CN116747256A - Application of compound SH in preparation of broad-spectrum coronavirus resistant medicament - Google Patents

Abstract

The invention provides an application of compound SH in preparing a broad-spectrum anti-coronavirus medicament, belonging to the technical field of biological medicines. The invention selects compound SH with nontoxic concentration to conduct broad-spectrum anti-coronavirus research, the compound SH can effectively inhibit the replication of beta group coronavirus (SARS-CoV-2, HCoV-OC 43) and alpha group coronavirus (HCoV-NL 63, HCoV-229E) in vitro, and the compound SH has broad-spectrum anti-coronavirus activity, lays a foundation for further developing broad-spectrum anti-coronavirus drugs, and has important development value and wide application prospect.

Description

Application of compound SH in preparation of broad-spectrum coronavirus resistant medicament

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of compound SH in preparation of broad-spectrum anti-coronavirus medicines.

Background

Coronaviruses (CoV) are the largest known genome, enveloped single-stranded positive-strand RNA viruses that cause infections of the respiratory, digestive and nervous systems in animals and humans. The ninth report from the international committee on viral classification divides it into four groups α, β, γ and δ. Wherein, the coronaviruses of the alpha and beta groups mainly infect mammals, and the coronaviruses of the gamma and delta groups mainly infect birds.

Coronaviruses can infect humans and many animals and can cause diseases ranging from common cold to severe acute respiratory syndrome. Six coronaviruses known to infect humans in the past 2020 include human coronavirus 229E (HCoV-229E), NL63 (HCoV-NL 63), HKU1 (HCoV-HKU 1), OC43 (HCoV-OC 43) and severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus, SARS-CoV) and middle east respiratory syndrome coronavirus (Middle east respiratory syndrome coronavirus, MERS-CoV) which cause symptoms of upper respiratory infections. The emerging new coronamutant strains, as well as the potential threat of unknown coronaviruses, place an urgent need to develop drugs with broad-spectrum anti-coronavirus effects.

Current coronavirus drugs are mainly directed to the study of highly pathogenic coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV), mainly comprising three types of immunomodulatory factors (interferon, ribavirin, etc.), viral entry inhibitors (monoclonal antibodies to S protein, polypeptides directed to the key fusion region of S protein, etc.), and inhibitors of viral replication (e.g., viral polymerase specific inhibitors, broad-spectrum protease inhibitors, etc.), but most are still in the research stage. The high-throughput screening of the drug library approved by the FDA or the drug library of small molecular compounds on the market is performed, so that the new application of the existing drugs is discussed, and the method becomes an important way for drug development. The drug reuse or old drug new use has numerous advantages, for example, candidate drugs already have data about pharmacological efficacy tests, functional targets, clinical safety and the like, are favorable for further toxicology evaluation, pharmacokinetic evaluation, preparation research and development and the like, can greatly reduce research and development risks, shorten research and development time and research and development cost, and are one of effective means for coping with epidemic outbreaks such as sudden virulent infectious diseases and the like.

Through "old drug new use" studies, a number of old drugs have been found to have an inhibitory effect on the replication of new coronaviruses, with adefovir, fampicvir, chloroquine, hydroxychloroquine, and the like entering clinical studies, especially adefovir being the first drug approved by the U.S. FDA for use in the treatment of novel coronavirus infections. The discovery of antibodies developed against the S protein of the receptor of a novel coronavirus recognition host cell and the discovery of small molecule inhibitors developed against key proteins in the replication process of novel coronaviruses such as 3CL protease and RNA-dependent RNA polymerase are main directions in the research of innovative drugs for resisting novel coronavirus infection. In addition, the traditional Chinese medicine plays an important role in preventing and treating novel coronavirus infection, and Jin Huaqing sensory particles, lotus herb plague clearing capsules, xuebijing injection, shuanghuanglian oral liquid, lung-heat clearing and toxin expelling decoction, damp-resolving toxin-vanquishing prescription, lung-diffusing toxin-vanquishing prescription and the like enter clinical researches and applications of novel coronavirus infection treatment.

