CA1247545A - Insulin derivatives modified in the b 30 position, processes for their preparation and their use, and pharmaceutical agents for the treatment of diabetes mellitus - Google Patents

Abstract

Abstract of the disclosure:

The invention relates to insulin derivatives of the formula I

Description

~Z475~5 . 2 --In ~he therapy of diabetes mell;tus today, ;n general preparations of the hypotensive hormone ;nsulin are admin;stered parenterally. The special nature o~
insulin and its metabolism mean tl1at the duration of action of a simple solution is only very short~ i~e.
that, for permanent control of the blood sugar in dia-betics, it is necessary to administer either a continuous ;nfus;on w;th metering apparatuses, mult;ple da;ly ;n-jections or a delayed act;on insulin preparation. Part;-cularly important principles with delayed action arestates of insulin which are sparingly soluble at the po;nt of ;n~ection te.~. crystalline or amorphous).
These ;nclude, for example, z;nc ;nsul;n crystals or protam;ne ;nsul;n crystals, wh;ch release ;nsul;n over a certain per;od as they slowly redissolve.
It has proven` extremely helpful in therapy to have ava;lable various insulin preparations ~hose charac-teristics of action meet the needs of the individual patient as closely as possible. In conjunction with non-opt;mum formulations, not only ;mmed;ate effects, suchas hyperglycem;a or hypoglycem;a, but aLso~ in particu-lar~ the l3te diabetic complicationsr including ret;no~

~ pathy, neuropathy~ nephropathy~ microang;opathy and ;~ macroanyiopathy, will be discussed.
' 5~S

Lack of insulin in the diabetic results in the body being no longer able to ach;eve its natural hormonal balance.
It ;s the object of the ;nvention to provide an ;nsul;n der;vative or an appropriate pharmaceut;cal agént by means of which tlle natural hormonal balance can be more closely approached in a d;abet;c state, and can be better ma;nta;ned than by the admin;stration of insulin ;n the hitherto convent;onal forms.
This object ;s achieved, in accordance with the invention, by insulin derivatives whose C-terminal amino-ac;d in the B 3U position ;s esterified, or by a pharma-ceutical agent ~Ihich conta;ns this insul;n der;vative as an act;ve compound.
Some human ;nsul;ns ester;f;ed ;n the 8 30 pos;-t;on, and processes for their preparation, have already been described, these stated esterif;ed human ;nsul;ns being intRrmediates in the semisynthesis of human insulin~
Thus, human inslJl1n-ThrB 30-oBut ;s disclosed in, for 20 example, Canadian Patents 1,145,697 and 145,698. Canadian Patent 1,162,868 discloses human ;nsulin-ThrB 30-OMe, human insulin-ThrB 30-oEt, human insulin-Thr~ 3-o-(Z,4,6-trimethylbenzyl) and human insul;n-ThrB 30tBut)-oBut.
Canadian Patent 1,175,37~ discloses human ~nsulin-(B 30) amide.
The invention relates to insulin derivatives of the formula I

.

7~45 ~ 4 --Alr s ~ s A21 lyA-chain Asn ~- 0~

S S ~I) S S
E~2 . l l B29 Rî~ R30 R3 in ~hich a~ R1 denotes H, R30 represents th~ rad;cal of a neutral, genet;cally S codable L-aminoacid ~hose OH group, ~here present, can be free or protected by a physioLog;cally acceptable group, and R31 denotes a physiologically acceptable neutral group SN blocking the carboxyl group, b) R1 denotes H-Phe, b1) R30 represents the radical of a neutral, genet~cally codable L-am~noacld, ~th the exceptlon o~ Thr, and any OH group present can be fre~ or protected by a phy iolog~cally acceptable group, and R31 j~ ~efined as-under a), b2) R30 represents Thr, and b2.1) the OH group of Thr~ 3~ is present ;n unprotected form, and R31 denotes a phys10logically acceptable neutral group -NRaRb or -ORC, where;n Ra and Rb are identical or different and represent tC1 -C6~ -a l kY ~ 3-cg) -cyc loa l ky l~ tC6~~1 0) -aryl, (C7-c~ aralkyL~ ~c3-c~)-heteroary~ or .

. .. ~ .

~7545 -(CH2~CH2-0-)mR in which m = 1 to about-120 and R = ~C1-C~ alkyl, and Ra may fur-therlnore be hydrogell, and these groups may furthermore be sub-stituted in the aryl part by one or more identical or differen~ substituents ~rom the ser;es comprlsing halogenS nitro, (C1~C4)-alkoxy, me.lylenedioxy and (C1 C4)-~lkyl~ or Ra and Rb together denote -CCH2~n- where n -- 4 - 6 and a methylene group may also be replaced by 0 or NH~ and Rc represents propyl, isopropyl~ butylf isobutyl, sec.-butyl~ (C5-C6)-alkyl, (C3-C8)-cycloalkyl, 6-C10)-arYl~ ~C-~-C11)-aralkyl, (C3-C~)-heteroaryl or (CH~-CH2-0-)rnR ;n which m ~ 1 to about 1~0 and R = tC1-C4)-alkyl~ and these groups may furthermore be subs~ituted in the aryl part by one or two identical or different subst;tuents from the se~ies comprisin~J halo~en, nitro, tC1-C4)-alkoxy, methylelled;oxy and (C1-C~)-alkyl, b2.~) ThrR 30 is present as the tert. butyl etller and R31 denotes a physiologically acceptable neutral group -NRaRb or -ORC, wherein Ra and Rb are defined as under b2~1) and R~ may furthermore denote hydro~en, and Rc represents (C1~C3)~alkyl, butyl. ;sobutyl, sec.-butyl, tCS~C6)-alkyl~ (C3~C~)-cycloalkyl, (C6~C10)-arYlr tC7-C11~-aralkyl~ tC3-Cg)-heteroaryl or (CH2 CH2-O~mR, where;n m and R are - as de~ined abo~e~ ar!d these ~roups may be sub~tituted in the aryl par. by one or more substi~uents from, the ~' ' . .

