CN115054585B

CN115054585B - Tablets containing Aidenafil citrate and preparation method and application thereof

Info

Publication number: CN115054585B
Application number: CN202210810256.3A
Authority: CN
Inventors: 宋更申; 刘超; 黄大卫; 余晓文; 马灿
Original assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Current assignee: Beijing Youcare Kechuang Pharmaceutical Technology Co ltd
Priority date: 2022-07-11
Filing date: 2022-07-11
Publication date: 2022-11-18
Anticipated expiration: 2042-07-11
Also published as: CN115054585A

Abstract

The application discloses a tablet containing citric acid aildenafil, a preparation method thereof and application of the tablet containing citric acid aildenafil in preparing a medicine for treating Alzheimer's disease. Through oral administration, the citric acid alidenafil can improve the pathological characteristics of the Alzheimer's disease, improve the learning, memory and perception abilities of mice with the Alzheimer's disease, and reduce the anxiety and the dysphoria degree, which indicates that the citric acid alidenafil has effective and stable treatment effect on the Alzheimer's disease.

Description

Tablets containing Aidenafil citrate and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an Aidinafei citrate tablet, and a preparation method and application thereof.

Background

Alzheimer's Disease (AD), also called senile dementia, is a degenerative disease of the central nervous system, with a hidden onset and a chronic progressive course, and is one of the most common types of senile dementia. It is mainly manifested as progressive memory disorder, cognitive dysfunction, personality change and language disorder, etc., and seriously affects social, working and life functions. The disease is more frequently seen in the elderly over 70 years old (the average age of 73 years old in men and 75 years old in women), and the symptoms of the disease are rapidly clarified after physical diseases, bone fractures or mental stimulation in a few patients. Women are more than men (the proportion of men and women is 1:3). The report of the alzheimer's disease association 2018 indicates that AD patients have reached 5000 ten thousand, and 1 new case is found every 3 seconds on average. Worldwide patients are expected to exceed 8000 million by 2030 and may exceed 1.5 million by 2050. Currently, there are only a limited number of drugs clinically used to alleviate the development of AD.

The etiology and pathogenesis of AD are not clarified, and characteristic pathological changes are extracellular senile plaques formed by deposition of beta amyloid (Abeta), neurofibrillary tangles in nerve cells formed by hyperphosphorylation of Tau protein, neuron loss with glia cell proliferation and the like.

The most studied of AD today are the immunoinflammatory response, cholinergic nerve damage and apoptosis. Recent studies have suggested that there is a genetic factor and genetic susceptibility in AD. First, inflammation is closely associated with the onset of AD; second, 90% of AD patients lack choline during nerve impulse transmission. Acetylcholine is an important neurotransmitter, participates in the transmission of nerve signals, and is closely related to learning and memory. Thirdly, the apoptosis rate of brain nerve cells of AD patients is about 40 times higher than that of normal people, so that the apoptosis of nerve cells can be one of the main causes of the decline of the central nervous system.

Regarding the pathogenesis of AD, there are currently several hypotheses: the oxidative stress hypothesis that excessive cellular oxidation causes damage to neurons; the amyloid beta polypeptide cascade hypothesis that a large amount of a β produced and accumulated may lead to a complex pathological cascade, ultimately leading to the occurrence of neurodegenerative diseases such as alzheimer's disease; the Tau protein hypothesis that excessive A beta may cause hyperphosphorylation of Tau protein, and finally cause reduction of the binding capacity of Tau protein and microtubules to cause instability of cytoskeleton, thereby forming neurofibrillary tangles and causing damage and death of nerve cells; glial cells release inflammatory factors such as interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), transforming growth factor-beta (TGF-beta), etc., which further activate the non-activated glial cells, make the inflammatory response more severe and damage neurons, and also make A beta aggregate to further expand the inflammatory response hypothesis, neurovascular hypothesis, metabolic hypothesis, etc. of the inflammatory response. Mutations at the gene level and other infectious causes, damage to the blood-brain barrier, etc., can all lead to the development of diseases.

All current drugs for treating AD only slow the worsening of symptoms and do not reverse the disease process. Treatment tends to alleviate the symptoms of AD as well as reduce degenerative neuropathy and cell death. Common medicines are classified into two categories, one is an acetylcholinesterase inhibitor and is beneficial to information transfer between nerve cells; another class of N-methyl-D-aspartate receptor antagonists, such as memantine, attempts to block neuronal damage caused by elevated glutamate concentrations. In addition, non-steroidal anti-inflammatory drugs can reduce the neuroinflammatory response caused by AD. Antiviral therapy may reduce AD-associated viral infections and the like mainly caused by herpes virus, hepatitis c virus, and the like.

Therefore, there is a need in the art to develop effective preventive and therapeutic agents for alzheimer's disease.

Disclosure of Invention

Therefore, the invention provides a medicament for effectively preventing and treating Alzheimer disease, namely a tablet containing citric acid alidenafil. The citric acid alidenafil, in particular the citric acid alidenafil crystal form H can treat the Alzheimer disease with high stability and effectively, and the treatment effect is obviously better than other PDE5 inhibitors and other citric acid alidenafil crystal forms. Accordingly, the present invention relates to the use of a tablet comprising the phosphodiesterase type 5 (PDE 5) inhibitor crystalline form H of citric acid, in the manufacture of a medicament for the treatment of alzheimer's disease.

PDE5 is a phosphodiesterase subtype with specific structure and function in tissues such as smooth muscle and platelet, and is mainly distributed in lung, pancreas, brain, corpus cavernosum penis, vascular smooth muscle cells, platelet, skeletal muscle cells and cardiac muscle cells. PDE5 can well regulate the contractile force of vascular smooth muscles, especially those of the penis and lung; PDE5 may also be involved in platelet aggregation via the NO-cGMP signaling pathway; meanwhile, PDE5 may play an important role in the transfer of cGMP signals in the brain.

PDE5 inhibitors include sildenafil (US 5250534A), tadalafil (US 6784179B 2), vardenafil (WO 1999024433) and avanafil (US 6656935B 2). The documents "progress of PDE5 inhibitors in ED research [ J ]. J.Zhonghua Male science journal, 2005, 11 (007): 552-555", "New use of PDE5 inhibitors in urology in Men's journal of China, 2013, 19 (8): 3" and "progress of PDE5 inhibitors in diseases related to cGMP signaling pathway [ J ]. Zhonghua pathophysiology journal, 2021, 37 (1): 7" etc. review the aspects of action mechanism, pharmacodynamics, safety, clinical application and new use of PDE5 inhibitors. Patent CN1127506C discloses a novel selective PDE5 inhibitor, idenafil. The citric acid alidenafil has various crystal forms, and patents CN101671337B, CN101671339B, CN101691372B, CN101671338B, CN101830903B, CN101698668B and CN112745323B report a crystal form A, a crystal form B, a crystal form C, a crystal form D, a crystal form O, a crystal form V and a crystal form H respectively.

The factors influencing the activity of the medicine are many, and comprise multiple factors of different prescriptions, different preparation methods, different crystal forms of raw medicines and the like. The raw material medicines are different in crystal form, (1) the physicochemical properties of the medicines are different, so that the dissociation degree, solubility, surface tension, melting point and the like of the medicines are different, and the medicine effect is further influenced; (2) the binding to the target is different, including the binding site to the target and the difficulty of the binding; (3) the structure-activity relationship thereof also changes; in addition, when the crystal forms are different, the chemical structures of the medicines are different, and the medicines can influence the activity of enzymes, participate or interfere the metabolism of cells, influence ion channels of cell membranes, change the release of active substances in vivo, and influence nucleic acid metabolism, immune function and physiological substance transportation in the process of biological action. Different crystal forms of the raw material medicines can cause great difference of water solubility and dissolution rate of the medicines and influence the bioavailability of the medicines.

For tablets, different tablet types, ingredients of excipients, and interactions of excipients with the drug substance can also affect drug efficacy. The adjustment of the ingredients and the addition ratio of different auxiliary materials can also affect the drug effect, but the specific influence cannot be obtained by conjecture according to the raw materials and auxiliary materials, because various auxiliary materials affect each other and the interaction between the auxiliary materials and the raw materials is different, the influence can influence the absorption, distribution, metabolism, excretion and the like of the drug in the body.

From the above, the exertion of the drug effect and the level of the drug effect are determined by the addition of comprehensive factors and are not fixed single influence factors, so the investigation process of the drug effect needs to be evaluated and tested by a targeted scheme. Different crystal forms and different dosage forms of the same compound can cause great difference of drug effects of the drug, and the drug effect can be determined only through a real experimental result.

The application discovers for the first time that various crystal forms of the citric acid alidenafil can be used for treating the Alzheimer disease with high stability and effectively, and particularly the treatment effect of the crystal form H is obviously superior to that of other PDE5 inhibitors and other crystal forms of the citric acid alidenafil. For example:

1) Of the different PDE5 inhibitors, including sildenafil citrate, avanafil, the compound IS00384, the compound NCX1741 and the citric acid ailnafil, the effect of citric acid ailnafil, especially form H, on the treatment of alzheimer's disease was best, significantly higher than the other inhibitors, which may be related to PDE5 inhibitor effect and selectivity on PDE5 inhibition. The inventor finds that the citric acid alidenafil has the strongest inhibiting effect on PDE5 and the highest selectivity, which is obviously higher than other inhibitors. The half-inhibitory concentration of citric acid alidenafil on PDE5 is 0.66nM, and the inhibitory effect on PDE5A is more than 1515 times that of other PDE subtypes. Thus, the inventors 'experiments demonstrate that different PDE5 inhibitors have widely varying effects on the treatment of alzheimer's disease.

2) Meanwhile, the found citric acid alidenafil crystal form H has the best effect on treating the Alzheimer disease in various crystal forms of citric acid alidenafil, including the crystal forms A, B, C, D, O, V and H, which is obviously higher than other crystal forms, and the effect is probably related to the stability difference of different crystal forms of tablets. The inventor finds that the crystal form H tablet has the highest stability, and the content of the crystal form H tablet can reach 98.10 percent, which is obviously higher than other crystal form tablets. Therefore, experiments prove that the effects of various crystal forms of the citric acid alidenafil on the treatment of the Alzheimer disease are greatly different.

3) Even if the same crystal form of a certain compound is prepared by different preparation processes, the citric acid alidenafil tablets obtained by different auxiliary materials have great difference in dissolution and stability. The dissolution property and stability of the citric acid alidenafil crystal form H tablet prepared by the application are obviously stronger than those of citric acid alidenafil crystal form H tablets (such as disclosed in CN 112745323B) and citric acid alidenafil crystal form A tablets (such as disclosed in CN 114028403A) disclosed in the prior art.

The method comprises the following specific steps:

the drug IS administered for 28 days by an oral administration mode to 7-month-old APP/PS1 transgenic AD model mice, and the tested drugs are PDE5 inhibitors sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741, an aildenafil citrate crystal form H, a crystal form A, a crystal form B, a crystal form C, a crystal form D, a crystal form O and a crystal form V, a citric acid aildenafil citrate crystal form A tablet prepared according to a CN114028403A prescription process, and a citric acid aildenafil crystal form H tablet prepared according to a CN112745323B prescription process. After administration, pathological changes of mouse hippocampal tissues are observed through Baens maze and open field experimental ethology detection and HE staining, the expression condition of mouse hippocampal Abeta-42 and degrading enzyme (NEP) genes thereof is detected through immunohistochemistry, and the therapeutic effect of each tested drug on AD mice is evaluated through detecting SOD (superoxide dismutase) activity, MDA (malondialdehyde) content and the like.

The result of the drug effect evaluation shows that the citric acid alidenafil crystal form H has the best treatment effect on AD mice and is obviously higher than other tested drugs. In the Baens maze experiment, the difference between the compound IS00384 group and the citric acid alidenafil crystal form H group in the AD mouse cave entrance time reaches 19.3 percent; compared with AD model blank mice, the AD mice in the open field experiment have the worst improvement effect of a compound IS00384 group or a compound NCX1741 group, and have the highest differences of 19.1%, 14.5%, 23.1%, 15.4%, 16.7% and 12.0% from a citric acid alidenafil crystal form H group.

In vitro activity experiments show that the Aidenafil citrate in the tested PDE5 inhibitors has the strongest inhibitory effect on the PDE5 and the highest selectivity which is obviously higher than that of other inhibitors. The semi-inhibitory concentration of sildenafil citrate on PDE5 IS 0.66nM, while the compound IS00384, the compound NCX1741, the sildenafil citrate and the avanafil are 5.5 times, 15.9 times, 2.0 times and 8.0 times respectively of the sildenafil citrate; the inhibitory capacity of the citric acid of the alidenafil on PDE5A IS 1515 times that of other PDEs subtypes, namely the selectivity IS 1515 times, the compound IS00384 IS 278 times, the compound NCX1741 IS 95 times, the citric acid of the alidenafil IS 770 times, the avanafil IS 190 times, and the inhibitor IS obviously lower than the citric acid of the crystal form H of the alidenafil. Compared with other PDE5 inhibitors, the citric acid alidenafil has the strongest effect of inhibiting PDE5 and the highest selectivity, thereby having the best treatment effect on AD mice.

The citric acid alidenafil has various crystal forms, including crystal forms A, B, C, D, O, V, H and the like, and in tablets prepared from different crystal forms, the crystal form H has the best treatment effect on AD mice, and the effect is obviously higher than that of other crystal forms. For example, in the bayns maze test, form a group differs by more than 13% from form H group; in an open field experiment, the AD mouse has total distance, average speed, activity times, central distance, central time, central activity time and the like, the difference between the crystal form A group with the worst effect and the crystal form H group respectively reaches 16.5%, 13.0%, 28.2%, 17.9%, 14.6% and 9.6%, and other crystal forms are also poorer than the crystal form H.

Through the stability inspection of different crystal form tablets, the tablet prepared by the crystal form H is most stable, the content can also reach 98.10% after 6 months under the conditions of high temperature and high humidity, the crystal form A is 90.37%, the crystal form A tablet prepared according to the formula process of CN114028403A is the lowest, only 86.12%, the crystal form H tablet prepared according to the formula process of CN112745323B is 94.57%, and other crystal forms are also obviously lower than the crystal form H, so the crystal form H tablet has the best treatment effect on AD mice.

The research result of the invention shows that the citric acid alidenafil crystal form H tablet has the most obvious curative effect on AD mice. For example, in the Baens maze experiment, the shortest time for entering holes is found in the citric acid alidenafil crystal form H group mice, which indicates that the AD model mice have the best learning and memory recovery after being treated. Compared with the AD model group, the mouse holing time is obviously reduced, and the holing time reduction ratio of each dosage group to the AD model group can respectively reach 31.2 percent, 35.9 percent and 43.2 percent, which are obviously higher than that of other PDE5 inhibitors and citric acid alidenafil crystal forms.

The cognitive ability and anxiety and dysphoria of the citric acid alidenafil crystal form H group AD mice in an open field experiment have the best improvement effect, and compared with other PDE5 inhibitors and other citrate alidenafil crystal forms, the treatment effect is obviously improved. Compared with the AD model group, the total mileage of the citric acid alidenafil crystal form H group is obviously reduced, and the dosage groups are respectively reduced by 19.1 percent, 21.9 percent and 23.7 percent compared with the AD model group; the average speed is obviously reduced, and each dosage group is respectively reduced by 15.9 percent, 23.2 percent and 23.2 percent compared with the AD model group; the activity frequency is obviously increased, and each dosage group is respectively increased by 30.8%, 43.6% and 41.0% compared with the AD model group; the central distance is obviously reduced, and each dose group is respectively reduced by 33.5%, 37.1% and 38.4% compared with the AD model group; the central time is obviously reduced, and each dosage group is respectively reduced by 20.1 percent, 27.8 percent and 34.0 percent compared with the AD model group; central activity time was also significantly reduced, with each dose group being 20.0%, 25.3% and 28.1% less than AD model group, respectively. These values are significantly higher than other PDE5 inhibitors and the other crystalline form group of aildenafil citrate.

The pathological detection result shows that the citric acid alidenafil crystal form H group has the best recovery effect on the hippocampus; compared with the AD model group, the citric acid alidenafil crystal form H dosage groups respectively reduce the expression quantity of Abeta-42 by 19.9 percent, 26.2 percent and 31.4 percent, and the expression quantity of NEP is respectively increased by 8.9 percent, 11.1 percent and 14.1 percent; SOD activity values are respectively improved by 4.1%, 6.0% and 8.5%, and MDA contents are respectively reduced by 8.5%, 10.5% and 10.5%. These values are all significantly higher than the other PDE5 inhibitors and the other crystalline form group of the citric acid alidenafil.

In vitro activity experiments show that the citric acid alidenafil has the strongest inhibiting effect on PDE5 in all PDE5 inhibitors, and is much higher than other inhibitors. For example, PDE5 is inhibited 15.9 times as much as compound NCX 1741; the citric acid alidenafil has the highest selectivity on PDE5, and the inhibiting effect on PDE5A is 1515 times that of other PDEs subtypes, namely, the selectivity is as high as 1515 times, and the compound NCX1741 with the lowest selectivity is only 95 times.

The stability investigation result of the citric acid alidenafil tablet shows that the stability of the form H tablet is the highest in all crystal forms and is obviously higher than that of other crystal form tablets, after 6 months of accelerated test, the content of the form H tablet can also reach 98.10%, the form A is only 90.37%, and the form A tablet prepared by the CN114028403A prescription process is only 86.12%.

