Use of a rapamycin composition

Abstract

This invention relates to the use of rapamycin, monolaurate and monomyristate as carriers, and water in the preparation of a medicament for treating vascular abnormalities of the skin of human subjects, by locally administering the medicament once daily to the abnormality at a ratio of 4 to 8 mg of rapamycin per square centimeter and / or 1 to 6 mg of rapamycin per square centimeter.

Description

Rapamycin Compositions The present invention relates to rapamycin compositions for administration to humans and to methods of producing or using rapamycin compositions. Rapamycin was isolated from a sample of Streptomyces hygroscopicus around 1972. The compound was initially developed as an antifungal agent and its production is described in U.S. Patent No. 3,929,992 to Ayerst McKenna and Harrison. Rapamycin has the following structural formula: . One problem with rapamycin is that it often requires long-term use and accumulates in the bloodstream to an undesirable extent over time, resulting in adverse side effects such as stomatitis, pneumonitis, hyperlipidemia, anemia, thrombocytopenia, renal toxicity, joint pain, and oedema. In addition, rapamycin is a drug classified by the U.S. FDA as Category C, and animal studies have shown adverse effects on developing fetuses, and high levels of rapamycin can be a potential problem for women during pregnancy. Objectives of the Invention An objective of a preferred embodiment of the present invention is to solve the above problems at least to some extent. While this applies to the preferred embodiment, it should be understood that the objectives of the present invention itself are not limited thereto, and it only provides a useful option for the public. Therefore, any objective, advantage, or benefit of any preferred embodiment should not be construed as a limitation on any claimed patent scope of a broader statement. Definitions When used in combination with respect to a combination of features or steps herein, the term "comprising" or its derivatives, such as "comprises", should not be construed as excluding the option of the presence of additional features or steps not yet mentioned. Thus, the term is inclusive rather than exclusive. First Aspect - Treatment Method According to the first aspect, the present invention is a method for treating vascular abnormalities of the skin of a human subject, comprising topically administering to the abnormality a drug (e.g., a composition) comprising: a) rapamycin; b) monolaurin and monomyristin as carriers; and c) water; once daily in the following ratio: d) 4 to 8 milligrams per square centimeter (mg / cm 2rapamycin; and / or e) 1 to 6 mg of rapamycin. Second Aspect - Use in Manufacturing According to the first aspect, the present invention is a use of a) rapamycin; b) monolaurate and myristate as carriers; and c) water in the preparation of a drug (e.g., a composition) for treating vascular abnormalities of the skin of a human subject, by topically administering once daily to the abnormality in the following proportions: d) 4 to 8 mg of rapamycin per square centimeter; and / or e) 1 to 6 mg of rapamycin. Alternatively, for the second aspect, the present invention is a use of a composition in the preparation of a drug for treating vascular abnormalities of the skin of a human subject, the composition comprising: a) rapamycin; b) monolaurate and myristate as carriers; and c) water; by topically administering the drug to the abnormality once daily in the following proportions: d) 4 to 8 mg of rapamycin per square centimeter; and / or e) 1 to 6 mg of rapamycin. Third Aspect - Composition for Treatment According to the third aspect, the present invention is a drug (e.g., a composition) comprising: a) rapamycin; b) monolaurate and myristate as carriers; and c) water; the drug being for treating vascular abnormalities of the skin of a human subject by topically administering once daily to the abnormality in the following proportions: d) 4 to 8 mg of rapamycin per square centimeter; and / or e) 1 to 6 mg of rapamycin. Fourth Aspect, Further Method of Treatment According to the fourth aspect, the present invention is a method of treating vascular abnormalities of the skin of a human subject, comprising topically administering a drug (e.g., a composition) to the abnormality once daily for at least 1, 2, 3, 4, 5, or 6 months, the drug (e.g., a composition) comprising: a) rapamycin, as the active ingredient; b) a carrier comprising monolaurate and myristate; and c) water, as the solvent; such that at the end of administration for at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin (if any) in the bloodstream of the subject does not exceed 1, 2, 3, 