Gaseous pharmaceutical composition for hypertension therapy

Abstract

To provide a novel pharmaceutical composition for hypertension therapy that can be applied to a wide range of hypertensive patients.SOLUTION: According to the present disclosure, a gaseous pharmaceutical composition for improving hypertension comprising a hydrogen gas is provided. The pharmaceutical composition according to the present disclosure can be used for improvement of a wide range of hypertension including essential hypertension.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to pharmaceutical compositions for treating hypertension, and more particularly to gaseous pharmaceutical compositions for treating hypertension comprising hydrogen gas. [Background technology]

[0002] The global prevalence of hypertension among adults is estimated at 1.13 billion. Hypertension is a strong risk factor for cardiovascular disease (CVD) and chronic kidney disease (CKD), occurring in more than 80% of CKD patients. Because CKD promotes hypertension, which may contribute to CKD progression, optimizing care during the predialysis phase of CKD is important. Despite this focus on hypertension treatment, more than 60% of hypertensive patients experience inadequate blood pressure control and do not achieve their blood pressure treatment goals.

[0003] Molecular hydrogen (H2) is a versatile gas with antioxidant and anti-inflammatory properties and has been a promising treatment option for ischemia-reperfusion injury in emergency and critical care settings, such as acute myocardial infarction, cardiac arrest, and hemorrhagic shock in animal models (Patent Document 1 and Non-Patent Documents 1-5).

[0004] The therapeutic effect of hydrogen on hypertension has also been suggested in patients with end-stage renal disease (Non-Patent Documents 6 and 7). Specifically, it has been reported that chronic hemodialysis patients who received hydrogen-enriched dialysate prepared by mixing dialysate concentrate with reverse osmosis water containing dissolved hydrogen produced by water electrolysis showed improved post-dialysis hypertension compared to conventional hemodialysis. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 021175 [Non-patent literature]

[0006] [Non-Patent Document 1] Journal of the American Heart Association 2012; doi: 10.1161 / JAHA. 112. 003459; Circulation.2014 Dec 9;130(24):2173-80 [Non-patent document 2] Hayashida,K.et al. Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury. Biochem.Biophys.Res.Commun.373,30-35;10.1016 / j.bbrc.2008.05.165(2008) [Non-patent document 3] Hayashida, K. et al. H(2) gas improves functional outcome after cardiac arrest to an extent to comparable therapeutic hypothermia in a rat model. J. Am.Heart Assoc.1,e003459;10.1161 / JAHA.112.003459(2012). [Non-patent document 4] Hayashida, K. et al. Hydrogen inhalation during normoxic resuscitation improves neurological outcome in a rat model of cardiac arrest independently of targeted temperature management. Circulation 130,2173-2180;10.1161 / CIRCULATIONAHA.114.011848(2014). [Non-patent document 5] Matsuoka, T. et al. Hydrogen gas inhalation inhibits progression to the “irreversible” stage of shock after severe hemorrhage in rats. J.Trauma Acute Care Surg.83,469-475;10.1097 / TA.0000000000001620(2017). [Non-patent document 6] Nakayama,M.et al. A novel bioactive haemodialysis system using dissolved dihydrogen(H2) produced by water electrolysis: a clinical trial. Nephrol.Dial.Transplant.25,3026-3033;10.1093 / ndt / gfq196(2010)) [Non-Patent Document 7] Nakayama,M.et al. Novel haemodialysis(HD) treatment employing molecular hydrogen(H2)-enriched dialysis solution improves prognosis of chronic dialysis patients: A prospective observational study. Sci.Rep.8,254;10.1038 / s41598-017-18537-x(2018) Summary of the Invention [Problem to be solved by the invention]

[0007] However, conventional technology is based on the premise that hydrogen is used in dialysis in a form in which it is dissolved in the dialysis fluid, and cannot be applied to hypertensive patients before dialysis (e.g., CKD patients) or to patients with hypertension not caused by renal failure. Therefore, an object of the present invention is to provide a novel pharmaceutical composition for treating hypertension that is applicable to a wide range of hypertensive patients, including those with essential hypertension, regardless of specific cause. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found for the first time that hypertension in hypertensive subjects, including those with genetic hypertension, can be improved by administering gaseous hydrogen (hydrogen) to the subject. That is, the present disclosure includes the following features.

[0009] [1] A gaseous pharmaceutical composition for improving hypertension, comprising: The pharmaceutical composition comprises hydrogen gas. characterized in that Pharmaceutical compositions.

[0010] [2] The gaseous pharmaceutical composition according to [1], The improvement of hypertension includes improvement of diastolic hypertension. Pharmaceutical compositions.