It IS thus clear that of the different PDE5 inhibitors, including sildenafil citrate, avanafil, the compound IS00384, the compound NCX1741 and the citric acid ailnafil, especially form H, IS the best for the treatment of alzheimer's disease, with the most significant improvement of the various symptoms, significantly higher than the other inhibitors, which may be related to the PDE5 inhibitor's PDE5 inhibitory effect and selectivity. The inventor finds that the citric acid alidenafil has the strongest inhibiting effect on PDE5 and the highest selectivity, which is obviously higher than other inhibitors. The half-inhibitory concentration of citric acid alidenafil on PDE5 is 0.66nM, and the inhibitory effect on PDE5A is over 1515 times that of other PDE subtypes. Thus, the inventors 'experiments demonstrate that the efficacy of different PDE5 inhibitors for the treatment of alzheimer's disease varies greatly (in particular in terms of the recovery of memory, cognitive ability, and the degree of improvement of anxiety and dysphoric condition in AD model mice).

Meanwhile, the inventor finds that in various crystal forms of the citric acid alidenafil, including the crystal forms A, B, C, D, O, V and H, the citric acid alidenafil crystal form H has the best effect on treating the Alzheimer's disease, is obviously higher than other crystal forms, and is possibly related to the stability difference of different crystal form tablets. The inventor finds that the stability of the crystal form H tablet is the highest, and after 6 months of accelerated test, the content of the crystal form H tablet can reach 98.10 percent, which is obviously higher than that of other crystal form tablets. Therefore, the experiments of the inventor prove that the effects of various crystal forms of the citric acid alidenafil on the treatment of the Alzheimer disease are greatly different.

Therefore, the citric acid alidenafil in various crystal forms can treat the Alzheimer disease with high stability and effectively, and particularly, the treatment effect of the crystal form H is obviously better than that of other PDE5 inhibitors and other crystal forms of citric acid alidenafil. The invention provides a new medicinal application of citric acid alidenafil crystal form H.

Drawings

FIGS. 1 (a) and 1 (b) are graphs showing the mean time to tunnel for each group of mice after administration of the Barnes maze experiment;

FIGS. 2 (a) and 2 (b) show the normal central activity time of the mice in each group in the open field experiment;

FIGS. 3 (a) and 3 (b) are HE staining (magnification × 400) of hippocampal tissue sections of experimental mice.

Detailed Description

The invention relates to the following technical scheme:

a tablet containing a phosphodiesterase type 5 (PDE 5) inhibitor is prepared from the following raw and auxiliary materials in parts by weight: 85-95 parts of phosphodiesterase type 5 (PDE 5) inhibitor, 147-160 parts of pregelatinized starch, 85-95 parts of calcium hydrophosphate, 5-12 parts of crosslinked sodium carboxymethylcellulose (added), 15-28 parts of polymethyl methacrylate, 100-120 parts of 70% ethanol, 3-5 parts of sodium stearyl fumarate, 7-13 parts of film coating premix and 50-65 parts of purified water.

In a preferred embodiment, the phosphodiesterase type 5 (PDE 5) inhibitor is selected from one or more of the following compounds: sildenafil citrate, avanafil citrate, IS00384 compound, NCX1741 compound and Aidenafil citrate.

In a preferred embodiment, the phosphodiesterase type 5 (PDE 5) inhibitor is sildenafil citrate.

In a preferred embodiment, the crystalline form of citric acid alidenafil includes form a, form B, form C, form D, form O, form V and form H.

In a preferred embodiment, the crystalline form of citric acid alidenafil is form H.

In a preferred embodiment, the invention relates to a tablet containing citric acid alidenafil, which is prepared from the following raw and auxiliary materials in parts by weight: the feed is prepared from the following raw and auxiliary materials in parts by weight: 85-95 parts of citric acid aildenafil, 147-160 parts of pregelatinized starch, 85-95 parts of calcium hydrophosphate, 5-12 parts of cross-linked sodium carboxymethylcellulose (added), 15-28 parts of polymethyl methacrylate, 100-120 parts of 70% ethanol, 3-5 parts of sodium stearyl fumarate, 7-13 parts of a film coating premix and 50-65 parts of purified water.

In a more preferred embodiment, the tablet is prepared from raw auxiliary materials selected from the following formulas in parts by weight:

prescription 1: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 9 parts of crosslinked sodium carboxymethylcellulose (added), 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

recipe 2: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 5 parts of cross-linked sodium carboxymethylcellulose (added), 15 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 3: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 12 parts of crosslinked sodium carboxymethylcellulose (added), 25 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 4: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of crosslinked sodium carboxymethylcellulose (added), 16 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water; alternatively, the first and second liquid crystal display panels may be,

prescription 5: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of crosslinked sodium carboxymethylcellulose (added), 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 6: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of crosslinked sodium carboxymethylcellulose (added), 12 parts of crosslinked sodium carboxymethylcellulose (added), 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 7: 92 parts of citric acid aildenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 12 parts of crosslinked sodium carboxymethylcellulose (added), 8 parts of crosslinked sodium carboxymethylcellulose (added), 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water.

In a more preferred embodiment, the crystalline form of citric acid alidenafil includes form a, form B, form C, form D, form O, form V and form H.

In a more preferred embodiment, the citric acid alidenafil is in crystalline form H.

The preparation method of the citric acid alidenafil tablet comprises the following steps:

1) Premixing

Sieving the raw materials according to the prescription amount, and sequentially putting the pregelatinized starch, the croscarmellose sodium (added internally) and the calcium hydrophosphate into a wet granulator for premixing;

2) Granulating

Adding an ethanol dispersion of polymethyl methacrylate for granulation;

3) Drying

Drying the wet granules, and controlling the moisture of the granules to be less than 5.0%;

4) Whole grain

Sieving and granulating the dried granules;

5) Total mixture

Adding croscarmellose sodium (added) and sodium stearyl fumarate into the granules after finishing, and mixing in a mixer;

6) Tabletting

Tabletting;

7) Coating

Coating to obtain the tablet.

The application provides an application of the tablet containing the citric acid alidenafil in preparing a medicine for treating Alzheimer's disease.

In addition, patent CN112745323B discloses crystalline form H of aildenafil citrate for the treatment of male erectile dysfunction. Based on the above, the application also provides the application of the tablets containing the Aidinafil citrate in preparing the medicines for treating erectile dysfunction.

Examples

Example 1: synthesis of Compounds IS00384 and NCX1741

1. Compound IS00384

The synthetic route is as follows:

compound IS00384 was synthesized according to the method in "Sawant et al, bioorg. Med. Chem. 23 (2015) 2121-2128". 30g 6-amino-1-hexanol (2) was dissolved in 500mL anhydrous Dichloromethane (DCM), stirred, 100g Diisopropylethylamine (DIPEA) was added, and 107g 4-ethoxy-3- (1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo [4,3-d was added]Pyrimidine-5-yl) benzenesulfonyl chloride (1), and stirring and reacting for 6 hours at the temperature of 25 ℃. TLC confirmed the reaction was complete, 500mL of water was added, the layers were separated, the organic phase was washed with 500mL of saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to give 109.2g of an off-white solid with a yield of 85.4%. MS (m/z) 492.20[ m ] +H] ⁺ ； ¹ H NMR (400MHz, DMSO-d6) δ：0.95 (t, 3H), 1.28-1.34(m, 6H), 1.45-1.60(m, 6H), 1.75 (m, 2H), 2.15(s, 3H), 2.78(t, 2H), 3.39(t, 2H), 3.63(t, 2H), 4.18(s, 1H), 4.22-4.27(m, 2H), 7.37(d, 1H), 7.88–7.94(m, 2H), 12.20(s, 1H)。

2. Compound NCX1741

The synthetic route is as follows:

the compound NCX1741 was synthesized according to the method of patent WO2020030489A 1. Avanafil (3) (100 g) was dissolved in anhydrous Dichloromethane (DCM), stirred, added with 4-Dimethylaminopyridine (DMAP) (25.7 g) and Dicyclohexylcarbodiimide (DCC) (51.6 g), added with 6-nitrooxycaproic acid (4) (44.3 g) and reacted at 25 ℃ for 15h. TLC confirmed the reaction was complete, 1L of water was added, the layers were separated, the organic phase was washed with 0.5L of saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, and distilled under reduced pressure to give 121.5g of a pale yellow solid with a yield of 90.1%. MS (M/z) 644.15 [ M + H ]] ⁺ ； ¹ H NMR(400MHz, DMSO-d6) δ：1.35-1.46(m, 2H), 1.56-1.69(m, 4H), 1.75-2.10(m, 4H), 2.32(t, 2H), 3.41-3.63(m, 4H), 3.69-3.74(m, 1H), 3.84(s, 3H), 3.93-4.52(m, 6H), 5.14(s, 1H), 7.08-7.52(m, 4H), 8.58(s, 1H), 8.75-8.82(m, 2H), 9.20(s, 1H)。

Example 2: preparation of citric acid alidenafil crystal forms H, A, B, C, D, O and V

1. Preparation of citric acid alidenafil crystal form H

Adding 1L N, N-dimethylformamide and 1.5kg of citric acid alidenafil into a reaction kettle, stirring, adding 30L of anhydrous ethanol, heating to reflux, performing hot filtration, supplementing 15L of anhydrous ethanol, cooling to 45 ℃ for 5 hours, keeping the temperature, stirring for 2 hours, slowly cooling to room temperature, and stirring for crystallization for 8 hours. Filtering, adding the obtained solid into 9L of anhydrous ethanol, stirring at room temperature for 5H, filtering, and vacuum drying at 35-40 deg.C to obtain 1.37kg of white powder, wherein the crystal form H of the Aidenafil citrate is determined by X-ray powder diffraction.

2. Preparation of citric acid alidenafil crystal form A, crystal form B, crystal form C, crystal form D, crystal form O and crystal form V

Respectively preparing citric acid alidenafil crystal form A, crystal form B, crystal form C, crystal form D, crystal form O and crystal form V according to the methods in CN101671337B, CN101671339B, CN101691372B, CN101671338B, CN101830903B and CN101698668B, and determining the citrate alidenafil crystal form A, the crystal form B, the crystal form C, the crystal form D, the crystal form O and the crystal form V as target crystal forms through X-ray powder diffraction.

Example 3: preparation of tablets

1. Recipe process optimization

Recipe 1: (Crystal form H of Aidenafil citrate)

1) Prescription composition (1000 tablets)

2) Preparation process

Pre-mixing: firstly, sieving a prescription amount of an Edinafil citrate raw material with an H crystal form by a 100-mesh sieve, and then sequentially adding the Edinafil citrate raw material, a prescription amount of pregelatinized starch, croscarmellose sodium (added internally) and calcium hydrophosphate into a wet granulator for premixing, wherein the stirring speed is 6rps, the cutting speed is 15rps, and the time is 15min;

and (3) granulating: adding an ethanol dispersion of polymethyl methacrylate for granulation: stirring speed 6rps, cutting speed 15rps, and time 5min;

and (3) drying: fluidized bed drying of the wet granules is carried outThe air temperature is 60 ℃, and the air inlet quantity is 60m ³ The moisture of the granules is controlled to be less than 5.0 percent;

straightening: sieving the dried granules with a 20-mesh sieve by using a swing type granulator;

total mixing: adding croscarmellose sodium (added) and sodium stearyl fumarate into the granulated granules in proportion, placing in a three-dimensional mixer, rotating at 12Hz, and mixing for 10min;

tabletting: tabletting by using a special-shaped punch, and controlling the hardness to be 12-15kg;

coating: the weight gain of the coating is controlled to be 1-3%.

Prescription 2: (Crystal form H of Aidenafil citrate)

Compared with the formula 1, the internal addition and external addition amount of the croscarmellose sodium is reduced from 9g to 5g, and the amount of the polymethyl methacrylate is reduced from 24g to 15g.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 5g of croscarmellose sodium (added internally); 5g of croscarmellose sodium (additionally added); 15g of polymethyl methacrylate; the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as the formula 1.

Prescription 3: (Aidinafil citrate crystal form H)

Compared with the formula 1, the internal addition and external addition amount of the croscarmellose sodium are increased from 9g to 12g, and the amount of the polymethyl methacrylate is increased from 24g to 25g.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 12g of croscarmellose sodium (added internally); 12g of croscarmellose sodium (additionally); 25g of polymethyl methacrylate; the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as the formula 1.

And (4) prescription: (Crystal form H of Aidenafil citrate)

Compared with the prescription 1, the cross-linked sodium carboxymethyl cellulose (internal addition): croscarmellose sodium (add): the polymethyl methacrylate was changed from 3.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 8g of croscarmellose sodium (added internally); 8g of croscarmellose sodium (additionally added); 16g of polymethyl methacrylate; the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as formula 1.

Prescription 5: (Aidinafil citrate crystal form H)

Compared with the prescription 1, the cross-linked sodium carboxymethyl cellulose (internal addition): croscarmellose sodium (add): polymethyl methacrylate was changed from 3.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 8g of croscarmellose sodium (added internally); 8g of croscarmellose sodium (additionally added); 24g of polymethyl methacrylate; the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as formula 1.

Prescription 6: (Aidinafil citrate crystal form H)

In comparison with formula 1, croscarmellose sodium (added internally): the croscarmellose sodium (plus) was changed from 1:1 to 2:3.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 8g of croscarmellose sodium (added internally); 12g of croscarmellose sodium (additionally); the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as formula 1.

Prescription 7: (Crystal form H of Aidenafil citrate)

Compared with the prescription 1, the cross-linked sodium carboxymethyl cellulose (internal addition): the croscarmellose sodium (plus) was changed from 1:1 to 3:2.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 12g of croscarmellose sodium (added internally); 8g of croscarmellose sodium (additionally added); the rest is the same as prescription 1.

2) Preparation process

The preparation process is the same as formula 1.

Prescription 8-10: comparative examples 1-3 (Crystal form H of Aidenafil citrate)

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials:

recipe 8: the croscarmellose sodium (internal addition and external addition) in the formula 1 is replaced by polyethylene glycol 2000, and the other steps are the same as the formula 1.

Prescription 9: the croscarmellose sodium in formula 1 (internal addition and external addition) is replaced by polyethylene glycol 4000, and the rest is the same as formula 1.

Prescription 10: the croscarmellose sodium (internal addition and external addition) in the formula 1 is replaced by sodium lauryl sulfate, and the other steps are the same as the formula 1.

2) Preparation process

The preparation process is the same as formula 1.

Prescription 11: comparative example 4 (crystalline form H of Aidinafil citrate)

1) Prescription composition (1000 tablets)

The formula 11 refers to a raw and auxiliary material formula of a patent CN112745323B to prepare the citric acid alidenafil tablet; the prescription composition is shown in the following table:

2) The preparation process comprises the following steps:

premixing: firstly, sieving a prescription amount of the EDANFEN citrate with an H crystal form by a 100-mesh sieve, and then sequentially adding the EDANFEN citrate, the crospovidone (internal addition) and the microcrystalline cellulose into a wet granulator for premixing, wherein the stirring speed is as follows: 6rps, cutting speed: 15rps for 15min;

and (3) granulating: adding povidone K30 ethanol solution for granulation: stirring speed: 6rps, cutting speed: 15rps for 5min;

and (3) drying: carrying out fluidized bed drying on the wet particles, wherein the air inlet temperature is as follows: 60 ℃ and 60m of air inlet volume ³ The moisture of the granules is controlled to be less than 5.0 percent;

total mixing: adding crospovidone (additionally added) and magnesium stearate into the granules after finishing the granules in proportion, placing the granules into a three-dimensional mixer, and rotating at the speed: 12Hz, mixing time: 10min;

tabletting: tabletting by adopting a special-shaped punch, and controlling the hardness to be 1215kg;

coating: the weight gain of the coating is controlled to be 13%.

Prescription 12: COMPARATIVE EXAMPLE 5 (crystalline form H of Aidenafil citrate)

The main difference from the prescription 1 is that the preparation process of the patent CN114028403A is adopted, namely, various medicinal components are directly mixed and granulated (the invention is the step-by-step addition of a disintegrating agent and an adhesive) to prepare the citric acid alidenafil tablet.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: the same as the prescription 1, wherein 18g of croscarmellose sodium is added internally.

2) Preparation process

The preparation method refers to a preparation method of citric acid alidenafil crystal form A tablets disclosed in patent CN 114028403A.

The process comprises the following steps: pulverizing raw materials, sieving with 80 mesh sieve, mixing with other materials, making soft mass with distilled water, granulating with 16 mesh sieve, drying at 40-45 deg.C in drying oven, grading with 16 mesh sieve, adding sodium stearyl fumarate into the dried granules, mixing, and tabletting.

Recipe 12 differs from recipe 1 mainly, see the following table:

prescription 13: comparative example 6 (Crystal form A of Aidenafil citrate)

The main difference from the prescription 1 is that the medicine prescription is different, and the addition modes of the disintegrant and the adhesive in the preparation process are different.

1) Prescription composition (1000 tablets)

Raw materials and auxiliary materials: 70g of citric acid alidenafil crystal form A, 5g of microcrystalline cellulose, 140g of lactose, 4000 g of PEG-4000, 1g of magnesium stearate, 30g of povidone K, 10g of croscarmellose sodium and a proper amount of distilled water, and the mixture is prepared into 1000 tablets.

2) Preparation process

The preparation method refers to the pharmaceutical ingredients and the preparation method of the citric acid alidenafil crystal form A tablet disclosed in patent CN 114028403A.

The process comprises the following steps: pulverizing PEG-4000 and crystalline form A of citric acid aildenafil together, sieving with 80 mesh sieve, mixing with other materials, making soft material with distilled water, granulating with 16 mesh sieve, drying at 40-45 deg.C in drying oven, grading with 16 mesh sieve, adding magnesium stearate into the dried granules, mixing, and tabletting.

