Introduction

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Prosaposin has been described as a neurotrophic factor. Previously, we defined the neurotrophic sequence within prosaposin as a 12-amino acid region in the N-terminal portion of the saposin C domain (O'Brien et al., 1994, 1995). A number of peptides have been synthesized that encompass this region and they are named prosaptides (O'Brien et al., 1995; Campana et al., 1996, 1998b; Hiraiwa et al., 1997a) or, as we name them herein, Prosaptide (trademark of Myelos Corporation) peptides. Prosaposin and Prosaptide peptides have been shown to stimulate neurite outgrowth and choline acetyl transferase activity in vitro (O'Brien et al., 1995; Kotani et al., 1996b; Qi et al., 1996, 1999) and to prevent neuronal cell death induced by serum deprivation (O'Brien et al., 1995; Kotani et al., 1996b). More recently, prosaposin and Prosaptide TX14(A) have been shown to prevent apoptosis of cerebellar granule cells (Tsuboi et al., 1998) and Schwann cells (Campana et al., 1999). In vivo, both prosaposin and an 18-amino acid prosaposin-derived peptide prevented the ischemia-induced death of hippocampal neurons and the associated cognitive impairment in gerbils (Sano et al., 1994; Kotani et al., 1996b) and in rats (Igase et al., 1999). Prosaposin also has been shown to reduce the lesion size and concomitant spatial learning deficit in a cortical stab wound model (Hozumi et al., 1999). In the periphery, prosaposin and Prosaptide peptides facilitate sciatic nerve regeneration (Kotani et al., 1996a), prevent paclitaxel-induced peripheral thermal hypoalgesia (Campana et al., 1998a), and ameliorate diabetic neuropathy in rats (Calcutt et al., 1997, 1999).

The amelioration of neurodegeneration in the peripheral nervous system (PNS) by prosaposin and Prosaptide peptides, in vivo, may be by direct action of prosaposin on neurons through neuroprotection and/or neurite outgrowth. Additionally, the effects may be due to the myelinotrophic action of prosaposin and prosaposin-derived peptides. Prosaposin and Prosaptide peptides have been shown to enhance the synthesis of sulfatide in Schwann cells and oligodendrocytes (Hiraiwa et al., 1997a), the expression of UDP-galactose:ceramide galactosyltransferase and the transcription of myelin protein zero (P₀) in Schwann cells (Hiraiwa et al., 1997a, 1999).

The molecular mechanism(s) that mediate the neurotrophic and myelinotrophic actions of prosaposin and Prosaptide peptides is under investigation. Prosaposin and Prosaptide peptides enhance the phosphorylation of extracellular signal-regulated kinases 1 and 2 in PC12 (Campana et al., 1996) and Schwann cells (Hiraiwa et al., 1997a; Campana et al., 1998b, 1999). Prosaptide peptides also stimulate phosphorylation of phosphotidylinositol-3-kinase (PI3K), the adaptor protein Shc, and Akt protein kinase in Schwann cells (Campana et al., 1998b, 1999). The inhibition of PI3K blocked the antiapoptotic effect of Prosaptide peptides in cerebellar granule cells (Tsuboi et al., 1998) and Schwann cells (Campana et al., 1999). In addition, inhibition of mitogen-activated protein kinase kinase diminished both the neuroprotective (Campana et al., 1999) and myelinotrophic (Campana et al., 1998b) actions of Prosaptide peptides. Furthermore, the phosphorylation of extracellular signal-regulated kinases 1 and 2 was blocked by the PI3K inhibitor wortmannin, supporting the hypothesis that a PI3K/MAP kinase pathway mediates the neurotrophic and myelinotrophic effects of prosaposin and peptides derived thereof. The activation of signal transduction molecules by, and the trophic actions of, prosaposin and Prosaptide peptides are mediated by a pertussis toxin-sensitive G_o protein-coupled receptor (Hiraiwa et al., 1997b; Campana et al., 1998b) and treatment of neuronal cells with Prosaptide peptides stimulates [³⁵S]guanosine-5'-O-(3-thio)triphosphate (GTPS) binding to cell membranes. Furthermore, pertussis toxin has been shown to inhibit Prosaptide peptide-induced sulfatide synthesis (Campana et al., 1998b) and neurite outgrowth (Misasi et al., 1998).

