Novo Nordisk 

Compound Sharing

Novo Nordisk 

Compound Sharing

||

Liver-preferential insulin analogue

Category
ID
Amount per vial
Insulin
NNC0123-0327
1300 nmol

Insulin is a peptide hormone acting as a key regulator of glucose homeostasis. Insulin binds to the insulin receptor (InsR), which exists in two alternatively spliced isoforms, InsR-A and InsR-B. The insulin receptor belongs to the same family of receptor tyrosine kinases as the IGF1 receptor (IGF1R) and insulin is therefore also able to bind to the IGF1R albeit with considerably lower affinity as compared to the insulin receptor.

NNC0123-0327 is a liver-preferential insulin analogue with a C20 fatty acid attached at position A22K. NNC0123-0327 is able to bind reversibly to serum albumin with high affinity (which reduces the trans-endothelial transport from plasma to periphery) while retaining a high InsR affinity in the presence of albumin compared to other acylated insulin analogues ensuring a high hepatic InsR-mediated clearance). Due to these properties, NN0123-0327 therefore displays its liver-preferential effects following peripheral administration.

Selected calculated properties for NNC0123-0327 and human insulin.
PropertyNNC0123-0327Human insulin
MW (Da)66075808
pI4.85.7
Fatty acidC20 diacid-
LinkergGlu-2xOEG-
Seqeunce substitutionsA22K, B29R, desB30-
Sum FormulaC296 H453 N71 O88 S6C257 H383 N65 O77 S6

2D sketch of NNC0123-0327. The B chain is lacking threonine at position B30, contains a B29 arginine substitution and there is an additional lysine a position A22 to which a fatty acid is attached via a gamma-Glu-2xOEG linker.
Graphic representation of the crystal structure of NNC0123-0327 crystallised under zinc free conditions (unpublished structure). The fatty acid, the linker and the lysine at position A22K are shown in yellow stick representations.

Receptor binding data for NNC0123-0327 (unpublished data).

The in vitro receptor binding affinity of NNC0123-0327 relative to human insulin (HI) is shown in the table below. The affinity was determined using human insulin receptor isoform A and also the human IGF1R as solubilized holoreceptors in the absence or presence of 1.5% (w/v) human serum albumin (HSA). Since albumin binding is a key mechanism for the design of NNC0123-0327, be aware that the apparent affinity and potency will be very dependent on whether the in vitro assays contain albumin or not.

ReceptorHSA concentration
[%]
Relative affinity
(mean +/- SD)
[% of HI]
Human InsR-ANo HSA165 +/- 26.5
1.511.3 +/- 1.1
Human IGF1RNo HSA570.3 +/- 47.4
1.59.1 +/- 0.1
InsR-A: insulin receptor isoform A; HI: human insulin; HSA: human serum albumin; IGF1R: IGF1 receptor

The pharmacokinetic properties of NNC0123-0327 in Wistar rats following an i.v. injection are listed in the table.

NNC0123-0327 displays a markedly protracted PK profile in vivo following a s.c. injection. Plasma PK parameters following i.v. dosing from a study in Wistar rats are given in the table below. For i.v. and s.c. studies in rats, dose levels of 1-5 nmol/kg and 5-30 nmol/kg, respectively, are recommended both for once and twice daily dosing studies. For the i.v. study in the table below, NNC0123-0327 was dissolved in 5 mM phosphate, 140 mM NaCl and 70 ppm polysorbate 20, pH 7.4. The PK parameters for NNC0123-0327 are determined in wake rats whereas the parameters for human insulin are determined in anaesthetized rats. The evaluation of the liver-preferential effects of NNC0123-0327 is described in the Edgerton et al. references listed in the reference section.

ParameterNNC0123-0327
Human insulin
Dose [nmol/kg)]51.2
T1/2 [h]0.830.28
MRT [h]
0.680.15
CL [mL/min/kg] 0.9622
Vz (mL/kg)
78623


CL: clearance; MRT: mean residence time; T1/2: half-life; Vz: volume of distribution

Human insulin (NNC0121-0308) is available as a reference compound to NNC0123-0327. Please indicate in the 'Purpose' field when you order NNC00123-0327 if you would like to have human insulin (NNC0121-0308) included in your shipment.

Peptides and proteins have a tendency to adhere to glass and plastic surfaces. This may at low concentration impact the actual amount in solution. To minimize this unspecific adherence, adding detergents or inert proteins like e.g., ovalbumin or other serum albumins to the solution can minimize this phenomenon. In case albumins are added to peptide/protein solutions, ensure that the albumins are free of any proteases, but be aware that it will affect the apparent potency and affinity in in vitro assays in case a fatty acid is attached to the compound. Recommended procedure for in vitro studies: dissolve the entire content of the vial by adding 4 mL 30 mM HEPES buffer pH 8. Gently rotate the vial until all content is dissolved. Avoid harsh shaking or stirring of the solution. Keep the stock solution at 4C overnight and make the desired number of aliquots (use low protein binding vials) of the stocks. Snap freeze the aliquots in liquid nitrogen and store them at minus 20C. When thawed, the stock solution should be stable for up to three weeks at 4C. Recommended procedure for in vivo studies: NNC0123-0327 and human insulin (NNC0121-0308) can be dosed in vivo in a zinc-free and phenol/m-cresol-free formulation vehicle. For NNC0123-0327 it may contain 8mM sodium phosphate, 240mM glycerol, pH 7.4 and for human insulin it may contain 10mM sodium phosphate, 140mM sodium chloride, pH 7.4. For both compounds, consider adding 0.007% polysorbate 20 if concentrations are so low that adsorption to vials may affect the concentration. If insulin oligomeric/multimeric properties are of interest, zinc and phenol/m-cresol can be added. Zinc is typically added as zinc acetate between 0 and 6 moles/mole of insulin analogue. The resulting tonicity should be recalculated. Formulations should be used fresh but can be stored for up to one week refrigerated.

Edgerton DS et al.
Insulin’s direct hepatic effect explains the inhibition of glucose production caused by insulin secretion
JCI Insight 2017; 2(6): e91863
Edgerton DS et al.
Targeting insulin to the liver corrects defects in glucose metabolism caused by peripheral insulin delivery
JCI Insight. 2019 Apr 4; 4(7): e126974