Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the insulin derivatives having a built-in glucose sensor are integrated in protracted acting, water-soluble aggregates of the derivatives in which the propensity to aggregation diminishes, and thereby the rate of absorption of the insulin is increased, as the concentration of glucose in the surrounding medium (e.g. tissue) is increased.
• compositions of insulin derivatives having a built-in glucose sensor are provided. If the concentration of glucose in the surrounding medium (e.g. tissue) is increased, the rate of dissolution of the insulin crystals is enhanced, and hence the rate of absorption increases.
• the surrounding medium e.g. tissue
• Soluble insulin derivatives having a lipophilic substituent linked to the ⁇ -amino group of a lysine residue in any of the positions B26 to B30 have been described in the literature. Such derivatives have a protracted profile of action after subcutaneous injection as compared to soluble human insulin, and this protracted action has been explained by a reversible binding to albumin in subcutis, blood and peripheral tissue.
• one way to obtain tight glucose control would be to couple a glucose sensor, positioned in the tissue of the patient, to a computer that controls an insulin pump.
• the pump is via a catheter connected to a needle inserted under the skin.
• Glucose sensors inserted in the tissue appears to get overgrown with fibrin, and it appears that non- invasive sensors, e.g. based on infrared optics, remain to be invented or developed. Attempts to develop systems for glucose dependent release of insulin from a depot has previously been described.
• the state of aggregation and the power of glucose to diminish this state can be demonstrated by gel filtration of the aggregated insulin derivatives in buffers containing varying concentrations of glucose in the eluents.
• the increased release of insulin derivative from subcutaneous depots can be demonstrated by the different levels of the insulin derivative in the plasma of pigs clamped at various blood glucose levels, e.g. 5 and 10 mM, after injection of the same dose of the insulin derivative.
• This new concept of glucose dependent insulin release complies with the convenience of the state of the art injection regimens of insulin therapy, and requires neither surgery nor the danger associated with storage of large implanted depots in the body.
• Fig. 1 shows that a steep correlation between the release of insulin and the glucose concentration is possible by the multiple interactions between insulin hexamers as compared to a mechanism involving just one bond.
• Fig. 2 shows the association and dissociation of glucose-binding insulin derivative 17a on a Biacore ® glucamine sensor chip.
• RU is Response Units.
• Fig. 3 shows the glucose displacement curves of a number of glucose-sensing insulin derivatives according to the invention from a Biacore ® glucamine sensor chip.
• Fig. 4 shows results from the aggregation test of Lys B29 (N ⁇ -( ⁇ -glutamyl-N ⁇ - lithocholoyl)-Dap B30 (N ⁇ -3-nitro-5-boronobenzoyl) human insulin (the title compound of Example 19), in a gel filtration assay on Bio-Gel P300 eluted at 37 °C by a) sodium chloride 100 mM, sodium phosphate 5 mM, preserved with sodium azide 0.01 % and hydrochloric acid added to pH 7.4 (solid line), b) sodium chloride 25 mM, sodium phosphate 5 mM, preserved with sodium azide 0.01 % and hydrochloric acid added to pH 8.0 (dash dot line), c) sodium chloride 25 mM, sodium
• insulin derivative refers to human insulin or an analogue thereof in which at least one organic substituent is bound to one or more of the amino acids.
• analogue of human insulin as used herein (and related expressions) is meant human insulin, in which one or more amino acid residues have been deleted and/or replaced by other amino acid residues, including non-codeable amino acid residues, or human insulin comprising additional amino acid residues, i.e. more than 51 in total.
• the amino acid sequence of human insulin is given La. in The Merck Index, 11th Edition, published in 1989 by Merck & Co., Inc., page 4888.
• spot is meant the amount of subcutaneous or intramuscularly injected or inhaled insulin composition, either in the form of crystalline compositions, such as NPH insulin and Lente insulin, or as solutions, such as albumin binding or soluble aggregating or acid solutions of neutral-precipitating, of insulin analogues or insulin derivatives.