4, or 5 ng / mL (and preferably does not exceed 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 ng / mL at the end of one or more months). For the avoidance of doubt, any value in ng / mL herein can apply to any time period specified herein. [Optional Features] The following optional features apply to all first, second, third, and fourth aspects of the present invention, in each case alone or in any combination with one or more of the following features or feature groups, unless they are mutually exclusive. [Dosage ratio] The drug is administered once daily or used for dosing at the following ratio: a) rapamycin at 5 to 7 mg per square centimeter; and / or b) rapamycin at 2 to 5 mg. The drug is administered once daily or used for dosing at the following ratio: c) rapamycin at about 6 mg per square centimeter; and / or d) rapamycin at 3 to 4 mg. The drug is administered for at least 1, 2, 3, 4, 5, or 6 months. The drug is administered or used for dosing such that at the end of at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin (if any) in the bloodstream of the subject does not exceed 1, 2, 3, 4, or 5 ng / mL. The drug is administered or used for dosing such that at the end of at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin (if any) in the bloodstream of the subject does not exceed 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 ng / mL. [Component content] The drug comprises 0.5 to 5 wt% of rapamycin. The drug comprises 0.5 to 1 wt% of rapamycin. The drug comprises approximately 0.5% of rapamycin. The drug comprises approximately 1% of rapamycin. [Treatment conditions] The vascular anomaly includes angiofibroma. The vascular anomaly includes facial angiofibroma. The vascular anomaly includes cutaneous vascular lesions. The vascular anomaly includes port-wine stain. The port-wine stain is treated after laser treatment. [Further features] The drug comprises approximately 7 wt% to 28 wt% or approximately 5 wt% to 10 wt% or approximately 7 wt% of monolaurate. The drug comprises approximately 7 wt% to 28 wt% or approximately 15 wt% to 25 wt% or approximately 21 wt% of myristate. The drug comprises: a) 5 wt% to 9 wt% of monolaurate + 19 wt% to 23 wt% of myristate; or b) 12 wt% to 16 wt% of monolaurate + 12 wt% to 16 wt% of myristate. The drug includes: a) about 7 wt% monolaurate + about 21 wt% myristate; or b) about 14 wt% monolaurate + about 14 wt% myristate. [Humectant] The drug includes a humectant. The humectant includes polyoxyethylene stearate. [Softening agent] The drug includes a softening agent. The softening agent includes one or more of propylene glycol, paraffin wax, lanolin, mineral oil, glycerol, lecithin, and sorbitol. [Buffer agent] The drug includes a buffer agent. The buffer agent includes one or more of anhydrous citric acid, sodium bicarbonate, and triethanolamine. [Chelating agent] The drug includes a chelating agent. The chelating agent includes one or more of disodium edetate, citric acid, and tetrasodium ethylenediaminetetraacetate (EDTA). [pH adjuster] The drug includes a hydroxide for pH adjustment. [Preservative] The drug includes a preservative. The preservative includes one or more of potassium sorbate, diazolidinyl urea, phenoxyethanol, and sodium hydroxymethylglycinate. [Water] The drug includes 58 wt% to 72 wt% water. [Other features] The drug includes: a) 0.5 wt% to 5 wt% rapamycin; b) a carrier including: 7 wt% to 28 wt% monolaurate; and 7 wt% to 28 wt% myristate; and c) water as a solvent. The drug includes 0.5 wt% to 5 wt% rapamycin, 5 wt% to 9 wt% monolaurate, and 19 wt% to 23 wt% myristate. The drug includes 0.5 wt% to 5 wt% rapamycin, 7 wt% monolaurate, and 21 wt% myristate. The drug includes 0.5 wt% or 1 wt% rapamycin, 7 wt% monolaurate, and 21 wt% myristate. The drug includes 0.5 wt% to 5 wt% of rapamycin, 12 wt% to 16 wt% of monolaurate, and 12 wt% to 16 wt% of monomyristate. The drug includes 0.5 wt% to 5 wt% of rapamycin, 14 wt% of monolaurate, and 14 wt% of monomyristate. The drug includes 0.5 wt% or 1 wt% of rapamycin, 14 wt% of monolaurate, and 14 wt% of monomyristate. The drug includes 0.5 wt% to 5 wt% of rapamycin, 17.5 wt% (±2 wt %) of monolaurate, and 10.5 wt % (±2 wt %) of monomyristate. The drug includes 0.5 wt% to 5 wt% of rapamycin, 17.5 wt% of monolaurate, and 10.5 wt% of monomyristate. The drug includes 0.5 wt% or 1 wt% of rapamycin, 17.5 wt% of monolaurate, and 10.5 wt% of monomyristate. The drug includes 0.5 wt% or 1 wt% of rapamycin, 21 wt% of monolaurate, and 7 wt% of monomyristate. The monolaurate is glyceryl monolaurate and the monomyristate is glyceryl monomyristate. The drug includes: a) 0.5 wt% to 5 wt% of rapamycin; b) a carrier including: 10 wt% to 18 wt% of monolaurate; and 