FR2669535A1 - Use of polysulphonated macromolecules as a medicament - Google Patents

Abstract

The invention relates to the use of polysulphonated macromolecules as medicaments, in particular as an antiviral agent. These macromolecules can be, in particular, polymers, polysaccharides or polypeptides.

Description

USE AS A MEDICINAL PRODUCT
OF POLYSULFONE MACROMOLECULES
The present invention relates to the use as a medicament of polysulfone macromolecules.

 According to the present invention, the term "polysulfone macromolecule" means molecules with a molecular mass of between 1,500 and 50,000 Dalton and preferably comprising at least 5, more preferably at least 7 sulphonic or sulphonate radicals, ie in acid form -SO3H or anionic -SO3, linked to the macromolecule by a carbon-sulfur bond.

More particularly, the present invention relates to the use of these polysulphonated macromolecules as an antiviral agent and in particular an active agent against the HIV 1 and HIV 2 viruses responsible for the
AIDS.

 According to the invention, the antiviral activity, in particular against the most important HIV1 virus, is obtained for macromolecules with a molecular mass between 2,500 and 10,000 Dalton.

 The preferred macromolecules according to the invention will have low toxicity, in particular with a CD50 value of greater than 1 mg / ml, more preferably greater than 2.5 mg / ml.

 In a preferred embodiment, the macromolecules according to the invention consist of the repetition or sequence of units or "monomeric" links, identical or different, sulfonated, preferably each comprising (e) at least one sulfonic radical or sulfonate.

 The term "monomer" is broadly defined and encompasses both conventional synthetic chemical monomers and amino acids or saccharide units.

 Thus, the present invention relates in particular to polysulphonated polymers but also to polysulphonated polypeptides or to polysulfone polysaccharides.

 Among the polymers, mention is made of homopolymers for example, poly (sulfonated styrene), poly (sulfonated ethylene), poly (sulfonated propylene), poly (sulfonated butylene), poly (urethane sulfonated), poly (acrylamide sulfone).

 Among the polymers, mention may also be made of biodegradable polymers of the sulphonated polylactide and polyglycolide type, that is to say, in general, the polymers resulting from the polymerization of monomers chosen from the intermolecular cyclic esters of sulphonated alpha hydroxycarboxylic acids.

 Of the polypeptides, homopolypeptides, i.e., poly (sulfonated amino acids), are preferred. For example, poly (sulfonated tyrosine), sulfonated poly (lysine), poly (sulfonated alanine), especially poly (cysteic acid) and sulfonated poly (phenylalanine) are exemplified.

 Among the polysaccharides, mention is made of sulphonated dextrins such as polysulphonated dextran, polysulfonated pentosan, polysulphonated mannan or mucopolysaccharides such as sulphonated heparins.

 Suitably, polysulfone cyclodextrins are used.

 The polysulfonated macromolecules according to the invention are commercially available being used for other applications or can be prepared if necessary by methods known to those skilled in the art (Journal of Organic Chemistry, Vol 55, nd 14 - July 6, 1990 "Synthesis of carbohydrate sulfonates and sulfonates Ester").

Thus, suitably, the sulphonic or sulphonate radical may be grafted directly, in particular on an aromatic ring or by substitution of a hydroxyl group of a monomeric unit of the macro molecule (with first substitution with a halogen then substitution of halogen by a sulfonate group via the reaction of
Strecker).

 The sulphonic or sulphonate radical may also be grafted indirectly onto the monomeric unit of the macromolecule, that is to say, in particular by means of the grafting of a sulphonated, aminoalkylsulphonated or alternatively acylsulphonated alkyl group.

pour m = 3 ou Cl(CH2)mSO3H pour m = 1 à 5, en milieu basique.The polysulfonation of the macromolecule may result from the sulfonation of monomeric units (including amino acid or saccharide) constituting it by reaction on an OH or amino group (-NH-) of said units with substitution of the hydrogen of said group by a group - (CH2) mSO3H with in particular m = 1 to 5, preferably 2 or 3. This reaction is made from the reagent

for m = 3 or Cl (CH2) mSO3H for m = 1 to 5, in basic medium.

en le substituant d'abord par

en faisant d'abord réagir ladite fonction hydroxyle avec le réactif de formule

suivi par une réaction de Strecker (substitution du C1 par SO 3 ou à partir d'une forme activée de l'acide sulfoacétique

Otherwise, the hydrogen of an OH group may be substituted by an acylsulfone group such as

substituting it first with

by first reacting said hydroxyl function with the reagent of formula

followed by a Strecker reaction (substitution of C1 with SO 3 or from an activated form of sulfoacetic acid

Ar is an aryl group especially phenyl especially from sulfobenzoic anhydride.

