Research article Design, synthesis, characterization

Annals of Drug discovery and Biomewdical Research Ann Drug disc Biomed Res 2014; 1(4): 234-253 www.annalsdbr.com

Research article

Design, synthesis, characterization and prolonged antihyperglycemic activity of some novel insulin conjugates Kiranmai M *a,, Prashantha Kumar B.R b,, Laxmi Adhikaryc a

Sree Dattha Institute of Pharmacy, Sheriguda, Ibrahimpatnam, Rangareddy 501 510, India. b J.S.S. College of Pharmacy, SS Nagar, Mysore 570 015, India. c

Biocon India Ltd., Bangalore, India.

Recieved: 5th Dec 2014. Accepted 27th Dec 2014. *Corresponding author: [email protected] Abstract: Background: The most recent strategy has been to acylate fatty acid residues to the insulin molecule which enhances its

affinity to

serum albumin to delay its activity. Herein, we report synthesis, analysis and SAR of certain novel insulins attached to fatty acid and hydrophobic moities. Results: Desthreoinsulin with succinimidyl caprate that is conjugate-3 has shown extended antihyperglycemic profile even up to 24 hours with single dose followed by conjugate 2 and 4. The extended action of conjugate 3 may be attributed to its enhanced affinity to serum albumin. It is interesting to point out that desthreoinsulin conjugates showed better antihyperglycemic activity than insulin conjugates. Conclusion: Among the tested insulin conjugates, conjugate 3 that is LysB29 (N- ε-decanoyl) des (B30) human insulin exhibited longer duration of antihyperglycemic activity and hence needs attention for further development.

Kiranmai M et al., Ann Drug disc Biomed Res 2014; 1(4)

neutral causing it to precipitate in the subcutaneous tissue; prolonged half life Introduction

(flat profile) to delay its activity by

An ideal insulin must possess long

binding insulin to a serum carrier [4-6].

duration of action, providing 24 h

Right from 1995 to till date reports have

control on blood glucose levels with

been showing promising action profiles

minimum

of long-acting insulin conjugates [7-10].

variability

in

absorbtion.

Traditionally available basal insulins

Zoltan vajo et al.,

have reported on

have been unsatisfactory for several

insulin conjugates prepared by acylating

reasons [1,2]. Insulin glargine was the

insulin with fatty acids such as palmitic

very first clinically available long acting

acid, myristic acid that are presently

recombinant human insulin [3]. The

undergoing preclinical and early clinical

most recent strategy has been to acylate

trails [11].

fatty acid residues to the

insulin

Insulin detemir (Figure 1) commonly

molecule which enhances its affinity to

called detemir differs from human

serum albumin.The results revealed

insulin

various advantages of fatty acid acylated

position B30, and a C14 fatty acid chain

insulins (FAAI) which include; no

(myristic acid) that has been attached to

pronounced peak of activity; delayed

the lysine at position B29 [12]. The

absorbtion by changing the insulin pH to

results also suggest that insulin detemir

structure with threonine in

A-chain COOH

NH 2

NH 2

Lys B B-chain

29 -X

R

Figure 1: Schematic representation of acylated human insulin: R denotes the fatty acid attached by an amide bond to the ε- amino group of LysB29, X denotes threonine, R is myristate moiety in case of detemir

235


Figure 2: Schematic representation of des (B30) human insulin acylated by lithocholic acid ligand at the ε-amino group of the side chain of LysB29.

has

significantly

glucose-lowering NPH(neutral

more

predictable

effect

protamine

than hagedorn)

increase

their

attempted

to

hydrophobicity, attach

hydrophobic

ring

insulin system

we with like

insulin and insulin glargine [13-16].

cyclohexane. The prime objective of our

Dorchy et al., have revealed that detemir

work was to attach above mentioned

has 98-99% albumin binding ability in

moieties to ε-amino group of lysine of

plasma [17]. Recently, Jonassen et al.,

insulin

reported that, NN344, lithocholic acid

conjugation was known to improve

acylated insulin conjugate (Figure 2)

duration

shows promising slow release action

bioavailability.