The compound SH, chinese also called as "Ji Ai Tesan", is an oral antiviral compound Chinese medicine, which is prepared from 5 Chinese herbal medicines of licorice, safflower, astragalus root, white mulberry root-bark, herba artemisiae capillaris, etc. according to the theory of Chinese medicine, according to the compatibility principle of 'monarch, minister, assistant and guide', extracts of the five medicines are reasonably compounded by different solvents. Compound SH is widely used in aids/aids patients in china and southeast asia. Clinical experiments show that SH is safe and effective, and can reduce the HIV viral load of 14% -35% of HIV positive patients when being singly used. In addition, SH and nucleoside analog reverse transcriptase inhibitors (NRTIs) combination therapy showed stronger antiviral activity than NRTIs alone. It has also been reported that SH binding to the protease inhibitor atazanavir enhances their inhibitory capacity. At present, no report on the preparation of coronavirus resistant medicines by using compound SH is yet seen.

Disclosure of Invention

In view of the above, the present invention aims to provide a new application of compound SH, in particular to an application of compound SH in preparing a broad-spectrum anti-coronavirus drug.

The invention provides application of compound SH in preparing a broad-spectrum anti-coronavirus medicament.

The invention provides application of compound SH in preparing inhibitors of broad-spectrum coronavirus replication.

Preferably, the coronavirus comprises an alpha group coronavirus and/or a beta group coronavirus.

Preferably, the group α coronavirus comprises HCoV-NL63 and/or HCoV-229E.

Preferably, the cell that inhibits replication of HCoV-NL63 is LLC-MK2;

the cell that inhibits replication of the HCoV-229E is MRC-5.

Preferably, the complex number of virus infection of HCoV-NL63 is 0.01, and the EC of compound SH ₅₀ 1.87. Mu.g/ml.

Preferably, when the complex number of virus infection of HCoV-229E is 0.01, the EC of compound SH ₅₀ 10.22. Mu.g/ml.

Preferably, the group β coronavirus comprises SARS-CoV-2 and/or HCoV-OC43.

Preferably, the cell inhibiting SARS-CoV-2 replication is Vero;

the cell inhibiting HCoV-OC43 replication is BHK-21.

Preferably, when the virus infection complex of SARS-CoV-2 is 0.01, the EC of compound SH ₅₀ 7.3. Mu.g/ml;

when the virus infection complex of HCoV-OC43 is 0.01, the EC of compound SH ₅₀ 4.4. Mu.g/ml.

The invention provides application of compound SH in preparing a broad-spectrum anti-coronavirus medicament. The test of the anti-coronavirus activity effect is carried out under the in vitro condition, and the result shows that the compound SH can effectively inhibit the replication of SARS-CoV-2 in Vero cells, and the half effective concentration EC of the drug with MOI=0.01 and 50% inhibition of the replication efficiency of SARS-CoV-2 is achieved ₅₀ 7.3. Mu.g/ml; the compound SH can effectively inhibit the replication of HCoV-OC43 in BHK-21 cells, and the half-effective concentration EC of the drug for inhibiting the replication efficiency of HCoV-OC43 with MOI=0.01 by 50 percent ₅₀ 4.4. Mu.g/ml; compound SH can effectively inhibit replication of HCoV-NL63 in LLC-MK2 cells, and half effective concentration of drug EC can inhibit replication of HCoV-NL63 with a replication efficiency of 0.01MOI by 50% ₅₀ 1.87. Mu.g/ml; compound SH can effectively inhibit the replication of HCoV-229E in MRC5 cells, so that the replication of HCoV-229E with MOI=0.01 is inhibited by 50 percentHalf effective concentration of the prepared medicine EC ₅₀ 10.22. Mu.g/ml. Meanwhile, the cytotoxicity detection experiment of the compound SH shows that the compound SH has CC in Vero, BHK-21, LLC-MK2 and MRC-5 cells ₅₀ Sequentially is>80. Mu.g/ml, 54.06. Mu.g/ml, 56.71. Mu.g/ml and 59.41. Mu.g/ml. The selection indices SI for the SH compound in Vero, BHK-21, LLC-MK2 and MRC-5 cells, which are important indicators for judging the effect of the drug, were calculated to be 10.9, 30.33, 12.83 and 5.81 for SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E, respectively. It can be seen that compound SH has good antiviral effect against various coronaviruses.

