Use of artemisia oil in medicaments for the treatment of side effect-related conditions caused by administration of tlr7 / 8 agonists

Abstract

Use of artemisia oil in a medicament for treating a side effect-related condition caused by the administration of a TLR7 / 8 agonist, in particular use of artemisia oil in the manufacture of a medicament for preventing, treating or ameliorating skin microflora imbalance caused by the administration of a TLR7 / 8 agonist on the body surface.

Description

Use of artemisinin oil in medications for treating side effects associated with the use of TLR7 / 8 agonists. Technical Field

[0001] This invention relates to the use of artemisia oil in the preparation of medicaments for the prevention, treatment or improvement of side effects associated with topical TLR7 / 8 agonists. Background Technology

[0002] Topical application of TLR7 / 8 agonists can treat a variety of diseases. For example, topical application of imiquimod cream can treat condyloma acuminata of the external genitalia and perianal region in adults; resimod combined with imiquimod can be applied topically as a potential adjuvant in the treatment of melanoma (Tambunlertchai, S.; Geary, SM; Salem, AK. Topically Applied Resiquimodversus Imiquimod as a Potential Adjuvant in Melanoma Treatment. Pharmaceutics 2022, 14, 2076). However, topical application of TLR7 / 8 agonists is limited by their side effects. There is an urgent need in clinical treatment for drugs that can prevent, treat, or improve symptoms associated with the side effects of TLR7 / 8 agonist administration. Artemisia naphta oil (AN) is the active ingredient obtained by further purification of the residues from the artemisinin extraction process. This invention has found that artemisia naphta oil can prevent, treat, or improve symptoms associated with the side effects of topical application of TLR7 / 8 agonists. Summary of the Invention

[0003] This invention relates to the use of artemisinin oil in the preparation of medicaments for the prevention, treatment, or improvement of conditions related to side effects caused by the administration of TLR7 / 8 agonists, and particularly to the use of artemisinin oil in the preparation of medicaments for the prevention, treatment, or improvement of skin microecological disorders caused by topical TLR7 / 8 agonists. The invention also includes pharmaceutical combinations of TLR7 / 8 agonists and artemisinin oil, and packaging of such pharmaceutical combinations.

[0004] In a first aspect of the invention, artemisia oil is provided for use in the preparation of a medicament for the prevention, treatment or improvement of symptoms related to side effects caused by administration of TLR7 / 8 agonists.

[0005] In some embodiments, the TLR7 / 8 agonist may be a 4-amino-1H-imidazo[4,5-C]quinoline derivative, motolimod or its derivatives, selgantolimod or its derivatives, vesatolimod or its derivatives, or TMX-202 or its derivatives, or a pharmaceutically acceptable salt thereof.

[0006] In some embodiments, the 4-amino-1H-imidazo[4,5-C]quinoline derivative is selected from: imiquimod, 852-A (PF-4878691), retsimide (R-848, retsimide), 3M-052, or a pharmaceutically acceptable salt thereof.

[0007] The TLR7 / 8 agonist described in this invention has the following structure:

[0008]

[0009]

[0010] In some implementations, side effects related to TLR7 / 8 agonists include, but are not limited to, skin microecological dysbiosis caused by topical application of TLR7 / 8 agonists. This skin microecological dysbiosis may include reduced diversity and richness of the skin flora. This skin microecological dysbiosis may include one or more of the following species becoming the dominant population: Muribacterium, Lactobacillus, Clostridium MBA03, Christensenellaceae R.7 group, Bacteroides, Thermovirga, M55.D21, Alloprevotella, Sporanaerobacter, and Fastidiosipila.

[0011] In some implementations, the application of artemisinin oil to subjects resulted in the restoration of homeostasis of skin microecological imbalance, which included downregulation of one or more of the following: MBA03, M55-D21, Christensenellaceae R.7 group, Thermovirga, Fastidiosipila, and Sporanaerobacter.

[0012] In some embodiments, artemisia oil is a composition for skin application, preferably a drug, cosmetic, or health product for skin application.

[0013] In some implementations, the drugs applied through the skin are selected from sprays, aerosols, emulsions, ointments, gels, solutions, or suspensions. Cosmetics applied through the skin include water, lotions, creams, serums, masks, cleansers, and essential oils.