[18]. Therefore, all these observations

Experimental

impress upon FAAI’s for showing



structure,

of

action

because,

with

lysine

enhanced

Chemistry

delayed and flat action profiles to

All the synthetic work was carried out

maintain normal blood glucose levels.

using analytical grade reagents and

Considering this, we have designed and

solvents. Reactions were monitored by

synthesized some of them for their

thin layer chromatography (TLC) carried

plausible antihyperglycemic activity.

out using aluminium cards precoated

In light of above background, it appears

(0.2 mm thickness) with silica gel

that, acylation of insulin at LysB29 with

(Merck) visualized under UV light.

fatty acid is essential for their long

Activated esters were concentrated by

duration of action, hence we have

using Heidolph Rotavac. The reactions

designed

conjugates

were monitored, purified and collected

attached with fatty acids. In order to

using Agilent 1100series HPLC. Final

some

insulin

236


reaction mixtures were concentrated by

mixture

using DNA 120 speed vac system. Mass

dichloromethane and added to triethyl

spectra of conjugates were obtained by

amine

using

LC-MS

succinimide (1d) (0.0352 M, 4.056 g)

working with electron spray ionization

was added to the above mixture and

principle on positive mode. ¹H NMR and

cooled to 0°C. The above reaction

13

C NMR spectra were recorded on

mixture was stirred over night. Then the

DRX-400 MHz Bruker NMR instrument

reaction mixture was washed with ice

using CDCl3 as solvent and TMS as

cold 1% HCl, water and with 1%

internal standard. Centrifugation was

NaHCO3

done

laboratory

concentrated to yield chloromyristate

centrifuge. Collected fractions of insulin

(1c) (92%). Completion of reaction was

conjugates were lyophilized by Thermo

monitored by TLC (ethyl acetate and

savant

methanol, 8:2) and then confirmed by 1H

Agilent

by

1100series

using

REMI

lyophilizer.

Post

enzymatic

was

redissolved

in

(0.054 M, 7.56 g). N-hydroxy

finally

13

filtered

and

C NMR. 1H NMR (CDCl3)

treatment all solutions were incubated at

NMR and

370C in BOD incubator.

δ in ppm : 0.845 (t, 3H, H-1), 1.245 to



Typical procedure

experimental to

prepare

N-

succinimidyl myristate N-succinimidyl myristate was prepared according to the step-1 of scheme1 as follows: Myristic acid (1a) (0.044 M, 10 g)

was

dissolved

in

100

ml

of

dichloromethane and cooled to 0°C. Catalytic amount of DMF (0.05 ml) was added to the above solution. Oxalyl chloride (1b) (0.0462 M, 5.82 g) was added slowly and stirred for 2-3 h under mild reflux conditions. Excess of oxalyl chloride and solvent was removed under reduced pressure by using rotavac. This

1.406 (m , 20H, H-2, H-3, H-4, H-5, H6, H-7, H-8, H-9, H-10, H-11), 1.744 (m, 2H, H-12), 2.59 (t, 2H, H-13), 2.80 to 2.821 (m, 4H, H-14, H-15).

13

C NMR

(CDCl3, 400 MHz) δ: 14.090 (C-1), 22.672 (C-2), 24.596 (C-3), 24.686 (C4), 25.582 (C-5), 28.782 (C-6), 29.074 (C-7), 29.231 (C-8), 29.337 (C-9), 29.421 (C-10), 29.540 (C-11), 29.624 (C-12), 30.930 (C-13), 31.905 (C-14,C15), 168.681 (C-18), 169.219 (C-16,C17). 

Typical procedure

experimental to

prepare

Conjugate 1 237


Desthreo insulin was conjugated with

phase: ethyl acetate and methanol, 8:2)

N-succinimidyl myristate according to

and then confirmed by 1H NMR and 13C

the step-2 of scheme 1 as follows:

NMR. 1H NMR (CDCl3) δ in ppm: 0.84

Desthreo insulin (0.069 mM, 0.4 g) was

(t, 3H, H-1), 1.25 to 1.40 (m, 10H, H-2,

dissolved in 5ml of 200 mM borate

H-3, H-4, H-5, H-6), 1.74 (m, 2H, H-7),

buffer (pH 8.2) and pH adjusted to 2.5

1.90 (m, 2H, H-8), 2.56 (t, 2H, H-9),

using 10 % HCl. The solution of 1c

2.82 to 2.85 (m, 4H, H-10, H-11).

