Pharmaceutical Preparation for Use in the Treatment of Systemic Inflammatory Response Syndrome (SIRS)
20230190793 · 2023-06-22
Inventors
Cpc classification
A61K33/20
HUMAN NECESSITIES
International classification
Abstract
The present invention describes a pharmaceutical preparation for use in the treatment of systemic inflammatory response syndrome (SIRS).
Claims
1. A pharmaceutical preparation for use in treatment of systemic inflammatory response syndrome (SIRS), containing a reactive chlorine compound as an active ingredient.
2. The pharmaceutical preparation for use according to claim 1, wherein the reactive chlorine compound comprises a peroxochloric acid, a dichloroperoxo acid, and/or a peroxochlorous acid or a pharmaceutically acceptable salt of these acids.
3. The pharmaceutical preparation for use according to claim 1, wherein the reactive chlorine compound comprises a molecular formula selected from the group consisting of HClO.sub.3, HClO.sub.4, and/or H.sub.2Cl.sub.2O.sub.6 or a pharmaceutically acceptable salt of these acids.
4. The pharmaceutical preparation for use according to claim 1, wherein the reactive chlorine compound comprises a structure of formula [O═ClOO]—, [O.sub.2ClOO]—, [O.sub.2ClOOClO.sub.2].sup.2−, and/or an anion of the reactive chlorine compound has a molecular formula Cl.sub.2O.sub.6.sup.2−.
5. The pharmaceutical preparation for use according to claim 1, wherein the reactive chlorine compound is obtained according to a method in which (a) chlorine dioxide is reacted with an aqueous or water-containing solution of hydrogen peroxide or another hydroperoxide or peroxide at a pH >=6.5, (b) the pH is lowered to 3 to 6 by addition of an acid, and (c) a gaseous free reactive chlorine compound is expelled with a cooled gas and collected in a basic solution with a pH >10.
6. The pharmaceutical preparation according to claim 1, wherein the reactive chlorine compound is obtained according to a method in which (a) chlorine dioxide is reacted with an aqueous or water-containing solution of hydrogen peroxide or another hydroperoxide or peroxide at a pH >=6.5, (b) the pH is lowered to 3 to 6 by the addition of an acid, (c) a gaseous free reactive chlorine compound is expelled with a cooled gas and collected in a basic solution with a pH >10, and (d) the collected reactive chlorine compound is incubated with chlorite at a pH of 6 to 8.
7. The pharmaceutical preparation according to claim 1, comprising a pharmaceutically acceptable carrier.
8. The pharmaceutical preparation according to claim 1, wherein a mass spectrum of the pharmaceutical preparation shows a signal at 189.0 m/z.
9. The pharmaceutical preparation according to claim 1, wherein a mass spectrum of the pharmaceutical preparation shows a signal at 83.2 m/z.
10. The pharmaceutical preparation according to claim 1, wherein a systemic inflammatory response syndrome (SIRS) is associated with a bacterial infection.
11. The pharmaceutical preparation according claim 1, wherein the pharmaceutical preparation contains a pharmaceutically active substance, which differs from the reactive chlorine compound of claim 1.
12. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation is provided in a sachet, which comprises at least two compartments for storage of at least two liquids, which can be opened mechanically, such that, after opening the compartments, the liquids can be mixed, wherein one of the compartments comprises a liquid reactive chlorine compound according to claim 1 and one of the compartments comprises a liquid for adjusting a pH to a physiological pH value.
13. A method of treatment of SIRS in a human subject in need thereof comprising concurrent or sequential administration of hemodialysis treatment and a pharmaceutical preparation according to claim 1 to the subject.
14. A combination preparation comprising separate packages of at least one pharmaceutical preparation according to claim 1 for the treatment of SIRS and at least one drug which differs from the reactive chlorine compound of claim 1.
