dceditor+in+chief lea smirčić duvnjak editorial board spomenka ljubić sandra vučković-rebrina...

Journal of Diabetes, Endocrinology and Metabolic Diseases

no.

p.p.

vol.

44

1

1-38

CONTENTS

ORIGINAL RESEARCH ARTICLES

EFFECTS OF MULBERRY AND JACKFRUIT LEAF EXTRACTS ON BLOOD GLUCOSE, OXIDATIVE STRESS AND DNA DAMAGE IN STZ/NA-INDUCED DIABETIC RATSS. A. Abd El-Azim, M. T. Abd El- Raheem, M. M. Said, M. A. Abdeen . . . . . . . . . . 3

REVIEW

PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9 INHIBITORS: NEW CLASS OF LIPID-LOWERING THERAPYT. Bulum, L. Duvnjak . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

CLINICAL OVERVIEW OF INSULIN GLARGINE 300 UNITS/mL (TOUJEO®)L. Smirčić Duvnjak, T. Bulum, T. Božek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

VUK VRHOVAC UNIVERSITY CLINIC, ZAGREB, DAMA - DIABETOLOGY ALUMNI MEDICAL ASSOCIATION

Vol.

44 N

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201

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SN 0

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79-0

08.6

7.43

2015

MEDICAL SCIENTIFIC JOURNAL OF THE VUK VRHOVAC INSTITUTE

UNIVERSITY CLINIC FOR DIABETES, ENDOCRINOLOGY AND METABOLIC DISEASES

SCHOOL OF MEDICINE, UNIVERSITY OF ZAGREB

CROATIAN MEDICAL ASSOCIATION, CROATIAN SOCIETY FOR ENDOCRINOLOGY ANDDIABETOLOGY

DIABETOLOGY ALUMNI MEDICAL ASSOCIATION

VUK VRHOVAC UNIVERSITY CLINIC, ZAGREB

VOLUME 44, NUMBER 1, 2015

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EDITOR-IN-CHIEF

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Key words: Artocarpus heterophyllus,

Morus alba,

gliclazide,

streptozotocin,

nicotinamide

SUMMARY

This study was conducted to evaluate the efficacy of

mulberry (MLE) and jackfruit (JLE) leaf extracts,

alone or in combination, compared with gliclazide, on

streptozotocin-nicotinamide-induced diabetic rats for

8 weeks. Results showed significant increase in plasma

glucose, HbA1C

%, TBARS, NOx and liver tissue DNA

damage levels in diabetic rats compared with normal

controls, whereas liver glycogen, GSH levels and

erythrocyte SOD and CAT activities were significantly

decreased in the former. Plasma glucose levels and

HbA1C

% were significantly decreased by treatment

with MLE or JLE, and normalized by gliclazide.

Alterations in plasma TBARS, GSH and NOx levels

were reverted to near normal with MLE and a mixture.

Moreover, the mixture restored the activities of

erythrocyte SOD and CAT, while JLE restored GSH

and CAT to normal values. All treatments resulted in

disappearance of DNA damage in diabetic livers.

Finally, we concluded that MLE or JLE treatment

exerted a therapeutic protective effect on diabetes.

INTRODUCTION

Diabetes mellitus (DM) is a group of metabolicdiseases characterized by hyperglycemia resultingfrom defects in insulin secretion, insulin action, orboth (1). In 2013, an astounding 382 million peoplewere estimated to have diabetes, with dramaticincreases seen in countries all over the world. Ifcurrent demographic patterns continue, more than 592million people will be affected with diabetes within ageneration (2).

Despite the availability of many antidiabeticmedicines on the market, diabetes and relatedcomplications continue to be major medical problems(3). The use of these drugs is accompanied with theirside effects (4). The search for more effective and saferhypoglycemic agents continues to be an important areaof research (3).

Some medicinal plants have been reported to beuseful in diabetes worldwide and have been usedempirically as antidiabetic and antihyperlipidemic

3Diabetologia Croatica 44-1, 2015

1 Department of Biochemistry, Faculty of Pharmacy, Cairo

University, Cairo, Egypt2 Biochemistry Division, National Organization for Drug Control

and Research (NODCAR), Giza, Egypt

Original Research Article

EFFECTS OF MULBERRY AND JACKFRUIT LEAF EXTRACTS

ON BLOOD GLUCOSE, OXIDATIVE STRESS AND DNA

DAMAGE IN STZ/NA-INDUCED DIABETIC RATS

S. A. Abd El-Azim1, M. T. Abd El- Raheem1, M. M. Said2, M. A. Abdeen1,2

Corresponding author: Marwa A. Abdeen, BS Pharm, Biochemistry

Division, National Organization for Drug Control and Research

(NODCAR), 6 Abou Hazem St., Madkour Station, Alharam St., P.O. Box-

29, Giza 12553, Egypt

E-mail: [email protected]

remedies (5). The medicinal plants may provide auseful source of new oral hypoglycemic compoundsfor the development of pharmaceutical entities or asdietary adjunct to existing therapy (6). Of thesemedicinal plants widely used in the field of herbalmedicine, Morus alba and Artocarpus heterophyllus

have been reported to possess several medicinalproperties including hypoglycemic effect (7-10).

Morus alba, known as mulberry, has long been usedas an antiphlogistic, diuretic, antihyperlipidemic andantidiabetic remedy in traditional medicine (3). Atoxicity study of Morus alba leaves revealed noremarkable acute or subacute toxicities (11). Mulberrywater extracts contain polyphenols, including gallicacid, chlorogenic acid, rutin and anthocyanins (12).Furthermore, the pipperidine alkaloid andglycoproteins from the Morus root bark and/or leafextract have been used as antidiabetic agents (4).

Artocarpus heterophyllus, commonly known as jak,is reported in Ayurveda to possess antibacterial, anti-inflammatory, antidiabetic, antioxidant and immuno -modulatory properties. It was found to be useful infever, boils, wounds, skin diseases, convulsions,diuresis, constipation, ophthalmic disorders, snakebite, etc. (13). The presence of phytoconstituents likephenolics, sterols, triterpenoids and carbohydratesindicates that the plant can be useful for treatingdifferent diseases (13). In previous studies, it wasreported that aqueous extract of Artocarpus

heterophyllus leaves possessed a significant oralhypoglycemic activity and was believed to improveglucose tolerance in normal subjects and diabeticpatients (14). Studies performed by Chandrika et al.

(15) showed the flavonoid fraction to have the highesthypoglycemic activity.

The present work aimed to study the possibleameliorative effects of Morus alba and Artocarpus

heterophyllus on experimentally induced type 2 DMcomparing their effects, alone or in combination, witha commercially known drug, gliclazide, as a referenceagent.

MATERIALS AND METHODS

Animals

The animal studies were implemented in compliancewith the policies outlined in the Guide for the Care andUse of Laboratory Animals, published by the USNational Institute of Health (NIH Publication No. 85-23, revised 1996). Experiments were carried out usingadult female Sprague-Dawley rats, weighing 150-180g, obtained from the animal house of NationalOrganization for Drug Control and Research(NODCAR), Giza, Egypt. Rats were housed in groupsof five per cage under controlled environmentalconditions of temperature and humidity and exposedto a 12-h light/dark cycle. Unless otherwise indicated,animals were fed on normal laboratory chow and weregiven tap water ad libitum throughout the expe -rimental period.

Induction of experimental diabetes

Experimental type 2 DM was induced in overnightfasted rats using a single intraperitoneal (i.p.) injectionof streptozotocin (STZ) (Sigma, St. Louis, MO, USA;65 mg/kg b.w.) dissolved in 0.09 M citrate buffer (pH4.5), immediately before use, 15 min after the i.p.administration of nicotinamide (NA) (16, 17) (FlukaChemie AG, Buchs, Switzerland; 110 mg/kg b.w.)with a modification in NA dose (18). Hyperglycemiawas confirmed by the elevated fasting plasma glucose(FPG) levels, determined on the 3rd and 7th day afterinjection of STZ/NA. Only rats that were found tohave moderate and stable hyperglycemia (170-250mg/dL) were considered diabetic and involved in theexperiment.

Experimental design

The animals were randomized into six groups asfollows: I) normal non-diabetic control (N) group; II)untreated diabetic (D) group; both received 1% Tween80 in distilled water as a vehicle (10 mL/kg b.w.); III)mulberry leaf extract-treated diabetic group(D+MLE); IV) jackfruit leaf extract-treated diabeticgroup (D+JLE); V) mixture-treated diabetic group

4

S. A. Abd El-Azim, M. T. Abd El- Raheem, M. M. Said, M. A. Abdeen / EFFECTS OF MULBERRY AND JACKFRUIT LEAF EXTRACTS

ON BLOOD GLUCOSE, OXIDATIVE STRESS AND DNA DAMAGE IN STZ/NA-INDUCED DIABETIC RATS

(D+MLE+JLE): received MLE, JLE or an equimixtureof MLE and JLE (500 mg/kg b.w.) dissolved in 1%Tween 80 in distilled water as a vehicle, respectively.each mL of all the previous extracts was standardizedto contain 50 mg extract; and VI) gliclazide-treateddiabetic group (D+GLZ): received gliclazide (14.4mg/kg b.w.) suspended in 1% Tween 80 in distilledwater as a vehicle.

All treatments were given orally for 8 weeks bygavage and the animals were monitored throughperiodic testing of fasting blood glucose and bodyweight weekly.

Materials

Gliclazide was kindly supplied as a raw material bythe Egyptian International Pharmaceutical IndustriesCompany (EIPICO), Egypt. A concentration of 14.4mg gliclazide/kg b.w. of rat was used, which isequivalent to 80 mg/b.w. therapeutic dose in human(19).

Chemicals for which the source is not noted were ofanalytical grade from Sigma, Sigma-Aldrich, Fluka,BDH, Park, Riedel-de Haën and MP Biomedicals.

Plant material collection

White mulberry leaves were collected from Morusalba tree grown in the agricultural land of the AppliedResearch Centre of Medicinal Plants (ARCMP),NODCAR, Giza, Egypt. Jackfruit leaves weresupplied from Al-Zohrea garden (Cairo, Egypt). Theplants were kindly identified as: Morus alba L.(Moraceae) and Artocarpus heterophyllus Lam.(Moraceae), respectively, and deposited in CairoUniversity Herbarium by Dr. Wafaa M. Amer, Botanyand Microbiology Department, Faculty of Science,Cairo University, Giza, Egypt.

