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Acceleration of recovery from sciatic nerve damage by the ACTH(4–9) analog Org 2766: Different routes of administration
Peptides, Vol. 8, pp. 1057--1059. ©Pergamon Journals Ltd., 1987. Printed in the U.S.A.
0196-9781/87 $3.00 + .00
Acceleration of Recovery From Sciatic Nerve Damage by the ACTH(4-9) Analog Org 2766: Different Routes of Administration AD J. A. M. DEKKER, MARTINUS M. PRINCEN, HENRIK DE NIJS, LEO G. J. DE LEEDE AND CHRIS L. E. BROEKKAMP Scientific: Development Group, Organon International BV, PO Box 20, 5340 B H Oss, The Netherlands R e c e i v e d 26 M a r c h 1987 DEKKER, A. J. A. M., M, M. PRINCEN, H. DE NIJS, L. G. J. DE LEEDE AND C. L. E. BROEKKAMP. Acceleration of recovery from sciatic nerve damage by the ACTH(4-9) analog Org 2766: Different routes af administration. PEPTIDES 8(6) 1057-1059, 1987.--Functional recovery following a sciatic nerve crush in rats was investigated by measuring the reflex withdrawal of the hindpaw to a hot air stream. The ACTH(4-9) analog Org 2766 accelerated recovery when administered subcutaneously (two-daily injections: l0/zg/animal; minipumps: 20--40~g/animal per 24 hr; biodegradable microspheres: 40 p~g/animal per 24 hr), but oral administration (1.5-20 mg/animal daily, in the drinking water; 1.5-15 mg/animal daily, by gavage) was not effective. Nerve regeneration
Nerve crush
Org 2766
Sensorimotor function
ACTH-Iike peptides have been demonstrated to improve recovery from peripheral nerve damage, when administered either intraperitoneally [8] or subcutaneously [1,2]. In particular, the ACTH-like peptide Org 2766 (Met(O2)-Glu-HisPhe-D-Lys-Phe) was effective in stimulating recovery when administered subcutaneously either as a long acting Zn.3(PO92 complex or in saline solution [1,2]. In contrast, daily oral administration of 500/~g of Org 2766 (dissolved in the drinking water) appeared to be ineffective. In the experiments described here, the efficacy of various routes of administration of Org 2766 on functional recovery from sciatic nerve damage in the rat was investigated. METHOD
Animals Female rats (Wistar, CPB-Wu), weighing 80-140 g at the start of the experiment, were obtained from TNO, Zeist, the Netherlands. They were housed five per cage and had free access to food (Hope Farms, Woerden, the Netherlands) and tap-water.
Surgery Animals were anaesthetized with 0.8 ml/kg Hypnorm a (Duphar, Weesp, the Netherlands). The right sciatic nerve was exposed at the level of the thigh and crushed approximately 6 mm below the sciatic notch with a haemostatic forceps (Mosquito, 10.0456, Stfpler, Utrecht, the Netherlands) for 30 sec. The wound was then sutured with silk (No. 1, Pfrimmer, Erlangen, FRG) or closed with wound clips.
Administration of the Peptide Org 2766 was administered subcutaneously (by two-daily injections, osmotic minipumps or biodegradable microspheres) or orally (via the drinking water or by gavage). Two-daily injections. The animals received subcutaneous injections of 0.5 ml saline or I0/xg Org 2766 dissolved in 0.5 ml saline, starting immediately after the operation. Osmotic minipumps. Org 2766 was dissolved in distilled water, filtered through a Millex a OR sterile filter (pore size=0.22 p.m, Millipore SA, Molsheim, France) into an osmotic minipump (Alzet R 2002, in vitro flow rate of 0.46 /zl/hr for 20 days) using a sterile syringe. The minipumps were equilibrated in sterile saline for 4 hr at 37°C and were implanted subcutaneously (in the scapular region) immediately after the operation. The minipumps were left in place during the experiment. Control animals received minipumps containing saline. Biodegradable microspheres, consisting of a l : l copolymer of lactic acid and glycolic acid with an intrinsic viscosity of 1.13 dl/g, were prepared by a solvent evaporation process. Forty milligrams of microspheres, containing 3% of Org 2766 and showing an in vitro release of 40/zg of Org 2766 per day for 14 days, were suspended in 0.5 ml of an isotonic mannitol solution, containing 0.05 /~g/ml benzalkonium chloride, and injected subcutaneously immediately after the operation. Control animals received microspheres without Org 2766. F o r oral administration, Org 2766 was either dissolved in the drinking water (a fixed volume of 22 ml, changed daily at the end of the light period, 6 p.m.) or given by gavage. In the
1057
D E K K E R E T AL.