[0011] [3] The gaseous pharmaceutical composition according to [2], The subject with hypertension is a subject with a diastolic blood pressure of 100 mmHg or more. Pharmaceutical compositions.

[0012] [4] The pharmaceutical composition according to any one of [1] to [3], The hypertension is essential hypertension or secondary hypertension. Pharmaceutical compositions.

[0013] [5] The pharmaceutical composition according to [4], The hypertension is essential hypertension. Pharmaceutical compositions.

[0014] [6] The pharmaceutical composition according to [4], The hypertension is secondary hypertension. Pharmaceutical compositions.

[0015] [7] The pharmaceutical composition according to [6], The secondary hypertension is hypertension caused by renal dysfunction. Pharmaceutical compositions.

[0016] [8] The pharmaceutical composition according to [7], The renal dysfunction is due to renal disease or due to nephrectomy or partial nephrectomy. Pharmaceutical compositions.

[0017] [9] The pharmaceutical composition according to any one of [6] to [8], The subject with secondary hypertension is a subject not undergoing dialysis. Pharmaceutical compositions.

[0018]

[10] The pharmaceutical composition according to any one of [1] to [9], The pharmaceutical composition further comprises oxygen gas. characterized in that Pharmaceutical compositions.

[0019]

[11] The pharmaceutical composition according to any one of [1] to

[10] , The pharmaceutical composition further comprises an inert gas. characterized in that Pharmaceutical compositions.

[0020]

[12] The pharmaceutical composition according to any one of [1] to

[11] , The hydrogen concentration in the pharmaceutical composition is 0.1% to 4.0% (v / v). characterized in that Pharmaceutical compositions.

[0021]

[13] The pharmaceutical composition according to any one of [1] to

[12] , The hydrogen concentration in the pharmaceutical composition is 1.0% to 2.0% (v / v). characterized in that Pharmaceutical compositions.

[0022]

[14] The pharmaceutical composition according to any one of [1] to

[13] , The hydrogen gas is provided by a hydrogen gas generator or a container containing hydrogen gas. characterized in that Pharmaceutical compositions.

[0023]

[15] The pharmaceutical composition according to

[14] , The hydrogen gas generator includes a hydrogen generating means by water electrolysis. characterized in that Pharmaceutical compositions.

[0024]

[16] The pharmaceutical composition according to

[14] , The container contains a mixed gas of hydrogen gas and nitrogen gas. characterized in that Pharmaceutical compositions.

[0025]

[17] The pharmaceutical composition according to any one of

[14] to

[16] , The hydrogen gas generating device or the container further comprises a control means, either on the device or container itself or on piping connected to the device or container, for monitoring and adjusting the amount of hydrogen gas supplied to the subject; characterized in that Pharmaceutical compositions.

[0026]

[18] A method for treating hypertension, comprising: administering hydrogen gas to a subject with hypertension; method.

[0027] Any combination of one or more of the above-mentioned aspects is also included within the scope of the present invention. [Effects of the Invention]

[0028] According to the present disclosure, a novel pharmaceutical composition for treating hypertension that can be applied to a wide range of hypertensive patients is provided. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 shows an overview of the experimental design used in the present examples. In the figure, the abbreviations and symbols represent the following: white arrow, 5 / 6 nephrectomy; black arrow, implantation of a telemetry transmitter; gray triangle, invasive hemodynamic monitoring via a femoral artery catheter; black triangle, heart rate variability analysis; white triangle, noninvasive hemodynamic measurement via the tail cuff method; gray bar, daily telemetry hemodynamic recording; LEW, Lewis rat; SHR, spontaneously hypertensive rat. [Figure 2] Figure 2 shows the effects of H inhalation early after partial nephrectomy on hemodynamics and renal function over a 4-week period. In the figure, the abbreviations represent the following: MAP, mean arterial pressure; BP, blood pressure. N = 3 per group. Data are expressed as mean ± SE; *P < 0.05, **P < 0.01 (ANOVA). [Figure 3] Figure 3 shows the effects of H inhalation early after partial nephrectomy on ambulatory blood pressure and renal function. The abbreviations in the figure are as follows: MAP, mean arterial pressure. N = 3 per group. Data are expressed as mean ± SE. *P = 0.04 (mixed-effects model). [Figure 4] Figure 4 shows the hemodynamic effects of delayed H inhalation in nephrectomized rats. In the figure, the abbreviations represent the following: BL, baseline; BP, blood pressure; bpm, heart rate (beats per minute). N = 17 in each blood pressure measurement group, and data are expressed as mean ± SE. [Figure 5] Figure 5 shows the effect of H2 on blood pressure variability in nephrectomized rats. In the figure, the abbreviations stand for: LEW, Lewis rat; TTI, implanted telemetry transmitter; ΔLFnu, normalized low frequency; ΔHFnu, normalized high frequency. [Figure 6]Figure 6 shows the hemodynamic effects of H inhalation in spontaneously hypertensive rats. In the figure, the abbreviations and symbols represent the following: double arrow, duration of intermittent gas inhalation; AUC, area under the curve; bpm, heart rate in beats per minute; h, time; HR, heart rate; and MAP, mean arterial pressure. N = 3 per group. Data are presented as mean ± SE; *P = 0.04 (paired t-test). [Figure 7] Figure 7 shows the hemodynamic effects of H inhalation in spontaneously hypertensive rats. In the figure, the abbreviations stand for: HF, high frequency; LF, low frequency; HFnu, normalized high frequency; LFnu, normalized low frequency. N = 3 per group. Data are presented as mean ± SE; *P = 0.04 (paired t-test). DETAILED DESCRIPTION OF THE INVENTION