Recipe 13 differs from recipe 1 mainly, see the following table:

2. evaluation of tablet Properties

1) Dissolution rate

The dissolution method comprises the following steps: second law of Chinese pharmacopoeia 2015 edition 0931

Rotating speed: 60 turns

Temperature of the medium: 37 +/-2 DEG C

Volume of medium: 1000mL

The determination method comprises the following steps: ultraviolet-visible spectrophotometry (chinese pharmacopoeia 2015 edition general rule 0401), wavelength: 292nm

The calculation method comprises the following steps: external standard method

The preparation method of the control solution comprises the following steps: taking a proper amount of citric acid alidenafil reference substance, precisely weighing, dissolving with water, and quantitatively diluting to a solution containing about 17 μ g of alidenafil per 1mL as a reference substance solution.

Taking 12 citric acid alidenafil tablets in each batch, taking 10mL of solution at 5min, 10min, 15min, 30min, 45min and 60min respectively by taking phosphate buffer solution with pH of 5.8 as dissolution medium, filtering, precisely measuring a proper amount of filtrate, measuring absorbance after dilution, calculating dissolution according to the absorbance, and the result is shown in Table 8:

2) Stability of

The prepared tablets were placed under high temperature (60 ℃. + -. 2 ℃), high light (4500 lx. + -. 500 lx) and high humidity (90%. + -. 5%) conditions for 10 days, and samples were taken to determine the content of the pharmaceutically active ingredient (in terms of alidenafil) and related substances, the contents and the maximum single impurity results are shown in Table 9.

3. Comparison of tablet Properties for different formulations

3-1, properties of tablet (Crystal form H of Aidenafil citrate) of formula 1 of the invention

The pharmaceutical ingredients in the prescription 1 are controlled to be 9 parts of croscarmellose sodium (added internally) and 24 parts of croscarmellose sodium (added externally); and croscarmellose sodium (added internally): croscarmellose sodium (add): polymethyl methacrylate = 3.

Specifically, the method comprises the following steps:

the dissolution experiment shows that the citric acid alidenafil tablet obtained by the formula is rapidly dissolved to 58.2 percent within 0 to 5min, the dissolution rate is 11.64 percent/min, and the dissolution effect is obvious; the dissolution rate can reach 75.8% within 15min; within 15 to 60min, the dissolution rate is rapidly increased to nearly 100 percent; the dissolution rate is high.

In the medicine tablet obtained by adopting the prescription, the content of the medicine active ingredient placed at high temperature is 99.40 percent, the maximum content of single impurity is 0.21 percent, and the change is not obvious;

the content of the active ingredients of the medicine placed under strong light is 99.46 percent, the maximum single impurity content is 0.18 percent, and the change is not obvious;

the content of the active ingredients of the medicine placed under high humidity is 99.37 percent, the maximum content of single impurities is 0.22 percent, and the change is not obvious;

the results of accelerated placing experiments under different conditions show that the citric acid alidenafil crystal form H tablet obtained by the formula 1 has good and obvious stability, has high content of active pharmaceutical ingredients in the tablet, and can meet the medication requirements of patients.

3-2, influence of the amount of Binder (polymethyl methacrylate) and disintegrant (croscarmellose sodium) on the Properties (crystalline form H of Aidenafil citrate)

The formula 2~5, the added amounts of binder (polymethylmethacrylate) and disintegrant (croscarmellose sodium) were varied, wherein,

prescription 2: 5 parts of croscarmellose sodium (added internally) and 15 parts of croscarmellose sodium (added externally) are respectively added;

prescription 3: the adding parts of the croscarmellose sodium (added internally) and the croscarmellose sodium (added externally) are 12 parts respectively, and the adding part of the polymethyl methacrylate is 25 parts;

prescription 4: 8 parts of croscarmellose sodium (added internally) and 16 parts of croscarmellose sodium (added externally) are respectively added;

prescription 5: the adding parts of the croscarmellose sodium (added internally) and the croscarmellose sodium (added externally) are respectively 8 parts, and the adding part of the polymethyl methacrylate is 24 parts.

Specifically, the method comprises the following steps:

in the citric acid alidenafil crystal form H tablet obtained by the prescription 2-5, the dissolution experiment shows that the citric acid alidenafil tablet obtained by the prescription can be rapidly dissolved to more than 50% (50% -58%) within 0-5 min, the dissolution rate is higher than 10%/min (10.12%/min-11.52%/min), and the dissolution effect is obvious; the dissolution rate can reach more than 70% in 15min, and the dissolution rate has obvious advantages; the dissolution rate is gradually increased to more than 95% -97% within 15-60min, the dissolution rate is close to 100%, the dissolution rate is high, and the dissolution rate is high.

The content of the active ingredients of the medicine tablet obtained by adopting the prescription 2-5 is kept above 99.30 percent and can reach 99.37 percent at most under the conditions of high temperature, strong light or high humidity; and the maximum single impurity content is controlled to be below 0.20-0.25%, the impurity change is not obvious, the stability is obvious, and the active substance ingredients in the tablet are kept at a high level.

And (3) knotting: when the addition amounts of the adhesive and the disintegrant in the citric acid alidenafil crystal form H tablet are controlled to be 5-12 parts of cross-linked sodium carboxymethylcellulose (added internally), 5-12 parts of cross-linked sodium carboxymethylcellulose (added externally) and 15-28 parts of polymethyl methacrylate, the citric acid alidenafil crystal form H tablet with less single impurity, high stability and good dissolution rate can be prepared.

Wherein, when the addition amounts of the croscarmellose sodium (added internally) and the croscarmellose sodium (added externally) in the formula are controlled to be 9 parts respectively and the addition amount of the polymethyl methacrylate is controlled to be 24 parts, the prepared citric acid alidenafil crystal form H tablet has the most obvious stability effect and the optimal dissolution performance.

3-3, effect of the amount of croscarmellose sodium (Internally) and croscarmellose sodium (externally) on Performance (Crystal form H of Aidenafil citrate)

Compared with the prescription 1, the prescription 6 and the prescription 7, the adding proportion of the croscarmellose sodium (added internally) and the croscarmellose sodium (added externally) is changed while the adding amount of the polymethyl methacrylate is kept at 24 parts; wherein:

in the formula 6, the addition amount of the croscarmellose sodium (added internally) is 8 parts, and the addition amount of the croscarmellose sodium (added externally) is 12 parts; i.e., croscarmellose sodium (add): croscarmellose sodium (plus) =2:3;

in the formula 7, the addition amount of the croscarmellose sodium (added internally) is 12 parts, and the addition amount of the croscarmellose sodium (added externally) is 8 parts; i.e., croscarmellose sodium (add): croscarmellose sodium (plus) =3:2.

Specifically, the method comprises the following steps:

by adopting the citric acid alidenafil crystal form H tablet obtained by the prescription 6, the dissolution rate of the citric acid alidenafil tablet obtained by the prescription is 9.74%/min within 0 to 5min; the dissolution rate can reach 64.7 percent within 15min; the dissolution rate is gradually increased to 92 percent within 15 to 60min, the dissolution rate is high, and the dissolution rate is high.

In the medicine tablet obtained by adopting the prescription, the content of the medicine active ingredient placed at high temperature is 99.29 percent, the maximum single impurity content is controlled at 0.25 percent, and the change is not obvious; the content of the active ingredients of the medicine placed under strong light is 99.31 percent, the maximum content of single impurity is only 0.21 percent, and the change is not obvious; the content of the active ingredients of the medicine placed under high humidity is 99.28 percent, the maximum content of single impurities is 0.24 percent, the change is not obvious, and the stability is good.

In the pharmaceutical tablets obtained by the formula 7, the citric acid alidenafil tablets obtained by the formula are dissolved in the range of 0 to 5min at a dissolution rate of 46.9 percent per minute, and the dissolution rate is 9.38 percent per minute; the dissolution rate can reach 63.8% within 15min; the dissolution rate is gradually increased to 92.3 percent within 15 to 60min, the dissolution rate is high, and the dissolution rate is high.

In the medicine tablet obtained by adopting the prescription, the content of the active ingredients of the medicine placed at high temperature is 99.31 percent, the maximum content of single impurity is 0.25 percent, and the change is not obvious; the content of the active pharmaceutical ingredients placed under strong light is 99.33%, the maximum single impurity content is only 0.19%, and the change is not obvious; the content of the active ingredients of the medicine placed under high humidity is 99.30 percent, the maximum content of single impurities is controlled to be 0.22 percent, the change is not obvious, and the stability is good.

From the comparison of the above data, when the dosage ratio of croscarmellose sodium (added) to croscarmellose sodium (added) in the formula content is (2~3): (3~2), the citric acid alidenafil crystal form H tablet with remarkable stability and dissolution rate can be prepared; the stability and dissolution are optimized when the ratio of the amount of croscarmellose sodium (added internally) to the amount of croscarmellose sodium (added externally) is 1:1.

3-4, effect of different disintegrant types on Performance (Crystal form H of Aidenafil citrate)

In the formulas 8-10, the internal crosslinked sodium carboxymethylcellulose and the external crosslinked sodium carboxymethylcellulose in the formula 1 are respectively adopted:

polyethylene glycol 2000 (formula 8, both internal croscarmellose sodium and external croscarmellose sodium replaced),

Polyethylene glycol 4000 (prescription 9, replacing inner croscarmellose sodium and outer croscarmellose sodium)

And

sodium dodecyl sulfate (prescription 10, both the inner croscarmellose sodium and the outer croscarmellose sodium are substituted).

Specifically, the method comprises the following steps:

in the pharmaceutical tablets obtained by adopting the method and the components of the prescription 8, the dissolution experiment shows that the dissolution rate of the citric acid alidenafil crystal form H tablet is only 39.7 percent within 0 to 5min, which is obviously lower than the dissolution rate (58.2 percent) of the tablet in the prescription 1 at the same time, and the dissolution rate in the previous period is very slow; the dissolution rate is only about 60% at 15min; within 15 to 60min, the dissolution rate is lower than 90 percent, and then the dissolution rate is slow; complete dissolution of the tablet cannot be realized, and the dissolution rate is poor.

In the citric acid alidenafil crystal form H medicine tablet obtained by adopting the prescription 8, the content of the medicine active ingredient placed at high temperature is 98.87 percent, and the maximum single impurity content is 0.37 percent; the content of the active ingredients of the medicine placed under strong light is 98.90 percent, and the maximum content of single impurities is 0.35 percent; the content of the active ingredient of the drug is 98.81% when the drug is placed under high humidity, and the maximum content of single impurity reaches 0.40%.

In the citric acid alidenafil crystal form H tablet obtained by adopting the method and the components of the prescription 9, the dissolution experiment shows that the citric acid alidenafil tablet is quickly taken out only by 38.3 percent within 0 to 5min, the dissolution rate is obviously lower than the dissolution rate (58.2 percent) in the prescription 1 at the same time, and the early-stage dissolution rate is very slow; the dissolution rate is only about 60% at 15min; the dissolution rate is kept at 89.7 percent and is lower than 90 percent within 15 to 60min, and then the dissolution rate is slow; complete dissolution of the tablet cannot be realized, and the dissolution rate is poor.

In the pharmaceutical tablet obtained by adopting the formula 9, the content of the pharmaceutical active ingredient placed at high temperature is 98.79 percent, and the maximum content of single impurity is 0.35 percent; the content of the active ingredients of the medicine placed under strong light is 98.88 percent, and the maximum content of single impurities reaches 0.33 percent; the content of the pharmaceutically active ingredient left under high humidity was 98.75%, and the maximum content of single impurities was as high as 0.39%.

In the citric acid alidenafil crystal form H medicine tablet obtained by adopting the method and the components of the prescription 10, the dissolution of the citric acid alidenafil tablet is only 38.0 percent within 0 to 5min, which is obviously lower than the dissolution (58.2 percent) in the prescription 1 at the same time, and the early-stage dissolution rate is very slow; the dissolution rate is only about 60% at 15min; within 15 to 60min, the dissolution rate is between 60.5 and 89.6 percent and is lower than 90 percent, and then the dissolution rate is slow; complete dissolution of the tablet cannot be realized, and the dissolution rate is poor.

In the pharmaceutical tablet obtained by adopting the formula 10, the content of the pharmaceutical active ingredient placed at high temperature is 98.75 percent, and the maximum content of single impurity is 0.40 percent; the content of the active ingredients of the medicine placed under strong light is 98.84 percent, and the maximum content of single impurities is 0.37 percent; the content of the pharmaceutically active ingredient left under high humidity was 98.75%, and the maximum content of single impurities was as high as 0.41%.

Compared with the impurity content of the formula 1, the impurities in the tablets prepared by the formulas 8 to 10 are obviously increased along with the prolonging of the standing time, and the active ingredients of the medicine are greatly reduced, so that the stability is poor.

And (3) knotting: in the formulas 8-10, polyethylene glycol 2000, polyethylene glycol 4000 and sodium dodecyl sulfate are adopted to replace the internal crosslinked sodium carboxymethylcellulose and the external crosslinked sodium carboxymethylcellulose in the formula 1, and the stability and the dissolution performance of the prepared drug tablet are far lower than the effect of the citric acid alidenafil crystal form H tablet.

3-5, influence of different medicament components (auxiliary material components and proportion) on performance (citric acid alidenafil crystal form H)

Compared with the prescription 1, the prescription 11 (refer to the patent CN112745323B medicament component and the preparation method) has the main difference that the auxiliary material components and the mixture ratio in the prescription are obviously different.

Specifically, the method comprises the following steps:

in the citric acid alidenafil crystal form H medicine tablet obtained by adopting the auxiliary material components and the mixture ratio in the prescription 11, the dissolution of the citric acid alidenafil tablet is only 44.3 percent within 0 to 5min, and the dissolution rate is 13.9 percent lower than the dissolution rate (58.2 percent) in the same time in the prescription 1; the dissolution rate is only about 58.2 percent in 15min; at 30min, the difference of the dissolution degree is the largest and reaches 19.9%; within 30 to 60min, the dissolution rate is 90.3 percent; and the dissolution degree of the prescription 11 is lower than that of the medicament of the prescription 1 in the same time; in contrast, formulation 1 has a significant dissolution advantage over formulation 11.

In the pharmaceutical tablet obtained by adopting the above formula 11, the content of the pharmaceutically active ingredient placed at high temperature is 99.01%, and the maximum content of single impurity is 0.33%; the content of the active ingredients of the medicine placed under strong light is 98.97 percent, and the maximum content of single impurities is 0.35 percent; the content of the pharmaceutically active ingredient left under high humidity was 99.05%, and the maximum content of single impurities was 0.36%. Under various standing conditions, the content of the active ingredients in the pharmaceutical tablet of the prescription 13 has no advantages; and the maximum single impurity content of the prescription 11 is not lower than the impurity content of the tablet of the prescription 1, and the stability is lower than the prescription 1 as a whole.

According to the content, the formula 11 adopts other auxiliary material components and proportions to replace the formula 1, and the dissolution performance and stability of the citric acid alidenafil crystal form H tablet prepared by the formula are not improved, so that the citric acid alidenafil crystal form H tablet prepared by the formula, the auxiliary material components and proportions has remarkable advantages in dissolution performance and stability.

3-6, influence of different preparation processes on the Properties (Crystal form H of Aidenafil citrate and Crystal form A of Aidenafil citrate)

The pharmaceutical ingredients and the addition proportion in the prescription 12 are the same as those in the prescription 1, and the prescription 12 is prepared by directly mixing all the pharmaceutical ingredients in the process of preparing a pharmaceutical preparation, adding water for granulation and tabletting according to the patent CN114028403A to prepare the citric acid alidenafil crystal form H tablet.

Specifically, the method comprises the following steps:

in the citric acid alidenafil crystal form H tablet obtained by adopting the method and the components of the prescription 12, the dissolution experiment shows that the dissolution rate of the citric acid alidenafil crystal form H tablet is only 35.1 percent within 0 to 5min, which is obviously lower than the dissolution rate (58.2 percent) in the prescription 1 at the same time, and the early-stage dissolution rate is very slow and is only 7.02 percent/min; the dissolution rate is only about 50% in 15min; within 15 to 60min, the dissolution rate reaches 89.5 percent, the dissolution effect is still obviously lower than 100 percent, the dissolution rate is slow, and the dissolution rate is poor.

In the pharmaceutical tablet obtained by adopting the formula 12, the content of the pharmaceutical active ingredient placed at high temperature is 98.9 percent, and the maximum content of single impurity reaches 0.36 percent; the content of the active ingredients of the medicine placed under strong light is 98.97 percent, and the maximum content of single impurities is 0.35 percent; the content of the active ingredients of the medicine placed at high temperature is 98.87 percent, and the maximum content of single impurity reaches 0.38 percent; compared with the impurity content of the formula 1, the impurities in the tablet prepared by the formula 9 are obviously increased along with the prolonging of the standing time, the active ingredients of the medicine are greatly reduced, and the stability is poor.

In the formula 13, the citric acid alidenafil crystal form A tablet disclosed in patent CN114028403A is referred to as a pharmaceutical ingredient and a preparation method thereof. The main difference from the prescription 1 is that the medicine prescription is different, and the addition modes of the disintegrant and the adhesive in the preparation process are different.

Specifically, the method comprises the following steps:

in the citric acid alidenafil crystal form A tablet obtained by adopting the method and the components of the formula 13, the dissolution experiment shows that the dissolution rate of the citric acid alidenafil tablet is only 34.0 percent within 0 to 5min, which is obviously lower than the dissolution rate (58.2 percent) of the citric acid alidenafil tablet in the formula 1 within the same time, and the early-stage dissolution rate is very slow and is only 6.8 percent/min; the dissolution rate is only about 50% at 15min; within 15 to 60min, the highest dissolution rate is 90.1 percent, the dissolution rate is slow, and the dissolution rate is poor.