[0014] In some embodiments, the volume concentration of artemisia oil in the composition is 0.011%-0.549%, preferably 0.055%-0.330%, more preferably 0.055%-0.165%, specifically selected from 0.055%, 0.110%, 0.165%, 0.220%, 0.275%, 0.330%, 0.385%, 0.440%, 0.495%, and 0.549%.

[0015] In a second aspect of the invention, a pharmaceutical composition is provided comprising a TLR7 / 8 agonist administered dermally and an artemisinin oil composition administered dermally. Preferably, the volume concentration of artemisinin oil in the artemisinin oil composition is 0.1%-5%, more preferably 0.5-3%, and more preferably 0.5-1.5%.

[0016] In a third aspect of the invention, a pharmaceutical combination package is provided, comprising a TLR7 / 8 agonist composition for dermal application and an artemisinin oil composition for dermal application; preferably, the volume concentration of artemisinin oil in the artemisinin oil composition is 0.1%-5%, more preferably 0.5-3%, and more preferably 0.5-1.5%.

[0017] In some embodiments, the drug combination package may contain the skin-administered TLR7 / 8 agonist composition and the skin-administered artemisinin oil composition in separate packages.

[0018] In some implementations, the drug combination package also includes instructions for use, which state that the artemisinin oil composition applied over the skin is used to improve symptoms associated with side effects caused by TLR7 / 8 agonists applied over the skin.

[0019] In some embodiments, the transdermal TLR7 / 8 agonist composition and the transdermal artemisinin oil composition are administered sequentially, simultaneously, or in a drug combination package. Preferably, the transdermal TLR7 / 8 agonist composition is used before the transdermal artemisinin oil composition.

[0020] In some embodiments, the drug combination package further includes instructions for use, which state that the artemisinin oil composition is used to prevent, treat or improve symptoms related to side effects caused by a transdermal TLR7 / 8 agonist, preferably further stating that the transdermal TLR7 / 8 agonist composition and the transdermal artemisinin oil composition are administered sequentially, simultaneously or in sequence; more preferably, stating that the transdermal TLR7 / 8 agonist composition is used before the transdermal artemisinin oil composition. Attached Figure Description

[0021] Figure 1 shows the rank abundance curves of skin microbiota sequencing results in mice treated with imiquimod cream.

[0022] Figure 2 shows the α-diversity analysis results of skin microbiota sequencing in mice treated with imiquimod cream.

[0023] Figure 3 shows the β-diversity analysis of skin microbiota sequencing results in mice treated with imiquimod cream.

[0024] Figure 4 shows the NMDS and PCA analysis of the skin microbiome of mice treated with imiquimod cream.

[0025] Figure 5 shows the differences in the abundance of dominant bacterial communities among the groups in mice treated with imiquimod cream. Detailed Implementation

[0026] The invention can be more readily understood by referring to the following additional embodiments of the invention and the detailed description of the examples included herein. It should be understood that the terminology used herein is provided for the purpose of describing particular embodiments only and is not intended to be limiting. Furthermore, it should be understood that unless expressly defined herein, the terminology used herein should be given its conventional meaning as known in the relevant art.

[0027] TLR7 / 8 agonist

[0028] TLR7 / 8 agonists specifically act on TLR7 / 8, including but not limited to 4-amino-1H-imidazo[4,5-C]quinoline derivatives, motolimod and its derivatives, selgantolimod and its derivatives, vesatolimod and its derivatives, or TMX-202 and its derivatives, or pharmaceutically acceptable salts thereof. The 4-amino-1H-imidazo[4,5-C]quinoline derivatives are selected from: imiquimod, 852-A (PF-4878691), resimod (R-848, retimiquimod), PF-4878691, and 3M-052, or pharmaceutically acceptable salts thereof.

