Appl Microbiol Biotechnol (2005) 67: 151159 DOI 10.

1007/s00253-004-1809-x

MINI-REVIEW

Gary Walsh

Therapeutic insulins and their large-scale manufacture

Received: 22 July 2004 / Revised: 5 October 2004 / Accepted: 19 October 2004 / Published online: 4 December 2004 # Springer-Verlag 2004

Abstract Biotechnological innovations over the past 25 years have underpinned the rapid development of a thriving biopharmaceutical sector. Therapeutic insulin remains one of the most commonly used products of pharmaceutical biotechnology and insulin-based products command annual global sales in excess of $4.5 billion. Innovations in its method of production and in particular the advent of engineered insulin analogues provide a fascinating insight into how scientific and technological advances have impacted upon the pharmaceutical biotechnology sector as a whole. Current insulin-based diabetes research is increasingly focused not on the insulin molecule per se, but upon areas such as the development of non-parenteral insulin delivery systems, as well as organ-/cell-based and gene therapy-based approaches to controlling the disease.

and the world market for insulin was valued at US $4.5 billion ($4,500 million) in 2002. The market is expected to reach $7.9 billion by 2007. Novo Nordisk, Lilly and Aventis represent major global insulin manufacturers. Novos diabetic care products had a net turnover of 2.5 billion in 2003, of which 2.3 billion represented insulin sales (Novo 2003). Lillys diabetic care products had aggregate worldwide sales of US $2.57 billion in 2003, with two insulin products (tradenames Humulin and Humalog, as described later) each commanding sales in excess of $1 billion. (Lilly 2003). Aventis estimated 2003 sales of its insulin product Lantus to be 487 million (Aventis 2003). Insulin therefore represents one of the single most significant biopharmaceutical product categories, both in terms of medical impact and market value.

The insulin market

According to the World Health Organization (WHO) some 171 million people suffer from diabetes mellitus, a figure that is likely to double by 2030. The condition kills 3.2 million people annually (WHO 2004). Insulin therapy is essential to the survival of those with type 1 diabetes (socalled insulin-dependent diabetes) and is used to control the symptoms/progression of a minority of those with the more commonly occurring type 2 diabetes (so-called non-insulin-dependent diabetes). The average daily insulin dosage is in the region of 1.42.1 mg (equivalent to 4060 U insulin activity). In the industrialized world alone this represents an annual requirement for 4,600 kg insulin product (Kjeldsen 2000). There are some 180 branded insulin products available worldwide (Owens et al. 2001)

Diabetes and the discovery of insulin

Diabetes mellitus was first described over 2000 years ago. Although easily identifiable by characteristic symptoms, it remained a fatal medical condition until the early 1920s (Sutcliffe and Duin 1992). A link between diabetes and pancreatic secretions was first recorded in the 1800s by Queen Victorias physician, who noted the presence of crystals in the pancreatic tissue of deceased diabetics. This link was confirmed by subsequent research illustrating that dogs developed diabetes within 2 days of having their pancreas removed (Sutcliffe and Duin 1992). The antidiabetic agent insulin was first discovered in 1921 by two Canadian physiologists, Frederick Banting and Charles Best, while working in the laboratories of John MacLeod at the University of Toronto (Banting and Best 1922). In the spring of 1921 Banting and Best first discovered the ability of extracts from the islets of Langerhans to revive diabetic dogs close to death. They carried out initial safety studies by injecting each other with the preparation, fortuitously at levels sufficiently low to avoid hypoglycaemia. In January 1922 they administered crude insulin preparations to a diabetic patient, 14-year-old Leonard Thomson, thus beginning the era of insulin therapy (Bliss 1993).

