The role of HEPES in pain control and reversal of demyelinating injury

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a hydrogen ion buffer which has a good buffering capacity in the pH range of 6.8~8.2 and can control a constant pH for a long time. Recently, HEPES can also as a composition of pain control agents, and reversing demyelinating damage.


The researchers described the use of HEPES and its derivatives as an analgesic, and anti-tumor agent for stabilizing cells, especially neuronal membranes, and reversing demyelinating damage. The HEPES composition is also used to treat withdrawal symptoms after treatment with antidepressants and selective serotonin inhibitors (SSRI); HEPES is also used as an analgesic for the treatment of pain associated with one or more cancers, and side effects after chemotherapy, including cognitive impairment.


The mechanism of action of HEPES includes:
(i) analgesic activity, possibly associated with prolonged anti-anandamide action, with no significant side effects compared to opioids;
(ii) anti-tumor activity may be related to previously discovered mechanisms using promethazine;
(iii) it is possible to stabilize cells, especially neuronal membranes, by modulating various ion channels;
(iv) Reversing demyelinating damage.
In an embodiment, HEPES and its derivatives are dissolved in sterile water, buffer, saline or other pharmaceutically acceptable carrier: (1) HEPES is dissolved in sterile water for oral, subcutaneous, parenteral, intravenous, intraperitoneal or intramuscular injection with administration of 10-100 mg/kg on a body weight basis once a day; (2) in combination with other carriers, such as an antidepressant selected from the group consisting of benzodiazepines, SSRI, serotonin-norepinephrine intake inhibitors (SNRI), noradrenergic and specific serotoninergic antidepressants (NaSSAs), norepinephrine (norepinephrine) reuptake inhibitors (NRI), norepinephrine-dopamine Reuptake inhibitors (NDRI), selective serotonin reuptake enhancers (SSRE), melatonin agonists, tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOI) and SSRI including citalopram tapropren, fluoxetine, fluvoxamine, paroxetine, sertraline, mermilidine or any combination thereof.

The product is administered on a weight basis and the minimum effective daily dose determined is 70~75 mg/kg. The material can be administered safely by oral or parenteral route. The inventors have observed in many cases that the drug can be administered by two routes without any significant side effects.


HEPES is a zwitterionic molecule that is commonly used as a buffer in animal and human cell cultures. Studies have shown that HEPES has the least cytotoxicity against all known buffers. At the same time, HEPES is a piperazine-based zwitterionic molecule. Piperazine compounds are derived from phenothiazine. Phenothiazine has been approved by the FDA as an anxiolytic and antipsychotic drug. Non-zwitterionic piperazine compounds have been approved by the FDA as anti-worm agent, other piperazine-based molecules, is approved as a food additive.


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The summary and comparation for synthesis of HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a hydrogen ion buffer which has a good buffering capacity in the pH range of 6.8~8.2 and can control a constant pH for a long time. The commonly used concentration is 10~50mmol/L, the general culture solution contains 20mmol/L HEPES can have a good buffer capacity. Then how to prepare it?


1. CN104803949A
(1) addition reaction of vinyl sulfonic acid or vinyl sulfonate with N-hydroxyethylpiperazine to prepare 4-hydroxyethylpiperazine Ethane sulfonate;
(2) using an acidifying agent to convert 4-hydroxyethylpiperazineethanesulfonate to 4-hydroxyethylpiperazineethanesulfonic acid and a salt of the corresponding acidifying agent to obtain an acidified mother liquor;
(3) the acidified mother liquor is crystallized to remove the salt of the corresponding acidifying agent to obtain the acidified mother liquor after the treatment;
(4) a soluble barium salt or a calcium salt is added to the acidified mother liquor to remove residual sulfate, and concentrated by evaporation to obtain a primary purification product of HEPES;
(5) The primary purified product is washed with a small molecular weight alcohol, and dried to obtain high purity HEPES.
The method not only can produce HEPES in high purity, but also has low purification cost, simple process, easy operation and no pollution.
2. CN104876892A
(1) condensation reaction of 2-hydroxyethylsulfonic acid or 2-hydroxyethylsulfonate with N-hydroxyethylpiperazine to obtain 4-hydroxyethyl Piperazine ethyl sulfonate;
(2) using an acidifying agent to convert 4-hydroxyethylpiperazineethanesulfonate to 4-hydroxyethylpiperazineethanesulfonic acid and a salt of the corresponding acidifying agent to obtain an acidified mother liquor;
(3) the acidified mother liquor is diluted with deionized water to 1~10 wt.%, and salt of the corresponding acidifying agent is removed by nanofiltration, and concentrated by evaporation to obtain a crude HEPES;
(4) The crude HEPES was washed and dried to obtain high-purity HEPES.

The method not only can produce HEPES with high purity, but also has low production cost, simple process, easy operation and high yield.