(0.084 mM, 0.0273 g) was dissolved in 3

NMR

ml of acetonitrile (preheated to 50°C).

1),22.448(C-2),24.751(C-3,C-4,C-

The p

H

(CDCl3,400

13

C

MHz)δ:13.889(C-

of the protein solution was

5),25.414(C-6),28.58(C-7), 29.001(C-8),

readjusted to 10.22 slowly by using 10

29.112(C-9), 30.736(C-10), 31.630(C-

% NaOH and stirred for 15min. To 2ml

11), 168.54(C-14), 169(C-12,C-13).

of this, ester solution was added and



Typical

experimental

stirred for 1h at 4°C to yield conjugate -

procedure

1(68.99 %).

Conjugate 2



Typical procedure

to

conjugated

prepare

experimental

Insulin

prepare

succinimidyl caprate according to the

N-

was

to

with

N-

step-2 of scheme 2 as follows: Insulin

succinimidyl caprate N-succinimidyl caprate was prepared

(0.172 mM, 1 g) was dissolved in 30 ml

according to the step-1 of scheme 2:

of 200 mM borate buffer and pH

Capric acid (2a) (0.01 M, 1.72 g),

adjusted to 2.5 by using 10% HCl. The

dicyclohexylcarbodiimide (2b) (0.01 M,

solution was stirred until the crystals

2.06 g), 1d (0.01 M, 1.15 g), dimethyl

were

amino pyridine (catalytic amount) were

inspection. Solution of active ester was

taken in a round bottomed flask and 100

prepared by dissolving ester (0.344 mM,

ml of dichloro methane was added to it.

0.092g) in 8.1 ml of acetonitrile (pre

This reaction mixture was stirred over

heated to 50°C).

night, filtered and concentrated to yield

solution was readjusted to 9.51 using

N-hydroxy

caprate

10% NaOH and solution was stirred at 4

(intermediate-2, 90%). Completion of

ºC for 15 min then 21.9 ml of

reaction was monitored by TLC (mobile

acetonitrile and ester solution were

succinimidyl

fully

dissolved

by

visual

pH of the protein

238


added. Solution was stirred at 4 0C for

(0.0198mM, 0.115g) was dissolved in

1h to yield conjugate-2 (54.99%).

10ml of 100mM borate buffer solution at



Typical procedure

experimental

pH 2.5. The solution was stirred until the

prepare

crystals were well dissolved by visual

to

inspection. pH of the insulin solution

conjugate-3 Desthreo insulin was conjugated with N-

was adjusted to 9.51 by using 10%

succinimidyl caprate according to the

NaOH and solution was stirred. 20µl of

scheme 3 as follows: Desthreo insulin

cyclohexanoyl chloride (3a) in 2ml of

(0.263 mM, 0.5 g) was dissolved in 20

100% acetonitrile was added to above

ml of 200 mM borate buffer and pH was

protein solution and stirred for 85 min at

adjusted to 2.5 by using 10% HCl. The

4°C to yield conjugate -4 (55.3%).

resultant

solution was stirred until



General

procedure

crystals were fully dissolved by visual

purification

inspection. Solution of 2c was prepared

conjugates [19]

of

for

synthesized

by dissolving the ester (0.454 mM,

Synthesized compounds were purified

0.1216 g) in 5.4 ml of acetonitrile (pre

by preparative HPLC and product

heated to 50ºC). pH of the protein

fractions were collected and pooled. The

solution was readjusted to 10.22 using

pooled fractions were analyzed by

10% NaOH and solution was stirred for

HPLC and lyophilized to get pure white

15min

40C,

at

then

14.6

ml

of

powder.

acetonitrile, solution of 2c were added and reaction mixture was stirred at 40C for 1h to yield conjugate-3 (85.5%). 

