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Sustained reduction of portal pressure by administration of octreotide-LAR in portal hypertensive rats
Hepatology Research 19 (2001) 108 – 119 www.elsevier.com/locate/ihepcom
Sustained reduction of portal pressure by administration of octreotide-LAR in portal hypertensive rats Katerina Kotzampassi a, Dimitrios Koufogiannis b, Efthimios Eleftheriadis a,* b
a Departments of Surgery, Uni6ersity of Thessaloniki, Thessaloniki, Greece Department of Medical Physics, Uni6ersity of Thessaloniki, Thessaloniki GR-546 55, Greece
Received 6 January 2000; received in revised form 27 April 2000; accepted 18 May 2000
Abstract Objecti6e: Recent studies have demonstrated octreotide as being potentially beneficial for the long-term management of cirrhotic portal hypertension. However, its short-term effects, requiring subcutaneous administration at least twice daily, make it inconvenient, practically, for long-term therapy. Hence, the current study was undertaken to evaluate the effects of the new long-acting-release octreotide formulation (octreotide-LAR) on portal pressure in a prehepatic portal hypertensive rat model. Methods: 14 days after portal vein stenosis or sham operation, rats were treated with either octreotide-LAR (0.25 mg/kg body weight, subcutaneously) or vehicle, after the baseline portal pressure measurements were obtained in each group. On days 10, 15, 18, 21, 24, 27 and 30 post-treatment, eight rats from each sub-group (portal vein stenosis plus octreotide-LAR, or vehicle, sham-operated plus octreotide-LAR or vehicle) were subjected to portal vein pressure measurement. Results: portal vein-stenosed rats exhibited a significantly higher portal vein pressure than sham-operated rats (13.82 9 2.18 vs. 5.829 1.08 mmHg, P= 0.0001). Octreotide-LAR treatment was found to significantly (P =0.001) reduce portal pressure in portal vein stenosed rats from the tenth post-injection day and thereafter to sustain reduction. Conclusion: the long-acting-release octreotide given in a single dose in a prehepatic portal hypertensive rat model diminishes and sustains diminished portal pressure for 20 days after the tenth post-injection day. These
Abbre6iations: PVS, portal vein stenosis. * Corresponding author. Fax: + 30-31-993496. E-mail address: [email protected] (E. Eleftheriadis). 1386-6346/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 0 ) 0 0 1 0 0 - 5
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findings seem promising for the portal hypertensive patient and further clinical studies are necessary. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Portal hypertension; Portal pressure; Octreotide; Octreotide-LAR; Experimental; Portal vein stenosis
1. Introduction Octreotide is a synthetic long-acting analogue of somatostatin, that has been found to have potential beneficial effects in the management of acute hemorrhage from esophageal varices [1,2] with no major side effects [3]. Acute experimental studies in animal models of portal hypertension have shown that single octreotide administration results in a dose-dependent decrease in portal pressure and portal vein tributary blood flow, associated with an increase in splanchnic vascular resistance, indicating vasoconstrictive action of octreotide [4–7]. Similar findings were reported after short-term administration of octreotide in portal hypertensive cirrhotic patients; hepatic and azygos blood flow were markedly decreased with minimal effects on systemic hemodynamics [8–11]. These promising effects have directed experimental research towards both the detailed mechanisms of its action in portal hypertension and its effects after long-term administration in portal hypertensive rats [12–16]. However, the necessity for repeated subcutaneous injections (two to three times daily) over a long period has made experimentation difficult. On the other hand, the need for an alternative or adjuvant to endoscopic therapy after variceal bleeding to reduce recurrent hemorrhage led to two randomized trials of octreotide for long-term management of cirrhosis, with encouraging results [17– 19]. However, the multiple daily injection dosage remains inconvenient for the patient and has the danger of patient’s non-compliance to the therapy. The long-acting-release formulation-octreotide-LAR-consisting of micro-spheres of poly-DL-lactide-co-glycolide-glucose containing octreotide has been developed to improve convenience, compliance and quality of life for the patient, by providing the same therapeutic advantages with less inconvenience and discomfort, since dosing is recommended once every 4 weeks [20,21]. Thus, the present experimental study was undertaken to evaluate the long-actingrelease octreotide treatment on portal pressure reduction in a prehepatic portal hypertensive rat model. 2. Material and method
2.1. Animals Two hundred and forty adult male Wistar rats weighing between 250 and 300 g were used in the study. The animals were housed together at a room temperature of 22 – 24°Celsius, with a 12-h light–dark cycle and were given free access to tap
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water and standard pellet rat diet until 16 h before the time of the hemodynamic study. The experimental protocol used was approved by the Governmental Animal Protection Committee and adhered to the European Community Guiding Principles for the care and use of animals.
2.2. Animal preparation Rats were divided into two groups; the portal vein stenosis group (PVS) and the sham-operated group (sham). Portal hypertension was induced according to the commonly used method. In brief, with rats under ether anesthesia, after a midline laparotomy, the portal vein was exposed free from surrounding fatty tissue and subjected to ligation (using 3 – 0 silk) together with a 20 gauge blunt-tipped needle lying alongside; subsequent removal of the needle yielded a calibrated stenosis of the portal vein. In sham-operated rats, the portal vein was exposed and isolated but no stenosis was performed.
