Method of inducing sleep with GHRH complementary peptide compositions

The present invention provides a method of inducing sleep in a host which includes administering to the host synthetic peptides, as well as fragments and analogs thereof, complementary to growth hormone releasing hormone.

BACKGROUND OF THE INVENTION 
This invention relates to a method of inducing sleep in a host which 
comprises administering synthetic Growth Hormone Releasing Hormone 
complementary peptides to the host. 
Growth Hormone Releasing Hormone (GHRH), also known as Growth Hormone 
Releasing Factor, is a hypothalamic peptide which positively regulates the 
synthesis and secretion of growth hormone in the anterior pituitary. GHRH 
was originally isolated and structurally characterized from human 
pancreatic tumors that caused acromegaly. Since then, GHRH has been 
isolated from several different species, including rat, pig, cow, man, 
sheep, and goat (Bohlen et al., Biochem Biophys. Res. Comm. 125: 
1005-1012, 1984). 
Both the function and nature of GHRH have been widely studied. 
The amino acid sequence of rat hypothalamic GHRH has been determined 
(Bohlen et al., 1984, supra). Also, the cDNA to rat GHRH has been reported 
(Mayo et al., Nature 314: 464-467, 1985). Human GHRH is reported to have 
high homology with rat GHRH and a GH-stimulating effect on the rat 
pituitary gland (Baird et al., Neuroendocrinology 42: 273-276, 1986). Ling 
et al., Biochem. Biophys. Res. Comm. 123: 854-861, (1984) tested the 
capacity of a series of C-terminal deleted analogs of synthetic human GHRH 
to release growth hormone and report that the minimal biologically 
important core of GHRH with full intrinsic activity comprises the fragment 
(3-21). 
Biro suggested (Biro, Medical Hypotheses 7:969-1007, 1981) that 
protein-protein interactions are based on binding of restricted portions 
of the proteins that are primarily formed by "informational complementary 
(ic)" amino acids. He also suggested that these specific complementary 
amino acids are encoded by complementary DNA sequences; further, 
interaction between complementary amino acid sequences would occur both in 
parallel and in antiparallel alignment of the peptides. Although Biro 
(1981) investigated only the 5'-3' direction, his data also support the 
significance of the 3'-5' direction by emphasizing the importance of 
palindrome nucleic acid sequences in encoding the specifically interacting 
peptide sequences. 
Model peptides designed to have minimal homology to the naturally occurring 
peptide but having the same hydropathic pattern have been demonstrated to 
exhibit biological activity (See, e.g., Kaiser and Kezdy, Science, 223: 
249-255, 1984). Blalock and Smith reported that codons for hydrophilic and 
hydrophobic amino acids on one strand of DNA are complemented by codons 
for hydrophobic and hydrophilic amino acids on the other DNA strand, 
respectively, and that codons for slightly hydrophilic ("uncharged") amino 
acids are complemented by codons for amino acids of the same character 
(Biochem. Biophys. Res Comm. 121: 203-207, 1984). These workers theorize 
that the two complementary strands of the DNA encode two peptides having 
hydropathic anti-complementarity. It has been reported that the 
hydropathic anti-complementarity of a number of amino acids (and hence 
that of the peptides) based on the genetic code occurs when complementary 
codons are read in the 5'-3' as well as in the 3'-5' direction (Bost et 
al., Proc. Natl. Acad. Sci. 82: 1372.1375, 1985a., Bost et al., Biochem. 
Biophys. Res. Comm. 128: 1372-1380, 1985b; Blalock and Bost, Biochem. J. 
234: 679-683, 1986). 
