WO2006080589A9

WO2006080589A9 - Sphingolipid-peg derivatives and composition for skin external use containing the derivatives

Info

Publication number: WO2006080589A9
Application number: PCT/KR2004/003382
Authority: WO
Inventors: Sungjin Park; You-A Hwang; Jinwook Kim; Changseo Park
Original assignee: Doosan Corp; Sungjin Park; You-A Hwang; Jinwook Kim; Changseo Park
Priority date: 2004-12-21
Filing date: 2004-12-21
Publication date: 2007-01-25
Also published as: WO2006080589A1

Abstract

Disclosed are sphingolipid-PEG derivatives, which comprise sphingolipid selected from the group consisting of N-acetylphytosphigosine, N-acetylsphiganine and N-acetylsphingadiene and polyethylene glycol, and compositions for skin external use containing the derivatives. The sphingolipid-PEG derivatives and the compositions exhibit an improved skin whitening effect. Further, the derivatives and compositions show an improved solubility of sphingolipid and a decreased toxicity and, accordingly, can be used broadly.

Description

[DESCRIPTION]

[Invent ion Tit Ie]

SPHINGOLIPID-PEG DERIVATIVES AND COMPOSITION FOR SKIN EXTERNAL USE CONTAINING THE DERIVATIVES

[Technical Field]

<i> The present invention relates to sphingolipid derivatives and a composition containing the derivatives. More specifically, the present invention relates to sphingolipid-polyethylene glycol derivatives formed by combining sphingolipid selected from a group consisting of N- acetylphytosphingosine, N-acetylsphinganine and N-acetylsphingadiene with polyethylene glycol and a skin whitening use of the derivatives.

[Background Art]

<2> Hyperpigmentation in the skin gives a serious mental stress from a skin esthetic view and also a serious hindrance to a social activity. In addition, according to social changes of that a white and delicate skin is a standard of beauty, developments of a whitening agent are actively performed. <3> Recent researches of the whitening agent are focused on controlling various factors participating in a melanin synthesis process. For example, the researches are progressed to a ultraviolet block, a control of cytokine or hormone, a control of an internal signal of melanocyte, an inhibition of activity of tyrosinase enzyme, an inhibition of movement of melanosome, and a quick desquamation of melanin pigmented skin cell.

<4> Sphingolipid is known as a material participating in a signal transduction in a cell and thus performing an important role in growth, differentiation and death of the cell. Ceramide is the most existing lipid in the skin and known as a material suppressing an evaporation of moisture from the skin and preventing an aging and a cutaneous disorder. In addition, it is known that phytosphingosine, which is a long chain base of sphingolipid, or acetylated derivatives thereof have excellent antibacterial and anti^¬ inflammatory effects and participate in biosynthesis of ceramide in the skin. However, a research of ceramide or sphingolipid derivatives in keratinocytes

i has been actively progressed, but a research of melanin synthesis is not currently much progressed.

<5> Kim, et al. (Kim, Dong-Seok, Cellular signaling 14 (2002) 779-785) performed a research on a participation in melanin synthesis using sphingolipid derivatives for the first time, and reported that ceramide derivatives (C2 ceramide) exhibit an excellent effect of suppressing melanin synthesis at 1 ~ 10 μ M, compared to kojic acid. In addition, it was reported that sphingosine-1-phosphate suppresses the melanin synthesis through a control of a signal transduction process of the melanin synthesis. Meanwhile, Doosan Corporation proved N-acetylphytosphingosine, which is a derivative of phytosphingosine, to have a whitening effect and filed the patent application regarding it (Korean Patent Application No.2002-72043).

<6> Meanwhile, the sphingolipid has a very low solubility, so that it has many restrains when it is used in many formulations.

<7> Polyethylene glycol is a polymer having a structure of HO- (CH₂CH₂O)UCH₂CH₂-OH, frequently used in foods or cosmetics and has an effect of retarding a degradation of an active material, so that it has been much used in a pharmaceutical prescription. The polyethylene glycol is an innoxious polymer, has a good solubility, and has no antigenicity and is easily removed in the human body, so that it is a suitable material for the purposes. Since the polyethylene glycol has hydroxy groups at both ends, monomethoxy polyethylene glycol (CH₃O-(CH₂CH₂O)nCH₂CH₂-0H) having one end inactivated is actually much used in PEGylation and those having the molecular weight of up to 30,000 Daltons are used. In order to bind monomethoxy polyethylene glycol to a medicine, a hydroxy group at one end having no methoxy connected thereto is activated and connected by using diverse chemicals.