2.3. Experimental protocol Fourteen days after portal vein stenosis the rats were ready for the experiment, since the hyperdynamic circulation accompanying portal hypertension was fully established [22]. At this time, under ether anesthesia, the initial (baseline) portal pressure, as well as mean arterial pressure measurements were performed in eight PVS rats and eight sham rats. Immediately thereafter, the remaining 112 PVS and the 112 sham-operated rats were randomly assigned to receive a single subcutaneous injection (0.25 mg/kg body weight) of the long-acting-release formulation of octreotide (octreotideLAR, Novartis, Hellas) or equal volume of vehicle, thus, four sub-groups were formed, i.e. PVS+LAR, PVS+ vehicle, sham+ LAR, sham+ vehicle. Portal vein pressure was then measured on the tenth post-injection day and on the 15th, 18th, 21st, 24th, 27th and 30th day in eight rats from each of the four sub-groups, every time. Additionally mean arterial pressure was evaluated in the 15th and the 30th day-groups.
2.4. Portal 6ein pressure measurements After ether anesthesia, the portal vein was exposed and pressure was measured by means of a direct puncture of the vein using a 23-G needle, under a constant perfusion of sterile sodium chloride 0.9% at a volume of 0.03 ml/min by the use of a hydraulic flow regulator (Infu 362 Datex, Swiss, Uhwiesen) according to the method described by Arndorfer et al. [23] and earlier applied by us for intravariceal pressure measurements [24]. This high-pressure low-compliance perfusion system was connected via an Elcomatic EM 750 (Harvard Apparatus, Ltd., Kent, UK) pressure transducer to a Palmer BioScience A100 (Sheerness, Kent, UK) amplifier, the pressure values registered by a chart recorder (Kipp Zonen BD11 model, Delft, Holland) on a
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paper running at 1 mm/s. The system was calibrated for pressure readings between 0 and 50 cm H2O, which was then converted to mmHg.
2.5. Statistical analysis All data, per treatment group and per study period were expressed as mean9 S.D. in mmHg. Analysis of variance (ANOVA) was then used for the statistical evaluation of the difference between treatment groups, as well as between different study periods within the same group of treatment. A P value less than 0.05 was considered as significant.
3. Results Portal vein-stenosed rats demonstrated a marked increase in portal pressure compared with that of sham-operated rats (13.829 2.18 vs. 5.829 1.08 mmHg, P=0.0001). Octreotide-LAR treatment, given after this baseline measurement, led to a highly significant decrease of portal pressure in PVS rats compared with the portal pressure of vehicle-treated rats (PVS+ vehicle group). This reduction was found to be in progress on the tenth post-injection day and stabilized after day 15. Octreotide-LAR treatment, given to sham-operated rats exhibited no effect on portal pressure at all (Table 1Fig. 1). Mean arterial pressure was found not to be significantly decreased in PVS versus sham operated rats (111 94 vs. 1229 3 mmHg). Octreotide-LAR led to an increase of MAP (measured on days 15 and 30) in PVS + LAR group from 111 9 4 to 119 94.5 mmHg compared with that of PVS + vehicle group, exhibiting no change. Octreotide-LAR or vehicle given to sham operated rats have no effect on MAP (1229 3 mmHg) at all. Table 1 Portal pressure measurementsa Day
PVS+LAR
PVS+vehicle
Sham+LAR
Sham+vehicle
10 15 18 21 24 27 30
0 (baseline) 10.9592.43c 8.649 1.07c 8.579 1.28c 8.52 91.20c 8.41 9 1.12c 8.209 1.10c 8.08 90.79c
13.829 2.18* 13.1692.15 12.49 9 1.88 12.02 91.48 12.049 1.71 11.66 9137 11.50 91.57 11.50 9 1.17
5.20 9 1.29 6.04 9 .0.81 5.69 9 1.18 6.07 9 1.17 5.87 9 1.07 5.69 9 1.06 5.56 9 1.03
5.82 9 1.08 6.07 9 1.17 5.70 9 1.20 5.82 9 1.21 5.57 9 1.12 6.07 9 1.17 5.70 9 0.93 5.709 1.20
a
Mean9 S.D. of portal pressure values throughout the 30 days period. Baseline measurements were performed before octreotide-LAR or vehicle treatment, thus portal pressure values are similar in LARand vehicle- treated groups, subjected to PLV or sham-operation. *, Represents significant difference (P=0.0001) in relation to sham-operated (baseline); c , represents significant difference (P= 0.001) in relation to vehicle-treated, within the same study-period.
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Fig. 1. Portal pressure measurements throughout the study-period. Closed squares represent PVS +vehicle group, open squares PVS +LAR group. Closed circles represent sham+ vehicle group, open circles sham + LAR group.