In other embodiments, the OH group of the macro molecule may be substituted by
an alkylsulphonated group such as - (CH2) mSO3H, the introduction of the alkylsulphonated chain being carried out by alkylation of an iodinated intermediate with the isopropyl mesylate anion followed by deprotection with ammonia or
an aminoalkylsulphonated group such as -NH (CH2) mSO3H, the incorporation of the aminoalkylsulphonated chain being carried out by conversion of a tosylate intermediate with the aminoalkylsulphonic acid.

avec de préférence n = 5 à 10 - R = H, acyle, benzoyle ou alkyle, - W = OH ou X-Y-SO3H avec de préférence au moins 5 unités dans lesquelles W = X-Y-SORH

avec R' = alkyle en C1-C7 ou aryle, notamment phényle - Y = un groupe hydrocarboné en C1-C20 tel que alkyle, aryle, éventuellement substitué par un groupe alkyle, aryle ou carboxylique et pouvant contenir un hétéroatome.Thus, when the polysaccharide is of the cyclic type, mention is made in particular of a sulfonated cyclodextrin of formula

with preferably n = 5 to 10 - R = H, acyl, benzoyl or alkyl, - W = OH or XY-SO3H with preferably at least 5 units in which W = XY-SORH

with R '= C 1 -C 7 alkyl or aryl, especially phenyl - Y = a C 1 -C 20 hydrocarbon group such as alkyl, aryl, optionally substituted by an alkyl, aryl or carboxylic group and which may contain a heteroatom.

Cyclodextrins of formula I are more particularly mentioned

<tb> with <SEP> W <SEP> = <SEP> - (CH2) <SEP> m5 <SEP> O3H, <SEP> -O- (CH2) m-S03H, <SEP> -O- <SEP> C- (CH <SEP>) <SEP> S03H,
<tb><SEP> O
<tb><SEP> -O- <SEP> C-Ar-SO3H, <SEP> -NHAlkSO3H, <SEP> with <SEP> of <SEP> preference <SEP> m <SEP> = <SEP> 1 <SEP > to <SEP> 5,
<Tb>
Alk which is a C1 to C20 hydrocarbon group such as an optionally substituted C1-C7 alkyl and Ar is an aryl especially phenyl.

The macromolecules according to the invention are used as antiviral agents. They are particularly active against HIV 1 and HIV 2 viruses that cause AIDS, SIV and FIV viruses (simian immunodeficiency and
Feline immunodeficency), HSV-1 and HSV-2 viruses responsible for herpes, cytomegalovirus, vaccinia virus (DNA virus) as well as polio virus, coxsackie B4 virus, Sindbis virus, Semliki Forest virus, Influenza virus, varicella virus, Para-Influenza virus, Respiratory syncytial virus, Junin virus, Tacaribe virus, Vesicular stomatitis virus, Reovirus, Moloney murine Sarcoma virus (RNA virus).

 It appears that the polysulfonated macromolecules according to the invention act by inhibiting the adhesion of the virus to the cell.

Some polysulfated polyanions have been described in the literature as possessing antiviral properties, in particular the
Dextran sulfate. However, it has been found that the polysulfonated macromolecules according to the invention possess this same property to a greater degree.

All the compounds according to the invention or their pharmaceutically acceptable salts when they are in a therapeutically effective amount are particularly useful against the HIV1 virus responsible for the
AIDS. They may be used for the preparation of a pharmaceutical composition for the treatment of AIDS.

pour m = 3 ou Cl-(CH2)m-CH2-SO3H pour m = 1 à 5, en milieu basique. Other advantages and features of the present invention will become apparent in the light of the following examples.
EXAMPLE 1 Polysulfonation of Macromolecules
1 - As we have seen, the sulfonation may consist in particular in the substitution of the hydrogen of an OH or -NH group of the "monomeric" units by a - (CH 2) m -SO 3 H group from the reagent

for m = 3 or Cl- (CH2) m -CH2-SO3H for m = 1 to 5, in basic medium.