Typical

experimental

procedure

to

prepare

conjugate-4 Insulin

was

conjugated

with

cyclohexanoyl chloride according to the scheme

4

as

follows:

Insulin

239


Table 1: List of Compounds Name

Molecular Weight

1a

Myristic acid

228

2a

Capric acid

172

3a

Cyclohexanoyl chloride

146.5

Intermediate-1 Intermediate-2

N- Hydroxy succinimidyl myristate N-Hydroxy succinimidyl caprate LysB29 (N- ε-tetradecanoyl) des(B30)human insulin

Conjugate 1 Conjugate 2 Conjugate 3 Conjugate 4



Parameters

used

volume-20µl,

268.44 5916.9

LysB29 (N- ε-decanoyl) human insulin LysB29 (N- ε-decanoyl)des (B30) human insulin

5963

LysB29 (N- ε-cyclohexanoyl) human insulin

5918

for

both

5852

It’s a technique for analyzing proteins by separating and detecting the mixture of

HPLC and LCMS Injection

325.44

rate-

fragments generated when a protein is

1ml/min, sop time-25min., column and

broken up with enzymes. The analysis

its temperature-C18 symmetry, 40°C,

can provide information about the

maximum pressure-400 bar, elution-

original protein's sequence and detect

gradient, buffer A-100% acetonitrile,

subtle

buffer B-0.1% triflouro acetic acid. All

when coupled with mass spectroscopy.

conjugation reactions were monitored by

Enzyme used here was glutamine C and

HPLC and confirmed by LC-MS. HPLC

it has specificity for aspartic acid and

and

all

glutamine amino acids. 200µg of each

conjugates including standard insulin is

synthesized conjugate was dissolved in

shown in Table 3.

200µl of 50 mM tris buffer at pH of 8.2

LC-MS

flow

information

for

differences

between

proteins

and 5 µl of glutamine C was added. The 

experimental

resultant solutions were incubated at

procedure for peptide mapping

37°C for 4h and then characterized using

Typical

240


LCMS and results were given in Tables

animal with blood glucose concentration

4-7. Figures 4-7 were showing the

above 250 mg/dl was considered to be

LCMS profiles of all four conjugates.

diabetic.



Pharmacology



Streptozotocin

overcome

the

hypoglycemia, which occurs during the induced

first

24h

following

streptozotocin

administration, diabetic rats were orally

diabetes in rats [20] Male wistar

To

rats of body weight

given 5% glucose solution. After the

150+25g were selected for this study.

confirmation of diabetes animals were

Rats were maintained under standard

divided in to five groups consisting of

environmental conditions (22-28

o

C

six

animals

each.

Animals

of

temperature, 60-70% relative humidity,

experimental groups ( I, II, III, IV) were

12 h dark/light cycle), fed with standard

administered a single subcutaneous dose

rat feed and water ad libitum. This study

of

was carried out in accordance with the

0.0045mg) and standard insulin (125

approved

milli units, 0.0045mg) was administered

protocol

animal

by

ethics

Institutional committee

(1330/ac/10/CPCSEA).

conjugates

(125

milli

units,

to group V. Blood samples were withdrawn from retro orbital plexus at 0, 1, 2, 3, 5, 7, 12

The animals were housed in large spacious hygienic cages during the course of experimental period. Prior to induction

of

diabetes

all

the

experimental animals were subjected to GTT (glucose tolerance test) and then diabetes was induced by single intra peritoneal injection of streptozotocin (45mg/kg) dissolved in freshly prepared citrate buffer (pH 4.5, 0.1 M) in over night fasted rats. 96 h after the injection,

and 24h after treatment, and analyzed for blood

glucose

concentration.