4. Discussion Bleeding from esophageal varices is the most common complication during the course of liver cirrhosis and is often the terminal event or an important contributory factor leading to the death of the patient. Thus, the need for prophylaxis to prevent variceal bleeding both in patients who have never bled and in those with a history of bleeding is pressing in the case of portal hypertension, as in few other clinical situations in medicine. Despite the central role of endoscopic techniques in controling acute variceal hemorrhage and reducing recurrent bleeding, underlying portal hypertension remains unaltered, accounting for a continued risk of bleeding even after variceal endoscopic eradication [25,26]. Consequently, there is an augmented interest in the use of vasoactive drugs as an alternative or as an adjuvant to endoscopic treatment. Similarly, in order to prevent the first bleeding episode vasoactive therapy seems to be an attractive proposition, since the benefit from prophylactic treatment appears to be too low to justify invasive therapeutic measures [27]. At this time, as the usefulness of b-blockers is limited by the adverse effects and recommendation of nitrates cannot be made unreservedly at this time [26–28] the most discussed agent of this category seems to be the synthetic analogue of somatostatin, octreotide. Given subcutaneously it results in a sustained reduction of portal pressure and azygos blood flow [8–10,29], by causing splanchnic arteriolar constriction [30] and inhibition of the vasoactive peptides contributing to the hyperdynamic circulatory syndrome of portal hypertension [31], without significant side-effects [25]. Several studies in humans and experimental animal models have evaluated the effect of octreotide on systemic hemodynamics during conditions with portal hypertension as a result of portal vein stenosis or cirrhotic liver disease [34,12,15,16]. These studies indicate that the peripheral vasodilation associated with portal hypertension is reversed by octreotide treatment, in the absence of changes in mean arterial pressure [10,11,15,16]. The mechanism responsible for this effect of octreotide acts through modulation of the release of vasoactive peptides from the
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gastrointestinal system during conditions with portal hypertension [12,16] although other data obtained from a common bile duct ligation rat model do not support that octreotide affects fasting plasma concentrations of glucagon and insulin [14]. Additionally, prolonged octreotide administration appears to involve little tachyphylaxis of its vasoconstrictive effect on the splanchnic circulation, so that the reduction in portal pressure elicited by the analogue is sustained; these advantages suggest octreotide to be potentially beneficial for the long-term management of cirrhotic portal hypertension patients, and this, indeed, appears to be the case [17– 19]. In a well-designed study [17], 32 cirrhotics, after cessation of their variceal bleeding episode, were randomly assigned to receive injection sclerotherapy or injection sclerotherapy plus octreotide (50 mg sc. bd). After 6 months of the combined treatment there was a significant reduction of the bleeding recurrence, and the number of elective injections required to obliterate varices, while survival was significantly improved. Furthermore, the combined treatment resulted in a sustained decrease in portal pressure, and improvement in liver function. Similarly, in a second study [18,19] 262 cirrhotic patients, after acute variceal bleeding, were randomized to receive octreotide 100 mg sc three times a day for 15 days or placebo in combination with b-blockers or sclerotherapy or both. Patients treated with octreotide showed a significant reduction in rebleeding episodes, blood transfusions and days in hospital. However, its short-term effects, requiring subcutaneous administration at least twice daily, make it inconvenient for long-term therapy in the majority of patients, although the recent development of a long-acting slow-release preparation (octreotide-LAR) is likely to make the therapy particularly desirable. A single subcutaneous injection of this preparation, when compared with the normal formulation of octreotide, results in stable plasma concentrations of octreotide for more than 4 weeks, as was found in human studies of its therapeutic use in the management of acromegaly [20,32–34] and in malignant carcinoid syndrome [21], the result of which is to limit the administration of the compound to once monthly. Therefore, the current study was designed to evaluate the effects of this new formulation of octreotide on portal hemodynamics in rats subjected to portal vein stenosis. Octreotide-LAR administered in a single dose of 0.25 mg/kg body weight was found to be significantly effective in decreasing portal vein pressure in the well-established model of portal vein stenosis. A comparable portal vein pressure reduction was also stated by Lin et al. [12] who use multiple daily subcutaneous injections of octreotide in the same rat model for 14 consecutive days. Thus octreotide-LAR seems to exhibit all the positive characteristics of the short-acting subcutaneous formulation and has the added advantage of slow drug release, which occurs by the cleavage of the polymer ester linkage to octreotide, primarily through tissue fluid hydrolysis [21]. However, there were important differences between our present and earlier studies. First of all, our study is the first, to our knowledge, in which octreotideLAR was tested to evaluate whether this new formula works towards reduction in portal hypertension in a continuous manner. For this purpose portal vein pressure
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was assessed every 3 days from day 10 up to day 30 post-injection; day 10 was selected to test the statement of other studies that this time-period is required to reach steady-state octreotide levels, as the release of the drug from the microspheres is slow, so the drug would not achieve its maximum efficacy in portal vein pressure reduction earlier, as is the case with growth hormones [20,32–34]. This finding was deduced in our study from the high S.D. values of portal vein pressure on day 10. Second, in this study a dose of 0.25 mg/kg body weight was used in rats, which is the equivalent of the human dose of 20 mg; in all the earlier experimental studies, whatever the purpose and the indications for its use, it was tested at doses up to 40 times higher [14,35]. In a recent experimental study, octreotide-LAR was given at a high dosage of 10 mg/kg body weight to test its effect on renal hemodynamics in a common bile duct ligation rat model, octreotide plasma levels 4 weeks later were found to be as high as 5809 90 and 20909 340 ng/l in controls and cirrhotics, respectively [14]. Although in our study no octreotide plasma levels were evaluated, its effect on portal pressure support the dose efficacy. Finally, the third point to be noted is that in our experiment octreotide-LAR was evaluated for long-term use on a more realistic rat model in which portal hypertension and the consequent hyperdynamic circulation were well established [22]. In the majority of earlier studies dealing with octreotide (or octreotide-LAR), it was injected immediately after portal vein ligation, i.e. just after acute portal hypertension induction [13,14], where neither systemic vasodilation nor liver injury had yet been established. The findings of the present study allow us to conclude that long-acting-release octreotide given in a single dose in a prehepatic portal hypertensive rat model sustains reduced portal pressure for 20 days after the tenth post-injection day. Although these findings seem very promising, as to whether octreotide-LAR would work as a valuable therapeutic option for the portal hypertensive patient, the results of the ongoing long-term clinical studies will provide the further necessary knowledge of the drug.