15. The combination preparation according to claim 14, wherein the drug, which differs from the reactive chlorine compound of claim 1, comprises at least an antibiotic, an antipyretic, a drug for the treatment of disseminated intravascular coagulation (DIC), an antibody, a cytokine, a chemokine, an antimicrobial peptide, a sphingomyelinase inhibitor, a statin, alpha-2-macroglobulin, thrombin-derived C-terminal peptide, sphingosine-1-phosphate, curcumin, ascorbic acid, resveratrol, melatonin, glycyrrhizin, and/or erythropoietin.
16. The pharmaceutical preparation of claim 6, wherein in step (d) the collected reactive chlorine compound is incubated with chlorite at a pH of approximately 7.
17. The pharmaceutical preparation according to claim 7, wherein the pharmaceutically acceptable carrier comprises water, wherein the water content is at least 90 wt. % and the pharmaceutical preparation is an aqueous solution.
Description
[0169] The following examples explain the invention in more detail, but are not in any way restrictive.
[0170]
[0171]
[0172]
[0173]
[0174]
EXAMPLE 1: PREPARATION OF DICHLORIC ACIDS
[0175] Drops of sulfuric acid (96%) are added carefully while stirring to a solution of 100 g anhydrous sodium chlorite in 200 mL water. With a strong gas flow (Ar, N.sub.2 or O.sub.2 or CO.sub.2-free air) the chlorine dioxide produced is expelled. The gas flow has to be so strong that the ClO.sub.2 content does not rise above 5 percent (risk of explosion). The ClO.sub.2-containing gas flow, is introduced into a solution of 15 mL 30% hydrogen peroxide in 35 mL water, which has previously been brought to pH 12 by the addition of 4M sodium hydroxide solution, via three washing bottles connected in series, each of which is filled with 30 mL of a 2 M NaClO.sub.2 solution with pH 11, in order to capture elemental chlorine. Instead of hydrogen peroxide also a solution of sodium perborate or sodium percarbonate or another peroxo compound can be used such as the H.sub.2O.sub.2 adduct of urea. During the introduction of gas the pH is checked by a glass electrode. By adding 4M NaOH, the pH is kept at 12 for the course of the reaction. The supplied hydroperoxide or supplied peroxo compound is used up when the introduction of gas leads to a permanent yellow coloration. The yellow solution is then decolored again with a drop of the solution of the oxidizing agent (e.g. H.sub.2O.sub.2).
[0176] The solution containing reactive chlorine is dropped while stirring to a solution of 500 g citric acid in 3 liters of water, which has been previously adjusted to pH 4.5 with 2 M sodium hydroxide solution. During the addition, the reactive chlorine compound formed is expelled by a powerful gas flow (N.sub.2 or O.sub.2). The gas flow should preferably be cooled. The hose connections should be as short as possible. The gas is collected for example in three wash bottles connected in series, each charged with 50 mL 0.1 M NaOH.
[0177] The contents of the wash bottles are combined and kept at pH >10.
[0178] For forming the dichloric acids which are preferably used according to the invention the pH is adjusted to 7 for example with hydrochloric acid and a 10-fold molar excess amount of sodium chlorite is added.
[0179] In a further embodiment, the pH is adjusted to 7 for example with hydrochloric acid and an equimolar amount of sodium chlorite is added for the formation of the dichloric acids which are preferably used according to the invention.
[0180] For storage, it is then preferred in each case if the pH is adjusted to approximately 10 to 13.
[0181] The total content of reactive chlorine anions is determined by potentiometric titration with 0.1 M HCl in a manner well known to the person skilled in the art. Here, different compounds can be determined on the basis of the pKa values of the different anions obtained over the titration curve.
[0182] The dichloric acids formed are present in solution in a mixture with a defined amount of chlorite and other reactive chlorine compounds.
[0183] The presence of the dichloric acids is detected by Raman spectroscopy.
EXAMPLE 2: ANALYTICAL DETERMINATIONS OF THE SOLUTION OBTAINED FROM EXAMPLE 1
1) pH Measurement:
[0184] The pH is determined by the single rod glass electrode. The product content and the state of equilibrium are dependent on the pH value.
