NZ238718A

NZ238718A - Insulin analogue and its preparation

Info

Publication number: NZ238718A
Application number: NZ238718A
Authority: NZ
Inventors: Jan Markussen; Liselotte Langkjaer; Kjeld Norris; Leo Boye Snel
Original assignee: Novo Nordisk As
Priority date: 1990-06-28
Filing date: 1991-06-26
Publication date: 1992-06-25
Also published as: DK155690D0; ZA914830B; IL98596A0; WO1992000321A1; AU8054391A; IE912247A1; EP0536245A1; PT98124A; JPH05508406A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £38718 238718 Priority DaU<s):. J&.Vj.J, c "**S Complete Sp®dilcatkm Filed: Class: (5)X ..1 f>.T\.Wv%.JaYvtv.... Publication Date: . P 0. Journal, No: 2 5 JUN 1992 HO DRAWINGS Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION NOVEL, PROTRACTED INSULIN ANALOGUES WE, NOVO NORDISK A/S, a Danish company of Novo Alle, 2 880 Bagsvaerd, DENMARK hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: t2 V\ - 1 - (followed by page la) 1991-06-14, ATS/VMN 3348.200-N2 23 8 7 ICL NOVEL, PROTRACTED INSULIN ANALOGUES Field of invention The present invention relates to novel insulin analogues with a prolonged insulin action, to a process for 5 the preparation of such insulin analogues and to injectable solutions containing the novel insulin analogues. Background of the invention Insulin analogues with a protracted insulin action have previously been described in EP 0194864A and EP 10 0254516A. In EP 0194864A protracted human insulin analogues wherein the C-terminal carboxyl group of the B-chain is blocked with an amido or ester residue and the amino acid residue in position A4, A17, B13 and B21 may be substituted 15 by Gin are described. EP 0254516A describes human insulin of the same type as in EP 0194486A but further being modified in the A21 position. Some of the above insulin analogues may, however, show a too low biological potency or the level of 20 prolongation may be too low for specific purposes. It is the purpose of the present invention to develop novel insulin analogues with prolonged insulin action not suffering from the above-mentioned drawbacks. Summary of the invention 25 It has according to the present invention surprisingly been found that introduction of a positive charge in the N-terminal end of the B-chain gives rise to insulin analogues with a highly prolonged insulin action as compared to human insulin and also a high in vivo potency. 30 In its broadest aspect the present invention is thus related to novel analogues of human insulin wherein at least one of the amino acid residues from Bl to B6 has been (followed by page 2) 1991-06-14, ATS/VMN yyr? 18 2 replaced by a basic amino acid residue, i.e. a lysine or arginine residue (Lys or Arg). For the purpose of improving the stability of the novel insulin analogues asparagine (Asn) in position A21 may furthermore be substituted with another amino acid residue. Also a further positive charge may be introduced by blocking the C-terminal carboxyl group in position B30 preferably by means of an amido or ester group. If the present insulin analogues are prepared by the socalled transpeptidition method (for details see later) it might furthermore be an advantage that the amino group linked to the C-terminal end of the Lys or Arg residue substituent is a proline residue. The invention is also related to a method for the preparation of the novel insulin analogues by which a biosynthetic precursor of the insulin analogue is converted into the insulin analogues by enzymatic and chemical conversion and to insulin solutions containing the novel insulin analogues. By "insulin analogues" as used herein is meant a compound having a molecular structure similar to that of insulin including the disulphide bridges between CysA7 and CysB7, and between CysA20 and CysB19 and an internal disulphide bridge between CysA6 and CysA11 and with insulin activity. Detailed description of the invention The present insulin analogues may be represented by the following formula I 1991-06-14, ATS/VMN 3348.200-NZ A(1-6)-Cys-A(8-19)-Cys-Z S S I I (I) s s I I xi"x2"x3~x4_x5~x6"cys"B(8~18)~cys-B(20-29)~Y 10 wherein