Preparation of aqueous plant extracts

Fresh leaves of both plants were washed and wiped,air-dried, coarsely powdered using cutter mill, addedto boiling double-distilled water and allowed to infuse.The extract was decanted, filtered under vacuum,

concentrated in rotary evaporator (Butchi RotavaporR-205, Switzerland) and then lyophilized (LyphilyzerSniffers Scientific b.v., Tilburg, Holland) (20).

The air-dried, powdered leaves of both plants weresubjected to preliminary phytochemical screening.The results revealed the presence of carbohydrates,tannins, glycosides, alkaloids and flavonoids inmulberry leaves and the presence of carbohydrates,flavonoids, glycosides, sterols, and tannins in jackfruit leaves.

Spectrophotometric determination of total

polyphenolics and total flavonoids in MLE and

JLE

Total polyphenolics content was colorimetricallyassayed according to the method of Aliyu et al. (21)using gallic acid as a standard. The concentration oftotal phenolics was calculated with reference to a pre-established standard calibration curve. The methodused to assay total flavonoids was that of Makky et al.

(22). The concentration of flavonoids was calculatedusing the equation of quercetin standard curve.

All spectrophotometric measurements in the currentstudy were performed using the UV-visiblespectrophotometer (Thermo-Nicolet evolution 100,England).

HPLC assay of flavonoids and phenolic

compounds in MLE and JLE

Flavonoids and phenolic compounds were HPLC-assayed according to the method of Mattila et al. (23),using a Hewlett Packard (series 1050) equipped withautosampling injector, solvent degasser, ultraviolet(UV) detector (set at 280 nm for phenolic deter -mination and 330 nm for flavonoid determination) andquaternary HP pump (series 1050). The columntemperature was maintained at room temperature.Separation was carried out using methanol andacetonitrile (2:1) as a mobile phase at a flow rate of 1mL/min. Authentic phenolics and flavonoids (SigmaCo.) were dissolved in mobile phase and injected into




HPLC. The retention time and the peak area were used

to calculate the phenolic and flavonoid concentrations

by data analysis using the Hewlett Packard software.

Determination of LD50

of MLE and JLE (acute

oral toxicity test)

Acute oral toxicity studies for both extracts and their

mixture were carried out as per Organization for

Economic Co-operation and Development (OECD)

Test Guideline 425. Mortality in each group within 24

h was recorded. The animals were further observed for

another 14 days for any signs of delayed toxicity.

Therapeutic dose was calculated using the equation:

therapeutic dose = LD50/10 (24).

Sample collection

At the end of the experiment, all animals from each

group were fasted overnight and blood was collected

according to the procedure described by Schermer

(25). Then, animals from each group were sacrificed

by decapitation; livers were removed for glycogen

assessment and analysis of total genomic DNA

damage.

Biochemical assays

Estimation of markers of diabetes

Glucose was determined in plasma according to the

enzymatic colorimetric method of Trinder (26) using

Audit Diagnostics kit (Cork, Ireland). Glycosylated

hemoglobin (HbA1c %) was determined in the

hemolysate using the Biosystems assay kit (Barcelona,

Spain), where the labile fraction is eliminated;

hemoglobins are retained by a cationic change resin.

HbA1c is specifically eluted after washing away the

hemoglobin A1a+b fraction (Hb A1a+b), and

quantified by direct photometric reading at 415 nm

(27). The method used in determination of liver

glycogen is that of Montgomery (28).

Estimation of biomarkers of lipid peroxidation

and oxidative stress and antioxidant enzymes

In plasma, the extent of lipid peroxidation wasestimated as the concentration of thiobarbituric acidreactive substances (TBARS) using the method ofBuege and Aust (29). In erythrocytes, reducedglutathione (GSH) concentration, Cu/Zn-superoxidedismutase (Cu/Zn-SOD) and catalase (CAT) activitieswere determined using the method described byBeutler et al. (30), Marklund and Marklund (31) andAebi (32), respectively, whereas serum nitric oxide(NOx) was determined using the method of Miranda et

al. (33).

Evaluation of the antimutagenic activity of the

tested plants by DNA fragmentation assay

The method of DNA fragmentation was performedaccording to the salting out extraction technique ofAljanabi and Martinez (34). The intensity of DNAbands was measured using the Biogene software(Biogene Software, France).

Statistical analysis

Biochemical results were expressed as mean ±standard error (SE) and statistically analyzed usingone-way ANOVA Statistical Package for the SocialSciences (SPSS) version 17.0 according to Snedecorand Cochran (35), followed by Tukey’s post-hoc testfor multiple comparisons. Differences were consi -dered to be statistically significant when p<0.05. Datawere graphically presented using Microsoft Excel(2007).

RESULTS

Spectrophotometric determination of total

polyphenolics and total flavonoids of MLE and

JLE

From the standard calibration curve, the totalphenolic content was calculated as gallic acid: 3.06and 1.83 mg/100mg dry extract in MLE and JLE,

6



respectively, whereas, total flavonoids were calculatedas quercetin: 0.409 and 0.46 mg/100mg dry extract inMLE and JLE, respectively.

HPLC assay of flavonoids and phenolic

compounds in MLE and JLE

As presented in Table 1, the flavonoids identified inMLE that showed the highest concentrations werehisperidin: 73.56 mg/100 g dry extract and rutin: 28.29mg/100 g dry extract, while in JLE the highestconcentrations were shown by vitexin: 163.23 mg/100g dry extract, hisperid: 26.74 mg/100 g dry extract andquercetin: 25.35 mg/100 g dry extract. Table 1 alsoshows that the lowest flavonoid concentrationidentified in MLE was that of narengenin: 0.46mg/100 g dry extract, while in JLE, 7-OH flavone:0.65 mg/100 g dry extract showed the lowestconcentration.

Table 2 shows that the highest concentrations ofphenolic compounds identified in MLE were those ofe-vanillic acid: 537.83 mg/100 g dry extract, 3-OH-tyrosol: 150.41 mg/100 g dry extract and benzoic acid:123.31 mg/100 g dry extract, while in JLE the highestphenolic compound concentrations were shown bychicoric acid: 161.12 mg/100 g dry extract, e-vanillicacid: 133.93 mg/100 g dry extract and protochatcuicacid: 128.76 mg/100 g dry extract. Table 2 also reveals

that the lowest phenolic compound concentrationidentified in MLE was that of gallic acid: 2.98 mg/100g dry extract, while in JLE cinnamic acid: 3.42 mg/100g dry extract, showed the lowest concentration.Vanillic acid was not identified in JLE but wasidentified in MLE: 9.22 mg/100 g dry extract.

Determination of LD50

and therapeutic dose

calculation

After administering the extracts orally to the rats indifferent gradual doses (1 to 5 g/kg b.w.), the animalsof the three groups showed no mortality or any toxicsymptoms during the first 24 hours or on the days afterfor as long as 2 weeks.




Flavonoids MLE (mg/100 g) JLE (mg/100 g)

Vitexin 2.54 163.23

Naringin 9.04 11.80

Rutin 28.29 9.17

Hisperidin 73.56 7.10

Rosmarinic 10.70 2.27

Quercetrin 9.92 25.35

Quercetin 1.86 3.76

Narengenin 0.46 1.80

Kaempferol 0.81 1.35

Hisperetin 3.77 26.74

Apegenin 0.69 7.42

7-OH flavone 0.51 0.65

Table 1. Concentrations of flavonoids identified in

mulberry (MLE) and jackfruit (JLE) leaf extracts using

HPLC

Phenolic compound MLE (mg/100 g) JLE (mg/100 g)

Gallic 2.98 5.01

Pyrogallol 92.26 91.90

3-OH-tyrosol 150.41 23.44

4-Amino-benzoic 4.08 10.99

Protochatcuic 51.92 128.76

Chlorogenic 27.48 25.74

Catechol 60.26 60.74

Epicatechin 26.28 27.81

Catechin 17.54 14.15

P-OH-benzoic 22.97 29.76

Caffeic 18.21 36.38

Vanillic 9.22 ----

Chicoric 59.45 161.12

Ferulic 12.44 30.83

Iso-ferulic 6.53 6.31

e-Vanillic 537.83 133.93

Reversetrol 8.82 3.56

Ellagic 29.58 33.04

Alpha-coumaric 19.64 6.37

Benzoic 123.31 62.47

3,4,5-Methoxy-cinnamic 8.26 19.74

Coumarin 10.20 5.44

P-coumaric 7.42 5.53

Cinnamic 6.23 3.42

Table 2. Concentrations of phenolic compounds

identified in mulberry (MLE) and jackfruit (JLE) leaf

extracts using HPLC

Therefore, no acute oral toxicity was proven for theextracts or their mixture up to the dose of 5 g/kg b.w.,and their LD50 were believed to be more than 5 g/kgb.w. Hence, studies were carried out with 1/10 of theLD50 as therapeutic dose (500 mg/kg) according toOECD-425 guidelines.

Determination of biochemical parameters

Plasma glucose, glycosylated hemoglobin and

liver glycogen levels

Data presented in Table 3 and Figure 1 indicate thatthe levels of fasting plasma glucose (FPG) of untreateddiabetic rats were significantly increased andamounted to 212.9% of the normal control values.Administration of different treatments partially

8



Group FPG (mg/dL) HbA1c

% Liver glycogen (mg/g liver)

N n=10 98.5±1.58 3.6±0.13 16.65±0.52

D n=9 209.7±9.66a 7.0±0.34a 8.10±0.54a

D + MLE n=9 143.8±6.37a,b 5.6±0.20a,b 13.10±0.98a,b

D +JLE n=10 128.3±5.97a,b 4.9±0.37a,b 12.26±0.63a,b

D + (MLE + JLE) n=10 125.5±5.15a,b 4.8±0.31a,b 12.40±0.54a,b

D + GLZ n=9 106.6±4.17b 4.5±0.34b 13.36±0.92a,b

Table 3. Mean ± standard error (SE) of fasting plasma glucose (FPG) levels, glycosylated hemoglobin percentage

(HbA1c %) and liver glycogen in differently treated groups at the end of the experimental period

N = normal control rats; D = diabetic control rats; D + MLE = mulberry leaves extract-treated diabetic rats; D + JLE = jackfruit leaves extract-treated diabetic rats; D + (MLE + JLE):

mixture-treated diabetic rats; D + GLZ = gliclazide-treated diabetic rats; p<0.05 (using one-way ANOVA followed by Tuckey’s HSD post-hoc test); asignificant difference from normal

control group; b significant difference from diabetic control group; n = number of animals in each group

Figure 1. Changes in fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c %) and liver glycogen levels

in STZ/NA-diabetic (D) rats and in diabetic rats treated for 8 weeks with either mulberry leaf extract (D+MLE),

jackfruit leaf extract (D+JLE), combined extracts [D+(MLE+JLE)] or gliclazide (D+GLZ).

