Stable concentrated insulin preparations for pulmonary delivery

Concentrated aqueous insulin formulations of high physical and chemical stability are disclosed. The formulations are highly suitable for pulmonary delivery.

FIELD OF THE INVENTION
 The present invention relates to concentrated aqueous insulin formulations
 of high physical and chemical stability and being suitable for pulmonary
 delivery.
 BACKGROUND OF THE INVENTION
 Diabetes is a general term for disorders in man having excessive urine
 excretion as in diabetes mellitus and diabetes insipidus. Diabetes
 mellitus is a metabolic disorder in which the ability to utilize glucose
 is more or less completely lost. About 2% of all people suffer from
 diabetes.
 Since the introduction of insulin in the 1920's, continuous strides have
 been made to improve the treatment of diabetes mellitus. To help avoid
 extreme glycaemia levels, diabetic patients often practice multiple
 injection therapy, whereby insulin is administered with each meal.
 In solution, the self-association pattern of insulin is a complex function
 of protein concentration, metal ions, pH, ionic strength and solvent
 composition. For the currently used soluble preparations containing U100
 insulin, zinc ions, isotonic agent and phenolic preservative, the
 following equilibria must be considered:
EQU 6 ln.revreaction.3 ln.sub.2
EQU 3 ln.sub.2 +2Zn.sup.2+.revreaction.ln.sub.6 (T.sub.6)
EQU T.sub.6.revreaction.T.sub.3 R.sub.3.revreaction.R.sub.6
 The known degradation patterns of insulin include a) fibril formation; b)
 deamidations at A18, A21 and B3; c) dimerisations via transamidation or
 Schiff-base formation; d) disulfide exchange reactions.
 According to Brange (Stability of Insulin, Kluwer Academic Press,1994),
 each of these degradation reactions proceed much faster in the monomeric
 state than in the hexameric state. Therefore, the most efficient means of
 stabilising insulin preparations is by pushing the above equilibrium as
 far to the right as possible. In addition to this general effect of mass
 action, the reactivity of selected residues is further modified depending
 on their direct involvement in the T.fwdarw.R conformational change. Thus,
 the reactivity of B3Asn is much lower in the R-state (when the residue
 resides in an .alpha.-helix) than in the T-state.
 The interconversion between T.sub.6, T.sub.3 R.sub.3 and R.sub.6
 conformations of the two zinc insulin hexamer is modulated by ligand
 binding to the T.sub.3 R.sub.3 and R.sub.6 forms. Anions such as chloride
 have affinity for the fourth coordination position in the metal ions of
 T.sub.3 R.sub.3 and R.sub.6, while preservatives such as phenol binds to
 hydrophobic pockets located near the surfaces of the T.sub.3 R.sub.3 and
 R.sub.6 forms (Derewenda, Nature 338, 594, 1989 and, Brzovic, Biochemistry
 33, 130557, 1994). By the use of Co.sup.2+ insulin it has been shown that
 the combined effect of anion and phenol binding is particularly efficient
 in stabilising the R.sub.6 state. (Brader, Trends Biochem. Sci. 30, 6636,
 1991 and; Bloom, J. Mol. Biol. 245, 324, 1995). Furthermore, for both
 Zn.sup.2+ - and Co.sup.2+ insulin it has been shown that phenol is much
 more efficient than m-cresol in inducing R-state in the insulin hexamer
 (Wollmer, Biol. Chem. Hoppe-Seyler 368, 903, 1987 and, Choi, Biochemistry
 32, 11638, 1993). High affinity phenol derivatives inducing R-state are
 7-hydroxy-indol ((Dodson, Phil. Trans. R. Soc. Lond. A 345, 153, 1993)
 resorcinol and 2,6- and 2,7-dihydroxy-naphtalen ((Bloom, J. Mol. Biol.
 245, 324, 1995).
 The physical denaturation of insulin is known as fibrillation. In the
 fibrillar state extended peptide chains are laying parallel or anti
 parallel and hydrogen bonded to each other, so-called .beta.-structure or
 .beta.-pleated sheets. Fibrils represent usually the lowest state of
 energy of the protein, and only harsh conditions such as strong base may
 enable a regeneration from this state to the native state of correctly
 folded protein. Factors that promote the rate of formation of fibrils are
 increasing the temperature, increasing the surface area between the liquid
 and the air phase and, for zinc-free insulin, increasing the
 concentration. For hexameric zinc-insulin the rate of fibril formation
 decreases with increasing concentration. The formation of fibrils is
 believed to proceed via monomerization of insulin. Fibrils of insulin have
 the appearance of gels or precipitates.