References [1] Jenkins SA, Shields R, Davies M, et al. A multicentre randomised trial comparing octreotide and injection sclerotherapy in the management and outcome of acute variceal haemorrhage. Gut 1997;41:526–33. [2] Sung JJ, Chung SC, Lai CW, et al. Octreotide infusion or emergency sclerotherapy for variceal haemorrhage. Lancet 1993;342(8872):637– 41. [3] McCormick PA, Chin J, Greenslade L, et al. Cardiovascular effects of octreotide in patients with hepatic cirrhosis. Hepatology 1995;21:1255 – 60. [4] Cerini R, Lee SS, Hadengue A, Koshy A, Girod C, Lebrec D. Circulatory effects of somatostatin analogue in two conscious rat models of portal hypertension. Gastroenterology 1988;94:703 – 8. [5] Pizcueta MP, Garcia-Pagan JC, Fernandez M, Casamitjana R, Bosch J, Rodes J. Glucagon hinders the effects of somatostatin on portal hypertension. A study in rats with partial portal vein ligation. Gastroenterology 1991;101:1710–5. [6] Jenkins SA, Baxter JN, Corbett WA, Shields R. The effects of a somatostatin analogue SMS 201-995 on hepatic haemodynamics in the cirrhotic rat. Br J Surg 1985;72:864 – 7.
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[7] Lebrec D. Animal models of portal hypertension. In: Okudsa K, Benhamou JP, editors. Portal Hypertension: Clinical and Physiological Aspects. Tokyo: Springer, 1991:101 – 13. [8] Jenkins SA, Nott DM, Baxter JN. Pharmacokinetics of octreotide in patients with cirrhosis and portal hypertension; relationship between the plasma levels of the analogue and the magnitude and duration of the reduction in corrected wedged hepatic venous pressure. HPB Surg 1998;11:13 – 21. [9] Zironi G, Rossi C, Siringo S, et al. Short- and long-term hemodynamic response to octreotide in portal hypertensive patients: a double-blind, controlled study. Liver 1996;16:225 – 34. [10] Eriksson LS, Brundin T, Soderlund C, Wahren J. Haemodynamic effects of a long-acting somatostatin analogue in patients with liver cirrhosis. Scand J Gastroenterol 1987;22:919 – 25. [11] Pringle SD, McKee RF, Garden OJ, Lorimer AR, Carter DC. The effect of a long-acting somatostatin analogue on portal and systemic haemodynamics in cirrhosis. Aliment Pharmacol Ther 1988;2:451–9. [12] Lin HC, Yang MC, Hou MC, et al. Effects of long-term administration of octreotide in portal vein-stenosed rats. Hepatology 1996;23:537 – 43. [13] Fort J, Oberti F, Pilette C, et al. Antifibrotic and hemodynamic effects of the early and chronic administration of octreotide in two models of liver fibrosis in rats. Hepatology 1998;28:1525 – 31. [14] Jonassen TE, Christensen S, Sorensen AM, et al. Effects of chronic octreotide treatment on renal changes during cirrhosis in rats. Hepatology 1999;29:1387 – 95. [15] Wang SS, Lee FY, Wu SL, et al. Effects of long-term administration of octreotide on sodium retention and atrial natriuretic peptide in carbon tetrachloride-induced cirrhotic rats. J Hepatol 1997;26:1128–34. [16] Sieber CC, Lee FY, Groszmann RJ. Long-term octreotide treatment prevents vascular hyporeactivity in portal-hypertensive rats. Hepatology 1996;23:1218 – 23. [17] Jenkins SA, Baxter JN, Critchley M, et al. Randomised trial of octreotide for long term management of cirrhosis after variceal haemorrhage. Br Med J 