10 wt% to 18 wt% of monomyristate; and c) water as a solvent. [All statements] The drug components listed in any of the foregoing aspects or alternatively can be ±5% or ±10% of the indicated values. For example, 0.5% of rapamycin means ±5% of rapamycin, or 1% of rapamycin means ±5% of rapamycin. Example Formulations 1 and 2 Two preferred embodiments of the present invention, namely Formulation 1 and Formulation 2, are used for the topical treatment of skin vascular abnormalities. Without limiting the scope of the present invention, these diseases can include human angiofibromas, such as facial angiofibromas or cutaneous vascular lesions. The formulations can also be used to treat port-wine stains, for example, to limit the possibility of spot regeneration after laser treatment. The substantial compositions of the formulations are shown in Table 1 below The composition according to the present invention, including Formulation 1 and Formulation 2, can be produced according to the following method. Generation of the aqueous pre-mixture The aqueous pre-mixture is produced as follows: ● Rapamycin is combined with propylene glycol by vortex mixing; ● Water is added while continuously vortex mixing; ● Subsequently, the mixture is heated to 70°C (±5°C); ● Citric acid, disodium EDTA, and potassium sorbate are added and vortex mixed until dissolved; ● Subsequently, the temperature of the mixture is adjusted to 50°C; and ● Polyoxyethylene (100) stearate is added. Generation of the oil-phase pre-mixture The oil-phase pre-mixture is produced as follows: ● Glycerol monolaurate and glycerol monomyristate are combined by low-speed vortex mixing and heated to 70°C (±5°C). Final mixture Both the aqueous and oil-phase pre-mixtures are combined at 70°C (±5°C) and thoroughly mixed until homogeneous. Then the mixture is slowly cooled at a rate of 1°C per minute and vortex mixed until a smooth, white, and iridescent cream is formed. When the cream is cooled to no more than 32°C, it is packaged in a 30 mL aluminum tube with a polypropylene screw cap. Example Formulations 3 and 4 For the same purpose, Formulations 3 and 4 are produced in the same manner as described for Formulations 1 and 2. They are listed in Table 2 below. Particle size characteristics Figures 1 and 2 show the characteristics of the rapamycin active ingredient in the 1% cream identified above as Formulation 2. Information is obtained by analyzing the formulation using a Malvern Morphologi GS3 image analyzer. This analyzer is actually an automated microscope that scans the formulation to detect the 3D shape characteristics of rapamycin particles. Referring to Figure 1, the CE diameter ( "circle equivalent diameter") value represents the particle size weighted by number. In other words, it represents the particle as a 2D shape converted to the closest applicable circle, and then the diameter of the circle is calculated. Referring to the specific symbols shown, D[n,0.10](μm): 3.17 means that based on the number of particles, 10% of the particles are 3.17 μm or smaller; D[n,0.16](μm): means that 16% are less than 3.83 μm; and D[n,0.50](μm): means that based on the number of particles, 50% are less than 8.33 μm, etc. Referring to Figure 2, the image shows the shape and relative size of the rapamycin particles in Formulation 2. Figures 1 and 2 show that rapamycin is not dissolved in the remainder of the composition but is suspended in the carrier or vehicle component. Trial - Rapamycin Blood Concentration Method A multicenter, double - blind, randomized, placebo - controlled, dose - response parallel study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines. The aim was to evaluate the efficacy of topical treatment of cutaneous vascular anomalies, including whether rapamycin transfers into the blood when topically applied via Formulation 1 or Formulation 2, and if so, the extent of transfer. The study consisted of 107 human participants aged 6 to 65 years, diagnosed with facial angiofibromas associated with tuberous sclerosis. The severity of the facial angiofibromas was mild or moderate. In other words, each subject had a score of 2 or 3 on the Investigator Global Assessment (IGA) scale. Participants were randomly assigned to one of three treatment groups: 0.5% rapamycin cream (Formulation 1; n = 36), 1% rapamycin cream (Formulation 2; n = 33), or placebo cream (i.e., the same excipient without active ingredient; n = 38). The formulations and placebo were applied topically to the angiofibromas once daily for 26 weeks at a rapamycin dose of 6 mg per square centimeter of skin. Given that different participants had different sizes of angiofibroma areas, they did not all receive the same amount of formulation. However, the