[0029] Skin microbiome and skin microbiome imbalance

[0030] The skin microbiome is composed of all microorganisms on the skin surface, along with skin cells, secretions, and the microenvironment. The microecological barrier formed by the human skin surface microbiome has been found to be crucial for skin health. Skin microecological homeostasis is an important factor in skin health. The skin microbiome can be measured using methods such as alpha diversity analysis, beta diversity analysis, non-metric multidimensional scaling (NMDS), principal component analysis (PCA), and the abundance of dominant microbiota. Other methods and techniques for measuring the skin microbiome are also known in this field. Skin microecological imbalance can manifest as reduced skin microbiota diversity and richness, altered dominant microbiota, and / or upregulation / downregulation of specific microbiota. It has been found that skin microbiome imbalance can promote the progression of inflammatory skin diseases (Prescott, SL, et al., The skin microbiome: impact of modern environment on skin ecology, barrier integrity, and systemic immune programming. World Allergy Organ J, 2017.10(1):p.29; Dreno, B., et al., The Skin Microbiome: A New Actor in Inflammatory Acne. Am J Clin Dermatol, 2020.21(Suppl 1):p.18-24).

[0031] Skin microbiome sequencing and data analysis

[0032] 16S rDNA is the most useful and commonly used molecular clock in bacterial systematics. It is found in a small number of species, is abundant (accounting for approximately 80% of bacterial RNA), has a moderate molecular size, and is present in all organisms. Its evolutionary clock properties are excellent, and it exhibits high conservation in structure and function, earning it the nickname "bacterial fossil." In most prokaryotes, rDNA has multiple copies; 5S, 16S, and 23S rDNA have the same copy number. Because of its moderate size (approximately 1.5 kb), 16S rDNA effectively reflects differences between different bacterial genera and its sequence is relatively easy to obtain using sequencing technology, making it widely accepted by bacteriologists and taxonomists.

[0033] This invention uses the Illumina NovaSeq PE250 system for 16S amplicon sequencing to obtain raw data. The raw data is then processed by removing adapters, filtering, deduplication, base correction, and removing chimeric sequences to obtain clean data that can be used for subsequent analysis.

[0034] The following are the specific methods for skin microbiome sequencing and data analysis.

[0035] DNA extraction method: Total genomic DNA was extracted from the samples using the CTAB method. DNA concentration and purity were determined on a 1% agarose gel, and the DNA was diluted with sterile water to 1 μg / μL for later use based on the concentration.

[0036] PCR amplification and purification methods: Diluted genomic DNA was used for PCR amplification. A 30 ng DNA sample and fusion primers were used for the PCR reaction. Forward primer (V3 region 341F): 5'-CCTAYGGGRBGCASCAG-3', reverse primer (V4 region 806R): 5'-GGACTACNNGGGTATCTAAT-3'. A 30 μl PCR reaction system was established: 10 ng DNA, 15 μl 2×PhusionMaster Mix buffer, 1 μl each of forward and reverse primers, and 2 μl double-distilled water. High-efficiency and high-fidelity enzymes (NewEngland Biolabs) were used for PCR to ensure amplification efficiency and accuracy. PCR amplification conditions: 98℃ pre-denaturation for 1 min; 98℃ denaturation for 10 s, 50℃ annealing for 30 s, 72℃ extension for 45 s, 30 cycles; 72℃ extension for 5 min. PCR products were stored at -20℃. The PCR products were mixed at equal concentrations and thoroughly mixed before being purified by agarose gel electrophoresis at 2% concentration using 1×TAE.

[0037] Library construction and sequencing methods: The library was constructed using the Illumina TruSeq DNA PCR-Free Library Preparation Kit. After the constructed library was quantified by Qubit and tested, it was sequenced using NovaSeq 6000.

[0038] Bioinformatics analysis methods: The obtained sequencing data were decomposed using barcode adapter sequences to obtain valid sequences. Then, the DADA2 plugin in Qiime (1.9.1) software was used to cluster, denoise, assemble, and dechirpify the original sequences, generating amplified characteristic sequences (operational taxonomic units, OTUs). The Uparse algorithm (Uparse v7.0.1001) was used to cluster all valid sequences from all samples, grouping them into OTUs with 100% identity. Representative sequences of OTUs were selected based on the algorithm's principles, choosing the sequence with the highest frequency as the representative sequence. Alpha diversity and beta diversity analyses were primarily performed using the Qiime (1.9.1) plugin.

[0039] Statistical analysis: For data that follow a normal distribution, express the data as mean ± standard deviation and use a paired-samples t-test to calculate the F-value; for data that do not follow a normal distribution, use the Mann-Whitney U test. All differences are considered statistically significant with P < 0.05.