12~'75~5 ser;es compris;ng halogen, n;tro, ~c1-C4)-alkoxy, methylenedioxy and (C1-C4) alkyl, b2.33 the OH group of ThrB 30 is protected by a physlolog;cally acceptable group from the ser;es com-5 prising ~C1-C3)-alkyl, butyl, isobutyl, sec.-butyl, (Cs-C6)-alkyl~ (C3 Cg)-cycloalkyl, (C6-C10)-aryl, (C8-C~ aralkyl, (C3-C9)-heteroar~yl, formyl, (C3-C6)-alkanoyl and (C7-C~ aroyl, which may be substituted in the aryl part as defined under b2.1) for Ra and Rb, or ;s protected by another physiolog;cally acceptable rad;cal conventicnally used in pept;de chem;stry, and R31 is as defined under a), or b2.4) the ~H ~roup of Thr~ 30 is protected by acetyl or bcnzyl, and R31 denotes a phys;olo~;cally flcceptable, neutral group NRaRb or -ORC, wherein Ra and Rb are as def;ned under b2~ J;th the exception of Rb =
-c10)~aryl., and where;n Rc j5 as def;ned under 20 b2~2) r and t~e;l phys;olog;cally acceptable salts.
The following L am;noac;ds are genetically cod-able~ , Ala, Ser, Thr, Val~ Leu, Ile, hsp, Asn, Glu, Gln, ~y~, Met, Ar~, Lys, H;sr Tyr~ Phe, Trp and Pro ~neutral aminoacicts are underlined).
Neutral groups ~N, ~Jhlch block the free carboxyl ~: function at the C-~erminal end of the e chain in the com . pounds according to the invention, are unders~ood as meaning ptlysiologically acceptable uncharged ~roups~

.

~Z9L~ 5 7 ~
espec;ally ester and amide groups, or other COOH protec-tive groups, as descr;bed ;n, for example, Bodanszky et al., Peptide Synthesis, 2nd edi~ion (1~76~ John Wiley Sons, preferably groups of the formula -NRaRb or -ORS, wherein Ra and Rb are identical or different and rep-- resent hydrogen, tC~-C6)~alkyl, (C~-C8) cycloalkyl, (C6-C10)-~rYlr (C7 C11) ar~lkyl, (C3~C9) hetero-aryl or -(CH2~CH2-O)rnR in which m - 1 to about 120 and R = ~C1-C~)-alkyl, and these groups may further~
more be subst;tuted in the alkyl part by one or rnore ident;cal or different substituents from the series com-pris;ng halogen, n;lror (C1-C4)-alkoxy, methylenedioxy and (C1-C~)-alkyl, or Ra and Rb together represent -CCtl2~n-~ wherein n - 4 - 6 and one methylene group nlay furth~r~ore be replaced by 0 or N~l, and Rc represents (C1~ C6)~alkyl,. (C3-C~)-cycloalltyl, (C6-C1n)-aryl., (C~ C11)-aralkyl, ~C-~-C9)-heteroaryl or the radical (CH2CIi2-0-)MR defined above, and aryl rad;cals rnay be subst;tuted as in the case of Ra and Rb.
Suitable OH protective groups for Ser, Thr or Tyr are physiologically acceptable groups froin the series comprising (C1-C6)-alkyl, (C3-C8)-cycloalkyl, (C6-10 aryl~ ~C8-C11)-aralkyl, (C3-C9)-heteroaryl (C1-C6~-alkanoyl and (C7-C11)-aroyl, or another physiolog;ally acceptable radical convent;onally used in pept;de chemistry (cf. for exarnple aodanszky et al., loc.
c;t~ ...).
In the above context, an~ also below, (Cj-C6)-alkyl is understood as rneaning, for example, methyl, ~29L7545 ethyl, propyl~ ;sopropyl, butyl, tert.-butyl, amyl or hexyl, (C3~Cg)~cycloalkyl as meaniny, for example, cyclopropyl, cyclobutyl, cyclohexyl, etc., (C6-C10)-aryl as meaningr for example, phenyl or naphthyl~ preferably phenyl, (C7~C~ aralkyl as rneaning, for exan~ple, benzyl, phenethyl and the like, (C3-C9)-heteroaryl as meaning, for example, pyridyl, pyrrolidyl, pyrimidinyl, morpholinyl~
pyrazinyl, ;midazolyl, ;ndolyl or quinolinyl, (C1-C6)~
alkanoyl as meaning, for example~ formyl, acetyl, pro-pionyl, butyryl~ etc., and (C7-C1~)-aroyl as meaning, for e~ample, benzoyl, naphthoyl or toluyl.
Insul;n der;vatives ~hich carry phenylalanine ;n position B1 are particularly preferred. Other preferred derivat;ves are those which have Ala, Thr or Ser in the B30 positior,~
The A chain and the cha;n (B 2 - 30) of the com-pounds accord1ng ~o the invent;on advantageously have the sequence of bovine o~ porc;ne insulin, but in particular of human insulin.
Other preferred insulin derivatives are ~hose in wh;ch R31 denotes alk-oxy or aralkoxy or ~OtC~l2C~z-O)In~.
Preferred OH protect;ve groups -for Ser, Thr or Tyr are (C1-C6)~alkyl, (C1-C6)~alkanoyl or (C7-C~ aralkyl~
. In the ser;es of insulin der;vat;ves accord;ng to thé invent;on, the follow;ng compounds may be ment;oned by way of e-xample, without restr;cting the ;nvent;on to these: ~