G. Walsh (*) Industrial Biochemistry Program, University of Limerick, Limerick City, Ireland e-mail: Gary.walsh@ul.ie Tel.: +353-61-202664 Fax: +353-61-202568

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Early commercial preparations

Immediately upon its discovery, scientists at the US pharmaceutical company Eli Lilly began collaborating with Banting and Best, culminating in the introduction of the first ever commercially available insulin product in 1922. In Europe two Danish companies (Nordisk Insulinlaboratorium and Novo Terapeutisk Laboratorium) also began producing insulin by the mid 1920s (the companies eventually merged in 1989, forming Novo Nordisk which, along with Lilly, remain the worlds largest producers of therapeutic insulins). Early commercial insulin preparations were generally produced by an acid-alcohol precipitation of pancreatic extracts from slaughterhouse animals. From the mid 1920s scientists attempted to increase product purity. The discovery in the early 1930s that zinc promoted insulin crystallization was a landmark in this regard (Scott 1934). Zinc crystallization, followed by a re-crystallization step, facilitated the production of what became known as conventional insulins. By todays standards conventional insulins were relatively crude preparations. In addition to proinsulin (the precursor polypeptide from which mature insulin is derived via in vivo proteolytic excision of the C or connecting peptide), such preparations harboured glucagon, somatostatin and low levels of proteases. Also present were modified forms of insulin itself, such as desamidoinsulin, generated by the acidalcohol-induced hydrolysis of asparagine residue amido groups. Such contaminants could potentially lead directly or indirectly to clinical complications. Bovine and porcine proinsulins are immunogenic in man, while protease contaminants could hydrolyse the insulin. The latter problem at least, may be minimized by formulating the final product at low pH, under which condition insulin is stable, while any proteases present are inactive. The development of large-scale chromatographic systems rendered possible the industrial-scale production of high-purity insulin preparations. Gel filtration chromatography in particular was found effective in removing contaminant high molecular mass proteins (in particular proteases, proinsulin and insulin dimers) from re-crystallized insulin preparations, and products purified in this way became known as single peak insulins. Industrial-scale systems used to produce single peak insulins included Pharmacias stack column system, which displays a total bed volume of 96 l. Two litres of crude insulin can be applied in a single 8-h run, which yields 50 g purified insulin product. An ion exchange step is now generally included (often prior to gel filtration) in order to further increase purity and such products are known as purified or highly purified insulins. In more recent years both FPLC- and HPLC-based preparative systems have been used to generate modern, high-purity product (Galloway and Chance 1994). Industrial-scale reverse-phase HPLC columns with bed volumes approaching 80 l have been commissioned and these can purify several 100 g of insulin in a single run. The increase in purity afforded by the highresolution characteristics of HPLC is particularly important

in the context of recombinant insulins (discussed subsequently), as any contaminants in such a product will be microbial in nature, and hence are likely to be highly immunogenic.

Insulins of human sequence

The full amino acid sequence of human insulin was deduced in 1960 (Nicol and Smith 1960). Comparative studies illustrated that the human protein differed in sequence from bovine insulin by three amino acids and from porcine insulin by a single amino acid. Although only weakly immunogenic in man, anti-insulin antibodies have been detected in the serum of some diabetics administered animal-derived insulins. This rendered desirable the development of a therapeutic product identical in sequence to native human insulin. Although technically feasible, direct chemical synthesis was economically unattractive (Sieber et al. 1974; Owens et al. 1991). Commercially feasible methods of enzymatically converting porcine insulin into a product identical to human insulin were developed and optimized in the 1970s and early 1980s (Morihara et al. 1979; Markussen 1980, 1982; Markussen et al. 1981). Porcine insulin contains an alanine residue at the C terminal of the B chain (position B30) whereas threonine is present at that position in human insulin. The enzymatic transpeptidation process takes advantage of the fact that proteases, when in mixtures of organic solvents and water, are capable of peptide bond synthesis (Homandberg et al. 1978). The process entails incubation of porcine trypsin with crude porcine insulin in an organicaqueous solvent medium, in the presence of a large excess of threonine ester. Following the transpeptidation reaction (in which the B30 alanine residue is selectively replaced by the threonine ester), the trypsin is removed by a gel filtration step while unconverted porcine insulin is removed by anion exchange chromatography. Subsequent cleavage of an ester group on the threonine B30 residue yields human insulin, which is further chromatographically purified. Commercial product produced by enzymatic means is termed Human insulin emp.