[0040] α-diversity analysis

[0041] The Wilcoxon test (for analyzing differences between two groups, the Kruskal-Wallis test is used) is employed to analyze whether the differences in α-diversity index between groups are significant. Indices for calculating community richness include Observed, Chao, and ACE. Observed represents the number of ASV species actually observed in the sample; Chao is an index that estimates the number of ASVs in the sample using the Chao1 algorithm; and ACE is an index that estimates the number of ASVs in the community. Indices for calculating community diversity include Shannon, Simpson, and J. Shannon is commonly used to reflect α-diversity (estimating microbial diversity in a sample); Simpson is used to quantitatively describe the biodiversity of a region; and J is the Pielou's evenness index, used to measure the proximity in quantity among different species in a sample.

[0042] β-diversity analysis

[0043] To assess the degree of difference in species abundance distribution among groups, distance analysis can be used in statistics. Algorithms such as Bray-Curtis, Unweighted_unifrac, and Weighted_unifrac are used to calculate the distance between pairs of samples, obtaining a distance matrix for further analysis and visualization. When analyzing intra-group differences in β-diversity, the Wilcoxon test (for two-group difference analysis) and the Kruskal-Wallis test (for multiple-group difference analysis) are generally used. The test results are presented as box plots, which visually reflect the evenness of the microbial community structure within each group. A higher intra-group β-diversity index indicates greater community differences among samples within the group and poorer repeatability, while a lower index indicates better repeatability within the group.

[0044] NMDS and PCA analysis

[0045] Non-metric multidimensional scaling (NMDS) is a data analysis method that simplifies research objects in a multidimensional space to a lower-dimensional space for location, analysis, and classification, while preserving the original relationships between objects. Distance is generally defined by the difference in the rank order of samples between groups. NMDS graphs are commonly used for beta analysis in microbial community studies. NMDS focuses on reflecting the ranking relationship of values ​​in the distance matrix, downplaying the absolute difference in values. With a large number of samples and species (a greater number of possible rankings than differences between values), the NMDS model can more accurately reflect the numerical ranking information of the distance matrix. Therefore, NMDS is more accurate when there are many samples or species. The quality of NMDS analysis results is measured using the stress coefficient. NMDS graphs typically provide the stress coefficient value of the model to determine whether the graph accurately reflects the true distribution of the data ranking. It is generally believed that when the stress coefficient is <0.2, it can be represented by a two-dimensional dot plot of NMDS, and its graph has certain interpretive significance; when the stress coefficient is <0.1, it can be considered a good ranking; when the stress coefficient is <0.05, it has good representativeness.

[0046] Principal Component Analysis (PCA) is a technique for simplifying data analysis. This method effectively identifies the most "primary" elements and structures within the data. By analyzing the distribution of bacterial communities in different samples, the similarity and differences between samples can be reflected. Using variance decomposition, the differences between multiple sets of data are reflected on a two-dimensional coordinate graph. The axes best reflect the two characteristic values ​​of the variance. In the graph, PC1 and PC2 represent the first and second principal components, respectively. The percentage after each principal component represents the explanatory power of that component for the differences in sample composition. Different colored points represent different groups; the closer two sample points are, the more similar their species composition. The scales on the X and Y axes represent relative distances and have no practical significance.

[0047] Abundance Rank Curve

[0048] Abundance grading curves can reflect two aspects: species abundance and species evenness. Species abundance is reflected by the length of the curve on the horizontal axis; the larger the range of the curve on the horizontal axis, the higher the species abundance. Species evenness is reflected by the shape (smoothness) of the curve; the flatter the curve, the higher the species evenness.

[0049] Drug combination

[0050] "Drug combination" refers to a fixed combination of a unit dosage form (e.g., capsule, tablet, or sachet), a non-fixed combination, or a set of components for combined administration. The components in the combination may be administered simultaneously, independently at the same time, or separately at time intervals that allow these combination partners to exhibit synergistic effects (e.g., synergistic or cumulative effects).