Bost et al., 1985a, supra, have reported that a peptide ("HTCA") 
corresponding to the complementary (5'-3') RNA sequence of ACTH (1-24) 
mRNA is capable of binding synthetic ACTH as determined by ELISA. Blalock 
and Bost, supra, have reported that both 3'-5' and 5'-3'complementary 
peptides bind .sup.125 I-ACTH in a solid-phase binding assay. Similar 
binding was reported for 5'-3' complementary peptides of .gamma.-endorphin 
(Bost et al., 1985a, supra). Antibodies raised against the complementary 
peptide, HTCA, have been reported to stimulate corticosterone secretion of 
adrenocortical cells in vitro (Bost et al., 1985a, supra). It is also 
reported that, using the same antibodies in immune affinity 
chromatography, the ACTH-receptor was purified and its molecular structure 
and .sup.125 I-ACTH binding characteristics were determined (Bost and 
Blalock, Molec. Cell. Endocrinol. 44: 1-9, 1986). According to Bost et 
al., 1985b, supra, messenger RNA sequences complementary to the mRNA 
sequences for the receptors of epidermal growth factor (EGF), 
interleukin-2 (IL- 2) and transferrin (TF) encode peptides having high 
homologies with the amino acid sequence of their respective ligands, if 
the transcription is carried out in 3'-5' direction. Gorcs et al., 
Peptides, 7: 1137-1145 (1986) report possible recognition the GnRH 
receptor by an antiserum against a peptide encoded by nucleotide sequence 
complementary to mRNA of a GnRH precursor peptide. 
GHRH peptides have been reported to have applications in the fields of 
animal husbandry, clinical medicine and basic research For example, it was 
determined that administration of human GHRH to lactating holstein cows 
increases the secretion of growth hormone consistently and causes an 
apparent increase in feed to milk conversion (Enright et al., J. Dairy 
Sci. 69: 344-351, 1986). GHRH peptides are useful in vitro e.g., as unique 
research tools for understanding how growth hormone secretion is regulated 
at the pituitary level and are also be useful in vivo, e.g., to treat 
symptoms related to growth hormone deficiencies to increase the rate and 
extent of growth in commercial animals, to increase milk yield in 
commercial animals. 
SUMMARY OF THE INVENTION 
The present invention provides a method of inducing sleep by use of 
biologically important synthetic peptides complementary to GHRH, and 
fragments and analogs thereof. 
One method of inducing sleep in a host in accordance with the present 
invention comprises the step of administering to a host a predetermined 
quantity of a composition comprising an effective amount of a synthetic 
peptide having the formula: 
EQU ( H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-Y 
wherein Y is OH or NH.sub.2 or a non-toxic salt thereof, in combination 
with a pharmacologically acceptable carrier therefor. This peptide is 
complementary in the 3'-5' direction to residues 14-25 of GHRH and is 
hereinafter referred to as 3'-5' peptide. 
Another method of inducing sleep in accordance with the present invention 
comprises the step of administering to a host a predetermined quantity of 
a composition comprising an effective amount of a synthetic peptide having 
the formula: 
EQU H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Leu-Pro-Val-His-Asp-P 
he-Val-Gln-Gln-Phe-Ala-Gly-Ile-Y 
wherein Y is OH or NH.sub.2 or a non-toxic salt thereof in combination with 
a pharmacologically acceptable carrier therefor. This peptide is 
complementary in the 5'-3 direction to residues 18-43 of GHRH (hereinafter 
referred to as 5'-3' CP). 
Fragments and analogs of the 3'-5' and 5'-3' peptides are also useful in 
the practice of the claimed invention. 
Thus, the GHRH complementary peptide for use in the present invention as 
well as fragments and analogs thereof, may be administered in vivo to 
hosts, to induce sleep.

DETAILED DESCRIPTION OF THE INVENTION 
The methods of the present invention use complementary peptides overlapping 
the sequence of human GHRH (18-25). As mentioned above, Ling et al. found 
a gradual decrease in relative GH-releasing potency of C-terminal deleted 
GHRH fragments until reaching hGHRH (1-27) (12% relative potency). A sharp 
decrease in biological activity was found with shorter fragments, e.g., 
hGHRH (1-24): 0.02% relative potency; hGHRH (1-19): No activity. The amino 
acid sequence of the 5'-3' CP was derived from the mRNA (Mayo et al., 
supra) for rat GHRH. 
