The stability of proteins used in medical treatment is an important concern in medicine. Protein degradation can be classified as physical or chemical degradation. Physical degradation is caused by a change in conformation that leads to aggregation of the protein and formation of protein fibrils. Chemical degradation of proteins entails a change in the pattern of covalent bonds between atoms, such as breakage or interchange of disulfide bridges, deamination or transamination of the protein. The present invention concerns the prevention of fibrillation and chemical degradation.
Administration of insulin has long been established as a treatment for diabetes mellitus. Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain (FIG. 1A). Although the structure of proinsulin has not been determined, a variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide (FIG. 1B). Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19; FIG. 1B) is thought to be coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin assembles to form soluble Zn2+-coordinated hexamers shortly after export from ER to the Golgi apparatus. Endoproteolytic digestion and conversion to insulin occurs in immature secretory granules followed by morphological condensation. Crystalline arrays of zinc insulin hexamers within mature storage granules have been visualized by electron microscopy (EM). Assembly and disassembly of native oligomers is thus intrinsic to the pathway of insulin biosynthesis, storage, secretion, and action (FIG. 2).
Insulin readily misfolds in vitro to form a prototypical amyloid. Unrelated to native assembly, fibrillation is believed to occur via an amyloidogenic partial fold (FIG. 1C). Factors that accelerate or hinder fibrillation have been extensively investigated in relation to pharmaceutical formulations. Zinc-free insulin is susceptible to fibrillation under a broad range of conditions and is promoted by factors that impair native dimerization and higher-order self-assembly. A storage form of insulin in the pancreatic β-cell and in the majority of pharmaceutical formulations is believed to be stabilized by axial zinc ions coordinated by the side chains of insulin amino acids, specifically the HisB10 residues. Formulation of insulin or insulin analogues as a zinc-stabilized hexamer retards but does not prevent fibrillation, especially above room temperature and on agitation.
Amino-acid substitutions in the A- and/or B chains of insulin have widely been investigated for possible favorable effects on the pharmacokinetics of insulin action following subcutaneous injection. Examples are known in the art of substitutions that accelerate or delay the time course of absorption. Such substitutions (such as AspB28 in the insulin analogue sold under the trademark NOVALOG® and [LysB28, ProB29] in the insulin analogue sold under the trademark HUMALOG®) can be and often are associated with more rapid fibrillation and poorer physical stability. Indeed, a series of ten analogues of human insulin for susceptibility to fibrillation, including AspB28-insulin and AspB10-insulin have been tested. All ten were found to be more susceptible to fibrillation at pH 7.4 and 37° C. than is human insulin. The ten substitutions were located at diverse sites in the insulin molecule and are likely to be associated with a wide variation of changes in classical thermodynamic stability. These results suggest that substitutions that protect an insulin analogue from fibrillation under pharmaceutical conditions are rare; no structural criteria or rules are apparent for their design. The present theory of protein fibrillation posits that the mechanism of fibrillation proceeds via a partially folded intermediate state, which in turn aggregates to form an amyloidogenic nucleus. In this theory, it is possible that amino-acid substitutions that stabilize the native state may or may not stabilize the partially folded intermediate state and may or may not increase (or decrease) the free-energy barrier between the native state and the intermediate state. Therefore, the current theory indicates that the tendency of a given amino-acid substitution in the insulin molecule to increase or decrease the risk of fibrillation is highly unpredictable.
Fibrillation, which is a serious concern in the manufacture, storage and use of insulin and insulin analogues for diabetes treatment, is enhanced with higher temperature, lower pH, agitation, or the presence of urea, guanidine, ethanol co-solvent, or hydrophobic surfaces. Current US drug regulations demand that insulin be discarded if fibrillation occurs at a level of one percent or more. Because fibrillation is enhanced at higher temperatures, diabetic individuals optimally must keep insulin refrigerated prior to use. Fibrillation of insulin or an insulin analogue can be a particular concern for diabetic patients utilizing an external insulin pump, in which small amounts of insulin or insulin analogue are injected into the patient's body at regular intervals. In such a usage, the insulin or insulin analogue is not kept refrigerated within the pump apparatus and fibrillation of insulin can result in blockage of the catheter used to inject insulin or insulin analogue into the body, potentially resulting in unpredictable blood glucose level fluctuations or even dangerous hyperglycemia. At least one recent report has indicated that lispro insulin (an analogue in which residues B28 and B29 are interchanged relative to their positions in wild-type human insulin; the product sold under the trademark HUMALOG®) may be particularly susceptible to fibrillation and resulting obstruction of insulin pump catheters.