<8> Lewis S. L. et al. (U.S. Patent No. 5,820,873, 1995) tried to synthesize polyethylene glycol-ceramide derivatives and to apply them to 1 iposome. [Disclosure] [Technical Problem]

<9> The object of the present invention is to prevent hyperpigmentation of melanin by using sphingolipid derivatives and thus suppressing an activity of tyrosinase, thereby preventing skin pigmentary symptoms such as melasma, ephelis, senile pigment spots and skin hyperpigmentation, etc. The other object of the invention is to provide a composition for skin external use having such effects.

[Technical Solution]

<10> In order to accomplish the object, there is provided a sphingolipid- polyethylene glycol derivative formed by combining sphingolipid selected from a group consisting of N-acetylphytosphingosine, N-acetylsphinganine and N- acetylsphingadiene with polyethylene glycol.

<1 1 > The sphingolipid-polyethylene glycol derivative is one of compounds having following formulas 1 to 3.

[Chemistry Figure 1]

<13>

[Chemistry Figure 2]

<15> [Chemi stry Figure 3]

<17>

<i8> In the above formulas, Ri is independently hydrogen, alkyl group of Ci-

₄0, alkenyl group of Ci-₄₀, alkynyl group, acyl group or aryl group of Ci-₄o. <19> When Ri is acyl group (COR2) , R2 is alkyl group, alkenyl group, alkynyl group or aryl group. <20> X is -NR3-, -0-, -S- or -Xi-alk-X₂-, wherein R3 is hydrogen, alkyl group, acyl group or aryl group of Ci-6, Xi and X₂ are independently amino group, amido group, carboxy group, cabamate group, carbonyl group, urea or phosphoro-, and alk is alkylene of Ci-6.

<2i> The sphingolipid-polyethylene glycol derivative of the invention has polyethylene glycol having a molecular weight of 550 ~ 10,000Da, more preferably 700-5,000Da and an end having a methoxy group replaced. <22> A composition for skin external use according to the invention contains the sphingolipid-polyethylene glycol derivative as described above as an effective ingredient. <23> The composition for skin external use according to the invention suppresses an activity of tyrosinase. <24> The composition for skin external use according to the invention is used for skin whitening. <25> In the composition for skin external use according to the invention, the composition is a cosmetics composition. <26> According to the invention, the composition for skin external use contains 0.005 ~ 10 wt.% of the sphingolipid-polyethylene glycol derivative. <27> A method of suppressing an activity of tyrosinase according to the invention uses the sphingolipid-polyethylene glycol derivative. <28> A method of suppressing a melanin synthesis according to the invention uses the sphingolipid-polyethylene glycol derivative.

[Advantageous Effects] <29> The sphingolipid-polyethylene glycol derivatives of the invention inhibit the melanin synthesis by suppressing the tyrosinase activity, thereby preventing and treating skin pigmentary symptoms such as melasma, ephelis, senile pigment spot, and skin hyperpigmentat ion, etc.

[Description of Drawings] <30> FIG. 1 is a photograph showing a melanin production inhibiting effect of a sphingolipid-polyethylene glycol derivative according to an embodiment of the invention; <3i> FIG. 2 is a graph showing a standard curve of a variation of a OD value (475 nm) according to the amounts of tyrosinase; and <32> FIG. 3 is a graph showing a tyrosinase activity inhibiting effect of a sphingolipid-polyethylene glycol derivative according to an embodiment of the invention. [Best Mode] <33> Hereinafter, preferred embodiments of the present invention will be described in detail. <34> The invention is characterized in that a whitening effect is increased by combining polyethylene glycol with sphingolipid. The inventors proved that sphingolipid-polyethylene glycol derivatives have an excellent whitening effect, compared to sphingolipid itself. <35> A typical example of the sphingolipid-polyethylene glycol of the invention is those that R₁ is independently hydrogen, alkyl group or acyl group, X is succinate, and the polyethylene glycol has a molecular weight of 750-5,000 and its one end has a methoxy group replaced.