Whereas strikingly positive effects of the prototypical prosaposin-derived peptide Prosaptide TX14(A) have been demonstrated in the peripheral nervous system of rats (Calcutt et al., 1997, 1999; Campana et al., 1998a), we have had limited success treating central nervous system (CNS) lesions in rats with i.c.v.-administered Prosaptide TX14(A) (K. Tsuboi, J. S. O'Brien, and C. Shults, unpublished observation; E. M. Taylor, J. S. O'Brien, and M. H. Tuszynski, unpublished observation). We have hypothesized that this may be due to enhanced degradation of Prosaptide TX14(A) in the CNS compared with the peripheral nervous system. It is known that the endothelial and epithelial cells of the blood-brain barrier are enriched with proteases (Brownlees and Williams, 1993; Brownson et al., 1994; Egleton and Davis, 1997), thus presenting both an enzymatic and physical barrier to the entry of proteins and peptides into the CNS. We have examined the in vivo stability of Prosaptide TX14(A) and shown that it is, indeed, rapidly degraded in vivo. In an attempt to design Prosaptide peptides with increased stability in vivo we have, based on considerations of known protease cleavage sites (Pardridge, 1991), designed three biologically active Prosaptide peptides; Prosaptide TX15-2 displays increased stability in brain.

In addition to examining stability in vivo, we have examined the biological activity of 15 novel Prosaptide peptides with neurite outgrowth and GTPS-binding assays and have sought to describe some of the sequence requirements for biologically active Prosaptide peptides. Finally, with the aim of developing Prosaptide peptides that are effective when given systemically, we have examined the ability of two prosaptides, Prosaptide TX14(A) and Prosaptide TX15-2, to cross the blood-brain barrier.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (250-300 g) were provided by Harlan Industries (San Diego, CA). All animal experiments were conducted in accordance with the National Institutes of Health Guide on Care and Use of Laboratory Animals.

Prosaptides. Anaspec (San Jose, CA) provided Prosaptide peptides at >95% purity. Prosaptide peptides were iodinated with iodobeads (Pierce, Rockford, IL) and Na¹²⁵I (NEN, Boston, MA) with 0.25 mCi/12.5 µg peptide and an 8-min labeling time. Iodinated peptides were purified from unincorporated ¹²⁵I with a 5-ml Sephadex G-10 column equilibrated in PBS. The radiopurity of iodinated peptides was assessed with HPLC. The specific activity of iodinated peptides was 5.5 to 6.5 mCi/mg. Human serum albumin and ^{99 M}Tc were purchased from MediPhysics (San Diego, CA) and albumin was labeled according to the manufacturer's instructions.

Neurite Outgrowth. Cell culture reagents were purchased from Life Technologies (Grand Island, NY). The mouse neuroblastoma cell line NS20Y was a generous gift from Drs. T. Taketomi and K. Uemura (Shinshi University, Matsumoto, Japan). Cells were maintained in complete media [Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal calf serum, 10 U/ml penicillin, 100 µg/ml streptomycin, and 1.1 mg/ml sodium pyruvate] at 37°C under humidified 5% CO₂.

Stimulation of GTPS Binding. SHSY5Y cells were the generous gift of Dr. Stephen Fisher (University of Michigan, Ann Arbor). The assay was performed as previously described (Thomas et al., 1995; Hiraiwa et al., 1997b). SHSY5Y cell membrane preparations (50-100 µg protein) were incubated with 125 µCi of [³⁵S]GTPS (NEN; 1000-1500 Ci/mmol). GDP (3 µM) was added to amplify the difference between ligand-stimulated and background binding. Unlabeled GTPS (10 nM) also was added to define nonspecific binding and this value was subtracted to give specific binding. All assays were performed in duplicate.

