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Appl Microbiol Biotechnol (2005) 67: 151–159

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 and the world market for insulin was valued at US $4.5
years have underpinned the rapid development of a thriving billion ($4,500 million) in 2002. The market is expected
biopharmaceutical sector. Therapeutic insulin remains one to reach $7.9 billion by 2007. Novo Nordisk, Lilly and
of the most commonly used products of pharmaceutical Aventis represent major global insulin manufacturers.
biotechnology and insulin-based products command an- Novo’s diabetic care products had a net turnover of €2.5
nual global sales in excess of $4.5 billion. Innovations in its billion in 2003, of which €2.3 billion represented insulin
method of production and in particular the advent of en- sales (Novo 2003). Lilly’s diabetic care products had ag-
gineered insulin analogues provide a fascinating insight gregate worldwide sales of US $2.57 billion in 2003, with
into how scientific and technological advances have im- two insulin products (tradenames Humulin and Humalog,
pacted upon the pharmaceutical biotechnology sector as a as described later) each commanding sales in excess of $1
whole. Current insulin-based diabetes research is increas- billion. (Lilly 2003). Aventis estimated 2003 sales of its
ingly focused not on the insulin molecule per se, but upon insulin product Lantus to be €487 million (Aventis 2003).
areas such as the development of non-parenteral insulin Insulin therefore represents one of the single most signif-
delivery systems, as well as organ-/cell-based and gene icant biopharmaceutical product categories, both in terms
therapy-based approaches to controlling the disease. of medical impact and market value.

The insulin market Diabetes and the discovery of insulin

According to the World Health Organization (WHO) some Diabetes mellitus was first described over 2000 years ago.
171 million people suffer from diabetes mellitus, a figure Although easily identifiable by characteristic symptoms, it
that is likely to double by 2030. The condition kills 3.2 remained a fatal medical condition until the early 1920s
million people annually (WHO 2004). Insulin therapy is (Sutcliffe and Duin 1992). A link between diabetes and
essential to the survival of those with type 1 diabetes (so- pancreatic secretions was first recorded in the 1800s by
called insulin-dependent diabetes) and is used to control the Queen Victoria’s physician, who noted the presence of
symptoms/progression of a minority of those with the more crystals in the pancreatic tissue of deceased diabetics. This
commonly occurring type 2 diabetes (so-called non-in- link was confirmed by subsequent research illustrating that
sulin-dependent diabetes). The average daily insulin dos- dogs developed diabetes within 2 days of having their
age is in the region of 1.4–2.1 mg (equivalent to 40–60 U pancreas removed (Sutcliffe and Duin 1992). The anti-
insulin activity). In the industrialized world alone this diabetic agent insulin was first discovered in 1921 by two
represents an annual requirement for 4,600 kg insulin Canadian physiologists, Frederick Banting and Charles
product (Kjeldsen 2000). There are some 180 branded Best, while working in the laboratories of John MacLeod at
insulin products available worldwide (Owens et al. 2001) 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
G. Walsh (*) by injecting each other with the preparation, fortuitously at
Industrial Biochemistry Program, University of Limerick, levels sufficiently low to avoid hypoglycaemia. In January
Limerick City, Ireland
e-mail: Gary.walsh@ul.ie 1922 they administered crude insulin preparations to a
Tel.: +353-61-202664 diabetic patient, 14-year-old Leonard Thomson, thus be-
Fax: +353-61-202568 ginning the era of insulin therapy (Bliss 1993).
152

Early commercial preparations in the context of recombinant insulins (discussed subse-