<36> The sphingolipid-polyethylene glycol derivative of the invention may be manufactured by reacting sphingolipid with polyethylene glycol previously activated in a solvent under a catalyst. <37> The manufacturing method is more specifically described as follows. Firstly, polyethylene glycol is activated. Succinic anhydride is added to the polyethylene glycol in the organic solvent and thus alcohol group is converted to carboxyl group. In the polyethylene glycol having carboxyl group replaced, an acid portion is activated using triethylamine and p- toluenesulfonylchloride, sphingolipid is added to the activated solution by a small amount, the added solution is warmed and thus the 24 hours reaction is completed.

<38> The compound made by the above method is extracted with an organic solvent such as chloroform or a mixing solvent of chloroform/methanol and purified with an adsorption-chromatography by silica gel.

<39> The composition of the invention is characterized in that it is a composition for skin external use. Particularly, the composition may be a cosmetics composition and is preferably water lotion, milk lotion, massage cream, nutri-cream, gel, pack or skin ropy type of cosmetics formulation.

<40> The composition of the invention contains 0.005 - 10 wt.% of the above sphingolipid-polyethylene glycol derivative. A whitening effect of the invention can be achieved when a content of the derivative is 0.005 wt.% or more, and a formulation stability is excellent when the content is 10 wt.% or less .

<4i> In the sphingolipid-polyethylene glycol derivatives of the invention, a molecular weight of the polyethylene glycol is preferably 550 ~ 10,000Da, more preferably 700 ~ 5,000Da. The range of the molecular weight is an optimal range to achieve an object to combine the polyethylene glycol with sphingolipid in the invention.

<42> In addition, the composition may be a transdermal administration type formulation such as lotion, ointment, gel, cream, patch or aerosol.

<43> The skin color of human is determined by various skin ingredients. Among them, melanin produced by melanocyte plays the most important role. The melanocyte of the skin is regulated to exhibit a certain degree of the skin color by an inherited character according to races. However, when it is stimulated by ultraviolet, stress, cytokine, and melanin synthesis inducing materials such as a melanocyte-stimulat ing hormone (MSH), melanin synthesis is increased and the synthesized melanin moves to adjacent keratinocytes via melanosomes and is finally subject to a process of desquamation from the skin.

<44> Generally, a temporary change of the skin color returns to the previous skin color through the above mechanism as time goes by. However, when the skin is exposed to sunlight for a long time, it often occurs that the skin color is irreversibly changed to a black.

<45> Melanin is synthesized in melanosome in the melanocyte, which is originated in endoplasmin reticulum, and regularly accumulated by an internal structure of the melanosome. Tyrosinase which converts tyrosine into melanin is produced in golgi apparatus and moves to the melanosome. In the melanosome prior to maturity, tyrosine causes an oxidation-reaction by tyrosinase and biosynthesis of melanin starts. The melanosom in which the melanin synthesis begins passes through about four steps of maturity processes and then generation of matured melanosome is completed. The matured melanosome moves to adjacent keratinocytes, and the skin color is determined according to the number, sizes and distributions of the melanin in the keratinocytes. The melanosome moves from a base layer of epidermis to keratinocyte, and to stratum corneum while protecting the keratinocyte. During moving, the melanosome is disintegrated, but the melanin maintains at its un- disintegrated state and is finally desquamated from the skin.

<46> The tyrosinase playing the most important role in the melanin biosynthesis is synthesized in the golgi apparatus, and then moves to the melanosome while being subject to a glycosylation process. After moving to the melanosome, it is converted into an activated form by phosphorylation.

<47> The melanin synthesis begins when the activated tyrosinase causes an oxidation-reaction using the tyrosine as a substrate. The tyrosine is converted into DOPA (dihydroxyphenylalanine) by tyrosinase, which is again converted into DOPA quinone by tyrosinase. The DOPA quinone couples with glutathione or cysteine and thus is converted into cysteinyl DOPA and finally into pheomelanin. On the other hand, after the DOPA quinone is converted into DOPAchrome, it goes through 5,6-dihydroxyindole then is converted into indole-5,6-quinone by an action of the tyrosinase, and finally into eumelanine. The tyrosinase participates in the last stage as well as in the early stage of the melanin synthesis reaction and thus is known as the most important enzyme of the melanin synthesis process. Many materials having an effect of inhibiting melanin formation were developed to inhibit an activity of tyrosinase. The composition of the invention contains also sphingolipid such as N-acetylphytosphingosine, etc. inhibiting the activity of tyrosinase.