1058 latter case the peptide was dissolved in 1 ml of distilled water and gavaged into the stomach immediately before the operation. Control animals received a similar volume of distilled water.
Test f o r Functional Recovery: The Hot Air Stream Method This test is described by Bijlsma et al. [1]. The hindpaw of a rat is exposed to a hot air stream until it is withdrawn or a maximum time of 3 sec has elapsed. A positive response is counted when the animal withdraws its paw within three sec (normal response times are within 1.1-1.5 sec). The animals are tested three times every week from day 8 onwards. If an animal responds on three consecutive test sessions, the first of these sessions marks the recovery day.
Statistical Analysis Differences between group means were evaluated statistically with Student's t-test. Recovery times of less than 10 days were excluded from analysis. A significance level of p<0.05 (two-sided) was adopted. RESULTS Functional recovery from sciatic nerve damage was measured by testing the responsiveness of the hindpaw to noxious stimulation by means of the hot air stream method. The time to recovery of this response is normally 19 days. As can be seen in Table 1, subcutaneous injection of Org 2766 (10 /zg/animal, every 48 hr, starting immediately after the operation) resulted in a statistically significant reduction of the total recovery period by 16%. Using the same route, but now with a constant delivery of the peptide by minipumps, releasing 20, 30 or 40/xg Org 2766 per 24 hr, again a significant reduction of the total recovery period of 12-16% was obtained (Table 1). Similarly, a constant delivery of 40/~g Org 2766 per 24 hr from subcutaneously injected biodegradable microspheres reduced the recovery period by 17%. As an alternative route, oral administration of Org 2766 was investigated. The peptide was either dissolved in the drinking water or given by gavage. As can be seen in Table 1, neither administration of Org 2766 in doses up to 20 mg/24 hr via the drinking water nor oral injection of the peptide in doses up to 15 mg/24 hr led to a reduction in the recovery period. DISCUSSION The neurotrophic effectivity of repeated subcutaneous injections of Org 2766 (10/zg/animal/48 hr) throughout the recovery period is in agreement with the data reported by Bijlsma et al. [l]. In the present study it is demonstrated that also other forms of subcutaneous administration are effective, notably the minipumps and the microspheres. The latter form of administration offers the advantage of a more simple treatment in the possible clinical use of Org 2766 in patients: one injection of microspheres immediately after the operation is as effective as repeated subcutaneous injections. Furthermore, the microspheres have the advantage over minipumps that they are completely biodegradable and therefore do not have to be removed. These microspheres have been shown to release the peptide for a period of 14 days, whereas the total recovery period is normally 5 days longer under the conditions used. Thus, in the later phase of the recovery no peptide was administered. The results lend support to the conclusion that
TABLE 1 THE EFFECTOF ORG 2766ON RECOVERYFROMSCIATIC NERVE DAMAGE Days to Recovery (% to Control)
Route of Administration SC: injection
control: Org 2766 5 /xg:
100+ 6 (8) 84--- 5 (9)*
SC: minipump
control: Org 2766 20 /zg: 30 p.g: 40 /xg:
100_ + 3 (19) 88--- 5 (9)* 84+ _ 7 (10)* 86--- 4 (10)t
SC: microspheres
control: Org 2766 40 /xg:
100--- 5 (7) 84_ + 4 (9)*
Oral: drinking water
control: Org 2766 1.5 mg: 4.5 mg: 20 mg:
100_+ 4 (20) 101-+ 4 (20) 101_+ 6 (20) 98_+12 (10)
control: Org 2766 1.5 mg: 15 mg:
100+_ 6 (10) 99_+ 4 (10) 108_+ 5 (9)
Oral: gavage
Values expressed as mean-+standard error (n). The time to recovery is approximately 19 days for the control animals. *p<0.05, tp<0.01 vs. control (Student's t-test). The amount of Org 2766 indicated is administered per 24 hr. Subcutaneous injections were given every 48 hr starting immediately after the operation; control animals received injections of saline. Minipumps releasing the given amount of peptide for 20 days were implanted SC after the operation; control animals received minipumps containing saline. Microspheres were injected in a suspension (0.5 ml) immediately after the operation. These microspheres showed an in vitro release of 40 ~g of Org 2766 per day for 14 days. Control animals received microspheres containing vehicle. Drinking water (fixed volume of 22 ml) was changed every day at the end of the light period; control animals received normal tap-water. For daily administration by gavage, Org 2766 was dissolved in 1 ml of distilled water; control animals received a similar volume of water.
