Preparation method of polydopamine-based nano composite material and application of polydopamine-based nano composite material in treatment of periodontitis

Abstract

The invention provides a preparation method and application of a nano composite material compounded with a metal material and amino acid on the basis of polydopamine nano particles. According to the material, three elements are combined, the antibacterial, anti-inflammatory and osteogenesis promoting effects are achieved through the synergistic effect so as to treat periodontitis, the ROS removing function in the periodontitis environment is achieved, the obvious anti-oxidation / anti-inflammatory effect is achieved, meanwhile, growth of periodontitis bacteria can be more efficiently inhibited after NO gas is released, and the periodontitis treatment effect is achieved. And the metal ions contained in the nano composite material can realize the repair of bone tissues, so that the nano composite material has remarkable advantages compared with single nano particles formed by elements.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically to a method for preparing a polydopamine-based nanocomposite material and its application in the treatment of periodontitis. Background Art

[0002] Periodontitis is one of the most common chronic infectious diseases in my country. According to statistics, the incidence of periodontitis in Chinese adults is as high as 80%. Periodontitis is the leading cause of tooth loss in adults, leading to gingival atrophy, loss of periodontal soft tissue attachment, and the formation of periodontal pockets, which can cause alveolar bone porousness. If left untreated, further exacerbation of periodontal inflammation can lead to loose teeth or even tooth loss, severely impacting patients' daily lives and work. Antibacterial treatment of periodontitis, eliminating periodontal tissue inflammation caused by periodontitis, and delaying the irreversible bone loss caused by late-stage periodontitis have always been key issues in clinical oral rehabilitation.

[0003] Polydopamine nanoparticles have excellent biosafety and can scavenge ROS to achieve antioxidant effects. Furthermore, their inherent properties, such as high photothermal conversion efficiency, combined with the characteristics of their drug-loaded scaffold, can exert antimicrobial infection functions. In recent years, new periodontitis treatments combining NIR irradiation with nanoparticles have become a research hotspot. However, previous nanoparticles composed of a single element may have functional limitations and insufficient targeting, making it difficult to meet the treatment needs of the complex pathological environment of periodontitis.

[0004] Therefore, it is very necessary to consider the pathological microenvironment of periodontitis and make targeted treatment needs, and to obtain a multi-element multifunctional composite nanomaterial with antibacterial, anti-inflammatory and osteopromoting effects for the treatment of periodontitis. Summary of the Invention

[0005] The purpose of the present invention is to provide a polydopamine-based nanocomposite material, a preparation method thereof, and an application in periodontitis. The invention aims to integrate metal ions having an osteogenesis implied and amino acids capable of releasing NO under light with polydopamine-based nanoparticles, and to use the prepared nanocomposite material for the treatment of periodontitis.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A polydopamine-based nanocomposite material is a nanocomposite material composed of metal strontium, amino acid arginine and polydopamine nanoparticles.

[0007] Furthermore, the polydopamine-based nanocomposite material has a diameter of 397.0 ± 19.7 nm and a zeta potential of -45.7 ± 0.2 mV.

[0008] The present invention also discloses a method for preparing the polydopamine-based nanocomposite material, comprising the following steps: S1: Mix 40 mL of anhydrous ethanol, 90 mL of deionized water, and 3 mL of 25% ammonia water and stir magnetically at room temperature to obtain solution A. Then, 500 mg of dopamine hydrochloride powder was weighed, added to 10 mL of deionized water, mixed, and then added to solution A. The reaction was continued by stirring at room temperature, followed by centrifugation, washing, and drying to obtain PDA nanoparticles. S2: 100 mg of PDA nanoparticles, 45 mg of SrCl2 powder, and 200 mL of deionized water were stirred at room temperature to obtain solution B. Then, 9 mg of ascorbic acid was dissolved in 10 mL of deionized water, and after mixing, it was added dropwise to solution B. Magnetic stirring was continued at room temperature. After the reaction was completed, the solution was centrifuged, washed, and dried to obtain PDA@Sr particles. S3: Take 20 mg of PDA@Sr nanoparticles and 20 mg of L-arginine powder and dissolve them in 50 mL of deionized water and 20 mL of Tris-HCl solution, react with magnetic stirring at room temperature, then centrifuge, wash, and dry to obtain the polydopamine-based nanocomposite material shown in the present invention.