2) Titration with 0.1 M HCl:
[0185] The titration is used for example for quantitatively determining the dichloric acid content or also the content of peroxochlorous acid or peroxochlorate.
[0186] 1 mL of the product solution is titrated potentiometrically with 0.1 M hydrochloric acid. Titration curves (pH vs. mL 0.1 M HCl) are recorded. From the acid consumption between pH 8.5 and 4.5 determined in the derivation of the titration curve, the content of anions of the corresponding acids in total is determined.
[0187] In a typical result 1 mL product solution gives a consumption of 0.72 mL 0.1 M HCl and thus a concentration of 0.072 M.
[0188]
[0189]
3) UV-Vis Absorption Spectrum:
[0190] The measurement of the UV spectrum is used to quantify the content of chlorite in the product solution. For comparison, spectra of a chlorite-containing and a chlorite-free product solution are shown in
[0191] In 1 cm quartz cuvettes the absorbance values are determined at 260 nm and 500 nm. From the difference A260-A500 and by means of the extinction coefficient for chlorite of c260 nm=140 M-1 cm-1 at 260 nm, the content of ClO.sub.2.sup.− ions can be determined.
[0192] Absorption at 360 nm indicates free chlorine dioxide (c360 nm=1260 M.sup.−1 cm.sup.−1).
4) Mass Spectroscopy
[0193] The ESI mass spectrometry was performed with a Bruker Esquire-LC spectrometer in standard MS mode. The sample was an aqueous product solution diluted with methanol before measurement. The scan range used was between 30 m/z and 400 m/z, with capillary exit −65 volts and skim −15 volts; the spectrum represents an average of 50 measurements.
[0194] The right arrow in
5) Ion Chromatography
[0195] All analyses were carried out with a modular ion chromatography system of the company Metrohm.
Pump: Metrohm IC 709 Pump
Detector: Metrohm 732 IC Detector
Suppressor: Metrohm 753 Suppressor Module
Column: Metrosep A 250
[0196] Flow rate: 1 ml/min
Injection volume: 20 μL
Eluent: 1 mM NaOH
[0197] Fresh solutions of reference substances of known concentration were prepared immediately before each measurement and then measured using the method described above with the specified eluent.
Retention Times of the Reference Substances:
[0198]
TABLE-US-00009 Substance Retention time [min] NaCl 13.21 NaClO.sub.2 12.30 NaClO.sub.3 16.26 NaClO.sub.4 4.36 NaOH 17.32 Na.sub.2CO.sub.3 21.98 Na.sub.2Cl.sub.2O.sub.6 19.77
[0199]
EXAMPLE 3 AND COMPARATIVE EXAMPLE 1
[0200] Experiments on mice to demonstrate the efficacy of reactive chlorine compounds to be used according to the invention. The solution of reactive chlorine compounds prepared in Example 1 and examined in more detail in Example 2 will be abbreviated to DPOCL in the following.
[0201] It is known from toxicological experiments that 45 mg DPOCL/kg does not cause any clinical symptoms. In order to test whether DPOCL protects from SIRS, the following experiments were carried out. These experiments are based on a CLP model, in which cecal ligation and puncture (CLP) is performed. This animal model is widely recognized and described in more detail by Wichterman et al. (Wichterman K A, Baue A E, Chaudry I H. Sepsis and septic shock—a review of laboratory models and a propsal. Journal of Surgical Research, 114:740-5, 1979). Furthermore, this method has been described above.
[0202] NMRI mice receive brief anesthesia with ketamine and xyiazine (ketamine 120 mg/kg KGW; xyiazine 16 mg/kg KGW). After the anesthetic has taken effect they are shaved on the abdominal side and disinfected. The abdominal cavity is opened with a 1 cm incision. The appendix is exposed. Distally, a portion is ligated and perforated with a 0.9 mm cannula. On applying light pressure a small amount of feces comes out. The appendix is pushed back into the abdomen. The peritoneum and musculature are closed with absorbable sutures and the skin is stapled with Michel wound staples. The procedure takes about 8 minutes. Immediately after the procedure and for the next 2 days, the animals are given the analgesic buprenorphine (0.1 mg/kg, s,c.) 2 times daily. Ketamine/xylazine keep the animal under anesthesia for about 45 minutes.