lL~47S~5 g Des-~he8 1-porcine insulin-(B 30)-OBut Des-Phe~ 1-human insulin-(B 30)-OBut Des-PheB 1-porcine insulin-(8 30)~0CH3 Des-Phe~ 1-human insulin-(B 30)~0BZL
S porcine insulir)~(B 30)-O~cyclohexyl human insulin-(B 30) 0~ CH3 bovine insul;n~(B 30)-OCH~
porc;ne insulin-(B 30)-O ~CH2-CH~-0 )20C~tl5 hurnan insulin-(B 3G)-NH-C2H
Des-ThrB 30-human insulin-ValB 3n 0- c H
Dcs-ThrB 30-hunlan insulin-ValB 30-NH2 human insulin-~B 30)-~ ~
human insulin (B 30)-NII-~C112~C~12~0)~-C~13 human insul1n-tl'~ 30)-N~Cll,~j)2 hunlarl insul;rl-~B 30)-OBZL
human ;nsul;n~(~ 30)-0-C~12-CH2~-hultlan insulin-ThrE~ 30~Et:loE~
human ;nsul;ll~TIIrB 30~CH~)oCH3 hunlan ;nsul;n~tB 30)-O~CC~ 5CH3 human ;nsul~n~Thr~Ac~O-I.t human ;nsul;n--rllrt~)OBut hulnan ;nsul;n-tB 3.0)-NI~
~ .
hyman insul;n-(B ~0) -o -~3 human ;nsul;n-ThrB30(FOR)-Opr

2~ human ;nsulin-(8 30~ -N~2 The ;nvention furthermore relates to a process ~or the preparation of insulin derivat;ves of the formula 754~

~, ~h~rein a) a Des-octapeptido (823-30)-insul~n of th~ for~ula II
~1 S - S A21 r-- r _I
S - I Gly A- chai~ Asn -OH
T

S ~; tlI) B2 ¦ ¦ ~22 ~n ~hich R1 d~not~s Ph~ or a ~ond, and st d~not~ an am~no protoct1ve ~roup, such ~g th~ t~rt.-butyloxyc~rbon-yl-tBos~, the tort. a~yloxycar~onyl tAoca or th~ ~thyl-~ulfonylethyloxyc3Pbonyl-tMsG) r~d~c~ h1ch c2n bo ~pl~ off by proton ~olvoly~i~ or by ~-ol~nae10n, i~
conden~ed ~lth ~ peptld~ of eh~ for~ula ~S

1~-Cly-Phe-~he-ryr(S2)-Thr(S~-Pro-L.~(S3~-R30-R3 ln ~h~ch R30 ~nd R31 h~v- th~ s~n~n~ t~n~d ~bovo, s2 r~pr~nt~ hydro~n~ ~l or aut, and S~ r~prosont~
a urcth3n~,~prot~c~1v~ group~ ~uCh ~ aoc~ Moc~ F~oc or Z, and any prot~ct~V~. ~roup3 pro~nt ~r~ 8pllt o~t 1n 5 ~Dnner ~h~ch 1~ kno~n p~r 9~ or e~ ~ 330-~n~ul1n of tha for~ul~ in ~h1ch Rt rop-ro~ont~ ~1 or H-PhQ and th~ C-t~ n~l end R30~R3 tog~ther repro~ent~ OH,. ~ r2actod t~1th ~ co~pound o~ th~
fornlula IV

H-~30_R31 t~V~

7S~5 in ~h;ch R30 and R31 have the mean;ngs def;ned above, in the presence of trypsin or of a trypsin-like endopep- I
t;dase, and any protective groups present are then spl;t o-ff ;n a manner wh;ch ;s known per se, and the compounds obtained by a) or b) are, i-f requ;red~ converted to théir phys;olog;cally acceptable salts.
Ir, process variant a), for example, the ~A1, ~B1 b;s~soc derivative of a Des-octapept;de~(B23-30j-insulin ;s reacted d;rectly with one equ;valent of a com-pound of the formula III, the procedure used being analo-gous to that described ;n U S. Patent 4,029r6~2, and the condensing agent used be;ng dicyclohexylcarbodiimide in slight excess, in the presence oF 1-hydroxybenzotriazole.
Since, ;n th;s process var;ant~ the carboxyl groups do not usually need to be protected, the ;nsul;n der;vat;ve is also normally not damaged either ;n the esterif;cat;on or in the alkal;ne hydrolys;s. Unreacted Des-octapeptide and a pept;de formed by condensation of IV w;th AspA21-OH differ in molecular size and in charge number and can therefore read;ly be separated by partition chromatography over SephadexRLH 20 or by gel chromato-graphy over SephadexRG 75 or G S0 superfine.
To elim;nate the ter~.-butyl protective groups, it is only necessary to treat the react;on product with trifluoroacet;c acid for 30 - 60 minutes a~ room ~empera-ture~ Th;s react;on does not damage the ;nsul;n der;va-t;ve. If a methylsulfonylethyloxycarronyl rad;cal ;s chosen as the N protect;ve group, spl;tt;ng off the latter by ~eliminat;on necessitates treatment ~;th an ~Z~75~5 alkali. The reaction conditions are such (e.g. 0.1 N
NaOH, 0C, 5 sec) that the insulin derivative is not damaged. The porc;ne ~A1, N~1-bis-soc-Des-B23-3o-octapeptide-insulin used as the starting material is pre-pared, for example, in the following manner:
- Porcine ;nsulin in a mixture of dimethylforrn-amide, dimethyl sulfoxide and water is reacted with an excess of tert.-butyloxycarbonyl-N-hydroxysucc;nim;de ester in the presence of N-ethylmorpholine~ This reac-tion gives the expected N~A1, ~B1 N~B29-tr;s~Boc ;nsul;n.
Small port;ons of tryps;n are then added to the solutiori of this compound in dimethylforlnamide and tris buffer (pH 7.5) until starting material is no longer detectable in the electrophoresis. lhe N~A1, No~1-bis~
~ocwDes-B23_30-octapeptide-;nsul;n is purified by par-t;tion chrornatography over SephadexR LH 20~
Th;s compound is then brought to react;on with one mole of the pep~ide of the formula III, wh;ch is pre-pared in a rnanner known per se by the methods of peptide chemistry, 1 - 2 moles of 1-hydroxybenzotriazole and about 0.9 mole of dicyclohexylcarbodiim;de in dirnethyl-formam;de at about pH 7 - 8 (cf. Chem. Ber. 103 ~1970), page 788).
The crude product is purified by partition chromatography, and is freed from the protective groups by treatment with trifluoroacetic acid/anisole at roorn temperature. ~fter prec;p;tation with ether~ isoelectr;c precipitation frorn water, and chromatography over ~, . .

