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hibited a linear kinetic process in both dosage regimens. Consequently, the oral bioavailability of 12% was determined by comparison of the dose-normalized AUC 0 –⬁ (189 g/ml 䡠 min, 100 mg/kg) of oral dose to the AUC 0 –⬁ (308 g/ml 䡠 min, 20 mg/kg) of iv injection of imperialine. This very low bioavailability suggested an extensive first-pass metabolism in oral administration of imperialine in rats. In summary, the developed novel HPLC–ELSD analytical method allowed us to successfully determine imperialine in the blood samples obtained from the dosed rats. This assay is simple and accurate and provides good reproducibility. The results of pharmacokinetic study demonstrated that imperialine is very rapidly absorbed orally and has a quick onset (15 min) and a short duration (t 1/ 2 of ⬃30 min) of action in rats. However, the low oral bioavailability may indicate a relative low therapeutic efficacy in oral ingestion of imperialine-containing herbal remedies. Further investigations into the correlation between antitussive activity and pharmacokinetic fate of imperialine and other active fritillaria alkaloids are currently under progress in our research team. Acknowledgment. This investigation was supported by the Research Grant Council (RGC) of Hong Kong for the Competitive Earmarked Grant (CUHK 4240/97M).
REFERENCES 1. Ministry of Public Health of the People’s Republic of China (1995) Pharmacopoeia of the People’s Republic of China, Beijing. 2. Li, S. L., Li P., Lin, G. Chan, S. W., and Ho, Y. P. (2000) J. Chromatogr. A 873, 221–228. 3. Xu, D. M., and Xu, Y. J. (1991) Chin. Traditional Herbal Drugs 8, 132–139. 4. Huang, K. C. (1999) The Pharmacology of Chinese Herbs, CRC Press, Boca Raton, FL. 5. Mir, G. N., and Ray Ghatak, B. J. (1965) Indian J. Exp. Biol. 3, 249 –252. 6. Chan, S. W., Kwan, Y. W., Lin, G., Ho, Y. P., and Li, P. (1998) PharmSci 1, S-369. 7. Li, S. L., Chan, S. W., Li, P., Lin, G., Zhou, G. H., Ren, Y. J., and Chiu, F. C. K. (1999) J. Chromatogr. A 859, 183–192. 8. Ding, K., Lin, G., Ho, Y, P., Cheng, T. Y., and Li, P. (1996) J. Pharm. Sci. 85, 1174 –1179. 9. Gunnarsson, T., Karlsson, A., Hansson, P., Johnson, G., Alling, C., and Odham, G. (1998) J. Chromatogr. B 705, 243–249. 10. Niemi, R., Taipale, H., Ahlmark, M., Vepsa¨la¨inen, J., and Ja¨rvinen, T. (1997) J. Chromatogr. B 701, 97–102. 11. Christie, W. W. (1985) J. Lipid Res. 26, 507–512. 12. Christie, W. W. (1986) J. Chromatogr. 361, 396 –399. 13. Christie, W. W. (1995) in Advances in Lipid Methodology (Christie, W. W., Ed.), Chap. 7, Vol. 1., pp. 239 –271, Oily Press, Ayr. 14. Hopia, A. I., and Ollilainen, J (1993) J. Liq. Chromatogr. 16, 2469 –2469.
A Double-Injection DNA Electroporation Protocol to Enhance in Vivo Gene Delivery in Skeletal Muscle Frank Hoover 1 and John Magne Kalhovde Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Norway Received June 2, 2000
Key Words: soleus; extensor digitorum longus; regeneration; gene transfer.