Z is Asn or another naturally occuring amino acid residue, is Phe, Lys or Arg, X2 is Val, Pro, Lys or Arg, X3 is Asn, Lys, Arg or Pro, X4 is Gin, Lys, Arg or Pro, X5 is His, Lys, Arg or Pro, X6 is Lys, Arg, Leu or Pro and Y is a threonine residue wherein the carboxyl group may be blocked 15 by an ester or amido group, with the proviso that at least one of Xj_, X2» X3, X4, X5 and Xg is Lys or Arg. Compared with human insulin, the change in charge is obtained by substituting one or more of the amino acid residues in position B1 to B6 with an arginine or lysine 20 residue. In addition, the C-terminal carboxyl group of the B-chain may be blocked by an ester group or amide group. If Z is not asparagine it may be a neutral amino acid, for example valine, glutamine, isoleucine, leucine, phenylalanine, tyrosine, methionine or preferably glycine, 25 serine, threonine or alanine. Z may also be an acidic amino acid, viz. glutamic acid or aspartic acid, or a basic amino acid, viz. lysine, arginine or histidine. Z is preferably glycine, alanine or serine. Examples of blocking groups of the C-terminal 30 carboxyl group in the B30 amino acid residue (threonine) are ester moities such as lower alkoxy with preferably not -more than 8 carbon atoms, preferably less than 5 carbon atoms. Preferred alkoxy groups are methoxy, ethoxy and tertiary butoxy. 35 The blocking group may also be an amido group with l. the formula -NR1!*2 wherein R1 and R2 are the same or //V ' * o , r\ < / v f c» 1991-06-14, ATS/VMN 3348.200-NZ 9X07 I t- o \J / 4 different and each represents hydrogen or alkyl with preferably up to 8 carbon atoms. R1 and R2 are preferably each hydrogen. Since compounds of formula I can be applied in the 5 clinic as solutions having a prolonged action, a decline in immunogenicity as compared to the commonly used suspensions of porcine or human insulins may occur. The degree of prolongation can be enhanced and controlled by the addition of zinc ions. 10 Parameters that may control the degree of prolongation of the insulin effect are the concentration of zinc and the choice of the compound of formula I. The range for preferred zinc content extends from 0 to about 2 mg/ml, preferably from 0 to 200 /xg/ml zinc and more preferably from 15 about 20 to 200 Aig/ml in a preparation containing about 240 nmole of a compound of formula I per ml. Using other concentrations of the compound of formula I, the content of zinc is to be adjusted correspondingly. The prolonged action of solutions of compounds of 20 formula I in the presence of zinc ions is ascribed to the low solubility of such compounds at neutral pH. The pH of the injectable solution of this invention should preferably be below the physiological pH, the upper limit being the pH where precipitation occurs. At the 25 physiological pH value, compounds of formula I of this invention have a low solubility. Stable solutions containing about 240 nmole/ml of compounds of formula I per ml have been obtained at pH about 5.5. The upper limit depends upon the constituents of the solution, i.e. isotonikum, preservative 30 and zinc concentration, and upon the choice of compound of formula I. There is no lower pH limit of the solutions and the chemical stability of the compounds of formula I where Z is different from asparagine, is high, even at pH 3. The preferred pH range for the injectable solutions of this 35 invention is from about 2.5 to 8.5, more preferred from about 1991-06-14, ATS/VMN 3348.200-NZ 238718 4.5 to 8. Especially preferred are pH ranges about 2.5 to 5.5, most prefered about 3 to 4.5. A furter aspect of this invention is that it provides improved flexibility for the patients. With two aqueous 5 solutions, one containing a compound of formula I and the other containing a zinc salt, the patient can obtain a desired degree of prolonged action and a desired profile by mixing the two solutions appropriately. Thus, the patient has, using two stock solutions, the possibility of choosing 10 one action and profile for the morning injection and another action and profile for the evening injection. Preferably, the