Gustavs Latkovskis^1,2,3, Vita Šaripo^1,2, Dana Vanaģele², Emma Sokolova¹, Ilona Vanaga⁴,

Andrejs Ērglis^1,2,3

¹ Latvijas Universitātes Medicīnas fakultāte

² Paula Stradiņa Klīniskā Universitātes slimnīca, Rīga, Latvija

³ Latvijas Kardioloģijas zinātniskais institūts

⁴ Latvijas Farmācijas un ķīmijas kompetences centrs

Ievads. Statīnu lietošanu var ierobežot miopātija, kas var izpausties kā skeleta muskuļu sāpes, vājums vai krampji. Tā kā nav zināms, kā šo blakni ārstēt, vairumam pacientu ir nepieciešams samazināt statīna devu, kas palielina kardiovaskulāro risku. Viens no miopātijas patoģenēzes iespējamiem mehānismiem ir statīnu izraisīts izoprenoīdu, to skaitā ubihinona, deficīts. Augu izcelsmes poliprenoli un koenzīms Q10 (CoQ10) jeb ubihinons varētu kompensēt šo vielu deficītu un mazināt miopātijas simptomus. Starptautiskā literatūrā nav publicēti pētījumi par poliprenolu ietekmi uz statīnu inducēto miopātiju.

Darba mērķis. Noskaidrot, vai dabīgas izcelsmes augu poliprenoli kopā ar CoQ10 spēj samazināt statīnu inducētās miopātijas simptomus un objektīvos rādītājus, kā arī noskaidrot to drošumu lietojot kopā ar statīniem.

Materiāli un metodes. Latvijas Kardioloģijas Centrā 2014. gadā 9 mēnešu laikā skrīninga rezultātā no 12 kandidātiem tika atlasīti 5 pacienti (2 sievietes un 3 vīrieši), kas atbilda statīnu inducētas miopātijas kritērijiem. Visi pacienti aņēma 4 mēnešu kursu ar skuju izcelsmes poliprenoliem (4 mg/diennaktī) un CoQ10 (100 mg /dn). Miopātija tika izvērtēta pēc subjektīviem simptomiem (vērtējuma skala 0-10 sāpēm, vājumam, krampjiem), kā arī pēc KFK, veloergometrijas un dinamometrijas datiem. Drošības izvērtēšanai tika veikta pilna asins aina, asins bioķīmiskās analīzes, un elektrokardiogrāfija.

Rezultāti. Pēc pacientu subjektīvā vērtējuma pašsajūtas uzlabošanās bija novērojama trīs gadījumos (60%) jau pēc 2 mēnešiem. No tiem diviem pacientiem bija papildus simptomu uzlabošanās nākamo 2 mēnešu laikā. Kopumā no četriem pacientam ar muskuļu sāpēm vienam pilnībā izzuda muskuļu sāpes, vienam muskuļu sāpes samazinājās būtiski, vienam – nedaudz. Sāpju skala 4 mēnešos samazinājās no vidēji 6,0 [3,5-7,75] līdz 3,5 [0,25-6,75] (p=0,109; n=4). Muskuļu sāpes miera stāvoklī no 3 pacientiem izzuda vienam. No četriem pacientiem ar muskuļu vājumu, tikai vienam simptomi mazinājās nedaudz un vienam izzuda. No diviem pacientiem ar muskuļu krampjiem, uzlabošanās bija vienā gadījumā. Vājums un krampji attiecīgi izmainījās no 6,5 [5,0-8,0] līdz 5,5 [1,25-7,5] (p=0,180; n=4) un no 5,0 [-] uz 3,0 [1,0-5,0] (p=0,317; n=2). Dinamometrijā roku spēks un veloergometrijā sasniegtā slodze būtiski nemainījās. KFK līmenis terapijas laikā būtiski nemainījās, lai gan bija ar tendenci pieaugt (no 134±80 līdz 147±53 U/l; p=0,662).