Skeletal muscle represents an easily accessible tissue that is amenable to transfection in vivo (1). Among the nonviral transfection methods, intramuscular injection of plasmid or “naked” DNA into muscle has received much attention since the first report by Wolff and colleagues (2). One advantage of this technique is that it provides stable epi-chromosomal expression over long periods of time (3, 4). Therapeutic application of this strategy already has shown promise in the preparation of DNA vaccines for use in livestock and humans (5, 6). However, despite optimization and modification, this method is hindered by a low level of transfection efficiency (about 1–2% of muscle fibers). Recently, direct intramuscular DNA injection in combination with electrical stimulation (in vivo electroporation) has resulted in increased numbers of transfected fibers and reporter gene activity (7–9). This method also directs long-term and stable transgene expression. One consequence of in vivo muscle electroporation is that regions of muscle become damaged and necrotic. This condition is temporary, since skeletal muscle has the ability to regenerate (10). Interestingly, intramuscular injection of plasmid DNA into myotoxin-induced regenerating rat skeletal muscle produces significantly higher levels of reporter gene expression compared to normal muscle (11). Here we report that a second dose of intramuscularly injected DNA into electroporated muscle will result in further increases in the number of muscle fibers transfected in vivo. This technique will provide a useful protocol to enhance transfection efficiency for gene expression studies and promoter analysis in vivo. 1
To whom correspondence should be addressed at University of Bergen, Center for Research in Virology, Bergen High Technology Center, P.O. Box 7800, 5020 Bergen, Norway. Fax: (47) 55 58 45 12. E-mail:
[email protected]. Analytical Biochemistry 285, 175–178 (2000) doi:10.1006/abio.2000.4730 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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NOTES & TIPS TABLE 1
Effects of Electrical Stimulation and Number of DNA Injections on Reporter Gene Activity in Innervated Soleus Muscle Method Direct single DNA injection Electroporation: single DNA injection Electroporation: double DNA injection
Mean ⫾ SD
n
Range
P value
39
4
21–109
n.d.
1144 ⫾ 743
6
102–1954
⬍0.03
5361 ⫾ 3001
6
2281–10747
to another region on the muscle prior to delivery of the next train. After 3 days, the muscles were exposed again and 50 g of plasmid DNA was injected intramuscularly. After a total of 10 days, muscles were removed and prepared for analyses. Results and Discussion
57 ⫾
Note. Adult rat solei were injected with 50 g of plasmid DNA encoding RSV luciferase. Following DNA injection, some animals were electrically stimulated. After 3 days, an additional 50 g dose of DNA was intramuscularly administered (electroporation: double DNA injection). Transfection efficiency was assessed in all sets 10 days later using a luciferase assay. Results are reported as the mean accompanied by the standard deviation. Abbreviations: SD, standard deviation; n, number of muscles; n.d., not determined.
Materials and Methods Animals and surgical procedures. Adult male Long Evans or Wistar rats (200 –300 g/body weight) were used in this study. Surgical operations were performed under Equithesin anesthesia (42.5 mg chloral hydrate and 9.7 mg pentobarbital in a 1-ml solution, 0.4 ml/100 g weight, ip). These experiments were approved under the guidelines established by the Norwegian Experimental Board and Ethical Committee for Animal Experiments. At the respective time point, animals were deeply anesthetized, and the soleus (SOL) 2 and extensor digitorum longus (EDL) muscles were removed. Following muscle extraction the animals were sacrificed by cervical dislocation. In vivo electroporation of skeletal muscle. The protocol used here was modified from that described by Mathiesen (9). Briefly, SOL or EDL muscles were exposed surgically and 50 g of plasmid DNA in physiological sterile saline (0.9% NaCl) was injected into the muscles. Following injection, an electrode (two uninsulated silver wires, 2.5 mm apart, each 1 cm long (diameter 0.6 mm)) was placed around the muscle. The stimulator (Haer 6 bp-as stimulator (Frederick Haer)) was used in the constant voltage mode. The output voltage was limited by the maximal current output (50 mA) and varied between ⫾25 and 45 V. The stimulation consisted of square wave pulses delivered in 10 –15 trains (with 2-s intervals) of 1000 pulses at 1000 Hz. Each pulse was bipolar and lasted 400 s. Following delivery of each train of pulses the electrode was moved 2 Abbreviations used: SOL, soleus; EDL, extensor digitorum longus; RSV, Rous sarcoma virus.