Both the 3'-5' and 5'-3' CP contain a 6 amino acid-long sequence that is 
identical with a sequence in rat GHRH and human GHRH in hydropathic nature 
(See FIG. 1b). If one aligns the amino acid sequences of the 3'-5' and 
5'-3' CP in antiparallel direction one can find a high homology in their 
amino acid distribution in the region that corresponds to GHRH (22-27) 
(FIG. 1b). The hydrophobic amino acids are the same in both the 3'-5' and 
5'-3' CP, while the hydrophilic ones are closely related but have a 
different charge (Asp vs. Gln). On the contrary, other peptides in the 
glucagon family have "uncharged" amino acids in position 25 (Trp in 
gastric inhibitory peptide and glucagon, Gly in secretin, Ser in 
vasoactive intestinal peptide [VIP]and peptide histidine-isoleucine-27 
[PHI-27]) and in position 22 (Tyr in VIP and PHI- 27). 
It is well known to those skilled in the art that certain fragments of an 
analogs of peptides will retain their biological activity. In fact, it has 
been reported that analogs and fragments of other sleep promoting peptides 
are also somnogenic. Thus, it is expected that fragments and analogs of 
both the 3'-5' CP and the 5'-3' CP will be useful in the practice of the 
present invention. 
It is expected that the substitution of D amino acid for L amino acids in 
both the 3'-5' CP and 5'-3' CP would result in a peptide having a 
sleep-promoting action, for example, L-ala could be replace by D-ala in 
both the 3'-5' CP and 5'-3' CP, or L-tyr could be replaced by D-tyr in the 
3'-5' CP. Such changes are often useful to reduce the rate of peptide 
breakdown, thereby reducing the amount needed for an effective somnogenic 
dose. By way of example, it has been reported that similar changes were 
made in the nonapeptide DSIP, and that it retained its somnogenic 
activity. See, e.g., Kovalzon, V. et al. Sleep 86. pp 172-184, ed. by 
Koella, W.P. et al.. Gustav Fischer Verlag. Stuttgart, N.Y. 1988., and 
Obal F. et al. Pharmacol. Biochem. Behav. 24: 889-894 (1986). 
It is also expected that phosphorylation of certain amino acid residues in 
both the 3'-5' and 5'-3' CP would result in sleep-promoting peptides. For 
example, if the ser residue of either the 5'-3' CP or the 3'-5' CP were 
phosphorylated, it is anticipated that such analogs will be somnogenic. By 
way of example, phosphorylation of the ser residue of DSIP results in a 
molecule that retains its ability to induce sleep (Oral Communication by 
S. Inoue, (Tokyo) at the Endogenous Sleep Factors Seminar, Nov. 11, 1988, 
Honolulu, Hi.). 
It is well known that fragments of peptides may retain the biological 
activity of the peptide. It is expected that removal of one or more amino 
acids from or addition of one or more amino acids to the amino terminal or 
carboxyl terminal of either the 5'-3' or the 3'-5' CP would result in 
somnogenic fragments. For example, the removal of val from the 5'-3' CP 
would most likely not alter biological activity. By parallel example, the 
fragment produced by removal of 3 amino acids from the carboxyl terminal 
of DSIP, has been reported to be biologically active (ref. 2, Obal et al. 
supra). Similarly, if amino acids are added to the carboxyl terminal of 
another somnogenic peptide, N-acetyl muramyl-L-alanyl-D-isoglutamine, to 
form, for example, 
N-acetyl-muramyl-L-alanyl-D-isoglutamyl-L-diaminopimelyl alanine, 
somnogenic activity is retained. (See, e.g., Krueger, J.M. et al., Brain 
Res. 403:249-257 (1987). 
Thus, selection of somnogenic analogs and fragments of the 3'-5' and 5'-3' 
CPs of the present invention can be accomplished by those skilled in the 
art without undue experimentation. 
The peptides for use in the present invention can be synthesized by any 
suitable method, such as by exclusively solid-phase techniques, by partial 
solid-phase techniques, by fragment condensation, by classical solution 
couplings, or by the employment of recently developed recombinant DNA 
techniques. 
The 12 amino acid long 3'-5' CP of GHRH and the 26 amino acid long 5'-3' CP 
of GHRH (See FIG. 1a) were synthesized at the Molecular Resource Center, 
Macromolecular Synthesis Laboratory, University of Tennessee, Memphis, Dr. 