In the pharmaceutical tablet obtained by adopting the formula 13, the content of the pharmaceutical active ingredient placed at high temperature is 98.7%, and the maximum content of single impurity is 0.41%; the content of the active ingredients of the medicine placed under strong light is 98.75 percent, and the maximum content of single impurities is 0.40 percent; the content of the active ingredient of the medicament placed under high humidity is 98.65 percent, and the maximum content of single impurities is as high as 0.44 percent; compared with the impurity content of the formula 1, in the tablet prepared by the formula 13, the impurities under different conditions are increased by more than one time along with the prolonging of the standing time, the impurities show a trend of being remarkably increased, the active ingredients of the medicine are remarkably reduced, and the stability is poor.

And (3) knotting: in the prescription 12 and the prescription 13, the croscarmellose sodium and the polymethyl methacrylate are adopted, and are directly mixed and then granulated by a wet method to prepare tablets; compared with the preparation method in the formula 1 of the invention by controlling the internal addition and external addition of the croscarmellose sodium, the citric acid alidenafil crystal form H tablet obtained by the preparation method of the invention has the advantages of obviously improved stability, faster and more obvious dissolution rate and dissolution effect.

In example 3, different formulations and preparation processes of the citric acid alidenafil crystal form H tablet are investigated by comparing the addition amounts (formulations 1 to 5) of (1) croscarmellose sodium (added), croscarmellose sodium (added) and polymethyl methacrylate, (2) the addition ratios (formulations 6 to 7) of (1) croscarmellose sodium (added) and (added) croscarmellose sodium (added) in the formulations, and (3) different disintegrants (formulations 8 to 10) in the formulations.

As a result, it was found that: the citric acid alidenafil crystal form H tablet obtained by distinguishing the external addition mode and the internal addition mode of the croscarmellose sodium and controlling the addition amount of the croscarmellose sodium (internal addition), the croscarmellose sodium (external addition) and the polymethyl methacrylate has obvious dissolution performance and stability effect; wherein the citric acid alidenafil crystal form H tablet obtained by the formula 1 has optimal dissolution performance and stability effect.

The citric acid alidenafil crystal form H (formula 11 CN112745323B) is obtained by comparing the auxiliary material components and the mixture ratio of different formulas, and the result shows that the citric acid alidenafil crystal form H tablet (formula 1) obtained by the preparation process is also obviously higher in dissolution and stability than the citric acid alidenafil crystal form H tablet obtained by formula 11 reference patent CN 112745323B.

The citric acid alidenafil crystal form H (formula 12 CN114028403A) is obtained by adopting a preparation process of directly and completely mixing and adding cross-linked sodium carboxymethylcellulose, and the result shows that the dissolution and the stability of the citric acid alidenafil crystal form H tablet (formula 1) obtained by the preparation process are also obviously higher than those of the citric acid alidenafil crystal form H tablet obtained by formula 12 patent CN 114028403A.

The citric acid alidenafil crystal form A (formula 13 CN114028403A) is obtained by comparing the patent CN114028403A and a preparation process of directly and completely mixing and adding cross-linked sodium carboxymethylcellulose, and the result shows that the citric acid alidenafil crystal form H tablet (formula 1) obtained by the preparation process is also remarkably higher in dissolution and stability than the citric acid alidenafil crystal form A tablet obtained by the patent CN114028403A (formula 13).

4. Preparation of tablets

According to the prescription process of the prescription 1, the citric acid alidenafil crystal form H tablet is prepared.

The raw material Aidinafil citrate crystal form H in the prescription 1 IS replaced by other raw materials, and a compound IS00384 tablet, a compound NCX1741 tablet, a citric Aidinafil citrate crystal form A tablet, a crystal form B tablet, a crystal form C tablet, a crystal form D tablet, a crystal form O tablet and a crystal form V tablet are obtained by the same preparation process.

According to the prescription 11: comparative example 4 (patent CN 112745323B) a citric acid alidenafil crystal form H tablet is prepared by a prescription process.

According to the prescription 13: comparative example 6 (patent CN 114028403A) a citric acid alidenafil crystal form A tablet is prepared by the prescription process.

Example 4: evaluation of drug efficacy

1. Alzheimer's Disease (AD) model building and animal grouping

1) Animals: the normal mice are C57/BL6J mice with the same background, and are 7 months old and half female and half male; AD model APP/PS1 mice, 7 months old, male and female half.

2) Positive control drug: memantine hydrochloride tablet

3) The tested drugs are: sildenafil citrate tablets, avanafil tablets, the compound IS00384 tablets prepared in example 3, the compound NCX1741 tablets, the citric acid aildenafil crystal forms H, A, B, C, D, O and V tablets, the citric acid aildenafil crystal form a prepared in patent CN114028403a, and the citric acid aildenafil crystal form H prepared in patent CN 112745323B. In order to simulate the storage and use of the conventional drugs, each of the test drugs was left at 25 ℃ and 60% humidity for 4 months, and then subjected to the administration treatment.

4) Animal grouping and dosing:

normal C57/BL6J mouse control group (without any intervention treatment) (N = 6);

AD model blank control group (without any intervention treatment) (N = 6);

AD model positive drug group (dose: 2.5 mg/kg/day, 1 dose/day/tube, oral administration, 4 weeks of continuous administration) (N = 6);

AD model low dose test drug group (dose: 5 mg/kg/day, 1 time/day/one, oral administration, 4 weeks of continuous administration) (N = 6);

dose test drug group in AD model (dose: 10 mg/kg/day, 1 time/day/one, oral administration, 4 weeks of continuous administration) (N = 6);

AD model high dose test drug group (dose: 15 mg/kg/day, 1 time/day/mouse, oral administration, 4 weeks of continuous administration) (N = 6).

2. Animal administration

Before administration: all animals are adaptively raised in an animal laboratory for 1 week, during the adaptive raising period, cognitive memory function behavior changes of mice in each group before drug treatment are observed through a Barnes maze experiment, and AD model mice after passing the behavioral test are randomly grouped for carrying out subsequent formal experiments.

Animal administration treatment: AD model mice were randomly grouped into additional control groups of 6 mice per group, with normal C57/BL6J mice. The normal C57/BL6J mouse control group and the AD model blank control group are not given any drug intervention treatment, and the other groups are respectively given corresponding drug (positive control drug memantine hydrochloride and low, medium and high dose test drugs) treatment, 1 time/day/mouse, oral administration and 4 weeks of continuous administration. The period of the behavioral test after 4 weeks of administration was continued for 1 week for a total period of 5 weeks.

3. Detecting the index

1) Body weight determination

Measuring for 1 time before grouping; the weight of each group of animals was recorded 2 times per week during the administration period, and the administration dose was adjusted; assay was performed 1 time before sacrifice.

2) Behavioristic test (Barnes maze, open field experiment)

After treatment, observing the behavior change of the learning and memory functions of each group of mice after drug treatment through a Barnes maze experiment (the Barnes maze experiment process comprises a learning training experiment and a exploration experiment for 7 days); the effect of autonomic activity and cognitive ability of each group of mice after drug treatment was observed by open field experiments.

3) Histopathological staining

The influence of the medicine on the pathological morphology of the hippocampal tissues of the APP/PS1 mouse is observed by adopting HE staining; immunohistochemical detection of mouse hippocampal Abeta-42 and its NEP gene expression.

4) Biochemical detection

SOD activity and MDA content in mouse serum are detected.

5) Data processing

Each group of experimental data is numerically calculated by computer software to obtain an accurate value (more than ten digits after the decimal point), and in order to reflect the visual simplicity of the table, the average values listed in the data tables 10 to 13 of the embodiment are approximate values after calculation.

4. Results

1) Body weight changes in mice

The body weight change trend of mice in each experimental group before and after administration is mild and has no abnormality, which indicates that the tested medicament has no influence on the body weight of the mice.

2) Effect of drugs on AD mouse behavior (Barnes maze and open field experiments)

a) Barnes maze experiment

Carrying out continuous 6-day Barens maze training experiments on the mice, freely exploring for 4min on the 7 th day, and recording the time for each mouse to enter the hole; after 4 weeks of continuous dosing, mice were trained and tested again for time to enter the hole. Table 10 lists the two tunnel times before and after dosing for mice, fig. 1 (a) and fig. 1 (b) are the mean tunnel times for each group of mice after dosing, statistical analysis between groups using the t-test program in EXCEL software, p <0.05, indicates a slight difference; p <0.01,. Indicates significant difference; p <0.001, indicates a highly significant difference.

As can be seen from table 10, for the experimental mouse group without administration, i.e., the normal mouse group and the AD model blank control group, the 1 st (before administration) average time to enter the hole was 149s for the normal mouse and 204s for the AD model blank control group; average time to tunnel for 2 nd (post-dose) normal mice 107s and 234s for ad model blank. The two-time hole-entering time is obviously longer than that of a normal mouse in the AD model blank control group, and the length of the AD model blank control group is 1.4 times and 2.2 times that of the normal mouse group, which shows that the memory capacity of the AD mouse is reduced, and a target hole needs to be found in a longer time.

The time to hole was reduced for each dose group compared to the AD model blank group. The citric acid alidenafil crystal form H group has the shortest hole entering time, the low dose group is 161s, the medium dose group is 150s, and the high dose group is 133s, so that the hole entering time is obviously reduced compared with the AD model group (p is less than 0.01). The reduction rate of the hole-entering time of each dose group compared with the AD model group is 31.2 percent, 35.9 percent and 43.2 percent respectively. The hole-entering time of the medium-dose group is equivalent to that of the positive control group, and the hole-entering time of the high-dose group is 13s less than that of the positive control group, so that the hole-entering time of the high-dose group is better than that of the positive control group.

Sildenafil citrate, avanafil, compound IS00384 and compound NCX1741 are respectively reduced by 26.9%, 23.1%, 21.4% and 20.1% in the low-dose group, 31.6%, 33.8%, 23.9% and 28.6% in the medium-dose group and 38.9%, 37.6%, 23.9% and 26.9% in the high-dose group compared with the AD model group. Compared with the citric acid alidenafil crystal form H group, the compound IS00384 and the compound NCX1741 have the largest difference and have obvious difference, wherein the difference between the high dose group and the citric acid alidenafil crystal form H group IS 19.3 percent and 16.3 percent respectively.

Compared with the AD model group, the hole entering time of the citric acid alidenafil other crystal form group (the crystal form A, B, C, D, O, V in sequence) is respectively reduced by 17.5%, 25.6%, 25.2%, 19.7%, 19.2% and 20.1% in the low dose group, 26.9%, 23.1%, 29.5%, 28.6%, 29.1% and 25.2% in the medium dose group, and 30.3%, 34.6%, 34.2%, 31.6%, 32.5% and 30.3% in the high dose group. The other crystal forms have reduced reduction compared with the crystal form H, wherein the phase difference of the crystal form A is the most, the phase difference of the low-dose group and the high-dose group is respectively 13.7 percent and 12.9 percent of that of the crystal form H, and the phase difference of the high-dose group of the crystal form V and the crystal form H is also 12.9 percent.

The punching time of different dosage groups of the crystal form A (CN 114028403A prescription process) is 196s, 189s and 187s respectively, and compared with the AD mouse model group, the reduction ratio is respectively as follows: 16.2%, 19.2% and 20.1% lower than those of the crystal form H group by 15.0%, 16.7% and 23.1% respectively, and lower than those of the crystal form A group (the prescription process of the application) by 1.3%, 7.7% and 10.2% respectively, which have significant differences.

The punching time of different dosage groups of the crystal form H (CN 112745323B prescription process) is 185s, 174s and 162s respectively, the reduction ratio of the crystal form H in comparison with the AD mouse model group is 20.9%, 25.6% and 30.8%, the reduction ratio of the crystal form H in comparison with the AD mouse model group is 10.3%, 10.3% and 12.4% respectively lower than that of the crystal form H in the prescription process of the application, and the obvious difference exists.

According to the Baens maze experiment, different PDE5 inhibitors comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein the memory function recovery effect of the crystal form H of the sildenafil citrate on AD model mice IS the best, and IS obviously higher than that of other tested medicaments. The hole-entering time IS reduced, the ratio of the citric acid alidenafil crystal form H IS the highest, the difference between the compound IS00384 and the compound NCX1741 high-dose group and the citric acid alidenafil crystal form H group can reach 19.3 percent and 16.3 percent, and the citric acid sildenafil and the alienafil group are lower than the citric acid alidenafil crystal form H group. This indicates that different PDE5 inhibitors have significant differences in therapeutic efficacy against alzheimer's disease.

The citric acid alidenafil crystal forms comprise the crystal forms A, B, C, D, O, V and H, wherein the average hole entering time of the crystal form H group is shortest, the learning and memory ability recovery effect on AD mice is best, and the crystal form H is obviously higher than that of other crystal forms. Under the condition of low dosage, the crystal form A with the worst effect has a hole-entering time reduction rate difference of 13.7 percent compared with the crystal form H, the crystal form V has a difference of 12.0 percent compared with the crystal form H, and other crystal forms have poor effects compared with the crystal form H.

The crystal form A tablets prepared by different prescription processes have obvious difference on the reduction ratio of the hole entering time of AD mice, the CN114028403A prescription process group is obviously lower than the prescription process group of the application, and the difference between the two can reach more than 10.0 percent.

Compared with the time for an AD mouse to enter a hole, the proportion of the crystal form H tablet prepared by the CN112745323B prescription process is reduced, and the difference is obviously reduced by more than 12 percent compared with the crystal form H tablet prepared by the method.

b) Open field experiment

The open field experiment is a common animal behavior experiment, can detect the spontaneous activity behavior and exploration behavior of a mouse, and is a method for evaluating the autonomous behavior of an experimental animal in a new and different environment and exploring the behavior and the tensity. Animals fear the new open environment and move mainly in the peripheral areas and less in the central area. The overall path and speed of movement of a mouse over an open field may reflect to some extent its spontaneous activity. The total movement distance, the movement times, the average speed, the central distance, the residence central time, the central movement time and the like of the mice are tested in the open field experiment, and specific results are shown in a table 11.

Total distance:

as can be seen from table 11, the normal mice have a small total distance because they feel strange to the surroundings in the new environment and influence their spontaneous activity. The AD model mouse has the degenerative disease of the nervous system, the perception capability is reduced, so the AD model mouse is not influenced by a new environment, and the total distance of the AD model mouse is increased compared with that of a normal mouse. The average number of the total courses of the normal mice is 25479mm, while the AD model mice reaches 41198mm, the increase is 15719mm, and the total course number is 1.6 times of that of the normal mice.

After the administration treatment of each administration group, the total number of courses is reduced compared with that of AD model mice. Wherein the average total distance of the citric acid alidenafil crystal form H group is reduced most, and the average distances of the low dose group, the medium dose group and the high dose group are reduced by 7867mm, 9037mm and 9756mm respectively, and have significant difference (p is less than 0.01). The reduction ratio of each dose group of the citric acid alidenafil crystal form H reaches 19.1 percent, 21.9 percent and 23.7 percent respectively, and the medium dose group and the high dose group are both higher than a positive control group (20.5 percent).

The average total mileage of sildenafil citrate, avanafil, the compound IS00384 and the compound NCX1741 in the low-dose group IS respectively reduced by 16.9%, 15.1%, 1.8% and 5.3% compared with that in the AD model group, the average total mileage of the medium-dose group IS respectively reduced by 18.8%, 20.1%, 11.2% and 2.8%, and the average total mileage of the high-dose group IS respectively reduced by 20.0%, 19.4%, 13.7% and 11.3%. The difference between the compound IS00384 group and the compound NCX1741 group and the citric acid alidenafil crystal form H group IS most obvious, the difference between the compound IS00384 low dose group and the compound NCX1741 medium dose group IS 17.3%, and the difference between the compound NCX1741 medium dose group and the compound NCX 00384 low dose group IS 19.1%. The difference between the sildenafil citrate and avanafil low-dose group and the Aidenafil citrate crystal form H group is smaller, and the difference between different dose groups is 1.8-4.3%.

The average total mileage of the citric acid alidenafil crystal form A, the citric acid alidenafil crystal form B, the citric acid alidenafil crystal form C, the citric acid alidenafil crystal form D, the citric acid alidenafil crystal form O and the citric acid alidenafil crystal form V is respectively reduced by 2.6%, 11.0%, 12.4%, 6.1%, 7.6% and 6.4% compared with the AD model group, the average total mileage of the citric acid alidenafil crystal form A, the average total mileage of the citric acid alidenafil crystal form B, the average total mileage of the citric acid alidenafil crystal form C, the average total mileage of the citric acid alidenafil crystal form D, the average total mileage of the citric acid alidenafil crystal form O and the average total mileage of the citric acid alidenafil crystal form V are respectively reduced by 8.8%, 10.5%, 15.9%, 11.7%, 12.2% and 13.7% compared with the AD model group, and the average dosage of the citric acid alidenafil crystal form V. Wherein the difference between the group A crystal form and the group H crystal form is the most obvious, and the difference between the low dose group, the medium dose group and the high dose group is 16.5 percent, 13.1 percent and 12.3 percent respectively.

The average total mileage of different dosage groups of the crystal form A (CN 114028403A prescription process) is respectively reduced by 0.7%, 4.0% and 5.3% compared with that of an AD model group, differs by 18.4%, 18.0% and 18.4% compared with that of a crystal form H group, is respectively reduced by 1.9%, 4.9% and 6.1% compared with that of the crystal form A (the prescription process of the application), and has obvious difference.

The average total mileage of different dosage groups of the crystal form H (CN 112745323B prescription process) is respectively reduced by 10.9 percent, 12.2 percent and 13.5 percent compared with that of an AD model group, and is respectively reduced by 8.2 percent, 9.7 percent and 10.2 percent compared with that of the crystal form H in the prescription process of the application, and the difference is obvious.