3. CN106905262A
(1) 2-hydroxyethylsulfonic acid or 2-hydroxyethylsulfonate and N-hydroxyethylpiperazine are subjected to a condensation reaction to obtain 4-hydroxyethylpiperazineethanesulfonate;
(2) adding an acidifying agent to 4-hydroxyethylpiperazineethanesulfonate, converting it to 4-hydroxyethylpiperazineethanesulfonic acid and a salt corresponding to the acidifying agent to obtain an acidified mother liquor;
(3) The acidified mother liquor is diluted with deionized water to a solution of 10-15wt.%, and extracted by a five-stage continuous countercurrent centrifugal extractor to extract a raffinate and extract an organic phase, the raffinate is aqueous solution of product and inorganic salt.
(4) the extracted organic phase is treated with reverse extraction, and recover the extracting agent, obtain impurities and by-products;
(5) the salt of the corresponding acidifying agent in raffinate in step 3 is removed with a nanofiltration membrane, and concentrated by evaporation to obtain a HEPES;
(6) Finally, the 4-hydroxyethylpiperazineethanesulfonic acid product was washed with absolute ethanol and dried to obtain high-purity HEPES.

The HEPES obtained by the invention has high purity, simple operation process, environmental protection process, low purification cost and high yield, which is suitable for industrial mass production.


4. CN108003117A

(1) adding a sodium sulfate solution with a mass fraction of 3%~5% in the anode chamber and the cathode chamber, and adding a sodium hydroxyethylpiperazine ethylsulfonate solution with a mass concentration of 10% to 30% in the liquid chamber, add pure water in the alkali chamber;
(2) Passing DC power supply for voltage regulation operation, control voltage is 15~25V, control current density is 10~20mA/cm2, device operating temperature is 20~35°C, flow control of each compartment is 30~40L/h, during the process of device operation, each compartment is circulated and cooled by the chilled brine in the circulating coil;
(3) Sampling and analyzing the solution in the alkali chamber and the liquid chamber at regular intervals. When the concentration of the solution in the alkali chamber is maintained, the device stops running, and finally the solution of HEPES is obtained, which was evaporated to obtain a solid crystal of HEPES.


HEPES obtained by the method satisfies the requirement of high purity, and can also recover the alkali liquor, thereby effectively avoiding the pollutant discharge and realizing the resource-saving utilization.


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Recipes of HEPES Buffer

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is a hydrogen ion buffer which has a good buffering capacity in the pH range of 6.8~8.2 and can control a constant pH for a long time. It can be used in a wide range of biochemical reactions, such as, in the research of organelles and highly denatured pH-sensitive proteins and enzymes, as well as biochemical diagnostic kit, DNA/RNA extraction kits and PCR diagnostic kit. Below we will list the common used recipes.


(1) HEPES buffer

Reagent	Amount to add (for 1 L)	Final concentration
NaCl	6.72 g	115 mM
CaCl2	133 mg	1.2 mM
MgCl2	114 mg	1.2 mM
K2HPO4	418 mg	2.4 mM
HEPES	4.77 g	20 mM
H2O	to 1 L

Adjust the pH to 7.4 with HCl or NaOH. HEPES buffer can be stored refrigerated for several weeks.
(2) HEPES Binding Buffer
10-mM HEPES, pH 7.4; 150 mM NaCl; 5 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2.

• Annexin V-FITC in HEPES binding buffer

Annexin V-FITC can be obtained from different companies (e.g., Clontech, Boehringer Mannheim, BioWhittaker). We recommend titrating it to determine optimal concentrations.

• PI in HEPES binding buffer

100 µg/mL propidium iodide, Dissolve propidium iodide in HEPES binding buffer.
(3) HEPES-KRH Buffer

Reagent	Final concentration
NaCl	116 mM
KCl	4 mM
MgCl2	1 mM
CaCl2	1.8 mM
Glucose	25 mM
HEPES acid	10 mM

Adjust pH to 7.4.

(4) HEPES-buffered saline buffer

Reagent	Final concentration
NaCl	150 mM
Tween-20	0.005% (v/v)
EDTA	3 mM
HEPES pH (7.4)	10 mM

(5) HEPES-Buffered Saline (HBS) Precipitation Buffer (2×)
50 mM HEPES, 280 mM NaCl. Adjust the pH to 7.10 (±0.05) with NaOH. Bring the final volume to 1 L in ddH2O. Filter-sterilize.
(6) HEPES Immunoprecipitation Buffer

Reagent	Final concentration
NaCl	10 mM
Na-β-glycerophosphate	20 mM
EDTA	1 mM
HEPES pH (7.5)	50 mM
Na3VO4	0.1 mM
NaF	50 mM
PMSF	1 mM
DTT	1 mM
NP-40	0.1%
Protease inhibitor cocktail

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Preparation of porous zinc oxide microspheres guided by HEPES

HEPES is abbreviated of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, which is a Good's buffer with pH buffer range of 6.8~8.2, and widely used in cell culture because of its good ability at maintaining physiological pH. It can also be used to prepare porous zinc oxide microspheres.