Results are expressed as percentage of normal control data ± SE; (a) significantly different from normal control group at p<0.05; (b) significantly different from diabetic control group

at p<0.05.

reversed the increase in FPG levels reaching 146%(MLE), 130.3% (JLE), and 127.4% (MLE+JLE). Onthe other hand, treatment with gliclazide normalizedFPG levels of diabetic rats.

Similar results were obtained for HbA1C% as shownby data compiled in Table 3 and given in Figure 1. TheHbA1C% levels of untreated diabetic rats were highlyincreased to reach 194.4% of normal control, while thetreatment with MLE, JLE, mixture or gliclazideameliorated this elevation, where the HbA1C% levelsreached only 155.6, 136.1, 133.3 and 125%,respectively.

As shown in Table 3 and Figure 1, liver glycogen offasted untreated diabetic animals decreased signi -ficantly to reach 48.6%. All treatments increased itslevels reaching 78.7%, 73.6%, 74.5% and 80.2% forMLE, JLE, mixture and gliclazide, respectively,whereas none of them returned the levels to normal.

Blood oxidative status

Table 4 and Figure 2 illustrate the effects of differenttreatments on plasma lipid peroxides measured asthiobarbituric acid reactive substances (TBARS),where they were highly elevated in untreated diabetic




Figure 2. Changes of plasma thiobarbituric acid reactive substances (TBARS), serum nitric oxide (NOx),

erythrocyte reduced glutathione (GSH), and the activities of Cu/Zn-superoxide dismutase (SOD) and catalase

(CAT) in STZ/NA-diabetic (D) rats and in diabetic rats treated for 8 weeks with either mulberry leaf extract

(D+MLE), jackfruit leaf extract (D+JLE), combined extracts [D+(MLE+JLE)] or gliclazide (D+GLZ).

Results are expressed as percentage of normal control data ± SE; (a) significantly different from normal control group at p<0.05; (b) significantly different from diabetic control group

at p<0.05.

Group TBARS (nmol/L) NOx (µmol/L) GSH (mg/dL) Cu/Zn-SOD (U/mg Hb) CAT (U/mg Hb)

N n=10 6.59±0.50 79.5±4.85 27.5±2.20 14.17±0.60 70.22±5.32

D n=9 15.45±1.03a 184.3±9.69a 15.0±1.26a 7.88±0.68a 37.7±2.70a

D + MLE n=9 9.20±0.81b 131.9±4.80a,b 22.3±1.55 10.01± 0.66a 65.2±5.90b

D +JLE n=10 10.90±0.73a,b 116.0±8.41a,b 24.1±2.37b 10.42±0.54ab 69.6±4.68b

D + (MLE + JLE) n=10 10.81±0.39a,b 98.7±7.3b 25.6±1.75b 14.86±0.55b 73.0±3.15b

D + GLZ n=9 11.08±0.73a,b 135.7±11.9a,b 16.9±0.99a 9.27±0.30a 50.60±4.1a

Table 4. Mean level ± standard error (SE) of plasma thiobarbituric acid reactive substances (TBARS), serum nitric

oxide (NOx), erythrocyte glutathione (GSH), Cu/Zn superoxide dismutase (Cu/Zn-SOD) and catalase (CAT) in

differently treated groups at the end of the experimental period

p<0.05 (using one-way ANOVA followed by Tuckey's HSD post-hoc test); asignificant difference from normal control group; bsignificant difference from diabetic control group; n =

number of animals in each group

rats compared to the normal control group.Administration of different extracts and gliclazidedecreased TBARS levels. Unexpectedly, only MLEtreatment went through normalizing TBARS levels.

Data shown in Table 4 and Figure 2 indicate themassive increase of serum NOx levels in untreateddiabetic rats, which reached approximately 2.3-foldthat of the normal control values. This wassignificantly decreased by all treatments, as theybecame only 1.65-, 1.45-, 1.24- and 1.7-fold for MLE,JLE, mixture and gliclazide, respectively. This alsoshows that only the mixture rather than any othertreatment succeeded in normalizing NOx values.

Table 4 and Figure 2 show the change in bloodoxidative status of diabetic treated and untreated rats,where blood GSH levels were remarkably decreased indiabetic untreated and returned to normal in JLE andmixture treated ones, but neither MLE nor gliclazidetreatment was capable of returning GSH values tonormal. However, MLE showed a better amelioratingeffect (81.1% of normal) than gliclazide (61.5% ofnormal), which showed no significant differencecompared with untreated diabetic rats.

As indicated in Table 4 and Figure 2, the Cu/Zn-SODlevels were markedly decreased in diabetic untreatedrats. Significant elevations of Cu/Zn-SOD levels wereshown by all treatments except for MLE andgliclazide, which caused no significant elevation inCu/Zn-SOD levels than the level in the group ofuntreated diabetic rats, where they reached only 70.6%and 65.4% of normal control, respectively, whereastreatment with JLE showed more amelioration (73.5%of normal control). A surprising ameliorating effect ofthe mixture, supporting the idea of having a synergisticactivity, was obvious, as it succeeded in raising Cu/Zn-SOD levels to more than that of normal controls(105%).

Table 4 and Figure 2 delineate the remarkabledecrease in erythrocyte CAT levels in untreateddiabetic rats compared to normal. Treatment withMLE, JLE or their mixture restored the CAT levelsback to normal. Although gliclazide treatmentincreased CAT levels, it was nonsignificant.

Analysis of total genomic DNA damage

The protective effects of different treatments on thetotal genomic DNA damage induced by STZ-inductionof diabetes in Sprague-Dawley rats after 8 weeks oftreatment are shown in Table 5 and Figure 3a and 3b.As shown in Figure 3a and 3b, severe damage wasobserved in the DNA of the untreated diabetic ratlivers (lane 2, L2), which represented the untreateddiabetic control group (D). We observed DNA releaseand migration in the form of smear shape indicatingnecrosis and also in the form of bands that indicatesapoptosis. These forms of released DNA (damage)disappeared in all lanes that represent the other treatedgroups, L3: D + MLE; L4: D + JLE; L5: D + (MLE +JLE) and L6: D + GLZ, which appeared nearly thesame as the normal control (L1: normal control).

DISCUSSION

In the present study, treatment of diabetic rats for 8weeks with hot water mulberry leaf extract (MLE)caused a significant decrease in FPG levels comparedto diabetic control. The current results were inaccordance with many previous studies (7,36-38).Morus alba is postulated to exert its hypoglycemicaction through α-glucosidase inhibitory activity, whichis attributed to the presence of D-glucose analogs 1-deoxynojirimycin and its derivatives (39). Moreover,other active constituents including piperridine alkaloidand glycoproteins from the Morus root bark and/orleaf extract have been used as antidiabetic agents (40),besides the presence of a variety of flavonoids andother phenolic compounds, many of which wereassayed in our study, including the potentially activeconstituents chlorogenic acid and rutin, which mightaccount for as much as half of the observedantidiabetic activity of Morus alba, as shown in aprevious study (41). Our findings also complied withthose of Mohammadi and Naik (3), who directlyrelated the improvement in the number of β cells aftertreatment with Morus alba extract to its antidiabeticactivity. This effect might also be attributed to rutin,which possesses stimulatory effects on the insulinsecretory response of the islets of Langerhans, inaddition to an ameliorative effect on the integrity of β

10






Group Intact DNA (4000 bp) 800 bp 600 bp 400 bp

N 250 - - -

D 147 205 203 146

D + MLE 255 - - -

D + JLE 240 - - -

D + (MLE+ JLE) 233 - - -

D + GLZ 254 - - -

Table 5. Protective effect of different treatments on total genomic DNA damage in Sprague-Dawley rats

N = normal control; D = diabetic control; D + MLE = mulberry leaves extract-treated diabetic rats; D + JLE = jackfruit leaves extract-treated diabetic rats; D + (MLE + JLE): mixture-

treated diabetic rats; D + GLZ = gliclazide-treated diabetic rats; bp: base pairs

Figure 3a. Digital photograph of DNA electrophoresis of liver tissues shows the protective effects of different

treatments against STZ, where, L1: normal control (N); L2: diabetic control (D); L3: D + MLE; L4: D + JLE; L5: D

+ (MLE+ JLE); L6: D + GLZ and M: DNA marker.

Figure 3b. Analytical peaks for DNA lanes which resemble the same lanes in Figure 3a using Biogene software.

cells (42). The current results also agreed with those ofHamdy et al. (4), who showed that Morus alba exertedits hypoglycemic effect by controlling oxidative stress,increasing glycogen levels, preventing anaerobicglycolysis and improving hepatic carbohydratemetabolism, by enhancing the activities of hexokinase,glucose-6-phosphate dehydrogenase and lactatedehydrogenase.

In the current work, treatment of diabetic rats for 8weeks with the aqueous extract of jackfruit leaves(JLE) partially reversed the elevated FPG levelstoward normal control values. This result compliedwith those of Shahin et al. (43) and Fernando andThabrew (44), who proved that the Artocarpus

heterophyllus aqueous extract could exert significantextra-pancreatic effects through inhibiting insulinaseactivity, thus inhibiting the breakdown of insulin in thecirculation. Another possible mechanism throughwhich JLE exerts its hypoglycemic action is the α-amylase inhibitory activity, which was previouslyreported, both in vitro and in vivo, in rat plasma (45).The α-glucosidase inhibitory activity of JLE may beattributed to the presence of considerable amounts ofvitexin flavonoid, which has already been reported topossess glucosidase inhibitory activity in vitro (46) orin vivo (47).

Regarding long term control of hyperglycemia, inour experimental model of diabetes, at the end of theexperimental period it was shown that glycatedhemoglobin (HbA1C %) was significantly elevated inuntreated diabetic group and this elevation wassignificantly counteracted by all treatments. Theseresults were consistent with those of Mohammadi andNaik (3) and Omar et al. (9). This could be attributedto the reported hypoglycemic effects of both plants.

