ C with stirring over 5 hours. The resulting suspension is filtered and then washed three times with 25 kg portions of 40-45 ⁰ C acetonitrile. The washed filter cake is transferred to drying trays and dried under vacuum with heating 40-45 °C over eight hours. This process yields approximately 37.5 kg (82%) of MMB4 diiodide.

(2) Dimethanesulfonate Resin Preparation

In a 100-gallon (380L) reactor, 172 kg of methanol is slowly charged to methanesulfonic acid (35.7 kg), maintaining the temperature at 20-40 ⁰ C. This solution is subsequently added to 77.5 kg of Dowex 550A (OH form), maintaining the temperature below 50 ⁰ C. The resultant resin/methanol/methanesulfonic acid slurry is then stirred at 25±5 ⁰ C for 2-2.5 hours and then filtered. The resin is washed in a plug flow manner with two-153 kg portions of methanol. A final wash of 35 kg of methanol is used to test for residual water; the in-process limit is no more than 0.4%. (3) Conversion ofMMB4 Diiodide to MMB4 Dimethane sulfonate

In a 100-gallon (380L) reactor, MMB4 diiodide, 10.3 kg, is dissolved in 204.5 kg of methanol with stirring by warming to 50+3 °C for 1-1.5 hours. While maintaining the temperature, half of the previously formed dimethanesulfonate resin is added and stirred at 50±3 ⁰ C for 2 to 2.5 hours. The solution is then filtered and the resin is washed with 20.5 kg of methanol. The filtrate and wash are combined and treated as described above with the remaining half of the resin. After the final filtration and washing, an in-process test is used to monitor iodide concentration. The wash and filtrate are combined and then reduced to a volume of 65-70 L under vacuum at a temperature less than 25 ⁰ C. After concentrating, 5.5 kg each of isopropanol and methanol are added followed by 98 kg of ethanol. The mixture is heated to reflux (approximately 72 ⁰ C) for 1-1.5 hour to achieve complete dissolution. Once clarity is achieved, the mixture is allowed to cool to 20+5 ⁰ C over approximately 9 hours to crystallize, followed by an additional hold time of 7-7.5 hours. The MMB4 dimesylate is then filtered and washed with a mixture of 4.5 kg ethanol and 2.3 kg of methanol. The filter cake is then dried at ambient temperature under vacuum for 8 hours. The typical yield is 5-5.7 kg or 55-63% of MMB4 dimethanesulfonate (Polymorph B). * * *

Example 3

A representative pharmaceutical formulation for MMB4 DMS is set forth below:

450 mg/mL of MMB4 DMS and 5mg/mL of benzyl alcohol in WFI is adjusted with an acetic acid solution to a pH of about 2.3. The following were then transferred to a 5mL volumetric flask: 25 mg benzyl alcohol (BA), 1.0 g "0.3% Acetic acid solution" and 2.25 g

MMB4 DMS. At this point, WFI water is added to dissolve the solids completely. The pH is then measured and adjusted with acetic acid solution to a pH of about 2.3. At this point one

brings the total volume to 5 mL with WFI water. This is then followed by filtering through a 0.2-micron syringe filter.