[0009] The present invention also discloses the application of the above-mentioned polydopamine-based nanocomposite material in the treatment of periodontitis, including the preparation of antibacterial drugs for treating periodontitis, scavenging ROS, reducing the expression of pro-inflammatory factors and promoting osteogenesis, and releasing NO gas under near-infrared light irradiation to enhance antibacterial properties.

[0010] The present invention also discloses a pharmaceutical composition comprising the polydopamine-based nanocomposite material and a pharmaceutically acceptable carrier.

[0011] The beneficial effects of the present invention are: The present invention obtains a novel polydopamine-based nanocomposite material through a simple synthesis method. Through the NO gas release ability, the antibacterial property is enhanced, showing a good effect of eliminating intracellular ROS, and exerting intracellular anti-inflammatory and osteogenic functions. After application in a rat periodontitis model, it also shows good in vivo anti-inflammatory, anti-inflammatory and osteogenic effects. This nanocomposite material obtained by photothermal response combined with gas therapy can better exert its multifunctional composite effect to treat periodontitis. The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1TEM results of the nanocomposite material of the present invention (scale: 500 nm); Figure 2 This is a graph showing the zeta potential of the nanocomposite material of the present invention; Figure 3 This is the XPS result diagram of the nanocomposite material of the present invention; Figure 4 This is a TGA result diagram of the nanocomposite material of the present invention; Figure 5 The photothermal images and corresponding temperature changes of the nanocomposite materials of the present invention with different nanoparticles (PDA, PDA@Sr, and PDA@Sr@Arg) at the same concentration (100 μg / mL) under near-infrared irradiation (1 W / cm²) over time; Figure 6 is the NO release capacity of the nanocomposite material of the present invention, wherein: A represents the NO release standard curve; B represents the NO release of nanocomposites under different conditions; Figure 7 The safety evaluation results of the nanocomposite material of the present invention in MC3T3-E1 cells and L929 cells are shown in Figure 5, wherein A represents MC3T3-E1 cells and B represents L929 cells; Figure 8 The effects of different treatment groups on intracellular ROS content, including: A is the fluorescence result, scale bar: 100 μm; B is the corresponding quantitative result graph; the green fluorescence intensity represents the ROS content; Figure 9 represents the regulation level of pro-inflammatory factors in the intracellular inflammatory state by different treatment groups; Figure 10 The regulatory levels of osteogenic factors in the osteogenic state induced by different treatment groups; Figure 11 A is the fluorescence results of live / dead staining of P.gingivalis bacteria in different treatment groups, scale: 100 μm, Figure 11 B is the corresponding quantitative result graph, where red represents dead bacteria and green represents live bacteria; Figure 12 The results of plate coating of P.gingivalis in different treatment groups are shown in Figure 1, where A is the result graph and B is the corresponding quantitative result graph; Figure 13 The results of in vivo bacterial plate coating in the rat periodontitis model treated with different treatment groups are shown in Figure 1, where A is the result graph and B is the corresponding quantitative result graph; Figure 14To evaluate the efficacy of different treatment groups in treating rat periodontitis models, including: A is the three-dimensional and two-dimensional Micro-CT images, and the yellow dashed line indicates the distance between the CEJ and ABC; B is the CEJ-ABC distance case; C is the alveolar bone BV / TV measurement result; D is the measurement result of alveolar bone Tb.N; E is the measurement result of alveolar bone Tb.Th; F is the measurement result of alveolar bone Tb.Sp; Figure 15 H&E staining results of the rat periodontitis model treated with different treatment groups (scale bar: 100 μm). DETAILED DESCRIPTION

[0013] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1

[0014] Preparation of polydopamine-based nanocomposites: (1) Preparation of PDA nanoparticles: First, take 40 mL of anhydrous ethanol, 90 mL of deionized water, and 3 mL of 25% ammonia water, put them in a beaker, place the beaker on a magnetic stirrer, and stir at room temperature for 2 hours; Next, 500 mg of dopamine hydrochloride powder was weighed and added to 10 mL of deionized water. After thorough mixing, the mixture was added to the above solution and magnetic stirring was continued at room temperature overnight. After the reaction was complete, the liquid was centrifuged (8000 rpm, 10 minutes) three times to collect the PDA nanoparticles.