average daily amount of rapamycin administered per group is shown in Table 3 below. Safety and efficacy were evaluated on Days 14, 56, 98, 140, and 182 (visits 1 - 5, respectively). The primary endpoint was based on the IGA score after 26 weeks of treatment. Secondary endpoints included the Facial Angiofibroma Severity Index (FASI) and percentage - based improvement reported by the subjects and clinicians. All enrolled participants were included in the final analysis. Results The degree of improvement at the end of the trial period is shown in Table 4 below. Participants whose IGA score did not improve by at least 1 point were not included in the above results. Figure 3 shows an example of reduction of facial angiofibroma in a person who received treatment with Formulation 2 above in the trial. This individual had an IGA score of 3 at the start of the trial and an IGA score of 2 at the 5th visit. Table 5 below reports the rapamycin content in the blood of different participants. The value of the number of participants "N" at each visit point is less than the number of people enrolled in each group because some people did not provide test samples. In some cases, this was due to the reluctance of pediatric participants (children) to donate blood at certain test points. For example, for visit point 1 with 1% rapamycin, the number of subjects tested was N = 26, compared to the total number of people in the group participating in the trial being N = 33. However, no rapamycin was detected in any participant who returned a test sample at any test stage, except for one participant in the 0.5% formulation group at test point 1 (day 14) with a rapamycin content of 1.1 ng / ml in the blood. In the trial, both Formulation 1 and Formulation 2 were well-tolerated and were effective in reducing facial angiofibromas in most participants. Notably, this was achieved without rapamycin entering the bloodstream, except for one exception noted in the day 14 test, but this did not recur in any subsequent test stage / visit. At 6 months, no rapamycin was detected in the blood of the participants tested. Given the expectation that rapamycin would enter the bloodstream, especially considering the length of time the drug was taken and the dosage administered, these results are striking. As a comparison, the commercial product HYFTOR™ gel was tested. This is a topical gel containing 0.2 wt% rapamycin for the treatment of tuberous sclerosis and similar diseases. This product was topically applied twice daily to the angiofibroma sites of 30 human participants. The average total daily dose per participant is shown in Table 6 below. Tests were conducted to determine the amount of rapamycin in the bloodstream during the administration of HYFTOR™ gel, and the results are shown in Table 7. Comparison Formulation 1 & 2 compared to HYFTOR™ gel As can be seen from Tables 3 and 6, Formulation 1 and 2 administered more rapamycin to the trial participants compared to HYFTOR™ gel. Especially in the case of Formulation 2, subjects aged ≥ 12 years received an average of 5.67 mg of rapamycin per day, while subjects in the same age group received a dose of 1.6 mg per day of HYFTOR™ gel. Although there was a larger amount of rapamycin in Formulation 2, no rapamycin could be detected in the system / bloodstream, while rapamycin was detected in the system / bloodstream in HYFTOR™ gel. This suggests that formulations based on monolaurate and monomyristate carriers can be used more safely to deliver large amounts of rapamycin to patients in need of high doses. In addition, the results indicate that even for those who require only a small amount of rapamycin, formulations based on monolaurate and myristate reduce the likelihood of rapamycin being picked up in the system / bloodstream. Disclosed As to the disclosure, this disclosure hereby discloses combinations of one or more of each item, feature or step mentioned herein with any other item, feature or step disclosed herein, in each case whether or not such combinations are claimed. Variation Although some preferred embodiments of the invention have been described by way of example, it is to be understood that modifications and improvements can be made without departing from the scope of the following claims. None Some preferred embodiments of the invention will be described with reference to the following drawings, in which: FIG. 1 shows the results obtained from a particle size analysis of a 1% rapamycin composition for topical application; FIG. 2 shows images of rapamycin particles in the same 1% composition; FIG. 3 provides "before and after" pictures of the skin of a person being treated for angiofibroma according to an embodiment of the invention.