1997;22(7119):1338– 41. [18] D’Amico G, Politi F, D’Antoni A, et al. Second study shows that octreotide may prevent early rebleeding in cirrhosis. Br Med J 1998;25(7140):1320. [19] D’Amico G, Politi F, Morabito A, et al. Octreotide compare with placebo in a treatment strategy for rebleeding in cirrhosis. A double blind, randomized pragmatic trial. Hepatology 1998;28:1206 – 14. [20] Gillis JC, Noble S, Goa KL. Octreotide long-acting release (LAR). A review of its pharmacological properties and therapeutic use in the management of acromegaly. Drugs 1997;53:681 – 99. [21] Rubin J, Ajani J, Schirmer W, et al. Octreotide acetate long-acting formulation versus open-label subcutaneous octreotide acetate in malignant carcinoid syndrome. J Clin Oncol 1999;17:600 – 6. [22] Sieber CC, Mosca PG, Groszmann RJ. Effect of somatostatin on mesenteric vascular resistance in normal and portal hypertensive rats. Am J Physiol 1992;262:G274– 7. [23] Arndorfer RC, Stef JJ, Dodds WJ, Linehan JH, Hogan WJ. Improved infusion system for intraluminal esophageal manometry. Gastroenterology 1977;73:23 – 7. [24] Eleftheriadis E, Kotzampassi K, Koufogiannis D. Modulation of intravariceal pressure with pentoxifylline: a possible new approach in the treatment of portal hypertension. Am J Gastroenterol 1998;93:2431–5. [25] D’Amico G, Pagliaro L, Bosch J. The treatment of portal hypertension: a meta-analytic review. Hepatology 1995;22:332–54. [26] Vargas HE, Gerber D, Abu-Elmagd A. Management of portal hypertension-related bleeding. Surg Clin North Am 1999;79:1–22. [27] Groszmann RJ. Beta-adrenergic blockers and nitrovasodilators for the treatment of portal hypertension: the good, the bad, the ugly. Gastroenterology 1997;113:1794 – 7. [28] Hwang SJ, Lin HC, Chang CF, et al. A randomized controlled trial comparing octreotide and vasopressin in the control of acute esophageal variceal bleeding. J Hepatol 1992;16:320 – 5. [29] Eriksson LS, Wahren J. Intravenous and subcutaneous administration of a long-acting somatostatin analogue: effects on glucose metabolism and splanchnic haemodynamics in healthy subjects. Eur J Clin Invest 1989;19:213–9. [30] Sieber CC, Mosca PG, Groszmann RJ. Effect of somatostatin on mesenteric vascular resistance in normal and portal hypertensive rats. Am J Physiol 1992;262:G274– 7.
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[31] Albillos A, Colombato LA, Lee FY, Groszmann RJ. Octreotide ameliorates vasodilatation and Na+ retention in portal hypertensive rats. Gastroenterology 1993;104:575 – 9. [32] Flogstad AK, Halse J, Haldorsen T, et al. Sandostatin LAR in acromegalic patients: a dose-range study. J Clin Endocrinol Metab 1995;80:3601 – 7. [33] Lancranjan I, Bruns C, Grass P, et al. Sandostatin LAR: a promising therapeutic tool in the management of acromegalic patients. Metabolism 1996;45:67 – 71. [34] Grass P, Marbach P, Bruns C, Lancranjan I. Sandostatin LAR (microencapsulated octreotide acetate) in acromegaly: pharmacokinetic and pharmacodynamic relationships. Metabolism 1996;45:27–30. [35] Comets E, Mentre F, Nimmerfall F, et al. Nonparametric analysis of the absorption profile of octreotide in rabbits from long-acting release formulation OncoLAR. J Controlled Release 1999;59:197–205.
.