Insulin fibrillation is an even greater concern in implantable insulin pumps, where the insulin would be contained within the implant for 1-3 months at high concentration and at physiological temperature (i.e. 37° C.), rather than at ambient temperature as with an external pump. Additionally, the agitation caused by normal movement would also tend to accelerate fibrillation of insulin. In spite of the increased potential for insulin fibrillation, implantable insulin pumps are still the subject of research efforts, due to the potential advantages of such systems. These advantages include intraperitoneal delivery of insulin to the portal circulatory system, which mimics normal physiological delivery of insulin more closely than subcutaneous injection, which provides insulin to the patient via the systemic circulatory system. Intraperitoneal delivery provides more rapid and consistent absorption of insulin compared to subcutaneous injection, which can provide variable absorption and degradation from one injection site to another. Administration of insulin via an implantable pump also potentially provides increased patient convenience. Whereas efforts to prevent fibrillation, such as by addition of a surfactant to the reservoir, have provided some improvement, these improvements have heretofore been considered insufficient to allow reliable usage of an implanted insulin pump in diabetic patients outside of strictly monitored clinical trials.
Resistance to fibrillation caused by heat or other causes would be particularly advantageous for insulin and insulin analogues in tropical and sub-tropical regions of the developing world. The major barrier to the storage and practical use of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 30° C. is accelerated fibrillation of the protein. The major reason for limitations to the shelf life of presently available pharmaceutical formulations of insulin and insulin analogues at temperatures above 10° C. is due to fibrillation of the protein. As noted above, fibrillation is of special concern for fast-acting or “mealtime” insulin analogues (such as the products sold under the trademarks HUMALOG® and NOVALOG®), particularly when these formulations are diluted by the patient and stored at room temperature for more than 15 days.
Modifications of proteins such as insulin are known to increase resistance to fibrillation but impair biological activity. For example, “mini-proinsulin,” is used to describe a variety of proinsulin analogues containing shortened linker regions such as a dipeptide linker between the A and B chains of insulin. Additional substitutions may also be present such as AlaB30 found in porcine insulin instead of ThrB30 as found in human insulin. This analogue is sometimes referred to as Porcine Insulin Precursor, or PIP. Mini-proinsulin analogues are frequently resistant to fibrillation but are impaired in their activity. In general, connecting peptides of length <4 residues block insulin fibrillation at the expense of biological activity; affinities for the insulin receptor are reported to be reduced by at least 10,000-fold. While such analogues are useful as intermediates in the manufacture of recombinant insulin, they are not useful per se in the treatment of diabetes mellitus. Therefore, a need exists for insulin analogues and other protein analogues that are resistant to fibrillation and that maintain at least a portion of their biological activity.
Development of fibrillation-resistant insulin analogues would not be expected to lead to proteins of indefinite shelf life due to eventual degradation of the protein by chemical modification. Whereas fibrillation represents a change in the structure and spatial relationships between insulin molecules by means of altered non-covalent interactions, chemical modification alters the pattern of covalent bonding between atoms in the insulin molecule. Examples of chemical degradation are breakage of disulfide bridges, formation of non-native disulfide bridges between insulin molecules to form covalent dimers and higher-order polymers, deamination of an asparagine side chain to form an aspartic-acid side chain, and rearrangement of aspartic acid to form iso-aspartic acid within the insulin molecule. Although the propensity of an insulin analogue to form fibrils is not correlated with its global thermodynamic stability, enhancing the thermodynamic stability of the insulin molecule has been established to protect the protein from chemical degradation. Therefore, among fibrillation-resistant analogues, a desirable property would also be enhanced thermodynamic stability to confer simultaneous protection from chemical degradation. The combination of resistance to fibrillation and resistance to chemical degradation would be expected to optimize the safe and effective use of an insulin analogue within the reservoir of an implantable insulin pump, the shelf life of an insulin analogue formulation at or above room temperature, and the routine use of an injectable insulin formulation by patients with diabetes mellitus in sub-tropical and tropical regions of the world.
Amino-acid substitutions in insulin have been investigated for effects on thermodynamic stability and biological activity. No consistent relationship has been observed between stability and activity. Whereas some substitutions that enhance thermodynamic stability also enhance binding to the insulin receptor, other substitutions that enhance stability impede such binding.