~ ~75~
~ 13 -Sephadex~ G 75 or G 50 superfine, the compound ;s electrophoret;caLly pure, and can be crystall;zed ;n a known manner~ The ;nsul;n derivat;ve thus obtained possesses full biolog;cal act;v;ty.
~es PheB1-insulir)s as starting compounds for the processes according to the ;nvent;on are disclosed in~ for a~,Q~ ~ete~fS ~ 3 or ~ /~3,3 example, ~ .
The Des-B30-insul;ns used as startin~ compounds ;n process var;ant b) are d;sclosed ;n, for example, EP-A--46~979 or Hoppe-Seylerls ~. Phys;ol. Chcm. 359 (1978), 799. The start;ng material of the formula IV which is used ;n variant b) ;s prepared in a manner known per se by the methods o-f peptide chemistry~
The Des-B 30-insulin and the compound of the i5 formula I~ are condensed with one another analogously to .
the procedure described in U.S. Patent 4,320,1g6, in the presence of trypsin or of a tryps;n-l;ke endopept;dase, ;n an aqueous organ;c solvent system at pll S - 9 and a temperature of 2~ to ~0Cn The ;nsul;n der;vat;ve ~0 obtained can be ;sola~ed by the conventional methods of peptide chemistry.
The compounds accord;ng to the invent;on may furthermore be synthesi~ed analogousl.y to the semisyn-thet;c procedures for the preparat;on of the known insul;n-(B 30~esters, whici1 procedures are descr;bed ;n, - e~ ,c~" ~f en?~5 /~ /6~ o, y9~
for exam~le, ~ K~{~ r----: hminoacid esters of the formula IV, ;n wh;ch R31 represents an ~lkylpolyoxyethyleneoxy cha;n, are prepared ~2~7~

analogously to the procedure described Canadian Patent 1,156,217.
Other compounds of the formula IV are known or can be preoared analogously to known processes.
- All of the stated insulin derivat;ves of the formula I have in common the fact that as a result of block;ng the (B 30)-carboxyl group, the molecule effec-tively receives an addit;onal posit;ve charge, ~h;ch shifts ;ts isoelectr;c po;nt toward the neutral point.
Depend;ng on the der;vat;ve, ;soelectr;c po;nts between 5.8 and 7.3 are measured ;n the ;soelectric focus;ng method. Thus, the derivatives are less soluble in the region of the neutral point than is natural ;nsul;n or pro;nsulin which have their isoelectr;c point, and hence the range of max;mum insolubility, a~ pH 5.4, but are usually present in solut;on in the reg1on of the neutral po;nt.
The solub;l;ty propert;es oF insulin and pro-insul;n ;n the range above the ;soelectr;c point, i.e. in the therapeutically particularly ;nterest;ng region of the neutral point, can be ;nfluenced by the addition of z;nc ;ons. In th;s context, zinc acts as a depot pr;n-c;ple by v;rtue of the fact that lt stab;l;zes the hexa-mer;c state of the insul;n and ;ts tendency to crystal-l;ze. lhese aggregates dissolve once again in subcutane-ous tissue.
Another familiar depot principle is the crystal-l;zation of the ;nsul;n or proinsulin as a complex with a basic protein, for example globin or protamine.
When the proinsulin is used in solution or ;n '; ,~
,-7~i4~

conjunction w;th one of the depot principles described, a further proteolytic degradation is requ;red in order to liberate fully active natural insulin. Intact proinsul;n has only about 1/8 of the biological activity of ;nsulin because, as is suggested, part of the b;ologically active - surface reg;on, the receptor-b;nding region, is masked by the C peptide present in the proinsulin. However, only homologous proinsulin, i.e. only that which possesses a human sequence, is suitable for diabetes therapy (cf. for e~le Canadian Patent 1~l7~l6o~eterologous proinsulin poss-esses significant immunogenicity. In this context, it is notew~orthy that human proinsulins, too, can exhibit variations in the C peptide moiety.
Surprisingly, it has now been found that, in con-trast to proinsulin, the insulin der;vatives of theformula I whose B chain ;s blocked at the C end have about the same biolo~;cal act;v;ty as an equimolar amount of natural insul;n~
The ;nvent;on furthermore relates to the use of ;nsulin der;vat;ves of the formula I ;n which R30 represents the radical of a neutral, genet;cally codable L-aminoacid whose OH group, where present, can be free or protected by a phys;ologically acceptable group~ and R31 denotes a phys;olog;cally acceptable neutral group SN blocking the carboxyl group, as remedies.
The invent;on also relates to med;caments for the treatment of d;abetes mellitus, cons;sting of a pharma-,, .
... .
.