As a II-VI semiconductor compound, zinc oxide has a band gap of 3.37 eV and an exciton binding energy of 60 meV. Due to its special piezoelectric and optical properties, zinc oxide is widely used in many fields such as solar cells, sensors, and voltage sensitive resistors, piezoelectric materials, antibacterial agents, and photocatalysis fields. The morphology and size of zinc oxide have an important influence on its properties and applications, especially porous zinc oxide microspheres, which exhibit low density and high specific surface properties in catalysts, gas sensors, drug delivery and other fields.

At present, different methods have been reported to synthesize porous zinc oxide microspheres, such as high temperature calcination, chemical vapor deposition, and chemical bath deposition. However, these methods have high reaction temperatures, are complicated to operate, and are difficult to control. The solvothermal method is widely used for the synthesis of zinc oxide micro/nano materials due to its simple equipment and mild reaction conditions. However, the synthesis of porous zinc oxide microspheres by solvothermal method often requires the addition of a templating agent or a porogen. After the completion of the reaction, further post-treatment is required to remove the templating agent, which increases the complexity of the process and environmental pollution.

In order to solve the above problems, the researchers [1] developed a preparation method for porous zinc oxide microspheres by using HEPES molecules, which has low cost and simple operation, and the obtained zinc oxide has uniform morphology, high specific surface area and multi-stage pore structure. The specific operations are as follows:

(1) 2~6 mmol of organic zinc salt (zinc acetate or zinc acetylacetonate) are sonicated in 30~70mL organic solvent (TEG, DEG, EG, DMF) for 10 minutes, then add 2~8 mmol HEPES;

(2) The mixed solution is placed in a stainless steel high pressure autoclave lined with 100 mL of polytetrafluoroethylene, and reacted at 150°C for 6 to 18 hours;

(3) The obtained product was washed by centrifugation at 10000 rpm/min for 10 minutes, and the supernatant was removed, repeat 5 times to remove the residual solvent and HEPES. The product was dried in a 60°C oven for 24 hours and then naturally cooled.

In this method, HEPES molecules play an important role in the formation of porous zinc oxide microspheres. After the addition of the HEPES molecule, it is adsorbed on the surface of the nucleus by electrostatic interaction with the zinc oxide seed crystal in the sol. The presence of the HEPES molecule blocks further clustering and aggregation of the nucleus. Porous zinc oxide microspheres are formed by the continued growth of the HEPES molecules under solvothermal conditions. The HEPES molecules and solvent in the surface and voids of the microspheres can be completely removed after multiple washings with deionized water.

HEPES (CAS 7365-45-9) molecules have the advantages of non-toxicity and environmental friendliness. The method is easy to operate, simple in equipment, low in synthesis temperature, low price in raw materials, good in repeatability, and suitable for industrial production. The prepared porous zinc oxide microsphere has the advantages of uniform size, multi-stage pore structure (pore size 4~30 nm) and large specific surface area (43.4~69.6m2/g), and can be used as photocatalyst and gas sensor.

Reference

[1]CHEN Rong, LI Qin, YANG Hao, Lv Zhong. A preparation method of porous zinc oxide microspheres guided by HEPES molecularly. 2014, CN103482682A.

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HEPES and PIPES buffer, what differentiates them?

In biochemical experiments, buffer solution plays an indispensable role, it can resist the influence of a small amount of strong acid and alkali and maintain the pH value closest to the physiological environment for the system. HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) buffer and PIPES buffer are both commonly used in biological experiments, both of which are Good's buffers and have similar structures. Many people often have doubts: Besides the structure, what is the difference between them?

Similarities between HEPES and PIPES buffers

HEPES and PIPES buffers, even all Good's buffers, have the following characteristics:

(1) pKa value between 6.0 and 8.0;

(2) High solubility in water;

(3) Membrane impermeability and not easy to penetrate biofilm;

(4) Limited impact on biochemical reactions, chemical and enzymatic hydrolysis, and no complex or precipitation with metal ions;

(5) Very low absorption of visible light and ultraviolet light;

(6) Ion concentration, solution composition and temperature have little effect on dissociation;

(7) Not participate or interfere with biochemical processes

What is the difference between them?

In summary, both HEPES buffer (CAS 7365-45-9) and PIPES are Good’s buffers, which do not form stable complexes with metal ions and are suitable for solution systems containing metal ions. However, there is also a certain difference between them. Therefore, when selecting the above buffer, we need to comprehensively consider the suitability of the experimental system and the difference in the nature of the two.

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