The liver glycogen level may be considered as thebest marker to assess the anti-hyperglycemic activityof any drug (42). In the present study, significantly lowfasting liver glycogen levels (approximately half) inthe untreated diabetic rats were observed. Thesefindings agreed with those of previous studies (42,48).The decrease in hepatic glycogen observed in thisstudy might be due to the inactivation of glycogensynthase system, which might have happened due tothe lack of insulin in type 2 diabetic state (49). In our

study, after 8 weeks of treating diabetic rats with MLEand/or JLE, a marked elevation in liver glycogenlevels was observed. These observations compliedwith those of Hamdy et al. (4) and Fernando andThabrew (44). The latter suggested that the increasedglycogen synthesis in liver and muscles of diabetic ratsfollowing administration of aqueous extract ofArtocarpus heterophyllus leaves could have beenmediated via increased concentration of insulinresulting from the inhibition of insulinase activity bythe extract (44). This shows the possible way ofantidiabetogenic action of these plant extracts that maybe through the improvement of glycogenesis processin muscles and liver (48).

There is evidence that diabetes induces changes inthe activities of antioxidant enzymes in varioustissues. In DM, oxidative stress seems mainly to bedue to an increased production of free radicals and/ora sharp reduction of antioxidant defense (50) and it islikely to be involved in the progression of pancreatic βcell dysfunction (51). The results of the current studyclearly showed a state of oxidative stress, obvious inthe untreated diabetic group, which showed significantincrease in TBARS and NOx levels and significantdecrease in enzymatic (SOD and CAT) andnonenzymatic (GSH) antioxidants. These findingswere in accordance with numerous studies thatrevealed lower antioxidant and enhanced peroxidativestatus in type 2 DM (52,53).

Streptozotocin-induced hyperglycemia resulted inincreased lipid peroxidation and protein carbonyls inred blood cells and other tissues (54). The assay ofTBARS measures malondialdehyde (MDA), the endproduct of lipid peroxidation, present in the sample, aswell as MDA generated from lipid hydroperoxides bythe hydrolytic conditions of the reaction (55). In thecurrent study, the TBARS levels of T2 diabetic ratswas found to be increased at the end of theexperimental period and this finding complied with theprevious findings reported by Nwanjo et al. (56) inSTZ diabetic rats. Nitric oxide is believed toparticipate in the regulation of the oxidation/reductionpotential of various cells and may be involved in eitherthe protection against or the production of oxidativestress within various tissues depending on its

12



concentration (57). In the present study, theconcentration of serum nitric oxide was significantlyincreased in the untreated diabetic rat group incomparison to the normal control group. Thesefindings are consistent with those of Yegin et al. (58).Increased NO activity could be attributed to increasednitric oxide synthase expression due to the highglucose level (59). Glutathione is the main source ofred blood cell antioxidant protection and its redoxstatus is often used as a measure of oxidative stress(60). In the present study, the erythrocyte GSH levelswere found to be significantly reduced in diabetic ratscompared with normal control. This result is consistentwith previous studies (61,62) in STZ/NA diabetic rats.Glutathione depletion could be attributed either to itsdecreased synthesis due to glycation of the keysynthesizing enzyme gamma-glutamylcysteinesynthetase (g-GCS), or increased degradation of GSHunder diabetic conditions. Moreover, hyperglycemiaresults in increased enzymatic conversion of glucoseto the polyalcohol sorbitol with concomitant decreasesin NADPH and glutathione (60). Antioxidantenzymes, including superoxide dismutase (SOD) andcatalase (CAT), constitute an important protectivemechanism against the ROS and their effectivenessvaries with the stage of development and otherphysiological aspects of the body. The current studyshowed a significant decrease in both SOD and CATactivities in erythrocytes of untreated diabetic ratscompared to normal control. These data are inagreement with previous studies (64,65). A possibleexplanation for the current results is reducedantioxidant protection in type 2 DM and/or greatlyincreased amounts of free radicals that overwhelm thedefense system (66). Another possible explanation isthat the decreased activity of Cu-Zn superoxidedismutase is related to either increased free radicalproduction causing oxidation followed by denaturingof the enzyme, or alternatively glycation of theenzyme with resulting inhibition of enzymatic activity(67). Watanab (68) attributed the reduction of CATactivity to the higher levels of H2O2 produced bypolymorphonuclear leukocytes in type 2 DM. Yan et

al. (69) investigated the inactivation of SOD and CATby sugars and the loss of antigenicity that wasmonitored by the loss of activity.

The present study showed that treatment of diabeticrats with JLE, as well as the mixture significantlyimproved SOD activity, reaching 73.5% and 105% ofnormal control, respectively. Although MLE caused anincrease in SOD activity that reached 70.6% of normalcontrol, this increase was not significant. The activityof catalase enzyme was also significantly improved bydifferent treatments reaching 93%, 99% and 104% ofnormal control for MLE, JLE and the mixture,respectively. Enhancement of antioxidant enzymaticactivities in vivo reflects the antioxidant potency ofboth plants.

Besides, the effects of these treatments on oxidationand lipid peroxidation biomarkers (GSH, SOD, CAT,TBARS and NOx) were found to be better than thoseof gliclazide. The results of ameliorating oxidativestress in Morus alba treated diabetic rats were inaccordance with those of previous studies (4,70). Theantioxidant effect of MLE might be attributed to thepresence of many flavonoids and phenoliccompounds, identified in MLE in this study, includinge-vanillic acid, 3-OH-tyrosol, benzoic acid,hisperedin, and rutin. Polyphenolic compounds wereshown to have radicals scavenging activity and anability to activate key antioxidant enzymes in vivo andthus breaking the vicious cycle of oxidative stress andtissue damage (71). The ameliorative effect ofmulberry-treatment on the oxidative state in vivo wasalso previously demonstrated (37,70). The in vitro

antioxidative properties were proven by Katsube et al.

(72) through LDL oxidation, 1,1-diphenyl-2-picrylhydrazyl (DPPH)3 radical scavenging, andFolin-Ciocalteu assays. Concerning JLE, the presenceof flavonoids and other phenolic compounds,including vitexin, hisperetin, quercetin, chicoric acid,e-vanillic acid and protochatcuic acid, identified inthis study, might have played an important role in theobserved antioxidant effects. Vitexin has been reportedto have a potent free radical scavenging activity (73).It was proven that 40 mg/kg dose of vitexin had thesame antioxidant capacity as vitamin E (74), whereasquercetin exerted antioxidant effects in STZ-inducedexperimental diabetes (75). Moreover, cyclo -heterophyllin and artonins A and B, prenyl flavonespreviously isolated from Artocarpus heterophyllus




Lam., serve as powerful antioxidants against lipidperoxidation when biomembranes are exposed tooxygen radicals (76).

In the current study, gliclazide also was shown topossess some antioxidant properties. However, itsameliorative effect on enzymatic SOD and CAT, andnonenzymatic antioxidant GSH, was not significant.On the other hand, it significantly decreased theelevation in TBARS and NOx caused in diabetic rats,but it failed to normalize it totally. These results are inaccordance with many studies that proved theantioxidant properties of gliclazide (77,78).

The liver plays a unique role in controllingcarbohydrate metabolism by maintaining glucoseconcentrations in a normal range over both short andlong periods of time (79). In 1980, Laguens et al. (80)reported the hepatotoxic effect of STZ independentlyof its diabetogenic action. In the current study, DNAanalysis by gel electrophoresis showed severe DNAdamage to the liver tissues of diabetic rats; this resultis in accordance with that of Kim et al. (81). LiverDNA damage observed in our study might haveresulted from the hepatotoxic effect of STZ. Thisresult was supported by the previous studiesperformed by Schmezer et al. (82), who observed thatSTZ had induced DNA strand breaks and mutations inthe liver and kidney of mice. Another explanation forthe observed DNA damage is the state of oxidativestress which is also known to cause massive DNAfragmentation. Moreover, oxidative damage to hepaticnuclear DNA increases in diabetic state and thisincrease was correlated with glycation stress (83).

In this study, treatment of STZ/NA diabetic rats for 8weeks either with MLE, JLE or their mixturesignificantly reduced the liver DNA damage. Since theDNA damage is most probably due to STZ-inducedoxidative stress, or the oxidative stress happened as aconsequence of diabetic state, it suggests that thisprotective effect against DNA damage was mostprobably due to the antioxidant properties previouslyproven for both plants. Gliclazide treatment alsosignificantly reduced DNA fragmentation in livers ofdiabetic rats and this result was in agreement with thatof Kim et al. (81) in STZ-diabetic mice. The reduction

in DNA fragmentation might be attributed to the freeradical scavenging activity reported for gliclazide(78).

Finally, the probable synergistic effect motivated theuse of a mixture of both plants in the treatment ofdiabetic rats in this study. However, the synergisticeffect of the mixture was highly pronounced only incase of elevating the diminished SOD levels and inlowering the elevated NOx levels in diabetic rats,while other parameters were very similar to or slightlybetter than with JLE. In our study, we observed thatJLE was always more effective than MLE, except fortwo cases, namely, TBARS and liver glycogen, inwhich MLE was more effective.

CONCLUSION

In conclusion, the current work demonstrated thattreatment with hot water extracts of mulberry leaves,jackfruit leaves or mixture of both is beneficial intreating diabetes mellitus, where they loweredhyperglycemia and oxidative stress, which, in turn,may have a consequent role in preventing theprogression of diabetic complications. The bioactivecomponent(s) responsible for the observed activitiesare not precisely known but it may be one or more ofthe phytochemical constituents established to bepresent in the leaf extracts. The presence of flavonoidsand other phenolic compounds in both extractssuggests that they might be the constituentsresponsible for these activities. Therefore, the presentstudy confirmed the results of earlier studies thatshowed the importance of using both these plants toameliorate diabetes and its complications. To the bestof our knowledge, this is the first study that comparedthe effect of these two plants to each other and to amixture of both, on a type 2 diabetic animal model.However, this study did not precisely define thecertain active constituent(s) that is/are responsible forthe hypoglycemic activities of both plants. Neither didit discover new mechanism(s) of action which mayunderlie these activities. Further mechanistic studiesare needed to identify the specific active constituentsresponsible for the biologic activities observed for

14



both plants. Clinical studies are also needed to provethe efficacy and safety of these medicinal plants inhumans.