Drawings

FIG. 1 shows the inhibitory effect of compound SH at different concentrations on SARS-CoV-2 virus replication; the abscissa is the drug concentration of compound SH, and the ordinate is the inhibition rate, wherein the curve fitted by the dark square points represents the virus replication inhibition efficiency of SARS-CoV-2;

FIG. 2 shows the inhibitory effect of compound SH at different concentrations on replication of HCoV-OC43 virus; the abscissa represents the drug concentration of compound SH, and the ordinate represents the inhibition rate, wherein the curve fitted by the dark square points represents the inhibition efficiency of virus replication of HCoV-OC 43;

FIG. 3 shows the inhibitory effect of compound SH at different concentrations on replication of HCoV-NL63 virus; the abscissa represents the drug concentration of compound SH, and the ordinate represents the inhibition rate, wherein the curve fitted by the dark square points represents the inhibition efficiency of virus replication of HCoV-NL 63;

FIG. 4 shows the inhibitory effect of compound SH at different concentrations on replication of HCoV-229E virus; the abscissa represents drug concentration of compound SH and the ordinate represents inhibition rate, wherein the curve fitted by the dark square points represents the inhibition efficiency of viral replication of HCoV-229E.

Detailed Description

The invention provides application of compound SH in preparing a broad-spectrum anti-coronavirus medicament.

The invention provides application of compound SH in preparing inhibitors of broad-spectrum coronavirus replication.

The present invention is not particularly limited to the compound SH, and conventional sources of compound SH known in the art may be used. In an embodiment of the invention, the compound SH is purchased from thailand zetta limited.

In the present invention, the coronavirus preferably comprises an alpha group coronavirus and/or a beta group coronavirus. The group α coronavirus preferably comprises HCoV-NL63 and/or HCoV-229E. The cell inhibiting replication of HCoV-NL63 is preferably LLC-MK2, and the complex of HCoV-NL63 has a viral infection of 0.01, and the compound SH has EC ₅₀ Preferably 1.87. Mu.g/ml. CC for HCoV-NL63 in cytotoxicity experiments ₅₀ 56.71. Mu.g/ml. The selection index SI of HCoV-NL63 is 30.33. The cell that inhibits replication of the HCoV-229E is preferably MRC-5. When the virus infection complex of HCoV-229E is 0.01, the EC of compound SH ₅₀ Preferably 10.22. Mu.g/ml. In cytotoxicity experiments, CC of HCoV-229E ₅₀ 59.41. Mu.g/ml. The HCoV-229E has a selection index SI of 5.81.

In the present invention, the group β coronavirus preferably comprises SARS-CoV-2 and/or HCoV-OC43. The cell for inhibiting SARS-CoV-2 replication is preferably Vero; when the virus infection complex of SARS-CoV-2 is 0.01, the EC of compound SH ₅₀ Preferably 7.3. Mu.g/ml. CC of SARS-CoV-2 in cytotoxicity experiments ₅₀ Is > 80. Mu.g/ml. The selection index SI of SARS-CoV-2 is 10.91. The cell that inhibits HCoV-OC43 replication is preferably BHK-21. When the virus infection complex of HCoV-OC43 is 0.01, the EC of compound SH ₅₀ Preferably 4.4. Mu.g/ml. In cytotoxicity experiments, CC of HCoV-OC43 ₅₀ 56.46. Mu.g/ml. The selection index SI of HCoV-OC43 is 12.83.

The dosage form of the drug or the inhibitor is not particularly limited in the present invention, and the dosage form of the drug or the inhibitor known in the art may be used.

The application of the compound SH provided by the invention in preparing broad-spectrum anti-coronavirus medicines is described in detail below with reference to examples, but they are not to be construed as limiting the scope of the invention.