Sustained reduction of portal pressure by administration of octreotide-LAR in portal hypertensive rats Katerina Kotzampassi a, Dimitrios Koufogiannis b, Efthimios Eleftheriadis a,* b
a Departments of Surgery, Uni6ersity of Thessaloniki, Thessaloniki, Greece Department of Medical Physics, Uni6ersity of Thessaloniki, Thessaloniki GR-546 55, Greece
Received 6 January 2000; received in revised form 27 April 2000; accepted 18 May 2000
Abstract Objecti6e: Recent studies have demonstrated octreotide as being potentially beneficial for the long-term management of cirrhotic portal hypertension. However, its short-term effects, requiring subcutaneous administration at least twice daily, make it inconvenient, practically, for long-term therapy. Hence, the current study was undertaken to evaluate the effects of the new long-acting-release octreotide formulation (octreotide-LAR) on portal pressure in a prehepatic portal hypertensive rat model. Methods: 14 days after portal vein stenosis or sham operation, rats were treated with either octreotide-LAR (0.25 mg/kg body weight, subcutaneously) or vehicle, after the baseline portal pressure measurements were obtained in each group. On days 10, 15, 18, 21, 24, 27 and 30 post-treatment, eight rats from each sub-group (portal vein stenosis plus octreotide-LAR, or vehicle, sham-operated plus octreotide-LAR or vehicle) were subjected to portal vein pressure measurement. Results: portal vein-stenosed rats exhibited a significantly higher portal vein pressure than sham-operated rats (13.82 9 2.18 vs. 5.829 1.08 mmHg, P= 0.0001). Octreotide-LAR treatment was found to significantly (P =0.001) reduce portal pressure in portal vein stenosed rats from the tenth post-injection day and thereafter to sustain reduction. Conclusion: the long-acting-release octreotide given in a single dose in a prehepatic portal hypertensive rat model diminishes and sustains diminished portal pressure for 20 days after the tenth post-injection day. These
Abbre6iations: PVS, portal vein stenosis. * Corresponding author. Fax: + 30-31-993496. E-mail address: [email protected] (E. Eleftheriadis). 1386-6346/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 3 8 6 - 6 3 4 6 ( 0 0 ) 0 0 1 0 0 - 5
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findings seem promising for the portal hypertensive patient and further clinical studies are necessary. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Portal hypertension; Portal pressure; Octreotide; Octreotide-LAR; Experimental; Portal vein stenosis
1. Introduction Octreotide is a synthetic long-acting analogue of somatostatin, that has been found to have potential beneficial effects in the management of acute hemorrhage from esophageal varices [1,2] with no major side effects [3]. Acute experimental studies in animal models of portal hypertension have shown that single octreotide administration results in a dose-dependent decrease in portal pressure and portal vein tributary blood flow, associated with an increase in splanchnic vascular resistance, indicating vasoconstrictive action of octreotide [4–7]. Similar findings were reported after short-term administration of octreotide in portal hypertensive cirrhotic patients; hepatic and azygos blood flow were markedly decreased with minimal effects on systemic hemodynamics [8–11]. These promising effects have directed experimental research towards both the detailed mechanisms of its action in portal hypertension and its effects after long-term administration in portal hypertensive rats [12–16]. However, the necessity for repeated subcutaneous injections (two to three times daily) over a long period has made experimentation difficult. On the other hand, the need for an alternative or adjuvant to endoscopic therapy after variceal bleeding to reduce recurrent hemorrhage led to two randomized trials of octreotide for long-term management of cirrhosis, with encouraging results [17– 19]. However, the multiple daily injection dosage remains inconvenient for the patient and has the danger of patient’s non-compliance to the therapy. The long-acting-release formulation-octreotide-LAR-consisting of micro-spheres of poly-DL-lactide-co-glycolide-glucose containing octreotide has been developed to improve convenience, compliance and quality of life for the patient, by providing the same therapeutic advantages with less inconvenience and discomfort, since dosing is recommended once every 4 weeks [20,21]. Thus, the present experimental study was undertaken to evaluate the long-actingrelease octreotide treatment on portal pressure reduction in a prehepatic portal hypertensive rat model. 2. Material and method
2.1. Animals Two hundred and forty adult male Wistar rats weighing between 250 and 300 g were used in the study. The animals were housed together at a room temperature of 22 – 24°Celsius, with a 12-h light–dark cycle and were given free access to tap
110
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water and standard pellet rat diet until 16 h before the time of the hemodynamic study. The experimental protocol used was approved by the Governmental Animal Protection Committee and adhered to the European Community Guiding Principles for the care and use of animals.
2.2. Animal preparation Rats were divided into two groups; the portal vein stenosis group (PVS) and the sham-operated group (sham). Portal hypertension was induced according to the commonly used method. In brief, with rats under ether anesthesia, after a midline laparotomy, the portal vein was exposed free from surrounding fatty tissue and subjected to ligation (using 3 – 0 silk) together with a 20 gauge blunt-tipped needle lying alongside; subsequent removal of the needle yielded a calibrated stenosis of the portal vein. In sham-operated rats, the portal vein was exposed and isolated but no stenosis was performed.
2.3. Experimental protocol Fourteen days after portal vein stenosis the rats were ready for the experiment, since the hyperdynamic circulation accompanying portal hypertension was fully established [22]. At this time, under ether anesthesia, the initial (baseline) portal pressure, as well as mean arterial pressure measurements were performed in eight PVS rats and eight sham rats. Immediately thereafter, the remaining 112 PVS and the 112 sham-operated rats were randomly assigned to receive a single subcutaneous injection (0.25 mg/kg body weight) of the long-acting-release formulation of octreotide (octreotideLAR, Novartis, Hellas) or equal volume of vehicle, thus, four sub-groups were formed, i.e. PVS+LAR, PVS+ vehicle, sham+ LAR, sham+ vehicle. Portal vein pressure was then measured on the tenth post-injection day and on the 15th, 18th, 21st, 24th, 27th and 30th day in eight rats from each of the four sub-groups, every time. Additionally mean arterial pressure was evaluated in the 15th and the 30th day-groups.