[0203] The symptoms of sepsis are assessed according to the following scheme, which is commonly used, wherein the symptoms of sepsis result from a change in behavior (separation, no grooming, passivity) and a drop in temperature:
TABLE-US-00010 Expected Time Clinical time period in intervals for Special model Symptoms • Score experiment¬ monitoring measures CLP no particular 0 Day 0 2 times Buprenorphine findings daily Slowed gait, 1 from day 1 2 times Buprenorphine; weight difference daily Food and water up to 10% on the cage floor additionally accessible Animal isolates 2 from day 1 2 times Buprenorphine; itself, temperature or 2 daily Food and water still above 32° C. on the cage floor (temperature additionally measured without accessible contact with an infrared thermometer on the ventral side), weight difference, over 10% Animal isolates 3 from day 1 3 times Buprenorphine itself and or 2 daily food and water temperature on the below 32° C.; reluctance to move;
Experiment 100
[0204] To determine the appropriate CLP mode, the distal 50% of the appendix of male NMRI mice was ligated and perforated with a 0.9 mm cannula 1 time (4 animals), 2 times (4 animals) or 3 times (8 animals). 0 of 8 animals survived three punctures, 3 of 4 animals survived 2 punctures and 2 of 4 animals survived 1 puncture.
Experiment 101
[0205] CLP with 3 punctures was performed on day 0 in 8 male NMRI mice (32-39 g) and (still under ketamine/xyiazine anesthetic) 4 mice were injected i.v. with 45 mg/kg DPOCL in 0.42 ml/10 g KGW, a further 4 mice were injected with an equivalent amount of buffer solution. The injections were well tolerated by the animals. On day 1, as expected, the animals already showed sepsis symptoms and were not to be exposed to further ketamine/xyiazine anesthesia, which (see above) lasts 30 to 60 minutes and cannot be antagonized. Therefore, anesthesia with midazolam, fentanyl and medetomidine was used on day 1, which was then antagonized with flumazenil, atipamezole and naloxone, because there is good experience with this anesthesia for other retrobulbar injections (in healthy mice). The CLP mice tolerated the combination of antagonizable anesthesia and {circumflex over ( )}1.5 ml i.v. poorly from day 1 and had to be killed on day 2, regardless of whether they had received DPOCL (4 animals) or PBS (4 animals).
Experiment 102
[0206] Also in this experiment CLP was performed on day 0 with 3 punctures in 8 NMRI mice (28-32 g) and (still under ketamine/xylazine anesthesia) 4 mice were injected i.v. with 45 mg/kg DPOCL in 0.42 ml/10 g KGW, a further 4 mice were injected with an equivalent amount of buffer solution. However, females were used in this experiment and isoflurane was used for anesthesia, as this brief anesthesia should be sufficient for retrobulbar injections. In fact, NMRI females survived CLP with isoflurane better, so that after 1 week 4 out of 4 DPOCL-treated animals were still alive and 2 out of 4 PBS-treated animals. However, even in the females, the high DPOCL volume on day 1, 2 and 3 was associated with some circulatory stress.
Experiment 103
[0207] As in experiment 102, CLP with 3 punctures was performed in 8 male NMRI mice (28-36 g) on day 0 and (still under ketamine/xyiazine anesthesia) 4 mice were injected i.v. with 45 mg/kg DPOCL in 0.42 ml/10 g KGW, a further 4 mice were injected with an equivalent amount of buffer solution. After good experience in experiment 102 isoflurane was again used for the further DPOCL injections. On day 8, 3 out of 4 animals treated with DPOCL and 1 out of 4 after PBS treatment were still alive. The males also had circulation problems due to the high injection volume.