<48> Each of eumelanin and pheomelanin synthesized in the melanocyte exists in a proper ratio different from each other according to races or parts in the human body, thereby causing a difference of the skin colors. The eumelanin exhibits black and brown colors, and the pheomelanin exhibits an orange color and is synthesized from two kinds of proteins, i.e., tyrosine and cysteine.

<49> The melanin functions to protect the skin from ultraviolet. It is a useful material to protect skin organs under the dermis and simultaneously to catch active oxygen and free radicals generated from the skin, thereby protecting the proteins and nuclei acids. However, when melanin having the useful functions as described above is abnormally produced and pigmented to the skin, melasma, ephelis, and skin dyspigmentation occur.

<50> When the composition of the invention is a transdermal administration type formulation of pharmaceutical composition, a preferred dosage is 0.001-1000 mg/body weight (kg) two times per a day, based on the composition containing 0.005 - 10 wt.% of N-acetylphytosphingosine-polyethylene glycol derivative. [Mode for Invention]

<5i> Hereinafter, preferred examples of the invention will be more specifically explained. However, the invention is not limited to such examples. <52> Examples

<53> <Manufacturing example 1: manufacture of sphingolipid-polyethylene glycol derivatives of the invention>

<54> 5 g (0.0067 mole) of methoxy polyethylene glycol was dissolved in pyridine solvent under atmosphere of nitrogen gas and 6.67 g (0.067 mole) of succinic anhydride was slowly added to the solution. After then, the solution was subject to a reaction at room temperature for 24 hours under a state that the light was blocked. After the completion of the reaction, pyridine was converted into a salt form by adjusting to pH 2 by adding hydrochloric acid, and was extracted and removed by adding chloroform and water. Similarly, an excessive succinic anhydride was also extracted and removed with the water layer. 1 g (0.0011 mole) of methoxy polyethylene glycol-carboxy1 acid obtained as above was dissolved in dichloromethane solvent under atmosphere of nitrogen gas and carboxyl group was activated by using 0.17 ml (0.0017 mole) of triethylamine and 2.11 g (0.011 mole) of p-toluenesulfonylchloride. After that, N-acetylphytosphingosine 0.22 g (0.0011 mole) was slowly added to the solution and then was subject to a reaction for 24 hours while warming. After the completion of the reaction, the reaction was stopped by extracting with distilled water, and purified with an adsorption-chromatography by silica gel, so that N-acetylphytosphingosine-polyethylene glycol derivative of the invention was obtained. The succinate, which was introduced in methoxy polyethylene glycol by reacting with succinic anhydride, was confirmed with IH NMR (δ = 2.6 ppm, t, 4H), and finally methylene (δ = 1.2 ppm, m, 48H and d = 0.9 ppm, t, 6H) of N-acetylsphingosine and ethoxy (δ = 3.6 ppm, m, 64H) and methoxy (δ = 3.3 ppm, s, 3H) of methoxy polyethylene glycol were confirmed. Accordingly, it was confirmed that the derivative having methoxy polyethylene glycol and N-acetylphytosphingosine coupled was synthesized.

<55>

<56> <Examples 1 to 6: manufacture of cream compositions>

<57> The inventors manufactured preferred embodiments of the invention as follows. The below examples 1 to 6 cream compositions contain different concentrations of N-acetylphytosphingosine-polyethylene glycol derivatives. Meanwhile, a cream composition according to a comparative example as a negative control group for comparing with the Examples 1 to 6 was also manufactured. <58> [Table 1]

<59> <6»> <60>

<67>

<68>

<69>

<70>

<71>

<72>

The inventors performed experiments for proving a whitening effect of sphingolipid-polyethylene glycol of the invention as follows.