• glucose sensor is meant a chemical group, capable of binding to or react- ing with glucose.
• the glucose sensor is part of the insulin molecule.
• the dissociation constant, K d of the sensor-glucose complex is usually in the range from 0.01 ⁇ M to 100 mM, for example from 1 ⁇ M to 20 mM or from 1 mM to 20 mM or from 1 mM to 100 mM.
• reversible glucose sensors are organic borates, preferably aryl boronates or other borates, where the attachment to an insulin derivative is via a carbon-boron bond.
• Alkyl boronates are oxidatively labile and often unstable (Snyder, Kuck and Johnson, J. Am. Chem. Soc 1938, 60, 105). Boronate sensors that bind glucose under physiological conditions are preferred. Simple aryl boronates, such as phenyl boronate, binds glucose only at relatively high pH, >9 (Shinkai and Takeuchi, Trends Anal. Chem. 1996, 15, 188). Acidic boronates, which bind glucose at physiologi- cai pH, are preferred. Examples of such boronate glucose sensors are aminomethyl- aryl-2-boronates (Bielecki, Eggert and Norrild, J. Chem.
• Reversible glucose sensors may also be peptides or pseudopeptides, optionally containing boronates.
• irreversible glucose binders are oxyamines and hydrazines, which react with glucose to form oximes and hydrazones (Veprek and Jezek, J. Peptide Sci. 1999, 5, 203; Peri, Dumy and Mutter, Tetrahedron 1998, 54, 12269).
• useful oxyamine functions are aminoxyacetic acid, AOA (Vilaseca et al. Bioconjugate Chem. 1993, 4, 515), and O-aminoserine, Ams (Spetzler and Hoeg-Jensen, J. Pept. Sci. 1999, 5, 582).
• the present invention is based on the discovery of soluble and aggregated forms of insulin derivatives, wherein the state of aggregation is being influenced by glucose.
• the aggregate is preferably soluble in water at neutral pH, in the range of 6.8 to 8.5.
• the soluble, aggregated forms of insulin derivatives dissoci- ates slowly after subcutaneous injection, making them suitable for a long-acting insulin composition, the advantage being that the composition contains no precipitate.
• soluble rather than suspended compositions are higher precision in dosing, avoidance of shaking of the vial or pen, allow- ance for a thinner needle meaning less pain during injection, easier filling of vials or cartridge and avoidance of a ball in the cartridge used to suspend the precipitate in the absence of air.
• the aggregated form can be observed for insulin derivatives under conditions where the hexameric unit is known to exist for most insulins.
• the aggregated form is composed of hexameric subunits, preferably of at least 4, more preferably 5 to 500, hexameric subunits.
• Any hexameric subunit of the aggregated forms of the compounds of this invention may have any of the known R 6 , R 3 T 3 , or T 6 structures, T 6 ' being the preferred form (Kaarsholm, Biochemistry 28, 4427-4435, 1989).
• Substances like Zn 2+ known to stabilise the hexameric unit are also found to stabilise the aggregated form of some insulin derivatives.
• compositions of glucose dependent aggregating insulin derivatives preferably comprises at least 2 zinc ions, more preferably 2 to 5 zinc ions, still more preferably 2 to 3 zinc ions, per 6 molecules of insulin derivative.
• compositions advantageously comprise at least 3 molecules of a phenolic compound per 6 molecules of insulin derivative.
• residues of Glu B13 provide binding sites for up to 3 Ca 2+ ions (Sudmeier et al., Science 212, 560-562, 1981 ).
• addition of Ca 2+ ions stabilises the hexamer and may be added to the pharmaceutical compositions, on the condition that the insulin derivative remains in solution.
• the disappearance half-time of the aggregate of the invention after subcutaneous injection in healthy human subjects, having normal blood glucose concentrations about 5 mM, is preferably as long as or longer than that of a human insulin NPH composition.