T. C. Cooper, Director. Applied Biosystems Model 430A, a fully automatic 
instrument-reagent system for solid phase peptide synthesis, was used. The 
Model 430A utilized an optimized system based on R. B. Merrifield's 
concept of solid phase peptide synthesis (Virender et al., Anal. Biochem., 
117: 147-157, 1981). Typically, solid phase synthesis occurs from the `C-` 
to the `N-terminal` of the peptide sequence. The alpha-carboxyl group of 
the C-terminal amino acid residue is covalently attached to an insoluble 
polystyrene resin bead through an organic linker. The alpha-amino group of 
this amino acid, and all the other amino acids used in synthesis are 
protected by an organic moiety. 
A general synthesis cycle consists of: deprotection of the resin-bound 
alpha-amino group, then washing, neutralization and washing of the resin. 
Next in the cycle is the formation of a peptide bond between the 
deprotected alpha-amino group and the activated carboxyl of the next 
alpha-amino protected amino acid of the sequence. This cycle is repeated 
until the desired sequence is complete. When synthesis is complete, the 
peptide is deprotected and cleaved from its polymer support; it is then 
separated from the resin and purified. 
The 3'-5' CP and 5'-3' CP, fragments thereof, or analogs thereof having 
well known substitutions and/or additions, as well as non-toxic salts of 
any of the foregoing, hereinafter collectively referred to as the active 
ingredient may be prescribed or administered to a host in accordance with 
the present invention to induce sleep. 
The amount of said active ingredient will, of course, depend upon the 
severity of the condition being treated, the route of administration 
chosen and the specific activity of the active ingredient, and ultimately 
will be decided by the attending physician or veterinarian. Such amount of 
active ingredient as determined by the attending physician or veterinarian 
is also referred to herein as an "effective" amount. In general, an amount 
of active ingredient from 0.01 to 1.0 nanomole per kg (body weight) is 
sufficient to induce slow wave sleep when administered 
intracerebroventricularly (icv). 
The active ingredient may be administered by any route appropriate to the 
condition being tested, e.g., orally, rectally, intravenously, 
intramuscularly, intraperitoneally, or intraventricularly. Preferably, the 
peptide is administered orally to the mammal being treated. It will be 
readily appreciated by those skilled in the art that the preferred route 
may vary. 
While it is possible for the active ingredient to be administered as the 
pure or substantially pure compound, it is preferable to present it as a 
pharmaceutical formulation or preparation. 
The formulations for use in the present invention, both for veterinary and 
for human use, comprise the 3'-5' CP and 5'-3' CP, or fragments or analogs 
thereof, as described above, together with one or more pharmaceutically 
acceptable carriers therefor and, optionally, other therapeutic 
ingredients The carriers must be "acceptable" in the sense of being 
compatible with the other ingredients of the formulation and not 
deleterious to the recipient thereof. Such carriers are well known to 
those skilled in the art of pharmacology. Desirably, the formulation 
should not include oxidation agents and other substances with which 
peptides are known to be incompatible. The formulations may conveniently 
be presented in unit dosage form and may be prepared by any of the methods 
well known in the art of pharmacy All methods include the step of bringing 
into association the active ingredient with a carrier which may constitute 
one or more accessory ingredients In general, the formulations are 
prepared by uniformly and intimately bringing into association the active 
ingredient with liquid carriers or finally divided solid carriers or both, 
and then, if necessary, shaping the product into the desired formulation. 
Formulations suitable for parenteral administration conveniently comprise 
sterile aqueous solutions of the active ingredient, which solutions are 
preferably isotonic with the blood of the recipient. Such formulations may 
be conveniently prepared by dissolving solid active ingredient in water to 
produce an aqueous solution, and rendering said solution sterile. The 
formulations may be presented in unit or multi-dose containers for 
example, sealed ampules or vials. 
Biological assays for sleep-promoting activity were performed on rabbits 
provided with chronically implanted ventricular guide tubes and four 
epidural screw electrodes for EEG. The animals were allowed at least one 
week to recover from surgery prior to their use for assays. Samples for 
testing were taken up in sterile artificial cerebrospinal fluid and a 
total of 50 ml solution was infused intraventricularly (ICV) at the rate 
of 25 ul/min through a No. 26 hypodermic needle inserted through a guide 
tube. Following the infusion and removal of the infusion probe the animals 
were left undisturbed for 6-8 hours while EEG and bodily movements were 
recorded. 