First generation recombinant human insulins

The advent of recombinant DNA technology provided an obvious alternative and more direct means of synthesizing insulin of human sequence. Moreover, recombinant-based production systems are independent of a supply of pancreatic tissue from slaughterhouse facilities. The pancreas of a single pig typically yields sufficient purified insulin to treat a single diabetic for 3 days; Walsh 1998). Product from such sources can no longer sustain the ever increasing demand for insulin, rendering recombinant production essential to meet current and future market requirements. Recombinant human insulin was first produced by scientists at City of Hope in collaboration with Genentech in 1978 (Chance and Frank 1993). The approach adopted

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Fig. 1 A generalized overview of the industrial-scale production of modern high-purity recombinant insulins via the proinsulin route (a). An industrial-scale preparative HPLC column (photograph courtesy of NovaSep, France) (b)

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entailed the separate expression of chemically synthesized nucleotide sequences coding for the insulin A and B chains in two Escherichia coli production strains. After independent purification the two chains were co-incubated under reaction conditions promoting formation of intact insulin via disulphide bond formation and early commercial recombinant insulins were produced by this chain combination procedure (Chance et al. 1999). An alternative mode of production entailed expression of a single chemically synthesized nucleotide sequence coding for native human proinsulin. The expressed polypeptide was then purified followed by proteolytic excision of the C-peptide in vitro (Fig. 1). This approach is preferred, largely as a consequence of the requirement for only a single fermentation and subsequent purification protocol and because it is a substantially more efficient process than the two-chain combination approach. This proinsulin route has been employed commercially since 1986 (Chance et al. 1999). In

collaboration with Genentech, Eli Lilly used this technology to produce Humulin, the first of several recombinant insulins to be approved for general medical use (Table 1). Recombinant human insulin has also been produced in engineered Saccharomyces cerevisiae since the mid 1980s (Markussen et al. 1986; Thim et al. 1986), and a substantial proportion of current recombinant commercial insulins are produced by expression-optimized yeast systems (Kjeldsen 2000; Owens et al. 1991) (Table 1). Initial experiments indicated that proinsulin was not secreted efficiently from S. cerevisiae, although a subsequently designed proinsulinlike molecule was. This engineered construct consists of the insulin A chain and a 29 amino acid B chain (lacking the C terminal B30 threonine), either directly fused or linked via a short synthetic (often AAK) C peptide. The cDNA sequence coding for this construct is fused with the S. cerevisiae -factor prepro-peptide signal leader sequence, which guides extracellular secretion, and is itself

Table 1 Recombinant human insulins/engineered insulins currently approved for general medical use in the USA and/or EU Product Humulin Novolin Description Recombinant human insulin produced in Escherichia coli Recombinant human insulin produced in Saccharomyces cerevisiae Recombinant human insulin produced in E. coli All contain recombinant human insulin produced in S. cerevisiae formulated as short/intermediate/ long-acting product Recombinant short-acting human insulin analogue produced in E. coli Recombinant short-acting human insulin analogue produced in E. coli Recombinant short-acting human insulin analogue produced in S. cerevisiae Recombinant short-acting human insulin analogue produced in S. cerevisiae Recombinant long-acting human insulin analogue produced in S. cerevisiae Recombinant rapid-acting insulin analogue produced in E. coli Structure Identical to native human insulin Identical to native human insulin Identical to native human insulin Identical to native human insulin Company Eli Lilly Novo Nordisk Approved 1982 (USA) 1991 (USA)

Insuman Actrapid/Velosulin/ Monotard/Insulatard/ Protaphane/Mixtard/ Actraphane/Ultratard Humalog (insulin Lispro)

Hoechst Novo Nordisk

1997 (EU) 2002 (EU)

Liprolog (insulin Lispro)

NovoRapid (insulin Aspart)