• the aggregate is composed of insulin derivatives, which have an albumin binding which is lower than that of Lys B29 (N ⁇ -tetradecanoyl) des(B30) human insulin.
• the lipophilic substituent is connected to the ⁇ -amino group of a lysine residue using an amino acid linker.
• the lipophilic substituent is advantageously connected to a lysine residue via a ⁇ - or an -glutamyl linker or via a ⁇ - or an ⁇ -aspartyl linker.
• the lipophilic substituent comprises the glucose sensor in the form of a borate group, an aryl boronate, an amino aryl boronate or a glucose binding peptide.
• the present invention furthermore provides novel insulin derivatives capable of forming aggregates, in which the degree of aggregation is inversely correlated to the glucose concentration.
• novel insulin derivatives capable of forming aggregates, in which the degree of aggregation is inversely correlated to the glucose concentration.
• These insulin derivatives may be provided in the form of aggregates in pharmaceutical compositions or, alternatively, they may be provided in a non- aggregated form in pharmaceutical compositions, in which case the aggregates form after subcutaneous injection of said compositions.
• the present invention furthermore is concerned with pharmaceutical compositions comprising an aggregate of insulin derivatives or non-aggregated insulin derivatives, which form aggregates after subcutaneous injection, the degree of ag- gregation being inversely correlated to the glucose concentration.
• pharmaceutical compositions comprising an aggregate of insulin derivatives or non-aggregated insulin derivatives, which form aggregates after subcutaneous injection, the degree of ag- gregation being inversely correlated to the glucose concentration.
• n is the number of glucose molecules required to break the polymeric insulin network, releasing the insulin hexamers from the network.
• the advantage of n being larger than 1 is apparent from Fig. 1 , which shows that increasing n from 1 to 6 increases the steepness of the curve for the fraction of free insulin hexamers over polymer, bound insulin hexamers.
• a faster release of insulin at a high glucose concentration, and a slower release at a low glucose concentration is possible by the multiple interactions between insulin hexamers than by a mechanism involving just one bond.
• the pharmaceutical composition according to the present invention comprises aggregates, a substantial fraction of which have a higher molecular weight than aldolase as determined by gel filtration using the medium of the composition as eluent.
• a pharmaceutical composition comprises both aggregating and rapid acting insulin analogues, the latter preferably being human insulin or one of the insulin analogues Asp B28 human insulin, Lys B28 Pro B29 human insulin, Gly A2 ⁇ Lys B3 ,lle B28 human insulin, Asp A21 ,Lys B3 ,lle B28 human insulin or des(B30) human insulin.
• Such a composition will provide both a rapid onset of action and a prolonged profile of action, the latter being influenced by the blood glucose concentration of the diabetic patient.
• the two insulins of the mixture form mixed hexamers both will be under influence of the blood glucose concentration.
• the pharmaceutical composition preferably comprises ag- gregating insulin and rapid acting insulin in a molar ratio of from 90:10 to 10:90.
• the insulin derivative containing a glucose sensing group is prepared as a crystalline NPH composition, using protamine to form the crystals, or as a crystalline Lente composition, using Zn 2+ -ions in the crystals. In these cases the rate of dissolution of the crystals is enhanced by the interaction between glucose and the glucose sensing group.
• the protracted insulin compositions are solutions having a pH value below physiological pH from which the insulin analogue will precipitate because of the rise in the pH value to physiological pH when the solution has been injected.
• Such analogues are described in EP 0 254 516 B1 (Novo Nordisk) and EP 0 368 187 B1 (Hoechst). These analogues have an amino acid residue in position A21 which is stable at pH values as low as practically useful in solutions to be injected. Examples of suitable amino acid residues at position A21 are glycine, serine or alanine.
• the insulins have mutations to increase the net charge of the molecule by about 2, e.g.
• Thr in position B27 can be substituted with Arg and Thr-OH in position B30 can be substituted with Thr-NH 2 or have additional basic residues, e.g. B31 -B32 Arg-Arg.