Secinājumi. Dabīgas izcelsmes poliprenolu un CoQ10 lietošana atsevišķiem pacientiem ar statīnu inducēto miopātiju var sekmīgi mazināt miopātijas subjektīvās izpausmes, pārsvarā muskuļu sāpes. Preliminārie dati norāda uz šo preparātu drošību. Nepieciešami pētījumi lielākās pacientu grupās, lai apstiprinātu iegūtos rezultātus.

Finansējums. Pētījuma sponsors SIA “Silv EXPO”.

Zita Freiberga¹, Baiba Jansone¹, Elga Poppela ¹, Zane Dzirkale¹, Ingrīda Māgure¹,

Lote Ansone¹, Kaspars Jēkabsons¹, Vladimirs Piļipenko¹, Jana Namniece¹, Raimonds Skumbiņš¹, Ulrika Beitnere¹, Uģis Klētnieks², Ilona Vanaga³, Ruta Muceniece¹, Vija Kluša¹

¹ Latvijas Universitāte, Medicīnas fakultāte

² SIA „Silvanols”, Latvija

³ SIA „Pharma and Chemistry Competence Centre of Latvia”

Ievads. Poliprenoli (PP) ir izdalīti no augiem, tai skaitā, no skujkokiem. PP ir uzrādījuši antioksidantu, hepatoprotektantu un membrānu aizsargājošu vielu īpašības (Pronin et al., 2013). Taču to ietekme uz CNS procesiem ir ļoti maz pētīta. Ir zināms, ka gan PP, gan holesterols un doliholi veidojas caur mevalonskābes atkarīgiem ceļiem ar 3-hidroksi-3- metilglutaril-koenzīma A (HMG-CoA) reduktāzes palīdzību. Šo enzīmu inhibē holesterola biosintēzes inhibītori statīni, kuru ilgstoša lietošana slimniekiem nelabvēlīgi ietekmē kognitīvās funkcijas (Schilling et al., 2014). Eksperimentālos datus par PP spēju izmainīt statīnu efektus līdz šim neesam atraduši.

Darba mērķis. Noteikt PP, atorvastatīna (AT), kā arī to kombinācijas ietekmi uz izmēģinājuma dzīvnieku (žurku) vispārējo aktivitāti, atmiņas procesiem un sāpju slieksni.

Materiāli un metodes. Pētījumā izmantoja Wistar līnijas sieviešu kārtas žurkas (230- 245 g), kurām 16 dienas vienreiz dienā per os ievadīja no egļu (Picea abies L.) skujām izolētus PP (A/S BioLat, Latvija), kā arī AT (Atoris, KRKA, Slovēnija). PP tika izšķīdināti rafinētā saulespuķu eļļā, savukārt AT ievadīja ūdens suspensijas veidā, tādēļ kontroles dzīvniekiem ievadīja abus šķīdinātājus. Tika izveidotas 4 eksperimentālās grupas (n=9-10 grupā): 1. kontroles grupa (fizioloģiskais šķīdums + eļļa), 2. grupa: PP devā 20 mg/kg, 3. grupa: AT 80 mg/kg un 4. grupa: PP + AT. Astotajā dienā kopš vielu ievadīšanas sākuma noteica pretsāpju efektu, izmantojot analgēzijas (tail flick) testu. No 13. līdz 15. dienai veica pasīvās izvairīšanās atmiņas testu (passive avoindance response). Dzīvnieku vispārējo aktivitāti pārbaudīja 15. dienā atvērtā lauka (open field) testā.

Rezultāti un diskusija. Analgēzijas testā AT ievadīšana pagarināja latento periodu, kas liecina par vielas pretsāpju jeb analgētisko iedarbību. Savukārt PP paši neuzrādīja pretsāpju efektu, kā arī neizmainīja atorvastatīna darbību. Atvērtā lauka testā ne PP, ne AT neuzrādīja ietekmi uz dzīvnieku vispārējo aktivitāti. Pasīvās izvairīšanās testa dati rāda, ka žurku atmiņa arī netiek ietekmēta no pārbaudītajām vielām un to kombinācijas.