zinc solution of this invention contains between about 2 /xg and 20 mg zinc per ml. Alternatively, both of the stock solutions may contain zinc, either in the same or different 15 concentrations, and/or both the stock solutions may contain a compound of formula I, either the same or different compounds. Perferably, the injectable solutions of this invention have a strength of between about 60 and 6000 nmole 20 of the compound of formula I per ml. Examples of novel insulin analogues according to the present invention are ArgB5,SerA21,ThrB30-NH2 human insulin ArgB5,ProB6,SerA21,ThrB30-NH2 human insulin 25 ArgB5,GlyA21,ThrB30-NH2 human insulin ArgB5,ProB6,GlyA21,ThrB30-NH2 human insulin ArgB2,SerA21,ThrB30-NH2 human insulin ArgB2,ProB3,SerA21,ThrB30-NH2 human insulin ArgB2,GlyA21,ThrB30-NH2 human insulin 30 ArgB2,ProB3,GlyA21,ThrB30-NH2 human insulin ArgB2,ArgB3,SerA21,ThrB30-NH2 human insulin ArgB2,Arg03,Ser^21 human insulin ArgB4,Pro05,Ser^21,ThrB30-NH2 human insulin ArgB4,ArgB5,ProB6,GlyA21,ThrB3° human insulin 1991-06-14, ATS/VMN 3348.200-NZ 9 7 -7 -s n 0k C O v, / I 0 6 ArgB3;GlyA21,ThrB30-NH2 human insulin Arg03,SerA21,ThrB30-NH2 human insulin Arg04/GlyA21,ThrB30-NH2 human insulin Arg84,SerA21/ThrB30-NH2 human insulin 5 ArgB1/ProB2,GlyA21,ThrB30-NH2 human insulin. The novel insulin analogues according to the present invention may be prepared by altering the proinsulin gene through replacement of codon(s) at the appropriate site in the native human proinsulin gene by codon(s) encoding the 10 desired amino acid residue substitute(s) or by synthesizing the whole DNA-sequence encoding the desired insulin analogue. The gene encoding the desired insulin analogue is then inserted into a suitable expression vector which when transferred to a suitable host organism, e.g. E. coli. 15 Bacillus or yeast, generates the desired product. The expressed product is then isolated from the cells or the culture broth depending on whether the expressed product is secreted from the cells or not. The novel insulin analogues may also be prepared by 2 0 chemical synthesis by methods analogue to the method described by Marki et al. (Hoppe-Seyler's Z. Physiol.Chem., 360 (1979), 1619-1632). They may also be formed from separately in vitro prepared A- and B-chains containing the appropriate amino acid residue substitutions, whereupon the 25 modified A- and B-chains are linked together by establishing disulphide bridges according to known methods (e.g. Chance et al., In: Rick DH, Gross E (eds) Peptides: Synthesis -Structure - Function. Proceedings of the seventh American peptide symposium, Illinois, pp. 721-728). 3 0 The insulin analogues may furthermore be prepared by a method analogue to the method described in EP 0163529A, the disclosure of which is incorporated by reference hereinto. By such a method an insulin precursor of human insulin wherein LysB29 is connected to GlyA1 by means of either a peptide 1991-06-14, ATS/VMN 3348 .200-NZ 2387 18 7 bond or a peptide chain of varying length with, correctly positioned disulphide bridges is expressed and secreted by yeast and then converted into human insulin by the so-called transpeptidation reaction. 5 The transpeptidation reaction is described in US patent specification No. 4,343,898 (the disclosure of which is incorporated by reference hereinto). In this reaction the peptide bond or peptide chain connecting Lys®29 and GlyA^ is exised and a threonine ester or threonine amide group is 10 coupled to the C-terminal end of LysB29. The novel insulin analogues may thus be prepared by a method wherein a biosynthetic insulin precursor with the following formula II 15 20 n (Qq-T)r- Al-A( 2-6)-Cys-A(8-19)-Cys-Z S S (II) Xi-X2-X3-X4-X5-x6-cys-B(8-18)-Cys-B(20-28)-B29 wherein Q is a peptide chain with q amino acid residues, q is 25 an interger from 0 to 33, T is Lys or Arg, r is 0 or 1 and X-p X2, X3, X4, X5, X6 and Z are defined as above, is reacted with a compound of the formula III HY (III) wherein Y is a protected threonine amino acid wherein the 3 0 carboxyl group is protected with an ester or amido group, using trypsin or trypsin like enzymes as a catalyst in a mixture of water and organic solvent. The ester or amido protecting group may then be cleaved off by acid or basic hydrolysis. 