 Insulin derivatives having truncations in the C-terminal of the B-chain,
 e.g. des-pentapeptide (B26-B30) insulin and des-octapeptide (B23-B30)
 insulin are more prone to form fibrils than human insulin. Insulin
 analogues which dissociate readily from the hexameric unit to the
 monomeric form, e.g. the AspB28 human insulin and the LysB28-ProB29 human
 insulin, are likewise more prone to form fibrils than human insulin.
 The native state of insulin is stabilised by bringing about the conditions
 that stabilises the hexameric unit, i.e. the presence of zinc ions (2-4
 zinc/hexamer), phenol (0.1-0.5% w/v) and sodium chloride (5-150 mM).
 Addition of agents that reduce the surface tension at the air-liquid
 interface further reduces the propensity to fibril formation. Thus,
 polyethylene glycol, polypropylene glycol and copolymers hereof with an
 average molecular weights of about 1800 have found use as stabilisers in
 concentrated insulin solutions for infusion pumps (Grau, 1982. In: Neue
 Insuline (Eds. Petersen, Schluter & Kerp), Freiburger Graphische Betriebe,
 pp. 411-419 and Thurow,1981: patent DE2952119A1). For a comprehensive
 review on the physical stability of insulin see Brange 1994, Stability of
 Insulin, Kluwer Academic Publisher, pp. 18-23.
 Most of the chemical degradation of insulin in preparations is due to
 reactions involving the carboxamide function of the asparagine residues,
 in particular residues B3 and A21. Hydrolysis of the amide groups leads to
 desamido derivatives, and transamidation involving an amino group from
 another molecule leads to covalently linked dimers and, after similar
 consecutive reactions, to trimers and higher polymers.
 In acid solution AsnA21 is the most reactive, leading to AspA21 insulin
 (Sundby, J. Biol. Chem. 237, 3406, 1962). In crude insulin of bovine and
 porcine origin, obtained by acid ethanol extraction, the most abundant
 dimers isolated were AspA21 -GlyA1 and AspA21-PheB1 linked (Helbig 1976,
 Insulindimere aus der B-Komponente von Insulinpraparationen, Thesis at the
 Rheinisch-Westfalischen Technischen Hochschule, Aachen).
 In neutral solution, which is the preferred embodiment of insulin
 preparations for injection therapy, AsnB3 is the most susceptible residue.
 Degradation products include AspB3 insulin, AspB3 -GInB4 isopeptide
 insulin, and dimers and higher polymers where AspB3 provides the carbonyl
 moiety of a peptide bond with an amino group of another molecule. For a
 comprehensive review on the chemical stability of insulin see Brange 1994,
 Stability of Insulin, Kluwer Academic Publisher, pp. 23-36. As for the
 physical stability conditions that stabilises the hexameric unit, i.e. the
 presence of zinc ions (2-4 zinc/hexamer), phenol (0.1-0.5% w/v) and sodium
 chloride (5-150 mM), decrease the rate of formation of degradation
 products during storage at neutral pH.
 A different type of polymerisation reaction is observed when the conditions
 that stabilises the hexameric unit is neglected. Thus, in the absence of
 zinc, phenol and sodium chloride, and using a temperature of 50.degree.
 C., disulfide-linked dimers and high molecular weight polymers are the
 prevailing products formed. The mechanism of formation is a disulfide
 interchange reaction, resulting from .beta.-elimination of the disulfides
 (Brems, Protein Engineering 5, 519, 1992).
 Solubility of insulin is a function of pH, metal ion concentration, ion
 strength, phenolic substances, solvent composition (polyols, ethanol and
 other solvents), purity, and species (bovine, porcine, human, other
 analogues). For a review see Brange: Galenics of Insulin, Springer-Verlag
 1987, p.18 and 46.
 The solubility of insulin is low at pH values near its isoelectric pH, i.e.
 in the pH range 4.0-7.0. Highly concentrated solutions of porcine insulin
 (5000 U/ml.about.30 mM) have been brought about at acid pH (Galloway,
 Diabetes Care 4, 366, 1981), but the insulin in the formulation is highly
 instable due to deamidation at AsnA21. At neutral pH highly concentrated
 solutions of zinc free insulin can be made, but these are unstable due to
 a high rate of polymerisation and deamidation at AsnB3. Porcine zinc
 insulin solutions at neutral pH comprising phenol have been reported
 physical stable at concentrations of 1000 U/ml at elevated temperature,
 but become supersaturated when the temperature is lowered to 4.degree. C.
