The present invention relates to an improved process for converting an insulin precursor into
an insulin compound, optionally via an insulin ester.
BACKGROUND OF THIS INVENTION
Insulin is a pancreatic hormone involved in the regulation of blood-glucose concentrations.
For example, human, porcine, and bovine insulin, insulin analogues and mixed insulins are
given to patients with insulin-dependent diabetes mellitus to control their blood-glucose
concentrations.
Porcine and bovine insulin are, usually, prepared from pancreas glands. Human
insulin can, semisynthetically, be prepared from porcine insulin. Alternatively, human insulin,
as well as many insulin analogues, can be prepared by genetic engineering. By genetic
engineering, which may, for example, be performed in bacteria or in yeast, an insulin
precursor is prepared which, thereafter, is to be converted into the desired product. This
conversion can be performed in different ways.
One possibility is the so-called transpeptidation where a peptide cleavage and a
peptide coupling takes place consecutively in the same reaction mixture, under the same
reaction conditions, vide, for example, US patent No. 4,343,898 (Novo Industri).
Another possibility is, in the first step, to cleave the insulin precursor, wofe, for
example, Hoppe-Seyler's Z. Physiol. Cliem. 359 (1978), 799, thereafter, to isolate the
intermediate product and, then, to perform the desired coupling in another reaction mixture
than that used in the first step, vide, for example. Nature 280 (1979), 412.
According to EP 87,238, a transpeptidation reaction is performed in a solvent system
comprising between about 75% and 97% (vol/vol) of at least one non-aqueous reaction
miscible solvent including at least about 50% (vol/vol) butane-1,4-diol.
According to US 4,579,820, the transpeptidation process is performed using an L-
specific serine carboxypeptidase enzyme, for example carboxypeptidase Y.
According to US 4,601,979 (Nordisk Insulinlaboratorium), the transpeptidation or only
the peptide coupling is performed in an aqueous reaction medium substantially free of
organic solvent.
According to WO 83/00504 (Nordisk insulinlaboratorium), a porcine product was
treated with carboxypeptidase A, the resulting des-alanine-B30 insulin product was
suspended in a lower alcohol, and this suspension was mixed with a solution of an L-
threonine ester and trypsin. In all the specific examples, the des-alanine-B30 insulin product
was isolated, either by freeze-drying or by precipitation.
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The object of this invention is to overcome or ameliorate at least some of the disadvantages
of the prior art. Hence, not all the more detailed objects mentioned below may be fully
overcome or ameliorated.
DEFINITIONS
The term "amino acid" as used herein, refers to amino acids which can be coded for by
nucleotide sequences. Analogously, this applies to the term amino acid residue which is an
amino acid from which hydroxy has been removed from a carboxy group and/or hydrogen
has been removed from an amino group.
Similarly, the terms peptide and peptide residue consists of amino acid residues.
Preferably, the peptide contains not more than 10 amino acid residues.
The term amino acid amide, as used herein, refers to an amino acid having an
optionally substituted C terminal carboxamide group.
The term peptide amide, as used herein, refers to a peptide having an optionally
substituted C terminal carboxamide group.
The term "insulin precursor", as used herein, refers to a polypeptide consisting of two
peptide chains (corresponding to the A and B chains of insulin and, hereinafter, designated
the A and B chains) which, similarly with insulin, are connected with each other via two
disulphide bridges (from one cysteine (Cys) residue to another cysteine residue) between
the two peptide chains and wherein, lilce in insulin, there is an disulphide bridge from one
cysteine residue in the A chain to another cysteine residue in the A chain. In this insulin
precursor there is, at least, one lysine or arginine residue in the B chain. Optionally, in this
insulin precursor, the A and B chains are connected with each other via a third peptide chain
(corresponding to the connecting peptide in insulin) between the C terminal end of the B
chain and the N terminal end of the A chain. In case the A and B chains are connected with
each other via this third peptide chain, lysine is present at the C terminal end of this third
peptide. Optionally, in this insulin precursor, a fourth peptide chain may be connected to the
N terminal end of the B chain. In case this fourth peptide chain is connected to the N
terminal end of the B chain, lysine is present at the C terminal end of this fourth peptide
chain. Furthermore, in this insulin precursor, there is an identity of the amino acid residues of
at least 80 %, preferably at least 85 %, more preferred at lest 90 %, and even more
preferred at least 95%, compared with human insulin, with the proviso that the third and
fourth peptide chains are to be disregarded for this calculation. In human insulin, there are
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disulphide bridges between Cys'^® and Cys*^\ between Cys^^ and Cys^^ and between
Cys*^° and Cys^^^ and there is lysine in the B29 position.