1991-06-14, ATS/VMN 8 Preferred compounds of formula III are Thr-NH2, Lys(Boc)-NH2, Thr(Bufc)-0But and Thr-0Bufc. Insulin preparations of this invention are prepared by dissolving a compound of formula I in an aqueous medium at 5 slightly acidic conditions, for example, in a concentration of 240 or 600 nmole/ml. The aqueous medium is made isotonic, for example, with sodium chloride or glycerol. Furthermore, the aqueous medium may contain zinc ions in a concentraion of up to about 30 /xg of Zn++ per nmol of compound of formula I, 10 buffers such as acetate, citrate and histidine and preservatives such as m-cresol, nipagin or phenol. The pH value of the final insulin preparation depends upon the number of charges that have been changed in the compound of formula I, the concentration of zinc ions, the concentration 15 of the compound of formula I and the compound of formula I selected. The pH value is adjusted to a value convenient for administration such as about 2.5 - 5.5, preventing precipitation. The insulin preparation is made sterile by sterile filtration. 20 The insulin preparations of this invention can be used similarly to the use of the known insulin preparations. Terminology The abbreviations used for the amino acids are those stated in J.Biol.Chem. 243 (1968), 3558. The amino acids are 25 in the L configuration. Unless otherwise indicated, the species of insulins stated herein is human. As used in the following text B(l-29) means a shortened B-chain of human insulin from PheB1 to Lys®29 and A(1-21) means the A-chain of human insulin. 30 The substitution(s) made in the huamn insulin molecule according to the practice of the invention is (are) indicated with a prefix referenced to human insulin. As an example Arg82 human insulin means a human insulin analogue wherein Arg has been substituted for Val in position 2 in the 3348.200-NZ p 7. - -7 1 Q J iJ 1991-06-14, ATS/VMN "twn 1 s 9 B-chain. ArgB2 ^ B( 1_29j -Ala-Ala-Lys-A(l-21) human insulin means a precursor for the forementioned insulin analogue wherein Arg has been substituted for Val in position 2 in the shortened B-chain and wherein the B(l-29) chain and the A(l-5 21) chain are connected by the peptide sequence Ala-Ala-Lys. Unless otherwise stated it is to be understood that the B(l-29) chain and the A(l-21) chain are connected by disulphide bridges between CysA7 and CysB7 and between CysA2° and CysB19, respectively, and that the A-chain contains an 10 internal disulphide bridge between cysA6 and CysA1, as in human insulin. Experimental part Example 1 ArgB2,ProB3,SerA21,ThrB30-NH2 human insulin, 15 ArgB5,SerA21,ThrB30-NH2 human insulin, ArgB4,GlyA21,ThrB30-NH2 human insulin and ArgB2,ProB3,GlyA21,ThrB3° human insulin ArgB3 jGly^1 ,ThrB30-NH2 human insulin 20 The above compounds were synthesized from the corresponding the following precursors: Arg82,ProB3jSer^21,B(l-29)-Ala-Ala-Lys-A(l-21) human insulin, Arg55,Ser^21,B(1-29)-Ala-Ala-Lys-A(1-21) human insulin, ArgB4^l}^21,B(l-29)-Ala-Ala-Lys-A(1-21) human insulin, 25 ArgB2,ProB3,GlyA21,B(l-29)-Ala-Ala-Lys-A(l-21) human insulin, and ArgB3,GluA21,B(l-29)-Ala-Ala-Lys-A(l-21) human insulin by tryptic transpeptidation in organic/aqueous solution in the presence of Thr-NH2 as described in EP 0194864A, Examples 3 0 4 and 6. Yields, charges relative to human insulin, rates of migration relative to insulin in DISC PAGE electrophoresis at pH 8.9 and deviations in amino acid compositions from human insulin appear from Table I. 1991-06-14, ATS/VMN 0k 10 3348.200-NZ 23 87 18 The insulin precursors were produced by a method analogous to the method described in EP 0163529A. The insulin precursors were recovered from the fermentation broths by adsorption of LiChroprep™ RP-18 as 5 described in Example 7 of EP 0163529A. The precursors were eluted from the column with 0.2 M KCl, 0.001 M HCl in 33% (v/v) ethanol. The insulin precursors were crystallized from the