A traditional approach to protecting insulin or insulin analogues from physical and chemical degradation is based on self-assembly of the protein in its native state to form dimers or higher-order oligomers. Because the process of fibrillation proceeds via a conformationally altered insulin monomer, sequestration of the monomer within a native assembly reduces the concentration of the susceptible monomer. Such assembly also enhances thermodynamic stability, retarding chemical degradation. In addition, insulin assembly damps conformational fluctuations, reducing the rates of both physical and chemical degradation. Because of these advantages, a common method of formulation is to form zinc-stabilized insulin hexamers, the predominant form of the protein in the products sold under the trademarks HUMALIN™ (Eli Lilly and Co.), HUMALOG™ (Eli Lilly and Co.), NOVALIN™ (Novo-Nordisk), and NOVALOG™ (Novo-Nordisk). Use of insulin hexamers complicates the treatment of diabetes mellitus by delaying absorption of the protein after subcutaneous injection.
Glycemic control by insulin replacement therapy, whether administered by external pump, intraperitoneal pump, or manual subcutaneous injection, is enhanced by rapidly absorbed insulin analogues. Rapid absorption enables the time course of insulin action at target tissues to more nearly coincide with the time course of absorption of nutrients after a meal, hence more closely approximating the physiological control of blood glucose concentration and metabolism in a healthy (non-diabetic) person. Absorption of insulin is delayed by its self-assembly as in conventional formulations of human insulin and animal insulins as zinc-stabilized insulin hexamers. A partial solution to this problem has been provided by the meal-time insulin analogues in which the hexameric formulation is destabilized by amino-acid substitutions in the insulin molecule (see above). This approach is not optimal as the substitutions impair the physical stability and thermodynamic stability of the insulin analogue, leading to elevated rates of fibrillation and chemical degradation. Such decreased stability requires continued formulation as a zinc hexamer, leading in turn to a delay in absorption relative to injection of a monomeric insulin analogue. To date, no monomeric analogues have been developed that exhibit sufficient physical stability and thermodynamic stability to allow their formulation and therapeutic use. Therefore, a need exists for insulin analogues that combine the favorable properties of retaining at least partial biological activity, remaining monomeric in solution at millimolar protein concentrations in buffers compatible with pharmaceutical formulations, resisting fibrillation and other forms of non-native protein aggregation, and exhibiting enhanced thermodynamic stability to delay chemical degradation.
Insulin analogues with affinities too low or too high for the insulin receptor may have unfavorable biological properties in the treatment of diabetes mellitus. Because clearance of insulin from the bloodstream is mediated primarily by interactions with the insulin receptor on target tissues, receptor-binding activities less than 25% would be expected to exhibit prolonged lifetimes in the bloodstream. Such delayed clearance would be undesirable in a fast-acting insulin analogue administered in coordination with food intake for the tight control of glycemia. Such reduced affinities would also decrease the potency of the insulin analogue, requiring injection of either a larger volume of protein solution or use of a more highly concentrated protein solution.
Conversely, insulin analogues with affinities for the insulin receptor higher than that of wild-type insulin may be associated with altered signaling properties and altered cellular processing of the hormone-receptor complex. A prolonged residence time of the complex between the super-active insulin analogue and the insulin receptor on the surface of a target cell or on the surface of an intracellular vescicle may lead to elevated mitogenic signaling. Enhanced mitiogenicity can occur if the amino-acid substitutions not only augment binding of the analogue to the insulin receptor, but also to the Type I IGF receptor. For these reasons, among fibrillation-resistant insulin analogues, it is desirable to have analogues whose affinities for the insulin receptor and IGF receptor are similar to those of wild-type human insulin.
A modification of insulin (substitution of HisB10 by Asp) has been described that enhances the thermodynamic stability of insulin and also augments its affinity for the insulin receptor by twofold. Because this substitution blocks the binding of zinc and prevents the assembly of insulin dimers into hexamers, it was investigated as a candidate fast-acting analog. Clinical development was stopped, however, when AspB10-insulin was found to exhibit increased mitogenicity, increased cross-binding to the insulin receptor, and elevated rates of mammary tumor formation on chronic administration to Sprague-Dawley rats. Because the otherwise favorable properties of AspB10-insulin and possibly other insulin analogues are confounded by these adverse properties, it would be desirable to have a design method to retain the favorable properties conferred by such substitutions while at the same time avoiding the adverse properties. A particular example would be re-design of the insulin molecule to retain the enhanced thermodynamic stability and receptor-binding properties associated with substitution of HisB10 by Asp without incurring increased cross-binding to the Type I IGF receptor or increased mitogenicity.