Secinājumi. AT uzrāda pretsāpju darbību, bet PP šo efektu neizmaina. Ne PP, ne AT, ne to kombinācija neietekmēja izmēģināju dzīvnieku vispārējo motoro aktivitāti un atmiņas procesus, kas norāda uz šo vielu drošību CNS līmenī.

Finansējums. Pētījums finansēts no Latvijas Farmācijas un ķīmijas kompetences centra projekta No. L-KC-11-0001 līdzekļiem. Projekts P29.

Raimonds Skumbiņš, Baiba Jansone, Zane Dzirkale, Kaspars Jēkabsons, Vladimirs Piļipenko, Ingrīda Māgure, Jana Namniece, Ulrika Beitnere, Elga Popela, Uģis Klētnieks, Ilona Vanaga, Ruta Muceniece, Vija Kluša

Latvijas Universitāte, Medicīnas fakultāte

SIA „Silvanols”, Latvija

SIA „Pharma and Chemistry Competence Centre of Latvia”

Ievads. Poliprenoli (PP) ir identificēti gandrīz visos dzīvajos organismos, kā arī pārtikā, augļos, dzērienos. Visbagātākais PP avots ir skujkoki. PP tāpat kā holesterols un doliholi ir substances, kas bioģenētiski kopīgi veidojas caur mevalonskābes atkarīgiem ceļiem, iesaistot 3-hidroksi-3-metilglutaril-koenzīma A (HMG-CoA) reduktāzi. Tādejādi vielas, piem., statīni, kas inhibē šo enzīmu, samazina ne tikai holesterola, bet arī doliholu un PP līmeni, kas var izraisīt muskuļu patoloģijas (mialģijas, krampjus u.c.). Mūsu koncepcija paredz, ka PP ievadīšana var protektēt šīs miopātijas, kompensējot doliholu deficītu.

Mērķis. Novērtēt PP un izvēlētā statīna – atorvastatīna (AT) – ietekmi uz muskuļu spēku un tonusu, kā arī pārbaudīt koncepcijas pareizību, pētot PP ietekmi uz AT efektiem žurkām.

Metodes. Pētījumi veikti uz Wistar sieviešu kārtas žurkām 230-245 g. PP, kas izolēti no egles skujām (Picea abies L.) saņemti no A/S BioLat, Latvija; atorvastatīns (Atorvastat) pirkts no KRKA, Slovēnija. PP izšķīdināti rafinētā saulespuķu eļļā, atorvastatīns pagatavots ūdens suspensijas veidā. Pārbaudāmās vielas un šķīdinātājus ievadīja vienreiz dienā per os 16 dienas. Žurkas tika sadalītas 8 grupās (n=9-10 grupā): kontroles grupā fizioloģiskais šķīdums + eļļa; 2.-4. grupās PP devās 1, 10 un 20 mg/kg; 5. grupā AT 80 mg/kg; 6.-8. grupās PP kombinācijā ar AT. Muskuļu spēku noteica 15. dienā ar satvēriena spēka (grip strength test) un 16. dienā ar stieplē karāšanās (wire hang test) testu. 16. dienā noteica arī muskuļu tonusu un koordināciju rotējošā stieņa (rotarod test) testā. 17.dienā žurkas anestezēja, punktēja sirdi, savāca asins paraugus kreatīnkināzes aktivitātes noteikšanai plazmā ar spektrofluorimetrisko metodi.

Rezultāti. Satvēriena spēka testā PP nevienā no testētām devām neuzrādīja no kontroles atšķirīgus rezultātus, taču AT žurkām muskuļu spēks samazinājās. PP devā 20 mg/kg protektēja šo AT efektu. Stieplē karāšanās testā žurkas, kurām ievadīti PP devās 1 un 10 mg/kg neietekmēja karāšanās laiku, kamēr devā 20 mg/kg tas pagarinājās divas reizes vs. kontrole. Toties AT karāšanās laiku samazināja vismaz trīs reizes vs. kontrole. PP visās devās ievērojami protektēja AT izraisīto muskuļu spēka samazināšanos. Rotējošā stieņa testā ne PP, ne AT neizmainīja muskuļu tonusu un koordināciju. Kreatīnkināzes aktivitātes izmaiņas (palielinājums par 25%) konstatēja tikai PP deva 20 mg/kg.