~LZ~5~

ceut;cally acceptable excipient and an insulin der;vat;ve o, the formula I as an act;ve compound.
These medicaments accord;n~ to the invent;on con-st;tute completely novel delayed-act;on pr;nc;ples wh;ch can be rendered effect;ve w;thout depot aux;l;ar;es, such as z;nc or protam;ne sul~ate. The depot act;on ;s attr;butable to an ;nherent physical princ;ple based on prote;n chem;stry: the poor solubility of the ;nsulin der;vative at its iscelectr;c point. It ;s pos~
sible tha~ its red;ssolut;on under physiological condi-tions is ach;eved by el;minat;on of the add;t;onal group or groups, for example by r,leans of enzymes w;th esterase activity. The part;cular ~roup or groups el;m;nated are e;ther pureLy phys;oloy;cal metabolites or readily meta-bol;zable, phys;olog;cally acceptable substances.
Furthermore, in contrast to intermediates whichare descr1bed in the literature and st;ll conta;n moieties of the heterologous C peptide, the ;nsulin derivatives~
as active compourlds in these novel medicaments, are not~
as a rule, nore h;ghly ;mmunogen;c than the correspond-;ng insulin itself~
The agents accordin~ to the invention contain, as active compounds, one cr more of the novel insulin derivatives of the ~ormula I.
They preferably have a pH bet~een 2.5 and ~5, and contain a suitable ;sotonic agent, a su;table pre-servat;ve and, if required~ a suitable buffer for a pH
range betweerl 5.0 and 8n5r A typical use form of the derivatives described ~;247S'~5 ~ 17 -comprises preparations wh;ch, below the isoelectric point, are present as solu~ions in a physio~og;cal~y acceptable excipient. The pH of the solution can be typically 4~5, and is therefore substantially higher than S that of acidic former insulins (typical pH 3.0). In certain cases, a more neutral injectable solution offers substant;al advantages in respect of its tolerationn Another typical use form comprises suspensions of ar,lorphous or crystalline precipitates of the deriva-tives describ~d~ in a phys;ologically acceptable excipi-ent at about neu~ral pH~
~ lowever, it is also possible to reinforce the poor solubil;ty in the physiolog;cal pH range, wh;ch ;s an ;nherent property of the deriva~ives~ by means of addit;onal depot pr;nciples, for example by the add;t;on -of zinc or protamil1e sulfate. rhe amount of z;nc added can be up to 100 1~g of Zn2+t100 insulin units, but is typically ahout SU ~g of Zn2~/100 insulin un;ts. The àrnount of pro~amine can be between 0.28 rn0 and 0.6 mg per 100 uni~s ~based on protamine sulfate). In this manner~
;t is possible to prepare preparat;ons which are active for a particularly long time and which will ha~e w;der application in future than to dater since, in particular, a base amount o-f insulin appears therapeutically advan-tageous. This discovery has in fact already been ~adein therapy with insulin metering apparatuses.
A suitable excip;ent ~hich is physiologically aeceptable and compat;ble with the insulin der;vative is a sterile aqueous solution which ;s rendered isotonic : L2~754S

~ith respect to blood in the usual manner, for example by means of glycerol, sodium chlor;de or glucose, and which addit;onally conta;ns one of the usual preservatives, for example phenol, m-cresol or a p-hydroxybenzoate. The 5 excipient can add;tionally contain a buffer substance, for example sodium acetate, sodium citrate or sodium phosphate~ The pH is adjusted us;ng dilute acids ttyp;cally HCl) or alkali solutions (typically NaOH).
The insulin derivatives can also be employed in the form of alkal; metal or ammonium salts in the agents accord;ng to the ;nvention. Any des;red amollnt of one or more insulin der;vat;ves of the formula I, or an insul;n der;vat;ve of the formula I, can be present ;n a m;xture of other insulin derivat;ves from amongst these, independently of one another and ;n each case in dis-solved, amorphous and/or crystalline form.
It is sometimes advantageous to add a su;table amount of a suitablè stabi l;zer to the preparat;on accord;ng to the ;nvent;on, the sa;d stabil;zer prevent-; 20 ;ng the precipitat;on of prote;n under thermal-mechan;cal load dur;ng contact ~;th var;ous mater;als. Such stab;-l;zers are d;sclosed in, for example, canadian Pate~t 1,146,069 or 1,19~,673.
In the agents accord;ng to the ;nvention, wh;ch can also conta;n one of the kno~n delayed-action prin-ciples, such as, for example, protamine sulfate, globin or zinc, in su;table amounts, a delayed-act;on pr;nc;ple of th;s type can be used in comb;nation ~ith the total ' ,' ' .~

~Z~7545 amount of active compound or witl1 some of th;s, or with one or more ;nsul;n derivatives of the formula I, in a m;xture. An agent can conta;n different insulin deriva-t;ves of the formula I ;n comb;nat;on with several d;fferent aux;l;aries hav;ng a delay;ng act;on~
In addition to containing the ;nsulin der;vatives of the formula I, the medicaments accord;ng to the ;nven-tion can also contain natural insulin and/or proinsulin and/or des Phe insulin, independently of one another and in each case in di~solved, amorphous andlor crystall;ne form~
Obv;ously~ therefore~ a large var;ety of act;on characteristics wl)ich can be very finely matched are achievable with -the therapeut;c agents according to the ;nvent;on; according to the comments Made at the outset, this should be associated with advances~ part;cularly with regard to late d;abet;c complications.
The examples wh;ch follow serve to illustrate the ;nvention further:
2 0 _r~ n Example 1:
Human insul;n-~B3û)-0-CH2~CH2-CH3 S g of porc;ne insulin are dissolved in 45 ml of d;methylformamide, 25 ml of d;methyl sulfoxide, O.S ml of N-ethylmorpholine and 2.5 ml of water~ 105 9 of tert.-butyloxycarbonyl-N-hydroxysucc;n;m;de are added at room temperature, wh;le st;rril1g, and ~he react;on ;s allowed to continue for 6 hours. It is then term;nated by add ng one drop of glacial acetic acid~ and the product is pre-cipitated with ether and filtered off. The residue is 7~S

