An ACTH-(4–9) analogue, Org 2766, improves recovery from acrylamide neuropathy in rats

European Journal of Pharmacology, 186 (1990) 181-187

181

Elsevier EJP 51504

An ACTH-(4-9) analogue, Org 2766, improves recovery from acrylamide neuropathy in rats R. E m i n e S p o r e l - O z a k a t 1, P h i l i p p a M. E d w a r d s 1, R o l a n d G e r r i t s e n V a n der H o o p 2 a n d W i l l e m H. G i s p e n 2 1 Bogazici University, Faculty of Arts and Sciences, Department of Biology, 80815 Bebek, lstanbul, Turkey, and 2 Department of Pharmacology, Rudolf Magnus Institute, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands Received 5 April 1990, revised MS received 8 June 1990, accepted 3 July 1990

Org 2766 is one of a series of melanocortins (ACTH and related peptides) that exert trophic influences on the central and peripheral nervous system of the rat. We used acrylamide neuropathy in rats as an experimental model of peripheral neuropathies of the dying-back type in order to assess the potential therapeutic efficacy of Org 2766 in this type of nerve damage. The peptide reversed the delayed persistent deficit in sensory conduction velocity without preventing the initial loss of motor coordination. The recovery of apparently normal coordination was unaffected by the peptide, but resistance to a second toxic challenge suggested that recovery was more complete in the peptide-treated rats. The finding that Org 2766 improved the quality of the repair following acrylamide neuropathy, together with previous studies showing beneficial effects in neuropathies caused by cisplatin or diabetes and after mechanical trauma, strongly suggests that Org 2766 may be beneficial in the treatment of various conditions in which the nervous system has sustained damage. Acrylamide; Melanocortins; Neuropathy (peripheral) 1. Introduction Melanocortins (ACTH, a-MSH, and related peptides) exert trophic influences on the central and peripheral nervous system in the rat (see reviews by: Strand and Smith, 1986; De Koning et al., 1988; Strand et al., 1989). The effectiveness of these peptides in enhancing nerve repair was first described for the recovery from mechanical trauma and was subsequently demonstrated in two unrelated conditions in which nerve damage occurs. Org 2766, a degradation-resistant ACTH-(4-9) analogue ( H - M e t ( O 2)-Glu-His-Phe-D-Lys-PheO H ) prevents the neurotoxic side-effects of cisplatin (an anti-tumour drug) both in man and

Correspondence to: W.H. Gispen, Rudolf Magnus Institute, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands.

rats when administered concurrently with cisplatin and reverses the development of neuropathy in diabetic rats (Gerritsen van der H o o p et al., 1988; 1990; Van der Zee et al., 1989). Thus, melanocortins may be generally useful in the treatment of neuropathies of diverse etiology, in which the final degree of disability is determined by the balance between neuronal damage and repair. In the present study, acrylamide neuropathy in rats was used as a model system to test the potential value of Org 2766 in the treatment of neuropathies. Acrylamide is a cumulative poison, and repetitive exposure to it results in peripheral neuropathy in both man and experimental animals (see reviews by: Spencer and Schaumburg, 1974a, b; Le Quesne, 1980), but the exact mechanism of action of acrylamide is not known. The neuropathy is of the dying-back type, in which there is a tendency for the initial changes to occur in the distal part of the nerve fibres (Fullerton and

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Barnes, 1966; Schaumburg et al., 1974; Suzuki and Pfaff, 1973). Sensory nerves are particularly susceptible, whereas motor nerves are affected to a lesser degree (Schaumburg et al., 1974; Sumner, 1980). The neuropathy produced by acrylamide is reversible and repair is similar to repair after crush, in that regeneration from proximal regions of the nerve results in functional recovery (Suzuki and Pfaff, 1973). Indeed, it has been shown by Fullerton and Barnes (1966) and Suzuki and Pfaff (1973) that regeneration of damaged nerves even occurs during intoxication. This is also the case in neurological disorders (such as ALS), where regeneration and degeneration occur concurrently (Wolhlfart, 1975). Quantitative functional and electrophysiological parameters were used to assess the influence of Org 2766 on the development of, and recovery from, acrylamide neuropathy. The results are discussed in relation to the possible mode of action of Org 2766.