[0208] The results of experiments 100, 102 and 103 are represented graphically in
Experiment 200: Determination of the Minimum DPOCL Dose Required
[0209] In experiment 200 further animals are treated with two lower doses of DPOCL after CLP, to determine whether lower doses of DPOCL also protect the animals.
Group 2.1
[0210] 18 mice receive directly after CLP 200 μl PBS retrobulbar i.v. on day 0, day 1, day 2 and day 3.
Group 2.2
[0211] 18 mice receive directly after CLP 15 mg DPOCL in 200 μl retrobulbar i.v. on day 0, day 1, day 2 and day 3.
Group 2.3
[0212] 18 mice receive directly after CLP 5 mg DPOCL in 200 μl retrobulbar i.v. on day 0, day 1, day 2 and day 3.
The survival of the groups is monitored at closer intervals.
Experiment 300: Determination of the Required Minimum Treatment Period
[0213] In experiment 300 the duration of the treatment is shortened by 1 day in each of the different subtrials to determine the minimum time DPOCL needs to be administered for the previously observed improvement of survival to occur.
Group 3.1
[0214] 18 mice receive directly after CLP×mg DPOCL in 200 μl retrobulbar i.v. on day 0, day 1, day 2 and day 3.
Group 3.2
[0215] 18 mice receive directly after CLP×mg DPOCL in 200 μl retrobulbar i.v. on day 0, day 1, and day 2.
Group 3.3
[0216] 18 mice receive directly after CLP×mg DPOCL in 200 μl retrobulbar i.v. on day 0 and day 1.
Group 3.4
[0217] 18 mice receive directly after CLP×mg DPOCL in 200 μl retrobulbar i.v. only on day 0.
[0218] The survival of the groups is monitored at closer intervals.
Experiment 400: Production of Cytokines and Chemokines (Mediators) 24 h after CLP
[0219] As a rule, successful SIRS therapy influences mediator production in the treated animals. Mediators can be determined in different tissues and compartments with and without further stimulation. A mediator measurement in the serum of septic animals 24 h after CLP is often used as a rough guide. IL-6 is determined as the main target variable, other mediators are secondary targets.
[0220] In addition, the bacterial load in the blood, peritoneal lavage, lungs, liver and kidneys is a determined as a further secondary target in these animals.
Group 4.1
[0221] 8 mice receive 200 μl PBS retrobulbar i.v. directly after CLP on the optimal days determined in experiment 4.
Group 4.2
[0222] 8 mice receive directly after CLP the optimal amount of DPOCL in 200 μl retrobulbar i.v. determined in experiment 2 at the optimal time points determined in experiment 3.
Experiment 500: Use of Evans Blue for Study of Vascular Permeability
[0223] A symptom of SIRS is DIC (disseminated intravascular coagulation) in combination with increased vascular permeability. This means that clotting factors and platelets are depleted and are no longer available to close off the endothelial damage that has occurred. The barrier function of the endothelium can be tested by injecting the dye Evans Blue i.v. and 30 min later perfusing the animals, removing organs (lungs, intestines), extracting the dye and detecting it photometrically. The more damage there is to the endothelium, the more dye will penetrate into the tissue.
[0224] The biometric design is taken over from experiment 4: group sizes n=8.
Group 5.1
[0225] 8 mice receive 200 μl PBS retrobulbar i.v. directly after CLP on the optimal days determined in experiment 4.
Group 5.2
[0226] 8 mice receive 200 μl DPOCL retrobulbar i.v. directly after CLP in the optimal amount determined in experiment 3 on the optimal days determine in experiment 4.
48 h after CLP all mice are injected with 200 μl Evans Blue retrobulbar, i.v. 30 min later the mice are anesthetized with ketamine/xylazine and perfused with PBS. The organs are removed and Evans Blue is determined.
[0227] The features of the invention disclosed in the above description, as well as in the claims, figures and embodiments, may be essential, both individually and in any combination, for the implementation of the invention in its various embodiments.