dissolved in 360 ml of dimethylformamide, and the solu-tion is diluted w;th 320 ml of tr;s buffer tO.05 M, 0.01 M of CaCl2~ pH 7.5). Port;ons of Z0 mg of trypsin are added at ;ntervals of 1 hour, at 36C.
S After a total of 12 additions, the pH is brought to ~.5 with ace~ic ac;d~ and the solu~ion is evaporated down. Subsequent purificat;on of the material on a SephadexR LH 20 column (~ x 200 cm) by means of par~
t;tion cllromatography us;ng the system n-butarlol/glacial acetic ac;dJwater (2:1:10) g;ves 3.25 g of ~A1, ~B1_ bis-Boc des-B23_30-octapeptide-jnsulin (porcine), in wh;ch the starting mater;al is no longer detectable in the acid;c and basic electrophoresisr Analysis of the substance for aminoacids gives the correct result~ When the ~OC groups are eliminated by way of a test, insulin activity is no lon~er found~ This material (3.25 g) is dissolved ;n 30 ml of dimethylformamide, together with 100 m~ of 1-hydroxybenzotriazole, 750 mg of HCl.Gly~Phe-Phe-Tyr(8ut)-Thr Pro-Lys(8Oc)-Thr(But)-OCH~CH2-CH3 and 0.5 ml of N-ethylmorpholine. 120 m~ of dicyclohexyl-carbodiimide are then added at room temperature, and the reaction mixture is stirred for 24 hours. The precipita~
ted dicyclohexylurea is f;ltered off, and the product is precipitated by adding ether.
The precipitate is fil~ered off, ~ashed with ether and dried. The substance is subjected to prelimin-ary pur;fication by partition chromatography over Sepha~
dexR LH 20, using the above system. ?.6 g of material from the princ;pal peak are isolated by precipitation ~2~75~5 ~;th acetone/ether. The dried, st;ll unprotected der;va-tive is allowed to react with a m;xture of 5 ml of tri-fluoroacetic acid and 1 ml of an;sole for 60 minutes at room temperature. The crude substance is then prec;pita ~ed from the ice-cooled solution by adding ether. The dried prec;p;tate is dissolved in water, the product is precipitated ~lith aqueous ammonia, and the mixture is centrifuged~ The product is pur;fied ;n 10% strength acet;c acid, over SephadexR G 5t) superfine or G 75~
Human insulin-(B30)-OCH2CH2CH3 can be isolated from thç fractions of the des;red peak by freeze-drying (yield after clystallization: 1.2 9). In the biological ~estf the insulin derivative thus obtained exhibits an act;vity equivalent to that of human insul;n~
The octapeptide oF the formula III is prepared accordin~ to the~ ~ollow;ng condensation diagram~ hy con-ventional methods of peptide condensation:

~47s~5 -- 22 ~
Synthe~ d~gr~N for thl? oct~p~pe1ds of th~r for~ul~ IaI
yr ' _ T' ~r ~ ~ro I Y~ 1 hr Z OH H-OBut .
DCU~ t t .

.H /Pd OBu But .. ROC But ~H H ~y ~}3ut , ~CH H O~le /~ f ~?r DCC~HOB DCC/H0~3 DCC/HOB

Z _ __ OEut ; ~ OMe i WC ~ Bu~C
. T~3 ~12/Pd H~Pd 2 __ . ~ OH I ~ OM~ Z- OH} / l ~ut . . DCC~ HOB . l)C~HO~t . ~ t ~ ~OC ~ut 2 _ _ ~_ ! - IOMe z ! 4Pr . NaOH . H2/Pd . Butl ~ 8,~t Z ~ ~ _ ~ C)H H- _ / _ I~OPr . DC~

Z ~t ~ ~ ~up .~ _~ ~_ ~2/Pd -- ! r H ~ __ ._ _ J ~ _--J !o Pr , A~lnoa~d ~nd ~lo~ntal an~ly~ corr~pori~ to th~ory ~Z475~i Medicaments Example 1-- Porcine insulin-(B 30)-OCH3 (prepared serni-syntheticalLy from des-B 30-porc;ne ;nsulin~, ~ormulated as a weakly acidic solution containing 40 IU per ml~ and ;ts depot act;v;ty:
14D5 mg of porcine insulin-(B 30)-OCH3 (27~5 IU~m~), 540~0 mg of crystalline dextrose (rnonohydrate) and 10~0 mg of methyl p-hydroxybenzoate are dissolved in a total volume oF 10 ml of water.
The pH is brought to 4~5 by adding 1 N HCl or 1 N NaOH.
When admin;stered in a dose of 0.4 IU/kg to rabbits~
.
a solution of this type exhibits a pronounced depot acti-v;ty. The area under the blood suyar curve is the same as that obtained w;th a standard preparat;on containin~ 40 IU/ml~

Example 2:

Human ;nsul;n-ThrB 30tBut)oBut ~pH = 6.~), pre-pared semisynthe~;cally from porc;ne insul;n, in a neutral for-mulation containing 40 IU per ml, and its depot activity:

1~8 rng of hulnan insulin-ThrB 30tBut) OBut (27 IU/mg) 21.0 mg of sodium dihydrogen phosphate dihydrate~

Z7.0 mg of m cresol and 160.0 mg of glycerol are dissolved in a total volume of 10 ml of water.

The pl~ is brought to 7.3 by adding 1 N HCl or 1 N NaOI~.