2.4. Portal 6ein pressure measurements After ether anesthesia, the portal vein was exposed and pressure was measured by means of a direct puncture of the vein using a 23-G needle, under a constant perfusion of sterile sodium chloride 0.9% at a volume of 0.03 ml/min by the use of a hydraulic flow regulator (Infu 362 Datex, Swiss, Uhwiesen) according to the method described by Arndorfer et al. [23] and earlier applied by us for intravariceal pressure measurements [24]. This high-pressure low-compliance perfusion system was connected via an Elcomatic EM 750 (Harvard Apparatus, Ltd., Kent, UK) pressure transducer to a Palmer BioScience A100 (Sheerness, Kent, UK) amplifier, the pressure values registered by a chart recorder (Kipp Zonen BD11 model, Delft, Holland) on a
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paper running at 1 mm/s. The system was calibrated for pressure readings between 0 and 50 cm H2O, which was then converted to mmHg.
2.5. Statistical analysis All data, per treatment group and per study period were expressed as mean9 S.D. in mmHg. Analysis of variance (ANOVA) was then used for the statistical evaluation of the difference between treatment groups, as well as between different study periods within the same group of treatment. A P value less than 0.05 was considered as significant.
3. Results Portal vein-stenosed rats demonstrated a marked increase in portal pressure compared with that of sham-operated rats (13.829 2.18 vs. 5.829 1.08 mmHg, P=0.0001). Octreotide-LAR treatment, given after this baseline measurement, led to a highly significant decrease of portal pressure in PVS rats compared with the portal pressure of vehicle-treated rats (PVS+ vehicle group). This reduction was found to be in progress on the tenth post-injection day and stabilized after day 15. Octreotide-LAR treatment, given to sham-operated rats exhibited no effect on portal pressure at all (Table 1Fig. 1). Mean arterial pressure was found not to be significantly decreased in PVS versus sham operated rats (111 94 vs. 1229 3 mmHg). Octreotide-LAR led to an increase of MAP (measured on days 15 and 30) in PVS + LAR group from 111 9 4 to 119 94.5 mmHg compared with that of PVS + vehicle group, exhibiting no change. Octreotide-LAR or vehicle given to sham operated rats have no effect on MAP (1229 3 mmHg) at all. Table 1 Portal pressure measurementsa Day
PVS+LAR
PVS+vehicle
Sham+LAR
Sham+vehicle
10 15 18 21 24 27 30
0 (baseline) 10.9592.43c 8.649 1.07c 8.579 1.28c 8.52 91.20c 8.41 9 1.12c 8.209 1.10c 8.08 90.79c
13.829 2.18* 13.1692.15 12.49 9 1.88 12.02 91.48 12.049 1.71 11.66 9137 11.50 91.57 11.50 9 1.17
5.20 9 1.29 6.04 9 .0.81 5.69 9 1.18 6.07 9 1.17 5.87 9 1.07 5.69 9 1.06 5.56 9 1.03
5.82 9 1.08 6.07 9 1.17 5.70 9 1.20 5.82 9 1.21 5.57 9 1.12 6.07 9 1.17 5.70 9 0.93 5.709 1.20
a
Mean9 S.D. of portal pressure values throughout the 30 days period. Baseline measurements were performed before octreotide-LAR or vehicle treatment, thus portal pressure values are similar in LARand vehicle- treated groups, subjected to PLV or sham-operation. *, Represents significant difference (P=0.0001) in relation to sham-operated (baseline); c , represents significant difference (P= 0.001) in relation to vehicle-treated, within the same study-period.
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Fig. 1. Portal pressure measurements throughout the study-period. Closed squares represent PVS +vehicle group, open squares PVS +LAR group. Closed circles represent sham+ vehicle group, open circles sham + LAR group.