pourra être obtenu à partir de la polytyrosine.
Avec notamment m = 1 à 5 de préférence 2 ou 3.1) Poly (sulfonated amino acid)
Thus in this way for example, the poly (sulfonated tyrosine) of formula

can be obtained from polytyrosine.
With in particular m = 1 to 5, preferably 2 or 3.

permet d'obtenir le poly(uréthane sulfoné) de formule

avec m = 1 à 5 de préférence 2 ou 3.2) Polysulfonated Polymers
Similarly, for example, formula polyurethane

allows to obtain the sulfonated polyurethane of formula

with m = 1 to 5, preferably 2 or 3.

R2 of the general formula is determined by the diol: which is used for the diisocyanate formation (in which polyurethane according to the shet

<tb><SEP> of <SEP> polyurethane, <SEP> while <SEP> than <SEP> R1 <SEP> depends <SEP> on
<tb> R = R1) <SEP> used <SEP> for <SEP> the <SEP> formation <SEP> of
<tb> a <SEP> next: <SEP> nO = C = NRN = C = O <SEP> + <SEP> nHO-R-OH
<Tb>

 - It is the NH groups that are used after for the grafting of the sulfonic function, for example via propane sultone.

Two examples are given below

Ref. Macromolecules, 1988, 21, 998-1004

Ref. Polymer, 1989, Vol. 30, 1204 and following.

3) Polysulfonated cyclodextrins
3-1) Cyclodextrin derivatives containing sulphonated functional groups can be prepared as described in US 3426011.

By reaction of the cyclodextrin with sodium 2-chloroethanesulfonate and pyridine in toluene, there is obtained a mixture of cyclodextrin derivatives in which a number of hydroxyl groups have been converted into sulfoethyl ether functions. Sulfopropyl ether derivatives may also be prepared by reaction with propanesultone and 50% sodium hydroxide according to the following scheme 1

<tb><SEP> dn <SEP> 7
<tb><SEP> 50 <SEP> o / o <SEP> NaOH
<tb> Pyridine, <SEP> Toluene <SEP> 50 <SEP> COH
<tb> Pyridine, <SEP> Toluene
<tb> acid <SEP> 2-Chloroethanesulfonic acid
<tb><SEP> -sq
<tb><SEP> ~ <SEP> rPz
<tb><SEP> on
<tb><SEP> n <SEP> c.a '(;; Ô5 <SEP> to <SEP> 10
<Tb>
SCHEME 1
3-2 Cyclodextrin derivatives can also be obtained by acyl sulfonation.These acyl derivatives result from a treatment of cyclodextrin by: - sulfobenzoic anhydride or
an activated form of sulfoacetic acid or
Chloroacetic acid chloride followed by
substitution reaction of halogen with an ion
sulphite (according to Strecker's reaction), according to
Figure 2 below

<SEP>? <SEP> OH
<tb><SEP> ~ I
<tb><SEP> Let <SEP> SO3 <SEP> ccI, CooHctj <SEP> \ <SEP> o <SEP>Â;; C
<tb><SEP> Ivel <SEP> 1 ~ Vh
<tb><SEP> Let <SEP> SO ~ <SEP> 3 - ((S <) <SEP> mCOOH ('2acti3ve) <SEP> \
<tb><SEP> c
<tb><SEP> Q <SEP> 50
<tb> (Ç <SEP> 3
<tb><SEP> È.
<Tb>

SCHEME 2
3-3) Substitution of an OH group by -SO3H.

The syntheses of toodyl cyclodextrins, mesylates and iodines are described. Hexakis (6-O-tosyl) -cyclodextrin and the analog heptakis (6-O-tosyl) -B cyclodextrin were prepared from the parent molecules by treatment with six or seven equivalents of tosyl chloride in pyridine. Peracetyl heptakis (6-iodo) -B cyclodextrin was obtained by reacting the heptakis totylate derivative with sodium iodide in acetic anhydride according to scheme 3 below.