Claims

1. The use of a) rapamycin; b) monolaurate and monomyristate as carriers; and c) water in the preparation of a medicament for treating vascular abnormalities of the skin of human subjects, said medicament being locally administered to said abnormality once daily in the following proportions: d) 4 to 8 mg of rapamycin per square centimeter; and / or e) 1 to 6 mg of rapamycin.

2. The use as described in claim 1, wherein, The drug is to be administered once daily to the abnormality in the following proportions: a) 5 to 7 mg of rapamycin per square centimeter; and / or b) 2 to 5 mg of rapamycin.

3. The use as described in claim 1, wherein, The drug is to be administered once daily to the abnormality in the following proportions: a) approximately 6 mg of rapamycin per square centimeter; and / or b) 3 to 4 mg of rapamycin.

4. The use as described in claim 1, wherein, The drug is administered to the patient for at least 1, 2, 3, 4, 5, or 6 months for the aforementioned abnormality.

5. The use as described in claim 1, wherein, The drug is administered to the abnormality such that, at the end of at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin in the bloodstream of the subject does not exceed 1, 2, 3, 4, or 5 ng / mL, if any.

6. The use as described in claim 1, wherein, The drug is administered such that, at the end of at least 1, 2, 3, 4, 5 or 6 months of administration, the concentration of rapamycin in the bloodstream of the subject, if any, does not exceed 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 ng / mL.

7. The use as described in claim 1, wherein, The drug comprises 0.5 to 5 wt% rapamycin.

8. The use as described in claim 1, wherein, The drug comprises 0.5 to 1 wt% rapamycin.

9. The use as described in claim 1, wherein, The drug comprises 0.5 wt% rapamycin.

10. The use as described in claim 1, wherein, The drug comprises 1 wt% rapamycin.

11. The use as described in claim 1, wherein, The vascular abnormalities include angiofibromas.

12. The use as described in claim 1, wherein, The vascular abnormalities include facial angiofibromas.

13. The use as described in claim 1, wherein, The vascular abnormalities include skin vascular lesions.

14. The use as described in claim 1, wherein, The vascular abnormalities include burgundy herniated spots.

15. The use as described in claim 14, wherein, The drug is used to treat burgundy herpes zoster that has been treated with laser.

16. The use as described in claim 1, wherein, The drug comprises 7 wt% to 28 wt% or 5 wt% to 10 wt% or 7 wt% of monolaurate.

17. The use as described in claim 1, wherein, The drug comprises 7 wt% to 28 wt% or 15 wt% to 25 wt% or 21 wt% of myristate ester.

18. The use as described in claim 1, wherein, The drug comprises: a) 5 wt% to 9 wt% of monolaurate + 19 wt% to 23 wt% of monomyristate; or b) 12 wt% to 16 wt% of monolaurate + 12 wt% to 16 wt% of monomyristate.

19. The use as described in claim 1, wherein, The drug comprises: a) about 7 wt% monolaurate + about 21 wt% monomyristate; or b) about 14 wt% monolaurate + about 14 wt% monomyristate.

20. The use as described in claim 18, wherein, The drug comprises 1 wt% rapamycin.

21. The use as described in claim 2, wherein, The drug includes a water-retaining agent.

22. The use as described in claim 21, wherein, The water-retaining agent includes polyoxyethylene stearate.

23. The use as described in claim 22, wherein, The drug includes a softener.

24. The use as described in claim 23, wherein, The softening agent includes one or more of propylene glycol, paraffin oil, lanolin, mineral oil, glycerin, lecithin, and sorbitol.