quently), as any contaminants in such a product will be
Immediately upon its discovery, scientists at the US phar- microbial in nature, and hence are likely to be highly
maceutical company Eli Lilly began collaborating with immunogenic.
Banting and Best, culminating in the introduction of the
first ever commercially available insulin product in 1922.
In Europe two Danish companies (Nordisk Insulinlabora- Insulins of human sequence
torium and Novo Terapeutisk Laboratorium) also began
producing insulin by the mid 1920s (the companies even- The full amino acid sequence of human insulin was de-
tually merged in 1989, forming Novo Nordisk which, along duced in 1960 (Nicol and Smith 1960). Comparative
with Lilly, remain the worlds largest producers of thera- studies illustrated that the human protein differed in se-
peutic insulins). quence from bovine insulin by three amino acids and from
Early commercial insulin preparations were generally porcine insulin by a single amino acid. Although only
produced by an acid-alcohol precipitation of pancreatic weakly immunogenic in man, anti-insulin antibodies have
extracts from slaughterhouse animals. From the mid 1920s been detected in the serum of some diabetics administered
scientists attempted to increase product purity. The dis- animal-derived insulins. This rendered desirable the devel-
covery in the early 1930s that zinc promoted insulin crys- opment of a therapeutic product identical in sequence to
tallization was a landmark in this regard (Scott 1934). Zinc native human insulin. Although technically feasible, direct
crystallization, followed by a re-crystallization step, fa- chemical synthesis was economically unattractive (Sieber
cilitated the production of what became known as “con- et al. 1974; Owens et al. 1991).
ventional insulins”. Commercially feasible methods of enzymatically con-
By today’s standards conventional insulins were rela- verting porcine insulin into a product identical to human
tively crude preparations. In addition to proinsulin (the insulin were developed and optimized in the 1970s and
precursor polypeptide from which mature insulin is derived early 1980s (Morihara et al. 1979; Markussen 1980, 1982;
via in vivo proteolytic excision of the “C” or “connect- Markussen et al. 1981). Porcine insulin contains an alanine
ing” peptide), such preparations harboured glucagon, so- residue at the C terminal of the B chain (position B30)
matostatin and low levels of proteases. Also present were whereas threonine is present at that position in human
modified forms of insulin itself, such as desamidoinsulin, insulin. The enzymatic transpeptidation process takes ad-
generated by the acid–alcohol-induced hydrolysis of as- vantage of the fact that proteases, when in mixtures of
paragine residue amido groups. Such contaminants could organic solvents and water, are capable of peptide bond
potentially lead directly or indirectly to clinical complica- synthesis (Homandberg et al. 1978). The process entails
tions. Bovine and porcine proinsulins are immunogenic in incubation of porcine trypsin with crude porcine insulin in
man, while protease contaminants could hydrolyse the an organic–aqueous solvent medium, in the presence of a
insulin. The latter problem at least, may be minimized by large excess of threonine ester. Following the transpeptida-
formulating the final product at low pH, under which condi- tion reaction (in which the B30 alanine residue is se-
tion insulin is stable, while any proteases present are inactive. lectively replaced by the threonine ester), the trypsin is
The development of large-scale chromatographic sys- removed by a gel filtration step while unconverted porcine
tems rendered possible the industrial-scale production of insulin is removed by anion exchange chromatography.
high-purity insulin preparations. Gel filtration chromatog- Subsequent cleavage of an ester group on the threonine B30
raphy in particular was found effective in removing con- residue yields human insulin, which is further chromato-
taminant high molecular mass proteins (in particular graphically purified. Commercial product produced by en-
proteases, proinsulin and insulin dimers) from re-crystal- zymatic means is termed “Human insulin emp”.
lized insulin preparations, and products purified in this way
became known as “single peak insulins”. Industrial-scale
systems used to produce single peak insulins included First generation recombinant human insulins
Pharmacia’s “stack” column system, which displays a total
bed volume of 96 l. Two litres of crude insulin can be The advent of recombinant DNA technology provided an
applied in a single 8-h run, which yields 50 g purified obvious alternative and more direct means of synthesizing
insulin product. An ion exchange step is now generally insulin of human sequence. Moreover, recombinant-based
included (often prior to gel filtration) in order to further production systems are independent of a supply of pan-
increase purity and such products are known as “purified” creatic tissue from slaughterhouse facilities. The pancreas
or “highly purified” insulins. In more recent years both of a single pig typically yields sufficient purified insulin to
FPLC- and HPLC-based preparative systems have been treat a single diabetic for 3 days; Walsh 1998). Product
used to generate modern, high-purity product (Galloway from such sources can no longer sustain the ever increasing
and Chance 1994). Industrial-scale reverse-phase HPLC demand for insulin, rendering recombinant production es-
columns with bed volumes approaching 80 l have been sential to meet current and future market requirements.
commissioned and these can purify several 100 g of insulin Recombinant human insulin was first produced by
in a single run. The increase in purity afforded by the high- scientists at City of Hope in collaboration with Genentech
resolution characteristics of HPLC is particularly important in 1978 (Chance and Frank 1993). The approach adopted
153