treatment during the initial period following the operation is critical for the facilitation of recovery by peptides [4]. With respect to alpha-MSH it was found that a treatment period of 8 days was necessary to obtain the full reduction of time to recovery [4]. In the case of Org 2766, however, the peptide should be given for a period longer than 8 days, but also here administration of the peptide in the first part of the recovery period was essential for stimulation of recovery [2]. It was recently shown that the dose-response curve of Org 2766 for the acceleration of sensory recovery has an inverted U-shape, with doses of 0.1 and 30/~g/animal (administered by two-daily subcutaneous injections) being less effective than 1 or 10 p~g/animal [3]. This raises the question whether higher or lower doses than those used in the present study would lead to a greater reduction in time to recovery. In the minipumps and microsphere experiments the minimum dose was chosen on the basis of the effectiveness of a subcutaneous injection of l0/zg (every 48 hr). This leads to a dose of 15/~g/24 hr, when a peak level effect after subcutaneous injection is taken into account: a pharmacokinetic model for uptake of Org 2766 in blood plasma after sub-
ROUTES OF ADMINISTRATION OF ORG 2766
1059
cutaneous administration shows that the peak level after a single injection can reach the level attained after a continuous administration of a 2.9 fold higher dose (C. Timmer, personal communication). The highest dose (40 /xg/24 hr) was chosen below the maximum of 45 /zg/24 hr, corresponding to the inactive higher dose for subcutaneous injections. Therefore, the doses used are in the upper end of the effective dose range for subcutaneous injections. However, it can not be excluded that either lower or higher doses would be more effective. This question can only be answered after a more extensive dose range has been investigated. In the present study no difference was observed between the effects of active doses (i.e., in the minipumps a 20 ~g dose is as effective as a 40/xg dose). This may indicate that the hot air stream method is not suited for the detailed study of dose-response relationships. On the other hand it should be noted that also in other tests for recovery relatively little difference is found between the effects of active doses. Doses of 1 and I0 tzg Org 2766 have an identical effect on recovery measured with an electric footshock test [3]. In a histological experiment, subcutaneous injections of 1, 10 and 100/zg alpha-MSH increased the number of newly formed sprouts to a similar extent (25%) [9]. The extrapolation to orally effective doses is even more difficult than that in the case of subcutaneous treatment. A daily dose of 0.5 mg Org 2766, dissolved in the drinking water, was ineffective in accelerating the recovery of sensorimotor functioning [2]. This finding in itself does not indicate whether the effective dose should be sought in a lower or higher range, but in other experiments the effective oral dose of Org 2766 was found to be in the milligram range: oral administration of 2 mg Org 2766 per animal per day facilitates behavioral recovery following lesions to the nucleus accumbens in rats [11]. Oral administration of a tablet containing 0.01-1 mg Org 2766 reduced CO2 induced amnesia for a passive avoidance response [7]. Although the subcutaneous effective dose for improving passive avoidance has been