Therefore, different PDE5 inhibitors comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein the crystal form H of the sildenafil citrate has the largest reduction ratio compared with the total distance of an AD model mouse, has the best perception capability recovery effect and IS obviously higher than other tested medicaments. Compared with the citric acid alidenafil crystal form H, the compound IS00384 and the compound NCX1741 have the difference of 19.1 percent in total mileage reduction. Other tested drugs also have lower reduction degree than the citric acid alidenafil crystal form H. This suggests that the effects of recovering the ability to sense AD mice are very different, although they are PDE5 inhibitors.

Tablets prepared from different crystal forms of the citric acid alidenafil comprise the crystal forms A, B, C, D, O, V and H, wherein the crystal form H has the best treatment effect and is obviously higher than other crystal forms. The effect of the crystal form A is 16.5 percent different from that of the crystal form H, other crystal forms are also poor than that of the crystal form H, and the curative effect of different crystal forms is greatly different when the tablets are orally taken and are prepared from the citric acid alidenafil.

Compared with the crystal form A tablet prepared by the method, the crystal form A tablet prepared by the CN114028403A formula process has the advantages that the reduction ratio of the total mileage of AD mice is obviously reduced, and the difference can reach 4.9% at most under different doses.

Compared with the time for an AD mouse to enter a hole, the proportion of the crystal form H tablet prepared by the CN112745323B prescription process is reduced, and the difference is obviously reduced by more than 10 percent compared with the crystal form H tablet prepared by the method.

The results of the total course of the open field experiment show that the citric acid alidenafil crystal form H has the highest reduction degree on the total course of an AD model mouse, has the best perception capability improvement effect and is obviously higher than other different PDE5 inhibitors and other citric acid alidenafil crystal forms; the effect of improving the perception capability of the AD model mouse by the crystal form A tablet prepared by the method is obviously better than that of the crystal form A tablet prepared by a CN114028403A prescription process; the effect of improving the perception capability of the AD model mouse by the crystal form H tablet prepared by the method is obviously better than that of the crystal form H tablet prepared by the CN112745323B prescription process.

Average speed:

the average speed of the normal mouse group is 42mm/s, the average speed of the AD model blank mouse group is 69mm/s, which is 1.6 times of that of the normal mouse, and the AD model mouse has increased dysphoria degree. The average speed of mice in each administration group is reduced compared with that of an AD model blank mouse group, the dysphoria degree is reduced, wherein the reduction value of the citric acid alidenafil crystal form H group is the largest, the average speeds of a low dose group, a medium dose group and a high dose group are respectively 58mm/s, 53mm/s and 53mm/s, the average speeds are equivalent to that of a positive control group, and the difference is obvious compared with that of the AD model blank control group (p is less than 0.01). The average speed of each dose group of the citric acid alidenafil crystal form H is respectively reduced by 15.9 percent, 23.2 percent and 23.2 percent compared with that of an AD model group.

The average speeds of sildenafil citrate, avanafil, compound IS00384 and compound NCX1741 in the low dose groups were 11.6%, 10.1%, 8.7% and 11.6% respectively, in the medium dose groups 14.5%, 8.7% and 17.4% respectively, and in the high dose groups 18.8%, 17.4%, 14.5% and 14.5% respectively, compared with the AD model group. Wherein the reduction amount of the compound IS00384 group IS the least, and the difference between the medium-dose group and the citric acid alidenafil crystal form H group IS up to 14.5%. The difference between the other tested sample groups and the citric acid alidenafil crystal form H group is 4.3-8.7%.

The citric acid alidenafil crystal form A, the citric acid alidenafil crystal form B, the citric acid alidenafil crystal form C, the citric acid alidenafil crystal form D, the citric acid alidenafil crystal form O and the citric acid alidenafil crystal form V, wherein the low dose group is respectively reduced by 5.8%, 7.2%, 10.1%, 14.5%, 15.9% and 5.8% compared with the AD model group, the medium dose group is respectively reduced by 10.1%, 13.0%, 10.1% and 11.6% compared with the AD model group, and the high dose group is respectively reduced by 14.5%, 11.6%, 17.4%, 14.5%, 18.8% and 15.9% compared with the AD model group. Wherein the difference between the dosage group in the crystal form A and the crystal form H group is as much as 13.0 percent, the difference between the dosage group in the crystal form D and the crystal form H group is 12.3 percent, and the difference between the high dosage group in the crystal form B and the crystal form H group is 11.8 percent.

The average speeds of different dosage groups of the crystal form A (CN 114028403A prescription process) are respectively reduced by 2.9%, 5.3% and 7.2% compared with the AD model group, the differences of the average speeds of the different dosage groups of the crystal form A (CN 114028403A prescription process) and the AD model group are respectively 13.0%, 17.9% and 15.9% compared with the crystal form H group, and the differences of the average speeds of the different dosage groups of the crystal form A (CN 114028403A prescription process) and the AD model group are respectively reduced by 2.9%, 4.8% and 7.2% compared with the crystal form A (CN 114028403A prescription process), and the differences are obvious.

The average speeds of different dosage groups of the crystal form H (CN 112745323B prescription process) are respectively reduced by 11.6%, 20.3% and 17.4% compared with the AD model group, and are respectively reduced by 4.3%, 2.9% and 5.8% compared with the crystal form H group of the prescription process of the application, and the average speeds are obviously reduced.

By comparing the average speed of the open field experiment, different PDE5 inhibitors comprise sildenafil citrate, avanafil citrate, a compound IS00384, a compound NCX1741 and crystal forms of the sildenafil citrate, wherein the crystal form H of the sildenafil citrate has the highest reduction degree on the average speed of an AD model mouse, has the best effect of relieving the morbidity characteristic of dysphoria and restlessness of the AD model mouse, and IS obviously superior to other tested drugs. The difference between the compound IS00384 group and the citric acid alidenafil crystal form H group IS up to 14.5 percent, and the reduction degree of other PDE5 inhibitor groups IS lower than that of the citric acid alidenafil crystal form H group, which shows that different PDE5 inhibitors have obvious difference on the treatment effect of the Alzheimer's disease.

The AD model mouse is treated by the citric acid alidenafil with different crystal forms, including the crystal form A, B, C, D, O, V and the H tablet, wherein the curative effect of the crystal form H is the best and is obviously higher than that of other crystal forms. On the average speed reduction degree, the difference between the crystal form A and the crystal form H reaches 13.0 percent, and other groups are also poorer than the crystal form H.

Compared with the crystal form A tablet prepared by the method, the crystal form A tablet prepared by the CN114028403A formula process has the advantages that the reduction ratio of the average speed of AD mice is obviously reduced, and the difference can reach 7.2 percent at different doses.

Compared with the average speed of AD mice, the crystal form H tablet prepared by the CN112745323B prescription process has a reduced proportion, and is obviously reduced compared with the crystal form H tablet prepared by the method, and the difference can reach 5.8 percent at most.

The result of the average speed of open field experiments shows that the crystal form H of the citric acid alidenafil has the highest reduction degree of the average speed of AD model mice and the best effect of improving the dysphoria state, and is obviously higher than other PDE5 inhibitors and other crystal forms of the citric acid alidenafil; the effect of improving the dysphoria state of an AD model mouse by using the crystal form A tablet prepared by the method is obviously better than that of the crystal form A tablet prepared by using a CN114028403A prescription process; the effect of improving the dysphoria state of the AD model mice by the crystal form H tablet prepared by the method is obviously better than that of the crystal form H tablet prepared by the CN112745323B prescription process.

The number of activities:

compared with normal mice, the activity times of the blank mice of the AD model are obviously reduced, the average activity times of the normal mice are 68, while the activity times of the blank mice of the AD model are only 39 and are reduced by 29 times compared with the normal mice, which indicates that the blank mice of the AD model are more anxious. The positive control group in the administration group is 50 times, the mouse activity times of each dose group of the citric acid alidenafil crystal form H group are the highest, are respectively 51 times, 56 times and 55 times, are respectively increased by 30.8 percent, 43.6 percent and 41.0 percent, are higher than the positive control group, and have obvious difference (p is less than 0.01) compared with the blank control group of the AD model.

The number of activities was also increased in the other administration groups compared to the AD model blank group. Sildenafil citrate, avanafil, compound IS00384 and compound NCX1741 the low dose component increased by 20.5%, 15.4%, 12.8% and 7.7%, respectively, the medium dose component increased by 28.2%, 23.1%, 25.6% and 23.1%, respectively, and the high dose component increased by 35.9%, 28.2%, 17.9% and 20.5%, respectively. Compared with the citric acid alidenafil crystal form H group, the difference of each dosage group of the compound NCX1741 is 23.1%, 20.5% and 20.5%, which has obvious difference.

In the citric acid alidenafil crystal form and other crystal form groups, the increment rate of each crystal form A dose group is the largest difference with that of the crystal form H, and the increment rates are respectively 25.7 percent, 28.2 percent and 25.6 percent.

The average activity times of different dosage groups of the crystal form A (CN 114028403A prescription process) are respectively increased by 2.6%, 7.7% and 10.3% compared with those of the AD model group, the difference between the average activity times of the crystal form A and the AD model group is respectively 28.2%, 35.9% and 30.8% compared with that of the crystal form H, and the difference between the average activity times of the crystal form A (CN 114028403A prescription process) and the AD model group is respectively 2.6%, 7.7% and 5.1% compared with that of the crystal form A (the application prescription process), so that the method has obvious difference.

The average activity times of different dose groups of the crystal form H (CN 112745323B prescription process) are respectively reduced by 23.1%, 28.2% and 33.3% compared with that of an AD model group, and are respectively reduced by 7.7%, 15.4% and 7.7% compared with that of the crystal form H group in the prescription process of the application, and the difference is obvious.

Compared with the activity times of an open field experiment, different PDE5 inhibitors comprise sildenafil citrate, avanafil citrate, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein the activity times of the crystal form H group of the sildenafil citrate are increased most compared with AD model mice, the anxiety condition of the AD model mice IS improved most, and the treatment effect IS obviously superior to other tested medicaments. Compared with the citric acid alidenafil crystal form H, the NCX1741 compound is lower by more than 20 percent in each dose group and has obvious difference, and other tested drug groups have no citric acid alidenafil crystal form H and have high increase ratio, which shows that different PDE5 inhibitors have great difference on the treatment effect of the anxiety symptoms of AD model mice.

Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, wherein the activity frequency increasing proportion of the crystal form H group is the highest, the worst is the crystal form A group, each dosage is lower than the crystal form H group by more than 25 percent, the difference is obvious, and other crystal form groups are also obviously lower than the crystal form H group. Therefore, the tablets prepared from different crystal forms of the citric acid alidenafil have great improvement difference on the anxiety degree of AD mice, and the crystal form H is obviously higher than other crystal forms.

Compared with the crystal form A tablet prepared by the method, the crystal form A tablet prepared by the CN114028403A formula process has the advantages that the increase proportion of the average activity times of AD mice is obviously reduced, and the phase difference can reach 7.7% under medium dosage.

Compared with the average activity times of AD mice, the crystal form H tablet prepared by the CN112745323B prescription process is reduced in proportion, and is obviously reduced compared with the crystal form H tablet prepared by the method, and the difference is up to more than 15%.

The activity frequency results of open field experiments show that the ratio of the citric acid alidenafil crystal form H to the activity frequency increase of an AD model mouse is the highest, the anxiety reducing effect is the best, and the treatment effect is obviously superior to other different PDE5 inhibitors and tablets prepared from citric acid alidenafil in different crystal forms; the effect of the crystal form A tablet prepared by the method on reducing anxiety of an AD model mouse is obviously better than that of the crystal form A tablet prepared by a CN114028403A prescription process; the effect of the crystal form H tablet prepared by the method on reducing anxiety of AD model mice is obviously better than that of the crystal form H tablet prepared by a CN112745323B prescription process.

The central route is as follows:

rodents spontaneously escape the central area during spatial activity and travel along the peripheral areas, theoretically when the cognitive abilities of the mouse are affected, if the cognitive abilities of the rodent to the new environment are poor, the residence time of the rodent in the central area is prolonged, and the corresponding distance in the central area is also prolonged.

As can be seen from Table 11, the mean central distance of the normal mice was 4857mm, and the mean central distance of the AD model blank group mice was 12041mm, which was greatly increased 2.5 times that of the normal mice. The central distance of mice was reduced in each dosing group compared to AD model blank group. The dosage groups of the citric acid alidenafil crystal form H in the administration group have the largest reduction amount and are all obviously reduced (p is less than 0.01), wherein the central activity path low dosage group is 8005mm, the middle dosage group is 7569mm, the high dosage group is 7412mm, which are all lower than that of the positive control group, and the reduction ratio of each dosage group compared with the AD model group respectively reaches 33.5%, 37.1% and 38.4%.

Sildenafil citrate, avanafil, the compound IS00384 and the compound NCX1741 were reduced by 25.5%, 26.6%, 18.1% and 21.6% in the low dose group, 30.0%, 29.4%, 24.2% and 25.2% in the medium dose group, and 31.1%, 31.6%, 23.5% and 25.8% in the high dose group, respectively. In the 4 groups, sildenafil citrate and avanafil citrate are better than the IS00384 and NCX1741 compounds, but are inferior to the form H of the sildenafil citrate. The reduction ratios of the compound IS00384 group with the worst effect, the low-dose group, the medium-dose group and the high-dose group are respectively 15.4 percent, 12.9 percent and 14.9 percent lower than that of the citric acid alidenafil crystal form H group, and the obvious difference IS achieved.

The most effective of the citric acid alidenafil crystal form other crystal form groups is the crystal form A group, each dosage group is respectively reduced by 15.6%, 20.5% and 23.9%, compared with the crystal form H group, the reduction amounts are respectively different by 17.9%, 16.6% and 14.5%, and the difference is obvious. The other crystal form groups are reduced by a lower proportion than the crystal form H group.

The average central paths of different dosage groups of the crystal form A (CN 114028403A prescription process) are respectively reduced by 8.6 percent, 11.3 percent and 18.0 percent compared with the AD model group, the differences of the isomorphous H group are respectively 25.0 percent, 25.9 percent and 20.4 percent, and the differences of the isomorphous A (the prescription process of the application) group are respectively 7.0 percent, 9.2 percent and 5.9 percent, which have obvious differences.

The average central distance of different dosage groups of the crystal form H (CN 112745323B prescription process) is respectively reduced by 18.0%, 22.5% and 26.9% compared with that of an AD model group, and is respectively reduced by 15.5%, 14.6% and 11.5% compared with that of the crystal form H group in the prescription process of the application, and the difference is obvious.

Compared with the central distance of the open field experiment, different administration groups of the PDE5 inhibitor comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and different crystal forms of the sildenafil citrate, wherein compared with AD model mice, the central distance of the crystal form H group of the sildenafil citrate IS reduced at the most, the cognitive ability improvement effect of the AD model mice IS the best, and the cognitive ability improvement effect of the AD model mice IS obviously higher than that of other groups. The worst IS the compound IS00384 and the compound NCX1741, the difference of each dose group IS up to 15.4 percent compared with the citric acid alidenafil crystal form H group, and other tested medicines are also inferior to the citric acid alidenafil crystal form H group.

The citric acid alidenafil different crystal form tablets comprise the crystal forms A, B, C, D, O, V and H, compared with AD model mice, the central distance reduction ratio of the citrate is the most in the crystal form H group, and is obviously higher than that in other crystal form groups. The crystal form A group is the least, the difference with the crystal form H group can reach 17.9 percent, and the reduction ratio of other crystal form groups is lower than that of the crystal form H group.

Open field experiment central distance data show that the citric acid alidenafil crystal form H has the highest reduction ratio to the central distance of an AD model mouse, the cognitive ability improvement effect is the best, and the treatment effect is obviously higher than other PDE5 inhibitors and other citric acid alidenafil crystal forms; the effect of the crystal form A tablet prepared by the method on improving the cognitive ability of the AD model mouse is obviously better than that of the crystal form A tablet prepared by a CN114028403A prescription process; the effect of the crystal form H tablet prepared by the method on improving the cognitive ability of the AD model mouse is obviously better than that of the crystal form H tablet prepared by a CN112745323B prescription process.

The central time:

the average central time of normal mice is 66s, and the average central time of AD model blank mice reaches 144s, which is 2.2 times of that of normal mice. The median time of mice was reduced in each dosing group compared to AD model blank group. The reduction value of each dosage group of the citric acid alidenafil crystal form H in the administration group is the largest and is obviously reduced (p is less than 0.01), the central activity time low dosage group is 115s, the medium dosage group is 104s, the high dosage group is 95s, wherein the medium dosage group and the high dosage group are lower than a positive control group (109 s), and the reduction ratio of each dosage group compared with an AD model group respectively reaches 20.1 percent, 27.8 percent and 34.0 percent. (FIGS. 2 (a) and 2 (b))

Compared with the AD model group, the sildenafil citrate, the avanafil citrate, the IS00384 compound and the NCX1741 compound are respectively reduced by 18.1%, 21.5%, 9.7% and 10.4% in the low dose group, 22.9%, 22.2%, 18.8% and 16.0% in the medium dose group, 27.1%, 29.2%, 22.9% and 17.4% in the high dose group, and the reduction ratios are lower than those of the crystalline form H group of the sildenafil citrate. This shows that the 4 PDE5 inhibitors have worse effect on AD model mice than crystalline form H of citric acid, the worst being compound NCX1741 group, and the different dose groups were respectively 9.7%, 11.8% and 16.7% worse than crystalline form H of citric acid, with significant differences.