Secinājumi. PP protektē AT izraisītu muskuļu spēka samazināšanos, tādējādi PP var rekomendēt pacientiem, kuriem nozīmēta ilgstoša AT terapija. Finansējums. Pētījums finansēts no Latvijas Farmācijas un ķīmijas kompetences centra projekta No. L-KC-11-0001 līdzekļiem. Projekts P29.

Baiba Jansone, Zane Dzirkale, Kaspars Jekabsons, Vladimirs Pilipenko, Ulrika Beitnere, Raimonds Skumbins, Zita Freiberga, Elga Poppela, Ugis Kletnieks, Ilona Vanaga, Karlis Kletnieks, Ruta Muceniece, Vija Klusa.

Chinese Journal of Pharmacology and Toxicology, 2015, Vol 29 Suppl 1, 113.

Abstract Long-chain polyprenols and dolichols are synthesised in a mevalonate-dependent pathway, similar to cholesterol. Therefore, statins, widely used cholesterol-lowering drugs, may halt the production not only of cholesterol but also of polyprenols and dolichols, leading to statin-induced myopathies. We hypothesised that administration of polyprenols may prevent statin-mediated changes in muscle strength. Our data demonstrated that atorvastatin (80 mg/kg per os for 16 days) considerably decreased muscle strength in normolipidemic female Wistar rats, while these impairments were protected by polyprenols at 1, 10 and 20 mg/kg doses administered per os for 16 days. Neither atorvastatin nor polyprenols influenced plasma cholesterol levels; however, 20 mg/kg polyprenols elevated creatine kinase activity by approximately 25%. The obtained data suggest that polyprenols have usefulness as a complement to long-term statin therapy to reduce muscle-related side effects.

Keywords Atorvastatin  Muscle strength Polyprenols Rats

Introduction In eukaryotic cells, cholesterol as well as long-chain polyprenols and dolichols are synthesised in a common mevalonate-dependent pathway. Therefore, these substances are considered to be biogenetically related compounds with different molecular structures and functions (Buhaescu and Izzedine 2007; Surmacz and Swiezewska 2011). Polyprenols as linear polymers are identified in almost all living organisms and are also found in the human diet in fruits as well as beverages such as tea, coffee and wine. Conifer tree needles are one of the richest sources of polyprenols. Free polyisoprenoid alcohols and their fatty acid esters are structural components of cellular membranes and modulate their physico-chemical properties such as fluidity and permeability (Chojnacki and Dallner 1988; Wang et al. 2008). The majority of data on polyprenols and polyprenyl phosphates had been focused on their ability to prevent toxic liver injury and restore disturbed hepatic functions by lowering serum cholesterol levels through effects on its biosynthetic pathway (Fedotova et al. 2012; Pronin et al. 2014) as well as by protecting unsaturated membrane lipids from oxidative free radicals (Bizzarri et al. 2003).

The widely used statins, cholesterol-lowering drugs, which, by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, may halt production not only of cholesterol but also of polyprenols and dolichols. In this context, statin-induced myopathies can be explained by isoprenoid depletion and hence by impaired protein prenylation and coenzyme Q10 deficiency (Buhaescu and Izzedine 2007; Littlefield et al. 2014; Pronin et al. 2014). However, the overall incidence such as myalgia, muscle aches, or cramps in clinical practice varies from 0.3% to 33% (Bays 2006; Wilkinson et al. 2014).

We hypothesised that polyprenols may protect against statin-induced changes in muscles, because statin-induced dolichol depletion could be compensated by polyprenols that might avert a dolichyl phosphate cycle deficiency. The present study aimed to investigate the effects of polyprenols (isolated from Picea abies L. spruce needles) to evaluate their influence on muscle strength/tone, plasma cholesterol levels and creatine kinase activity in normolipidemic atorvastatin-treated rats.