Acknowledgments

We would like to thank Dr. Eslam M. El-Garawanyfor assisting with the DNA analysis technique and Dr.Zeinab Yousef Ali for assisting with some biochemical

techniques. We would also like to thank Dr. Wafaa M.Amer for identification of plants under study and Dr.Amira Ali, Dr. Marwa Ali and Dr. Haidy Omar forassistance in phytochemical studies.




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Key words: proprotein convertase subtilisin kexin 9,

PCSK9,

LDL-cholesterol,

hypercholesterolemia,

cardiovascular disease

SUMMARY

Elevated LDL-cholesterol level is a major risk factor

for cardiovascular disease (CVD) and atherosclerosis.

LDL-cholesterol is cleared from circulation by the

LDL receptor (LDL-R). Proprotein convertase

subtilisin kexin 9 (PCSK9), an enzyme secreted into

the plasma by the liver, binds the LDL-R in complex

which is then subject to endocytosis and degradation

in lysosomes. PCSK9 inhibitors prevent LDL-R

degradation, the LDL-R recycling is preserved, with a

consequent increase in receptor density on the

hepatocyte surface and LDL-cholesterol clearance. In

recent years, human monoclonal antibodies against

PCSK9 have been identified as an innovative lipid-

lowering strategy. Clinical trials confirmed that

treatment with PCSK9 antibodies combined with

statins, as well as in monotherapy produces profound

reductions in total, LDL-cholesterol and

lipoprotein(a), with a similar level of safety and

survival benefit compared with no anti-PCSK9

treatment. Confirmation of these findings in ongoing,

long-term trials will establish the role of these novel

agents in reducing the risk of CVD.

INTRODUCTION

Hypercholesterolemia is the most important riskfactor for atherosclerosis and cardiovascular disease(CVD) (1). Inherited autosomal dominanthypercholesterolemia (ADH) is associated withmarkedly elevated levels of cholesterol and occurs at afrequency of 1/500 worldwide, and it is linked toheterozygous dominant mutations in the genesencoding the low density lipoprotein receptor (LDL-R) with over 1600 mutations identified (2). Statinshave been the first-line drugs for loweringcholesterol since the late 1980s. Statins inhibit therate-limiting step of cholesterol synthesis catalyzed byhydroxylmethylglutaryl coenzyme A reductase(HMGCoA reductase) reducing the incidence ofatherosclerosis. They upregulate the transcriptionfactor SREBP-2, which then stimulates the expressionof LDL-R resulting in increased LDL-cholesteroluptake by hepatocytes, and consequently lowering


1Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic

Diseases, Merkur University Hospital, Zagreb, Croatia2School of Medicine, University of Zagreb, Zagreb, Croatia

Review

PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9

INHIBITORS: NEW CLASS OF LIPID-LOWERING THERAPY

T. Bulum1, L. Duvnjak1,2

Corresponding author: Tomislav Bulum, MD, PhD, Vuk Vrhovac Clinic for

Diabetes, Endocrinology and Metabolic Diseases, Merkur University

Hospital, Dugi dol 4a, HR-10000 Zagreb, Croatia


circulating levels of LDL-cholesterol (3). Althoughstatins reduce cardiovascular events by 25%-40%,they often lead to suboptimal levels of LDL-cholesterol, especially in patients with ADH, and arenot well tolerated due to unwanted side effects (4, 5).Other drugs are also being considered to managehyperlipidemia such as fibrates, ezetimibe,colesevelam, torcetrapib, avasimibe, implitapide, andniacin. Fibrates decrease fatty acid and triglyceridelevels by stimulating the peroxisomal β-oxidationpathway, ezetimibe selectively inhibits intestinalcholesterol absorption, cholestyramine, colestipol andcolesevelam sequester bile acids, torcetrapib inhibitscholesterol ester transfer protein, avasimibe inhibitsacyl-CoA:cholesterol acyltransferase, implitapideinhibits microsomal triglyceride transfer protein, andniacin modifies lipoproteins (6).

Many bioactive secretory proteins, includingpolypeptide hormones and enzymes, are initiallyproduced as inactive precursors, and conversion of aninactive secretory precursor into active product(s) iscatalyzed by a special group of proteases denoted asproprotein convertases (7). Proprotein convertasesubtilisin kexin 9 (PCSK9) was first reported in early2003 (8). It is highly expressed in liver hepatocytes

and found to represent the third familialhypercholesterolemia gene, with the LDL-R andapolipoprotein B (ApoB) genes being the other two (9,10). After the discovery of PCSK9, its relationship toCVD has been well documented (11, 12). Moreover,heterozygote African nonsense loss-of-functionmutation of PCSK9 correlates with a remarkable 88%reduction in CVD risk when compared withnoncarriers (13). In addition, because mammals cansurvive and stay healthy without PCSK9, it was foundthat 2 complete loss-of-function mutations caused anamazingly low level of circulating LDL-cholesterol ofabout 0.4 mmol/L (14, 15). Since PCSK9 binds thehepatocyte-derived LDL-R, disrupting this protein-protein interaction prevents LDL-R degradation, andraising the LDL-R levels lowers LDL-cholesterollevels and protects from the development ofatherosclerosis (16, 17).

22

T. Bulum, L. Duvnjak / PROPROTEIN CONVERTASE SUBTILISIN KEXIN 9 INHIBITORS: NEW CLASS OF LIPID-LOWERING

THERAPY

Figure 1. LDL-cholesterol metabolism in the liver without PCSK9 binding to the LDL receptor: LDL receptor is

recycled and more LDL-cholesterol can be removed from the blood.

LDL-R: LDL receptor; LDL-c: LDL-cholesterol

BIOLOgy, ExTRACELLULAR AND

INTRACELLULAR ACTIvITIES Of

PCSK9 IN ANIMAL MODELS

The human 22-kb gene PCSK9 is located on thesmall arm of chromosome 1p32 and contains 12 exonsand 11 introns and encodes a 692–amino acid (aa)proteinase K-like serine protease6 named PCSK9 (10,18). In the adult, PCSK9 is highly expressed in liverhepatocytes and less in the small intestine and kidney,and has no other substrate than itself, and its activity isrelated to its binding to specific target proteins and toescort the resulting complex toward intracellulardegradation compartments (19). The first PCSK9target to be identified is the LDL-R at the surface ofliver hepatocytes (20, 21). After binding to the LDL-R,the PCSK9-LDL-R complex inhibits normal processthat has resulted in the acidic pH of endosomes andcauses allosteric dissociation of the LDL-R and itsrecycling to the cell surface, as well as direction of theLDL-cholesterol to the lysosomes for degradation(Figure 1) (22). In contrast, the complex PCSK9-LDL-R does not dissociate at acidic pH but is rather moretightly associated and then moved to lysosomes fordegradation (Figure 2) (23).

PCSK9 targets the LDL-R for degradation via twopathways: an intracellular one from the trans Golginetwork directly to lysosomes implicating clathrinlight chains and an extracellular one requiring clathrinheavy chain-mediated endocytosis of the cell surfacecomplex PCSK9-LDL-R (24, 25). The cell surfaceendocytosis of the PCSK9-LDL-R complex is adominant degradation pathway and the level ofhepatocyte cell surface LDL-R decreases, which isconfirmed in mouse KO models, leading to theaccumulation of LDL-cholesterol in plasma (13, 16,26, 27). Moreover, in cases of high levels of mutantsof PCSK9, this can result in acceleratedatherosclerosis and cardiovascular disease (28, 29).The molecular mechanisms regulating the sorting ofthe prosegment PCSK9-LDL-R complex toendosomes/lysosomes by the intracellular pathwaysare poorly defined. It seems that the subcellulartrafficking of the PCSK9-LDL-R complex leading toits degradation requires the presence of the C-terminalcys-his–rich domain (CHRD) segment of PCSK9, butnot the cytosolic tail of the LDL-R (30, 31). It issuggested that as yet undefined molecular PCSK9partners regulate the trafficking of the prosegmentPCSK9-LDL-R complex towards lysosomes for



THERAPY

Figure 2. LDL-cholesterol metabolism in the liver with PCSK9 binding to the LDL receptor: LDL receptor is

degraded and less LDL-cholesterol can be removed from the blood.

LDL-R: LDL receptor; LDL-c: LDL-cholesterol; PCK9: proprotein convertase subtilisin kexin 9

degradation (32). Recently, the critical requirement forthe M2 domain of the CHRD in the extracellularpathway has been described, but not for theintracellular one (33). However, both the intracellularand the extracellular LDL-R degradation activities ofPCSK9 require the presence of the CHRD because thelack of this domain does not traffic to lysosomes andis likely recycled to the cell surface (33, 34). Inaddition, Annexin A2 (AnxA2), a functional inhibitorof PCSK9 that targets the CHRD, also acts as anendogenous regulator of LDL-R degradation inextrahepatic tissues (35). No specific inhibitor of theintracellular function of PCSK9 has yet been identifiedbecause studies suggest that the twointracellular/extracellular pathways show differentdynamic subcellular co-localization patterns ofPCSK9 and LDL-R (36).

Transgenic mice overexpressing PCSK9 under anApoe promoter are viable, fertile and severelyhypercholesterolemic, and develop acceleratedatherosclerosis with larger plaque size when fed aregular chow diet (21, 26). The circulating LDL-cholesterol in these mice is only 5-fold increased,although the LDL-R is not detected in the liver,compared with 15-fold increase in LDLR-knockoutmice, suggesting that the LDL-R are expressed inextrahepatic tissues and those LDL-R are not regulatedby cicrulating PCSK9 (7, 26). Pig model of a humanPCSK9 mutant also had markedly reduced levels ofhepatic LDL-R and severe hypercholesterolemia, andthese minipigs developed spontaneous progressiveatherosclerotic lesions (37). In contrast, completePCSK9-knockout mice are viable and fertileexhibiting severe hypocholesterolemia with a 40%-80% drop in total cholesterol and LDL-cholesterolcompared to liver-specific PCSK9-knockout mice thatexhibit about 27% less circulating cholesterol levels,indicating that liver PCSK9 contributes to about 70%of the cholesterol phenotype (26, 27, 38). Moreover,PCSK9-knockout mice have reduced plasma levels ofsphingomyelin and ceramides, known risk factors forCAD (39). Since PCSK9 may target other receptors, inPCSK9-knockout mice VLDL-R is also upregulatedand associated with a 2-fold decrease in postprandialtriglyceride levels, suggesting a role for PCSK9 in

triglyceride metabolism, possibly via its ability toenhance the degradation of VLDL-R (40). CirculatingPCSK9 also regulates the levels of its cell surfacereceptors in adipose tissue and lack of PCSK9increases the surface expression of VLDL-R thatfacilitates triglyceride hydrolysis and free fatty aciduptake in visceral adipocytes (41). VLDL-R isupregulated in perigonadal depots of PCSK9-knockout mice, and perigonadal fat is part of thevisceral adipose tissue, which correlates directly withmetabolic disease and CAD.