The other crystal forms of the citric acid alidenafil have poorer effects than the crystal form H, wherein the high dose group of the crystal form A is 14.6 percent lower than the high dose group of the crystal form H, and the medium dose group of the crystal form O is 13.2 percent lower than the medium dose group of the crystal form H.

The average central time of different dosage groups of the crystal form A (CN 114028403A prescription process) is respectively reduced by 9.0%, 13.2% and 14.6% compared with that of an AD model group, the difference of the same crystal form H group is respectively 11.1%, 14.6% and 19.4%, and the difference of the same crystal form A (the prescription process of the application) group is respectively 7.6%, 8.3% and 4.9%, and the difference is obvious.

The average total mileage of different dosage groups of the crystal form H (CN 112745323B prescription process) is respectively reduced by 16.0 percent, 20.1 percent and 23.6 percent compared with that of an AD model group, and is respectively reduced by 4.2 percent, 7.6 percent and 10.4 percent compared with that of the crystal form H group of the prescription process of the application, and the difference is obvious.

Compared with the central time of an open field experiment, different PDE5 inhibitor administration groups comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein compared with an AD model mouse, the crystal form H group of the sildenafil citrate has the highest central time reduction ratio and the best cognitive ability improvement effect on the AD model mouse, and IS obviously superior to other groups. The central time reduction ratio of the citric acid alidenafil crystal form H group can reach 34.0 percent at most, while the compound NCX1741 group under the same dosage is only 17.4 percent, the difference between the two is 16.6 percent, and other tested drug groups are lower than the citric acid alidenafil crystal form H group.

Tablets prepared from different crystal forms of the citric acid alidenafil comprise the crystal forms A, B, C, D, O, V and H, the highest time reduction ratio to the central region of an AD model mouse after administration is the crystal form H group, and the crystal form H group is obviously higher than other crystal form groups. The worst is the crystal form A group and the crystal form O group, the difference between the two crystal form experimental groups and the crystal form H group is as high as 14.6 percent and 13.2 percent, and other crystal form groups are lower than the crystal form H group.

The effect of the crystal form A tablet prepared by the method on improving the cognitive ability of the AD model mouse is obviously better than that of the crystal form A tablet prepared by a CN114028403A prescription process, and the phase difference can reach 8.3 percent at most; the effect of the crystal form H tablet prepared by the method on improving the cognitive ability of AD model mice is obviously better than that of the crystal form H tablet prepared by a CN112745323B prescription process, and the phase difference can reach up to 10.4 percent.

Central activity time:

the average central activity time of normal mice was 63s, and the ad model blank mice was 135s, which was greatly increased. The dosage groups are reduced, and the central activity time (p is less than 0.01) of each dosage group of the citric acid alidenafil crystal form H can be obviously reduced, wherein the medium dosage group is 101s and is the same as the positive control group; the high dose group was 94s, lower than the positive control group. Compared with the AD model group, the central activity time reduction rate of each dosage group of the citric acid alidenafil crystal form H is respectively 20.0 percent, 25.3 percent and 28.1 percent.

Compared with the AD model group, the central activity time of sildenafil citrate, avanafil, the compound IS00384 and the compound NCX1741 in the low-dose group IS respectively reduced by 16.3%, 18.5%, 9.6% and 10.4%, the central activity time of the medium-dose group IS respectively reduced by 20.0%, 20.7%, 18.4% and 13.3%, the central activity time of the high-dose group IS respectively reduced by 23.7%, 25.9%, 20.7% and 16.3%, and the reduction ratio of the high-dose group IS lower than that of the crystalline form H of the sildenafil citrate. Wherein, different dosage groups of the compound NCX1741 are respectively 9.6 percent, 12.0 percent and 11.9 percent worse than the citric acid alidenafil crystal form H group.

The other crystal forms of the citric acid alidenafil have poorer effects than the crystal form H, wherein the crystal form A high-dose group with the largest difference is 9.6 percent lower than the crystal form H high-dose group, and the crystal form V high-dose group is 11.1 percent lower than the crystal form H high-dose group, so that the citric acid alidenafil has obvious difference.

The average central activity time of different dosage groups of the crystal form A (CN 114028403A prescription process) is respectively reduced by 6.7 percent, 10.4 percent and 14.8 percent compared with that of an AD model group, the difference between the isomorphous H group and the AD model group is respectively 13.3 percent, 14.9 percent and 13.3 percent, and the difference between the isomorphous A (the prescription process of the application) group and the AD model group is respectively 7.4 percent, 8.1 percent and 3.7 percent, and the difference is obvious.

The average central activity time of different dose groups of the crystal form H (CN 112745323B prescription process) is respectively reduced by 12.6 percent, 17.8 percent and 20.0 percent compared with that of an AD model group, and is respectively reduced by 7.4 percent, 7.5 percent and 8.1 percent compared with that of the crystal form H group in the prescription process of the application, and the difference is obvious.

Compared with the central activity time of an open field experiment, different PDE5 inhibitor administration groups comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein compared with an AD model mouse, the crystal form H group of the sildenafil citrate has the highest central activity time reduction ratio and the best cognitive ability improvement effect on the AD model mouse, and IS obviously higher than other experimental groups. The most effective crystalline form H of citric acid, aildenafil, was 12% higher than the least effective compound NCX1741, and the other drug tested were all lower than the crystalline form H of citric acid, aildenafil.

The citric acid alidenafil in different crystal forms comprises a crystal form A, B, C, D, O, V and a crystal form H, wherein the reduction ratio of the central activity time of the crystal form H group to that of the AD model mouse is the highest and is obviously higher than that of other crystal form groups. The worst is that the difference between the crystal form A group and the crystal form V group is 9.6 percent and 11.1 percent respectively, and other crystal form groups are lower than the crystal form H group.

The effect of the crystal form A tablet prepared by the method on improving the cognitive ability of an AD model mouse is obviously better than that of the crystal form A tablet prepared by a CN114028403A prescription process, and the phase difference can reach 8.1 percent at most.

The effect of the crystal form H tablet prepared by the method on improving the cognitive ability of the AD model mouse is obviously better than that of the crystal form H tablet prepared by the CN112745323B prescription process, and the phase difference can reach 8.1 percent at most.

Open field experimental results show that AD model placebo mice do not have their spontaneous activity affected by the new environment, indicating that their perception is reduced, and that the degree of agitation is increased, being more anxious. After the administration treatment, the total distance of mice in each administration group is reduced, the average speed is reduced, the activity times are reduced, the central distance is reduced, the central time is reduced, and the central activity time is reduced, which indicates that all tested drugs can improve the cognitive ability of AD model mice, reduce the dysphoria and anxiety degree, and have certain curative effect on AD mice. In the administration group, all indexes of the citric acid alidenafil crystal form H are obviously improved, and the treatment effect is equivalent to or superior to that of a positive control.

The best effect of different PDE5 inhibitors, such as sildenafil citrate, avanafil, the IS00384 compound, the NCX1741 compound and the crystalline form of the Aidenafil citrate on the recovery of cognitive ability, irritability and anxiety of AD model mice IS the Aidenafil citrate crystalline form H. The curative effect of the citric acid alidenafil crystal form H group is obviously higher than that of other PDE5 inhibitor groups, for example, the total distance of the open field experiment is reduced, and the citric acid alidenafil crystal form H group is up to 19.1 percent higher than a compound NCX 1741; the average speed IS reduced by a proportion, the difference of the dosage group in the compound IS00384 and the dosage group in the citric acid alidenafil crystal form H IS 14.5 percent; the proportion of the increase of the activity times IS that the dosage of the citric acid alidenafil crystal form H low-dosage group IS 23.1 percent more than that of the compound NCX1741, and the dosage of the citric acid alidenafil crystal form H high-dosage group IS 23.1 percent more than that of the compound IS00384 high-dosage group; the central distance reduces the proportion, and each dosage group of the citric acid alidenafil crystal form H IS respectively 15.4 percent, 12.9 percent and 14.9 percent higher than that of the corresponding dosage group of the compound IS 00384; the central time is reduced by 16.7 percent, the citric acid alidenafil crystal form H high-dose group is more than the compound NCX1741 high-dose group; the central activity time decreased in proportion, 11.9% more in the high dose group of crystalline form H of aildenafil citrate than in the high dose group of compound NCX 1741. The curative effect of the citric acid alidenafil crystal form H is obviously higher than that of other PDE5 inhibitors.

Different tablets prepared from the citric acid alidenafil crystal forms comprise the crystal forms A, B, C, D, O, V and H, and after oral administration, the crystal form H tablet has the highest recovery effect on the perception capability and the anxiety and the dysphoria of AD mice under each dose, and is obviously higher than other crystal forms. For example, the total course of the open field experiment decreased by a factor of 16.5% in the form a low dose group compared to the form H low dose group; the average rate decreases by a ratio that is 13.0% worse for the dosage group in form a than for the dosage group in form H; the proportion of increased activity times, 28.2% higher in the form H than in the form a; the central distance is reduced by a ratio, and the difference of each dosage group of the crystal form A and the crystal form H is respectively 17.9 percent, 16.6 percent and 14.5 percent; the central time is reduced by a proportion, the high dosage group of the crystal form H is 14.6 percent more than that of the high dosage group of the crystal form A, and the medium dosage group of the crystal form H is 13.2 percent more than that of the medium dosage group of the crystal form O; the central activity time decreased by a ratio of 9.6% and 11.1% more for the form H high dose group than for the form a and form V high dose groups, respectively. The therapeutic effect of the crystal form H tablet is obviously higher than that of other crystal form tablets.

The citric acid alidenafil crystal form A tablet prepared by the method has a remarkably better effect on improving AD model symptoms than a crystal form A tablet prepared by a CN114028403A prescription process, wherein the total distance of an open field experiment is reduced by a proportion, the crystal form A tablet prepared by the method is 6.1% higher than the crystal form A tablet prepared by a CN114028403A prescription process, the average speed reduction proportion is 7.2% higher, the average activity frequency increase proportion is 7.7% higher, the central distance reduction proportion is 9.2% higher, the central time reduction proportion is 8.3% higher, and the central activity time reduction proportion is 8.1% higher. The tablet of the citric acid alidenafil crystal form A prepared by different methods has great difference in the treatment effect on AD mice, and the treatment effect of the tablet prepared by the method is obviously higher than that of the tablet prepared by the CN114028403A prescription process.

The citric acid alidenafil crystal form H tablet prepared by the method has a remarkably better effect on improving AD model symptoms than a crystal form H tablet prepared by a CN112745323B prescription process, wherein the total route of an open field experiment is reduced by a proportion, the crystal form H tablet prepared by the method is 10.2% higher than the crystal form H tablet prepared by a CN112745323B prescription process, the average speed reduction proportion is 5.8% higher, the average activity frequency increase proportion is 15.4% higher, the central route reduction proportion is 15.5% higher, the central time reduction proportion is 10.4% higher, and the central activity time reduction proportion is 8.1% higher. The citric acid alidenafil crystal form H tablets prepared by different methods have great difference in the treatment effect on AD mice, and the treatment effect of the tablets prepared by the method is obviously higher than that of the tablets prepared by the CN112745323B prescription process.

It follows that different PDE5 inhibitors differ greatly in cognitive ability and recovery from anxiety and dysphoria in AD model mice. In all tested drug groups, the citric acid alidenafil crystal form H group has the highest degree of improving the symptoms of the AD model and is obviously higher than other PDE5 inhibitors. The compound IS00384 and the compound NCX1741 have larger treatment effects than the citric acid alidenafil crystal form H, which shows that although the compound IS00384 and the compound NCX1741 are the same as PDEs inhibitors, not all the PDEs inhibitors have obvious curative effect on the Alzheimer disease.

Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, and the curative effect of the crystal form H on the treatment effect of AD model mice is the best by oral administration, and the curative effect is obviously higher than that of other crystal forms; the citric acid alidenafil crystal form A tablet prepared by the method has a remarkably better treatment effect on AD mice than a tablet prepared by a CN114028403A prescription process; the treatment effect of the crystal form H tablet prepared by the method on AD model mice is obviously better than that of the crystal form H tablet prepared by a CN112745323B prescription process.

3) Pathological examination

a) Hippocampus japonicus region structure

And (3) fixing the hippocampal tissues of mice in a normal control group, an AD model blank control group, a positive control group, a compound IS00384 group, a compound NCX1741 group and a citric acid idenafil crystal form H group by 4% paraformaldehyde, dehydrating, embedding paraffin, slicing, staining the tissue slices by HE, and observing pathological morphological changes of skin tissues by using microscopy.

FIGS. 3 (a) and 3 (b) show that the hippocampal structure of normal mice group is normal, and no obvious damage to nerve cells is seen; the structure of the hippocampus of the blank group of the AD model has no obvious change, abnormal nerve cells can be seen only in a local area, the cells are subjected to solid shrinkage and necrosis, cavities appear around the cells, the blood vessels of the hippocampus are obvious in congestion, and the female is obvious in lesion compared with the male; the necrotic cells in the hippocampus of the positive control group are obviously reduced, and the hyperemia of small blood vessels is improved.

In the citric acid alidenafil crystal form H group, the damage of hippocampal nerve cells in each female dose group has a certain improvement phenomenon, the low, medium and high female doses have a certain treatment effect, the three groups have no obvious difference, the nerve cell nucleus solidification phenomenon still exists in a sample in each dose group, and the treatment effect is relatively better in comparison with the low and medium male doses.

In the compound IS00384 group, local damage of hippocampus can be still seen in the low-dose group, no obvious damage IS seen in the hippocampal nerve cells in the female middle and high-dose groups, and the damage to the nerve cells IS obvious in individual samples of male low and middle doses.

The samples of the compound NCX1741 group, the low dose group and the medium dose group still have a small amount of cell nucleus shrinkage; the congestion of small blood vessels is improved as a whole.

In conclusion, the citric acid alidenafil crystal form H group hippocampal nerve cells have the best improvement effect, and are superior to the compound IS00384 group and the compound NCX1741 group.

b) A beta-42 and NEP gene expression in hippocampus

Amyloid beta (a β) is a polypeptide characterized by 39-43 amino acid residues, is the major component of amyloid plaques in the brains of alzheimer's patients, and is formed by the cleavage of Amyloid Precursor Protein (APP). A β -42 is considered to be the most pathogenic subtype, and A β -42 tends to form plaque-like deposits. Because the deposited A beta fibers have neurotoxicity, the deposited A beta fibers can cause neuron degeneration and even apoptosis, increase the activity of brain colloid cells, stimulate in-vivo inflammatory reaction and the like, cause a series of pathological injuries around the A beta fibers and finally form the structure of senile plaques. Neprilysin (NEP) is the major a β degrading enzyme in the brain, and NEP is down-regulated in alzheimer's patients, so the amount of NEP directly affects a β deposition.

The expression levels of hippocampal Α β -42 and NEP in AD mice were determined by immunohistochemistry, and the results are shown in table 12:

expression level of A.beta.42:

as can be seen from Table 12, the mean optical density (IOD/Area) value of A.beta.42 was 0.110 for the normal control group of 6 mice and 0.191 for the AD model blank group, which is an increase of 73.6%; the positive control group is 0.136, which is reduced by 28.8% compared with the blank control group of the AD model. In each administration group, the average optical density value of Abeta-42 of the citric acid alidenafil crystal form H group is the lowest, and the low dose group, the medium dose group and the high dose group are respectively reduced by 19.9 percent, 26.2 percent and 31.4 percent compared with the AD model group, and are all obviously reduced (p is less than 0.01), wherein the high dose group is superior to the positive control group.

Sildenafil citrate, avanafil, the compound IS00384 and the compound NCX1741 were reduced by 17.3%, 17.8%, 5.8% and 6.3% in the low dose group, 20.9%, 22.0%, 9.9% and 17.8% in the medium dose group, and 25.7%, 27.7%, 15.2% and 20.9% in the high dose group, respectively, compared with the AD model group. The reduction of the compound IS00384 IS the least, and the difference of each dosage group IS 14.1 percent, 16.2 percent and 16.2 percent respectively compared with the citric acid alidenafil H group, and the difference IS obvious. The dose groups of the compound NCX1741 are 13.6 percent, 8.4 percent and 11.5 percent lower than the citric acid alidenafil H group respectively.

The difference of different citric acid alidenafil crystal forms is large, the reduction of the crystal form H group is maximum, the reduction of the crystal form A group is minimum, the crystal form A low dose group, the crystal form A medium dose group and the crystal form A high dose group are respectively 14.7 percent, 19.4 percent and 18.3 percent lower than the crystal form H group, and the crystal form V dose groups are respectively 10.5 percent, 13.6 percent and 15.2 percent lower than the crystal form H group, and are all obviously reduced.

The dosage of different crystal form A (CN 114028403A prescription process) groups is respectively reduced by 1.6%, 3.1% and 6.8% compared with the AD model group, the difference of the same crystal form H group is respectively 18.3%, 23.0% and 24.6%, and the difference of the same crystal form A (prescription process) group is respectively 3.7%, 3.7% and 6.3%, which have obvious difference.

The average total mileage of different dose groups of the crystal form H (CN 112745323B prescription process) is respectively reduced by 7.1%, 13.8% and 16.6% compared with that of an AD model group, and is respectively reduced by 12.8%, 12.4% and 14.8% compared with that of the crystal form H group of the prescription process of the application, and the difference is obvious.

From the expression level of the Abeta-42, different PDE5 inhibitors including sildenafil citrate, avanafil citrate, the IS00384 compound, the NCX1741 compound and the Aidenafil citrate in each crystal form have the largest reduction ratio in the Aidenafil citrate crystal form H group compared with AD model mice, which IS obviously higher than other experimental groups, and the Addenafil citrate crystal form H has the best effect of clearing Abeta-42 after administration. The beta-42 expression of the citric acid alidenafil crystal form H can be reduced by 31.4 percent at most, while the beta-42 expression of the compound IS00384 and the compound NCX1741 are reduced by 15.2 percent and 19.9 percent at high dose, the effect IS obviously lower than that of the citric acid alidenafil crystal form H, and the reduction degree of the beta-42 expression of other administration groups IS also lower than that of the citric acid alidenafil crystal form H.

Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, wherein the crystal form H group has the best effect and the highest reduction ratio which is obviously higher than other crystal form groups. The dosage of the crystal form A and the crystal form V is respectively 18.3 percent lower than that of the crystal form H and 15.2 percent lower than that of the crystal form H under the high dosage, and the reduction ratio of other crystal form groups is also lower than that of the crystal form H.

The citric acid alidenafil crystal form A tablet prepared by the prescription process is obviously higher than the tablet prepared by the CN114028403A prescription process in the reduction ratio, and the difference between the citric acid alidenafil crystal form A tablet and the tablet is up to 6.3 percent.

The citric acid alidenafil crystal form A tablet prepared by the prescription process is obviously higher than the tablet prepared by the CN112745323B prescription process in the reduction ratio, and the difference between the citric acid alidenafil crystal form A tablet and the tablet is up to 14.8 percent.

NEP expression level:

average optical density (IOD/Area) values for NEP, 0.156 for normal control, 0.135 for ad model blank control, and 0.150 for positive control. NEP of each administration group is increased, wherein the NEP of the citric acid alidenafil crystal form H group has the highest rising value, the NEP values of the low dose group, the medium dose group and the high dose group can respectively reach 0.147, 0.150 and 0.154, and the NEP is obviously increased compared with the AD model group (p is less than 0.01), wherein the medium dose group is equivalent to the positive control group, and the high dose group is higher than the positive control group. The NEP expression quantity of each dose group is respectively increased by 8.9 percent, 11.1 percent and 14.1 percent compared with that of the AD model group.

The sildenafil citrate, the avanafil, the IS00384 compound and the NCX1741 compound are respectively increased by 3.7%, 3.0%, 0.7% and 0.7% in the low-dose group, 6.7%, 5.9%, 7.4% and 3.7% in the medium-dose group, 10.4%, 11.1%, 10.4% and 8.9% in the high-dose group, and the difference of the increasing ratio IS 3.0% -8.1% in the comparison with the Aidenafil citrate crystal form H group.

The NEP value increment of different crystal form groups of the citric acid alidenafil has some differences, wherein the increment is the most under each dosage of the crystal form H group, and compared with the AD model group, the increment ratios of the low dose group, the medium dose group and the high dose group are respectively 8.9 percent, 11.1 percent and 14.1 percent. In other crystal form groups, the increment of the crystal form A is the least, and different dosage groups are respectively 5.2 percent, 7.4 percent and 8.1 percent less than those of the crystal form H group.

Different dosage groups of the crystal form A (CN 114028403A prescription process) are respectively increased by 0.7%, 0.7% and 1.5% compared with the AD model group, the difference of the same crystal form H group is respectively 8.1%, 10.4% and 12.6%, and the difference of the same crystal form A (the prescription process of the application) group is respectively 3.0%, 3.0% and 4.4%, which have obvious difference.

Different dosage groups of the crystal form H (CN 112745323B prescription process) are respectively increased by 1.9%, 7.3% and 10.6% compared with the AD model group, and are respectively reduced by 7.0%, 3.8% and 3.5% compared with the crystal form H group in the prescription process of the application, and the difference is obvious.

According to NEP expression, different PDE5 inhibitors comprise sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein the increase ratio of the crystal form H group of the sildenafil citrate relative to the blank group of the AD model IS the largest, the maximum can reach 14.1%, and the increase ratio IS obviously higher than that of other experimental groups. The low dose group of the compound IS00384 and the compound NCX1741 IS 8.1 percent lower than the citric acid alidenafil crystal form H group, and other tested drug groups are also lower than the citric acid alidenafil crystal form H group.

Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, wherein the crystal form H group has the best effect, and the increase ratio is obviously higher than that of other crystal form groups. The increment of the crystal form A is the least, the crystal form A is respectively 5.2 percent, 7.4 percent and 8.1 percent lower than that of the crystal form H under low dose, medium dose and high dose, and other crystal form groups are also lower than those of the crystal form H.

The citric acid alidenafil crystal form A tablet prepared by the prescription process is increased by 4.4 percent in proportion than the tablet prepared by the CN114028403A prescription process, and the difference is obvious; the citric acid alidenafil crystal form H tablet prepared by the prescription process is 7.0 percent higher than the tablet prepared by the CN112745323B prescription process, and has obvious difference.

From the results of A beta-42 and NEP data, PDE5 inhibitors can reduce the expression level of A beta-42 and increase the expression level of NEP, thereby reducing the accumulation of A beta and improving the learning, memory, cognitive ability and the like of AD model mice. Different PDE5 inhibitors including sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, wherein the sildenafil citrate has the best effect in the H group, and compared with the AD model group, the A beta-42 reduction ratio and the NEP increase ratio are the highest and are obviously higher than those of other PDE5 inhibitors. The A beta-42 reduction ratio of the compound IS00384 in the low dose group, the medium dose group and the high dose group has differences of 14.1%, 16.2% and 16.2% compared with the citric acid alidenafil H, and the compound NCX1741 in the low dose group, the medium dose group and the high dose group has differences of 13.6%, 8.4% and 11.5% compared with the citric acid alidenafil H. The NEP increasing proportion of the compounds IS00384 and NCX1741 in the low dose group differed by 8.1% compared with the citric acid alidenafil H group. Therefore, it is known that, although both PDE5 inhibitors are effective, their effects on the expression of a β -42 and NEP are greatly different, and thus their therapeutic effects on alzheimer's disease are greatly different.

The citric acid alidenafil has different crystal forms, including crystal forms A, B, C, D, O, V and H, wherein the A beta-42 reduction ratio and the NEP increase ratio of the crystal form H group are the highest and are obviously higher than those of other crystal form groups. The Abeta-42 reduces the difference between the high-dose group of the crystal form A and the high-dose group of the crystal form V and the high-dose group of the crystal form H by 18.3 percent and 15.2 percent, and the NEP increases the ratio, namely, the middle-dose group and the high-dose group of the crystal form A are respectively 7.4 percent lower and 8.1 percent lower than those of the crystal form H. Therefore, the tablets prepared from different crystal forms of the citric acid alidenafil have great difference in the expression of Abeta-42 and NEP, and have obvious difference in the treatment effect of the Alzheimer disease.

The A beta-42 reduction and NEP increase ratio of the citric acid alidenafil crystal form A tablet prepared by the prescription process are higher than those of the citric acid alidenafil crystal form A tablet prepared by the CN114028403A prescription process. Wherein the decrease proportion of the Abeta-42 is different from 6.3 percent at most, the increase proportion of the NEP is different from 4.4 percent at most, and the difference is obvious. The citric acid alidenafil crystal form H tablet prepared by the prescription process has higher Abeta-42 reduction and NEP increase ratio than the citric acid alidenafil crystal form H tablet prepared by the prescription process of CN 112745323B. Wherein the decrease ratio of Abeta-42 is 14.8% at the highest, and the increase ratio of NEP is 7.0% at the highest.

Therefore, the treatment effect of the tablets prepared by the prescription process of the application on the Alzheimer disease is obviously better than that of the tablets prepared by the prescription processes of CN114028403A and CN 112745323B.

c) Influence of drug on SOD (superoxide dismutase) activity and MDA (malondialdehyde) content in mouse serum

SOD is one of the important antioxidant enzymes for eliminating free radicals in the body, and is a main enzyme of an antioxidant system in the body. MDA is used as an intermediate product in the lipofuscin formation process, the content of MDA can indirectly reflect the generation condition of free radicals in a body and the lipid peroxidation degree of tissue cells of the body, and oxidative damage is an important pathogenesis of Alzheimer's disease. The mouse SOD activity and MDA content data are shown in Table 13.

SOD activity:

the data in table 13 show that the average SOD activity value is 56.148 in normal mice, 49.381 in AD model mice is reduced by 12.1%, the SOD activity values of each administration group are improved compared with the AD model group, wherein the SOD activity value with the largest increase range is the crystalline form H group of the citric acid aildenafil, the SOD activity values under different doses are 51.389U/mL, 52.329U/mL and 53.565U/mL, and are obviously improved (p is less than 0.01) compared with the AD model group, the SOD activity values under different doses are respectively improved by 4.1%, 6.0% and 8.5%, and the high dose group is equivalent to the positive control group.

Sildenafil citrate, avanafil, the compound IS00384 and the compound NCX1741 in the low dose groups were increased by 3.1%, 1.3%, 1.5% and 1.0% respectively, in the medium dose groups by 4.6%, 3.3%, 1.5% and 1.3% respectively, and in the high dose groups by 6.0%, 5.3%, 3.3% and 1.3% respectively, compared with the AD model control group. Compared with the citric acid alidenafil crystal form H group, the compound IS00384 and the compound NCX1741 have larger differences, the differences of the medium dose group are respectively 4.5 percent and 4.7 percent, and the differences of the high dose group are respectively 5.2 percent and 7.1 percent.

The difference between the citric acid alidenafil other crystal form group and the crystal form H group is the crystal form A group with the largest difference, the differences between the low dose, the medium dose and the high dose are respectively 3.7 percent, 4.7 percent and 5.2 percent, and the difference between the other crystal form group and the crystal form H group is 0.3 to 3.9 percent.

Different dosage groups of the crystal form A (CN 114028403A prescription process) are respectively increased by 0.2%, 0.3% and 0.4% compared with the AD model group, the difference of the isomorphous H group is respectively 3.8%, 5.6% and 8.0%, and the difference of the isomorphous A (prescription process of the application) group is respectively 0.1%, 0.9% and 2.8%, which have obvious difference.

Different dosage groups of the crystal form H (CN 112745323B prescription process) are respectively increased by 0.6 percent, 1.7 percent and 3.3 percent compared with the AD model group, and the differences of the crystal form H group and the AD model group are respectively 3.5 percent, 4.3 percent and 5.2 percent, and the obvious differences are provided.

According to the determination result of the SOD activity value, different PDE5 inhibitors comprising sildenafil citrate, avanafil citrate, a compound IS00384, a compound NCX1741 and crystal forms of the sildenafil citrate have the highest rising proportion of the SOD activity value of an Aidenafil citrate crystal form H group and can reach 8.5 percent compared with an AD model mouse, and the result shows that the Aidenafil citrate crystal form H has the best treatment effect on the AD model mouse and IS obviously superior to other administration groups. The SOD activity values of the compound IS00384 and the compound NCX1741 are increased by 5.2 percent and 7.1 percent at most compared with the citric acid alidenafil crystal form H group, and the increasing ratios of other administration groups are lower than the citric acid alidenafil crystal form H group.

The citric acid alidenafil has different crystal forms including the crystal forms A, B, C, D, O, V and H, wherein the SOD activity value of the crystal form H group is increased in the largest proportion, the effect is the crystal form A group with the worst effect, each dosage is 3.7 percent, 4.7 percent and 5.2 percent lower than that of the crystal form H group, and other crystal form groups are also lower than those of the crystal form H group.

The SOD activity value increase proportion of the citric acid alidenafil crystal form A tablet prepared by the prescription process is obviously higher than that of the tablet prepared by the CN114028403A prescription process.

The citric acid alidenafil crystal form H tablet prepared by the prescription process has a SOD activity value increase ratio which is obviously higher than that of the tablet prepared by the CN112745323B prescription process.

MDA content:

the MDA content detection result shows that the average value of OD of a normal mouse is 1.834, the average value of an AD model mouse is 2.124, the rise amplitude is 15.8 percent, and the MDA content of the AD mouse is greatly increased. Compared with an AD model group, the average value of MDA content OD of each administration group is reduced, wherein the citric acid alidenafil crystal form H group is the lowest, the different dosage groups are 1.944, 1.902 and 1.900 respectively, the dosage groups are reduced by 8.5%, 10.5% and 10.5% respectively compared with the AD model group, and the dosage groups are obviously reduced (p is less than 0.01) compared with the AD model group, wherein the medium dosage group and the high dosage group are lower than a positive control group.

In other administration groups, the differences between the compound IS00384 group and the NCX1741 group and the citric acid alidenafil crystal form H group are larger, the differences between the compound IS00384 group and the citric acid alidenafil crystal form H group are respectively 6.1%, 2.6% and 4.5% under each dosage, and the differences between the compound NCX1741 group are respectively 7.0%, 5.8% and 5.8%.

In the other crystal form groups of the citric acid alidenafil, the difference between the crystal form A group and the crystal form H group is the largest, the difference is respectively 5.3 percent, 4.7 percent and 4.8 percent under each dosage, and the difference between the other crystal form groups is 1.3 to 3.9 percent.

The dosage groups of the crystal form A (CN 114028403A prescription process) are respectively reduced by 1.6%, 2.4% and 3.9% compared with the AD model group, the difference of the same crystal form H group is respectively 6.9%, 8.1% and 6.7%, and the difference of the same crystal form A (prescription process) group is respectively 1.5%, 3.4% and 1.9%, which have obvious difference.

The dosage groups of the crystal form H (CN 112745323B prescription process) are respectively reduced by 5.8%, 8.3% and 9.1% compared with the AD model group, and the differences of the crystal form H and the AD model group are respectively 2.6%, 2.2% and 1.4%, and the difference is obvious.

According to the MDA content detection result, different PDE5 inhibitors including sildenafil citrate, avanafil citrate, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate have the highest MDA content reduction ratio in the crystal form H group of the sildenafil citrate compared with an AD model mouse, and are obviously higher than those in other administration groups. Compared with the citric acid alidenafil crystal form H group, the differences between the compounds IS00384 and NCX1741 can reach 6.1 percent and 7.0 percent at most, and the differences are obvious.

The citric acid alidenafil has different crystal forms, including crystal forms A, B, C, D, O, V and H, wherein the MDA content reduction ratio of the crystal form H group is the largest and can reach more than 10%, and the crystal form A group with the lowest reduction ratio.

The citric acid alidenafil crystal form A tablet prepared by the prescription process has the MDA content reduction ratio which is obviously higher than that of the tablet prepared by the CN114028403A prescription process, and the difference between the citric acid alidenafil crystal form A tablet and the tablet can reach 3.4 percent at most.

The citric acid alidenafil crystal form H tablet prepared by the prescription process has the MDA content reduction ratio which is obviously higher than that of the tablet prepared by the CN112745323B prescription process, and the difference between the citric acid alidenafil crystal form H tablet and the tablet can reach 2.6 percent at most.

From the above, the PDE5 inhibitor can increase the activity of SOD and reduce the content of MDA, thereby improving the symptoms of AD model mice. Different PDE5 inhibitors, including sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and various crystal forms of the sildenafil citrate, compared with an AD model group, the SOD activity increasing ratio and the MDA content reducing ratio of the crystal form H of the sildenafil citrate are the highest. The SOD activity value increases in proportion, and the difference between the compound IS00384 and the compound NCX1741 in the high-dose group can reach 5.2 percent and 7.1 percent compared with the citric acid alidenafil crystal form H group. The MDA content IS reduced, and the difference between the compound IS00384 and the compound NCX1741 IS up to 6.1 percent and 7.0 percent compared with the citric acid alidenafil crystal form H group. This indicates that not all PDE5 inhibitors have the same effect in increasing SOD activity and reducing MDA, and thus the efficacy on alzheimer's disease will vary greatly.

Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, wherein the crystal form H has the best effect of improving SOD activity and reducing MDA content, the crystal form A group is the worst, and the difference between the crystal form A group and the crystal form H group is more than 5 percent, and the difference is obvious. Therefore, the crystalline form of citric acid alidenafil is also very important for the therapeutic effect.

The citric acid alidenafil crystal form A and crystal form H tablets prepared by different prescription processes have obvious difference on the influences of SOD activity and MDA content, the SOD activity can be obviously increased and the MDA content can be obviously reduced by using the prescription process of the application, and the effect is obviously higher than that of the crystal form A tablets prepared by the CN114028403A prescription process and the crystal form H tablets prepared by the CN112745323B prescription process.

Example 5: in vitro Activity assay

The results of the drug effect evaluation show that the symptoms of the AD model mice are greatly improved by different PDE5 inhibitors. Of the different PDE5 inhibitors, including sildenafil citrate, avanafil, the compound IS00384, the compound NCX1741 and the citric acid ailnafil, the effect of citric acid ailnafil, especially form H, on the treatment of alzheimer's disease was the best, significantly higher than the other inhibitors.

In this regard, the inventors speculate that this may be related to the PDE5 inhibition and selectivity of different PDE5 inhibitors. To verify this, the inventors further examined the inhibitory effect of different PDE5 inhibitors on PDE enzymes, and carried out in vitro PDE enzyme activity inhibition tests on 5 tested drugs, i.e., compound IS00384, compound NCX1741, crystalline form H of sildenafil citrate, sildenafil citrate and avanafil. The results show that the citric acid alidenafil crystal form H has highest inhibitory activity to PDE5A and half Inhibitory Concentration (IC) ₅₀ ) The concentration of the inhibitor is 0.66nM, and the other 4 inhibitors are 5.5 times, 15.9 times, 2.0 times and 3.5 times of the citric acid alidenafil crystal form H respectively. The citric acid alidenafil crystal form H has the strongest selectivity on PDE5, and the inhibition effect is 1515 times that of other PDE, namely the selectivity on PDE5 is as high as 1515 times. Therefore, among the different PDE5 inhibitors, including sildenafil citrate, avanafil, compound IS00384, compound NCX1741, and alidenafil citrate, alidenafil citrate inhibits PDE5 most strongly, has the highest selectivity, and IS significantly higher than other inhibitors.