Slow wave sleep (SWS) was scored in two ways: (i) by conventional 
subjective scoring of the duration of SWS from polygraph records and (ii) 
by digital print-out of integrated mean rectified cortical slow waves 
(1/2-4 Hz). thus obtaining a measure of the amplitude was well as duration 
of delta wave EEG activity. Control animals were also assayed at the same 
time. 
The results of these assays demonstrate that both the 3'-5' and the 5'-3' 
CP have the capacity to enhance sleep. Thus, after administration of 
either of these substances, the recipient animal spent significantly more 
time in SWS and rapid-eye-movement sleep. Although excess sleep was 
observed, this sleep appeared normal, in that animals continued to cycle 
through the various states of vigilance. The temperatures of the animals 
remained normal. In addition, their behavior was normal in that they could 
be aroused if they were asleep. They continued to eat, drink and groom 
during periods of spontaneous awakenings. 
The invention will be further understood with reference to the following 
examples which are purely exemplary in nature and are not meant to be 
utilized to limit the scope of the invention. 
EXAMPLE 1 
Synthesis of 3'-5'CP and 5'-3' CP 
The syntheses of 3'-5' CP amide, with the sequence: 
EQU H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-NH.sub.2 
and the 5'-3' CP amide, with the sequence: 
H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Leu-Pro-Val-His-Asp-P 
he-Val-Gln-Gln-Phe-Ala-Gly-Ile-NH.sub.2 
were performed on an Applied Biosystems Model 430A Peptide Synthesizer 
which is totally microprocessor controlled, utilizing software version 
1.2. This machine is a solenoid controlled, gas-driven (prepurified 
nitrogen or argon) synthesizer. The aminomethyl resin was purchased from 
Applied Biosystems, Inc. and had an amino acid substitution of about 
0.6-0.7 millimoles/gm. of resin. This resin consists of 1% cross-linked 
polystyrene to which had been attached a phenylacetamidomethyl (PAM) 
group. The carboxyl terminal amino acid, leucine or isoleucine, in these 
cases, was attached to the PAM resin and contained an amino terminal 
blocked with the TBOC (t-butoxylcarbonyl)-protecting group. 
After deprotection of this group on the machine utilizing trifluroacetic 
acid, the peptide was built in a stepwise manner. TBOC-protected amino 
acids were purchased in pre-weighed amounts (approximately 2 mmoles) in 
sealed cartridges from Applied Biosystems. The chemical forms of the amino 
acids used in these peptides are listed below: 
______________________________________ 
t-BOC-L-Aspartic acid(O-Benzyl) 
t-BOC-L-Glutamine 
t-BOC-L-Asparagine t-BOC-L-Glutamate (O-BZ) 
t-BOC-L-Alanine t-BOC-L-Glycine 
t-BOC-L-Arginine (Tosyl) 
t-BOC-L-Serine (Benzyl) 
t-BOC-L-Leucine H.sub.2 O 
t-BOC-L-Histidine (Tos) 
t-BOC-L-Isoleucine t-BOC-L-Valine 
t-BOC-L-Phenylalanine 
t-BOC-L-Proline 
______________________________________ 
O-Benzyl, Benzyl and Tosyl (p-toluenesulfonyl) refer to the type of amino 
and hydroxyl protecting groups present on the amino acid derivatives. 
Briefly, the resin-bound amino acid is deprotected by the addition of 
trifluroacetic acid, neutralized and washed extensively T-BOC protected 
amino acids are dissolved in suitable solvents and transferred to the 
activator vessel of the instrument DCC (Dicyclohexylcarbodiimide) is then 
added to the dissolved amino acid, and a symmetric anhydride is formed, 
called a PSA (protected symmetric anhydride). A by-product of the 
reaction, dicyclohexylurea, forms a precipitate. The equation for this 
reaction is given below: 
##STR1## 
There are three exceptions to the use of amino acid PSA s in the Model 430A 
cycles; asparagine, glutamine and arginine are coupled as 
1-hydroxybenzotriazole (HOBT) esters. These esters are utilized because 
symmetric anhydrides of these amino acids are unstable and undergo 
unacceptable side reactions. In addition to being HOBT-esters, these amino 
acids are double coupled, that is, two cartridges of these amino acids are 
required for each cycle. After the initial coupling, the resin is washed, 
then the coupling is repeated, to increase the yields. 