[0015] (2) Preparation of PDA@Sr nanoparticles: First, 100 mg of PDA nanoparticles, 45 mg of SrCl2 powder, and 200 mL of deionized water were weighed, placed on a magnetic stirrer, and stirred at room temperature for 2 h.

[0016] Next, 9 mg of ascorbic acid was weighed and dissolved in 10 mL of deionized water. After thorough mixing, the solution was slowly added dropwise to the above solution, and magnetic stirring was continued at room temperature for 6 hours. After the reaction was complete, the solution was centrifuged (8000 rpm, 10 minutes) and washed three times before collecting the PDA@Sr nanoparticles.

[0017] (3) Preparation of PDA@Sr@Arg nanoparticles: 20 mg of PDA@Sr nanoparticles and 20 mg of L-arginine powder were weighed and dissolved in 50 mL of deionized water and 20 mL of Tris-HCl solution, and magnetically stirred at room temperature overnight. After the reaction was completed, the solution was centrifuged by a centrifuge (8000 rpm, 10 min), washed three times, and finally the PDA@Sr@Arg nanoparticles were collected. Example 2

[0018] Characterization of PDA@Sr@Arg nanocomposites: The micro-morphology of the surface of the PDA@Sr@Arg nanocomposites prepared in Example 1 was observed using TEM; zeta surface potential, XPS energy spectrum, and TGA were detected, and the results are shown in Figures 1-4 ; Figure 1 The results show that the nanoparticles exhibit a typical spherical structure, and the diameter of the PDA@Sr@Arg nanocomposites is 397.0 ± 19.7 nm; Figure 2 The results show that the zeta potential of PDA is -52.4 ± 0.8 mV, while the zeta potentials of PDA@Sr and PDA@Sr@Arg are -45.2 ± 1.8 mV and -45.7 ± 0.2 mV, respectively, which indicates that the loading of Sr2+ and Arg affects the zeta potential of PDA, resulting in a change; As shown in Figure 3 , C, N and O elements were detected in PDA, while Sr element was only detected in PDA@Sr and PDA@Sr@Arg, which further confirms the successful loading of Sr; From Figure 4 , the weight loss rates of PDA, PDA@Sr and PDA@Sr@Arg are 65.7%, 62.9% and 56.9%, respectively. The difference in weight loss rate between PDA@Sr and PDA@Sr@Arg is due to the loading of Arg. Example 3

[0019] Performance detection of PDA@Sr@Arg nanocomposites: The performance of the prepared nanocomposites was detected by photothermal experiment and NO release. First, the photothermal experiment placed different nanoparticles (concentration was 100 μg / mL) under near-infrared (NIR) irradiation (808 nm, 1 W / cm²) for a certain time for evaluation.

[0020] Through Figure 5The results show that at the same concentration, the temperature of PDA, PDA@Sr and PDA@Sr@Arg increases with the increase of irradiation time, while the temperature of PBS does not change significantly. After 15 minutes of NIR irradiation, the temperature of PDA, PDA@Sr and PDA@Sr@Arg increases to 49.2°C, 48.5°C and 45.3°C, respectively, showing similar trends. This indicates that the prepared nanocomposites have the ability of photothermal conversion.

[0021] The NO release detection is established by using commercial NaNO2 (1, 3, 5, 7 and 9 μM) to establish a standard curve. Then, the NO release of different groups is measured by Griess reagent, including: 100 μg / mL of PDA@Sr+NIR, PDA@Sr@Arg, PDA@Sr@Arg+NIR and PDA@Sr@Arg+NIR (50, 100 and 200 μg / mL).