Engineered: inversion of native B28B29 proline lysine sequence Engineered: inversion of native B28B29 proline lysine sequence Engineered: B28 proline replaced by aspartic acid Engineered: B28 proline replaced by aspartic acid Engineered: devoid of B30 threonine and a C14 fatty acid is covalently attached to B29 lysine Engineered: B3 asparagine is replaced by a lysine and B29 lysine is replaced by glutamic acid Engineered: A 21 asparagine replaced by glycine and B chain elongated by two arginines

Eli Lilly

1996 (USA and EU) 1997 (EU)

Eli Lilly

Novo Nordisk

1999 (EU)

Novolog (insulin Aspart)

Novo Nordisk

2001 (USA)

Levemir (insulin Detemir)

Novo Nordisk

2004 (EU)

Apidra (insulin Glulisine)

Aventis Pharmaceuticals

2004 (USA)

Lantus (insulin Glargine; Optisulin)

Recombinant long-acting human insulin analogue produced in E. coli

Aventis Pharmaceuticals

2000 (USA and EU)

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enzymatically removed during the process. Expression levels of up to 80 mg/l were initially obtained, although process optimisation has likely increased this value. The single chain insulin precursor is then recovered by ion exchange chromatography, with subsequent conversion to human insulin by a trypsin-mediated transpeptidation reaction carried out in an aqueousorganic solvent media, in the presence of excess threonine ester (Kjeldsen 2000). The product then undergoes further purification via a combination of crystallization and chromatography. More recent innovations in this system centre around optimization of the secretory efficiency from yeast. A notable success in this regard has been achieved by engineering a stabilizing interaction between an aromatic amino acid introduced into the C peptide and a phenol-binding site within the hydrophobic core of the molecule. By stabilizing the precursor molecule the secretory efficiency was increased fivefold (Kjeldsen et al. 2002).

Table 2 General pharmacokinetic characteristics of representative short-, intermediate- and long-acting native and engineered insulins Category Onset Peak activity Duration (time after (time after administration) administration) 30 min1 h 25 h 68 h

Insulin pharmacokinetics and formulation

Healthy humans typically secrete insulin continuously at a low basal level, with rapid but transient increases triggered by elevated blood glucose levels. Typically endogenous insulin levels peak 1 h after consumption of a meal, returning to basal levels within a further 2 h. Conventional insulin therapy cannot accurately reproduce this endogenous secretion pattern. When present in blood at physiologically normal levels (ca. 1010 M), biologically active insulin exists in monomeric form. Insulin is present in commercial formulations at far higher concentrations (ca. 103 M). At such concentrations it exists primarily in oligomeric form, as zinc-containing insulin hexamers (Blundell et al. 1972). The hexamers comprise three identical dimers co-ordinated to the zinc ions and the strongest subunit interactions lie between the dimerizing monomers. These complexes must first dissociate in order to be absorbed from the site of injection into the bloodstream. As a result, subcutaneously injected insulins have a slower onset and longer duration of action when compared to post-meal endogenous secretions (Kang et al. 1991) (Table 2). Peak plasma concentrations are not witnessed for up to 2 h, with levels remaining elevated for up to 5 h. In order to best mimic physiological patterns, conventional insulins are usually administered 30 min or more before mealtimes and the diabetic should not subsequently alter the planned mealtime. In addition to such fast- (short-) acting products, commercial product can also be formulated in order to retard the rate of insulin entry into the bloodstream from the site of injection (Table 2). Such slow- (long-) acting insulins may also be administered to diabetics in order to mimic low (base line) endogenous insulin secretion patterns. Long-acting insulins are usually prepared by formulating soluble insulins so as to generate insulin suspensions. This further prolongs the rate of dissociation into individual insulin monomers post injection. Insulin suspensions may be generated by addition of zinc (promoting zinc-insulin

Native insulin; short-acting formulation Native insulin; intermediateaction formulation Native insulin; long-acting formulation Humalog (engineered, rapid acting) Novorapid (engineered, rapid acting) Lantus (engineered, long acting)

2h

412 h

Up to 24 h

4h

1020 h

Up to 36 h 35 h

20 min

3090 min

15 min

30120 min

35 h

1h

24 h

crystal formation) or protamines (basic polypeptides to which insulin complexes).