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)
154

entailed the separate expression of chemically synthesized collaboration with Genentech, Eli Lilly used this technol-
nucleotide sequences coding for the insulin A and B chains ogy to produce Humulin, the first of several recombinant
in two Escherichia coli production strains. After indepen- insulins to be approved for general medical use (Table 1).
dent purification the two chains were co-incubated under Recombinant human insulin has also been produced in
reaction conditions promoting formation of intact insulin engineered Saccharomyces cerevisiae since the mid 1980s
via disulphide bond formation and early commercial re- (Markussen et al. 1986; Thim et al. 1986), and a substantial
combinant insulins were produced by this “chain combina- proportion of current recombinant commercial insulins are
tion” procedure (Chance et al. 1999). An alternative mode produced by expression-optimized yeast systems (Kjeldsen
of production entailed expression of a single chemically 2000; Owens et al. 1991) (Table 1). Initial experiments
synthesized nucleotide sequence coding for native human indicated that proinsulin was not secreted efficiently from
proinsulin. The expressed polypeptide was then purified S. cerevisiae, although a subsequently designed proinsulin-
followed by proteolytic excision of the C-peptide in vitro like molecule was. This engineered construct consists of
(Fig. 1). This approach is preferred, largely as a con- the insulin A chain and a 29 amino acid B chain (lacking
sequence of the requirement for only a single fermentation the C terminal B30 threonine), either directly fused or
and subsequent purification protocol and because it is a linked via a short synthetic (often AAK) C peptide. The
substantially more efficient process than the two-chain cDNA sequence coding for this construct is fused with
combination approach. This “proinsulin route” has been the S. cerevisiae α-factor prepro-peptide signal leader se-
employed commercially since 1986 (Chance et al. 1999). In quence, 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 Description Structure Company Approved

Humulin Recombinant human insulin Identical to native human Eli Lilly 1982 (USA)
produced in Escherichia coli insulin
Novolin Recombinant human insulin Identical to native human Novo Nordisk 1991 (USA)
produced in Saccharomyces insulin
cerevisiae
Insuman Recombinant human insulin Identical to native human Hoechst 1997 (EU)
produced in E. coli insulin
Actrapid/Velosulin/ All contain recombinant human Identical to native human Novo Nordisk 2002 (EU)
Monotard/Insulatard/ insulin produced in S. cerevisiae insulin
Protaphane/Mixtard/ formulated as short/intermediate/
Actraphane/Ultratard long-acting product
Humalog (insulin Lispro) Recombinant short-acting human Engineered: inversion of Eli Lilly 1996 (USA
insulin analogue produced in native B28–B29 proline– and EU)
E. coli lysine sequence
Liprolog (insulin Lispro) Recombinant short-acting human Engineered: inversion of Eli Lilly 1997 (EU)
insulin analogue produced in native B28–B29 proline–
E. coli lysine sequence
NovoRapid (insulin Aspart) Recombinant short-acting human Engineered: B28 proline Novo Nordisk 1999 (EU)
insulin analogue produced in replaced by aspartic acid
S. cerevisiae
Novolog (insulin Aspart) Recombinant short-acting human Engineered: B28 proline Novo Nordisk 2001 (USA)
insulin analogue produced in replaced by aspartic acid
S. cerevisiae
Levemir (insulin Detemir) Recombinant long-acting human Engineered: devoid of B30 Novo Nordisk 2004 (EU)
insulin analogue produced in threonine and a C14 fatty
S. cerevisiae acid is covalently attached
to B29 lysine
Apidra (insulin Glulisine) Recombinant rapid-acting insulin Engineered: B3 asparagine Aventis 2004 (USA)
analogue produced in E. coli is replaced by a lysine and Pharmaceuticals
B29 lysine is replaced by
glutamic acid
Lantus (insulin Glargine; Recombinant long-acting human Engineered: A 21 asparagine Aventis 2000 (USA
Optisulin) insulin analogue produced in replaced by glycine and Pharmaceuticals and EU)
E. coli B chain elongated by two
arginines
155