[0022] Among them, the light group is exposed to near-infrared irradiation (1 W / cm 2 ) for 15 minutes, and the concentration of generated nitric oxide (NO) is detected. The specific operation is to uniformly disperse the nanoparticles in PBS, and after the light group is irradiated, 60 μL of supernatant is quickly added to the Griess reagent, and the azo compound formed is detected by the enzyme label at 540 nm. The results are shown in Figure 6 As can be seen, except for PDA@Sr@Arg, PDA@Sr does not detect the generation of NO under near-infrared irradiation, and PDA@Sr@Arg does not generate NO without near-infrared irradiation, indicating that the NO gas of PDA@Sr@Arg is excited by near-infrared irradiation. Example 4

[0023] Biocompatibility of nanocomposites: MC3T3-E1 cells and L929 cells were used to evaluate the effect of nanocomposites on cell activity.

[0024] The cells were seeded in a 96-well plate and cultured overnight, and the medium was replaced with a medium containing different concentrations (0, 10, 20, 50, 100 and 200 μg / mL) of nanocomposites. Cells cultured with different concentrations of nanocomposites were used as experimental groups, and cells without seeding only with medium were used as blank groups.

[0025] After 24 hours, 100 uL of CCK-8 reagent with a concentration of 10% was added to each well, and after incubation in the dark for 1 h, the absorbance at 450 nm was measured by the enzyme label. The results are shown in Figure 7As shown, although cell viability at 100 μg / mL for PDA@Sr and PDA@Sr@Arg differed from that at 0 μg / mL, cell viability remained above 97.9% ± 2.0%, 91.0% ± 0.8%, and 88.6% ± 0.9% for the PDA, PDA@Sr, and PDA@Sr@Arg groups, respectively. Cell viability decreased when the concentration increased to 200 μg / mL. Therefore, the prepared nanocomposites exhibited good biosafety, and 100 μg / mL was selected as the nanoparticle concentration for further experiments. Example 5

[0026] Antioxidant, anti-inflammatory and osteogenic effects of nanocomposites: (1) Intracellular ROS levels were detected using a dichlorodihydrofluorescein diacetate (DCFH-DA) detection kit. According to the experimental instructions, the cell density was 1 × 10 5 At 4 hr, cells were treated with 100 μg / mL of different nanocomposites overnight and induced with 100 μM H2O2 for 30 minutes as a control group. Afterwards, the cells were incubated with fresh solution containing DCFH-DA for 0.5 h in a dark environment and then observed under an inverted fluorescence microscope.

[0027] like Figure 8 As shown, green fluorescence (green fluorescence intensity represents ROS levels) was almost absent in the normal group, while significant green fluorescence was observed in the control group (H2O2). The fluorescence intensity of the PDA and PDA@Sr treated groups decreased, indicating that both materials possessed some ROS scavenging ability. However, PDA@Sr@Arg exhibited a more efficient ROS scavenging ability.

[0028] (2) The cells were divided into experimental groups and RNA was routinely extracted from each group. The RNA levels of TNF-α, IL-1β and IL-6 were detected using a real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) test system to evaluate the anti-inflammatory effect of the nanocomposite. The specific operation was as follows: 1 μg / mL LPS was added to L929 cells to induce them into an inflammatory state, and then the culture medium was replaced for further treatment. Seven groups were set up in the experiment: Normal group (no treatment), LPS group (only LPS intervention), PDA group, PDA@Sr group and PDA@Sr@Arg group (all nanocomposite groups contained LPS induction, and the nanocomposite concentration was 100 μg / mL). After overnight treatment, the cells and supernatant were collected for subsequent experiments.

[0029] The results are as follows Figure 9As shown in the figure, compared with the normal group, the expression levels of inflammation-related genes (TNF-α, IL-1β, and IL-6) in the LPS group were significantly increased after LPS induction. The PDA and PDA@Sr treatment groups significantly reduced the expression levels of TNF-α, IL-1β, and IL-6, and PDA@Sr@Arg had the most significant downregulation effect on these genes.