pool by successive additions of water (1 volume per volume of pool), solid trisodiura citrate to obtain a molarity 10 of 0.05 M and finally zinc acetate to obtain a molarity of 0.006 M. The pH value was adjusted to 6.8 and the mixture was left overnight at 4'C. The crystals were isolated by centrifugation, washed with water and dried in vacuo. Protected amino acids and protected peptides for 15 enzymatic semisynthesis were either prepared by standard methods or purchased (custom synthesis) from either Nova Biochem or Bachem, both Switzerland. Table I Substitution in Yield Charge relative human insulin % to human insulin at pH 7 Arg82,Pro83,Ser^21, 27 +2 ThrB30-NH2 10 ArgB5,SerA21, 54 +2 ThrB30-NH2 ArgB4,GlyA21, 48 +2 ThrB30,NH2 ArgB2,ProB3,GlyA21, 57 +2 15 ThrB30-NH2 1 v£> H* I O C\ I Rate of migration Deviations in amino at pH 8.9, acid compositions % relative to from human insulin > human insulin after acid hydro- w lysis ^ residues/mole 3 55 +1 Arg, +1 Pro, +1 Ser, -2 Asp, -1 Val t-* 55 +1 Arg, +1 Ser, ^ -1 Asp, -1 His 55 +1 Arg, +1 Gly, -1 Glu, -1 Asp 55 +1 Arg, +1 Pro, +1 Gly, -1 Val, -2 Asp roc Chip oog I SJZ ^*tS3 cx> 1991-06-14, ATS/VMN 3348.200-NZ 2 3 ~ "7 ° 12 Sterile injectable solutions of the above compounds for testing of the degree of prolonged action were made using 1.6% (w/v) glycerol as the isotonicum, and 0.26% (w/v) phenol as the preservative. The concentration of zinc ions was 8, 80 5 or 160 yxg/ml. The pH values of the solutions were adjusted sufficiently off the isoelectric point of the analogues to keep the solutions clear upon storage at 4*C. The solutions contained 240 nmole/ml of the tested compounds. The concentration of 240 nmole/ml was verified by HPLC. 10 Injectable solutions containing 240 nmole/ml of the compounds stated in Table II and having the pH of 3 and content of zinc stated therein were then made. The prolongation of the hypoglycemic effect was tested according to British Pharmacopoeia 1980, A 142, in 15 fasted rabbits. Each test solution was administered subcutaneously in a dosis of 14.3 nmole per rabbit in 12 animals weighing 3 - 4 kg, and the course of the hypoglycemia was followed for 6 hours. For comparison the fast acting preparation, Actrapid™ human insulin was included in the 20 tests. The results of the tests are shown in Table II giving the percentage of glucose after 1, 2, 4 and 6 hours (h) . Results from determination of biological potencies Mouse Blood Glucose Assay (MBG) and Free Fat Cell Test (FFC) are listed in Table III. 1991-06-14, ATS/VMN 3348.200-NZ 13 Table XI Compound Zn++ Substitutions in human insulin pq/nl ArgB5,SerA21,ThrB30-NH2 8 ArgB5,SerA21,ThrB30-NH2 80 ArgB5,SerA21,ThrB30-NH2 160 Arg02,Pro®3,SerA21,ThrB30-NH2 8 ArgB2,ProB3,SerA21,ThrB30-NH2 80 Actrapid™ human insulin 7 238718 Glucose in percent of initial Ih 2h 4h 6h 59 64 96 94 85 92 97 95 76 89 96 94 58 67 73 72 73 66 64 61 53 53 92 97 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Table III Biological potency relative to human insulin. (British Pharmacopoeia 1980, A141 - A142). 10 15 MBGA Potency relative Confidence Substitutions in to human limits, % human insulin insulin, % (P = 0.05) ArgB5,SerA21,ThrB30-NH2 76 86-68 ArgB2 proB3,serA21, ThrB -NH2 56 49-64 ArgB2.ProB3,GlyA21, ThrB36-NH2 ArgB4,GlyA21,ThrB30-NH2 66 60-72 20 1 FFC Potency relative to Confidence human limits, % insulin, % (p = 0.05) 56 54-58 50 47-52 59 57-61 40 38-41 \o VO »—1 I 0 cn 1 > Ui 00 OOm o z OO 1991-06-14, ATS/VMN 3348.200-NZ 238718 15 5EfcfKS_ WHAT >?WE CLAIM IS: -

1. Human insulin analogues having the following formula I 5 S S 1 A(1-6)-Cys-A(8-19)-Cys-Z S S 10 I | (I) S S I I Xl""X2~X3~X4~X5~X6~Cys~B(8"18) ~Cys-B ( 20-29) -Y wherein Z is Asn or another naturally occuring amino acid 15 residue, is Phe, Lys or Arg, X2 is Val, Pro, Lys or Arg, X3 is Asn, Lys, Arg or Pro, X4 is Gin, Lys, Arg or Pro, X5 is His, Lys, Arg or Pro, X6 is Lys, Arg, Leu or Pro and Y is a threonine residue wherein the carboxyl group may be blocked by an ester or amido group, with the proviso that at least 20 one of X^, X2, X3, X4, Xg and Xg is Lys or Arg.