<Tb>

<SEP> 5
<tb><SEP> s
<tb><SEP>'OH<SEP> 3
<tb><SEP> 3 <SEP> Z
<tb><SEP> OH <SEP> n
<tb><SEP> CHLORIDE <SEP> FROM <SEP> TOSYLE
<tb><SEP> YRIDINE
<tb> L) os <SEP> NaI
<tb><SEP> 6 <SEP> 6
<tb><SEP> 5 <SEP> Nal <SEP> s
<tb><SEP>'<SEP> OH <SEP> AC2O <SEP>'<SEP> OAc
<tb><SEP> 3 <SEP> z <SEP> 3 <SEP> z
<tb><SEP> OH <SEP> OAc <SEP> n
<Tb>
SCHEME 3
These derivatives can be used as intermediates for the synthesis of sulfonated cyclodextrin analogues according to scheme 4.

Indeed, the introduction of an alkylsulfonated chain can also be achieved by alkylation of a 6-iodo cyclodextrin with the anion isopropylmesylate followed by deprotection with ammonia according to the method described in J. Org-Chem. 1990, 55, 4231.

SCHEMA 4
3-4) The incorporation of an aminoalkylsulfonated chain can be carried out by transformation of tosylate cyclodextrins with 2-amino-ethanesulphonic acid (or its salt), with N- (2-aminoethyl) -2 aminoethanesulphonic acid. (or its salt) or by a derivative of the acid
L-cysteic according to scheme 5 below:

- le polyacrylamide sulfoné de formule

lucide (poly (2-acrylamido -2 méthyl -1 propane sulfonique))
référence 19, 1973-3 de la firme ALDRICH.With for example
R = - (CH2) 2SO H
R = -CH-CH2-SO3H
COOR
EXAMPLE 5 4) Other Examples of Polysulfonation of Macromolecules: Polyalkylene Sulfonates of Formula

the sulphonated polyacrylamide of formula

lucid (poly (2-acrylamido-2-methylpropanesulphonic acid))
reference 19, 1973-3 of the firm ALDRICH.

a polymer with a biodegradable skeleton of formula

This polymer can be obtained in particular by oxidative degradation of a polysaccharide
It is a degradation of the polysaccharide (for example dextran) by sodium periodate. The bonds 2-3 and 3-4 are broken under these conditions, the carbon 3 disappears in the form of formic acid and the other two are oxidized to aldehydes. A reduction with borohydride makes it possible to obtain the corresponding diol. This process is described in "The
Polysaccharides "Vol.1 p.81 and following Academic Press 1982, Scheme

dextran

Then: .So a direct sulfonation (via an intermediate Strecker reaction) according to the scheme

<tb> HO + H2-CHOCH-iH <SEP> Direct <SEP> Sulfonation <SEP> H-O + H2-CFHOCH + H
<tb><SEP> HH2 <SEP> l <SEP> H2 <SEP> via <SEP> a <SEP><SEP> Reaction of <SEP> H2H
<tb><SEP> OH <SEP> OH <SEP> Strecker <SEP> O3 <SEP> i:
<tb><SEP> 503
<Tb>
Sulfone propane sulfonation according to the scheme

- la polylysine sulfonée

avec m = 1 à 5 de préférence 2 à 3.<tb> HO + H2-1CHOCH- + H <SEP> Sulfonation <SEP> to <SEP><SEP> W <SEP> = HZH-OHC <SEP> -H
<tb><SEP> 2 <SEP> C :: H2 <SEP> propane <SEP> sultone <SEP> CH2 <SEP> CH2
<tb><SEP> OH <SEP> OH
<tb><SEP> I <SEP> I
<tb><SEP> (cl2) 3 <SEP> (cl2) 3
<tb><SEP> I <SEP> I
<tb><SEP> so3 <SEP> 503 <SEP> n
<tb> - sulfonated polyphenylalanine

- the sulfonated polylysine

with m = 1 to 5, preferably 2 to 3.

- le poly(mannose pyranosyl sulfoné) ou mannan polysulfonate

- le poly(xylose pyranosyl sulfoné) ou pentosan polysulfonate

(CH2) 4-NH- (CH2) m-SO3- poly (glucose pyranosyl sulfonated) or dextran polysulfonate

poly (sulfonated mannose pyranosyl) or mannan polysulfonate

poly (xylose pyranosyl sulfone) or pentosan polysulfonate

EXAMPLE 2 Anti-HIV Activity of Different Polystyrene Polymers
polysulfonate.

des différents poids moléculaires suivants : 1800, 5400, 8000, 18 000, 35 000, 46 000 et 100 000.Formula

different molecular weights: 1800, 5400, 8000, 18,000, 35,000, 46,000 and 100,000.