[0030] (3) The experimental steps were the same as the previous step. MC3T3-E1 cells were cultured in osteogenic induction medium for 7 days before PCR experiments. Figure 10 It can be seen that the expression of osteogenesis-related genes, including ALP, Runx2, and OPG, were upregulated by PDA@Sr and PDA@Sr@Arg. Example 6

[0031] Verification of in vitro antibacterial ability: Subgingival plaque biofilm is a key initiator of periodontal disease. Porphyromonas gingivalis (P. gingivalis) is a typical periodontal pathogen. P. gingivalis was co-cultured with PDA@Sr@Arg to test its bactericidal potential. Groups were divided into a control group (no treatment), a PDA@Sr@Arg group, and a PDA@Sr@Arg+NIR group. After treatment, the antibacterial effect of P. gingivalis was determined.

[0032] (1) The antibacterial ability of PDA@Sr@Arg was detected by live / dead staining. Figure 11 As shown, the PDA@Sr@Arg+NIR group exhibited more red fluorescence compared to the control and PDA@Sr@Arg groups. This is because NIR stimulates the production of NO, indicating that NO release in the PDA@Sr@Arg+NIR group significantly increased the proportion of red staining in the dead bacteria, demonstrating its most significant antibacterial activity.

[0033] (2) The antibacterial ability of PDA@Sr@Arg was verified by bacterial plate coating. Figure 12 As shown, the number of P. gingivalis was significantly reduced after co-culture of PDA@Sr@Arg under NIR irradiation compared with the Control group. Example 7

[0034] Validation of therapeutic ability in a rat periodontitis model: Thirty healthy SD male rats aged 6-8 weeks (weighing 180-200 g) were randomly divided into 6 groups (n=5) after 1 week of adaptive feeding: Normal group, Periodontitis group, PDA group, PDA@Sr group, PDA@Sr@Arg group, and PDA@Sr@Arg+NIR group.

[0035] Rats were anesthetized with 2% sodium pentobarbital. Sterile ligatures (0.2 mm) were placed around the cervical area of ​​the maxillary second molars bilaterally in all five groups except the Normal group for 3 weeks to establish an experimental periodontitis model. During the 3-week experiment, rats were injected with PBS (Normal and Periodontitis groups), PDA (100 μg / mL) (PDA group), PDA@Sr (100 μg / mL) (PDA@Sr group), and PDA@Sr@Arg (100 μg / mL) (PDA@Sr@Arg group and PDA@Sr@Arg+NIR group) at the ligature site every 3 days. The PDA@Sr@Arg+NIR group was irradiated with NIR (1 W / cm2) after each injection. 2 , 15 minutes) until the end of the experiment and subsequent experiments.

[0036] (1) In vivo antibacterial ability detection At the end of treatment, the treated area was probed with a sterile cotton swab and the sample was immediately placed in 1 mL of sterile saline. 100 μL of the solution from the corresponding experimental group was evenly spread on Columbia blood agar plates. The plates were then incubated at 37°C for 48 hours, and the number of colonies was observed and counted.

[0037] The results are as follows Figure 13 As shown in the results, a large number of bacteria were observed in the periodontitis group, while the bacterial survival rate was significantly reduced in the PDA@Sr@Arg+NIR group. These results indicate that PDA@Sr@Arg under NIR irradiation has significant in vivo antibacterial ability and can kill bacteria in periodontal infection sites, and its antibacterial effect is better than that of other nanomaterial groups.

[0038] (2) Micro-CT analysis was performed. The maxilla was separated and fixed with 10% formalin for 24 hours, and then analyzed using multimodal 3D visualization software. Figure 14As shown in A and B, observing the second molar area, the distance between ABC and CEJ was the longest in the Periodontitis group compared with the Normal group, indicating significant alveolar bone loss. At the same time, bone parameters in the Periodontitis group, such as bone volume per unit tissue volume (BV / TV), trabecular thickness (Tb. Th), and trabecular number (Tb. N), decreased, while trabecular spacing (Tb. Sp) increased. These results demonstrate the successful establishment of the periodontitis model ( Figure 14 Compared with the periodontitis group, the PDA@Sr@Arg+NIR group significantly improved the values ​​of the above indicators (BV / TV, Tb. Th, and Tb. N) and reduced the distance between the ABC and CEJ and the Tb. Sp value, demonstrating that PDA@Sr@Arg under NIR irradiation is more effective in inhibiting bone loss than other nanomaterials.