In Vivo Stability Studies. Rats were anesthetized with 65 mg/kg sodium pentobarbital (Abbott Laboratories, Santa Clara, CA) and the right jugular vein and left carotid artery exposed. ¹²⁵I-peptide (2 × 10⁶) was injected into the jugular vein in a volume of 100 µl of PBS. At various times after injection, serum and brain were collected. Peptides were extracted from brain by homogenization in 5 ml of ice-cold extraction solution. Prosaptide TX14(A) was extracted with 56% acetonitrile in 0.1% trifluoroacetic acid (TFA); Prosaptide TX15-2 was extracted with 87.5% ethanol in 0.25 M HCl containing 10 mM each of N-ethylmaleimide, 1,10-phenanthroline, EDTA, and D-thyroxine; Prosaptide prosaptide Q, and Prosaptide prosaptide S were both extracted with 56% acetonitrile in 0.1% TFA containing 10 mM N-ethylmaleimide, 1,10-phenanthroline, EDTA, and D-thyroxine. Homogenates were centrifuged and supernatants collected. Supernatants and serum were lyophilized overnight, resuspended in 1 ml 0.1% TFA, filtered, and applied to a C18 reversed phase HPLC column. Fractions (1 ml) were collected and radioactivity counted. Processing controls were prepared by adding ¹²⁵I-prosaptide (1 × 10⁶ cpm) to blood or brain in vitro and then processing them as described above. Such controls enabled correction of the in vivo degradation for any degradation caused by processing.

Blood-Brain Barrier Transport. Transport experiments were conducted according to the methods of Blasberg et al. (1983) and Patlak et al. (1983), as described by Banks and Kastin (1993). ¹²⁵I-peptides (2 × 10⁶ cpm) and ^{99 M}Tc-albumin (2 × 10⁶; plasma marker) were injected together i.v. as described above. At various times, serum and brain were collected and radioactivity measured with a gamma counter. The log radioactivity in serum was plotted against time and from this, exposure times were calculated as previously described (Blasberg et al.,1983; Patlak et al., 1993; Banks and Kastin, 1993). The brain/blood ratios for ¹²⁵I and ^{99 M}Tc were calculated and the ¹²⁵I ratios were corrected for the amount of serum present in brain as measured with ^{99 M}Tc-albumin (¹²⁵I  ^{99 M}Tc). These corrected ¹²⁵I brain/blood ratios were plotted against the exposure time and the rate constant for influx (K_i) of Prosaptide peptides into brain was taken from the slope of the line. To examine the specificity of Prosaptide peptide transport into brain, 10 µg of unlabeled Prosaptide peptide was included in the injection mixture in some experiments, which was equivalent to 100-fold molar excess.

	Results

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Figure 1 shows the 14-amino acid sequence of the neurotrophic region of prosaposin and the consensus sequence obtained from our previous examination of interspecies amino acid conservation (O'Brien et al., 1995). Based on this information and known enzyme cleavage sites, we investigated the sequence requirements for bioactive, stable prosaposin-derived peptides. The Prosaptide peptides synthesized for these investigations are listed in Table 1.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Sequence of the neurotrophic region from human prosaposin and the consensus sequence based on interspecies conservation. Redrawn with permission from O'Brien et al. (1995).

All Prosaptide peptides that are designated as biologically active promoted neurite outgrowth in NS20Y cells (Table 1). The minimal length required for biological activity was 11 amino acids from L18 of the N-terminal of saposin C. With respect to the consensus sequence shown in Fig. 1, mutation of N22 to D22 (Prosaptide 14 M1) abolished biological activity in all assays. L18 could be changed to isoleucine (Prosaptide TX15-2) without loss of bioactivity; however, changing L18 to S18 (Prosaptide 14 M2) abolished biological activity in all assays. I28 can be changed to L28 [Prosaptide TX14(A), 12A, 11A, and 11] or S28 (Prosaptide 14 M3) and these Prosaptide peptides retained biological activity in all assays.

Investigation of other amino acid positions revealed that K17 can be changed to D-alanine [Prosaptide TX14(A), TX14(C), TX14(D), TX15-1, TX15-2, prosaptide S, and prosaptide Q] and I19 can be changed to a D-leucine (Prosaptide 12A) with no change in biological activity. Mutation of K23 to alanine [Prosaptide TX14(A)], serine (Prosaptide prosaptide S), or glutamine (Prosaptide prosaptide Q) caused no loss of biological activity; however, changing K23 to glycine [Prosaptide TX14(C), TX14(D), and TX15-3] caused a complete loss of biological activity. L29 could be changed to Y29 [Prosaptide TX14(A) and 12A], to S29 (Prosaptide TX15-2, prosaptide S, and prosaptide Q) or to G29 (Prosaptide 14 M3) without loss of biological activity. These observations show that whereas there is a requirement for at least three amino acids after E25 for biological activity (compare Prosaptide 11 to Prosaptide 10 and 9), the exact nature of these amino acids is not critical.