Engineered (second generation) recombinant insulins

First generation recombinant insulins display an amino acid sequence identical to native human insulin and aim to compete with semi-synthetic human insulin and animalderived insulin products. However, the ability to chemically synthesize genes of altered nucleotide sequence (or techniques such as site-directed mutagenesis) facilitates the straightforward synthesis of insulin analogues displaying altered amino acid sequences. Thus far several such insulin analogues have been engineered to display either an accelerated or prolonged duration of action and many have been approved for general medical use (Tables 1, 2). Fast-acting insulins Most attempts to develop a fast-acting insulin analogue centred around altering the amino acid residues whose side chains contribute to oligomerization, particularly to dimer formation. The principle dimerization interactions involve residues B8, 9, 12, 13, 16 and 2328. Protein engineering experiments concentrated primarily upon a subset of these residues (B9, 12 and 2628), believed to lie on the periphery or fully outside the insulin receptor-binding region. The main engineering strategies pursued focused upon making amino acid substitutions which introduced charge repulsion at the monomermonomer surface, although sub-

156 Fig. 2ac Native human insulin and the engineered product insulin Lispro differ in amino acid sequence by the positioning of two amino acids. Native insulin contains a proline (P) residue at position 28 and a lysine (K) at position 29 of the B chain (a). The relative positioning of these two amino acids is inverted in the case of insulin Lispro (b). Illustration uses standard oneletter abbreviations for amino acids. The three dimensional structure of insulin Lispro (in the presence of phenol, zinc and chloride ions) has been determined by X-ray diffraction studies (c). Structure courtesy of the Protein Data Bank (PDB) (http://www.pdb.org/. PDB ID: 1LPH; from Ciszak et al. 1995 Structure 3:615)

stitutions leading to altered steric hinderance, hydrophobic interactions or removal of metal binding sites were also pursued (Brange et al. 1988, 1990). Several such engineered products were shown to inhibit oligomer formation, and/or promote a more rapid disassociation of oligomers, forming monomers. When compared to classic rapid-acting insulin preparations, these were absorbed 23 times faster after subcutaneous injection (Brange et al. 1988; Vora et al. 1988; Kang et al. 1990). Insulin Lispro was the first such engineered product to gain regulatory approval (Chance et al. 1999). This fastacting insulin analogue displays an identical amino acid sequence to native human insulin except for an inversion of the natural prolinelysine sequence at positions 28 and 29 of the B chain (Fig. 2). The rationale for this sequence alteration is rooted in studies of insulin-like growth factor 1 (IGF-1) which bares a strong structural resemblance to insulin (or, more accurately, to proinsulin). Up to 50% of residues within the IGF-1 A and B domains are identical to those in comparative positions in the insulin A and B chains. However, IGF-1 displays a significantly decreased propensity to self-associate when compared to insulin. Scientists noted that the sequence lysineproline found in the B28 and B29 positions of IGF-1 is reversed in

insulin. It was hypothesized that, if this sequence difference underlined the differing self-association characteristics, then inversion of the sequence in insulin should reduce the latters propensity to self-associate. Direct experimentation proved this to be the case (Chance et al. 1999; Frank et al. 1995). The dimerization constant for insulin lispro is 300 times less than for unmodified human insulin (Frank et al. 1995). Structurally this appears to occur as the sequence inversion leads to local conformational changes at the C termini of the B-chains, eliminating two critical hydrophobic interactions as well as weakening two terminal betasheet hydrogen bonds that stabilize the dimer (Ciszak et al. 1995). Insulin Lispro is produced commercially in a manner quite similar to the proinsulin route used to produce recombinant native human insulin (Fig. 1). A synthetic nucleotide sequence coding for LysB28ProB29 human proinsulin is expressed in a commercial strain of E. coli. After fermentation and extraction the insulin Lispro is proteolytically excised from the proinsulin by treatment with trypsin and carboxypeptidase B (Frank 1981). It is then extensively purified by a combination of high-resolution chromatographic methods. Usually included in the final formulation is M-cresol (preservative and stabilizer), zinc oxide