Blood

glucose levels were measured by glucose oxidation method using standard glucose kits (Ranbaxy chemicals). Values are presented as means± S.E.M. Statistical

difference

between

the

treatment and the controls were tested by one way analysis of variance (ANOVA) followed

by

Dunnet’s

multiple

comparison tests.

the blood glucose levels were measured by glucose oxidation method. Each

241


Intermediate 2 on conjugation with pure

Results and discussion Synthesis

of

N-

insulin (Step-2, Scheme 2) in borate

succinimidyl myristate was achieved via

buffer gave LysB29 (N- ε-decanoyl)

a two-step synthetic route starting from

human

myristic acid (1a) (Step-1, Scheme 1).

conjugating des (B30) human insulin

Reaction of myristic acid (1a) in

with intermediate 2 in borate buffer gave

dichloromethane with oxalyl chloride

LysB29 (N- ε-decanoyl) des (B30)

(1b) in presence of dimethyl formamide

human insulin (Scheme 3, Conjugate-

(DMF) gave chloromyristate (1c) that

3). Conjugating human insulin in borate

was redissolved in dichloromethane and

buffer with cyclohexanoyl chloride gave

made

N-hydroxy

LysB29 (N- ε-cyclohexanoyl) human

succinimide (1d) in presence of triethyl

insulin (Scheme 4, Conjugate 4).

amine resulting in the formation of N-

Conjugation of des (B30) human insulin

succinimidyl myristate. Intermediate 1

with cyclohexanoyl chloride failed to

on conjugation with pure desthreo

provide considerable yield hence has

insulin (Step-2, Scheme 1) in borate

been exempted from the study. List of

buffer gave LysB29 (N- ε-tetradecanoyl)

starting materials, intermediates and

des (B30) human insulin (Conjugate 1)

synthesized compounds has been given

which was considered as insulin basal

in Table 1.

standard to compare the activity.

All these conjugates were screened in

to

intermediate

react

Synthesis

of

with

1,

intermediate-2,

N-

vivo

insulin

in

order

(Conjugate-2).

to

evaluate

their

succinimidyl caprate was achieved from

pharmacological

capric acid (2a) (Step-1, Scheme 2).

antihyperglycemic activity has been

Reaction

in

evaluated at a subcutaneous dose of 125

N-hydroxy

milliunits which exhibited significant

of

capric

dichloromethane

acid

with

(2a)

activity.

On

succinimide (1d) and N, N-dicyclohexyl

extended

carbodiimide (2b) in presence of DMAP

compared to standard human insulin at

at

room

succinimidyl

temperature caprate

cyclohexanyl urea (2c).

and

antihyperglycemic

Their

activity

gave

N-

the same dose. Biological activity results

N,

N-

have been given in Table 2 and in vivo profiles in Figure 3. The in vivo profile of conjugate 1 was taken as standard to 242


compare the action of other conjugates.

antihyperglycemic profile even up to 24

All conjugates have shown prolonged

hours with single dose followed by

action. Conjugates 1, 2, 3 and 4 have

conjugate 2 and conjugate 4. The

shown flat profiles between 4-12 h, 5-12

extended action of conjugate 3 may be

h, 7-24 h and 5-12 h, respectively.

attributed to its enhanced affinity to

Furthermore

the

desthreoinsulin

conjugate

with

of

serum albumin. It is interesting to point

succinimidyl

out

that

desthreoinsulin

caprate (fatty acid

showed

acylated) that is conjugate-3 has shown

activity than insulin conjugates.

high

significance

and

Retention Time, RT (minutes) Area under the curve,AUC (mAU*s) Calculated Mol. weight/Mol. weight of molecular ion (Daltons)

antihyperglycemic

extended Insulin Standard

Parameters

better

conjugates

Desthreo Insulin

Conjugate 1

2

3

4

12.181

6.013

15.989

16.764

19.866

8.533

45,482

8,797.76

2,522.36

2734.81

18,660

19,008.2

5808/ 1452

5707/ 1427

5917/ 1480.1

5962/ 1491.5

5861/ 1466.1

5918/ 1480.4

Table 3: HPLC and LC-MS data

Table 4: Peptide mapping results of Conjugate – 1*

Fragment

Location

Expected Molecular weight (Da)

Linked Molecular weight (Da)

Observed Molecular weight (Da)

Chain Fragment

1

A(1-4)

416.2

NIL

439.2

GIVE

2

A(5-17)