• Sites enabling the attachment of a glucose sensor are the N-terminal amino groups of glycine A1 and phenylalanine B1 and the ⁇ -amino group of lysine B29.
• One or more additional or alternative lysine residues may be incorporated for this purpose, e.g. in position B3 or B28.
• the glucose sensor may be incorporated as part of the peptide chain, preferably in the C-terminal part of the B-chain.
• the pharmaceutical composition preferably further comprises a buffer substance, such as a phosphate, for example sodium phosphate, glycine or glycylglycine buffer, an isotonicity agent, such as sodium chloride or glycerol, and phenol and/or m- cresol as a preservative.
• a buffer substance such as a phosphate, for example sodium phosphate, glycine or glycylglycine buffer
• an isotonicity agent such as sodium chloride or glycerol
• phenol and/or m- cresol as a preservative.
• mannitol or sorbitol can be added as isotonicity agents and the resulting interaction with the glucose sensor can be utilized to adjust stability and the release profile of the composition.
• the sodium chloride, used as isotonic agent, the zinc- and optionally calcium ions, which promote and stabilize the hexamer formation are particularly important since they facilitate the aggrega- tion of the insulin derivative in the composition and thereby effectively prolong the time of disappearance from the site of injection.
• a pharmaceutical composition according to the invention preferably comprises chloride ions in a concentration of 5 to 150 mM.
• the concentration of the glucose-sensing insulins of the present invention is generally in the range from 0.1 to 15 mM for example from 0.1 to 2 mM.
• the amount of zinc contained in the compositions is 0.3-0.9% by weight relative to the insulin derivative.
• Phenolic compounds like phenol or m-cresol or mixtures thereof are suitably applied in a total concentration of from 5 to 50 mM, and chloride ions in a concentration of from 10 mM to 100 mM.
• the present invention furthermore relates to a method of treating diabetes mel- litus comprising administering to a person in need of such treatment an effective amount of water-soluble aggregates of insulin derivatives according to the invention or effective amount an insulin derivative according to the invention, capable of forming water-soluble aggregates upon subcutaneous injection, aggregate size depending on the glucose concentration.
• the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific human insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the human insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.
• Biacore surface plasmon resonance
• SPR surface plasmon resonance
• the dextran can be chemically modified by immobilization of small molecules, peptides, or proteins. The binding of the com- pound to be tested to the dextran or modified dextran is measured in real time which allows kinetic measurements.
• glucamine is immobilized on a carboxylate surface by a standard amine coupling method.
• the glucamine-modified surface binds the glucose- sensing insulin 17a as illustrated in Fig. 2.
• the response can be quantified and plotted as a competition curve from which the EC50 can be determined, see Fig. 3. Under the conditions used (low binding), EC50 is a good estimate of the value of the dissociation coefficient, Kd.
• Table 1 The experimental conditions used in the above experiments are 0.1 M NaCl, 0.1 M phosphate, pH 7.4, 25 °C.
• the insulin activity of the insulin derivatives of the invention can be demon- strated by their binding to an insulin receptor preparation.
• Scintiplates (Wallac) are coated with Goat antimouse IgG and an insulin receptor antibody is added, followed by solubilized human insulin receptor.
• the binding of the insulins of the invention to the insulin receptor is measured by competition with 125 l-TyrA14 human insulin and scintillation counting. Results obtained with insulin derivatives according to the invention are presented in Table 2.
• the formation of glucose-dependent, high molecular weight soluble aggregates may be demonstrated by gel filtration using a column of the polyacrylamide gel Bio-Gel P300 (BIO-RAD) in a neutral aqueous eluent comprising from 20 to 140 mM sodium chloride, 5 mM sodium phosphate at pH 7.4 or higher and a glucose concentration varying from 0 to 20 mM or higher, e.g. from 0 to 100 mM.
• the gel filtration may be performed with a lower sodium chloride concentration.
• the buffer system described was chosen to mimic the conditions in malian tissue in vivo, in order to be able to detect derivatives changing their state of aggregation under conditions similar to those after the subcutaneous injection.