shown to vary from 10 ng/animal [5] to 5 mg/kg [6] in different experiments, Rigter et al. [7] found that in a similar experiment the subcutaneous effective doses are 1000-10,000 fold lower than the effective oral doses. The oral dose used in the experiments described here was obtained by multiplying the minimal effective subcutaneous dose by a factor 1000 and taking into account the constant level factor of approximately 3. Since 0.5/zg per 24 hr has been reported as a minimal effective subcutaneous dose [1,3], this leads to an oral dose of 1.5 mg/24 hr. The highest oral dose used in this study was obtained by multiplying the minimal effective dose after administration through minipumps by 1000. As shown in Table 1, oral administration of these doses dissolved in the drinking water did not result in accelerated recovery of sensorimotor functioning. The possible degradation of Org 2766 in the drinking water was investigated by HPLC analysis of samples of the drinking water taken after 24 hr. At least 77% of the original amount of Org 2766 was recovered as the intact peptide. Since most of the drinking water is consumed after 24 hr, it can be estimated that less than 12% of the peptide is lost before administration. Therefore it is unlikely that degradation plays a major part in the ineffectiveness of this route of administration. Breakdown prior to administration is avoided by gavaging Org 2766 into the stomach. Furthermore, administration by gavage rather than via the drinking water will lead to a peak in plasma levels of the peptide [10]. However, also this type of oral administration did not affect the recovery of sensorimotor functioning. Therefore, on the basis of the present data, subcutaneous rather than oral administration of Org 2766 should be considered optimal for the acceleration of recovery from sciatic nerve damage. The effectiveness of a subcutaneous injection of microspheres containing Org 2766 offers the possibility of a relatively simple administration of this neurotrophic peptide in future clinical experiments.
REFERENCES
1. Bijlsma, W. A., F. G. I. Jennekens, P. Schotman and W. H. Gispen. Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: structure-activity study. Eur J Pharmacol 76: 73-79, 1981. 2. Bijlsma, W. A., P. Schotman, F. G. I. Jennekens, W. H. Gispen and D. de Wied. Enhanced recovery of sensorimotor function in rats is related to the melanotropic moiety of ACTH/MSH neuropeptides. Eur J Pharrnacol 92: 231-236, 1983. 3. de Koning, P., J. H. Brakkee and W. H. Gispen. Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. J Neurol Sci 74: 237246, 1986. 4. Edwards, P. M., C. E. E. M. van der Zee, J. Verhaagen, P. Schotman, F. G. I. Jennekens and W. H. Gispen. Evidence that the neurotropic action of alpha-MSH may derive from its ability to mimick the actions of a peptide formed in degenerating nerve stumps. J Neurol Sci 64: 333-340, 1984. 5. Fekete, M. and D. de Wied. Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH4-10 and [D-Pher] ACTH4-10 on active and passive avoidance behavior of rats. Pharmacol Biochem Behav 16: 387-392, 1982.
6. Martinez, J. L., B. J. Vasquez, R. A. Jensen, B. SoumireuMourat and J. L. McGaugh. ACTH4--9 analog (ORG 2766) facilitates acquisition of an inhibitory avoidance response in rats. Pharmacol Biochem Behav 10: 145-147, 1979. 7. Rigter, H., R. Janssens-Elbertse and H. van Riezen. Reversal of amnesia by an orally active ACTH 4-9 analog (Org 2766). Pharmacol Biochem Behav 5: Suppl 1, 53-58, 1976. 8. Strand, F. L. and T, T. Kung. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1: 135-138, 1980. 9. Verhaagen, J., P. M. Edwards, F. G. I. Jennekens and W. H. Gispen. Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts. Peptides 8: 581-585, 1987. I0. Verhoef, J. and A. Witter. In vivo fate of a behaviorally active ACTH4-9 analog in rats after systemic administration. Pharmacol Biochem Behav 4: 583-590, 1976. 11. Wolterink, G. and J. M. van Ree. Org 2766 accelerates functional recovery of impaired motor activity due to lesions in the nucleus accumbens in rats. Abstract for meeting: 'Neuropeptides and Brain Function,' May 28-30, 1986, Utrecht, the Netherlands.