 (Brange and Havelund in Artificial Systems for Insulin Delivery, Brunetti
 et al. eds, Raven Press 1983).
 In order to reduce the inconvenience of insulin injections much attention
 has been given to alternative routes of administration (for an overview
 see Brange and Langkjaer in Protein Delivery: Physical Systems, Sanders
 and Hendren, eds., Plenum Press 1997). Pulmonary delivery seems to be the
 most promising of these (Service, Science 277, 1199. 1997). Insulin can be
 given aerolised in the form of dry powder or as nebulised droplets from an
 insulin solution. The efficacy might be enhanced by coached breathing
 (Gonda, U.S. Pat. No. 5,743,250) and addition of an absorption enhancer
 (Baekstroem, U.S. Pat. No. 5,747,445) or protease inhibitors (Okumura,
 Int. J. Pharm. 88, 63, 1992).
 The bioavailability of a nebulised concentrated insulin solution (500 U/ml)
 was shown to be 20-25% as compared to a subcutaneous injection (Elliot,
 Aust. Paediatr. J. 23, 293, 1987). By using 30-50 .mu.l insulin solution
 per puff the insulin solution need to be 5-20 times more concentrated than
 the usual concentration of 0.6 mM. By using a single dose container, e.g.
 a blister pack (Gonda, U.S. Pat. No. 5,743,250), the demand for a
 preservative is abolished. Most insulin formulations are preserved by the
 toxic, mucose irritating and unpleasant odorous phenol and m-cresol.
 However, omitting phenols will cause stability problems. In addition to
 the bacteriostatic efficacy, the phenols act as physico-chemical
 stabilisers of insulin in combination with zinc ions. So, it is preferred
 that formulations of insulin for inhalation are made with a minimum
 concentration of phenol or that phenol has been replaced by more
 acceptable substitutes.
 DESCRIPTION OF THE INVENTION
 Definitions
 By "analogue of human insulin" (and similar expressions) as used herein is
 meant human insulin in which one or more amino acids have been deleted
 and/or replaced by other amino acids, including non-codeable amino acids,
 or human insulin comprising additional amino acids, i.e. more than 51
 amino acids.
 By "derivative of human insulin" (and similar expressions) as used herein
 is meant human insulin or an analogue thereof in which at least one
 organic substituent is bound to one or more of the amino acids.
 By "phenolic molecule" as used herein is meant phenol or any derivative
 thereof such as m-cresol or chloro-cresol.
 Brief Description of the Invention
 It is an object of the present invention to provide a concentrated insulin
 formulation for pulmonary delivery having an acceptable physical and
 chemical stability.
 This object has unexpectedly been accomplished by providing an insulin
 formulation in which the concentration of chloride is kept below 50 mM,
 and in which the concentration of other anions such as phosphate is
 minimised.
 Accordingly, the present invention relates to an aqueous insulin
 formulation comprising: 3 to 20 mM of human insulin or an analogue or a
 derivative thereof, less than 50 mM of chloride, less than 10 mM of any
 anions other than chloride and acetate, 2 to 5 Zn.sup.2+ ions per six
 molecules of insulin, and at least 3 phenolic molecules per six molecules
 of insulin.
 Preferred Embodiments
 The insulin formulation according to the present invention preferably
 comprises 3 to 15, more preferably 4 to 15 mM, still more preferably 5 to
 15 mM, even more preferably 6 to 15 mM of human insulin or an analogue or
 a derivative thereof.
 In certain advantageous embodiments, the formulation of the invention
 comprises about 3 mM, about 6 mM, about 9 mM, about 12 mM, or about 15 mM
 of human insulin or an analogue or a derivative thereof.
 When the insulin formulation of the invention is to be administered from
 multi-dose containers a preservative effect is desired and it may thus
 advantageously contain up to 50 mM of phenolic molecules. Surprisingly,
 however, adequate stability is obtained by using a relatively low
 concentration of phenolic molecules such as 3 to 12 phenolic molecules per
 six molecules of insulin, preferably 3 to 9 phenolic molecules per six
 molecules of insulin. A low concentration of phenolic molecules can be
 used when no or little preservative action is needed such as in
 single-dose containers. A further advantage of using a low amount of
 phenolic molecules is an increased convenience for the patient.