[0030] The present disclosure relates to a pharmaceutical composition for improving hypertension (hereinafter also referred to as the pharmaceutical composition according to the present disclosure).

[0031] Hypertension refers to a condition in which blood pressure is consistently high. Specifically, hypertension is diagnosed when, at any time during repeated blood pressure measurements, the systolic blood pressure exceeds the reference value of 140 mmHg (also referred to herein as "systolic hypertension") and / or the diastolic blood pressure exceeds the reference value of 90 mmHg (also referred to herein as "diastolic hypertension").

[0032] In the present disclosure, "improvement of hypertension" refers to reducing at least one of systolic hypertension or diastolic hypertension to below the reference value during or for at least a certain period after administration of a pharmaceutical composition according to the present disclosure.

[0033] In another embodiment of the present disclosure, "ameliorating hypertension" includes at least improving diastolic hypertension for at least a period of time during or after administration of a pharmaceutical composition according to the present disclosure.

[0034] In the present disclosure, the "at least a certain period of time" may vary depending on the severity of symptoms in the subject, age, sex, and the administration concentration and administration period of the pharmaceutical composition of the present disclosure. For example, the "at least a certain period of time" may be at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, more preferably at least 72 hours, more preferably at least 96 hours, more preferably at least 120 hours, more preferably at least 144 hours, or more preferably at least 168 hours from the end of administration of the pharmaceutical composition of the present invention.

[0035] Without being bound by any theory, it is believed that the pharmaceutical composition according to the present disclosure improves hypertension by suppressing sympathetic nervous function and promoting parasympathetic nervous activation in a subject to which the composition is administered due to hydrogen gas.

[0036] The pharmaceutical composition according to the present disclosure is characterized in that it is a gaseous pharmaceutical composition containing hydrogen gas. Furthermore, since the pharmaceutical composition according to the present disclosure is a gaseous pharmaceutical composition, it is characterized in that it is continuously administered to a subject over a certain period of time. In the present disclosure, hydrogen atoms include all of their isotopes, i.e., protium (P or 1 H), deuterium (D or 2 H), and tritium (T or 3 H). Therefore, molecular hydrogen may include P2, PD, PT, DT, D2, and T2. In a preferred embodiment of the present disclosure, 99% or more of the hydrogen gas contained in the pharmaceutical composition according to the present disclosure is P2, which is natural molecular hydrogen.

[0037] The pharmaceutical composition according to the present disclosure may further comprise oxygen gas, which may be premixed with hydrogen gas to form a mixed gas, or may be mixed with hydrogen gas immediately before or at the time of administration to a subject.

[0038] The pharmaceutical composition according to the present disclosure may further contain an inert gas. The inert gas is used for the purpose of preventing explosion and adjusting the concentration of hydrogen gas or oxygen gas, and therefore may exist in the form of a mixed gas with hydrogen gas and / or oxygen gas. Inert gases that can be used in the pharmaceutical composition according to the present disclosure include, but are not limited to, nitrogen gas, helium gas, argon gas, etc. In one embodiment of the present disclosure, inexpensive nitrogen gas is used as the inert gas.

[0039] In the pharmaceutical composition according to the present disclosure, the concentration of hydrogen gas may be, but is not limited to, any concentration between 0.1 and 4.0% (v / v). The lower limit of the hydrogen gas concentration is set as the minimum concentration at which the effect of improving hypertension can be exerted. Therefore, the minimum concentration at which hypertension can be improved can be appropriately set depending on the severity of the subject's condition, the presence or absence of diseases that cause hypertension, gender, age, etc. In one embodiment, the lower limit of the hydrogen gas concentration can be selected between 0.1 and 1.0%, for example, 0.5%. On the other hand, the upper limit of the hydrogen gas concentration is set from the perspective of safety, since the lower explosion limit of hydrogen in air is 4%. Therefore, the upper limit of the hydrogen gas concentration can be selected from any concentration of 4% or less, for example, 3.0%, 2.5%, or 2.0%, as long as safety is ensured.