We transfected SOL with 50 g of plasmid DNA containing the RSV promoter (9) driving a luciferase reporter construct using established intramuscular DNA direct injection (two animals bilateral transfection, n ⫽ 4) and electroporation (six animals bilateral, n ⫽ 12) protocols (2, 9). After 3 days (d3) we intramuscularly injected an additional 50 g of luciferase DNA into the right SOL of each of the animals that had been electroporated (n ⫽ 6). Seven days later (d10), SOL were removed from all animals and luciferase assays were performed using a commercially available kit (Promega). These experiments were performed on the same days with the same preparation of DNA. The mean luciferase values presented in Table 1 show that both electroporation methods yield higher luciferase values compared to direct intramuscular injection. In
FIG. 1. A second injection of DNA following electroporation transfects muscle fibers. (A) Whole-mount view of a soleus muscle after 10 days following electroporation. On Day 0 (d0) the muscle had been electroporated and followed by intramuscular injection of a GFP DNA construct after 3 days. (B) Soleus and EDL muscles were exposed, injected with sterile physiological saline, and stimulated electrically. After 3 days muscles were reexposed and injected with 50 g of a RSV--galactosidase plasmid. Following 10 days, muscles were removed and analyzed. Whole-mount view showing -galactosidase staining in electroporation-induced regenerating muscles from solei (left) and EDL (right).
177
NOTES & TIPS TABLE 2
Comparison of Regeneration Method on Number of Transfected Fibers in Skeletal Muscle Method
Muscle
Mean ⫾ SD
n
Range
P value
Electroporation Electroporation Marcain
Soleus EDL Soleus
177 ⫾ 87 72 ⫾ 48 105 ⫾ 48
6 6 5
96–305 20–136 30–158
⬍0.02 n.d.
Note. Solei and EDL were stimulated electrically with 0.9% NaCl or induced to regenerate by Marcain injection. After 3 days, 50 g of -galactosidase DNA was intramuscularly injected into the regenerating muscles. After 10 days, muscles were removed and processed for Lac-Z histochemistry, and the number of transfected blue fibers was counted. Results are reported as the mean accompanied by the standard deviation. Abbreviations as in Table 1.
addition, the additional injection of DNA 3 days after electroporation resulted in a significant increase in luciferase reporter gene activity compared to electroporation alone (n ⫽ 6, P ⬍ 0.03, Wilcoxin signed-rank test for nonparametric paired comparisons). This increase in reporter gene activity was reproducible and occurred in every instance. These results suggested that the second delivery of DNA increased the transfection efficiency fibers of skeletal muscle in vivo. We posited that the increase in reporter gene activity was due to newly transfected fibers resulting from the electroporation-induced damage. To investigate this possibility and quantify this effect, we electroporated SOL and EDL with sterile physiological saline (no plasmid DNA). After 3 days, muscles were reexposed and injected with 50 g of a plasmid containing the Rous sarcoma virus (RSV) promoter driving the expression of -galactosidase (12). Seven days later (d10), muscles were removed, processed for -galactosidase activity (13), and analyzed for positive fibers. We observed many positive fibers in each muscle (Fig. 1), supporting the notion that additional fibers were being transfected with the second dose of DNA following electroporation. Furthermore, we have observed reporter gene activity resulting from the second injection for periods greater than 1 month. In addition, we determined that under the conditions described here, SOL muscles contained a significantly larger number of transfected myofibers compared to the EDL (n ⫽ 6, P ⬍ 0.02, Wilcoxin signed-rank statistical test). The reasons for this difference are unknown, but it is suggested that different muscles may require different parameters leading to efficient and high levels of transfection with the electroporation method. The mean number of transfected fibers observed using the electroporation-induced regeneration (177) appeared to be greater than that obtained with the myotoxin Marcain (64, see Vitadello et al. (11)). To test this, we injected Marcain into SOL as described and after 3 days we injected 50 g of plasmid DNA encoding the -galactosidase gene. After 10 days, we removed muscles, processed them for the -galactosidase reaction, and after inspection, calculated a mean of 105 positive blue fibers (Table 2). This indicated that the electroporation-induced regen-