enzymatically removed during the process. Expression Table 2 General pharmacokinetic characteristics of representative
levels of up to 80 mg/l were initially obtained, although short-, intermediate- and long-acting native and engineered insulins
process optimisation has likely increased this value. The Category Onset Peak activity Duration
single chain insulin precursor is then recovered by ion (time after (time after
exchange chromatography, with subsequent conversion to administration) administration)
human insulin by a trypsin-mediated transpeptidation re-
action carried out in an aqueous–organic solvent media, in Native insulin; 30 min–1 h 2–5 h 6–8 h
the presence of excess threonine ester (Kjeldsen 2000). The short-acting
product then undergoes further purification via a combina- formulation
tion of crystallization and chromatography. Native insulin; 2h 4–12 h Up to
More recent innovations in this system centre around intermediate- 24 h
optimization of the secretory efficiency from yeast. A no- action
table success in this regard has been achieved by engi- formulation
neering a stabilizing interaction between an aromatic amino Native insulin; 4h 10–20 h Up to
acid introduced into the C peptide and a phenol-binding site long-acting 36 h
within the hydrophobic core of the molecule. By stabiliz- formulation
ing the precursor molecule the secretory efficiency was in- Humalog 20 min 30–90 min 3–5 h
creased fivefold (Kjeldsen et al. 2002). (engineered,
rapid acting)
Novorapid 15 min 30–120 min 3–5 h
Insulin pharmacokinetics and formulation (engineered,
rapid acting)
Healthy humans typically secrete insulin continuously at a Lantus 1h – 24 h
low basal level, with rapid but transient increases triggered (engineered,
by elevated blood glucose levels. Typically endogenous long acting)
insulin levels peak 1 h after consumption of a meal, re-
turning to basal levels within a further 2 h. Conventional
insulin therapy cannot accurately reproduce this endoge- crystal formation) or protamines (basic polypeptides to
nous secretion pattern. When present in blood at physio- which insulin complexes).
logically normal levels (ca. 10−10 M), biologically active
insulin exists in monomeric form. Insulin is present in
commercial formulations at far higher concentrations (ca. Engineered (second generation) recombinant insulins
10−3 M). At such concentrations it exists primarily in oligo-
meric form, as zinc-containing insulin hexamers (Blundell First generation recombinant insulins display an amino acid
et al. 1972). The hexamers comprise three identical dimers sequence identical to native human insulin and aim to
co-ordinated to the zinc ions and the strongest subunit compete with semi-synthetic human insulin and animal-
interactions lie between the dimerizing monomers. These derived insulin products. However, the ability to chemi-
complexes must first dissociate in order to be absorbed cally synthesize genes of altered nucleotide sequence (or
from the site of injection into the bloodstream. As a result, techniques such as site-directed mutagenesis) facilitates the
subcutaneously injected insulins have a slower onset and straightforward synthesis of insulin analogues displaying
longer duration of action when compared to post-meal altered amino acid sequences. Thus far several such insulin
endogenous secretions (Kang et al. 1991) (Table 2). Peak analogues have been engineered to display either an ac-
plasma concentrations are not witnessed for up to 2 h, with celerated or prolonged duration of action and many have
levels remaining elevated for up to 5 h. In order to best been approved for general medical use (Tables 1, 2).
mimic physiological patterns, conventional insulins are
usually administered 30 min or more before mealtimes and
the diabetic should not subsequently alter the planned Fast-acting insulins
mealtime. In addition to such “fast-” (short-) acting prod-
ucts, commercial product can also be formulated in order to Most attempts to develop a fast-acting insulin analogue