After the PSA is prepared, the amino acid is transferred to the 
concentrator vessel, where it is purged with nitrogen to remove volatile 
dichloromethane (DCM). N,N-Dimethylformamide (DMF) is then added to the 
amino acid. Individual PSA's have varying stabilities in DCM/DMF mixtures, 
so the temperatures are carefully regulated by the program. HOBT-esters 
are not purged since they are unstable. 
Certain individual amino acids require special treatment. Histidine is one 
such amino acid. It is purchased as the DCHA (dicyclohexylamine) salt and 
passed through a suitably prepared AG-50-X8(H.sup.+) ion exchange column 
in DCM just before use. T-BOC-his(Tos) is unstable in DCM at room 
temperature, and thus must be placed on the machine within 4 hours of use. 
Two other alterations in the cycle are required with histidine. Since the 
amino acid is supplied as a solution, no DCM is delivered to the 
cartridge, and the purge cycle is shortened to 6.5 minutes because of its 
instability. 
As the amino acids are activated and the solvents are evaporated and 
changed, the resin is treated with trifluoroacetic acid (TFA) in DCM 
twice, then washed with DCM to remove some of the TFA. The resin is then 
neutralized with diisopropylethylamine (DIEA) in DMF and washed with DMF. 
DMF is the solvent of choice for coupling of activated amino acids to the 
growing peptide chain. 
After the peptide is complete, the T-BOC group is then removed with the 
standard deprotection step and washed with DCM. 
Synopsis of one single cycle 
Addition of the first amino acid residue of the first peptide, isoleucine, 
was carried out in the following manner: 
1. The amino acid cartridge was punctured with a needle assembly and 
approximately 3 ml of dichloromethane (DCM) was delivered to the powdered 
amino acid. The solution was mixed with nitrogen bubbles for approximately 
2 minutes, and the solution transferred to the activator vessel. 
2. One millimole of 0.5 M dicyclohexylcarbodiimide (DCC) in dichloromethane 
was delivered to the activator vessel followed by gas purging to mix. The 
by-product of this reaction, dicyclohexylurea, begins to precipitate 
almost immediately. After 8 minutes, the solution is filtered through a 
glass frit, and delivered to the concentrator vessel. 
3. During the activation and concentration cycles, the resin, in the 
reaction vessel, was treated in the following way: 
a. 33% trifluoroacetic acid (TFA) in DCM for 2.5 minutes 
b. 50% TFA in DCM for 18 minutes 
c. Three DCM washes 
d. 10% DIEA in DMF for 3 minutes 
e. Five DMF washes 
The deprotection steps are identical to the above for all amino acids. 
4. In the concentrator vessel, the DCM solution was purged with nitrogen 
gas for a total of about 16 minutes, and approximately 4 ml of DMF was 
added. The temperature was automatically controlled at 15.degree. C. or 
below After the last DMF wash of the resin, the activated amino acid in 
DMF was delivered to the reaction vessel and coupled for about 25 minutes 
with vigorous vortexing. Other amino acids utilizing this coupling time 
are histidine, leucine, isoleucine, phenylalanine and proline. Others such 
as aspartic acid, glycine, serine and alanine use about 18 minutes. 
Arginine and asparagine are coupled twice for 42 minutes each. 
5. As the peptide chain lengthens, longer coupling times are required and 
are automatically incorporated into the compiled program as it progresses. 
6. After coupling was completed, the resin was drained and washed five 
times with DCM. Activation of the next amino acid and deprotection of the 
resin for the next cycle was begun. 
Synopsis of a double couple cycle 
1. Two amino acid cartridges were required for the double couple cycles of 
argining and asparagine. These were placed one after another in the 
guideway of the Model 430A synthesizer. 