2. Materials and methods

2.1. Animals and dosing Female Wistar rats weighing 150-200 g at the start of the experiment were used. Animals received an aqueous solution of acrylamide (50 m g / m l per kg) i.p. every 48 h, 7 or 8 times. Org 2766 was dissolved in 0.1% BSA/0.05 N HC1 and diluted 1 : 20 with a 0.9% NaC1 solution to a final concentration of 50 /Lg/ml. Animals received 50 I*g/kg s.c. every 48 h, starting on the same day as the acrylamide treatment or 24 h after the last acrylamide injection. Control rats received saline (0.9%) instead of acrylamide and 0.1% BSA/0.05 N HC1 diluted 1 : 2 0 with 0.9% NaCl in place of Org 2766.

In short, the rats were dropped from a horizontal position at a height of 30 cm. The position of the forth digit of each hindlimb upon landing was marked and the distance between the two measured. This was repeated a total of 4 times for each rat, and the mean values were calculated. The print lengths were obtained from records of the walking pattern as described by De Medinacelli et al., (1982) except that the plantar surfaces of the hind feet were painted with indigo mixed with glycerol and the rats walked through a confined passage lined with semi-absorbent paper. The passage led, at an upward slope of 10 °, to a dark goal box. A total of four prints for each rat were measured, and the mean values were calculated. 2.3. Electrophysiology Electrophysiological measurements were carried out under general anaesthesia induced with H y p n o r m ® (Duphar, Weesp, NL) containing fluanisone (10 m g / m l ) and fentanyl citrate (0.2 m g / m l ) administered s.c. (1 m g / k g ) . The conduction velocities of sensory and motor components of the sciatic and tibial nerves between the ankle and the thigh were estimated by the method of Stanley (1981) as modified by De Koning and Gispen (1987). The method depends on the difference in latency of the muscle action potential in the hind paw evoked by stimulation at the ankle or the thigh. The muscle action potential evoked by direct excitation of motor fibres (M-response) is well separated from the delayed H-response elicited by excitation of Ia-afferents, which excite c~-motoneurons in the spinal cord via a monosynaptic pathway. This allows measurement of both motor and H-related sensory conduction velocities from the mixed nerve.

2.2. Motor coordination 3. Results

In order to asses quantitatively the effect of acrylamide on motor coordination, two tests were carried out: the landing foot spread and the print length analysis. The landing foot spread (LFS) was measured as described previously (Edwards and Parker, 1977).

3.1. Motor coordination Administration of acrylamide (50 m g / m l per kg, seven or eight doses) LFS values, which returned to normal about 25-30 days after the last

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covered f r o m a c r y l a m i d e n e u r o p a t h y were nonetheless m o r e susceptible to a s e c o n d exposure, as a r a p i d a n d m o r e p r o n o u n c e d increase in L F S was seen following s u b s e q u e n t e x p o s u r e to a lower dose of a c r y l a m i d e (fig. 2). R a t s t r e a t e d with Org 2766 d u r i n g the initial e x p o s u r e to a c r y l a m i d e a n d r e - e x p o s e d to the toxin s h o w e d fewer a b n o r m a l i ties t h a n rats n o t given the p e p t i d e (fig. 2). T h e p r i n t length was the o n l y p a r a m e t e r altered in the w a l k i n g p a t t e r n of i n t o x i c a t e d a n i m a l s (other p a r a m e t e r s are n o t shown). Print length values i n c r e a s e d in the t r e a t e d animals, b u t the shape of the curve was n o t altered b y p e p t i d e t r e a t m e n t (fig. 3). Print length values c o u l d n o t be m e a s u r e d on d a y s w h e n n e u r o p a t h y was m o s t severe, b e c a u s e the a n i m a l s were n o t able to walk.

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Fig. 2. The effect of Org 2766 on sensitivity to a second exposure to acrylamide. The 'recovered' rats from a previous intoxication received acrylamide (50 mg/ml per kg i.p., every 48 h) as a second exposure during the period shown by the arrow. In the initial exposure to acrylamide, one group had received peptide and the other vehicle together with acrylamide, as indicated in fig. 1. Values given are the means + S.E.M. * Indicates a significant difference between the two groups (P = 0.05, analysis of variance with a supplemental t-test).