<73>

<74> <Experimental example 1: inhibiting effects of sphingolipid- polyethylene glycol derivatives on the melanin production>

<75> Melanin production inhibiting effects of each of N- acetylphytosphingosine-polyethylene glycol derivative, N-acetylsphinganine- polyethylene glycol derivative, N-acetylsphingadiene-polyethylene glycol derivative, N-acetylphytosphingosine, N-acetylsphinganine, N- acetylsphingadiene and kojic acid were measured. The above materials were dissolved in DMSO (Dimethyl sulfoxide) and then used. In addition, cells strains to be used in this example were prepared by culturing B16F10 Melanoma which is a cell of skin cancer of mouse in DMEM (Dulbecco^' s Modified Eagle Medium) containing 10% FBS (Fetal bovine serum) and antibiotics in a CO₂ incubator supplied with an air containing 5% CO₂, at 37°C . <76> A method of measuring melanin content is as follows. B16F10 mouse

3 melanoma cells were inoculated on 24-well multi-plate with 2x 10 cells/well and cultured in 5% CO₂ incubator for 24 hours. After pre-culturing, test materials were diluted in DMEM containing no serums, treated to the cell according to the concentrations thereof and then cultured for 96 hours. After washing with PBS, 0.85 N KOH was added and dissolved with ultrasonic waves, and an absorbance was measured at 475 nm and thus an amount of melanin was measured. On one hand, eumelanin and pheomelanin were subject to a fractional quantitative analysis using a high speed chromatography and production percents for a control group were obtained. The results are shown in table 2. The resultant values are production percents of melanin and indicate production percents (%) for a non-treated group.

<77>

<78> [Table 2] <79>

As can be seen from the above result, the concentration inhibiting the melanin synthesis of about 90% in all samples was about 100 μ M in the case of the Kojic acid. To the contrary, sphingolipid-polyethylene glycol derivative had a melanin synthesis inhibiting effect of about 90% at 1O yM which is 1/10 compared to the kojic acid. Although it is not described in this example, it was found that arbutin had an equal level of an inhibiting effect at 10 times concentration of the kojic acid and at 100 times of the compound of the invention. Further, as can be seen from the table, the sphingolipid-polyethylene glycol derivative had a melanin synthesis inhibiting effect twice or more as much as the sphingolipid.

<80> Meanwhile, the inventors photographed that the cell strains used in this experiment was collected using a centrifugation. This is shown in Fig. 1. In Fig. 1, the color of the lower part indicates a degree of melanin synthesis. As shown in Fig. 1, it can be seen that N-acetylphytosphingosine- polyethylene glycol derivative inhibited the melanin synthesis and thus color of the cell lump was not changed to a black. That is, since the color is more lighter compared to the N-acetylphytosphingosine, it could be seen that the N-acetylphytosphingosine-polyethlene glycol derivative had a more excellent effect of inhibiting the melanin priduction.

<81>

<82> <Experimental example 2: inhibiting effect of N-acetylsphingosine- polyethylene glycol derivatives on tyrosinase activity >

<83> The inventors performed an experiment in order to examine the effect of N-acetylphytosphingosine-polyethylene glycol derivatives on tyrosinase activity as follows.

<84> In this experiment, N-acetylphytosphingosine-polyethylene glycol derivative was dissolved in DMSO (Dimethyl sulfoxide) as in the experimental example 1. In addition, cells strains to be used in the invention were prepared by culturing B16F10 Melanoma which is a cell of skin cancer of mouse in a CO2 incubator supplied with an air containing 5% CO2, at 37°C using DMEM

(Dulbecco' s Modified Eagle Medium) containing 10% FBS (Fetal bovine serum) and antibiotics. A method of measuring a tyrosinase activity inhibition is as follows. B16F10 mouse melanoma cells were inoculated on 96-well multi-plate

4 with Ix 10 cells/well and cultured in 5% CO2 incubator for 24 hours. After

pre-culturing, test materials were diluted in DMEM containing no serums, treated to the cell according to their concentrations and then cultured for 96 hours. Then, they were washed twice with PBS. 1% Triton- X-100/PBS was added to it. After dissolving with ultrasonic waves, 5 μ 1 of 10 mM L-Dopa previously prepared to be 37^°C was added and reacted for 30 minutes at 37°C . Then, an absorbancy was measured at 475 nm. The results are shown in Fig. 3. The resultant values are tyrosinase activities and indicate activity (%) for a non-treated group. Fig. 2 shows a standard curve of tyrosinase. <85> As can be seen from Fig. 3, it could be seen that N- acetylphytosphingosine-polyethylene glycol derivatives had an effect of inhibiting about 50% of tyrosinase enzyme activity at about 10 μ M. Equally to the results of the experiment of melanin synthesis, this showed that N- acetylphytosphingosine-polyethylene glycol derivatives having concentrations of 1/10 of kojic acid and 1/100 of arbutin had an outstandingly excellent inhibitory effect, compared to the arbutin or kojic acid.