In Anxa2-knockout mice (Annexin A2 is a naturalextrahepatic inhibitor of PCSK9), plasma PCSK9doubles and LDL-R decreases by about 50% inextrahepatic tissues such as adrenals, small intestineand colon (35, 42). PCSK9 also regulates the LDL-Rprotein levels in intestinal cell lines and has a role inthe transintestinal reverse cholesterol excretion fromthe small intestine (43, 44). PCSK9 is highlyexpressed in pancreatic β-cell lines and PCSK9-knockout mice express more LDL-R in pancreatic isletcells, are glucose-intolerant and may be at risk todevelop diabetes mellitus (8, 45).

PCSK9 INHIBITORS – CLINICAL TRIALS

The animal model of the loss-of-function mutationsin PCSK9 that resulted in markedly reduced LDL-cholesterol plasma levels facilitates investigation of anew class of lipid-lowering therapies that mightprevent CAD (46). Each LDL-R normally recyclesapproximately 150 times and monoclonal antibodybinding and inhibition of PCSK9 prevents lysosomaldegradation of the LDL-R. The LDL-R recycling ispreserved, with a consequent increase in receptordensity on the hepatocyte surface and LDL-cholesterolclearance. International guidelines have promotedincreasingly lower treatment goals for LDL-cholesterol to reduce cardiovascular risk (1, 47).Although statins have acceptable efficacy and safetyprofiles, more than one half of cardiovascular eventsare not prevented by these drugs, and up to 40% ofpatients receiving statins are not able to reach targetLDL-cholesterol levels with current guidelinerecommendations (48). While some of those patients

24


THERAPY

were treated with submaximal doses, had side effectsand pharmacological interactions, a limited additional6% decrease in LDL-cholesterol was observed withdoubling of the statin dose (49). In recent years,human monoclonal antibodies against PCSK9 havebeen identified as an innovative lipid-loweringstrategy. Many monoclonal antibodies includingevolocumab, alirocumab and bococizumab have beendeveloped within the past 5 years. These PCSK9inhibitors, administered subcutaneously in monthly orsemimonthly injections, combined with statins, as wellas in monotherapy, provided benefits in reducingatherogenic lipid fractions in several phase 3 studies(50-52).

Results of a meta-analysis of 24 randomized trialsthat evaluated the effects of PCSK9 antibodies in 10159 adults with hypercholesterolemia, previouslytreated with statins who had not met target LDL-cholesterol goals or who did not tolerate statins,showed that PCSK9 inhibition, compared withplacebo or ezetimibe control groups, led to a 47%reduction in LDL-cholesterol (50). Compared toezetimibe, LDL-cholesterol reduction observed withalirocumab and evolocumab was distinctly greaterwith an average additional 36% reduction in LDL-cholesterol from baseline. Moreover, the reduction inlipoprotein(a), another lipid fraction that contributes toatherosclerotic plaque formation, was 25% on averagecompared with placebo or ezetimibe. The results alsoshowed that therapy with PCSK9 antibodies wasassociated with lower odds of all-cause mortality andmyocardial infarction but nonsifgnificant reduction incardiovascular mortality. Results were robust insensitivity analyses that were stratified by intensity ofbackground statin therapy or by comparator (placeboor ezetimibe). However, these results warrant cautiousinterpretation because the included trials were of smallor moderate size with short follow up periods andsmall total number of events. There was also astatistically significant 30% reduction in the odds ofincreased creatine kinase levels with the use of PCSK9antibodies compared with no anti-PCSK9 treatmentand the rates of serious adverse events did notsignificantly differ with versus without anti-PCSK9treatment, confirming the overall comparative safety

of the drug. In contrast to this meta-analysis wherereduction in cardiovascular mortality was notstatistically significant, OSLER trials 1 and 2 showeda statistically significant reduction in a combinedcardiovascular end point with evolocumab (53). Inaddition, the ODYSSEY LONG TERM study, whichincludes the largest patient population studied over 78weeks, demonstrated a reduction in myocardialinfarction with PCSK9 antibody therapy (54).However, PCSK9 antibodies have been testedpredominantly in adults without the acute coronarysyndrome who have hypercholesterolemia.

Since statins (and ezetimibe) may induceupregulation of PCSK9 attenuating their LDL-cholesterol lowering effect, PCSK9 inhibitors may beparticularly helpful in patients who do not achieve theexpected LDL-cholesterol level with maximallytolerated statins or in those with statin intolerance andwith very high baseline LDL-cholesterol levels(patients with familial hypercholesterolemia) (55, 56).In support to these data, the use of PCSK9 antibodiescombined with statins provided benefits in terms ofreducing atherogenic lipid fractions in patients withhyperlipidemia (LAPLACE-2) or heterozygousfamilial hypercholesterolemia (RUTHERFORD-2)(57, 58). Moreover, in GAUSS and GAUSS-2 trials,which included predominantly statin-intolerantpatients, treatment with evolocumab resulted in up to50% reduction in the LDL-cholesterol level (59).Fourteen trials comprising 4378 patients wereincluded in the analysis of HDL-cholesterol andshowed 6.3% increase in HDL-cholesterol with theuse of PCSK9 antibodies compared to placebo orezetimib (50). Ten studies comprising 5357 patientswere included in the analysis of total cholesterol andshowed a 39% reduction with the use of PCSK9antibodies compared to placebo and 24% reductioncompared to ezetimib. Recently, the US Food andDrug Administration (FDA) granted approval toPCSK9 inhibitor alirocumab for heterozygous familialhypercholesterolemia and for patients with clinicalatherosclerotic cardiovascular disease, while theEuropean Medicines Agency (EMA) hasrecommended approval of the PCSK9 inhibitor



THERAPY

alirocumab for individuals who cannot lower theirhigh LDL-cholesterol levels with statins or whocannot tolerate statins.

Four large cardiovascular outcomes trials exploringthe ability of PCSK9 antibodies to affect clinicaloutcomes are ongoing. Since PCSK9 antibodies havebeen tested predominantly in adults without the acutecoronary syndrome, the FOURIER study is assessingwhether treatment with evolocumab compared withplacebo reduces recurrent cardiovascular events in 27500 adults with established CAD (60). TheODYSSEY outcomes trial will investigate the effect ofalirocumab on major adverse cardiovascular events inabout 18 000 patients with heterozygous familialhypercholesterolemia, established coronary heartdisease, or a coronary heart disease risk equivalent,receiving treatment with statins at the maximumtolerated dose (61). In post hoc analysis of 2341patients after 78-week follow up alirocumab reducedthe level of LDL-cholesterol by 61% compared toplacebo and there was evidence for reduction in therate of cardiovascular events with alirocumab (62).Two cardiovascular outcome studies withbococizumab investigating the change in LDL-cholesterol, in some subjects to the levels well belowthe current guideline recommended targets, in morethan 22 000 patients, are in the phase 3 program (50).

CONCLUSION

Since the discovery of PCSK9 in 2003 and itsimplication in LDL-cholesterol regulation,investigations have shown that PCSK9 is a keyregulator of LDL-cholesterol metabolism thatpromotes hepatic degradation of LDL-R and reducesLDL-cholesterol clearance. The initial clinical trialexperience with PCSK9 inhibitors has confirmed thattreatment with PCSK9 antibodies produces profoundreductions in total, LDL-cholesterol andlipoprotein(a), with a similar level of safety andsurvival benefit compared with no anti-PCSK9treatment. However, PCSK9 inhibitors are monoclonalantibodies that are high molecular mass proteinscomplex to manufacture and potentially immunogenic,are high cost and unsuited for oral delivery.Confirmation of these findings in ongoing, long-term,pivotal trials with prespecified cardiovascular endpoints should provide additional data on the safety ofthis innovative therapy and will help establish the roleof these novel agents in reducing the incidence ofcardiovascular disease.

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THERAPY

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49. Blom DJ, Hala T, Bolognese M, Lillestol MJ, TothPD, Burgess L, et al.; DESCARTES Investigators.A 52-week placebo-controlled trial of evolocumabin hyperlipidemia. N Engl J Med 2014;370:1809-1819.



THERAPY

50. Navarese EP, Kolodziejczak M, Schulze V, GurbelPA, Tantry U, Lin Y, et al. Effects of proproteinconvertase subtilisin/kexin type 9 antibodies in adults with hypercholesterolemia: a systematic reviewand meta-analysis. Ann Intern Med 2015;163:40-51.

51. Stein EA, Mellis S, Yancopoulos GD, Stahl N,Logan D, Smith WB, et al. Effect of a monoclonalantibody to PCSK9 on LDL cholesterol. N Engl JMed 2012;366:1108-1118.

52. Cannon CP, Cariou B, Blom D, McKenney JM,Lorenzato C, Pordy R, et al. Efficacy and safety ofalirocumab in high cardiovascular risk patientswith inadequately controlled hypercho -lesterolaemia on maximally tolerated doses ofstatins: the ODYSSEY COMBO II randomizedcontrolled trial. Eur Heart J 2015;36:1186-1194.

53. Sabatine MS, Giugliano RP, Wiviott SD, Raal FJ,Blom DJ, Robinson J, et al. Efficacy and safety ofevolocumab in reducing lipids and cardiovascularevents. N Engl J Med 2015;372:1500-1509.

54. Mihaylova B, Emberson J, Blackwell L, Keech A,Simes J, Barnes EH, et al.; Cholesterol TreatmentTrialists’ (CTT) Collaborators. The effects oflowering LDL cholesterol with statin therapy inpeople at low risk of vascular disease: meta-analysis of individual data from 27 randomisedtrials. Lancet 2012;380:581-590.

55. Davignon J, Dubuc G. Statins and ezetimibemodulate plasma proprotein convertase subtilisinkexin-9 (PCSK9) levels. Trans Am Clin ClimatolAssoc 2009;120:163-173.