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Fig. 4. The effect of Org 2766 on the reduction in H-related sensory nerve conduction velocity induced by acrylamide. Animals received acrylamide (50 m g / m l per kg i.p. every 48 h, in total seven doses) injected concurrently with either Org 2766 ( 5 0 / ~ g / k g s.c., 14 injections) ([], right, n = 7) or vehicle (r~, left, n = 6 ) . Values given are the means+S.E.M. A N O V A R for repeated measures indicated a significant group effect over the period studied (F(1,11)= 6.394, P = 0.028). * Indicates a significant difference (P < 0.03 post hoc testing) between the two groups.

seven doses of acrylamide persisted as late as 88 days after acrylamide dosing was stopped (fig. 4). The effect of Org 2766 was therefore studied at

Fig. 6. The effect of Org 2766 on the reduction in H-related sensory nerve conduction velocity induced by acrylamide. Animals received acrylamide (50 m g / m l per kg i.p. every 48 h, in total seven doses). Twenty-four hours after the last acrylamide dose, the animals were injected with Org 2766 (50 p , g / k g s.c., every 48 h, 14 injections) ([2], right, n = 7) or vehicle ([~, n = 8). Control animals received saline and vehicle (El, left, n =10). Values given are means_+S.E.M. A N O V A R for rep e a t e d measures indicated a significant group effect over the p e r i o d s t u d i e d ( F ( 2 , 1 9 ) = 11.882, P < 0.001). * Indicates a significant difference (P < 0.0015, post hoc testing) between the control and the intoxicated groups.

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Fig. 5. The effect of Org 2766 on the reduction in H-related conduction velocity induced by acrylamide. Animals received acrylamide (50 m g / m l per kg i.p. every 48 h, in total eight doses) injected concurrently with either Org 2766 (50 /~g/kg s.c. 20 injections), (r~, right, n = 9) or vehicle ([:3, n = 7). Control animals ([], left, n = 6) received saline instead of acrylamide and were concurrently injected with vehicle. Values given are the means_+S.E.M. A N O V A R for repeated measures showed a significant effect of treatment (F(2,19) = 25.456, P < 0.001). * Indicates a significant difference (P < 0.05, post hoc testing) from controls. ~ Indicates a significant difference (P < 0.005, post hoc testing) between the peptide- and vehicle-treated groups.

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Fig. 7. The effect of Org 2766 on the reduction in motor conduction velocity induced by acrylamide. Animals received (50 m g / m l per kg i.p. every 48 h, in total eight doses) injected concurrently with either Org 2766 ( 5 0 / ~ g / k g s.c., 20 injections) (m, right, n = 8) or vehicle (r-q, n = 6). Control animals received saline and vehicle ([2], left, n = 6). Values given are the means_+ S.E.M. A N O V A R for repeated measures indicated a significant group effect over the period studied (F(2,19)=9.835, P = 0.001). * Indicates a significant decrease (P < 0.006, post hoc testing) between the control and vehicle group. ~ Indicates a significant difference (P = 0.05, post hoc testing) between the peptide treated and the vehicle group.

185

late time points when a deficit in conduction velocity would be expected. The delayed reduction in H-related sensory conduction induced by seven or eight doses of acrylamide was reversed in animals treated with Org 2766 (figs. 4, 5). It should be noted that this peptide effect was observed long after (72 days in fig. 4) the peptide treatment had been stopped. When, however, initiation of peptide treatment was delayed until 24 h after the last acrylamide injection, the beneficial effect could not be seen (fig. 6). H-related sensory conduction velocity values of 'late'-treated and non-treated animals were both still subnormal 92 days after the last dose of acrylamide. Motor nerve conduction velocity was slightly reduced by eight doses of acrylamide, but the reduction was less marked than the reduction in sensory conduction and a significant peptide effect was only observed on day 82 (fig. 7). A lower dose of acrylamide did not significantly reduce motor conduction velocity (data not shown).