[0040] In the pharmaceutical composition according to the present disclosure, the concentration of oxygen gas can be in the range of 21% to 99.9% (v / v) assuming that the concentration of hydrogen gas is 0.1 to 4.0% (v / v).

[0041] In the pharmaceutical composition of the present disclosure, the concentration of the inert gas is set within a range that maintains the appropriate concentration of hydrogen gas and / or oxygen gas and ensures the explosion-proof effect of these gases. Therefore, a person skilled in the art can appropriately set the concentration of the inert gas depending on the concentration of the hydrogen gas and / or oxygen gas used. For example, when the inert gas is nitrogen gas, the concentration of such an inert gas can be arbitrarily selected within the range of 0 to 78.9% (v / v).

[0042] The gas concentrations used throughout this specification indicate the content at 20°C and 101.3 kPa.

[0043] The pharmaceutical composition according to the present disclosure may further contain other atmospheric gases such as carbon dioxide, air, or anesthetic gases, as long as the effect of hydrogen gas is not impaired.

[0044] The pharmaceutical composition according to the present disclosure can be administered to a subject by inhalation using an inhalation device, for example. Such an inhalation device can include, but is not limited to, an inhalation mask. The inhalation mask preferably covers the subject's mouth and nose simultaneously to ensure administration of an appropriate concentration to the subject.

[0045] In one embodiment of the present disclosure, the pharmaceutical composition according to the present disclosure is provided in a form that can be administered directly to a subject. For example, in this embodiment, the pharmaceutical composition according to the present disclosure is provided in the form of a mixed gas prepared by pre-mixing hydrogen gas and an inert gas, as well as oxygen gas for breathing and any other gas at appropriate concentrations.

[0046] In another aspect of the present disclosure, the pharmaceutical composition according to the present disclosure is provided in a form that is prepared immediately before or at the time of administration to a subject. For example, in this embodiment, the pharmaceutical composition according to the present disclosure is provided by connecting a container containing a mixed gas of hydrogen gas and an inert gas and a container containing oxygen gas to an inhalation mask via piping, and delivering the mixed gas to a patient at a flow rate that provides a concentration appropriate for administration to the subject. In one aspect of the present disclosure, the container may be a portable gas cylinder or, for example, a large storage tank installed outdoors. The gas may be stored in a container in the form of compressed gas, or may be stored in a liquefied form, such as liquefied hydrogen gas, in a liquid gas container (LGC). In another example of the present disclosure, the hydrogen gas may be supplied from a hydrogen gas generator. Such a generator may include any hydrogen generating means known to those skilled in the art, including, but not limited to, hydrogen generating means using water (e.g., purified water, alkaline water such as potassium hydroxide) electrolysis, hydrogen generating means using hydrogen storage alloys (e.g., magnesium, vanadium), hydrogen generating means using heating or degassing of hydrogen-dissolved water, hydrogen generating means using ammonia decomposition, hydrogen generating means using steam reforming of hydrocarbons (e.g., methane), hydrogen generating means using methanol / ethanol reforming, hydrogen generating means using water decomposition with a catalyst (e.g., titanium oxide), and hydrogen generating means using a chemical reaction between water and a metal hydride (e.g., alkaline earth metal hydride, alkali metal hydride, typically magnesium hydride). In the present disclosure, a hydrogen generating means using water electrolysis is particularly preferred.

[0047] In another aspect of the present disclosure, the pharmaceutical composition of the present disclosure is provided by supplying hydrogen gas to a sealed chamber such that the concentration of the gas is maintained constant. For example, in this embodiment, the pharmaceutical composition of the present disclosure is provided by supplying a mixture of hydrogen gas and an inert gas to a sealed chamber containing a subject at a flow rate such that the hydrogen concentration in the sealed chamber is maintained at a suitable concentration.

[0048] The subjects to which the pharmaceutical compositions of the present disclosure are administered are not particularly limited, and may include humans as well as rodents such as mice, rats, and rabbits, and non-human mammals such as monkeys, cows, horses, and goats.

[0049] In one embodiment of the present disclosure, the subject to which the pharmaceutical composition of the present disclosure is administered is a human subject. In a specific embodiment of the present disclosure, the subject to which the pharmaceutical composition of the present disclosure is administered is a subject (e.g., a human subject) with a diastolic blood pressure of 100 mmHg or more.