 The insulin formulation according to the invention preferably contain less
 than 40 mM, more preferably less than 30 mM of chloride, still more
 preferably 5 to 20 mM of chloride, in order to secure optimal stability.
 In a particular embodiment the insulin may comprise a low amount of
 phosphate buffer, preferably up to 5 mM of phosphate.
 Insulin formulations of the invention comprising 2 to 4 Zn.sup.2+ ions,
 preferably 2.2 to 3.2 Zn.sup.2+ ions per six molecules of insulin, are
 very stable.
 Insulin formulations of the invention comprising 3 to 5 Zn.sup.2+ ions,
 preferably 3.5 to 5 Zn.sup.2+ ions per six molecules of insulin, are also
 suitable.
 Surprisingly, it is possible to add relatively high concentrations of
 zwitterions such as glycylglycine and glycine to the insulin formulation
 of the invention without decreasing the solubility of insulin.
 Glycylglycine acts as a buffer at neutral pH and furthermore increase the
 dissolution rate of zinc insulin at neutral to basic pH due to a
 moderately zinc chelating effect. Also, glycylglycine may act as a
 scavenger for amine reactions during the storage period. Thus, in a
 preferred embodiment the insulin formulation of the invention further
 comprises 5 to 150 mM of a zwitterionic amine, preferably glycylglycine or
 glycine.
 In a preferred embodiment the insulin formulation of the invention further
 comprises 5 to 50 mM of trishydroxymethylaminomethan which acts as a
 buffer at neutral pH and as a scavenger for amine reactive compounds.
 In another preferred embodiment the insulin formulation of the invention
 comprises sodium ions as cations. The sodium ion has a low salting out
 effect.
 In another preferred embodiment the insulin formulation of the invention
 comprises potassium or a mixture of potassium and sodium ions as cations.
 Potassium ions in a concentration higher than the plasma concentration of
 4-5 mM increase the transport of insulin through the lungs.
 In another preferred embodiment potassium ion in a concentration more than
 4-5 mM is used in combination with a mild bronchodilator such as menthol.
 In another preferred embodiment the insulin formulation of the invention
 comprises between 0.001% by weight and 1% by weight of a non-ionic
 surfactant, preferably tween 20 or Polox 188. A nonionic detergent can be
 added to stabilise insulin against fibrillation during storage and
 nebulisation.
 In another preferred embodiment the insulin formulation of the invention
 comprises 1 mM to 10 mM of an anionic surfactant, preferably sodium
 taurocholate, in order to further increase the bioavailabilty of insulin.
 In a preferred embodiment the insulin used is human insulin.
 In another preferred embodiment the insulin used is an analogue of human
 insulin wherein position B28 is Asp, Lys, Leu, Val or Ala and position B29
 is Lys or Pro; or des(B28-B30), des(B27) or des(B30) human insulin.
 The preferred analogues of human insulin are those in which position B28 is
 Asp or Lys, and position B29 is Lys or Pro, preferably Asp.sup.B28 human
 insulin or LyS.sup.B28 PrO.sup.B29 human insulin.
 In another preferred embodiment the insulin is selected from the group of
 soluble long-acting insulin derivatives such as derivatives of human
 insulin having one or more lipophilic substituents, preferably acylated
 insulins.
 The insulin derivative according to this embodiment is preferably selected
 from the group consisting of B29-N.sup..epsilon. -myristoyl-des(B30) human
 insulin, B29-N.sup..epsilon. -palmitoyl-des(B30) human insulin,
 B29-N.sup..epsilon. -myristoyl human insulin, B29-N.sup..epsilon.
 -palmitoyl human insulin, B28-N.sup..epsilon. -myristoyl Lys.sup.B28
 Pro.sup.B29 human insulin, B28-N.sup..epsilon. -palmitoyl Lys.sup.B28
 Pro.sup.B29 human insulin, B30-N.sup..epsilon. -myristoyl-Thr.sup.B29
 Lys.sup.B30 human insulin, B30-N.sup..epsilon. -palmitoyl-Thr.sup.B29
 Lys.sup.B30 human insulin, B29-N.sup..epsilon.