retard the rate of insulin entry into the bloodstream from the centred around altering the amino acid residues whose side
site of injection (Table 2). Such “slow-” (long-) acting chains contribute to oligomerization, particularly to dimer
insulins may also be administered to diabetics in order to formation. The principle dimerization interactions involve
mimic low (base line) endogenous insulin secretion patterns. residues B8, 9, 12, 13, 16 and 23–28. Protein engineering
Long-acting insulins are usually prepared by formulating experiments concentrated primarily upon a subset of these
soluble insulins so as to generate insulin suspensions. This residues (B9, 12 and 26–28), believed to lie on the pe-
further prolongs the rate of dissociation into individual riphery or fully outside the insulin receptor-binding region.
insulin monomers post injection. Insulin suspensions may The main engineering strategies pursued focused upon
be generated by addition of zinc (promoting zinc-insulin making amino acid substitutions which introduced charge
repulsion at the monomer–monomer surface, although sub-
156
Fig. 2a–c Native human insulin
and the engineered product in-
sulin 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 one-
letter abbreviations for amino
acids. The three dimensional
structure of insulin Lispro (in
the presence of phenol, zinc and
chloride ions) has been deter-
mined 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 insulin. It was hypothesized that, if this sequence difference
interactions or removal of metal binding sites were also pur- underlined the differing self-association characteristics,
sued (Brange et al. 1988, 1990). Several such engineered then inversion of the sequence in insulin should reduce the
products were shown to inhibit oligomer formation, and/or latter’s propensity to self-associate. Direct experimentation
promote a more rapid disassociation of oligomers, forming proved this to be the case (Chance et al. 1999; Frank et al.
monomers. When compared to classic rapid-acting insulin 1995). The dimerization constant for insulin lispro is 300
preparations, these were absorbed 2–3 times faster after times less than for unmodified human insulin (Frank et al.
subcutaneous injection (Brange et al. 1988; Vora et al. 1995). Structurally this appears to occur as the sequence
1988; Kang et al. 1990). inversion leads to local conformational changes at the C
Insulin Lispro was the first such engineered product to termini of the B-chains, eliminating two critical hydropho-
gain regulatory approval (Chance et al. 1999). This fast- bic interactions as well as weakening two terminal beta-
acting insulin analogue displays an identical amino acid sheet hydrogen bonds that stabilize the dimer (Ciszak et al.
sequence to native human insulin except for an inversion of 1995).
the natural proline–lysine sequence at positions 28 and 29 Insulin Lispro is produced commercially in a manner
of the B chain (Fig. 2). The rationale for this sequence quite similar to the proinsulin route used to produce re-
alteration is rooted in studies of insulin-like growth factor 1 combinant native human insulin (Fig. 1). A synthetic
(IGF-1) which bares a strong structural resemblance to nucleotide sequence coding for LysB28–ProB29 human pro-
insulin (or, more accurately, to proinsulin). insulin is expressed in a commercial strain of E. coli. After
Up to 50% of residues within the IGF-1 A and B domains fermentation and extraction the insulin Lispro is proteoly-
are identical to those in comparative positions in the insulin tically excised from the proinsulin by treatment with trypsin
A and B chains. However, IGF-1 displays a significantly and carboxypeptidase B (Frank 1981). It is then extensively
decreased propensity to self-associate when compared to purified by a combination of high-resolution chromato-
insulin. Scientists noted that the sequence lysine–proline graphic methods. Usually included in the final formula-
found in the B28 and B29 positions of IGF-1 is reversed in tion is M-cresol (preservative and stabilizer), zinc oxide
157