These polymers are marketed in particular by the firm
MACHEREY-NAGEL.

 1. Results on MT-4 cells infected with the HIVI virus.

 The products at different concentrations were placed in the presence of MT4 cells (3 × 10 4 cells per microtray well), infected or not with 100 CCID50 of HIV-1 virus (strain HTLV-IIIB). After 5 days of incubation at 37 ° C, the number of living cells was determined by the MTT method, based on the reduction by living cells of 3 '- (4,5-dimethylthiazol-2-yl) -2 5-diphenyltetrazoliumbromide, yellow, formazan, blue.

CD50 = cytotoxic dose 50 = dose that kills 50% of the cells.

ED50 = effective dose 50 = dose that protects 50% of the cells
viral infection.

These two values can be easily obtained by measuring cell viability. CD50
SI = selectivity index =
ED50
TABLE 1

<tb> Macromolecules <SEP> (PM) <SEP> CD-50 <SEP> ED-50 <SEQ> SI
<tb><SEP> ug / ml <SEP> Imi <SEP>
<tb><SEP> SPS <SEP> 1600 <SEP> 1000 <SEP> 0.56 <SEP> 1788
<tb><SEP> SPS <SEP> 5400 <SEP> 1000 <SEP> 0.12 <SEQ> 8644
<tb><SEP> SPS <SEP> 8000 <SEP> 450 <SEP> 0.15 <SEP> 2987
<tb><SEP> SPS <SEP> 18000 <SEP> 219 <SEP> 0.66 <SEP> 333
<tb><SEP> SPS <SEP> 35000 <SEP> 624 <SEP> 0.63 <SEP> 998
<tb><SEP> SPS <SEP> 46000 <SEP><SEP><SEP> 167 <SEP> 2.14 <SEP> 78
<tb><SEP> SPS <SEP> 100000 <SE> 140 <SE> 3.74 <SEP> 37
<tb><SEP> DS <SEP> 5000 <SEP> 1000 <SEP> 0.39 <SEP> 2564
<tb><SEP> PVAS <SEP> 1000 <SEQ> 0.18 <SEP> 5555
<tb> SPS:: polystyrene sulfonate of different PM.

DS 5000: dextran sulfate of PM 5000 D.

PVAS: polyvinyl alcoholsulfate MW 20000 D.

Reference for DS and PVAS values: Antimicrobial Agents and Chemotherapy, 1990, 34, 1, pp. 134-138.

 As a rule, the SPS PM 5 400 gives the best results.

2) Inhibition of giant cell formation
Protocol
1 μl of complete RPMI medium, containing the test products at the desired concentrations, are added to each well of a 96-well plate.

2- 50 μl per well of Molt-4 cells at 1106 cells / ml are added
3- 50 μl per well of HUT72 cells infected with HTLVIIIb or LAV2 virus at 10 6 cells / ml are added.

 Before adding the cells, they are washed twice.

 4- Incubation is carried out at 37 ° C. in the presence of 5% of CO 2 for 20 hours to 24 hours.

 After incubation, the cells are resuspended, the giant cells are counted.

 The absence of giant cells reveals an active product.

Result
SPS 5400 completely inhibits giant cell formation at a concentration of lpg / ml.

mercialisé par la firme ALDRICH (référence 27, 842-4).EXAMPLE 3: Antiviral activity of polyethylene sulfone (PESS) of formula

marketed by the firm ALDRICH (reference 27, 842-4).