[0051] Drug combination packaging

[0052] In some embodiments of the present invention, the pharmaceutical combination is packaged in the same package, which may also include instructions for use, stating that the artemisinin oil composition is used for the prevention, treatment, or improvement of side effects related to a skin-administered TLR7 / 8 agonist, preferably further stating that the skin-administered TLR7 / 8 agonist composition and the skin-administered artemisinin oil composition are administered sequentially, simultaneously, or in sequence; more preferably, stating that the skin-administered TLR7 / 8 agonist composition is used before the skin-administered artemisinin oil composition. In some embodiments of this application, the skin-administered TLR7 / 8 agonist composition and the skin-administered artemisinin oil composition are packaged separately, and the package may also include instructions for use, stating that the artemisinin oil composition is used for the prevention, treatment, or improvement of side effects related to a skin-administered TLR7 / 8 agonist, preferably further stating that the skin-administered TLR7 / 8 agonist composition and the skin-administered artemisinin oil composition are administered sequentially, simultaneously, or in sequence; more preferably, stating that the skin-administered TLR7 / 8 agonist composition is used before the skin-administered artemisinin oil composition.

[0053] Artemisia annua oil composition

[0054] In some embodiments of the present invention, the volume concentration of artemisia annua oil in the artemisia annua oil composition is 0.1%-5%, 0.25-4.75%, 0.5%-4.5%, 0.75%-4.25%, 1%-4%, 1.25%-3.75%, 1.5%-3.5%, 1.75%-3.25%, 2%-3%, 2.25%-2.75%, or 2.5%. In a preferred embodiment of the present invention, the volume concentration of artemisia annua oil is 0.5-3%. In a preferred embodiment of the present invention, the volume concentration of artemisia annua oil in the artemisia annua oil composition is 0.5-1.5%.

[0055] Example 1 Extraction and preparation of test samples of artemisia annua oil

[0056] The petroleum ether mother liquor remaining after artemisinin production was used to extract Artemisia annua (Artemisia annua) extract (oil extract). The extraction process is as follows:

[0057] The first step is to turn on the microwave heater to heat the mother liquor, and use vacuum distillation to evaporate the solvent in the mother liquor to obtain a concentrate. After cooling to room temperature, the extract concentrate is obtained.

[0058] The second step is to take the extract concentrate obtained in the previous step, add ethanol, shake to mix and dissolve, let stand, and remove the surface wax to obtain the initial oil ethanol solution of the extract.

[0059] The third step involves placing the round-bottom flask containing the above solution in a heater, turning on the microwave heater to heat the mother liquor, and then distilling under reduced pressure to recover the ethanol and obtain the primary oil extract.

[0060] Fourth step: Place the initial oil extract in a round-bottom flask, add distilled water, place it in a microwave heater, turn on the microwave heater and heat to boiling, collect the water vapor containing oil; then add distilled water again, repeat the above distillation process, collect the oil-containing aqueous solution; continue heating, reduce pressure and collect the distilled oil, and combine it with the oil-containing aqueous solution.

[0061] Step 5: Let the oil-containing aqueous solution stand overnight to separate the layers, discard the aqueous layer, and obtain crude extract of Artemisia annua (Artemisia argyi) (oil extract);

[0062] Step 6: Heat the crude extract (oil extract) of Artemisia annua (Artemisia argyi), add silica gel while hot, stir, let stand, and then filter while hot to obtain the finished product.

[0063] Take the artemisia oil prepared in step 6 and prepare 0.5%-5% artemisia oil test samples according to Table 1.

[0064] Table 1 Composition of Artemisia annua oil test sample

[0065]

[0066]

[0067] Example 2

[0068] Thirty-six BALB / c mice were randomly divided into four groups using a random number table: a blank control group, a model group, a water intervention group, a base water intervention group, a 0.5% artemisinin oil intervention group, a 1.0% artemisinin oil intervention group, a 1.5% artemisinin oil intervention group, a 3.0% artemisinin oil intervention group, and a 5.0% artemisinin oil intervention group, with four mice in each group. Except for the control group and the normal control group, all mice in the other groups underwent dorsal hair removal, covering an area of ​​approximately 2 cm × 3 cm. 42 mg of 5% imiquimod cream was applied once daily for 7 consecutive days. After successful model establishment, different concentrations of artemisinin oil were administered via spray according to the corresponding group, with intervention lasting for two weeks.