• decreasing the concentration of sodium chloride, or increasing the pH value precisely to obtain aggregates having a molecular weight close to the molecular weight of aldolase the possibility of observing glucose influence is better.
• HPLC High Performance Liquid Chromatography.
• MALDI-MS Matrix Assisted Laser Desorption lonisation Mass Spectrometry.
• Lys B29 (N ⁇ -lithocholoyl)-N-phenyl-B29-benzylamide-2-boronic acid des(B30) human insulin, 1.
• N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• Lys B29 (N ⁇ -lithocholoyl)-N-phenyl-B30-(benzylamide-2-boronic acid) human insulin, 3.
• related compounds can be obtained by substituting N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue, for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• Lys B29 (N ⁇ -lithocholoyl)-N'-methyl-N'-(benzyl-2-boronic acid)-2-amino-N-methyl-B30- ethylamide human insulin, 4.
• related compounds can be obtained by substituting N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue, for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• Lys B29 (N ⁇ -lithocholoyl)-N'-(benzoyl-3-borno-5-nitro)-2-amino-N-phenyl-B30-ethylamide des(B30) human insulin, 5.
• related compounds can be obtained by substituting N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue, for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• Lys B29 (N ⁇ -lithocholoyl)-2-(pyridinium-3-boronic acid)-acetyl-2-amino-N-phenyl-B30- ethylamide des(B30) human insulin, 6.
• the resulting amine was coupled to the carboxylic acid group of LysB29 in des(B30) human insulin using achromobacter lyticus protease (Morihara and Ueno, Biotech. Bioeng. 1991 , 37, 693). Subsequently, the ⁇ -amino group of LysB29 was acylated selectively using N-hydroxysuccinimidyl lithocholate (US 5,646,242) to give structure 6.
• Lys B29 (N ⁇ -tetradecanoyl)-B29-anilide-3-boronic acid des(B30) human insulin, 7.
• Aniline-3-boronic acid was coupled to the carboxylic acid group of LysB29 in des(B30) human insulin using achromobacter lyticus protease (Morihara and Ueno, Biotech. Bioeng. 1991 , 37, 693). Subsequently, the ⁇ -amino group of LysB29 was acylated selectively using N-hydroxysuccinimidyl tetradecanoylate (US 5,646,242) to give structure 7.
• Lys B29 (N ⁇ -lithocholoyl)-Ams B30 human insulin, 8.
• Lys B29 (N ⁇ -cholanoyl-3-boronic acid) des(B30) human insulin, 10.
• related compounds can be obtained by substituting N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue, for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• related compounds can be obtained by substituting N-hydroxysuccinimidyl lithocholate with another N-hydroxysuccinimidyl ester of an acid having a lipophilic acid residue, for example hyocholic acid, hyodeoxycholic acid or che- nodeoxycholic acid.
• Des(B30) human insulin (1 g) was dissolved in 50 ml 0.05 M boric acid by adjusting the pH to 10.2 with 1 N NaOH and placed in a thermostat at 15°C.
• To the solution was added 61 mg of Boc-AOA-OSu dissolved in 50 ml acetonitrile.
• the reaction was stopped after 1 h by addition of 19 ml 0.2 N ethanolamine, pH 9.0.
• the product was pre- cipitated by addition of water to a total volume of 250 ml, adjusting the pH to 5.5 with HCI and cooling the solution to -20°C.
• the precipitate was isolated by centrif ugation at -10°C and dried in vacuo.
• Mass spectrometry revealed the parent insulin compound, the monoacylated insulin, and diacylated insulin.
• the dried product was treated for 1 h at room temperature with 10 ml trifluoroacetic acid plus 0.3 ml triisopropylsilane.
• the reac- tion mixture was added dropwise to 100 ml of cold diethyl ether; and the precipitate formed was isolated and dried in vacuo.