4. Discussion

The trophic influences of melanocortins (ACTH and related peptides) on the central and peripheral nervous system have been known for some time. We report for the first time that an ACTH-(4-9) analogue, Org 2766, improved recovery from acrylamide neuropathy, which served as a model of a dying-back type of peripheral neuropathy. Org 2766 reversed the delayed persistent deficit in sensory conduction velocity but did not prevent the initial loss of motor coordination, as measured by landing foot spread and print length analysis. Normal motor coordination was reached 2-3 weeks after intoxication was stopped and this recovery was unaffected by the peptide. It has been suggested that some of the clinical symptoms of acrylamide neuropathy result from central nervous system damage, notably the loss of Purkinje cells (Cavanagh and Nolan, 1982). The rapid reversal of the initial symptoms, i.e. LFS and print length parameters, indicates that these functional abnormalities result from peripheral nerve pathology, rather than being due to loss of CNS neurons as described by Cavanagh and Nolan (1982). Gipon

et al. (1977) have also suggested that the deficit in motor coordination, as measured by rotarod performance, is correlated with peripheral nerve degeneration. The peripheral nerves in the 'recovered' rats clearly remained abnormal, since a reduced H-related sensory nerve conduction velocity was still observed after 109 days and could be permanent. The increased sensitivity to a second exposure to acrylamide also suggests a poor quality of repair. It is these residual deficits that are reduced by treatment with Org 2766. A similar pattern was observed for the effect of Org 2766 on the recovery from crush lesions; a deficit in H-related sensory nerve conduction velocity was observed in non-treated animals up to 214 days after the lesion (De Koning and Gispen, 1987). Treatment with Org 2766 starting on the day of crush and carried out at 48 h intervals for 8 days prevented the long-term deficit. Following cisplatin intoxication, recovery of H-related sensory conduction velocity was apparently complete (Gerritsen van der Hoop et al., 1988). However, as with acrylamide, these rats were more sensitive to a second exposure to the neurotoxin and treatment with Org 2766 during the initial intoxication prevented this effect. The greater sensitivity of sensory, as opposed to motor, nerves to acrylamide confirms previous reports (Sumner and Asbury, 1975; Hopkins and Gilliat, 1971). The selectivity is observed even between sensory and motor nerves of similar diameter and length (Sumner and Asbury, 1975). The relative insensitivity of the motor system to acrylamide decreased the possibility of detecting a beneficial peptide effect on motor conduction velocity in our experiments. Acrylamide has been reported to cause preferential degeneration of large diameter fibres, which are the fastest conducting and which include the Ia afferent system. Previous studies have attributed decreases in maximal conduction velocities in mixed peripheral nerves to a selective loss of these rapidly conducting fibres (FuUerton and Barnes, 1966; Hopkins and GiUiat, 1971). Using the H-reflex to measure conduction velocities, which enables Ia afferents to be measured selectively, we observed reduced conduction velocities

186 as a result of acrylamide intoxication. Our experiments do not distinguish between a selective loss of the fastest conducting Ia fibres and a decrease in the conduction velocity of individual fibres. Both are likely to occur. It has been shown that there is a selective loss of large diameter fibres of the Ia afferents (Sumner and Asbury, 1975) during intoxication. During recovery, distal degenerated axons are replaced by regenerated axons which have characteristic small diameters and short internodal lengths (Fullerton and Barnes, 1966). Both these characteristics of regenerated axons reduce conduction velocities. Furthermore, in proximal regions of affected nerves, decreased axonal diameters have also been reported (Gold et al., 1985). Thus a decrease in the conduction velocity of individual fibres would be expected during recovery, and the observed beneficial effects of Org 2766 during recovery indicates an action on repair processes. Org 2766 reverses the reduced sensory nerve conduction velocity observed in streptozotocin-induced diabetic rats and in cisplatin (an antitumour drug)-intoxicated rats (Van der Zee et al., 1989; Gerritsen van der Hoop et al., 1988). These actions of the peptide have been correlated with an increased proportion of large diameter myelinated fibres (Van der Zee et al., 1989; Gerritsen van der Hoop et al., 1989). Other studies on the effect of melanocortins on peripheral nerve regeneration have shown that there is a critical post-lesion period during which the melanocortin treatment is effective (Edwards et al., 1984). Treatment initiated 10 days after crush is totally ineffective. This may account for our finding that Org 2766 was ineffective when treatment was initiated 24 h after termination of intoxication. The total amount of acrylamide required to produce peripheral neuropathy can only be administered in divided doses, because of the acute toxicity of the agent. In divided dosing regimes, there is evidence that a repair response, i.e. regeneration, occurs during the intoxication period (Fullerton and Barnes, 1966). Therefore in our experiment, by analogy with the critical effective period following crush, peptide treatment initiated 24 h after cessation of intoxication may be too late to assist the repair of nerves damaged