[0039] (3) Histological staining analysis. After the experiment, the maxilla of the SD rat was isolated and the soft tissue was removed. The samples were fixed with 4% paraformaldehyde for 24 hours and then dehydrated. The samples were decalcified, paraffin-embedded, and blocked. H&E staining was then performed. Figure 15 H&E staining in the Figure 3 shows the inflammatory response of the periodontal tissues in each group after treatment. In the Normal group, protruding gingival papillae, normal gingival epithelium, regularly arranged periodontal ligament fibers, normal epithelial attachment and alveolar ridge height were visible between the molars. In contrast, the Periodontitis group showed decreased attachment at the ligation site, disappearance of the gingival papilla structure between the molars, shrinkage of the junctional epithelium, a large number of infiltrating inflammatory cells, and a significant decrease in alveolar bone height, indicating that the Periodontitis group had persistent inflammation and severe alveolar bone absorption. In the PDA@Sr@Arg+NIR group, the junctional epithelium was tightly attached to the cementum surface, there was less epithelial inflammatory cell infiltration in the gingival sulcus, and the alveolar bone height was only slightly decreased, indicating that the inflammation was effectively controlled and alveolar bone absorption was inhibited.

[0040] In summary, PDA@Sr@Arg nanocomposites have been shown to effectively promote tissue healing in periodontitis states, better exert antibacterial effects through NO gas, and inhibit inflammation.

[0041] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0042] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A polydopamine-based nanocomposite material, characterized in that: The polydopamine-based nanocomposite material is a nanocomposite material of composite metal strontium, amino acid arginine and polydopamine nanoparticles.

2. The polydopamine-based nanocomposite material according to claim 1, wherein The polydopamine-based nanocomposite material has a diameter of 397.0 ± 19.7 nm and a zeta potential of -45.7 ± 0.2 mV.

3. A method for preparing the polydopamine-based nanocomposite material according to claim 1 or 2, characterized in that: The following steps are involved: S1: Mix 40 mL of anhydrous ethanol, 90 mL of deionized water, and 3 mL of 25% ammonia water and stir magnetically at room temperature to obtain solution A. Then, 500 mg of dopamine hydrochloride powder was weighed, added to 10 mL of deionized water, mixed, and then added to solution A. The reaction was continued by stirring at room temperature, followed by centrifugation, washing, and drying to obtain PDA nanoparticles. S2: 100 mg of PDA nanoparticles, 45 mg of SrCl2 powder, and 200 mL of deionized water were stirred at room temperature to obtain solution B. Then, 9 mg of ascorbic acid was dissolved in 10 mL of deionized water, and after mixing, it was added dropwise to solution B. Magnetic stirring was continued at room temperature. After the reaction was completed, the solution was centrifuged, washed, and dried to obtain PDA@Sr particles. S3: Take 20 mg of PDA@Sr nanoparticles and 20 mg of L-arginine powder and dissolve them in 50 mL of deionized water and 20 mL of Tris-HCl solution, react with magnetic stirring at room temperature, then centrifuge, wash, and dry to obtain the polydopamine-based nanocomposite material shown in the present invention.

4. A use of the polydopamine-based nanocomposite material according to claim 1, characterized in that: Used to treat periodontitis.

5. The use according to claim 4, characterized in that Preparation of antibacterial drugs for treating periodontitis.

6. The use according to claim 4, characterized in that Clear ROS, reduce the expression of pro-inflammatory factors and promote osteogenesis.

7. The use according to claim 4, characterized in that The polydopamine-based nanocomposite material releases NO gas under near-infrared light irradiation to enhance antibacterial properties.

8. A pharmaceutical composition comprising the polydopamine-based nanocomposite material according to claim 1 or 2 and a pharmaceutically acceptable carrier.

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