Example 1

Cytotoxicity detection of compound SH

This example relates to four coronaviruses, SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E, respectively, whose sensitive cell lines are Vero, BHK-21, LLC-MK2 and MRC-5, respectively. In order to test the safe drug concentration of compound SH, the toxicity of compound SH in these four sensitive cell lines was tested using the CCK-8 method in this example. The detection principle is as follows: cell Counting Kit-8 (abbreviated as CCK-8) is a detection reagent based on WST-8 and widely applied to cell proliferation and cytotoxicity. WST-8 chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazolium monosodium salt is a compound similar to MTT that is reduced by a dehydrogenase in mitochondria to an orange formazan product with high water solubility in the presence of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate. The more and faster the cells proliferate, the darker the color; the greater the cytotoxicity, the lighter the color, and for the same cells, the shade of color is proportional to the number of living cells, so that cell proliferation and toxicity analysis can be directly performed using this property. The light absorption value is measured at the corresponding wavelength by using an enzyme-labeled instrument, and the color shade is proportional to the number of living cells within a certain cell number range. The number of living cells is judged according to the measured absorbance value (OD value), and the greater the OD value is, the stronger the cell activity is, and the smaller the drug toxicity is when the drug toxicity is measured.

The specific operation is as follows: the four cells of Vero, BHK-21, LLC-MK2 and MRC-5 were each prepared according to a 3X 10 protocol ⁴ Cells/well were inoculated in 96-well plates and incubated with DMEM containing 10% fetal bovine serum for 16 hours to a cell density of 80%; then the culture solution is sucked and removed, and the culture solution is replaced by a DMEM culture medium containing 2% fetal calf serum; span Ai Tesan (Compound SH, available from Thailand Zetai Co., ltd.) is prepared by adding 10 ml DMSO to 100mg, vortex mixing and dissolving to 10mg/ml as stock solution, and refrigerating at 4deg.C for use. In application, 2% fetal bovine serum DMEM is diluted to 100, 20, 4, 0.8, 0.16, 0.032 μg/ml working solution, and the cells are added, and four groups of medicines with each concentration are arranged in parallel, and a blank control group (without medicines and cells) and a cell control group (without medicines) are arranged. Placing at 37deg.C, 5% CO ₂ Culturing in incubator for 72 hr, discarding supernatant, adding CCK-8 working solution into each well, incubating at 37deg.C for 1-4 hr, and using multifunctional enzyme-labeled instrumentThe absorbance was measured at a wavelength of 450 nm. The inhibition of cellular activity was calculated according to formula I:

cell activity inhibition ratio (%) = (drug group-blank group)/(cell control group-blank group) ×100% formula I

Calculating average value and standard deviation fitting curve by Graphpad Prism 8 software, and calculating cytotoxicity CC of compound SH after converting drug concentration into logarithmic value ₅₀ 。

The results are shown in Table 1, CC of Compound SH in Vero, BHK-21, LLC-MK2 and MRC-5 cells ₅₀ Respectively is>80. Mu.g/ml, 54.06. Mu.g/ml, 56.71. Mu.g/ml and 59.41. Mu.g/ml.

TABLE 1 cytotoxicity of Compound SH in different cells

Example 2

In vitro anti-coronavirus activity effect detection of compound SH

1 viral infection and pharmaceutical action

Vero, BHK-21, LLC-MK2 and MRC-5 cells were isolated according to 1X 10 ⁴ Cells/well were inoculated in 96-well plates and cultured with DMEM containing 10% fetal bovine serum for 16 hours to a cell density of 80%; then, the cell culture solution is sucked and removed, and the cell culture solution is replaced by a DMEM culture medium containing 2% fetal calf serum; when evaluating the effect of compound SH on resisting SARS-CoV-2 virus, adding compound SH into corresponding Vero cell holes according to the dosages of which the final concentrations are 80, 20, 5, 1.25, 0.31 and 0.08 mug/ml respectively; when evaluating the anti-HCoV-OC 43, HCoV-NL63 and HCoV-229E virus effects of the driver Ai Tesan, compound SH is added into corresponding cell holes according to the dosages of 100, 20, 4, 0.8, 0.16 and 0.032 mug/ml of final concentration respectively, and corresponding cell control holes without adding medicines and virus control holes only with viruses are arranged at the same time; within 1 hour after the addition of the drug, SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E were added to the corresponding sensitive cell wells at a volume of 10. Mu.l per well to give a Multiplicity of viral infection of 0.01 (multiplexing ofInfection, MOI=0.01), placed at 37 ℃,5% CO ₂ Supernatants were collected after 48 hours of incubation in incubators.