during all but the last few days of the intoxication period. We currently are investigating whether Org 2766 can reserve neuropathies induced by other toxins. The ability of Org 2766 to improve repair in neuropathies with different biochemical origins suggests that the peptide is acting on general repair processes, as is also seen after mechanical damage. The potential value of Org 2766 in the treatment of human neuropathies has been shown in a recent clinical trial in which the peptide had beneficial effects in cisplatin-treated patients (Gerritsen van der Hoop et al., 1990). At present, the mechanism of action of acrylamide and Org 2766 is not understood. However, it is striking that these two agents have opposing effects on both conduction velocity and regenerative sprouting (Morgan-Hughes et al., 1974; Kemplay and Cavanagh, 1984; Verhaagen et al., 1987; Tanii et al., 1987). Progress in understanding these phenomena is therefore of value not only for the development of effective therapeutic agents but also for advancing our knowledge of the repair processes themselves.

Acknowledgements Part of this study was made possible by a Research Fellowship from the European Science Foundation. Grant No. SVF/87/002. Org 2766 was a kind gift from Organon Int. BV, Oss, The Netherlands. We would like to extend our thanks to Dr. Tuncay Altug of DETAM (Experimental Animal Unit, Istanbul) for supplying experimental animals for some of these experiments and to Paul Van der Most for preparation of the figures.

References Cavanagh, J.B. and C.C. Nolan, 1982, Selective loss of Purkinje cells from the rat cerebellum caused by acrylamide and the responses of fl-glucuronidase and fl-galactosidase, Acta Neuropathol. 58. 210. De Koning, P., P.M. Edwards and W.H. Gispen, 1988, Melanocortins: a potential role in the therapy of neurological disorders, in: Post-LesionNeural Plasticity, ed. L. Flohr (Springer-Verlag, Heidelberg) p. 595. De Koning, P. and W.H. Gispen, 1987, Org 2766 improves functional and electrophysiologic aspects of regenerating sciatic nerve in the rat, Peptides 8, 415.

187 De Medinacelli, L., W.J. Freed and R.J. Wyatt, 1982, An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks, Exp. Neurol. 77, 634. Edwards, P.M. and V.H. Parker, 1977, A simple, quantitative, sensitive and early assessment of the degree of disability of rats with acrylamide induced neuropathy by measurement of landing foot spread, Toxicol. Appl. Pharmacol. 40, 589. Edwards, P.M., C.E.E.M. Van der Zee, J. Verhaagen, P. Schotman, F.G.I. Jennekens and W.H. Gispen, 1984, Evidence that the neuropathic actions of a-MSH may derive from its ability to mimic the actions of a peptide formed in degenerating nerve stumps, J. Neurol. Sci. 64, 334. Fullerton, P.M. and J.M. Barnes, 1966, Peripheral neuropathy in rats produced by acrylamide, Br. J. Industr. Med. 23, 210. Gerritsen van der Hoop, R., P. De Koning, E. Boven, J.P. Neijt, F.G.I. Jennekens and W.H. Gispen, 1988, Efficacy of the neuropeptide Org 2766 in the prevention and treatment of cisplatin induced neurotoxicity in rats, European J. Cancer Clin. Oncol. 24, 637. Gerritsen van der Hoop, R., J.P. Neijt, F.G.I. Jennekens and W.H. Gispen, 1989, Protection from cisplatin induced neuropathy in rats by the ACTH(4-9) analog Org 2766, in: Proc. Peripheral Nerve Development and Regeneration, VoL 19, ed. D. Pleasure (Liviana Press, Springer Verlag, Heidelberg) p. 145. Gerritsen van der Hoop, R., C.J. Vecht, M.E.L. Van den Burg, A. Elderson, W. Haanstra, W. Boogerd, J.J. Heijmans, E.P. Vries, J.C. Van Houwelingen, F.G.I. Jennekens, W.H. Gispen and J.P. Neijt, 1990, An ACTH(4-9) analog prevents cisplatin induced neurotoxicity in ovarian cancer patients, N. Engl. J. Med. 232, 89. Gipon, L., P. Schotman, F.G.I. Jennekens and W.H. Gispen, 1977, Polyneuropathies and CNS protein metabolism. I. Description of the acrylamide syndrome in rats, Neuropathol. Appl. Neurobiol. 3, 115. Gold, B.G., J.W. Griffin and D.L. Price, 1985, Slow axonal transport in acrylamide neuropathy: Different abnormalities produced by single-dose and continious administration, J. Neurosci. 5, 1755. Hopkins, A.P. and R.W. Gilliat, 1971, Motor and sensory conduction velocity in the baboon: normal values and changes during acrylamide neuropathy, J. Neurol. Neurosurg. Psychiat. 34, 415. Kemplay, S. and J.B. Cavanagh, 1984, Effects of acrylamide and some other sulphydryl reagents on spontaneous and pathologically induced terminal sprouting from motor end plates, Muscle Nerve 7, 101. Le Quesne, P.M., 1980, Acrylamide, in: Experimental Clinical Neurotoxicology, eds. P.S. Spencer and H.H. Schaumburg (Williams and Wilkins, Baltimore) p. 309.