[0050] In another specific embodiment of the present disclosure, the subject to which the pharmaceutical composition of the present disclosure is administered is a subject (e.g., a human subject) with essential hypertension. In the present disclosure, "essential hypertension" should be interpreted as understood by those skilled in the art, and includes all cases of hypertension that do not have a specific underlying disease. For example, essential hypertension includes, but is not limited to, familial hypertension, hypertension due to lifestyle habits, etc.

[0051] In another specific embodiment of the present disclosure, the subject to which the pharmaceutical composition of the present disclosure is administered is a subject (e.g., a human subject) with secondary hypertension, a disease or condition that can cause secondary hypertension, including, but not limited to, renal dysfunction.

[0052] In certain embodiments of the present disclosure, the disease or condition causing secondary hypertension is a subject (e.g., a human subject) with renal dysfunction. The renal dysfunction may be due to any renal disease, such as chronic renal failure, acute renal failure, or may be due to nephrectomy or partial nephrectomy.

[0053] In yet another embodiment of the present disclosure, the subject to which the pharmaceutical composition of the present disclosure is administered is a subject (e.g., a human subject) with impaired renal function who is not already undergoing dialysis.

[0054] The pharmaceutical composition according to the present disclosure is administered continuously to a subject over a certain period of time. The administration time of the pharmaceutical composition according to the present disclosure is not particularly limited as long as it is a time period during which the pharmaceutical composition according to the present disclosure can exert its effect of improving hypertension. A person skilled in the art can appropriately determine an appropriate time period depending on the severity of symptoms in the subject, age, sex, administration concentration of the pharmaceutical composition according to the present disclosure, etc. Such a time period may be, but is not limited to, for example, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, or longer.

[0055] Furthermore, the number of times the pharmaceutical composition according to the present disclosure is administered is not limited and may be administered once or multiple times. The administration interval and the number of times the pharmaceutical composition according to the present disclosure is administered can be appropriately set depending on the symptoms of the patient.

[0056] The pharmaceutical composition according to the present disclosure can be provided by a device for providing the pharmaceutical composition according to the present disclosure. The device according to the present disclosure includes at least a means for providing hydrogen gas, typically a container for storing hydrogen gas or the hydrogen gas generator, and hydrogen gas, which is an active ingredient of the pharmaceutical composition according to the present disclosure, is provided by the container or the hydrogen gas generator.

[0057] The device according to the present disclosure preferably further comprises a pipe connected at one end to the means for providing hydrogen gas. The pipe serves as a hydrogen gas distribution means for delivering hydrogen gas to a subject for inhalation, and the other end is connected directly to an inhalation means for inhaling the gas or to a gas mixing device for mixing with other gases such as oxygen gas. In a preferred embodiment, the means for providing hydrogen gas further comprises a control means for monitoring and adjusting the amount of hydrogen gas supplied to the subject. Alternatively, the control means may be further provided on the pipe connected to the means for providing hydrogen gas.

[0058] The device according to the present disclosure preferably further includes a gas mixing device. The gas mixing device is a means for mixing hydrogen with other gases so that the hydrogen gas from the means for providing hydrogen gas has a concentration appropriate for administration to a subject, and is typically connected to an oxygen gas storage container or an oxygen gas generator via piping. In a preferred embodiment, the gas mixing device further includes a means for monitoring and adjusting the hydrogen concentration in the mixed gas, a means for monitoring and adjusting the oxygen concentration in the mixed gas, and / or a control means for monitoring and adjusting the flow rate of the mixed gas to the subject.

[0059] In another embodiment, the device according to the present disclosure can be used in conjunction with a ventilator, in which the gas mixing device is connected to the ventilator, and oxygen gas delivered from the ventilator is mixed with hydrogen gas in the gas mixing device and then introduced into the inspiratory line, or mixed with hydrogen gas introduced into the inspiratory line and then returned to the ventilator as exhaled gas.

[0060] The terms used in this specification are used to describe particular embodiments and are not intended to limit the invention.

[0061] Furthermore, the term "comprise" used in this specification intends that the stated items (components, steps, elements, or numbers, etc.) are present, unless the context clearly dictates otherwise, and does not exclude the presence of other items (components, steps, elements, or numbers, etc.).

[0062] The entire disclosures of the documents cited in this specification should be considered to be incorporated herein by reference, and a person skilled in the art can incorporate the relevant disclosure contents of those prior art documents as part of this specification, in accordance with the context of this specification, without departing from the spirit and scope of the present invention.

[0063] The present disclosure will be explained in more detail below by showing examples, but the present disclosure is not limited to the examples shown below. [Example]

[0064] In this example, we investigated whether H2 inhalation therapy is effective in preventing and / or treating hypertension in a rat model of hypertension.