The results are presented in Table 2 below:
TABLE 2

<tb><SEP> Anti-HIV <SEP> Results
<tb> Polymer <SEP> CD <SEP> 50 <SEP> ED <SEP> 50 <SEP> IF <SEP> Virus
<tb> PESS <SEP> 19.26 <SEP> mg / ml <SEP> 0.15 <SEP>, ug / ml <SEP> 129474 <SEP> HIV <SEP> 1
<tb> DS <SEP> 5000 <SEP> 2.5 <SEP> mg / ml <SEP> 0.39 <SEP> pg / ml <SEP> 6410 <SEP> HIV <SEP><SEP> I <SEP>
<tb> PVAS <SEP> 2.5 <SEP> mg / ml <SEP> 0.18 <SEP> pg / ml <SEP> 13889 <SEP> HIV <SEP> 1
<tb> PESS <SEP> 13.6 <SEP> mg / ml <SEP> 0.076 <SEP> tug / ml <SEP> 178525 <SEP> HIV <SEP> 2
<tb> D55000 <SEP> 2.5 <SEP> mg / ml <SEP> 0.07 <SEP> g / ml <SEP> 35714 <SEP> HIV <SEP> 2
<tb> PVAS <SEP> 2.5 <SEP> mg / ml <SEP> 0.28 <SEP> pg / ml <SEP> 8928 <SEP> HIV <SEP> 2
<Tb>

Claims (16)

 1. Drug use of polysulfonated macromolecules.  2. Use as medicaments of polysulfone polymers, polysaccharides or polypeptides, or of their polysulphonate salts.  3. Medicament according to claim 1 or 2, characterized in that the polymer, polysaccharide or polypeptide has a molecular weight of between 1,500 and 50,000, preferably between 2,500 and 10,000.  4. Medicament according to one of claims 1 to 3 characterized in that the macromolecules consist of the repetition of identical or different monomeric units of which at least 5 comprise at least one sulfonic or sulfonate group.  5. Medicament according to one of claims 1 to 4 characterized in that they are selected from poly (sulfonated styrene), poly (ethylene sulfonated), polyl (sulfonated propylene), poly (urethane sulfonated), poly (sulfonated acrylamide ).  6. Medicines according to one of claims 1 or 5 characterized in that they are selected from poly (amino acids sulfonated).  7. Medicaments according to claim 6 characterized in that they are chosen from poly (sulfonated tyrosine), poly (sulfonated lysine), poly (sulfonated alanine), poly (L-cysteic acid), poly (sulfonated phenylalanine).  8. The medicament as claimed in one of claims 1 to 3, characterized in that it is a polysaccharide such as a dextrin or a polysulphonated heparin.  9. Medicament according to one of claims 1 to 3, characterized in that the macromolecules correspond to the following formulas

 10. Medicament according to one of claims 1 to 4 characterized in that it consists of a macromolecule of formula

<Tb> <tb> <SEP> so-3 <SEP> / <SEP> O-CH-CH-OCH. <tb> <SEP> 50 <SEP> 3 <tb> SO ~ <SEP> #CHH <SEP> n, <SEP> (SOH23) m <SEP> n <SEP> with <SEP> m <SEP> = <SEP> 0 <SEP> to <SEP> 3 , <tb> gCH2- <SEP>. <SEP> ACH2-CH! <SEP> 3 <SEP>, -CH-2; <Tb> <tb> <SEP> SO <SEP> SO ~ <SEP> SO ~ <tb> <SEP> CH <SEP> CH  <SEP> 50 <SEP> 3 <SEP> I <SEP> j <SEP> n  11. Medicament according to claim 6, characterized in that it consists of a macromolecule of formula

 with m = 1 to 5, preferably 2 to 3.  12. Medicament according to one of claims 1 to 7 characterized in that it is a polysulfonated cyclodextrin.  13. Medicament according to claim 12, characterized in that it is a cyclodextrin of formula

 with preferably n = 5 to 10 - R = H, acyl, benzoyl or alkyl, - W = OH or X-Y-SO 3 H with preferably at least 5 units in which W = X-Y-SO 3 H

 with R '= C 1 -C 7 alkyl or aryl, especially phenyl - Y = a C 1 -C 20 hydrocarbon group such as alkyl, aryl, optionally substituted by an alkyl, aryl or carboxylic group and which may contain a heteroatom.  14. Medicament according to claim 13, characterized in that it is a sulphonated cyclodextrin of formula (I) in which

Ar is an aryl, especially a phenyl.  with Alk which represents a C1-C20 hydrocarbon group and  with preferably m = 1 to 5,

 15. Antiviral composition characterized in that it comprises a polysulfonated macromolecule according to one of claims 1 to 14 or a pharmaceutically acceptable polysulfonate salt thereof in a therapeutically active amount.  16. Use of the polysulfonated macromolecules according to one of claims 1 to 14 or their pharmaceutically acceptable polysulfonate salts in the preparation of a pharmaceutical composition useful for the treatment of AIDS related to the HIV virus.