1487.6

2968.3

1487.1

QCCTSICSLYQLE

4

B(1-13)

1481.7

NIL

NIL

FVNQHLCGSHLVE

3 5 6

A(18-21) B(14-21) B(22-29)

512.2 866.4 1013.5+211

1376.6 NIL 211

1377 NIL 1224.5

NYCN ALYLVCGE RGFFYTPK

Myristate moiety is attached to B-29(lysine) of desthreo insulin *Refer Figure 3

243


Table 5: Peptide mapping results of Conjugate – 2# Expected Molecular weight (Da)


Observed molecular weight (Da)

Chain Fragment

A(1-4)

416.2

NIL

439.2

GIVE

2

A(5-17)

1487.6

2968.3

1487.1

QCCTSICSLYQLE

4

B(1-13)

1481.7

NIL

NIL

FVNQHLCGSHLVE

1377 NIL 1270.8

NYCN ALYLVCGE RGFFYTPKT

Fragment

Location

1

A(18-21) 512.2 1376.6 3 B(14-21) 866.4 NIL 5 B(22-30) 6 1115.6+155 155 Caprate moiety is attached to B-29(lysine) of insulin # Refer figure 4

Table 6: Peptide mapping results of Conjugate – 3+ Expected Molecular weight (Da)



Chain Fragment

A(1-4)

416.2

NIL

417.1

GIVE

2

A(5-17)

1487.6

2968.3

1487.6

QCCTSICSLYQLE

4

B(1-13)

1481.7

NIL

1483.1

FVNQHLCGSHLVE

3 5

A(18-21) B(14-21)

512.2 866.4

1376.6 NIL

1377.7 NIL

NYCN ALYLVCGE

6

B(22-29)

1013.5+155

155

1169.7

RGFFYTPK

Fragment

Location

1

Caprate moiety is attached to B-29(lysine) of desthreo insulin +Refer figure 5

Table 7: Peptide mapping results of Conjugate– 4 Expected Molecular weight (Da)



Chain Fragment

A(1-4)

416.2

NIL

439.2

GIVE

2

A(5-17)

1487.6

2968.3

1487.2

QCCTSICSLYQLE

4

B(1-13)

1481.7

NIL

NIL

FVNQHLCGSHLVE

3

A(18-21)

512.2

1376.6

1377.7

NYCN

5

B(14-21)

866.4

NIL

NIL

ALYLVCGE

6

B(22-30)

1115.6+111

111

1226.7

RGFFYTPKT

Fragment

Location

1

Cyclohexanoyl moiety is attached to B-29(lysine) of insulin Refer figure 6

244


Table 2: Antihyperglycemic effect of insulin conjugates and human insulin Groups 0 1

246.03

30 min

1h

2h

3h

4h

5h

6h

7h

12 h

24 h

201.53

131.45

124.10

121.93

105.92

109.83

104.40

102.63

98.067

81.983

+0.711** +1.167** +0.671** +1.252** +1.054** +3.056** +0.784** +0.715** +3.637** +0.668** +0.951** 2

291.17

286.02

197.95

198.09

180.922

171.58

147.26

138.97

146.02

149.27

160.38

+0.578** +0.318** +0.618** +0.446** +0.577** +0.746** +0.381** +0.118** +0.591** +0.693** +1.150** 3

255.90

273.87

231.59

221.35

214.20

191.71

185.99

155.69

139.19

138.19

137.28

+0.575** +0.891** +0.556** +0.443** +0.646** +0.556** +0.418** +0.438** +0.493** +0.525** +0.514** 4

271.67

220.57

240.81

221.26

208.33

201.54

178.38

171.11

169.80

169.08

148.75

+0.803** +0.688** +0.374** +0.388** +0.378** +0.303** +0.506** +0.804** +0.222** +0.279** +0.566** Insulin

270.52

245.67

98.92

125.11

145.89

171.37

201.80

222.45

201.17

266.17

301.11

Std.

+0.272** +0.438** +0.158** +0.318** +0.349** +0.261** +0.302** +0.320** +0.413** +0.361** +0.304**

Values were expressed as Mean+SEM (n=6) where ** p