[0069] Table 2. Mouse grouping information

[0070] Grouped Experiments Sample Type Sample Array 1 Blank Control Group Skin Swabs 4 Groups 2 Model Group Skin Swabs 4 Groups 3 Model + Base Water Skin Swabs 4 Groups 4 Model + 0.5% Artemisia annua Oil Skin Swabs 4 Groups 5 Model + 1% Artemisia annua Oil Skin Swabs 4 Groups 6 Model + 1.5% Artemisia annua Oil Skin Swabs 4 Groups 7 Model + 3% Artemisia annua Oil Skin Swabs 4 Groups 8 Model + 5% Artemisia annua Oil Skin Swabs 4 Groups 9 Model + Water Skin Swabs 4 surface

[0071] Abundance ranking curves showed that the sample microbiota had high abundance and good species homogeneity, and that the abundance of mouse skin microbiota was significantly upregulated after intervention with artemisinin oil. See Figure 1 for the test results.

[0072] Alpha diversity analysis showed that the richness and diversity of skin microbiota at the lesion sites of mice in the model group were significantly upregulated after intervention with artemisinin oil. Specifically, intervention with 0.5% artemisinin oil significantly increased the diversity of skin microbiota, while intervention with 1.5% artemisinin oil significantly increased the richness of microbiota. See Figure 2 for the test results.

[0073] β-diversity analysis revealed significant differences in skin microbiota among the groups. See Figure 3 for the test results.

[0074] NMDS and PCA analyses showed that artemisinin oil intervention did indeed cause significant changes in the skin microbiota of mice. See Figure 4 for the test results.

[0075] Differential analysis of the abundance of dominant bacterial communities showed that the dominant bacterial communities in the nine grouped samples were mainly 10 species, including Muribacteraceae, Lactobacillus, Clostridium MBA03, Christensenellaceae R.7 group, Bacteroides, Thermovirga, M55.D21, Alloprevotella, Sporanaerobacter, and Fastidiosipila. As shown in Figure 5, MBA03, M55-D21, Christensenellaceae R.7 group, Thermovirga, Fastidiosipila, and Sporanaerobacter were all significantly downregulated after Artemisia annua oil intervention. Unbound by theoretical constraints, artemisinin oil intervention promoted both the abundance and diversity of skin flora in the model group mice. Furthermore, the intervention effect of artemisinin oil on the model group mice was related to the inhibition of pathogenic bacteria such as Christenellaceae R.7 group and Fastidiosipila, as well as antibiotic-resistant host flora such as Sporanaerobacter and MBA03.

[0076] The human skin concentrations of 0.5%, 1.0%, 1.5%, 3.0%, and 5.0% artemisinin oil, after being converted to human concentrations by dividing the mouse skin concentrations by a factor of 9.1, are 0.055%, 0.110%, 0.165%, 0.330%, and 0.549%, respectively.

[0077] Although the invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the invention includes all such alternatives, modifications, and variations falling within the spirit and scope of the appended claims. All disclosures, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety.

Claims

1. Use of artemisia annua oil in the preparation of compositions for the prevention, treatment, or improvement of skin microecological dysbiosis induced by imiquimod administration, wherein the artemisia annua oil is a composition administered through the skin, and the volume concentration of artemisia annua oil in the composition is 0.110%; the skin microecological dysbiosis includes a decrease in the diversity and richness of the skin flora, wherein the skin microecological dysbiosis is characterized by the dominance of Clostridium MBA03, Kristensenaceae R-7, Thermobacterium leynei, M55.D21, Coccidioides spp., and Coxella spp.; the application of artemisia annua oil to the subject produces a homeostatic restoration of the skin microecological dysbiosis, wherein the homeostatic restoration is characterized by the downregulation of Clostridium MBA03, M55.D21, Kristensenaceae R-7, Thermobacterium spp., Coxella spp., and Sporanaerobacter spp.

2. The use as described in claim 1, characterized in that, The composition is a drug or cosmetic applied through the skin.

3. The use as described in claim 2, characterized in that, The drug applied through the skin is selected from sprays, aerosols, emulsions, ointments, gels, solutions, or suspensions; the cosmetic applied through the skin is selected from water, lotion, cream, serum, mask, cleanser, and essential oil.

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