Materials and methods Female Wistar rats weighing 230-245 g were obtained from the Laboratory of Experimental Animals, Riga Stradins University, Riga, Latvia. Animals were housed in plastic cages (5 per cage) with food and water ad libitum and kept in a controlled laboratory environment (temperature 22° C, humidity 50-60 %, 12 h light/dark cycle). Female rats were used because the female sex is considered to be a risk factor to obtain statin-induced adverse effects (Sathasivam and Lecky 2008). All efforts were made to minimise animal suffering and to reduce the number of animals used. Experiments were conducted in accordance with the EU Directive 2010/63/EU and local laws and policies on the protection of animals used for scientific purposes. Animal protocol was approved by the Animal Ethics Committee of Food and Veterinary Services, Riga, Latvia. Rats were randomly divided into 8 groups (n=9-10 per group).

Atorvastatin (Atoris) was purchased from KRKA, Slovenia. Commercially available purified polyprenols C55 - C95 (from Picea abies L. spruce needles) were purchased by JSC BioLat, Latvia. Blood cholesterol level measuring strips for the Accutrend GCT meter were purchased from Roche Diagnostics, Switzerland. Commercial Creatine Kinase Activity Assay kits (MAK116) were from Sigma-Aldrich, St. Louis, MO, USA.

Experimental animals were treated with polyprenols (at the doses of 1, 10 and 20 mg/kg) that were dissolved at 2 mL/kg in refined sunflower oil. Atorvastatin tablets were ground to powder, suspended in saline and administered at 80 mg/kg in a volume of 10 ml/kg. The drugs were administered once daily orally (per os) via orogastric cannula for 16 consecutive days. Control animals received saline (10 mL/kg per rat) and refined sunflower oil (2 mL/kg per rat). Treated groups received polyprenols and atorvastatin individually or their combination.

Assessment of muscle strength was performed in grip strength and wire hang tests, muscle tone in rotarod test. A rat grip strength meter (model 47105, Ugo Basile, Italy) was used to assess forelimb strength. On day 8, animals were pre-trained for six training trials to establish a reliable assessment of gripping ability, and on day 15, the grip strength test was performed. The grip strength meter automatically determined and recorded the maximum force displayed by each animal in grams. The mean value of five consecutive measurements for each animal was calculated. Rats were allowed to recover for 30 s between the measurements.

The wire hang apparatus consisted of a stainless steel wire (90 cm length, 3 mm in diameter) that was fixed horizontally between two vertical supports and 60 cm above a soft padded surface. On day 16, a wire hang test was performed. The rat was forced to grasp the central position of the wire with its forepaws. The latency (s) to fall from the wire on to a flat soft pad was measured. When the latency time was over 120 s, the rat was released from the wire, and the time was recorded as 120 s. The trial was conducted three times for each rat, and the longest duration was the value used for evaluation. A resting pause between the consecutive attempts was 3 min.

Locomotor coordination and tone were measured in rats on an accelerating rat rotarod apparatus (model 47700, Ugo Basile, Italy). A day before the beginning of the treatment, rats were pre-trained for five rotarod test trials. Each time the rat fell off the rotarod during the training trials, it was immediately placed back onto the treadmill to achieve 5 min stability. On day 16, the animals performed the rotarod task. The gradually accelerating rotor mode was used to increase the speed slowly from 4 to 40 rpm over a 5-min period. The trial ended when the rat fell from the rod or after 5 min, which was used as the maximal test time. An hour break was given between four consecutive trials. The time (as latency in seconds to fall) and the rotarod treadmill speed were automatically registered. The results are expressed as the mean values of four trials.