When administered in a dose of 0.4 IU/kg to rabb;ts, a suspens;on of this type exhibits a pronounced ~Z~7~S
- ~4 -` depot act;vity.
Example 3:
Human ;nsul;n-tB 30)--NH2, prepared sem;syn-thet;cally frorn porc;ne ;nsulin, in the form of a crystal-l;ne NPH preparat;on containing 40 IU/ml, and its greatlydelayed action:
14.5 mg of lluman ;nsulin (B 30)-NH2 (27.5 IU/M9), 1.3 mg of protam;ne sulfate, 21.0 mg of sod;um d;hydrogen phosphate dihydrate, 15.0 mg of m-cresol, 6.0 mg of phenol and 160.0 mg of glycerol are d;ssolved ;n water to g;ve a total volume of 10 ml.
The pll is brouyht to 7.3 by add;ng 1 N HCl or 1 N NaOH.
When adm;n;stered ;n a dose of 0.4 IU/kg to rabb;ts, a crystal suspens;on of th;s type exhib;ts a greatly delayed act;on.
Example 4 M;xture of human ;nsulin-Arg~31-011 and human insul;n-ThrB 30ButtoBut), both prepared semisyl1-thetically frorn porcine insulin, ;n the form of a zinc-containing suspension w;th 40 IU/ml, and i~s greatly delayed action:
7.3 mg of human insulin-Arg-~31-OH t27.5 IU/rng), 7.4 mg of human insulin-ThrB 30B~It~oBut) (27.0 IU/mg) 0.46 mg of anhydrous zinc chLoride, 14.0 mg of sodium acetate, 10.0 mg of methyl p-hydroxybenzoate and ~47~;~S

80.0 mg of sodium chloride are d;ssolved ;n water to give a total volume of 10 ml.
The pH ;s brought to 7.0 by add7ng 1 N HCl or 1 N NaOH.
S When adm;n;s~ered ;n a dose of 0~4 lU/kg to rabbits, a suspension of this type exh;bits a greatly delayed act;on~
Exampl_ 5~
Human insul;n-(B 30)-0-(CH2CH2-0~)2oC2H5, prepared sem;synthet;cally from porc;ne ;nsul;n~ in the form of a neutral preparat;on conta;n;ng 100 IU/ml~ and ;ts delayed action:
41.7 m~ of human insulin~(B 30~0-(CH2CH2 0 ~20C2l15 (24.1) IlJ/mg~, 1~.0 m~ of sod;um acetater 10.0 mg of methyl p-hydroxybenzoate and ~0.0 mg of sodiurn chloride are d;ssolvecl ;n water to g;ve a total volume of 10 ml.
The ~H is brought to 7.0 by add;ng 1 N HCl or 1 N NaOH.
In rabbits, a suspension of th;s type exh;bits a delayed action.

Claims (32)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of an insulin derivative of the formula I

(1) wherein a) R1 represents H, R30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R31 represents a physiologically acceptable neutral group SN blocking the carboxyl group, or b) R1 represents H-Phe, b1) R30 represents the radical of a neutral, genetically codable L-aminoacid, with the exception of Thr, and any OH group present can be free or protected by a physiologically acceptable group, and R31 is defined as under a), or b2) R30 represents Thr, and b2.1) the OH group of ThrB30 is present in unprotected form, and R31 represents a physiologically acceptable neutral group -NRaRb or -ORc, wherein Ra and Rb are identical or different and represent (C1-C6)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)aryl, (C7-C11)-aralkyl, (C3-C9)-heteroaryl or -(CH2-CH2-O-)mR
in which m = 1 to about 120 and R = (C1-C4)-alkyl, and Ra may furthermore be hydrogen, and these groups may furthermore be substituted in the aryl part by one or more identical or different substituents from the series comprising halogen, nitro, (C1-C4) alkoxy, methylenedioxy and (C1-C4)-alkyl, or Ra and Rb together represent [CH2]n-where n = 4 - 6 and a methylene group may also be replaced by O or NH, and Rc represents propyl, isopropyl, butyl, isobutyl, sec.-butyl, (C5-C6)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)-aryl, (C7-C11)-aralkyl, (C3-C9)-heteroaryl or (CH2-CH2-O)-mR in which m = 1 to about 120 and R =
(C1-C4)-alkyl, and these groups may furthermore be substituted in the aryl part by one or two identical or different substituents from the series comprising halogen, nitro, (C1-C4)-alkoxy, methylenedioxy and (C1-C4)-alkyl, b2.2) ThrB30 is present as the tert.-butyl ether and R31 represents a physiologically acceptable neutral group -NRaRb or -ORC, wherein Ra and Rb are defined as under b2.1) and Rb may furthermore represent hydrogen, and Rc represents (C1-C3)-alkyl, butyl, isobutyl, sec.-butyl, (C5-C6)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)-aryl, (C7-C11)-aralkyl, (C3-C9)-heteroaryl or (CH2-CH2-O-)-mR, wherein m and R are as defined above, and these groups may be substituted in the aryl part by one or more substituents from the series comprising halogen, nitro, (C1-C4)-alkoxy, methylenedioxy and (C1-C4)-alkyl, b2.3) the OH group of ThrB30 is protected by a physiologically acceptable group from the series comprising (C1-C3)-alkyl, butyl, isobutyl, sec.-butyl, (C5-C6)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)-aryl, (C8-C1l)-aralkyl, (C3-C9)-heteroaryl, formyl, (C3-C6)-alkanoyl and (C7-C11)-aroyl, which may be substituted in the aryl part as defined under b2.1) for Ra and Rb, or is protected by another physiologically acceptable radical conventionally used in peptide chemistry, and R31 is as defined under a), or b2.4) the OH group of ThrB30 is protected by acetyl or benzyl, and R31 represents a physiologically acceptable,neutral group -NRaRb or -ORc, wherein Ra and Rb are as defined under b2.1), with the exception of Rb = (C7-C10)-aryl, and wherein Rc is as defined under b2.2), and its physiologically acceptable salts, which comprises a) condensing a Des-octapeptide (B 23-30)-insulin of the formula II