0196-9781/87 $3.00 + .00
Acceleration of Recovery From Sciatic Nerve Damage by the ACTH(4-9) Analog Org 2766: Different Routes of Administration AD J. A. M. DEKKER, MARTINUS M. PRINCEN, HENRIK DE NIJS, LEO G. J. DE LEEDE AND CHRIS L. E. BROEKKAMP Scientific: Development Group, Organon International BV, PO Box 20, 5340 B H Oss, The Netherlands R e c e i v e d 26 M a r c h 1987 DEKKER, A. J. A. M., M, M. PRINCEN, H. DE NIJS, L. G. J. DE LEEDE AND C. L. E. BROEKKAMP. Acceleration of recovery from sciatic nerve damage by the ACTH(4-9) analog Org 2766: Different routes af administration. PEPTIDES 8(6) 1057-1059, 1987.--Functional recovery following a sciatic nerve crush in rats was investigated by measuring the reflex withdrawal of the hindpaw to a hot air stream. The ACTH(4-9) analog Org 2766 accelerated recovery when administered subcutaneously (two-daily injections: l0/zg/animal; minipumps: 20--40~g/animal per 24 hr; biodegradable microspheres: 40 p~g/animal per 24 hr), but oral administration (1.5-20 mg/animal daily, in the drinking water; 1.5-15 mg/animal daily, by gavage) was not effective. Nerve regeneration
Nerve crush
Org 2766
Sensorimotor function
ACTH-Iike peptides have been demonstrated to improve recovery from peripheral nerve damage, when administered either intraperitoneally [8] or subcutaneously [1,2]. In particular, the ACTH-like peptide Org 2766 (Met(O2)-Glu-HisPhe-D-Lys-Phe) was effective in stimulating recovery when administered subcutaneously either as a long acting Zn.3(PO92 complex or in saline solution [1,2]. In contrast, daily oral administration of 500/~g of Org 2766 (dissolved in the drinking water) appeared to be ineffective. In the experiments described here, the efficacy of various routes of administration of Org 2766 on functional recovery from sciatic nerve damage in the rat was investigated. METHOD
Animals Female rats (Wistar, CPB-Wu), weighing 80-140 g at the start of the experiment, were obtained from TNO, Zeist, the Netherlands. They were housed five per cage and had free access to food (Hope Farms, Woerden, the Netherlands) and tap-water.
Surgery Animals were anaesthetized with 0.8 ml/kg Hypnorm a (Duphar, Weesp, the Netherlands). The right sciatic nerve was exposed at the level of the thigh and crushed approximately 6 mm below the sciatic notch with a haemostatic forceps (Mosquito, 10.0456, Stfpler, Utrecht, the Netherlands) for 30 sec. The wound was then sutured with silk (No. 1, Pfrimmer, Erlangen, FRG) or closed with wound clips.
Administration of the Peptide Org 2766 was administered subcutaneously (by two-daily injections, osmotic minipumps or biodegradable microspheres) or orally (via the drinking water or by gavage). Two-daily injections. The animals received subcutaneous injections of 0.5 ml saline or I0/xg Org 2766 dissolved in 0.5 ml saline, starting immediately after the operation. Osmotic minipumps. Org 2766 was dissolved in distilled water, filtered through a Millex a OR sterile filter (pore size=0.22 p.m, Millipore SA, Molsheim, France) into an osmotic minipump (Alzet R 2002, in vitro flow rate of 0.46 /zl/hr for 20 days) using a sterile syringe. The minipumps were equilibrated in sterile saline for 4 hr at 37°C and were implanted subcutaneously (in the scapular region) immediately after the operation. The minipumps were left in place during the experiment. Control animals received minipumps containing saline. Biodegradable microspheres, consisting of a l : l copolymer of lactic acid and glycolic acid with an intrinsic viscosity of 1.13 dl/g, were prepared by a solvent evaporation process. Forty milligrams of microspheres, containing 3% of Org 2766 and showing an in vitro release of 40/zg of Org 2766 per day for 14 days, were suspended in 0.5 ml of an isotonic mannitol solution, containing 0.05 /~g/ml benzalkonium chloride, and injected subcutaneously immediately after the operation. Control animals received microspheres without Org 2766. F o r oral administration, Org 2766 was either dissolved in the drinking water (a fixed volume of 22 ml, changed daily at the end of the light period, 6 p.m.) or given by gavage. In the
1057
D E K K E R E T AL.