2. The first amino acid cartridge was punctured with a needle assembly and 
approximately 4 ml of HOBT (1-Hydroxybenzotriazole) in 
N,N-Dimethylformamide (DMF) (2 mmole) was delivered to the cartridge to 
dissolve the amino acid Asparagine requires the addition of 0.3 ml of DCM, 
and arginine requires 1.5 ml of DCM for complete dissolution. After mixing 
(6.5 min for asparagine, 8 minutes for arginineJ), the solution was 
transferred to the activator vessel. 
3. The HOBT-ester double couple cycles all employ the same transfer 
process. The HOBT/amino acid mixture is added to 4 ml (2 mmoles) of DCC 
(dicyclohexylcarbodiimide) in the activator vessel. After precipitation of 
DCU, the HOBT-ester is transferred to the concentrator vessel after being 
filtered through a glass frit. 
4. The solution is then directly transferred to the reaction vessel, 
without gas purging. 
5. During the previously described activation phase, the resin was 
deprotected using the following schedule: 
a. 33% TFA in DCM for 2.5 minutes 
b. 50% TFA in DCM for 18 minutes 
c. Three DCM washes 
d. Five DMF washes 
f. Begin first coupling periodj 
6. Coupling then takes place for about 42 minutes with vigorous vortexing. 
The resin is then washed according to the following schedule: 
g. Three DMF washes 
h. 10% DIEA in DMF for 45 seconds 
i. One DMF wash 
j. Three DCM washes 
7. The second amino acid cartridge is prepared in the same manner as the 
first, and coupled f.degree. r another 42 minutes. The resin is then 
drained, washed with DMF, then 5 times with DCM. 
EXAMPLE 2 
Administration of 5'-3' CP and changes in Vigilance 
Operation and experimental procedure. Adult male New Zealand White 
Pasteurella-free rabbits, weighing 3-4 kg, were purchased from Myrtle's 
Rabbitry (Thompson Station, Tenn.). Under ketamine-xylazine (3.5-5.0 mg/kg 
iv) anesthesia, animals were provided with chronically implanted EEG 
electrodes, a glass-bead thermistor, and a cerebral ventricular guide tube 
(Krueger, J.M et al., Am. J. Physiol. 251 (Regulatory Integrative Comp. 
Physiol. 20): R591-R597, 1986; and Walter J.D. et al., Am. J. Physiol., 
250 (Regulatory Integrative Comp. Physiol. 19): R96-R103, 1986). Briefly, 
stainless steal screws were implanted over the frontal parietal, and 
occipital cortex; and a small (1 mm diam) 50-k.OMEGA. thermistor (Fenwall 
Electronics) was placed 3 mm into the parietal cortex. Wires from Amphenol 
plugs (no. 223-1509) were soldered to the screws for EEG recordings and to 
the thermistors for brain temperature (T.sub.br) recordings. The 
ventricular guide tube was implanted 4 mm lateral to the bregma; during 
the implantation procedure, pressure at the tip of the infusion needle was 
monitored and used as an aid to locate the ventricle (See, e.g., Krueger, 
J.M et al., Am. J. Physiol., 246 (Regulatory Integrative Comp. Physiol. 
15): R994-R999, 1984). Dental acrylic (DuzAll) was then used to insulate 
the leads and to secure the guide tube and EEG/thermistor plugs to the 
skull. A topical antibiotic (Bacitracin, Lilly) was applied to the 
incision, and 150,000 U of Duracillin (Lilly) were administered 
intramuscularly. At least 1 week was allowed for recovery. 
Animals were housed in rooms on a 12:12-h light-dark cycle (0600-1800 h 
light) maintained at 21 .+-.2.degree. C. The day before an experiment 
animals were brought to experimental chambers (Hotpack model 352600) for 
an overnight acclimation period. Each experimental chamber was also kept 
at 21 .+-.2.degree. C. on a 12:12-h light-dark cycle. Food and water were 
available ad libitum at all times. At the top of the recording chamber, 
BRS/LVE electrical contact swivel was fixed; this allowed the rabbits free 
movement during recording periods. From the other end of the swivel a 
cable led to a Grass polygraph model 7D. Before each recording period the 
rabbits were connected to the recording cable for a 1-h habituation 
period, but data were not collected during this time. 
