2020-03-16 10:00:46
Minghui Wang， Ao Niua, Zhibo Gonga, Zhengwen Xua, Lili Lia, Bo Lia, Jinxin Wang
Over 85% of strokes are ischemic which occurs when a blood vessel in the neck or brain is blocked. The ischemic penumbra is the area surrounding the ischemic core, neuron cells suffer from moderate ischemia and dysfunction but not death, which is potentially reversible when reperfusion happens. Therefore, neuroprotective agent that promote neuron survive and stabilize penumbra is a promising strategy for ischemic stroke therapy.
Danshen is a widely used Chinese herbal medicine in treating vascular diseases such as hypertension and ischemic stroke.2 Tanshinone II-A is the major lipophilic pharmacological constituent of Danshen. The neuroprotective efficacy of tanshinone II A in stroke has been confirmed in both transient and permanent focal cerebral ischemia mice models.3–4 However, the high lipophilicity and extreme aqueous insolubility of tanshinone IIA limit its further application.
To explore the potential clinical application of tanshinone IIA, our initial thought was to modify tanshinone IIA to improve the water solubility and drug release profile. Based on the tanshinone IIA structure-activity relationship and chemical structure modification position, we chose Przewaquinone A, which is also one of the most important ingredients of Danshen and has neuroprotective effects (Fig. 1). The only difference between Przewaquinone A and tanshinone II A is a methoxy group in position 17. As with tanshinone II-A, the water solubility of przewaquinone A is also unsatisfying.
Polyethylene glycol (PEG), which has been widely used to improve the pharmacokinetic behavior of drugs in prodrug design, is usually applied as an effective way for improving the pharmacokinetics of natural products with biological activities.6–14 Most importantly, PEG has been proven safe and approved by the FDA for systemic human use.15
Firstly, we conjugated przewaquinone A with PEG with different spacers, including L-glycine (L-Gly), L-phenylalanine (L-Phe), L-alanine (L-Ala) and two tripeptides (Gly-Glu-Gly, Gly-Asp-Gly). The preparation of the conjugates was started from the introduction of carboxyl to both terminals of PEG through a method introduced by Bersani et al.16 (Scheme 1). Meanwhile, tanshinone IIA was oxidized to obtain the przewaquinone A (Scheme 2).
We chose amino acids and tripeptides as spacers because of their relative appropriate drug release velocity.17 Furthermore, amino acids could contribute to the cell penetrability of nature compounds because PepT1.18 3a–5d have two przewaquinone A (2) molecules and three different spacers respectively. Compounds 6a–7d involved two different tripeptides to further increase the przewaquinone A ratio in the conjugates (Scheme 3.).
All final conjugates were characterized by FT-IR. For instance, the typical FT-IR spectra of carboxylic-PEG 2000 (1a) and przewaquinone A-Phe-PEG 2000 conjugate (5a) were shown in Fig. 2. In the spectrum of PEG 2000, the broad bands located at 3482 cm−1 and 3126 cm−1 are, respectively, due to the adsorption of OeH stretching vibrations of
hydroxyl ends on carboxylic-PEG. Several new IR signals, like the absorption bands at 1737 cm−1 and 1627 cm−1, attributed to the carboxyl groups of 5a (Fig. 2). 3a and 4b were also identified by MALDI-Tof. The theoretical average weights of 3a and 4b are 2698.8 and 4726.8, which are very close to the MALDI-Tof results.
Water solubility of these prodrugs enhanced remarkably by introducing PEG into przewaquinone A (averagely increased more than 107 times) (Table 1). There was no large difference between conjugates with different PEG molecular weights with the same amino acid linker. Although higher molecular weight of PEG means more hydrogen- bonding acceptors, its lower drug loading capability may lead to the UV value lower, which was applied to calculate solubility.