25. The use as described in claim 22, wherein, The drug includes a buffer.

26. The use as described in claim 25, wherein, The buffer includes one or more of anhydrous citric acid, sodium bicarbonate, and triethanolamine.

27. The use as described in claim 25, wherein, The drug includes chelating agents.

28. The use as described in claim 27, wherein, The chelating agent includes one or more of disodium edetate, citric acid, and tetrasodium ethylenediaminetetraacetate.

29. The use as described in claim 27, wherein, The drug includes a hydroxide for pH adjustment.

30. The use as described in claim 29, wherein, The drug includes preservatives.

31. The use as described in claim 30, wherein, The preservatives include one or more of potassium sorbate, diazoalkylurea, phenoxyethanol, and sodium hydroxymethylglycine.

32. The use as described in claim 1, wherein, The drug comprises 58 wt% to 72 wt% water.

33. The use as described in claim 1, wherein, The drug comprises: a) 0.5 to 5 wt% rapamycin; b) a carrier comprising: 7 to 28 wt% monolaurate; and 7 to 28 wt% monomyristate; and c) water as a solvent.

34. The use as described in claim 1, wherein, The drug comprises 0.5 wt% to 5 wt% of rapamycin, 5 wt% to 9 wt% of monolaurate, and 19 wt% to 23 wt% of monomyristate.

35. The use as described in claim 34, wherein, The drug comprises 0.5 wt% to 5 wt% rapamycin, 7 wt% monolaurate, and 21 wt% monomyristate.

36. The use as described in claim 34, wherein, The drug comprises 1 wt% rapamycin, 7 wt% monolaurate, and 21 wt% monomyristate.

37. The use as described in claim 1, wherein, The drug comprises 0.5 wt% to 5 wt% rapamycin, 12 wt% to 16 wt% monolaurate, and 12 wt% to 16 wt% monomyristate.

38. The use as described in claim 1, wherein, The drug comprises 0.5 wt% to 5 wt% rapamycin, 14 wt% monolaurate, and 14 wt% monomyristate.

39. The use as described in claim 1, wherein, The drug comprises 0.5 wt% or 1 wt% rapamycin, 14 wt% monolaurate, and 14 wt% monomyristate.

40. The use as described in claim 1, wherein, The drug comprises 0.5 wt% to 5 wt% rapamycin, 17.5 wt% ± 2 wt% monolaurate, and 10.5 wt% ± 2 wt% monomyristate.

41. The use as described in claim 1, wherein, The drug comprises 0.5 wt% to 5 wt% rapamycin, 17.5 wt% monolaurate, and 10.5 wt% monomyristate.

42. The use as described in claim 1, wherein, The drug comprises 0.5 wt% or 1 wt% rapamycin, 17.5 wt% monolaurate, and 10.5 wt% monomyristate.

43. The use as described in claim 1, wherein, The drug comprises 0.5 wt% or 1 wt% rapamycin, 21 wt% monolaurate, and 7 wt% monomyristate.

44. The use as described in claim 1, wherein, The monolaurate is glyceryl monolaurate and the monomyristate is glyceryl monomyristate.

45. The use as described in claim 1, wherein, The vascular abnormalities include angiofibromas, cutaneous vascular lesions, or burgundy herpes zoster.

46. ​​The use as described in claim 1, wherein, The drug comprises: a) 0.5 wt% to 5 wt% rapamycin; b) a carrier comprising: 10 wt% to 18 wt% monolaurate; and 10 wt% to 18 wt% monomyristate; and c) water as a solvent.

47. The use as described in claim 2, wherein, The drug is administered to the abnormality such that, at the end of at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin in the bloodstream of the subject does not exceed 1, 2, 3, 4, or 5 ng / mL, if any.

48. The use of a) rapamycin as an active ingredient; b) a carrier comprising monolaurate and monomyristate; and c) water as a solvent in the preparation of a medicament for treating vascular abnormalities of the skin of a human subject, comprising topically applying the medicament to the abnormality once daily for at least 1, 2, 3, 4, 5, or 6 months, such that at the end of at least 1, 2, 3, 4, 5, or 6 months, the concentration of rapamycin in the bloodstream of the subject, if any, does not exceed 1, 2, 3, 4, or 5 ng / mL.

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