The term "amino acid ester", as used herein, refers to an amino acid carrying a C
terminal carboxy protecting group and, optionally, a hydroxy protecting group.
The term "peptide ester", as used herein, refers to a peptide wherein at least the C
terminal carboxy group carries a carboxy protecting group. Optionally, any hydroxy group is
protected and, optionally, the e-amino group of any lysine residues is derivatised, preferably
with a hydrophobic group, for example an acyl group having at least 10 carbon atoms.
Preferably, the peptide ester contains not more than 10 amino acid residues.
The term nucleophile compound, as used herein, refers an amino acid ester, an
amino acid amide, a peptide, a peptide ester, and a peptide amide. In any of these amino
acid esters, amino acid amides, peptides, peptide esters, and peptide amides, the amino
group in any lysine group is, optionally, derivatised, preferably with a hydrophobic group, for
example, an acyl group having at least 10 carbon atoms.
The term "insulin compound", as used herein, refers to insulin from any species such
as porcine insulin, bovine insulin, and human insulin and salts thereof such as zinc salts, and
protamin salts. Furthermore, the term "insulin compound", as used herein, refers to what
could briefly be designated "insulin analogues". Insulin analogues, as used herein, refers to
insulin compounds wherein one or more of the amino acid residues have been exchanged
with another amino acid residue and/or from which one or more amino acid residue has
been deleted and/or from which one or more amino acid residue has been added, provided
that said insulin analogue has a sufficient insulin activity. Examples of insulin analogues are
described in the following patents and equivalents thereto: US 5,618,913; EP 254,516; EP
280,534; US 5,750,497; and US 6,011,007. Examples of specific insulin analogues are
insulin aspart (i.e., [Asp^^^] human insulin), insulin lispro (i.e., [Lys^^^Pro^^®] human insulin),
and insulin glargin (i.e., [Gly*^\Arg^^\Arg^^^] human insulin). The term "insulin analogue", as
used herein also covers what could be designated insulin derivatives, i.e., compounds which
a skilled art worker would generally considers derivatives of insulin, vide general textbooks,
for example, insulin having a substituent not present in the parent insulin molecule.
Examples of insulin derivatives are insulins or insulin analogues having an optionally
substituted carboxamide group. Also compounds which can be considered being both an
insulin derivative and an insulin analogue are herein covered by the term insulin analogue.
Examples of such compounds are described in the following patents and equivalents
thereto: US 5,750,497 and US 6,011,007. Hence, a further example of a specific insulin
analogue is insulin detemir (i.e., des-Thr^^° human insulin y Lys^^^ tetradecanoyi). The insulin
compounds prepared by this invention have an anti-diabetic activity sufficiently high to be
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used to treat diabetic patients. The anti-diabetic activity can be determined using the so-
called free fat cell assay.
The term pH value, as used herein, refers to the value measured with a pH meter by
immersing a calomel combination glass electrode connected to the pH meter directly in the
solution, the pH value of which is to be measured. The pH meter is calibrated with an
aqueous standard buffer.
BRIEF DESCRIPTION OF THE FIGURES
SEQ ID NO.: 1 is the peptide moiety Glu-(Glu-Ala)3-Pro-Lys-; SEQ ID NO.: 2 is the peptide
moiety Glu-Glu-Gly-Glu-Pro-Lys-; and SEQ ID NO.: 3 is the peptide moiety Gly-Phe-Phe-
Tyr-Thr-Lys-Pro-Thr.
BRIEF DESCRIPTION OF THIS INVENTION
The present invention relates to a process for preparing insulin compounds. These insulin
compounds can be used as medicaments. In a preferred embodiment of this invention,
insulin compounds having threonine (Thr) in the C terminal end of the 8 chain are prepared.
Any sl