4. Discussion Bleeding from esophageal varices is the most common complication during the course of liver cirrhosis and is often the terminal event or an important contributory factor leading to the death of the patient. Thus, the need for prophylaxis to prevent variceal bleeding both in patients who have never bled and in those with a history of bleeding is pressing in the case of portal hypertension, as in few other clinical situations in medicine. Despite the central role of endoscopic techniques in controling acute variceal hemorrhage and reducing recurrent bleeding, underlying portal hypertension remains unaltered, accounting for a continued risk of bleeding even after variceal endoscopic eradication [25,26]. Consequently, there is an augmented interest in the use of vasoactive drugs as an alternative or as an adjuvant to endoscopic treatment. Similarly, in order to prevent the first bleeding episode vasoactive therapy seems to be an attractive proposition, since the benefit from prophylactic treatment appears to be too low to justify invasive therapeutic measures [27]. At this time, as the usefulness of b-blockers is limited by the adverse effects and recommendation of nitrates cannot be made unreservedly at this time [26–28] the most discussed agent of this category seems to be the synthetic analogue of somatostatin, octreotide. Given subcutaneously it results in a sustained reduction of portal pressure and azygos blood flow [8–10,29], by causing splanchnic arteriolar constriction [30] and inhibition of the vasoactive peptides contributing to the hyperdynamic circulatory syndrome of portal hypertension [31], without significant side-effects [25]. Several studies in humans and experimental animal models have evaluated the effect of octreotide on systemic hemodynamics during conditions with portal hypertension as a result of portal vein stenosis or cirrhotic liver disease [34,12,15,16]. These studies indicate that the peripheral vasodilation associated with portal hypertension is reversed by octreotide treatment, in the absence of changes in mean arterial pressure [10,11,15,16]. The mechanism responsible for this effect of octreotide acts through modulation of the release of vasoactive peptides from the
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gastrointestinal system during conditions with portal hypertension [12,16] although other data obtained from a common bile duct ligation rat model do not support that octreotide affects fasting plasma concentrations of glucagon and insulin [14]. Additionally, prolonged octreotide administration appears to involve little tachyphylaxis of its vasoconstrictive effect on the splanchnic circulation, so that the reduction in portal pressure elicited by the analogue is sustained; these advantages suggest octreotide to be potentially beneficial for the long-term management of cirrhotic portal hypertension patients, and this, indeed, appears to be the case [17– 19]. In a well-designed study [17], 32 cirrhotics, after cessation of their variceal bleeding episode, were randomly assigned to receive injection sclerotherapy or injection sclerotherapy plus octreotide (50 mg sc. bd). After 6 months of the combined treatment there was a significant reduction of the bleeding recurrence, and the number of elective injections required to obliterate varices, while survival was significantly improved. Furthermore, the combined treatment resulted in a sustained decrease in portal pressure, and improvement in liver function. Similarly, in a second study [18,19] 262 cirrhotic patients, after acute variceal bleeding, were randomized to receive octreotide 100 mg sc three times a day for 15 days or placebo in combination with b-blockers or sclerotherapy or both. Patients treated with octreotide showed a significant reduction in rebleeding episodes, blood transfusions and days in hospital. However, its short-term effects, requiring subcutaneous administration at least twice daily, make it inconvenient for long-term therapy in the majority of patients, although the recent development of a long-acting slow-release preparation (octreotide-LAR) is likely to make the therapy particularly desirable. A single subcutaneous injection of this preparation, when compared with the normal formulation of octreotide, results in stable plasma concentrations of octreotide for more than 4 weeks, as was found in human studies of its therapeutic use in the management of acromegaly [20,32–34] and in malignant carcinoid syndrome [21], the result of which is to limit the administration of the compound to once monthly. Therefore, the current study was designed to evaluate the effects of this new formulation of octreotide on portal hemodynamics in rats subjected to portal vein stenosis. Octreotide-LAR administered in a single dose of 0.25 mg/kg body weight was found to be significantly effective in decreasing portal vein pressure in the well-established model of portal vein stenosis. A comparable portal vein pressure reduction was also stated by Lin et al. [12] who use multiple daily subcutaneous injections of octreotide in the same rat model for 14 consecutive days. Thus octreotide-LAR seems to exhibit all the positive characteristics of the short-acting subcutaneous formulation and has the added advantage of slow drug release, which occurs by the cleavage of the polymer ester linkage to octreotide, primarily through tissue fluid hydrolysis [21]. However, there were important differences between our present and earlier studies. First of all, our study is the first, to our knowledge, in which octreotideLAR was tested to evaluate whether this new formula works towards reduction in portal hypertension in a continuous manner. For this purpose portal vein pressure
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was assessed every 3 days from day 10 up to day 30 post-injection; day 10 was selected to test the statement of other studies that this time-period is required to reach steady-state octreotide levels, as the release of the drug from the microspheres is slow, so the drug would not achieve its maximum efficacy in portal vein pressure reduction earlier, as is the case with growth hormones [20,32–34]. This finding was deduced in our study from the high S.D. values of portal vein pressure on day 10. Second, in this study a dose of 0.25 mg/kg body weight was used in rats, which is the equivalent of the human dose of 20 mg; in all the earlier experimental studies, whatever the purpose and the indications for its use, it was tested at doses up to 40 times higher [14,35]. In a recent experimental study, octreotide-LAR was given at a high dosage of 10 mg/kg body weight to test its effect on renal hemodynamics in a common bile duct ligation rat model, octreotide plasma levels 4 weeks later were found to be as high as 5809 90 and 20909 340 ng/l in controls and cirrhotics, respectively [14]. Although in our study no octreotide plasma levels were evaluated, its effect on portal pressure support the dose efficacy. Finally, the third point to be noted is that in our experiment octreotide-LAR was evaluated for long-term use on a more realistic rat model in which portal hypertension and the consequent hyperdynamic circulation were well established [22]. In the majority of earlier studies dealing with octreotide (or octreotide-LAR), it was injected immediately after portal vein ligation, i.e. just after acute portal hypertension induction [13,14], where neither systemic vasodilation nor liver injury had yet been established. The findings of the present study allow us to conclude that long-acting-release octreotide given in a single dose in a prehepatic portal hypertensive rat model sustains reduced portal pressure for 20 days after the tenth post-injection day. Although these findings seem very promising, as to whether octreotide-LAR would work as a valuable therapeutic option for the portal hypertensive patient, the results of the ongoing long-term clinical studies will provide the further necessary knowledge of the drug.
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