56. Ason B, Tep S, Davis HR Jr, Xu Y, Tetzloff G,Galinski B, et al. Improved efficacy for ezetimibeand rosuvastatin by attenuating the induction ofPCSK9. J Lipid Res 2011;52:679-687.

57. Raal FJ, Stein EA, Dufour R, Turner T, Civeira F,Burgess L, et al. PCSK9 inhibition withevolocumab (AMG 145) in heterozygous familialhypercholesterolaemia (RUTHERFORD-2): arandomised, double-blind, placebo-controlled trial.Lancet 2015;385:331-340.

58. Robinson JG, Nedergaard BS, Rogers WJ, FialkowJ, Neutel JM, Ramstad D, et al. Effect ofevolocumab or ezetimibe added to moderate- orhigh-intensity statin therapy on LDL-C lowering inpatients with hypercholesterolemia: theLAPLACE-2 randomized clinical trial. JAMA2014;311:1870-1882.

59. Stroes E, Colquhoun D, Sullivan D, Civeira F,Rosenson RS, Watts GF, et al. Anti-PCSK9antibody effectively lowers cholesterol in patientswith statin intolerance: the GAUSS-2 randomized,placebo-controlled phase 3 clinical trial ofevolocumab. J Am Coll Cardiol 2014;63:2541-2548.

60. Further cardiovascular outcomes research withPCSK9 inhibition in subjects with elevated risk(FOURIER). ClinicalTrials.gov: NCT01764633.Accessed at https://clinicaltrials.gov/ct2/show/NCT01764633 on 1 August 2015.

61. ODYSSEY outcomes: evaluation ofcardiovascular outcomes after an acute coronarysyndrome during treatment with alirocumabSAR236553 (REGN727). ClinicalTrials.gov:NCT01663402. Accessed at https://clinicaltrials.gov/ct2/show/NCT01663402 on 1 August 2015.

62. Robinson JG, Farnier M, Krempf M, Bergeron J,Luc G, Averna M, et al. Efficacy and safety ofalirocumab in reducing lipids and cardiovascularevents. N Engl J Med 2015;372:1489-1499.

30


THERAPY

Key words: new insulin glargine 300 units/mL,

constant and prolonged PK/PD profile,

type 1 and type 2 diabetes mellitus

SUMMARY

New insulin glargine 300 units/mL (Gla-300) is a

basal insulin that provides the same number of units as

Gla-100 in a third of the volume. Compared with Gla-

100, Gla-300 has shown more constant and prolonged

pharmacokinetic (PK)/pharmacodynamic (PD)

profiles. Gla-300 has been shown to achieve similar

glycemic control with less nocturnal hypoglycemia

compared with Gla-100, and lower hypoglycemia at

any time of day. The EDITION clinical program also

provides evidence for less weight gain with Gla-300

than with Gla-100. The PK/PD profiles of Gla-300

may allow more flexibility in the timing of doses.

Based on these findings, Gla-300 could be optimal

treatment option for a range of people with type 1 and

type 2 diabetes mellitus.

INTRODUCTION

Based on the current knowledge about the treatmentof type 2 diabetes mellitus (T2DM), it isrecommended to initiate insulin therapy using a basalinsulin. T2DM is a pathophysiologically progressivedisease requiring intensification of therapy over time.Consequently, some patients will eventually needintensified insulin therapy, consisting of anintermediate- or long-acting insulin at bedtime forfasting glucose control and a short-acting mealtimeinsulin (administered before breakfast, lunch anddinner) for the control of postprandial hyperglycemia.This so-called basal-bolus insulin regimen is thestandard of care for all people with type 1 diabetesmellitus (T1DM), with long-acting insulin analoguesbeing used much more commonly than humanintermediate-acting insulins, due to their more evenprofile of action leading to a reduced risk ofhypoglycemia.

After the failure of an oral antihyperglycemic drug(OAD) combination therapy, basal insulin treatment isrecommended to reduce the level of fastinghyperglycemia, which occurs in people with T2DM asa consequence of increased hepatic glucose productionwhen the quantity of endogenous insulin is no longersufficient to suppress gluconeogenesis adequately (1).


1Vuk Vrhovac Clinic for Diabetes, Endocrinology and Metabolic

Diseases, Merkur University Hospital, Zagreb, Croatia2School of Medicine, University of Zagreb, Zagreb, Croatia

Review

CLINICAL OVERVIEW OF INSULIN GLARGINE 300 UNITS/mL

(TOUJEO®)

L. Smirčić Duvnjak1,2, T. Bulum1, T. Božek1

Corresponding author: Prof. Lea Smirčić Duvnjak, MD, PhD, Vuk Vrhovac

Clinic for Diabetes, Endocrinology and Metabolic Diseases, Merkur

University Hospital, Dugi dol 4a, HR-10000 Zagreb, Croatia


Hypoglycemia is the most frequent adverse reaction ofinsulin therapy. Insulin therapy aimed at reducingglycemic values is associated with an increased risk ofhypoglycemia. The fear from hypoglycemia remains amajor obstacle to achieve adequate glycemic control inboth insulin-experienced and insulin-naïve patientswith type 1 and type 2 diabetes mellitus.

The even and peakless profile of long-acting insulinanalogues mimics the physiological basal pancreaticinsulin secretion, thereby reducing the risk ofhypoglycemia compared to long-acting human insulin(neutral protamine Hagedorn, NPH), with asatisfactory HbA1c-lowering effect.

The ideal basal insulin should be administered oncedaily and ensure a slower, more even insulin release,with little glycemic variability, an evenly distributed24-hour effect and a low risk of hypoglycemia.

PHARMACOKINETICS AND

PHARMACODyNAMICS Of INSULIN

gLARgINE 300 UNITS/mL

Insulin glargine is a human insulin analogue with lowsolubility at neutral pH. Insulin glargine is completelysoluble at pH 4. Following injection into subcutaneoustissue, the acidic solution is neutralized, leading to theformation of a precipitate from which small amountsof insulin glargine are continuously released (2).

Insulin glargine 100 units/mL (Lantus®) is indicatedfor the treatment of diabetes mellitus in patients whoneed insulin to improve their glycemic control.

Insulin glargine 300 units/mL (Toujeo®) has the samemolecular composition as insulin glargine100 units/mL but shows a more even and prolongedpharmacokinetic profile, enabling an evenlydistributed daytime glucose-lowering effect with highreproducibility of action, i.e. less variability. Theseadvantages result from structural enhancement of thesubcutaneous microprecipitate (Figure 1). Themicroprecipitate surface area has been reduced and itscompactness increased, thus facilitating a prolongedrelease of insulin glargine from insulin glargine300 units/mL precipitate compared to insulin glargine100 units/mL (3-6).

As observed in euglycemic clamp studies in patientswith T1DM (a crossover study in 18 patients), theglucose-lowering effect of insulin glargine300 units/mL after a subcutaneous injection was morestable and prolonged compared to insulin glargine100 units/mL. At clinically relevant doses, the effect ofinsulin glargine 300 units/mL was sustained beyond24 hours (up to 36 hours) (7).

In view of the above, insulin glargine 300 units/mL isnot bioequivalent to insulin glargine 100 units/mL andthe two are not interchangeable (Figure 2).

In a continuous glucose monitoring study conductedin 59 patients with T1DM, the mean glucose profilewas consistent in patients treated with insulin glargine300 units/mL compared to those treated with insulinglargine 100 units/mL, regardless of whether the doseswere administered in the morning or in the evening(6).

After a subcutaneous injection of insulin glargine inhealthy subjects and diabetic patients, insulin serumconcentrations indicated a slower and significantlymore prolonged absorption, without peak values,compared to human NPH insulin. The concentrationswere consistent with the time profile of thepharmacodynamic activity of insulin glargine. Thetherapeutic steady-state is reached after 3 to 4 days ofdaily administration of insulin glargine 300 units/mL.After subcutaneous administration of insulin glargine300 units/mL, the intra-subject variability, defined asthe coefficient of variation for 24-hour insulinexposure, was low at steady-state (17.4%). In humansubcutaneous tissue, insulin glargine is partiallymetabolized at the carboxyl termini of the beta-chainto form active metabolites, M1 (21A-Gly-insulin) andM2 (21A-Gly-des-30B-Thr-insulin). Pharmacokineticand pharmacodynamic findings indicate that the effectof a subcutaneous insulin glargine injection isprincipally based on exposure to M1. In the majorityof subjects, insulin glargine and M2 wereundetectable, and when detected, their concentrationwas independent of the dose or the insulin glargineformulation administered. The elimination half-life ofinsulin glargine 300 units/mL after subcutaneous

32

L. Smirčić Duvnjak, T. Bulum, T. Božek / CLINICAL OVERVIEW OF INSULIN GLARGINE 300 UNITS/mL

(TOUJEO®)



(TOUJEO®)

Figure 1. The more sustained release of insulin glargine from the Gla-300 (insulin glargine 300 units/mL)

precipitate compared to Gla-100 (insulin glargine 100 units/mL) is attributable to the reduction in injection

volume by two thirds, which results in a smaller precipitate surface area.

Figure 2. Insulin concentration (INS), glucose infusion rate (GIR) and blood glucose (BG) after multiple doses of

Gla-100 (insulin glargine 100 units/mL) and Gla-300 (insulin glargine 300 units/mL).

administration is determined by the rate of absorptionfrom the subcutaneous tissue. The half-life is 18-19 hours, regardless of the dose (5).

CLINICAL EffICACy

The efficacy and safety of the new insulin glargine300 units/mL was evaluated in a comprehensiveEDITION clinical program, which encompassed awide range of T2DM patients (n=2474), includinginsulin-naïve patients treated with OADs(EDITION3), patients previously treated with basalinsulin in combination with OADs (EDITION2) andpatients on intensified insulin therapy consisting of abasal insulin and a short-acting mealtime insulin(EDITION1) (8-10). In such a large number of T2DMpatients participating in the EDITION program,insulin glargine 300 units/mL proved to be just aseffective in improving glycemic control over 6 monthsas insulin glargine 100 U/mL.

However, in the EDITION clinical program, insulinglargine 300 units/mL demonstrated superiority overinsulin glargine 100 units/mL with respect to the

number of confirmed (≤3.9 mmol/L) and/or severe

hypoglycemic events (relative difference: 14%) at any

time of day (over 24 hours) (Figure 3).