Morgan-Hughes, J.A., S. Sinclair and J.H.J. Durston, 1974, The pattern of peripheral nerve regeneration induced by crush in rats with severe acrylamide neuropathy, Brain 97, 235. Schaumburg, H.H., H.M. Wisniewski and P.S. Spencer, 1974, Ultrastructural studies of the dying-back process. I. Peripheral nerve terminal and axon degeneration in systemic acrylamide intoxication, J. Neuropathol. Exp. Neurol. 2, 260. Spencer, P.S. and H.H. Schaumburg, 1974a, A review of acrylamide neurotoxicity. Part I. Properties; uses and human exposure, Can. J. Neurol. Sci. 1, 143. Spencer, P.S. and H.H. Schaumburg, 1974b, A review of acrylamide neurotoxicity. Part II. Experimental animal neurotoxicity and pathologic mechanisms, Can. J. Neurol. Sci. 1, 151. Stanley, E.F., 1981, Sensory and motor nerve conduction velocities and the latency of the H-reflex during growth of the rat, Exp. Neurol. 71,497. Strand, F.L., K.J. Rose, J.A. King, A.C. Segara and L.A. Zuccarelli, 1989, ACTH modulation of nerve development and regeneration, Prog. Neurobiol. 33, 45. Strand, F.L. and C.M. Smith, 19~6, LPH, ACTH, MSH and motor systems, in: Neuropeptides and Behaviour, Vol. 1, eds. D. De Wied, W.H. Gispen and T.B. Greidanus (Pergamon Press, Oxford) p. 245. Sumner, A.J., 1980, Axonal polyneuropathies, in: The Physiology of Peripheral Nerve Disease, ed. A.J. Sumner (W.B. Saunders, Philadelphia) p. 340. Sumner, A.J. and A.K. Asbury, 1975, Physiological studies of the dying-back phenomenon: muscle stretch afferents in acrylamide neuropathy, Brain 98, 91. Suzuki, K. and L. Pfaff, 1973 Acrylamide neuropathy in rats. An electron microscopic study of degeneration and regeneration. Acta Neuropathol. 24, 197. Tanii, H., S. Kato and K. Hashimoto, 1987, Inhibition of neurite outgrowth from retinal explant culture by acrylamide and related compounds, Semin. Toxicity Mech. 1, 117. Van der Zee, C.E.E.M., R. Gerritsen van der Hoop and W.H. Gispen, 1989, Beneficial effects of the peptide Org 2766 (ACTH 4-9 analog) in the treatment of peripheral neuropathy in streptozocin-diabetic rats, Diabetes 38, 225. Verhaagen, J., P.M. Edwards, F.G.I. Jennekens, P. Schotman and W.H'. Gispen, 1987, Early effects of an ACTH(4-9) analog on regenerative nerve sprouting demonstrated by the use of neurofilament-binding antibodies isolated from serum raised by a-MSH immunization, Brain Res. 404, 142. Wolhlfart, G., 1975, Collateral regeneration from residual motor nerve fibers in amyotrophic lateral sclerosis, Neurology 7, 124.