Rabbits were then briefly taken out of the experimental cages and given the 
3'-5' or 5'-3' CP (the "test substance"). [When animals received 
intracerebroventricular (ICV) injections, appropriate amounts of test 
substances (0.1-10.0 ul) were diluted to 50 ul with artificial CSF [3 mM 
KCL, 1.15 mM CaCl.sub.2 and 0.96 mM MgCl.sub.2 in pyrogen-free saline 
(PFS), 1.55 mM NaCl (Abbott)]; these solutions were slowly infused over 2 
min. Immediately after injection, colonic temperatures (T.sub.co) were 
measured using a calibrated thermistor probe (Yellow Springs Instruments) 
inserted 10 cm into the colon; then the rabbits were returned to the 
experimental cages for a 6-h recording period. After the recording period 
T.sub.co was measured again. Injection (ICV) took place between 0900 and 
1000 h. 
EEG, ratios of .theta./.delta. cortical EEG activity, T.sub.br and motor 
movement were recorded from animals to define the state of vigilance (see 
below). Cortical EEG signals were fed into a Buxco (Sharon, Conn.) DL.24 
EEG analyzer, and the rectified average voltages in the 0.5. to 3.5-Hz 
(.delta.), 4-to 7.5-Hz (.theta.), 8- to 12.5-Hz (.alpha.), and 13- to 
25-Hz(.beta.) frequency bands were printed on paper each minute. In 
addition the ratios of .theta./.delta. voltages were computed, and these 
values were continuously recorded simultaneously with the EEG on polygraph 
paper. 
The Grass and Buxco amplifiers, filters and averager were calibrated using 
sine waves of known peak-to-peak voltage and frequency. To evaluate 
T.sub.br implanted thermistors were calibrated by reference to T.sub.co ; 
this method assumes that T.sub.br follows T.sub.co within a constant 
range. Thus, when an animal received a pyrogenic substance, T.sub.co was 
taken before and after fever developed while T.sub.br was simultaneously 
recorded. The difference between the two T.sub.br on the polygraph paper 
was assumed to be equal to the difference between the two T.sub.co ; this 
allowed quantitation of T.sub.br at other times Rabbit body movements were 
monitored using a Grass tremor transducer (model SPAI) attached to the 
recording cable. 
Polygraph recordings were analyzed visually to determine periods of W, SWS 
and rapid-eye-movement (REM) sleep. The recordings were divided into 12-s 
epochs; each epoch was classified as either W, SWS or REM sleep as 
follows. W was characterized by low-voltage EEG, high incidence of body 
movement, midlevel range of .theta./.delta. ratios, and a decreasing 
T.sub.br after REM sleep episodes or increasing T.sub.br after SWS 
episodes. SWS was identified by increased EEG slow-wave voltage, little or 
no body movements, low .theta./.delta. ratios, and a decreasing T.sub.br 
REM sleep was identified by a low-voltage EEG, phasic body movements, high 
.theta./.delta. ratios, and a relatively rapid increase in T.sub.br. 
The percentage of time spent in SWS and REM sleep was determined for each 
hour (FIGS. 1 and 2) and for the total recording period (Tables 1 and 2). 
Printed average voltage was used to calculate hourly mean voltage in each 
frequency band; only values for the .delta.-frequency band are presented 
(Table 1 and FIG. 1), since values for the other frequency bands were not 
affected by test substances. In addition, maxium values for .delta.-wave 
voltages during SWS episodes were determined after rTNF treatment. These 
values were obtained by first identifying the 12 maximum 1-min printed 
average voltages for each rabbit, then checking the polygraph record to 
make sure these were associated with periods of SWS rather than with 
movement artifact. The values for each rabbit for both control and 
experimental conditions were averaged; for different experimental groups 
the means .+-. SE of these averages were then determined (Table 1). 
Student s t tests for paired data were used for comparison between data 
obtained from the same animals under experimental and control conditions. 
A significance level of P&lt;O.05 was used. 
It is understood that the examples and embodiments described herein are for 
illustrative purposes only, and that various modifications or changes in 
light thereof that will be suggested to persons skilled in the art are to 
be included in the spirit and purview of this application and the scope of 
the approved claims.