2. Inhibition effect of compound SH on SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E by fluorescent quantitative RT-PCR detection

The corresponding viral target genes of SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E were detected by using the absolute fluorescence quantitative RT-PCR method to reflect the replication level of the virus.

Viral RNA was extracted according to the Instructions of the Sesamum indicum RNA nucleic acid extraction Kit and RT-PCR was performed using the One Step Prime Script RT-PCR Kit (RR 064A). The primer probe sequences for detecting each virus are as follows:

the primer probe sequence for detecting SARS-CoV-2 is as follows:

the upstream primer sequence (q-SARS-CoV-2-F) is:

5’-CCCTGTGGGTTTTACACTTAA-3’(SEQ ID NO:1)；

the downstream primer sequence (q-SARS-CoV-2-R) is:

5’-ACGATTGTGCATCAGCTGA-3’(SEQ ID NO:2)，

the probe sequence (q SARS-CoV-2-probe) is:

5’-FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3’(SEQ ID NO:3)。

the primer probe sequence for detecting HCoV-OC43 is as follows:

the upstream primer sequence (q-OC 43-F) is:

5’-GCTCAGGAAGGTCTGCTCC-3’(SEQ ID NO:4)；

the downstream primer sequence (q-OC 43-R) is:

5’-TCCTGCACTAGAGGCTCTGC-3’(SEQ ID NO:5)，

the probe sequence (q-OC 43-probe) is:

5’-FAM-TTCCAGATCTACTTCGCGCACATCC-TAMRA-3’(SEQ ID NO:6)；

the primer probe sequence for detecting HCoV-NL63 is as follows:

the upstream primer sequence (q-NL 63-F) is:

5’-AGGACCTTAAATTCAGACAACGTTCT-3’(SEQ ID NO:7)，

the downstream primer sequence (q-NL 63-R) is:

5’-GATTACGTTTGCGATTACCAAGACT-3’(SEQ ID NO:8)，

the probe sequence (q-NL 63-probe) is:

5’-FAM-TAACAGTTTTAGCACCTTCCTTAGCAACCCAAACA-TAMR A-3’(SEQ ID NO:9)。

the primer probe sequence for detecting HCoV-229E is as follows:

the upstream primer sequence (q-229E-F) is:

5’-CGCAAGAATTCAGAACCAGAG-3’(SEQ ID NO:10)，

the downstream primer sequence (q-229E-R) is:

5’-GGCAGTCAGGTTCTTCAACAA-3’(SEQ ID NO:11)，

the probe sequence (q-229E-probe) is:

5’-HEX-CCACACTTCAATCAAAAGCTCCCAAATG-TAMRA-3’(SEQ ID NO:12)。

the reaction system is as follows: 10. Mu.L of 2 XOne Step SYBR RT-PCR Buffer III, 0.5. Mu. L Takara Ex Taq HS, 0.5. Mu. L PrimeScript RT Enzyme Mix II, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 5. Mu.L of RNA template, and make up to 25. Mu.L with sterile double distilled water.

The reaction parameters are as follows: 42 ℃ for 5min and 95 ℃ for 10 s; and (3) circulating for 40 times at 95 ℃ for 5s and 60 ℃ for 30s, and collecting fluorescent signals after extension. And 4 times of repetition are carried out on each sample, the CT value of the sample is counted, and the measured CT value of the sample is substituted into a standard curve to calculate the virus copy number in the sample. Viral replication inhibition was calculated according to formula II:

viral replication inhibition (%) = (viral control-drug control)/viral control x 100% formula II.