Selected peptides were further examined for their ability to stimulate the binding of GTPS to cell membranes prepared from the SHSY5Y neuroblastoma cell line (Fig. 2). The biologically active Prosaptides TX14(A), TX15-1, TX15-2, prosaptide S, prosaptide Q, 12A, and 11A stimulated GTPS binding by 50% (Fig. 2). The biologically inactive Prosaptides TX14(C), TX14(D), 14 M1, TX15-3, and 10 did not stimulate GTPS binding to SHSY5Y cell membranes.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Stimulation of specific GTPS binding to SKNMC cell membranes by Prosaptide peptides. SKNMC cell membranes were incubated with 10 ng/ml selected peptides in the presence of [35S]GTPS. All assays were performed in duplicate. Data shown are means ± S.E.

Figure 3 shows potential enzyme cleavage sites within the wild-type neurotrophic region of prosaposin and the Prosaptide TX14(A) sequences. Preliminary data showed that, in vitro, Prosaptide TX14(A) is more stable than the wild-type prosaptide; this is presumably due to the presence of a D-alanine in the second position (H.-C. Chan and J. S. O'Brien, unpublished data). Such a substitution has been shown in other peptides to reduce the susceptibility to aminopeptidase N activity (Pardridge, 1991). Figure 4A shows the in vivo stability of Prosaptide TX14(A) in serum. Within 2 min, the proportion of radioactivity that could be identified as intact Prosaptide TX14(A) had decreased to ~65%. Degradation continued until, at 60 min, <30% of Prosaptide TX14(A) was intact. In brain, Prosaptide TX14(A) degraded rapidly. By 10 min, <30% of the radioactivity in brain was present as intact Prosaptide TX14(A) and by 60 min, there was no detectable Prosaptide TX14(A) in brain. Prosaptide TX15-2, with a sequence based on wild-type prosaposin, had no internal A, which is a potential target of dipeptidyl dipetidase, nor any internal L, which is a target of both enkephalinase and angiotensin-converting enzyme (Pardridge, 1991). The in vivo stability of Prosaptide TX15-2 also is shown in Fig. 4. It was rapidly degraded in serum and, at 10 min, <10% was intact (Fig. 4A). However, in brain, the proportion of intact Prosaptide TX15-2 increased over time (Fig. 4B). At 2 min, ~20% was intact whereas at 60 min, nearly 50% of radioactivity in brain was identified as intact Prosaptide TX15-2. The increased degradation of Prosaptide TX15-2 in serum compared with ProsaptideTX14(A) may have been due to the presence of two lysines (K23 and K26) that were not present in Prosaptide TX14(A); lysine is a target of trypsin (Pardridge, 1991).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Putative sites of proteolytic degradation in Prosaptide wild-type (A) and Prosaptide TX14(A) (B).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   In vivo stability of Prosaptide TX14(A) (A), Prosaptide TX15-2 (B), Prosaptide prosaptide S (C), and Prosaptide prosaptide Q (D) in serum () and brain (). Iodinated Prosaptides TX14(A), TX15-2, prosaptide S, and prosaptide Q were injected i.v. At various times after injection, serum and brain were taken and processed for HPLC analysis. The percentage of intact Prosaptide peptide was calculated from the elution profile of radioactivity, corrected for degradation that occurred during processing, and plotted against time. The equations of the lines are A, serum: y = axb, where a = e4.4 and b = 0.4, r2 = 0.69, n = 5 and brain: y = 24.ln(x) + 93, r2 = 0.95, n = 5; B, serum: y = 13.ln(x) + 60, r2 = 0.51, n = 5 and brain: y = 48x + 22, r2 = 0.84, n = 5; C, serum: y = axb, where a = e5.7 and b = 1.0, r2 = 0.86, n = 5 and brain: y = 30.ln(x) + 112, r2 = 0.84, n = 5; D, serum: y = axb, where a = e4.5 and b = 0.5, r2 = 0.99, n = 5 and brain: y = axb, where a = e4.0 and b = 0.6, r2 = 0.99, n = 5.

In an attempt to decrease serum degradation and preserve brain stability, we designed Prosaptide prosaptide S and Prosaptide prosaptide Q, based on Prosaptide TX15-2. Neither Prosaptide prosaptide S nor Q displayed the increased brain stability of Prosaptide TX15-2 (Fig. 4B). However, Prosaptide prosaptide S may have had delayed degradation in serum compared with Prosaptide TX15-2 and TX14(A) (Fig. 4A). At 2 min, Prosaptide prosaptide S was ~90% intact and at 10 min ~45% of the radioactivity was present as intact Prosaptide prosaptide S.