erating gene transfer method transfected more muscle fibers than the Marcain method. The application of electrical fields to enhance eukaryotic gene delivery in vivo is in its infancy. Recently, several independent parametric studies have applied electroporation technology to skeletal muscle in vivo and report high and sustainable levels of reporter gene activity (7–9). We have modified the electroporation protocol by adding an additional injection of DNA 3 days following electroporation. Taken together we interpret our results to suggest that two transfections can occur as a consequence of electroporation. The first transfection occurs immediately following electroporation (9), while the second transfection occurs in regenerating muscle damaged by the electroporation. Although the magnitude of the additional reporter gene activity is variable, it was always higher than the controls, suggesting that more fibers were indeed transfected. The utility of this technique will likely be beneficial for the study of weak cis-acting elements in vivo, DNA vaccine preparation, and the overexpression of secreted proteins in transfected muscle in vivo (14). In summary, this electroporation technique applied to skeletal muscle in vivo can greatly aid gene expression paradigms for both basic and applied biological research. Acknowledgments. We are grateful to Drs. Iacob Mathiesen and Terje Lømo for constructive suggestions during the preparation of this work and for their comments on previous versions of the manuscript. This work was conducted at the University of Oslo in the laboratories of Dr. Terje Lømo and supported by the Sophie and Leif Torp Fund (Frank Hoover) and a European Union Biotechnology grant (BIO4 CT96 0216, Terje Lømo).
REFERENCES 1. Blau, H. M., and Springer, M. L. (1995) Muscle-mediated gene therapy. N. Engl. J. Med. 333, 1554 –1556. 2. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Ascardi, G., Jani, A., and Fengler, P. L. (1990) Direct gene transfer into mouse muscle in vivo. Science 247, 1465–1468. 3. Wolff, J. A. (1997) Naked DNA transport and expression in mammalian cells. Neuromusc. Disord. 7, 314 –318. 4. Wolff, J. A., Ludtke, J. J., Acsadi, G., Williams, P., and Jani, A.
178
5. 6.
7. 8.
9.
NOTES & TIPS
(1992) Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum. Mol. Genet. 1, 363–369. Beard, C. W., and Mason, P. W. (1998) Out on the farm with DNA vaccines. Nature Biotechnol. 16, 1325–1328. Wang, R., Doolan, D. L., Le, T. P., Hedstrom, R. C., Coonan, K. M., Charoenvit, Y., Jones, T. R., Hobart, P., Margalith, M., Ng, J., Weiss, W. R., Sedegah, M., de Taisne, C., Norman, J. A., and Hoffman, S. L. (1998) Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science 282, 476 – 480. Aihara, H., and Miyazaki, J. (1998) Gene transfer into muscle by electroporation in vivo. Nature Biotechnol. 16, 867– 870. Mir, L. M., Bureau, M. F., Rangara, R., Schwartz, B., and Scherman, D. (1998) Long-term, high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle. C. R. Acad. Sci. III 321, 893– 899. Mathiesen, I. (1999) Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther. 6, 508 –514.
10. Bischoff, R. (1994) in Myology (Engle, A. G., and Franzini-Armstrong, C., Eds.), pp. 97–118, McGraw-Hill, New York. 11. Vitadello, M., Schiaffino, M. V., Picard, A., Scarpa, M., and Schiaffino, S. (1994) Gene transfer in regenerating muscle. Hum. Gene Ther. 5, 11–18. 12. Kisselev, O., Pronin, A., Ermolaeva, M., and Gautam, N. (1995) Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex. Proc. Natl. Acad. Sci. USA 92, 9102–9106. 13. Sanes, J. R., Rubenstein, J. L., and Nicolas, J. F. (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J. 5, 3133–3142. 14. Rizzuto, G., Cappelletti, M., Maione, D., Savino, R., Lazzaro, D., Costa, P., Mathiesen, I., Cortese, R., Ciliberto, G., Laufer, R., La Monica, N., and Fattori, E. (1999) Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc. Natl. Acad. Sci. USA 96, 6417– 6422.