1058 latter case the peptide was dissolved in 1 ml of distilled water and gavaged into the stomach immediately before the operation. Control animals received a similar volume of distilled water.
Test f o r Functional Recovery: The Hot Air Stream Method This test is described by Bijlsma et al. [1]. The hindpaw of a rat is exposed to a hot air stream until it is withdrawn or a maximum time of 3 sec has elapsed. A positive response is counted when the animal withdraws its paw within three sec (normal response times are within 1.1-1.5 sec). The animals are tested three times every week from day 8 onwards. If an animal responds on three consecutive test sessions, the first of these sessions marks the recovery day.
Statistical Analysis Differences between group means were evaluated statistically with Student's t-test. Recovery times of less than 10 days were excluded from analysis. A significance level of p<0.05 (two-sided) was adopted. RESULTS Functional recovery from sciatic nerve damage was measured by testing the responsiveness of the hindpaw to noxious stimulation by means of the hot air stream method. The time to recovery of this response is normally 19 days. As can be seen in Table 1, subcutaneous injection of Org 2766 (10 /zg/animal, every 48 hr, starting immediately after the operation) resulted in a statistically significant reduction of the total recovery period by 16%. Using the same route, but now with a constant delivery of the peptide by minipumps, releasing 20, 30 or 40/xg Org 2766 per 24 hr, again a significant reduction of the total recovery period of 12-16% was obtained (Table 1). Similarly, a constant delivery of 40/~g Org 2766 per 24 hr from subcutaneously injected biodegradable microspheres reduced the recovery period by 17%. As an alternative route, oral administration of Org 2766 was investigated. The peptide was either dissolved in the drinking water or given by gavage. As can be seen in Table 1, neither administration of Org 2766 in doses up to 20 mg/24 hr via the drinking water nor oral injection of the peptide in doses up to 15 mg/24 hr led to a reduction in the recovery period. DISCUSSION The neurotrophic effectivity of repeated subcutaneous injections of Org 2766 (10/zg/animal/48 hr) throughout the recovery period is in agreement with the data reported by Bijlsma et al. [l]. In the present study it is demonstrated that also other forms of subcutaneous administration are effective, notably the minipumps and the microspheres. The latter form of administration offers the advantage of a more simple treatment in the possible clinical use of Org 2766 in patients: one injection of microspheres immediately after the operation is as effective as repeated subcutaneous injections. Furthermore, the microspheres have the advantage over minipumps that they are completely biodegradable and therefore do not have to be removed. These microspheres have been shown to release the peptide for a period of 14 days, whereas the total recovery period is normally 5 days longer under the conditions used. Thus, in the later phase of the recovery no peptide was administered. The results lend support to the conclusion that
TABLE 1 THE EFFECTOF ORG 2766ON RECOVERYFROMSCIATIC NERVE DAMAGE Days to Recovery (% to Control)
Route of Administration SC: injection
control: Org 2766 5 /xg:
100+ 6 (8) 84--- 5 (9)*
SC: minipump
control: Org 2766 20 /zg: 30 p.g: 40 /xg:
100_ + 3 (19) 88--- 5 (9)* 84+ _ 7 (10)* 86--- 4 (10)t
SC: microspheres
control: Org 2766 40 /xg:
100--- 5 (7) 84_ + 4 (9)*
Oral: drinking water
control: Org 2766 1.5 mg: 4.5 mg: 20 mg:
100_+ 4 (20) 101-+ 4 (20) 101_+ 6 (20) 98_+12 (10)
control: Org 2766 1.5 mg: 15 mg:
100+_ 6 (10) 99_+ 4 (10) 108_+ 5 (9)
Oral: gavage
Values expressed as mean-+standard error (n). The time to recovery is approximately 19 days for the control animals. *p<0.05, tp<0.01 vs. control (Student's t-test). The amount of Org 2766 indicated is administered per 24 hr. Subcutaneous injections were given every 48 hr starting immediately after the operation; control animals received injections of saline. Minipumps releasing the given amount of peptide for 20 days were implanted SC after the operation; control animals received minipumps containing saline. Microspheres were injected in a suspension (0.5 ml) immediately after the operation. These microspheres showed an in vitro release of 40 ~g of Org 2766 per day for 14 days. Control animals received microspheres containing vehicle. Drinking water (fixed volume of 22 ml) was changed every day at the end of the light period; control animals received normal tap-water. For daily administration by gavage, Org 2766 was dissolved in 1 ml of distilled water; control animals received a similar volume of water.