An even greater relative difference (31%) in the

number of confirmed (≤3.9 mmol/L) and/or severe

hypoglycemic events favoring insulin glargine

300 units/mL compared to insulin glargine

100 units/mL was observed during night time (00:00-

06:00 AM) (Figure 3).

In general, these effects on the risk of hypoglycemia

have been consistently observed in the clinical

program throughout the treatment period, i.e. both in

the titration and the dose maintenance periods (11).

DOSINg

Insulin glargine 300 units/mL is administered once

daily at any time, but always at the same time of day.

The dosing regimen of insulin glargine 300 units/mL

(dose and timing) should be adjusted individually. The

recommended starting dose in T2DM patients is

0.2 units/kg daily, followed by individual dose

adjustments. In the elderly (≥65 years), progressive

deterioration of renal function may result in gradual

and steady decreases in insulin requirements. Insulin

requirements may also be lower in patients with renal

and hepatic impairment. When switching from an

intermediate- or long-acting insulin to insulin glargine

300 units/mL, the basal insulin dose may have to be

modified and concomitant anti-hyperglycemic drugs

adjusted (2).

Switching from once-daily basal insulins, including

insulin glargine 100 units/mL, to once-daily insulin

glargine 300 units/mL can be done unit-to-unit based

on the previous basal insulin dose. When switching

from twice-daily basal insulins to once-daily insulin

glargine 300 units/mL, the recommended initial dose

of insulin glargine 300 units/mL is 70%-80% of the

total daily dose of the basal insulin that is being

discontinued (2).

34


(TOUJEO®)

Figure 3. Cumulative mean number of confirmed (≤3.9

mmol/L) and/or severe events (A) during the day (24

h) and at night (00:00-05:59 AM) (B) based on the

pooled analysis of EDITION1, EDITION2 and

EDITION3. GLA-100 (insulin glargine 100 units/mL);

GLA-300 (insulin glargine 300 units/mL)

SAfETy

Insulin glargine 300 units/mL is not the insulin ofchoice for the treatment of diabetic ketoacidosis. Insuch cases, intravenous administration of regularinsulin is recommended. In case of inadequateglycemic control or a predisposition to hyper- orhypoglycemia, prior to considering dose modificationthe patient’s adherence to the prescribed dosingregimen, injection sites and the proper injectiontechnique, as well as all other relevant factors shouldbe evaluated. Given that insulin glargine 100 units/mLand insulin glargine 300 units/mL are neitherbioequivalent nor interchangeable, switching from oneformulation to the other may require a dosemodification and should be performed under closemedical supervision (2).

There are no data on pregnancies exposed to insulinglargine from controlled clinical trials.

Insulin glargine has also been investigated in acomprehensive cardiovascular outcome study entitledORIGIN (Outcome Reduction with Initial GlargineINtervention). This multicentre, randomized clinicalstudy included T2DM patients (N=12,537) at a highcardiovascular risk (12). Patients were randomized(1:1) to either insulin glargine or the standard of care.The first co-primary efficacy outcome was the time tothe first occurrence of cardiovascular death, nonfatalmyocardial infarction or nonfatal stroke, whereas thesecond co-primary efficacy outcome was the time tofirst occurrence of any of the first co-primary events,or a revascularization procedure, or hospitalization forheart failure. Insulin glargine did not alter the relativerisk of cardiovascular disease and cardiovascularmortality when compared to the standard of care.

CONCLUSION

Based on the clinical and pharmacological indicators,insulin glargine 300 units/mL is an advancement in thetreatment of T1DM and T2DM in patients who requireeither initiation or transition of insulin therapy. In bothT1DM and T2DM, it is important to meet basal insulinrequirements using a peakless long-acting insulinanalogue, which minimizes the risk of hyperglycemia

and hypoglycemia (8-10, 13, 14). It is preciselyhypoglycemia that is an important obstacle to achievediabetes control goals, and insulin glargine300 units/mL, being a long-acting basal insulin of lowvariability and prolonged even activity, facilitatesglycemic control with a low risk of hypoglycemia. Ina broad population of T2DM patients, insulin glargine300 units/mL provides comparable glycemic control tothat provided by glargine-100, with less hypoglycemiaat any time of day (24 h), and a more pronouncedreduction in hypoglycemia during the night, andimportantly during the titration period (first 8 weeks)(11).

The flexibility in dosing time allows for insulinglargine 300 units/mL to be administered up to 3 hoursbefore or after the usual dosing time, thus ensuringfurther improvement in the patient quality of life whilemaintaining a good glycemic control.

In addition, in patients treated with insulin glargine300 units/mL, the mean observed weight change at theend of a 6-month period in the clinical program wasless than 1 kg. Given that insulin therapy is usuallyassociated with weight gain, this result, indicating analmost weightneutral effect of insulin glargine300 units/mL, is an important additional treatmentbenefit, especially in patients with T2DM.



(TOUJEO®)

REfERENCES

1. Approaches to Glycemic Treatment. Diabetes Care2016;39(Suppl 1):S52-S59.

2. Toujeo (insulin glargine 300 units/mL) – Summaryof Product Characteristics, May 2015.(www.ema.europa.eu)

3. Steinstraesser A, Schmidt R, Bergmann K,Dahmen R, Becker RH. Investigational newinsulin glargine 300 U/mL has the samemetabolism as insulin glargine 100 U/mL.Diabetes Obes Metab 2014;16:873-876.

4. Becker RHA, Dahmen R, Bergmann K, LehmanA, Jax T, Heise T. Insulin glargine 300 units·mL−1

provides a more even activity profile andprolonged glycemic control at steady statecompared with insulin glargine 100 units·mL−1.Diabetes Care 2015;38:637-643.

5. Shiramoto M, Eto T, Irie S, Fukuzaki A, TeichertL, Tillner J, et al. Single-dose new insulin glargine300U/mL provides prolonged, stable glycaemiccontrol in Japanese and European people with type1 diabetes. Diabetes Obes Metab 2015;17:254-260.

6. Becker RHA, Nowotny I, Teichert L, Bergmann K,Kapitza C. Low within- and between-dayvariability in exposure to new insulin glargine 300U/mL. Diabetes Obes Metab 2015;17:261-267.

7. Jinnouchi H, Koyama M, Amano A, Takahashi Y,Yoshida A, Hieshima K, et al. Continuous glucosemonitoring during basal-bolus therapy usinginsulin glargine 300−1 and glargine 100 U mL−1 inJapanese people with type 1 diabetes mellitus: acrossover pilot study. Diabetes Ther 2015;6:143-152.

8. Riddle MC, Bolli GB, Ziemen M, Muehlen-Bartmer I, Bizet F, Home PD. New insulin glargine300 units/mL versus glargine 100 units/mL inpeople with type 2 diabetes using basal andmealtime insulin: glucose control andhypoglycemia in a 6-month randomized controlledtrial (EDITION 1). Diabetes Care 2014;37:2755-2762.

9. Yki-Järvinen H, Bergenstal R, Ziemen M,Wardecki M, Muehlen-Bartmer I, Boelle E, et al.New insulin glargine 300 units/mL versus glargine100 units/mL in people with type 2 diabetes usingoral agents and basal insulin: glucose control andhypoglycemia in a 6-month randomized controlledtrial (EDITION 2). Diabetes Care 2014;37:3235-3243.

10. Bolli GB, Riddle MC, Bergenstal RM, Ziemen M,Sestakauskas K, Goyeau H, et al. New insulinglargine 300 U/mL compared with glargine 100U/mL in insulin-naïve people with type 2 diabeteson oral glucose-lowering drugs: a randomizedcontrolled trial (EDITION 3). Diabetes ObesMetab 2015;17:386-394.

11. Matsuhisa M, Koyama M, Cheng X, Shimizu S,Hirose T. New insulin glargine 300 U/mL:glycaemic control and hypoglycaemia in Japanesepeople with type 1 diabetes mellitus (EDITION JP1). Diabetologia 2014;57(Suppl 1):S400-S400.

12. The ORIGIN Trial Investigators. Basal insulin andcardiovascular and other outcomes indysglycemia. N Engl J Med 2012;367:319-328.

13. Ritzel R, Roussel R, Bolli GB, Vinet L, Brulle-Wohlhueter C, Glezer S, et al. Patient-level meta-analysis of the EDITION 1, 2 and 3 studies:glycaemic control and hypoglycaemia with newinsulin glargine 300 U/mL versus glargine 100U/mL in people with type 2 diabetes. DiabetesObes Metab 2015;17:859-867.

14. Bergenstal RM, Bailey TS, Rodbard D, Ziemen M,Guo H, Muehlen-Bartmer M, et al. Insulin glargine300 U/mL vs 100 U/mL: glucose profiles ofmorning vs evening injections in adults with type 1diabetes mellitus measured with continuousglucose monitoring (CGM). Diabetologia2014;57(Suppl 1):S388-S388.

36


(TOUJEO®)

38

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MANUSCRIPTS. Original manuscripts in English should betypewritten, double-spaced and pages numbered consecutivelyin the following order: Title page containing the title of the article, the name(s) of theauthor(s) including the first name and highest academicdegree(s), the name of department and institution in which thework was done, and the address for correspondence, includingtelephone and/or fax number and e-mail address. Summary and Key Words should be written on a separate page.

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· Journals Pepeonik-Rogulja Z, Pinter S, Metelko Z. Canthe age at diagnosis of type 1 diabetes mellitus affect thecourse of diabetic retinopathy. Diabetologia Croatica2004;33:97-101.

· Books International Diabetes Federation. Diabetes Atlas,3rd ed. Brussels: International Diabetes Federation; 2006.

· Chapter in Book Reaven GM. Insulin resistance and itsconsequences: type 2 diabetes mellitus and the insulinresistance syndrome. In: LeRoith D, Taylor SI, OlefskyJM, editors. Diabetes mellitus: a fundamental and clinicaltext. 3rd ed. Philadelphia: Lippincott Williams & Wilkins;2004. pp. 899-915.

· Electronic Material Gazzaruso C, Garzaniti A,Giordanetti S, Falcone C, Fratino P. Silent coronary arterydisease in type 2 diabetes mellitus: the role oflipoprotein(a), homocysteine and apo(a) polymorphism.Cardiovasc Diabetol [Internet] 2002 Nov [accessed 2002Dec 9];1(5). Available from: URL:http://www.cardiab.com/content/1/1/5

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