3. Fitting the curve and calculating EC ₅₀

Calculating the mean value of inhibition efficiency and standard deviation fitting curve graph by using Graphpad Prism 8 software, converting drug concentration into logarithm, and calculating EC of compound SH ₅₀ . The inhibition effects of compound SH on four coronaviruses of SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E are shown in figure 1, figure 2, figure 3 and figure 4 respectively.As shown in FIG. 1, compound SH can effectively inhibit the replication of SARS-CoV-2 in Vero cells, and the half-effective concentration EC of the drug can inhibit the replication of SARS-CoV-2 with MOI=0.01 by 50 percent ₅₀ 7.3. Mu.g/ml; as shown in FIG. 2, compound SH can effectively inhibit replication of HCoV-OC43 in BHK-21 cells, and half-effective concentration EC of drug for inhibiting replication efficiency of HCoV-OC43 by 50% with MOI=0.01 ₅₀ 4.4. Mu.g/ml; as shown in FIG. 3, compound SH is effective in inhibiting replication of HCoV-NL63 in LLC-MK2 cells, and half-effective concentration of drug EC is effective in inhibiting replication of HCoV-NL63 at 50% efficiency of 0.01MOI ₅₀ 1.87. Mu.g/ml; as shown in FIG. 4, compound SH can effectively inhibit replication of HCoV-229E in MRC5 cells, and half-effective concentration EC of drug for 50% inhibition of replication of HCoV-229E with MOI=0.01 ₅₀ 10.22. Mu.g/ml.

4. Calculation of the selection index SI

By drug toxicity CC ₅₀ Concentration of drug effect EC ₅₀ The ratio of (a) can be calculated to obtain a selection Index SI (SI), which is an important indicator for judging the efficacy of a drug. Wherein the index is selected>1, the medicine is effective and safe, the larger the SI value is, the safer the medicine is, and the more obvious the effect is. Cytotoxicity test results CC obtained according to embodiment 1 ₅₀ And half-effective concentration of the virus-inhibited drug EC obtained in example 2 ₅₀ The selection index SI is further calculated according to formula III.

Selection index si=cc ₅₀ /EC ₅₀ Formula III

The selection indexes SI of the compound SH in Vero, BHK-21, LLC-MK2 and MRC-5 cells for SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E are respectively 10.9, 30.33, 12.83 and 5.81. The result shows that the compound SH has good antiviral effect on four human coronaviruses of SARS-CoV-2, HCoV-OC43, HCoV-NL63 and HCoV-229E.

TABLE 2 Compound SH broad-spectrum anti-coronavirus effect study

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Application of compound SH in preparing broad-spectrum anti-coronavirus medicine.

2. Application of compound SH in preparing inhibitor of broad-spectrum coronavirus replication.

3. The use according to claim 1, wherein the coronavirus comprises an alpha group coronavirus and/or a beta group coronavirus.

4. The use according to claim 3, wherein the group α coronavirus comprises HCoV-NL63 and/or HCoV-229E.

5. The use according to claim 4, wherein the cell inhibiting replication of HCoV-NL63 is LLC-MK2;

the cell that inhibits replication of the HCoV-229E is MRC-5.

6. The use according to claim 5, wherein the complex of HCoV-NL63 virus infection is 0.01, the EC of compound SH ₅₀ 1.87. Mu.g/ml.

7. The use according to claim 5, wherein the complex of HCoV-229E has an EC of compound SH at a viral multiplicity of infection of 0.01 ₅₀ 10.22. Mu.g/ml.

8. The use according to claim 3, wherein the group β coronavirus comprises SARS-CoV-2 and/or HCoV-OC43.

9. The use according to claim 8, wherein the cell that inhibits SARS-CoV-2 replication is Vero;

the cell inhibiting HCoV-OC43 replication is BHK-21.

10. The use according to claim 9, wherein the compound SH has an EC at a complex number of viral infections of SARS-CoV-2 of 0.01 ₅₀ 7.3. Mu.g/ml;

when the virus infection complex of HCoV-OC43 is 0.01, the EC of compound SH ₅₀ 4.4. Mu.g/ml.