treatment during the initial period following the operation is critical for the facilitation of recovery by peptides [4]. With respect to alpha-MSH it was found that a treatment period of 8 days was necessary to obtain the full reduction of time to recovery [4]. In the case of Org 2766, however, the peptide should be given for a period longer than 8 days, but also here administration of the peptide in the first part of the recovery period was essential for stimulation of recovery [2]. It was recently shown that the dose-response curve of Org 2766 for the acceleration of sensory recovery has an inverted U-shape, with doses of 0.1 and 30/~g/animal (administered by two-daily subcutaneous injections) being less effective than 1 or 10 p~g/animal [3]. This raises the question whether higher or lower doses than those used in the present study would lead to a greater reduction in time to recovery. In the minipumps and microsphere experiments the minimum dose was chosen on the basis of the effectiveness of a subcutaneous injection of l0/zg (every 48 hr). This leads to a dose of 15/~g/24 hr, when a peak level effect after subcutaneous injection is taken into account: a pharmacokinetic model for uptake of Org 2766 in blood plasma after sub-
ROUTES OF ADMINISTRATION OF ORG 2766
1059
cutaneous administration shows that the peak level after a single injection can reach the level attained after a continuous administration of a 2.9 fold higher dose (C. Timmer, personal communication). The highest dose (40 /xg/24 hr) was chosen below the maximum of 45 /zg/24 hr, corresponding to the inactive higher dose for subcutaneous injections. Therefore, the doses used are in the upper end of the effective dose range for subcutaneous injections. However, it can not be excluded that either lower or higher doses would be more effective. This question can only be answered after a more extensive dose range has been investigated. In the present study no difference was observed between the effects of active doses (i.e., in the minipumps a 20 ~g dose is as effective as a 40/xg dose). This may indicate that the hot air stream method is not suited for the detailed study of dose-response relationships. On the other hand it should be noted that also in other tests for recovery relatively little difference is found between the effects of active doses. Doses of 1 and I0 tzg Org 2766 have an identical effect on recovery measured with an electric footshock test [3]. In a histological experiment, subcutaneous injections of 1, 10 and 100/zg alpha-MSH increased the number of newly formed sprouts to a similar extent (25%) [9]. The extrapolation to orally effective doses is even more difficult than that in the case of subcutaneous treatment. A daily dose of 0.5 mg Org 2766, dissolved in the drinking water, was ineffective in accelerating the recovery of sensorimotor functioning [2]. This finding in itself does not indicate whether the effective dose should be sought in a lower or higher range, but in other experiments the effective oral dose of Org 2766 was found to be in the milligram range: oral administration of 2 mg Org 2766 per animal per day facilitates behavioral recovery following lesions to the nucleus accumbens in rats [11]. Oral administration of a tablet containing 0.01-1 mg Org 2766 reduced CO2 induced amnesia for a passive avoidance response [7]. Although the subcutaneous effective dose for improving passive avoidance has been