1. Source of PDE

Except that PDE6 was purchased, all isoforms were self-purified.

2. PDE expression and purification Process

Using PDE5A as an example, the PDE protein expression and purification process is described.

1) Plasmid transformation

mu.L of the recombinant plasmid PET15b-PDE5A (535-860) was transferred into thawed E.coli competent cells (E.coli) BL21 (codon), mixed by inversion and then left on ice for 30min. After heat shock at 42 ℃ for 90s, it was placed on ice for 3min. Adding 700 μ L LB medium without antibiotic, and incubating at 37 deg.C for 50min. Centrifuging for 2min, removing 400 μ L of supernatant, blowing the rest liquid with pipette, mixing, coating on plate, and incubating at 37 deg.C overnight.

2) Bacterial culture

Monoclonal colonies were picked and cultured overnight in 100mLLB medium while the strain was preserved at-80 ℃. The bacterial liquid is added into 500mL of corresponding culture medium according to the proportion of 1:8, and 0.4% of glucose and antibiotics are added at the same time, and the mixture is cultured by a shaking table at the constant temperature of 37 ℃. Adding 0.1mMIPTG to the required density of the thalli to induce the over-expression of the target protein, setting the low temperature, culturing for 40-48 hours, centrifuging for 30min, and collecting the thalli.

3) Bacterial lysis and disruption

The bacteria were resuspended in nickel column lysis buffer at a ratio of bacteria weight to lysate volume of 1:6. After the bacteria were resuspended until no obvious clumps were present, the cells were placed in an ice box for ultrasonication and the bacteria were again disrupted again and again by a cell disruptor for 2 times under the protection of 4 ℃ condensate water. Finally, the mixture is centrifuged for 30min at 4 ℃ by a high-speed centrifuge, and the supernatant is removed.

4) Nickel column affinity chromatography

Adding the supernatant into a well-balanced nickel affinity column, and slowly flowing out after the liquid and the filler are fully mixed. Washing with Ni-bufferA and Ni-bufferB, eluting the target protein with Ni-bufferC C, and collecting by tube. The concentration of the collected liquid was measured with an ultraviolet spectrophotometer, and samples were prepared for SDS-PAGE electrophoresis, identified and the high concentration collection tubes were pooled.

5) Obtaining the target protein by enzyme digestion

The recombinant protein purified by the nickel column needs to be digested by Thrombin to remove His tags. The pooled proteins were added to Thrombin together with 2.5mM CaCl ₂ After mixing, 1mM EDTA was added to terminate the reaction.

6) Anion exchange chromatography

Adding the protein after enzyme digestion into a well-balanced column. Eluting the target protein by using buffer solution, and collecting by tubes. The concentration of the eluate in each tube was determined and SDS-PAGE was run to identify the protein purity in each tube.

7) Molecular sieve chromatography

Enriching a protein sample obtained after anion exchange column chromatography, adding the concentrated protein solution into a loading column, and setting a sample introduction program and elution speed time to ensure that the column pressure is less than 0.25Mpa. Eluting with molecular sieve buffer solution, and collecting eluate in different tubes according to the peak value of the real-time UV280 curve of the instrument. The concentration of the eluate in each tube is measured, and the eluate with high concentration should be subjected to purity identification by SDS-PAGE. And finally, concentrating the target protein eluent with higher purity to a protein concentration of about 10mg/mL, and subpackaging.

Other methods of PDE protein expression and purification are as above.

3. Enzyme Activity assay

The PDE protein was removed from the cryorefrigerator and thawed on ice. Radioisotope substrate [8- ³ H]cAMP and [8- ³ H]Diluting the raw solution of the-cGMP by 100 times with purified water to be used as mother solution, PDE5A according to 54. Mu.L of PDE5AssayBuffer and 4. Mu.L of [ alpha ], [ ³ H]-ratio of cGMP mother liquors the substrate mixture is formulated. The PDE protein was diluted to a gradient concentration with the corresponding AssayBuffer.

After preparing each reagent, 58. Mu.L of the prepared substrate mixture, 2. Mu.L of the test drug or DMSO, and 40. Mu.L of the diluted gradient protein were sequentially added to the reaction tube, wherein the control group was AssayBuffer replaces enzyme solution, stands for reaction for 15 minutes at room temperature, and then 200 mu L0.2MZnSO is added in sequence ₄ And 200. Mu. L0.2MBa (OH) ₂ The solution is vortexed, fully mixed and then centrifuged at high speed. And immediately absorbing the supernatant after centrifugation, transferring the supernatant into a scintillation tube added with scintillation fluid in advance, and performing vortex oscillation to fully and uniformly mix the supernatant and the scintillation fluid. Sequentially placing the test pieces into a test rack, setting a program, and measuring the reading by using a liquid scintillation counter.

The activity data of the target protein at 7-9 different inhibitor concentrations were determined as described above. Each reaction was repeated at least 3 times and averaged. The rate of hydrolysis of the protein in the inhibitory activity of the test compound on the enzyme was calculated using microsoft excel software (hydrolysis = 1-CPMDMSO/CPMctrl), and the conversion of the protein in the presence of each concentration of the compound was calculated (conversion = 1-cpminihibitor/CPMctrl). From the rate of hydrolysis of the protein and its conversion in the presence of the compound to be determined, the relative activity of the protein at the inhibitor concentration (relative activity = conversion/hydrolysis) can be calculated, giving the inhibition of the protein by this compound (inhibition = 1-relative activity). The data were processed using GraphPadPrism software and IC was calculated by fitting the inhibition data obtained for 7-9 concentration points using non-linear regression ₅₀ 。

4. Results of the experiment

As can be seen from Table 14, crystalline form H of Aidinafei citrate has the highest inhibitory activity against PDE5A and the half Inhibitory Concentration (IC) ₅₀ ) IS 0.66nM, the compound IS00384, the compound NCX1741, sildenafil citrate and avanafil are 5.5 times, 15.9 times, 2.0 times and 8.0 times of the citric acid of the crystalline form H of the aildenafil respectively; the citric acid alidenafil crystal form H has the highest inhibitory activity to PDE6C, IC ₅₀ The value was 9.1nM, compound IS00384, compound NCX1741, sildenafil citrate and avanafil are 4.5 times, 59 times, 1.9 times and 48 times, respectively, that of the sildenafil citrate.

The 5 tested drugs have low inhibitory effect on other PDE subtypes, and IC ₅₀ The value is 1. Mu.M or more. The 5 tested medicines are shown to have selectivity on inhibiting different PDE subtypes, the inhibiting effect of citric acid alidenafil crystal form H on PDE5A and PDE6C IS 1515 times and 110 times of that of other PDE subtypes respectively, the inhibiting effect of compound IS00384 IS 278 times and 25 times of that of compound NCX1741 IS 95 times and 1.9 times of that of compound, the inhibiting effect of citric acid sildenafil IS 770 times and 59 times of that of compound, and the inhibiting effect of avanafil IS 190 times and 2.3 times of that of compound. The citric acid alidenafil crystal form H has the highest selectivity on PDE 5.

From the data, different PDE5 inhibitors comprise citric acid alidenafil crystal form H, a compound IS00384, a compound NCX1741, citric acid sildenafil, avanafil and the like, wherein the citric acid alidenafil crystal form H has the strongest inhibition effect on PDE5 and the highest selectivity. The inhibitor has inhibiting effect and selectivity on PDE5, and the crystal form H of the citric acid alidenafil can respectively reach 15.9 times of that of a compound NCX1741 and 8.0 times of that of avanafil.

This may be associated with the above-demonstrated significantly improved inhibitory effect of crystalline form H of idenafil citrate on alzheimer's disease: the citric acid alidenafil crystal form H has the best effect on the learning and memory ability and cognitive ability of AD model mice and the recovery effect on symptoms such as anxiety, dysphoria and the like, and is obviously higher than other PDE5 inhibitors.

Example 6: stability study of Aidenafil citrate tablets

The efficacy evaluation shows that in various crystal forms of the citric acid alidenafil, including the crystal forms A, B, C, D, O, V and H, the citric acid alidenafil crystal form H has the best effect on treating the Alzheimer's disease and is obviously higher than other crystal forms.

The inventor speculates that the difference of the symptom improvement effect of the tablets of the citric acid alidenafil in different crystal forms on AD model mice is probably related to the difference of the properties of the tablets prepared in different crystal forms. In this regard, the inventors further examined the differences in stability of different crystalline form tablets. The result shows that the tablet of the crystal form H has the highest stability, and the content can reach 98.10 percent which is obviously higher than that of other crystal form tablets.

The citric acid alidenafil crystal form H, crystal form a, crystal form B, crystal form C, crystal form D, crystal form O and crystal form V tablets prepared in example 2 were subjected to an accelerated stability test in a constant temperature and humidity chamber for 6 months. The test conditions are as follows: the temperature is 40 +/-2 ℃, the relative humidity is 75 +/-percent (RH), and the samples are respectively taken in 0, 1, 2, 3 and 6 months for content detection. The results are as follows:

as can be seen from Table 15, the content of the tablets prepared from different crystal forms of the citric acid alidenafil is greatly different after an accelerated test for 6 months, wherein the crystal form H tablet is the most stable, the content of the tablet after the accelerated test for 6 months is 98.10%, the stability of the crystal form A tablet is the worst, the content of the tablet is only 90.37%, and the content of the other crystal form tablets is lower than that of the crystal form H. The content of the crystal form A tablet prepared by the CN114028403A prescription process is 88.12 percent after the accelerated test is 6 months, and the crystal form A tablet is 4.25 percent lower than the crystal form A tablet prepared by the prescription process and 11.98 percent lower than the crystal form H tablet. The content of the crystal form H tablet prepared by the CN112745323B prescription process is 94.57% after 6 months of accelerated test, and is 3.53% lower than that of the crystal form H tablet prepared by the prescription process.

According to stability test results, the citric acid alidenafil tablets with different crystal forms comprise a crystal form H, a crystal form A, a crystal form B, a crystal form C, a crystal form D, a crystal form O and a crystal form V, wherein the stability of the crystal form H tablet is the highest and is obviously higher than that of the other crystal forms. Compared with the crystal form H, the content of other crystal form tablets is obviously reduced after an accelerated test, and the reduction amplitude is 4.43-7.88%. The content of the crystal form A tablet prepared by the CN114028403A prescription process is obviously lower than that of the crystal form A tablet prepared by the prescription process. The content of the crystal form H tablet prepared by the CN112745323B prescription process is obviously lower than that of the crystal form H tablet prepared by the prescription process.

Thus, the best effect of crystalline form H of citric acid on the treatment of alzheimer's disease may be related to the strongest stability of form H among the numerous forms. After the AD model mouse is treated by oral administration, the symptoms are greatly improved, and the treatment effect of other crystal form tablets on the AD model mouse is obviously lower than that of the crystal form H due to the reduction of active pharmaceutical ingredients.

In a word, the drug effect evaluation result of the application shows that the oral citric acid alidenafil crystal form H tablet has obvious improvement effect on the learning and memory ability, the cognitive ability and the dysphoria state of the APPswe/PS1 delta 9 transgenic AD mice. Different PDE5 inhibitors including sildenafil citrate, avanafil, a compound IS00384, a compound NCX1741 and an alidenafil citrate in various crystal forms have greatly different treatment effects on AD model mice, and the crystal form H of the alidenafil citrate has the best effect and IS obviously superior to other PDE5 inhibitors. Different citric acid alidenafil crystal form tablets comprise crystal forms A, B, C, D, O, V and H, wherein the crystal form H has the best effect of improving the symptoms of AD model mice, and the curative effect is obviously better than that of other crystal forms. The citric acid alidenafil crystal form A tablet prepared by the prescription process has a remarkably better effect than the crystal form A tablet prepared by the CN114028403A prescription process. The citric acid alidenafil crystal form H tablet prepared by the prescription process has a remarkably better effect than the crystal form H tablet prepared by the CN112745323B prescription process.

The pathological analysis result shows that the citric acid alidenafil crystal form H has certain improvement phenomenon on the damaged nerve cells in the hippocampal region of the AD model mouse, and the effect is better than that of other PDE5 inhibitors; the inhibitor is better than other PDE5 inhibitors in reducing A beta-42 expression and increasing NEP expression, and is better than other crystal forms of citric acid alidenafil; the citric acid alidenafil crystal form H is also superior to other PDE5 inhibitors and other citric acid alidenafil crystal forms in the aspects of increasing SOD activity and reducing MDA content; the citric acid alidenafil crystal form A tablet prepared by the prescription process is obviously better than the crystal form A tablet prepared by the CN114028403A prescription process in the aspects of increasing SOD activity and reducing MDA content. The citric acid alidenafil crystal form H tablet prepared by the prescription process is obviously better than a crystal form A tablet prepared by a CN112745323B prescription process in the aspects of increasing SOD activity and reducing MDA content.

The effect of the citric acid alidenafil, especially form H, on the treatment of alzheimer's disease is the best, significantly higher than that of other inhibitors, which may be related to the PDE5 inhibitor's PDE5 inhibitory effect and selectivity. In vitro activity detection results show that the inhibition effect of the citric acid alidenafil crystal form H on PDE5 and the selectivity of the citric acid alidenafil crystal form H on PDE5 inhibition are obviously higher than those of other inhibitors.

Meanwhile, in various crystal forms of the citric acid alidenafil, including the crystal forms A, B, C, D, O, V and H, the citric acid alidenafil crystal form H has the best effect on treating the Alzheimer's disease and is obviously higher than other crystal forms, which is probably related to the stability difference of different crystal form tablets. In fact, the stability of the citric acid alidenafil crystal form H tablet is greatly higher than that of other crystal forms, and the content of the crystal form H tablet is obviously higher than that of other crystal forms.

The citric acid alidenafil crystal form A tablet prepared by the prescription process has obviously better effect on treating Alzheimer's disease than the crystal form A tablet prepared by the prescription process of CN 114028403A. The citric acid alidenafil crystal form H tablet prepared by the prescription process has obviously better effect on treating Alzheimer's disease than the crystal form A tablet prepared by the prescription process of CN 112745323B.

Therefore, the invention provides a medicine for effectively preventing and treating Alzheimer disease, namely the citric acid alidenafil crystal form H. The citric acid alidenafil crystal form H can be used for treating the Alzheimer disease with high stability and effectively, has a treatment effect remarkably superior to other PDE5 inhibitors and other citric acid alidenafil crystal forms, and has a good application prospect in preparation of medicaments for preventing and treating the Alzheimer disease.

Claims

1. A tablet containing Aidinafil citrate is prepared from the following raw and auxiliary materials in parts by weight: 85-95 parts of citric acid aildenafil, 147-160 parts of pregelatinized starch, 85-95 parts of calcium hydrophosphate, 5-12 parts of added croscarmellose sodium, 15-28 parts of polymethyl methacrylate, 100-120 parts of 70% ethanol, 3-5 parts of sodium stearyl fumarate, 7-13 parts of a film coating premix and 50-65 parts of purified water.

2. The tablet of claim 1, wherein the crystalline form of Aidenafil citrate is selected from form A, form B, form C, form D, form O, form V or form H.

3. The tablet of claim 2, wherein the crystalline form of Aidinafei citrate is form H.

4. The tablet of claim 1, which is prepared from raw and auxiliary materials selected from the following formulas in parts by weight:

prescription 1: 92 parts of Aidinafil citrate, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 9 parts of internally added cross-linked sodium carboxymethyl cellulose, 9 parts of externally added cross-linked sodium carboxymethyl cellulose, 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 2: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 5 parts of internally added croscarmellose sodium, 5 parts of externally added croscarmellose sodium, 15 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 3: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 12 parts of internally added croscarmellose sodium, 12 parts of externally added croscarmellose sodium, 25 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

and (4) prescription: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of internally added croscarmellose sodium, 8 parts of externally added croscarmellose sodium, 16 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 5: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of internally added croscarmellose sodium, 8 parts of externally added croscarmellose sodium, 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 6: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 8 parts of internally added croscarmellose sodium, 12 parts of externally added croscarmellose sodium, 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of a film coating premix and 60 parts of purified water; alternatively, the first and second electrodes may be,

prescription 7: 92 parts of citric acid alidenafil, 152 parts of pregelatinized starch, 90 parts of calcium hydrophosphate, 12 parts of internal crosslinked sodium carboxymethylcellulose, 8 parts of external crosslinked sodium carboxymethylcellulose, 24 parts of polymethyl methacrylate, 105 parts of 70% ethanol, 3.5 parts of sodium stearyl fumarate, 8.5 parts of film coating premix and 60 parts of purified water.

5. The tablet of claim 4 wherein the crystalline form of Aidenafil citrate is selected from form A, form B, form C, form D, form O, form V or form H.

6. The tablet according to claim 5, wherein the crystalline form of Aidenafil citrate is form H.

7. A process for the preparation of a tablet according to any one of claims 1 to 6, comprising the steps of:

1) Premixing

Sieving the prescribed dose of citric acid alidenafil, and sequentially putting the pregelatinized starch, the internally added cross-linked sodium carboxymethyl cellulose and the calcium hydrophosphate into a wet granulator for premixing;

2) Granulating

Adding the ethanol dispersion of polymethyl methacrylate for granulation;

3) Drying

4) Whole grain

Sieving and granulating the dried granules;

5) Total mixture

Adding additional croscarmellose sodium and sodium stearyl fumarate into the granules, and mixing in a mixer;

6) Tablet press

Tabletting;

7) Coating

Coating to obtain the tablet.

8. Use of a tablet according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of alzheimer's disease.

9. Use of a tablet according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of erectile dysfunction.