 -(N-palmitoyl-.gamma.-glutamyl)-des(B30) human insulin,
 B29-N.sup..epsilon. -(N-lithocholyl-.gamma.-glutamyl)-des(B30) human
 insulin, B29-N.sup..epsilon. -(.omega.-carboxyheptadecanoyl)-des(B30)
 human insulin and B29-N.sup..epsilon. -(.omega.-carboxyheptadecanoyl)
 human insulin.
 The most preferred insulin derivative is B29-N.sup..epsilon.
 -myristoyl-des(B30) human insulin or B29-N.sup..epsilon.
 -(N-lithocholyl-.gamma.-glutamyl)-des(B30) human insulin.
 The above soluble long acting insulin derivatives are albumin binding and
 have been designed to provide a constant basal supply of insulin
 (Markussen, Diabetologia 39, 281, 1996). Subcutaneous administration once
 or twice daily secures the required basal delivery of insulin, whereas for
 pulmonary administration several daily inhalations are recommended,
 preferably in connection with meals.
 The insulin derivatives have a protracted onset of action and may thus
 compensate the very rapid increase in plasma insulin normally associated
 with pulmonary administration. By careful selection of the type of
 insulin, the present invention enables adjustment of the timing, and in
 order to obtain the desired insulin profile.
 In a particular embodiment of the present invention, the insulin
 formulation comprises an insulin analogue or human insulin as well as an
 insulin derivative.
 The phenolic molecules in the insulin formulation are preferably selected
 from the group consisting of phenol, m-cresol, chloro-cresol, thymol, or
 any mixture thereof.
 The insulin preparation of the present invention preferably has a pH value
 in the range of 7 to 8.5, more preferably 7.4 to 7.9.

This invention is further illustrated by the following examples which,
 however, are not to be construed as limiting.
 EXAMPLE 1
 2.5 ml of a 21 mM insulin stock solution was made by dissolving 337 mg zinc
 free human insulin in 1237 .mu.l water and adding 263 .mu.l of 0.1 M
 ZnCl.sub.2 and 637 .mu.l water before adjusting pH with 38 .mu.l of 0.2 M
 NaOH and finally adding water to 2.5 ml, calculating the specific volume
 of insulin as 0.7 .mu.l/mg. A preparation of 15 mM was then made by adding
 350 .mu.l of 0.16 M m-cresol, 175 .mu.l of 0.32 M phenol and salt or
 detergent to the concentrations shown in Table 1 and thereafter diluted by
 medium to 12, 9, 6, 3 and 0.6 mM and stored at 5.degree. C.
 EXAMPLE 2
 Zinc insulin was dispersed in water (1:10) on icebath, added glycylglycine
 (7/15) equivalent and sodium hydroxide (3.1 equivalent) and stirred slowly
 overnight at 5.degree. C. 0.1 equivalent of zinc chloride and detergent
 was then added, pH adjusted to 7.5 by 0.8 equivalent of hydrochloric acid
 and volume adjusted before adding phenol and water and finally diluting
 the 15 mM preparation with medium containing sodium chloride,
 glycylglycine and detergent to obtain 12, 9, 6, and 3 mM of human insulin.
 (Table 2 and 3).
 The results are presented in the following Tables 1 to 3.
 The data of Table 1 show that even a small amount of phosphate (e.g. 5 mM)
 reduce the stability of insulin, and substituting sodium chloride by
 trihydroxymethylaminomethan hydrochloride also tends to decrease the
 solubility of insulin. Contrary to salts the zwitterions glycylglycine and
 glycine increased the solubility of insulin, and it was possible to add
 unexpectedly high concentrations of the zwitterions glycylglycine and
 glycine without deteriorating the stabilising effect on insulin.
 Glycylglycine acts as a buffer at neutral pH and furthermore increases the
 dissolution rate of zinc insulin at neutral to basic pH due to a
 moderately zinc chelating effect. Glycylglycine may also act as a
 scavenger for amine reactions during storage. Addition of the non-ionic
 detergents tween 20 and poloxamer 188 up to 1% by weight and 3 mM of the
 anionic detergent sodium taurocholate did not reduce the stability at
 5.degree. C. storage.
 An evaluation of the effect of a phenolic substance added equimolar to
 insulin is shown in table 2. Three of the phenolic molecules increase the
 physical stability from 6 to 15 mM of insulin or more at low temperature
 and reduce the formation of polymers at elevated temperature by a factor
 of 2-3 (at low chloride concentration). In another set of experiments
 (table 3) the relative amount of phenol or chloro-cresol is varied from 0
 to 2 per insulin at increasing chemical stability.