• the compound 15 was purified by RP- HPLC at pH 4.0 using a gradient from 20 to 60% ethanol. Mw found by MALDI-MS: 5778 (theoretical value: 5780).
• Example 18 Lys B29 (N ⁇ -( ⁇ -glutamyl-N 0l -lithocholoyl),Orn B30 (N ⁇ -3-nitro-5-boronobenzoyl) human insulin, 18
• Lys B29 N ⁇ -( ⁇ -glutamyl-N ⁇ -lithocholoyl),Dap B30 (N ⁇ -3-nitro-5-boronobenzoyl) human insulin, 19
• the Dap B30 analogue of 17 was prepared by a method corresponding to the method used for the preparation of 17.
• the Dab B30 analogue of 20 was prepared by a method corresponding to the method used for the preparation of 20.
• This amino acid derivative was coupled to the carboxylic acid group of LysB29 in des(B30) human insulin using achromobacter lyticus protease (Morihara and Ueno, Biotech. Bioeng. 1991 , 37, 693) and the methyl ester was saponified to give 23a (yield: 30 %. Mw found by ESMS: 6006 (theoretical value: 6005)).
• Lys B29 N ⁇ -( ⁇ -glutamyl-N ⁇ -lithocholoyl),Lys B30 (N ⁇ -4-boronobenzenesulfonyl) human insulin, 24
• Lys B29 N ⁇ -( ⁇ -glutamyl-N ⁇ -lithocholoyl),Lys B30 (N ⁇ -2,5-difluoro-4-boronobenzenesulfonyl) human insulin, 25
• the Lys B30 (N ⁇ -4-borono-2,5-difluoro-benzenesulfonyl) analogue of 23 was prepared by a method corresponding to the method used for the preparation used for 23.
• Lys B29 N ⁇ -( ⁇ -glutamyl-N O! -lithocholoyl
• Lys B30 N'-(3-nitro-5-borono-benzoyl)-1 ,4- phenylendiamine) human insulin amide
• N-(te/ ⁇ -Butyloxycarbonyl)-phenylenediamine (Aldrich) was reacted with N- succinimidyl-3-nitro-5-pinacolboronobenzoate (see example 16) in THF.
• the Boc-group was removed using TFA to give N'-(3-nitro-5-borono-benzoyl)-1 ,4-phenylendiamine, tri- flouroacetate:
• This aniline derivative was coupled to the carboxylic acid group of LysB29 in des(B30) human insulin using achromobacter lyticus protease (Morihara and Ueno, Biotech. Bioeng. 1991 , 37, 693) to give 26a (yield: 4%. Mw found by ESMS: 5990 (theoretical value: 5990)).
• the ⁇ -amino group of LysB29 was acylated selectively using ⁇ -N-hydroxysuccinimidyl ⁇ -methyl glutamyl-N ⁇ -lithocholate (US 5,646,242) and the methyl ester group was saponified to give structure 26 (yield: 25%. Mw found by ESMS: 6478 (theoretical value: 6477)).
• N-tert-Butyloxycarbonyl-piperazine (Aldrich) was reacted with 2- (pinacolborono)benzyl bromide in ether and TEA.
• the Boc-group was removed and the amine was coupled to N ⁇ -tett-butyloxycarbonyl ⁇ -tert-butyl aspartate using carbon- yldiimidazole in DMF.
• the resulting aspartate was treated with TFA followed by metha- nol and trimethylsilyl chloride, 10:1 , to give ⁇ -(N'-(2-boronobenzyl)piperazine)) methyl aspartate, dihydrochloride:
• a pharmaceutical composition comprising a solution of 600 nmol/mL of Lys B29 (N ⁇ -( ⁇ - glutamyl-N ⁇ -lithocholoyl),Dap B30 (N ⁇ -3-nitro-5-boronobenzoyl) human insulin, synthesized according to Example 19. 10 mg of insulin derivative 19 was suspended in 600 ⁇ L water on ice bath and dissolved by addition of 10 ⁇ L 1 N sodium hydroxide.

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