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(stabilizer), glycerol (tonicity modifier) and a phosphatebased buffer. Insulin Aspart is a second fast-acting commercial insulin analogue (Table 1). It differs from human insulin in that the proline residue at position 28 of the B chain is replaced by aspartic acid. This alteration increases inter-chain charge repulsion, thereby decreasing the propensity of individual insulin molecules to self-associate and facilitating rapid entry into the blood from the site of injection (Brange et al. 1988; Frank 1991). More recently a third rapid-acting insulin analogue has been approved under the tradename Apidra (Table 1). Because of their relatively short duration of action these rapid-acting insulin analogues are usually administered in combination with intermediate/long-acting insulins. Slow-acting insulins Although appropriate formulation has been employed in the past to generate long-acting insulins, engineered insulin analogues displaying prolonged duration of action have also been developed. Insulin Glargine is one such product approved for general medical use (Table 1). This differs from native insulin in that the C terminal asparagine residue of the A chain has been replaced with a glycine and the C terminal of the B chain has been elongated by two arginine residues. The net effect is to increase the isoelectric point (pI) from 5.4 towards more neutral values. The pI represents the pH at which a protein displays a net zero electric charge and consequently is least soluble. The product, expressed in E. coli and produced via the proinsulin route, is formulated at pH 4. At this pH it is fully soluble. However, upon subcutaneous injection, the insulin experiences more neutral pH values and thus appears to precipitate in the subcutaneous tissue. Insulin re-solubilizes very slowly from the precipitate, resulting in a longer duration of release into the bloodstreamsufficiently long to support a single daily dosage regime. Levemir (tradename) is an alternative long-acting insulin analogue developed by Novo Nordisk, which gained marketing approval within the European Union in June 2004. The analogue differs from native human insulin in that it is devoid of the threonine B30 residue, and a C14 fatty acid group is covalently attached via the side-chain of lysine B29. These modifications enable the insulin to bind reversibly to albumin, both at the injection site and in plasma. This in turn ensures constant release of product, giving it a duration of action of up to 24 h (Kurtzhals et al. 1997; Owens et al. 2001; Havelund et al. 2004). A key clinical feature of this insulin analogue is that it appears to display lower within-subject variability as compared to other longacting insulin formulations (Heise et al. 2004). Investigation of toxico-pharmacological properties of insulin analogues have also revealed some interesting results. For example, comparative-binding studies (Kurtzhals et al. 2000) reveal that the rapid-acting analogues insulin Lispro and insulin Aspart display similar affinities to that of native insulin for binding to the IGF-1 receptor. Insulin

Glargine, on the other hand, displays a six- to eightfold increase in IGF-1 receptor affinity whereas insulin Detemir displays a reduced affinity for the IGF-1 receptor. Such variations in IGF-1 receptor binding affinities may be of potential long-term significance, as they appear to correlate with product mitogenic potency.

Summary and future directions

Over the past 1015 years innovation in insulin therapy has largely focused upon the development of engineered insulins with altered pharmacokinetic properties, and a range of both fast- and slow-acting insulin analogues are now routinely used by millions of diabetics worldwide. While further insulin analogues will no doubt be developed, the focus of innovation in diabetic therapy is shifting somewhat. Alternative delivery systems Traditionally insulin administration entailed subcutaneous injection via syringe. Insulin pens in effect offer alternative means of achieving subcutaneous administration as do implanted or external insulin pumps (Steil et al. 2004) Several other potential delivery systems are also under development (Cefalu 2004; Owens et al. 2003; Ghilzai, 2003). These include the jet injector, which uses highpressure air to propel a fine spray of insulin through the skin. Transdermal delivery using insulin patches is also under investigation, but inhaled insulin delivery systems using aerosol devices appear to hold the most immediate therapeutic promise (Pillai and Panchagnula 2001). Macromolecules are absorbed from the lung surprisingly well, with reported bioavailabilities ranging from a few percent up to values approaching 50% in some instances (Patton et al. 1999; Patton 1996). High pulmonary bioavailibility likely stems from the lungs large surface area, thin diffusion layer, relatively low rate of cell surface clearance and the presence of proteolytic inhibitors. Additional advantages include availability of reliable nebulizer-based delivery systems and avoidance of first pass metabolism. Exubera is one such inhalable dry powder insulin formulation currently being evaluated by Pfizer and Aventis in conjunction with Nektar Therapeutics. Currently additional safety trials are ongoing subsequent to the diagnosis of pulmonary fibrosis in one patient participating in phase III clinical trials. One recent study also found that intrasubject variability values for insulin half life, terminal elimination rate constant and mean residence time were significantly less for inhaled insulin as compared to product administered by subcutaneous injection (Kapitza et al. 2004). Additional efforts to develop alternative insulin delivery systems (particularly nasal and oral systems) are ongoing (Hinchcliffe and Illum 1999; Carino and Mathiowitz 1999). Oralin and HIM2 represent two such products currently undergoing clinical trials. Oralin is an insulin formulation administered via an inhaler device. The insulin is absorbed