Assessment of cholesterol levels and creatine kinase activity was done at the end of the study, on day 17. Rats were anesthetised with ketamine (100 mg/kg) and xylazine (10 mg/kg), and blood for cholesterol level and creatine kinase (CK) activity measurement was collected by cardiac puncture. A few drops of the collected blood were used to measure the blood cholesterol levels using the Accutrend GCT meter (Roche Diagnostics, Switzerland) expressed in mM/L. For CK activity determination, the blood was collected into tubes containing heparin. The tubes were immediately centrifuged at 3000 rpm for 10 min and stored at - 80° C until the CK assay was performed. CK determination in rat blood plasma samples was performed by standard spectrophotometric analysis according to the manufacturer’s instructions (Sigma-Aldrich, USA) using commercial Creatine Kinase Activity Assay (MAK116) kits. The absorbance was read at 340 nm at 37° C using a spectrophotometer (INFINITE M200 PRO NanoQuant, Tecan Group Ltd., Switzerland). The data were calculated in units/L. One unit of CK activity was defined as the amount of enzyme that transfers 1.0 µmol phosphate from phosphocreatine to adenosine diphosphate (ADP) per min at pH 6.

GraphPad Prism 6 software (GraphPad Software Inc., USA) was used for the statistical analysis. All of the data are expressed as the means ± S.E.M. Differences among experimental groups were analysed by one-way ANOVA followed by Uncorrected Fisher’s LSD post-test for in vivo tests, and by Kruskal-Wallis followed by the Dunn’s Multiple Comparison test for the biochemical data. Significance was assumed at P<0.05.

Results In the grip strength test (Fig. 1A), administration of polyprenols at all tested doses did not have an effect on rat grasping strength compared with the control group. When treated with atorvastatin at 80 mg/kg, rats exhibited a reduction in grasping strength vs. control. Polyprenols administered at 20 mg/kg reversed the atorvastatin effect by increasing grasping strength to the control level. Rat hanging time (Fig. 1B) was not altered by polyprenol administration at 1 and 10 mg/kg doses, while 20 mg/kg prolonged hanging time by approximately 2-fold vs. the control group. Atorvastatin (80 mg/kg) significantly (by approximately 3-fold) decreased hanging time compared with the control group, indicating that atorvastatin reduced muscle strength. Polyprenols at all tested doses significantly prolonged the atorvastatin-induced reduction in rat hanging time, thereby restoring muscle strength to the control level. In an accelerating rotarod test (data not shown), polyprenols at all doses, atorvastatin at 80 mg/kg and concomitant polyprenol and atorvastatin administration did not influence rat falling latency.

Blood cholesterol levels were not changed in any tested group (data not shown). A significant increase (by approximately 25%) in plasma creatine kinase activity was observed after administration of polyprenols at 20 mg/kg (but not in 1 and 10 mg/kg) compared with the control group (Fig. 2). Creatine kinase activity after atorvastatin (80 mg/kg) treatment or concomitant polyprenol (20 mg/kg) administration was comparable to that of the control group.

Discussion Most reviewed data about polyprenols and their biological effects and mechanisms are related to their hepatoprotection due to cholesterol-lowering, antioxidant, and membrane-protecting properties(Cantagrel and Lefeber 2011; Hartley and Imperiali 2012; Pronin et al. 2014). Recent report (Milenkovic et al. 2013) demonstrated that polyprenols might interact with cellular signalling cascades to regulate transcription factor activity and gene expression, particularly by influencing microRNAs.