[0065] Materials and Methods [animal] Male Lewis rats (8 weeks old, weighing 250–300 g) and 10-week-old male spontaneously hypertensive rats (SHR / Izumo rats; hereafter referred to as SHR) were used (CLEA Japan). Rats were provided with water and standard diet ad libitum and were not fasted prior to the experiment. Rats were housed under standardized temperature (22 ± 1°C) and humidity (55 ± 5%) conditions with a 12-h:12-h light / dark cycle. Rats were allowed to acclimate to the experimental conditions for at least 1 week prior to the experiment. This study was approved by the Institutional Animal Care and Use Committee (Keio University, Tokyo, No. 13002-4; and Nippon Veterinary and Life Science University, Tokyo, No. 30K-61). Rats were randomly assigned to the H2 or control group.

[0066] [Telemetry transmitter implantation] To monitor continuous blood pressure fluctuations, rats were implanted with a telemetry transmitter. Specifically, rats were anesthetized with isoflurane, and the left groin was disinfected with 1% chlorhexidine. An approximately 1.5 cm incision was made in the left groin to expose the left femoral artery, and a telemetry transmitter (HD-S10, Physiotel HD Telemetry, Data Science International) catheter was inserted into the left femoral artery. The tip of the transmitter catheter was positioned in the abdominal aorta caudal to the renal artery bifurcation. The transmitter body was inserted into a subcutaneous pocket created in the left lower back, and the skin was sutured. All surgical procedures were performed aseptically.

[0067] [Invasive and non-invasive blood pressure measurement] Invasive blood pressure measurements were performed 4 weeks after gas inhalation. An arterial catheter (PE50, Natsume) was inserted into the left femoral artery under isoflurane inhalation, and arterial blood pressure was measured (DX-360, Nihon Kohden). Approximately 24 hours after the final gas inhalation, blood pressure and heart rate were measured noninvasively in 5 / 6 nephrectomized rats using a tail cuff pressure measurement system (BP-98A, Softron). Hemodynamic parameters were measured three times, and the median values ​​were used as representative values.

[0068] [Heart rate variability analysis] Heart rate variability (HRV) was analyzed using telemetry system software (Ponemah Ver. 6.3, Data Science International). Frequency-domain analysis (500 Hz sampling rate; very low frequency (VLF) (0.05-0.25 Hz); low frequency (LF) (0.25-1.0 Hz); and high frequency (HF) (1.0-3.0 Hz)) was performed using the first minute (approximately 300-400 beats) of arterial pressure waveform data every 5 minutes. For HRV analysis, data recorded during the first 15 minutes were excluded, and valid data from the first minute of each 5-minute block was used. LF and HF reflected sympathetic and parasympathetic components, respectively (Akselrod, S. et al. Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control. Science 213, 220-222 (1981); Pagani, M. et al. Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympatho-vagal interaction in man and conscious dog. Circ. Res. 59, 178-193 (1986); and Montano, N. et al. Power spectrum analysis of heart rate variability to assess the changes in sympathovagal balance during graded orthostatic tilt. Circulation 90, 1826-1831 (1994)).The LF / HF ratio and HF / (LF + HF) were used as indices of sympathetic and parasympathetic nervous activity, respectively (Heart rate variability: standards of measurement, physiological interpretation, and clinical use. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Circulation 93, 1043-1065 (1996); and Pagani, M. et al. Relationship between spectral components of cardiovascular variabilities and direct measures of muscle sympathetic nerve activity in humans. Circulation 95, 1441-1448 (1997)).

[0069] [Creation of partial nephrectomy model] To induce renal hypertension, a 5 / 6 nephrectomy was performed using a previously described method with slight modifications (Nephrology (Carlton) 19, 552-561; 10.1111 / nep.12279 (2014); J. Am. Soc. Nephrol. 4, 2023-2031 (1994); and Kidney Int. 40, 29-34; 10.1038 / ki.1991.175 (1991)). Briefly, rats were anesthetized with isoflurane inhalation (induction at 4% and maintenance at 1.5%), and an approximately 4 cm upper midline laparotomy was performed. Under a microscope, the branches of the left renal artery were identified and selectively ligated with 7-0 silk as close as possible to the left kidney, resulting in a gross infarction of approximately two-thirds of the left renal cortex. In the first pilot study (Figures 1a and 2), we performed resection of the infarcted portion of the kidney using a high-temperature cautery pen (AA11, Bobby, Tennessee, USA). The right renal artery, renal vein, and ureter were then ligated with 4-0 silk, and the right kidney was resected. The abdominal wall and skin were then closed with 4-0 nylon sutures. The anesthesia time was uniformly 30 minutes for all rats.