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into the bloodstream through the buccal mucosa (Generex 2004). Hexyl-insulin monoconjugate 2 (HIM2) consists of recombinant human insulin which contains a polyethylene glycol moiety plus alkyl linker attached to insulin via lys B29. Early clinical trials showed that HIM2 administered orally in a gelatin capsule provided some control over postprandial hyperglycaemia in patients with type 1 diabetes (Clement et al. 2004). Alternatives to insulin: transplants and gene therapy More radical approaches to diabetes treatment seek to bypass the need for insulin administration altogether. These include pancreatic transplantation (or a variant procedure entailing transplantation of isolated insulin-producing islet cells), stem cell therapy (Peck and Ramiya 2004) and gene therapy. Although first undertaken in the late 1960s, intact pancreatic allotransplantation (and later pancreatic islet transplantation) has met with only modest success. Complications include the requirement for subsequent chronic immunosuppression to maintain transplant viability and the shortage of donor pancreatic tissue (Lee and Bae 2000). Innovations such as (protective) encapsulation of islet cells in alginate, polyacrylate or other polymer-based microspheres have proven less than completely successful, as have the development of insulin-loaded hydrogels, responsive to glucose concentrations (Lee and Bae 2000; Kost and Langer 1991). However, research efforts in these areas continue (Roche et al. 2003). Attempts to stimulate the regeneration of insulin-producing islet cells represent an interesting variation upon islet cell-based therapeutic approaches. Research undertaken by Transition Therapeutics (http://www.transitiontherapeutics.com) indicate that administration of a combination of epidermal growth factor and gastrin may stimulate the regeneration of pancreatic insulin-producing islet cells. While phase I clinical trials were completed this year, later stage clinical trials are required to assess the genuine therapeutic potential of this approach. The use of stem/progenitor cells induced to differentiate into insulin-producing adult cells would provide an alternative cell-based therapeutic route. While such an approach would overcome issues such as a chronic shortage of donor pancreatic tissue, the ability to manipulate and control stem cell differentiation is at an embryonic stage. This approach, therefore, is of long-term rather than shortor intermediate-term potential (Efrat 2004). Gene therapy-based approaches to diabetes treatment continue to be the focus of intensive research efforts (Nett et al. 2003; Yoon and Jun 2002; Levene and Leibowitz 1999). Transient expression at least of the insulin gene has been clearly demonstrated upon its introduction into liver and skeletal muscle cells (Yoon and Jun 2002). Serious obstacles to a clinically useful gene therapy-based treatment approach, however, remain. These include:lack of appropriate glucose-based regulatory control of insulin synthesis and secretion in target cells; lack of the enzymatic machinery required for proinsulin processing in target cells.

Current and future research efforts in this area will focus mainly upon overcoming such obstacles. Overall, transplantation and gene therapy-based approaches to treating or curing diabetes remain beset by serious technical difficulties. This renders likely the continued use of purified therapeutic insulin preparations as the mainstay approach to (insulin-dependent) diabetes treatment for many years to come.

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