shown to vary from 10 ng/animal [5] to 5 mg/kg [6] in different experiments, Rigter et al. [7] found that in a similar experiment the subcutaneous effective doses are 1000-10,000 fold lower than the effective oral doses. The oral dose used in the experiments described here was obtained by multiplying the minimal effective subcutaneous dose by a factor 1000 and taking into account the constant level factor of approximately 3. Since 0.5/zg per 24 hr has been reported as a minimal effective subcutaneous dose [1,3], this leads to an oral dose of 1.5 mg/24 hr. The highest oral dose used in this study was obtained by multiplying the minimal effective dose after administration through minipumps by 1000. As shown in Table 1, oral administration of these doses dissolved in the drinking water did not result in accelerated recovery of sensorimotor functioning. The possible degradation of Org 2766 in the drinking water was investigated by HPLC analysis of samples of the drinking water taken after 24 hr. At least 77% of the original amount of Org 2766 was recovered as the intact peptide. Since most of the drinking water is consumed after 24 hr, it can be estimated that less than 12% of the peptide is lost before administration. Therefore it is unlikely that degradation plays a major part in the ineffectiveness of this route of administration. Breakdown prior to administration is avoided by gavaging Org 2766 into the stomach. Furthermore, administration by gavage rather than via the drinking water will lead to a peak in plasma levels of the peptide [10]. However, also this type of oral administration did not affect the recovery of sensorimotor functioning. Therefore, on the basis of the present data, subcutaneous rather than oral administration of Org 2766 should be considered optimal for the acceleration of recovery from sciatic nerve damage. The effectiveness of a subcutaneous injection of microspheres containing Org 2766 offers the possibility of a relatively simple administration of this neurotrophic peptide in future clinical experiments.
REFERENCES
1. Bijlsma, W. A., F. G. I. Jennekens, P. Schotman and W. H. Gispen. Effects of corticotrophin (ACTH) on recovery of sensorimotor function in the rat: structure-activity study. Eur J Pharmacol 76: 73-79, 1981. 2. Bijlsma, W. A., P. Schotman, F. G. I. Jennekens, W. H. Gispen and D. de Wied. Enhanced recovery of sensorimotor function in rats is related to the melanotropic moiety of ACTH/MSH neuropeptides. Eur J Pharrnacol 92: 231-236, 1983. 3. de Koning, P., J. H. Brakkee and W. H. Gispen. Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. J Neurol Sci 74: 237246, 1986. 4. Edwards, P. M., C. E. E. M. van der Zee, J. Verhaagen, P. Schotman, F. G. I. Jennekens and W. H. Gispen. Evidence that the neurotropic action of alpha-MSH may derive from its ability to mimick the actions of a peptide formed in degenerating nerve stumps. J Neurol Sci 64: 333-340, 1984. 5. Fekete, M. and D. de Wied. Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH4-10 and [D-Pher] ACTH4-10 on active and passive avoidance behavior of rats. Pharmacol Biochem Behav 16: 387-392, 1982.
6. Martinez, J. L., B. J. Vasquez, R. A. Jensen, B. SoumireuMourat and J. L. McGaugh. ACTH4--9 analog (ORG 2766) facilitates acquisition of an inhibitory avoidance response in rats. Pharmacol Biochem Behav 10: 145-147, 1979. 7. Rigter, H., R. Janssens-Elbertse and H. van Riezen. Reversal of amnesia by an orally active ACTH 4-9 analog (Org 2766). Pharmacol Biochem Behav 5: Suppl 1, 53-58, 1976. 8. Strand, F. L. and T, T. Kung. ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve. Peptides 1: 135-138, 1980. 9. Verhaagen, J., P. M. Edwards, F. G. I. Jennekens and W. H. Gispen. Pharmacological aspects of the influence of melanocortins on the formation of regenerative peripheral nerve sprouts. Peptides 8: 581-585, 1987. I0. Verhoef, J. and A. Witter. In vivo fate of a behaviorally active ACTH4-9 analog in rats after systemic administration. Pharmacol Biochem Behav 4: 583-590, 1976. 11. Wolterink, G. and J. M. van Ree. Org 2766 accelerates functional recovery of impaired motor activity due to lesions in the nucleus accumbens in rats. Abstract for meeting: 'Neuropeptides and Brain Function,' May 28-30, 1986, Utrecht, the Netherlands.