Figure 5 shows the serum clearance and blood-brain barrier permeability of Prosaptide TX14(A). Figure 6 shows the serum clearance and transport of Prosaptide TX15-2 across the blood-brain barrier. Prosaptide TX14(A) and TX15-2 were cleared from serum in a linear manner over time with a half-life of 4.2 ± 0.3 and 2.5 ± 0.3 min, respectively. Prosaptide TX14(A) crossed the blood-brain barrier with a K_i of 1.25 × 10³ ml g¹ min¹, whereas Prosaptide TX15-2 crossed with a K_i of 4.86 × 10³ ml g¹ min¹. Passage of neither peptide was inhibited by the inclusion of excess unlabeled peptide in the injection mix (Figs. 5B and 6B); in the presence of excess unlabeled peptide the K_i was 1.13 × 10³ and 6.60 × 10³ ml g¹ min¹ for Prosaptides TX14(A) and TX15-2, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Serum clearance of Prosaptide TX14(A) (A) and transfer of Prosaptide TX14(A) across the blood-brain barrier (B). Iodinated peptides were injected i.v. At various times after injection, the radioactivity was measured in serum and brain. From the serum clearance, exposure time was calculated. The 125I brain/blood ratio was corrected for 99 MTc brain/blood ratio and plotted against exposure time. Ki in the absence () or presence () of unlabeled Prosaptide TX14(A) was calculated from the slope of the line.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Serum clearance of Prosaptide TX15-2 (A) and transfer of Prosaptide TX15-2 across the blood-brain barrier (B). Iodinated peptides were injected i.v. At various times after injection, the radioactivity was measured in serum and brain. Exposure times were calculated from the serum clearance. The 125I brain/blood ratio was corrected for 99 MTc brain/blood ratio and plotted against exposure time. Ki in the absence () or presence () of unlabeled Prosaptide TX15-2 was calculated from the slope of the line.

	Discussion

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In this article, we presented data on the minimal sequence requirements and amino acid sequence determinants of prosaptide biological activity. In addition, we measured the degradation, in serum and brain, of Prosaptide TX14(A), the prototypical Prosaptide peptide, and of three newly designed peptides, one of which, Prosaptide TX15-2, demonstrated increased stability in brain. Finally, we showed that both Prosaptide TX14(A) and TX15-2 crossed the blood-brain barrier. This work provides a basis for the design of Prosaptide peptides for the treatment of CNS disorders.

Previously, we described a 12-amino acid prosaposin-derived peptide (18-29) that stimulated neurite outgrowth in NS20Y cells (O'Brien et al., 1995). Subsequently, Qi and coworkers (1996) found that a 10-amino acid peptide from 22 to 31 (NKTEKEILDA) demonstrated neuritogenic activity. Based on these two reports, Qi et al. (1996) proposed that the minimal length was likely to be a 7- to 11-amino acid sequence incorporating amino acids 22 to 29. Herein, we largely confirm this hypothesis and describe an 11-amino acid peptide (18-28) that demonstrates neuritogenic activity (Prosaptide 11: LIDNNKTEKIL). In addition, this peptide prevents cell death in serum-deprived NS20Y cells and stimulates binding of GTPS to cell membranes, activities possessed by prosaposin, saposin C, and longer prosaposin-derived peptides. Collectively, these results suggest that the minimal length required for Prosaptide peptide activity may be <11 amino acids.