In the present study, we tested polyprenols at 1, 10 and 20 mg/kg per os for 16 days. Atorvastatin was chosen as the model compound. The mid-range atorvastatin dose of 80 mg/kg and the 16-day treatment regimen were selected from the literature (Madsen et al. 2008). We demonstrated that atorvastatin significantly decreased muscle strength in both tests (grip strength and wire hang test). In particular, a dramatic atorvastatin effect was observed in the wire hang test when muscle weakness was approximately 3-fold vs. control. Polyprenols at all tested doses significantly protected against atorvastatin-induced alterations by restoring muscle strength. The mechanisms underlying atorvastatin myopathies are mostly explained by its direct influence on the mevalonate pathway leading to the inhibition of cholesterol biosynthesis and endogenous polyisoprenoid production, resulting in reduced concentrations of metabolites that are necessary for cellular processes. Thus, the deficiency of such molecules as ubiquinone (provides mitochondrial electron transport), isoprenoid pyrophosphates (protein prenylation) and dolichol (protein glycosylation) is essential for the development of statin-induced myopathy (Baker 2005; Manoukian et al. 1990). Other data revealed that atorvastatin (10 mg/kg for 2 months in rats) might down-regulate the expression of proteins that are essential for skeletal muscle function (Camerino et al. 2011). Polyprenols at 20 mg/kg per se considerably increased rat hanging time in the wire hang test. Concurrently, neither atorvastatin nor polyprenols influenced muscle tone and coordination in the accelerating rotarod test, indicating that these substances have no central muscle relaxing effect.

Because our experiments were performed in normolipidemic rats, atorvastatin did not influence cholesterol levels, as it was predicted. Atorvastatin also did not alter creatine kinase activity. Cholesterol levels were also not influenced by polyprenols however, surprisingly, polyprenols (20 mg/kg) elevated (by approximately 25 %) creatine kinase activity. At this dose polyprenols showed the increased muscle strength.

Indeed, we cannot currently explain this phenomenon, particularly why creatine kinase activity remained at control values when treated with both polyprenols and atorvastatin (when polyprenols restored muscle tone in atorvastatin-treated animals). One speculation can be that by increasing creatine kinase activity, polyprenols may intensify intracellular energy transportation and ATP generation, leading to normalisation of atorvastatin induced impairment of energy processes.

In summary, one may conclude that polyprenols may act as successful protectors of atorvastatin-induced muscle weakness. These data suggest that the combination of polyprenols with atorvastatin may be helpful for reducing muscle-related side effects in patients receiving a long-term atorvastatin therapy.

Acknowledgements The study was financially supported by Pharma and Chemistry Competence Center of Latvia, Ltd. Grant No. L-KC-11-0001 with the co-financing of the European Regional Development Fund. Project P29: "The conifer isoprene alcohol biological activity studies in pathology models". Authors thank Elga Poppela and Jana Namniece for technical assistance in experiments.

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Ilona Vanaga, Ilva Nakurte, Ewa Swiezewska, Ausma Korica, Ugis Kletnieks, Ruta Muceniece

Pharma and Chemistry Competence Centre of Latvia, Ltd, Riga, Latvia

Faculty of Chemistry, University of Latvia, Riga, Latvia

Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland

Biolat JSC, Salaspils, Latvia

Faculty of Medicine, University of Latvia, Riga, Latvia

Background. Polyprenols (PPs) are natural long-chain isoprenoid alcohols of the general formula H-(C5H8)n-OH where n is the number of isoprene units. PPs play an important function acting as natural bioregulators and precursors of dolichols which are their 2,3-dihydro derivatives. Efforts to provide detailed insight into various dolichol- or polyprenol-dependent glycosylation events were hampered by challenges associated with either accumulating sufficient material for biochemical characterization or by difficulties associated with the structural elucidation of these molecular species. Today, liquid chromatography coupled with mass spectrometry offers a powerful analytic tool.

Aim of the study was to investigate structure of PPs isolated from Picea abies L. spruce needles.

Methods. PPs were extracted from the Norway spruce (Picea abies (L.) needles using different organic solvents with a consecutive distillation of the solvent. Molecular structure of PPs was determined by ultra performance liquid chromatography time of flight (UPLC-TOF) method and number of the isoprene units (n) and quantity of PPs were analyzed by using the standard PPs.

Results. The end product was chromatographically isolated with purity > 95%. Obtained spruce needle PPs consisted of a mixture of PP homologues with isoprene units number n=12 to 22, mainly from 13 to 19 (from C65H106O to C95H114O).

Conclusion. PPs extracts are of high purity and they are free of organic solvents. Spruce needles contain mainly medium-chain length PPs from C65 (Pren-13) to C95 (pren-19).