Drug loading capability shows no big difference for 3a–5d. However, 6a–6d and 7a–7d have significantly higher drug payload. That could be easily explained by the strategy of using amino acid with two carboxy acids group to load more 2. In addition, drug loading ability decreased as the increase of PEG molecular weight in each group, which could be explained by two reasons. Firstly, terminal concentration would decrease if the molecular weight of the PEG increases. That will decrease the conjugate rate of PEG and przewaquinone A. Secondly, basing on the calculation formula, which is przewaquinone A% = (mprzewaquinone A/prodrug) × 100%, larger polymeric molecular weight leading to lower theoretical przewaquinone A ratio was reasonable.
In vitro hydrolysis assay in human plasma of these polymers was carried out to evaluate the drug release behavior of PEGylated przewaquinone A by incubating in human plasma at 37 °C. 3a, 3d, 4a, 4d, 5a, 5d and 7d were selected to compare the influence of molecular weight of PEG and amino acid spacers on drug release properties.
The results were shown in Fig. 4. Firstly, for 3a, 3d, and 4a, 4d, conjugates with higher molecular weight have a slower drug releasing rate, which can also be observed for other polymer conjugates.19 It may because the actual molar of primary drug would decrease if the molecular weight of PEG increases. Hydrolyzation hindrance in these polymers also decreased due to the steric hindrance.17 For example, glycine connected conjugate (3a) has higher releasing rate than phenylalanine connected conjugate (5a). Moreover, types of hydrolytic enzyme in human plasma may also has great influence on the releasing properties of conjugates. That could explain why some spacers with larger hin- drance still had higher releasing rates.
Then, we chose the prodrug 3a and przewaquinone A to examine the in vivo release behavior of our prodrugs. After injection, concentration of przewaquinone A and the 3 was monitored by HPLC. However, only compound 3 was detected in plasma after prodrug 3a administered intravenously (Fig. 5a). This means that the in vivo amide bond of prodrug 3a was much more easily to be broken than ester bond (Fig. 6). Przewaquinone A (2) was detected after 5 mins (Fig. 5b). 3a and 2 reached to their highest concentration in nearly 240 mins and 5 mins respectively, suggesting that 3a was released from prodrug in a sustained manner over a long period of time without a burst, which verified the effectiveness of our prodrug strategy.
To further confirm the effectiveness of PEG conjugates on protecting neuron in vivo, a murine model of focal cerebral ischemia was induced by intra-luminal transient middle cerebral artery occlusion (tMCAO). Compound 3a was administered through caudal vein after reperfusion at doses of 4, 8 and 16 mg/kg daily (QD) for 3 days. Gigaton (15 mg/kg) was selected as the positive control. The neurological deficit was evaluated using Bederson's score.20–21 As a result, the application of compound 3a (16 mg/kg) led to a significant reduction of the observed neurological deficit after 72 h compared to the model group as well as the ignition group (Fig. 8B). Consistent with the improved neurological function, there was a 31.4% reduction in the infarct volume in rats treated with compound 3a (16 mg/kg) compared to the model, and significantly less than the gigaton group (Fig. 8C). Meanwhile, the water content of 3a (16 mg/kg) decreased significantly compared to the model and positive control (Fig. 8D). Overall, the neuroprotective activity of 3a is stronger than that of ignition, indicating 3a has potential for further development as a neuroprotective drug.
In summary, we designed and synthesized a series of novel PEG- amino acid-przewaquinone conjugations. A based on the prodrug strategy. In vivo drug release assay confirmed that 3a could release active przewaquinone A in a sustained manner after intravenous injection, which proved that PEG prodrug might be an effective way to extend the half-life of some potent natural products. Meanwhile, compound 3a was efficacious in a mice tMCAO model, indicating that 3a could be a promising candidate for neuroprotective drugs. These data greatly proved that the PEG conjugation strategy is a quite effective way to improve the pharmacokinetics even the in vivo activity of many natural products. We believe that this meaningful and potential strategy could bring light on resolving the related important problems of numerous valuable natural products on clinical use in the future.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.
Acknowledgements
This work was supported financially by the Key Research & Development Program in Jiangsu [NO. BE2015683]; the Introduction Program of Leading Scientific and Technological Entrepreneurship in Nanjing [NO. 2013B14007].
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