[0070] [Gas inhalation] H2 gas (1.3% H2 + 21% O2 + 77.7% N2) and control gas (21% O2 + 79% N2) were pre-filled into gas cylinders at the factory. The H2 and control groups inhaled the pre-mixed H2 and control gases. All animals inhaled the assigned gas at 10 L / min for 1 hour in an established anesthesia box, followed by an additional 3 minutes to purge the box (i.e., a total of 63 minutes). In the nephrectomized model and SHR, gas inhalation was repeated daily for 4 and 2 weeks, respectively.

[0071] [Statistical analysis] Descriptive statistics were presented as mean ± standard error of the mean. Comparisons were performed using analysis of variance, unpaired t-test, paired t-test, or Mann-Whitney U test, as appropriate. Mixed-effects models were used to analyze repeated-measures variables. All tests were two-sided, and a P value of <0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc.).

[0072] result [Preventive effect of daily 1-hour H2 gas inhalation on hypertension caused by 5 / 6 nephrectomy] To evaluate the antihypertensive effect of inhaled H, Lewis rats (8 weeks old; N = 3 per group) were inhaled 1.3% H or control gas daily for 1 hour in spontaneously breathing chambers immediately after 5 / 6 nephrectomy (Figure 1a). Single-stage 5 / 6 nephrectomy, performed using microsurgery by experienced surgeons, yielded reproducible results with very little error, as demonstrated by minimal deviations in renal function (Figure 3) and a 100% survival rate. Four weeks after 5 / 6 nephrectomy, arterial blood pressure was significantly reduced in the H group compared with the control group (mean arterial pressure (MAP) 94.2 ± 10.3 mmHg, 134.1 ± 3.3 mmHg, P = 0.02) (Figure 2a-c).

[0073] To investigate the time course required for H gas to exert its therapeutic effect, telemetry-implanted rats (8 weeks old; 3 rats per group) underwent a 5 / 6 nephrectomy and blood pressure fluctuations were continuously monitored (Figure 1b). During the first gas inhalation immediately after the 5 / 6 nephrectomy, blood pressure in the H gas group decreased compared with the control group (117.4 ± 1.8 mmHg vs. 125.6 ± 4.7 mmHg, P = 0.04). However, once inhalation was stopped, the difference in blood pressure between the two groups quickly disappeared (Figure 3a). Before the start of inhalation on the second day, there was no difference in blood pressure between the two groups. After the first week of inhalation, pre-inhalation blood pressure began to decrease in the H group compared with the control group (Figure 3b).

[0074] [Therapeutic effect of daily 1-hour H2 inhalation on established hypertension after 5 / 6 nephrectomy] To evaluate the therapeutic effect of 1-hour inhalation of H gas on established hypertension, Lewis rats (8 weeks old; N = 17 per group) underwent a 5 / 6 nephrectomy followed by a 3-week recovery period (Fig. 1c). Three weeks after the 5 / 6 nephrectomy, blood pressure increased equally in both groups. Although there was no statistically significant difference, blood pressure began to decrease 1 week after the start of H inhalation. This blood pressure-lowering effect of H gas continued throughout the inhalation period (Fig. 4a-d).

[0075] [Effects of H2 inhalation on the autonomic nervous system] To examine the antihypertensive effect of H2 in more detail, we performed chronic, continuous blood pressure monitoring noninvasively using a wireless implantable telemetry system (Figure 5a). Gas inhalation began on the day of 5 / 6 nephrectomy and continued for 4 weeks. Again, no difference was observed in the time course of changes in renal function between the two groups. A longer gas inhalation period resulted in a significant decrease in blood pressure in the 5 / 6 nephrectomy + H2 group compared with the control 5 / 6 nephrectomy group.

[0076] To examine the effects of H2 therapy on autonomic nervous system activity, we performed spectral analysis of blood pressure fluctuations. Spectral components were calculated in normalized units (nu). Low-frequency (LF) power indicates sympathetic dominance, while high-frequency (HF) power indicates parasympathetic dominance. The increase in LF power and the decrease in HF power over time associated with 5 / 6 nephrectomy tended to be attenuated by H2 inhalation, but the results were not significant when compared as continuous variables over a 4-week time course. On the other hand, when comparing the changes from baseline (day 0) to 4 weeks after the start of H2 inhalation, the increase in LF power and the decrease in HF power observed in the control 5 / 6 nephrectomy group were significantly attenuated in the 5 / 6 nephrectomy + H2 group (Figure 5b, c).