<86>

<87> <Experimental example 3: whitening effect of cream composition of the invention on the human skin>

<88> The inventors performed this experiment to examine whether the composition of the invention actually exhibits a whitening effect on the human skin. It was measured whitening effects of the cream compositions of the examples 1 to 6, and a whitening effect of the comparative example 1 was also measured to compare with the effects.

<89> For 20 healthy male persons, opaque tape having 1.5 cm diameter of perforation was stuck to the region of the arm of each of the examinees. Then, UVB having 1.5 to 2 times of minimal erythema dose of each examinee was applied to induce blackening of the skin. After that, the cream compositions of the examples 1 to 6 and the cream composition of the comparative example 1 were respectively applied, and light and darkness of the skin was measured using a spectrophotometer after two months. Each of test materials was applied two times every day at morning and evening.

<90> The effects were judged by calculating a L value indicating the light and darkness of the skin. <91> Δ L = L value on a last day at which the test material was applied -L value before applying the test material.

<92> The results are shown in table 3. <93> <94> [Table 3] <95>

<96>

<97> As can be seen from the results, it could be confirmed that the sphingolipid-polyethylene glycol derivatives of the invention exhibited very excellent whitening effects according to its concentration.

[Industrial Applicability]

<98> As described above, the sphingolipid-polyethylene glycol derivatives of the invention inhibit the melanin synthesis by suppressing the tyrosinase activity, thereby preventing and treating skin pigmentary symptoms such as melasma, ephelis, senile pigment spot, and skin hyperpigmentation, etc.

Claims

[CLAIMS] [Claim 1]

<100> A sphingolipid-polyethylene glycol derivative formed by combining sphingolipid selected from a group consisting of N-acetylphytosphingosine, N- acetylsphinganine and N-acetylsphingadiene with polyethylene glycol. [Claim 2]

<101> The sphingolipid-polyethylene glycol derivative according to claim 1, wherein the derivative is one of compounds having following formulas 1 to 3.

<102> [Chemistry Figure 1]

<104>

<105>

[Chemistry Figure 2]

<107> [Chemistry Figure 3]

<109>

<110> , wherein Ri is independently hydrogen, alkyl group of Ci-40, alkenyl group of C1-4Q, alkynyl group, acyl group or aryl group of Ci-40,

<111> when R₁ is acyl group (COR2), R2 is alkyl group, alkenyl group, alkynyl group or aryl group, <ii2> and X is -NR₃-, -0-, -S- or -Xi-alk-X₂-, wherein R3 is hydrogen, alkyl group, acyl group or aryl group of Ci-6, X₁ and X2 are independently amino group, amido group, carboxy group, cabamate group, carbonyl group, urea or phosphoro-, and alk is alkylene of C₁^.

[Claim 3]

<ii3> The sphingolipid-polyethylene glycol derivative according to claim 2, wherein the polyethylene glycol has a molecular weight of 550 ~ 10,000Da.

[Claim 4]

<ii4> The sphingolipid-polyethylene glycol derivative according to claim 3, which has polyethylene glycol having a molecular weight of 700 ~ 5,000Da and an end having a methoxy replaced.

[Claim 5]

<ii5> A composition for skin external use containing the sphingolipid- polyethylene glycol derivative according to any one of claims 1 to 4 as an effective ingredient.

[Claim 6]

<ii6> The composition according to claim 5, wherein the composition suppresses a tyrosinase activity.

[Claim 7]

<ii7> The composition according to claim 5, wherein the composition is a skin whitening composition.

[Claim 8]

<ii8> The composition according to claim 5, wherein the composition is a cosmetics composition.

[Claim 9]

<ii9> The composition according to claim 5, wherein the composition contains 0.005 ~ 10 wt.% of the sphingolipid-polyethylene glycol.

[Claim 10] <i20> A method of suppressing a tyrosinase activity using the sphingolipid- polyethylene glycol derivative according to any one of claims 1 to 4.

[Claim 11]

<i2i> A method of suppressing a melanin synthesis using the sphingolipid- polyethylene glycol derivative according to any one of claims 1 to 4.