2. Human insulin analogues according to claim 1, wherein X-j^ is Phe.

3. Human insulin analogues according to claim 1 or 2 wherein Z is Gly, Ala or Ser. 25

4. Human insulin analogues according to claim 1 wherein X^ is Phe, X2 is Arg or Lys, X3 is Pro, X4 is Gin, X5 is His, X6 is Leu, Z is Gly, Ala or Ser and Y is Thr-NH2 or Thr.

5. Human insulin analogues according to claim 1 30 wherein is Phe, X2 is Val, X3 is Asn, X4 is Gin, X5 is His, Xg is Arg or Lys, Z is Gly, Ala or Ser and Y is Thr-NH2 or Thr.

6. Injectable solutions with prolonged insulin action containing a human insulin analogue with the general formula 35 I 1991-06-14, ATS/VMN 3348.200-NZ #» ? 3 B 7 "1 S 16 rs-s~i A(1-6)-Cys-A(8-19)-Cys-Z f T i s I S s I I Xl~X2"X3~X4"X5~X6"Cys"B(8~18)"Cys-B(20-29)-Y (I) 10 wherein Z is Asn or another naturally occuring amino acid, X1 is Phe, Lys or Arg, X2 is Val, Lys or Arg, X3 is Asn, Lys, Arg or Pro, X4 is Gin, Lys, Arg or Pro, X5 is His, Lys, Arg or Pro, X6 is Lys, Arg, Leu or Pro and Y is a threonine residue wherein the carboxyl group may be blocked by an ester 15 or amido group, with the proviso that at least one of X1# X2, X-j, X4, X5 and Xg is Lys or Arg, together with conventional auxiliary agents, such as buffers, preservatives and agents making the solution isotonic.

7. A method for making insulin analogues with the 20 following formula I 25 A(1-6)-Cys-A(8-19)-Cys-Z S S I I S S I I Xl"X2"X3~X4"X5~X6"Cys~B(8"18)"Cys"B(20~29)~Y (I) 30 wherein Z is Asn or another naturally occuring amino acid residue, X-^ is Phe, Lys or Arg, X2 is Val, Pro, Lys or Arg, X3 is Asn, Lys, Arg or Pro, X4 is Gin, Lys, Arg or Pro, X5 is His, Lys, Arg or Pro, X6 is Lys, Arg, Leu or Pro and Y is a threonine residue wherein the carboxyl group may be blocked 35 by an ester or amido group, with the proviso that at least 1991-06-14, ATS/VMN 17 3348.200-NZ 23 8 7 1 8 one of Xj_, X2, X3, X4, X5 and Xg is Lys or Arg, wherein an insulin percursor with the following formula II n •(Qq-T)r- Al-A(2-6)-Cys-A(8-19)-Cys-Z S S I I 10 S s I I Xl"X2"X3"X4""X5"X6~Cys"B(8"18)"Cys_B(20"28) ~B29 (II) wherein Q is a peptide chain with q amino acid residues, q is an interger from 0 to 33, T is Lys or Arg, r is 0 or 1 and 15 Xx, X2, X3, X4, X5, X6 and Z are defined as above, is reacted with a compound of the formula III HY (III) wherein Y is a protected threonine amino acid residue wherein the carboxyl group is protected with an ester or amido group, 20 using trypsin or trypsin like enzymes as a catalyst in a mixture of water and organic solvent, whereupon the protecting group if desired is cleaved off by acid or basic hydrolysis. NOVO NORDISK A/S j t AwIkuj yBy Their Attorneys V BALDWIN SON & CAREY T26 JUNi<S9! F * </div>