We have investigated the effect of amino acid identity on the biological activity of Prosaptide peptides. It is known that mutation of N21 to D21 abolishes the biological activity of a 22-amino acid peptide (O'Brien et al., 1995). Herein, we show, by changing N22 to D22 (Prosaptide 14 M1), that N22 is also essential for biological activity of Prosaptide peptides. In contrast, Qi et al., (1999) showed that mutation of N21 to D21 in the full-length saposin C protein did not effect the ability of saposin C to stimulate neurite outgrowth. Further analysis of sequence requirements showed that mutation of L18 to S18 (Prosaptide 14 M2) caused a loss of biological activity. Again, there is disagreement with data presented by Qi et al. (1996); they showed that a peptide comprising residues 22 to 31 of saposin C, and therefore not containing L18, was bioactive. In this study, we found that another amino acid that is sensitive to mutation is K23. Whereas the biological activity of prosaptides was unchanged if K23 was changed to alanine, serine, or glutamine, it was abolished if K23 was changed to glycine. These results show that receptor binding or activation most likely requires an amino acid in this position with more than a simple hydrogen side group. Qi et al. (1999) have suggested that K23 is not required based on interspecies comparisons. The lack of agreement between our results and those of Qi et al. (1996, 1999) suggests that the sequence requirements for bioactivity of Prosaptide peptides may be stricter than that of the full-length saposin C or prosaposin molecules. Certainly, the effect of specific mutations is context dependent. Currently, we investigating the strict consensus residues for bioactivity by deletion and "alanine-walking" experiments.

We assessed the biological activity of Prosaptide peptides with neurite outgrowth, cell death prevention, and GTPS-binding assays. All of the prosaptides that stimulated neurite outgrowth and prevented cell death also stimulated GTPS binding. This suggests that a common site of interaction with the cell may mediate the full range of Prosaptide peptide actions. This site of interaction is presumably the putative prosaposin receptor described thus far as a G_o protein-coupled receptor (Hiraiwa et al., 1997b; Campana et al., 1998b).

The lack of a robust effect of Prosaptide TX14(A) in rat models of CNS degeneration (K. Tsuboi, J. S. O'Brien, and C. Shults, unpublished data; E. M. Taylor, J. S. O'Brien, and M. H. Tuszynski, unpublished data) contrasts with the robust effects seen in the PNS. We hypothesized that this was due to a preferential degradation of Prosaptide TX14(A) in the CNS. The data presented herein demonstrated that Prosaptide TX14(A) is rapidly degraded in the brain. Serum, which is presumably the source for Prosaptide TX14(A) effects in the PNS, is associated with a slower degradation rate.

Using available information on the molecular targets of a number of blood-brain barrier proteases, we designed Prosaptide TX15-2. Prosaptide TX15-2 apparently became enriched in brain over time, whereas it was rapidly degraded in serum. This suggests that although it is susceptible to serum proteases, it is less susceptible to blood-brain barrier proteases and once it enters the brain it may be protected from further degradation. This result prompted us to design Prosaptide prosaptide S and Prosaptide prosaptide Q. Unfortunately, these peptides did not display the same level of stability in brain as Prosaptide TX15-2.

There is some evidence that prosaposin-derived peptides may be effective in the treatment of CNS disorders. An 18- amino acid peptide prevents nerve damage and associated cognitive impairment in both gerbil and rat models of ischemia when administered i.c.v. (Kotani et al. 1996b; Igase et al., 1999). The peptide used has the sequence LSELIINNATEELLIKGL and so contains putative sites for degradation by enkephalinase, endopeptidase, dipeptidyl dipeptidase, and aminopeptidase N. It seems likely that this peptide is degraded in the CNS, however, this is yet to be confirmed. The fact that this peptide ameliorates ischemic brain damage in gerbils and rats may indicate that it is not degraded or that the rate of degradation is slower than that of the Prosaptide peptides described herein. Alternatively, assuming that degradation of the peptide occurs, amelioration of ischemic damage may require small concentrations of Prosaptide peptide.

Systemic treatment of CNS disorders is highly desirable and passage of any potential therapeutic across the blood-brain barrier is a requirement for the systemic treatment of the CNS. Herein, we showed that both Prosaptide TX14(A) and TX15-2 cross the blood-brain barrier by a mechanism that was not inhibited by excess unlabeled peptide. This suggests that Prosaptide peptides are transported across the blood-bran barrier by a nonspecific mechanism. However, it is possible that 10 µg of unlabeled peptide per animal was not enough to inhibit a specific mechanism.

Whereas the rate of influx of Prosaptide TX15-2 was ~5 times faster than that for Prosaptide TX14(A), it is unclear whether this rate is entirely reflective of Prosaptide TX15-2 transport across the barrier. The time course of the uptake experiments is 10 min and during this time much degradation of Prosaptide TX15-2 occurred in serum. Therefore, some of the uptake of radioactivity may be uptake of metabolites. In conclusion, the data presented forms a strong basis for the design of stable blood-brain barrier-permeable Prosaptide peptides for the treatment of CNS disorders.