[0077] [Hemodynamic effects of inhaled H2 in spontaneously hypertensive rats] Spontaneously hypertensive rats (SHRs) are a well-established model of genetic hypertension widely used in hypertension research (Okamoto, K. & Aoki, K. Development of a strain of spontaneously hypertensive rats. Jpn. Circ. J. 27, 282–293 (1963); and Rubattu, S., Struk, B., Kreutz, R., Volpe, M. & Lindpaintner, K. Animal models of genetic hypertension: what can we learn for human hypertension? Clin. Exp. Pharmacol. Physiol. 22, 386–393 (1995)). To examine the consistency of the blood pressure-lowering effect of H2 in other models of hypertension, we investigated the therapeutic effect of H2 gas on hypertension in SHRs (Fig. 1d). SHRs (10 weeks old) were randomly divided into H2 or control groups (N = 3 in each group) and inhaled the assigned gas for 2 weeks. At 10 weeks of age, blood pressure in SHRs was already as high as approximately 150 mmHg at MAP. In the control group, blood pressure and heart rate continued to increase over time. However, in the H2 group, this increase in blood pressure and heart rate was suppressed in week 2 compared to week 1 (Figure 6a-d). After 2 weeks of H2 gas inhalation, blood pressure and heart rate remained low in the following week (week 3), even when H2 gas inhalation was discontinued. Using frequency analysis to evaluate autonomic nervous function, we found that during the H2 inhalation period, SNA (LF / HF) decreased and the para-SNA (HF / LF+HF) ratio increased in week 2 compared to week 1 (Figure 7a-d). The effects of H2 gas on autonomic nervous function disappeared within the first week after the end of inhalation. [Industrial Applicability]

[0078] According to the pharmaceutical composition of the present disclosure, the pharmaceutical composition of the present disclosure can be used to improve a wide range of hypertension, including essential hypertension.

Claims

1. A gaseous pharmaceutical composition for improving hypertension, comprising: The pharmaceutical composition comprises hydrogen gas. characterized in that Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, The improvement of hypertension includes improvement of diastolic hypertension. Pharmaceutical compositions.

3. 3. The pharmaceutical composition of claim 2, The subject with hypertension is a subject with a diastolic blood pressure of 100 mmHg or more. Pharmaceutical compositions.

4. 4. The pharmaceutical composition according to any one of claims 1 to 3, The hypertension is essential hypertension or secondary hypertension. Pharmaceutical compositions.

5. 5. The pharmaceutical composition of claim 4, The hypertension is essential hypertension. Pharmaceutical compositions.

6. 5. The pharmaceutical composition of claim 4, The hypertension is secondary hypertension. Pharmaceutical compositions.

7. 7. The pharmaceutical composition of claim 6, The secondary hypertension is hypertension caused by renal dysfunction. Pharmaceutical compositions.

8. 8. The pharmaceutical composition of claim 7, The renal dysfunction is due to renal disease or due to nephrectomy or partial nephrectomy. Pharmaceutical compositions.

9. 9. The pharmaceutical composition according to any one of claims 6 to 8, The subject with secondary hypertension is a subject not undergoing dialysis. Pharmaceutical compositions.

10. 10. The pharmaceutical composition according to any one of claims 1 to 9, The pharmaceutical composition further comprises oxygen gas. characterized in that Pharmaceutical compositions.

11. 11. A pharmaceutical composition according to any one of claims 1 to 10, The pharmaceutical composition further comprises an inert gas. characterized in that Pharmaceutical compositions.

12. 12. A pharmaceutical composition according to any one of claims 1 to 11, The hydrogen concentration in the pharmaceutical composition is 0.1% to 4.0% (v / v). characterized in that Pharmaceutical compositions.

13. 13. A pharmaceutical composition according to any one of claims 1 to 12, comprising The hydrogen concentration in the pharmaceutical composition is 1.0% to 2.0% (v / v). characterized in that Pharmaceutical compositions.

14. 14. A pharmaceutical composition according to any one of claims 1 to 13, comprising The hydrogen gas is provided by a hydrogen gas generator or a container containing hydrogen gas. characterized in that Pharmaceutical compositions.

15. 15. The pharmaceutical composition of claim 14, The hydrogen gas generator includes a hydrogen generating means by water electrolysis. characterized in that Pharmaceutical compositions.

16. 15. The pharmaceutical composition of claim 14, The container contains a mixed gas of hydrogen gas and nitrogen gas. characterized in that Pharmaceutical compositions.

17. 17. A pharmaceutical composition according to any one of claims 14 to 16, comprising The hydrogen gas generating device or the container further comprises a control means, either on the device or container itself or on piping connected to the device or container, for monitoring and adjusting the amount of hydrogen gas supplied to the subject; characterized in that Pharmaceutical compositions.

18. 1. A method of treating hypertension, comprising: administering hydrogen gas to a subject with hypertension; method.

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