Acknowledgements. Financial support from Pharma and Chemistry Competence Centre of Latvia, Ltd. Grant No. L-KC-11-0001 with the co-financing of the European Regional Development Fund. Project P29: "The conifer isoprene alcohol biological activity studies in pathology models".

Ilona Vanaga^1,4, Baiba Jansone², Gustavs Latkovskis^3,3a,3b, Vita Saripo³, Vija Klusa², Ruta Muceniece², Kaspars Jekabsons², Karlis Kletnieks⁴

¹ Pharma and Chemistry Competence Centre of Latvia Ltd., Riga, Latvia

²Department of Pharmacology, Faculty of Medicine, University of Latvia, Riga, Latvia

³Pauls Stradins Clinical University Hospital, Riga, Latvia

^3aLatvian Research Institute of Cardiology, University of Latvia, Riga, Latvia

^3bChair of Internal Medicine, University of Latvia, Riga, Latvia

⁴Silv EXPO Ltd., Riga, Latvia

Background. PPs are natural long-chain isoprenoid alcohols, which play an important role as natural bio regulators and precursors of dolichols. PPs and dolichols are synthesised in a mevalonate-dependent pathway, similar to cholesterol. Statins - widely used cholesterol-lowering drugs, tend to halt the production not only of cholesterol but also of PPs and dolichols, which may lead to statin-induced myasthenia and myopathies.

Aim of the study was to investigate the effects of PPs (isolated from Picea abies L. spruce needles) on blood biochemical parameters in Wistar female rats per se and in combination with Atorvastatin after various performed locomotor tests, and also in model of statin-induced myopathies in humans

Materials & Methods. PPs were extracted from the Norway spruce (Picea abies (L.) needles and isolated by column chromatography with purity ≥ 95%. Female Wistar rats weighing 230-245 g were treated with PPs at the doses of 1, 10 and 20 mg/g, Atorvastatin 80 mg/kg and a combination of PPs and Atorvastatin for 16 days during which period various tests were performed (open field, grip, wire and rotarod. Figure «Rat experimental design»). Measurements of cholesterol levels and creatine kinase (CK) activity in Wistar female rats was done on day 17.

Human trial was an open-label, one-center prospective pilot study, which included 11 patients (5 women and 6 men) matching strict statin-induced myopathy criteria. All patients received supplementation of PPs (4mg/day) in combination with Coenzyme Q10 (100 mg/day) for 2 months. The study was carried out at the Latvian Centre of Cardiology between March 2014 and August 2015.

Results. In rats creatine kinase (CK) level (administrating 20 mg/kg of PPs) was elevated by approx. 25%. Because the rats were normolipidemic cholesterol levels were unchanged by the administration of either PPs or Atorvastatin. Neither PPs, nor Atorvastatin or combination thereof changed rat locomotor activity in open field. Atorvastatin induced 3 times shorter rat hanging time in grip and wire tests compared to the control group. PPs at 20 mg/kg improved hanging time twice compared to the control group.

In human trials no changes of ECG and dynamometry were observed at 2 months. None of the analysed biochemical parameters changed significantly in humans at 2 months with the exception of CK levels, which increased by 65%. PP and CoQ10 use in most patients (80%) with statin induced muscle symptoms, as reported by subjective expression, successfully alleviated the symptoms.

Discussion. Although exact mechanism is to be elucidated, our data suggest that PPs in higher doses elevate CK due to reinforcement of the intracellular transport and ATP production by promoting energy normalization process, worsened by statin activity.

Conclusions. The safety of PPs in this clinical setting seems acceptable, but needs further investigation in larger human trials. From all the data of our carried out studies, both in vivo and clinical, we conclude that PPs can be considered potentially beneficial for relief of statin-induced myasthenia or myopathy.

Acknowledgements. Financial support was from Pharma and Chemistry Competence Centre of Latvia, Ltd. grant No. L-KC-11-0001 with the co-financing of the European Regional Development Fund. Project P29: "The conifer isoprene alcohol biological activity studies in pathology models".