(stabilizer), glycerol (tonicity modifier) and a phosphate- Glargine, on the other hand, displays a six- to eightfold
based buffer. increase in IGF-1 receptor affinity whereas insulin Detemir
Insulin Aspart is a second fast-acting commercial insulin displays a reduced affinity for the IGF-1 receptor. Such
analogue (Table 1). It differs from human insulin in that the variations in IGF-1 receptor binding affinities may be of
proline residue at position 28 of the B chain is replaced by potential long-term significance, as they appear to correlate
aspartic acid. This alteration increases inter-chain charge with product mitogenic potency.
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. Summary and future directions
1988; Frank 1991). More recently a third rapid-acting in-
sulin analogue has been approved under the tradename Over the past 10–15 years innovation in insulin therapy
Apidra (Table 1). Because of their relatively short duration has largely focused upon the development of engineered
of action these rapid-acting insulin analogues are usually insulins with altered pharmacokinetic properties, and a
administered in combination with intermediate/long-acting range of both fast- and slow-acting insulin analogues are
insulins. now routinely used by millions of diabetics worldwide.
While further insulin analogues will no doubt be devel-
oped, the focus of innovation in diabetic therapy is shifting
Slow-acting insulins somewhat.

Although appropriate formulation has been employed in


the past to generate long-acting insulins, engineered insulin Alternative delivery systems
analogues displaying prolonged duration of action have
also been developed. Insulin Glargine is one such product Traditionally insulin administration entailed subcutaneous
approved for general medical use (Table 1). This differs injection via syringe. Insulin pens in effect offer alternative
from native insulin in that the C terminal asparagine residue means of achieving subcutaneous administration as do
of the A chain has been replaced with a glycine and the C implanted or external insulin pumps (Steil et al. 2004)
terminal of the B chain has been elongated by two arginine Several other potential delivery systems are also under
residues. The net effect is to increase the isoelectric point development (Cefalu 2004; Owens et al. 2003; Ghilzai,
(pI) from 5.4 towards more neutral values. The pI rep- 2003). These include the jet injector, which uses high-
resents the pH at which a protein displays a net zero electric pressure air to propel a fine spray of insulin through the
charge and consequently is least soluble. The product, skin. Transdermal delivery using insulin patches is also
expressed in E. coli and produced via the proinsulin route, under investigation, but inhaled insulin delivery systems
is formulated at pH 4. At this pH it is fully soluble. How- using aerosol devices appear to hold the most immediate
ever, upon subcutaneous injection, the insulin experiences therapeutic promise (Pillai and Panchagnula 2001). Macro-
more neutral pH values and thus appears to precipitate in molecules are absorbed from the lung surprisingly well,
the subcutaneous tissue. Insulin re-solubilizes very slowly with reported bioavailabilities ranging from a few percent
from the precipitate, resulting in a longer duration of release up to values approaching 50% in some instances (Patton
into the bloodstream—sufficiently long to support a single et al. 1999; Patton 1996). High pulmonary bioavailibility
daily dosage regime. likely stems from the lung’s large surface area, thin dif-
Levemir (tradename) is an alternative long-acting insulin fusion layer, relatively low rate of cell surface clearance and
analogue developed by Novo Nordisk, which gained mar- the presence of proteolytic inhibitors. Additional advan-
keting approval within the European Union in June 2004. tages include availability of reliable nebulizer-based deliv-
The analogue differs from native human insulin in that it is ery systems and avoidance of first pass metabolism.
devoid of the threonine B30 residue, and a C14 fatty acid Exubera is one such inhalable dry powder insulin formu-
group is covalently attached via the side-chain of lysine lation currently being evaluated by Pfizer and Aventis in
B29. These modifications enable the insulin to bind re- conjunction with Nektar Therapeutics. Currently additional
versibly to albumin, both at the injection site and in plasma. safety trials are ongoing subsequent to the diagnosis of
This in turn ensures constant release of product, giving it a pulmonary fibrosis in one patient participating in phase III
duration of action of up to 24 h (Kurtzhals et al. 1997; clinical trials. One recent study also found that intrasubject
Owens et al. 2001; Havelund et al. 2004). A key clinical variability values for insulin half life, terminal elimination
feature of this insulin analogue is that it appears to display rate constant and mean residence time were significantly
lower within-subject variability as compared to other long- less for inhaled insulin as compared to product adminis-
acting insulin formulations (Heise et al. 2004). tered by subcutaneous injection (Kapitza et al. 2004).
Investigation of toxico-pharmacological properties of Additional efforts to develop alternative insulin delivery
insulin analogues have also revealed some interesting re- systems (particularly nasal and oral systems) are ongoing
sults. For example, comparative-binding studies (Kurtzhals (Hinchcliffe and Illum 1999; Carino and Mathiowitz 1999).
et al. 2000) reveal that the rapid-acting analogues insulin Oralin and HIM2 represent two such products currently
Lispro and insulin Aspart display similar affinities to that of undergoing clinical trials. Oralin is an insulin formulation
native insulin for binding to the IGF-1 receptor. Insulin administered via an inhaler device. The insulin is absorbed
158

into the bloodstream through the buccal mucosa (Generex Current and future research efforts in this area will focus
2004). Hexyl-insulin monoconjugate 2 (HIM2) consists of mainly upon overcoming such obstacles. Overall, trans-
recombinant human insulin which contains a polyethylene plantation and gene therapy-based approaches to treating or
glycol moiety plus alkyl linker attached to insulin via lys curing diabetes remain beset by serious technical difficul-
B29. Early clinical trials showed that HIM2 administered ties. This renders likely the continued use of purified ther-
orally in a gelatin capsule provided some control over apeutic insulin preparations as the mainstay approach to
postprandial hyperglycaemia in patients with type 1 di- (insulin-dependent) diabetes treatment for many years to
abetes (Clement et al. 2004). come.

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