(II) wherein R1 represents Phe or a bond, and S1 represents an amino protective group, which may be the tert.-butyloxycarbonyl-(Boc), the tert.-amyloxy-carbonyl-(Aoc) or the methylsulfonylethyloxy-carbonyl-(Msc) radical, which can be split off by proton solvolysis or by .beta.-elimination, with a peptide of the formula III

H-Gly-Phe-Phe-Tyr(S2)-Thr(S2)-Pro-Lys(S3)-R30-R31 (III) wherein R30 and R31 have the meanings defined above, S2 represents hydrogen, Bzl or But, and S3 represents a urethane protective group which may be tert.-butyloxycarbonyl (BOC), methoxycarbonyl (MOC), Fluorene-methyoxycarbonyl (FMOC), or benzyloxycarbonyl (Z), and any protective groups present are split off, or b) reacting a Des-B30-insulin of the formula I, wherein R1 represents H or H-Phe and the C-terminal end R30-R31 together represents OH, with a compound of the formula IV

H-R30-R31 (IV) wherein R30 and R31 have the meanings defined above in the presence of trypsin or of a trypsin-like endopeptidase, and any protective groups present are then split off, and the compound obtained by a) or b) is, if desired, converted to its physiologically acceptable salts.

2. The process as claimed in claim 1, wherein R31 represents alkoxy or aralkyloxy, which are defined as in claim 1.

3. The process as claimed in claim 1, wherein R1 represents H-Phe, R30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R31 represents a physiologically acceptable neutral group SN blocking the carboxyl group, and its physiologically acceptable salts.

4. The process as claimed in claim 1, wherein represents H.

5. The process as claimed in claim 4, wherein R31 represents alkoxy or aralkyloxy, which are defined as in claim 1.

6. The process as claimed in claim 1, 2 or 3, wherein R30 represents Ala, Thr or Ser.

7. The process as claimed in claim 4 or 5, wherein R represents Ala, Thr or Ser.

8. The process as claimed in claim 1, 2 or 3, wherein the A chain and the chain (B2-29) have the sequence of human insulin.

9. The process as claimed in claim 4 or 5, wherein the A chain and the chain (B2-29) have the sequence of human insulin.

10. The process as claimed in claim 1, 3 or 4, wherein R3 represents -O-(CH2-CH2-O-)mR, wherein m and R
are as defined in claim 1.

11. The process as claimed in claim 3, 4 or 5, wherein R31 represents (C1-C6)-alkoxy or (C7-C11)-aralkoxy.

12. The process as claimed in claim 1, 2 or 3 wherein any OH group which may be present in R30 is free or protected by (C1-C6)-alkyl, (C1-C6)-alkanoyl or (C7-C11)-aralkyl.

13. The process as claimed in claim 4 or 5 wherein any OH group which may be present in R30 is free or protected by (C1-C6)-alkyl, (C1-C6)-alkanoyl or (C7-C11)-aralkyl.

14. A process as claimed in claim 1, which further comprises preparing a medicament, which comprises bringing an active compound of the formula I into a suitable dosage form.

15. The process as claimed in claim 14, wherein a pharmaceutically acceptable excipient and an active compound of the formula I are included.

16. The process as claimed in claim 14, wherein the medicament has a pH between 2.5 and 8.5, and which includes a suitable isotonic agent and a suitable preservative, and wherein the insulin derivative of the formula I is present in one or both of dissolved form and in suspension.

17. The process as claimed in claim 14, wherein a suitable buffer having a pH between 5.0 and 8.5 is included.

18. The process as claimed in claim 14, wherein between 0 and 100 µg of zinc/100 I.U. are included.

19. The process as claimed in claim 14, wherein the active compound of the formula I is present in the form of an alkali metal salt or of the ammonium salt.

20. The process as claimed in claim 14, wherein any desired amount of one or more insulin derivatives of the formula I, or an insulin derivative of the formula I, are present in a mixture of other insulin derivatives from amongst these, independently of one another and in each case is dissolved, amorphous or crystalline form.

21. The process as claimed in claim 14, wherein a suitable amount of an auxiliary having a delaying action is included.

22. The process as claimed in claim 14, wherein a suitable amount of an auxiliary having a delaying-action is included and this delayed-action principle is used in combination with the total amount of active compound or with some of this, or with one or more insulin derivatives of the formula I, in a mixture.

23. The process as claimed in claim 14, wherein different insulin derivatives of the formula I are included in combination with several different auxiliaries having a delaying-action.

24. A medicament consisting of a pharmaceutically acceptable excipient and an active compound of the formula I as defined in claim 1.

25. A medicament as claimed in claim 24, which has a pH between 2.5 and 8.5, contains a suitable isotonic agent and a suitable preservative, and wherein the insulin derivative of the formula I is present in one or both of dissolved form and in suspension.

26. A medicament as claimed in claim 24, which contains a suitable buffer and has a pH between 5.0 and 8.5.

27. A medicament as claimed in claim 24, which contains between 0 and 100 µg of zinc/100 I.U.

28. A medicament as claimed in claim 24, wherein the active compound of the formula I is present in the form of an alkali metal salt or of the ammonium salt.

29. A medicament as claimed in claim 24, wherein any desired amount of one or more insulin derivatives of the formula I, or an insulin derivative of the formula I, are present in a mixture of other insulin derivatives from amongst these, independently of one another and in each case in dissolved, amorphous or crystallin form.

30. A medicament as claimed in claim 24, which contains a suitable amount of an auxiliary having a delaying-action.

31. A medicament as claimed in claim 24, which contains a suitable amount of an auxiliary having a delaying-action wherein this delayed-action principle is used in combination with the total amount of active compound or with some of this, or with one or more insulin derivatives of the formula I, in a mixture.

32. A medicament as claimed in claim 24, which contains different insulin derivatives of the formula I
in combination with several different auxiliaries having a delaying action.