 EXAMPLE 3
 441 mg B29-N.sup..epsilon. -(N-lithocholyl-.gamma.-glutamyl)-des(B30) human
 insulin (143 nmol/mg) was suspended in 5 ml water at 0.degree. C. and 220
 .mu.l 1 N NaOH added. After dissolution of the insulin analog 295 .mu.l
 0.1 M ZnCl.sub.2 was added and the solution stirred until a temporary
 precipitate was dissolved. 315 .mu.l 0.32 mM phenol and 98 .mu.l 0.5 M
 glycylglycine and 70 .mu.l 1% Tween 20 were subsequently added and pH
 measured to 7.60. Finally 693 .mu.l water was added and the solution was
 passed through a sterile 0.22 .mu.m Millex.RTM.-GV filter unit to obtain 7
 ml 9 mM B29-N.sup..epsilon. -(N-lithocholy-.gamma.-glutamyl)-des(B30)
 human insulin. The solution remained stable after 3 months at 5.degree. C.
 TABLE 1
 Stability of solutions of human insulin at conventional phenol/cresol
 concentrations (used for multiple dose containers) as a function of salt
 concentration, ion charge, and detergent concentration.
 Physical stability of
 solution at 5.degree. C.
 Excipient Maximal concentration without
 0.5 Zn.sup.2+ /insulin precipitation for 4 months. Test
 phenol and cresol 16 mM solutions were 0.6, 3, 6, 9, 12
 pH 7.5 and added (mM): and 15 mM insulin, respectively.
 reference (norm. dissolution*.sup.)) 3-6
 reference (low ion strength) 12
 NaCl 10 15
 NaCl 20 12
 NaCl 40 6
 NaCl 60 &lt;3
 NaH.sub.2 PO.sub.4 5
 +NaCl 20 6
 +NaCl 25 6
 +NaCl 37.5 6
 +NaCl 50 6
 glycylglycine 7 15
 glycylglycine 12 15
 glycylglycine 24 15
 glycylglycine 48 15
 glycylglycine 72 15
 glycylglycine 96 15
 glycylglycine 120 15
 glycine 10 15
 glycine 20 15
 glycine 40 15
 glycine 60 15
 glycine 80 15
 glycine 100 15
 trishydroxymethylaminomethan**.sup.) 7 12
 tris 12 9
 tris 24 9
 tris 48 3
 tween 20 0.05% 15
 tween 20 0.2% 15
 tween 20 1% 15
 tween 20 5% &lt;3
 Polox 188 0.2% 12
 Polox 188 1% 12
 sodium taurocholate 3 12
 sodium taurocholate 15 9
 *.sup.) addition of 1 .mu.l 1N hydrochloric acid per mg insulin
 corresponding to about 6 equivalents of chloride.
 **.sup.) neutralised by hydrochloric acid
 TABLE 2
 Stability of human insulin at equimolar concentrations of
 phenolic preservatives.
 Chemical
 Excipient Physical stability of stability
 0.5 Zn.sup.2+ /insulin, NaCl 15 solution at 5.degree. C. at 37.degree. C.
 mM, Maximal stable concen- %
 glycylglycine 7 mM, tration without precipita- polymer/week
 tween 20 0.01%, pH 7.5 tion for 3 months at 3, 6, 3 and 15 mM
 and equimolar; 9, 12, 15 mM insulin insulin
 cresol 15 0.55 0.56
 phenol 15 0.37 0.39
 chlor-cresol 15 0.51 0.40
 thymol 9 0.85 1.25
 reference (without phenolics) 6 0.94 1.49
 TABLE 3
 Stability of human insulin at varied concentrations of
 phenolic preservatives.
 Excipient Chemical stability
 0.5 Zn.sup.2+ /insulin, NaCl 15 at 37.degree. C.
 mM, glycylglycine 7 mM, Equivalent phenolic com- % polymer/week
 tween 20 0.01%, pH 7.5 pound per insulin mole- 3 and 9 mM
 and cule insulin
 reference 0 0.99 1.43
 phenol 0.33 0.69 0.96
 phenol 0.67 0.52 0.55
 phenol 1 0.46 0.38
 phenol 2 and 1.33 0.27 0.26
 chloro-cresol 0.33 0.66 0.93
 chloro-cresol 0.67 0.48 0.58
 chloro-cresol 1 0.30 0.30
 chloro-cresol 2 and 1.33 0.13 0.18