J Pharm Biomed Anal.2017 Feb;135:126-132

Structure and Pharmaceutical Formulation Development of a New Long-acting Recombinant Human Insulin Analog Studied by NMR and MS

Elżbieta Bednarek,a Jerzy Sitkowski,a Wojciech Bociana, Piotr Borowiczb, Grażyna Płucienniczak,b Dorota Stadnikb, Weronika Surmacz-Chwedorukb, Beata Jaworska,a and Lech Kozerski*a

aNational Medicines Institute , 00-725 Warsaw, Poland,

bInstitute of Biotechnology and Antibiotics, Warsaw, Poland

 

 Correspondence to Professor Dr. Lech Kozerski

National Medicines Institute , Chełmska 30/34, 00-725 Warsaw

e-mail; l.kozerski@nil.gov.pl

phone; +48 (22) 8514372 ext 318 

 

Abstract 

Although last generations antidiabetic drugs are becoming more efficient yet the administration of human insulin (HIS) or its various analogues obtained by rationally designed sequence mutations is still the most effective way of pharmacotherapy in cases of acute type 1 diabetes. There has been developed commercial preparations containing modified human insulin with long-acting or rapid-acting properties [1] but still there is a need to obtain new human insulin analogues for better control of glucose level and minimize the risk for hypoglycemia in diabetics therapy.

As a result of our earlier studies [2], [3], [4] in this area we have designed a promising human insulin analog SK3R (serine and positively charged arginine residues have been added which elongate the C-terminus of the A-chain (A22) and B-chain (B31), respectively, and lysine has been used to replace the asparagine at position B3).

A monomer structure of a novel human insulin analog A22S-B3K-B31R (SK3R)

(Fig.1) has been characterized by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS) established in the same medium. The composition of the oligomer ensemble for neat insulins and pharmaceutical formulation in water was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion coefficient Di x 10-10m2s-1, whose value is a population averaged of individual coefficients for species in oligomeric ensemble. The nanospray ESI MS experiment was used to establish the masses of oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were established and compared to insulin glargine characterized by the same profile of action in diabetics. These properties are compared with human insulin (HIS) which is a standard reference for novel recombinant insulins.

 

 

Fig 1. The ensemble of 20 structures from molecular modeling, based on NMR derived experimental constraints, representing molecular structure of SK3R insulin monomer in water/acetonitrile ( 65/35 vol% ) solution. Black string represents the backbone structure of human insulin used as a reference. 

 

Highlights

  • The study of a molecular structure and pharmaceutical formulation of novel recombinant human insulin analog SK3R was undertaken on a molecular level.
  • Oligomerization of a neat insulin and a pharmaceutical form was monitored using NMR derived translational diffusion parameters and nanospray ESI MS.
  • The oligomerization process of insulin during development of pharmaceutical formulation with routinely used excipients has been studied using translation diffusion coefficient Di x 10-10m2s-1 established in water solution.
  • The SK3R insulin has a long-acting, flat, almost no “peakless” course of regulating glucose in time and shows no fluctuations on concentration during long-term administration.

 

References:

[1] M.A. Weiss, Design of ultra-stable insulin analogues for the developing world. Journal of Health Specialities 1 (2013 ) 59   – 70.
[2] P. Borowicz, A. Płucienniczak, J. Mikołajczyk, T. Głąbski, D. Kurzynoga, D. Mikiewicz-Syguła, A. Wojtowicz-Krawiec, M. Zieliński, M. Kęsik-Brodacka, V. Adamczewska-Cecuda, I. Sokołowska, G. Płucienniczak, D. Stadnik, J. Antosik, J. Pstrzoch, J. Bernat, W. Sławiński, T. Pawlukowiec, J. Stępniewski, and M. Bogiel, Novel insulin analogues of prolonged activity, Institute of Biotechnology and Antibiotics, Patent No.  WO 2010/002283, Poland.
[3] P. Borowicz, E. Bednarek, W. Bocian, J. Sitkowski, B. Jaworska, J. Mikołajczyk, T. Głąbski, D. Stadnik, W. Surmacz, M. Bogiel, and L. Kozerski, Recombinant A22(G)-B31 (R)-human insulin. A22 addition introduces conformational mobility in B chain C-terminus. J Biomol NMR 52 (2012) 365-70.
[4] P. Borowicz, W. Bocian, J. Sitkowski, E. Bednarek, D. Mikiewicz-Sygula, D. Kurzynoga, D. Stadnik, W. Surmacz-Chwedoruk, W. Koźminski, and L. Kozerski, Biosynthetic engineered B28(K)-B29(P) human insulin monomer structure in water and in water/acetonitrile solutions. Journal of Biomolecular NMR 55 (2013) 303-9.