Method for modulating hemopoiesis

FIELD OF THE INVENTION 
The invention relates to a biologically active peptide; a novel 
pharmaceutical composition containing the peptide; a method for preparing 
the peptide; and uses of the peptide. 
BACKGROUND OF THE INVENTION 
Extracts from the thymus gland are known as regulators of immune processes. 
For example, U.S. Pat. No. 5,070,026 to Morozov et al. (corresponding to 
Swiss patent No. CH 659,586) teaches a thymus gland preparation containing 
polypeptides of varying composition and their use in stimulating 
immunological activity and enhancing reparative processes and hemopoiesis. 
Goldstein et al. (Proc. Natl. Acad. Sci. U.S.A., 74(2):725-729, February 
1997) teaches the isolation of thymosin, which comprises various 
biologically reactive polypeptides isolated from the thymus gland, and its 
use in enhancing immunological responses. U.S. Pat. No. 4,910,296 to Birr 
et al. and European Patent application Ser. No. 89/102569 to Lattanzi 
teach a composition containing human Thymosin Alpha 1 and fragments 
thereof which have immunoregulatory and immunostimulatory properties. 
Thymic extracts consist of complexes of polypeptides. Their production from 
natural sources is limited by the complexity of the process, the 
relatively small yields of active substances, and the large variability of 
the physical, chemical characteristics and biological properties of the 
products. In addition, unwanted components which are present in natural 
thymic preparations sometimes cause side effects. 
These problems create a demand for the identification and preparation of 
synthetic peptides. Several peptides with immunoregulatory properties have 
been synthesized (see, for example, SU 1,582,393; EP 230,052; U.S. Pat. 
No. 4,190,646; U.S. Pat. No. 5,008,246; and U.S. Pat. No. 5,013,723). Many 
scientific laboratories have tried to develop methods for preparing 
synthetic derivatives of natural peptides, which are more active than 
their natural analogs (see, for example, EP 0,136,720, 1984; EP 0,137,904, 
1984). 
Australian Patent No. AU-B-29308/89 (corresponding to WO 08906134) teaches 
the preparation of Glu-Trp and its use for treating immune deficiency 
conditions. WO 9308815 to Khavinson et al. discloses the peptide Glu-Trp 
and cyclic monomers and polymers thereof, for use in the treatment of 
immunosuppression. Semina et al. (Radiatsionnaya Biologiya Radioekologiya 
33(3), 1993; WO 8906134) have shown that the levorotary (L) enantiomer of 
the dipeptide H-Glu-Trp-OH acts as an immunostimulant and can induce the 
proliferation of cells. 
However, the known synthetic peptides do not always have the necessary 
immunoregulatory properties, and effectiveness. Furthermore, many of them 
are effective only in large doses that can cause side effects. 
SUMMARY OF THE INVENTION 
The present inventors have synthesized a series of highly active compounds 
of the general formula X-Glu-Trp-Y, and have found that these peptides 
have immunoregulatory properties that are superior to the peptides of the 
prior art. The identified peptides have the ability to effectively 
modulate the immune system of humans and animals, in relatively low doses, 
and with little or no side effects. The present inventors have further 
found that these peptides have hemopoietic properties. The peptides are 
prepared using a method which provides high yields with simple and 
efficient steps. 
Broadly stated, the present invention relates to a peptide of the formula I 
EQU X--Glu--Trp--Y, (I) 
wherein X is H, Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, 
D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, His, Lys, Arg 
.gamma.-aminobutyric acid, or .xi.-aminocaproic acid; Y is Gly, Ala, Leu, 
Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, 
D-Pro, D-Tyr, D-Phe, D-Trp, Arg, .gamma.-aminobutyric acid, 
.xi.-aminocaproic acid, --OH, NH.sub.2, N.sub.2 H.sub.3, or a mono- or 
di-substituted amide (C.sub.1 -C.sub.3); with the proviso that when X is 
H, Y is not --OH. 
Preferred peptides of the invention have the sequence H-Ile-Glu-Trp-OH, 
His-Glu-Trp-OH, H-Glu-Trp-NH.sub.2, H-Glu-Trp-Arg, Lys-Glu-Trp-OH, 
Arg-Glu-Trp-OH, H-Glu-Trp-Tyr, Lys-Glu-Trp-Tyr, H-Glu-Trp-N.sub.2 H.sub.3, 
H-Glu-Trp-Gly, or Val-Glu-Trp-OH. In a further preferred embodiment, the 
peptide of the present invention is H-Ile-Glu-Trp-OH ["Neogen"]. 
The invention also relates to analogs and derivatives of the peptides of 
the invention and cyclized peptides. The term "peptide" or "peptides" used 
herein includes these analogs, derivatives and cyclized peptides. 
The present invention further relates to a process for preparing a peptide 
of the formula I. 
In another embodiment, the invention provides a method of regulating the 
immune system and/or hemopoiesis in an animal comprising administering to 
the animal an effective amount of the peptide of the invention. In one 
embodiment, the invention provides a method of stimulating the immune 
system of an animal. In yet another embodiment, the invention provides a 
method of restoring hemopoiesis in an animal with impaired hemopoiesis. 
In yet another embodiment the invention provides a method of treating 
hemopoietic disorders, for example, without limitation to immune 
cytopenia, multiple myeloma, chronic lymphoid leukosis, lymphocytic 
lymphomas, lymphosarcomas and in particular to .beta.-cellular lymphoid 
leukosis. 
In another embodiment the invention provides a method for treating immune 
and/or hemopoietic disorders such as cancer in an animal comprising 
administering to the animal a peptide of the invention, preferably in 
combination with cytostatic treatment such as a cytostatic agent. Most 
preferably the peptide is H-Ile-Glu-Trp-OH and the cytostatic treatment 
comprises the administration of either oxyurea or heat. 
Preferably the peptide is administered in an amount of 0.001 to 0.1 mg/kg 
by weight of the animal. 
The invention still further relates to a pharmaceutical composition 
comprising one or more peptides of the invention and a pharmaceutically 
acceptable carrier, the use of a peptide of the Formula I to regulate the 
immune system, and a method of regulating the immune system. 
Other, features and advantages of the present invention will become 
apparent from the following detailed description. It should be understood, 
however, that the detailed description and the specific examples, while 
indicating preferred embodiments of the invention are given by way of 
illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION 
I. Peptides of the Invention 
As mentioned previously, the present invention relates to a peptide of the 
formula I: 
EQU X--Glu--Trp--Y, (I) 
wherein X is H, Gly, Ala, Leu, Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, 
D-Leu, D-Ile, D-Val, D-NVal, D-Pro, D-Tyr, D-Phe, D-Trp, His, Lys, Arg 
.gamma.-aminobutyric acid, or .xi.-aminocaproic acid; Y is Gly, Ala, Leu, 
Ile, Val, NVal, Pro, Tyr, Phe, Trp, D-Ala, D-Leu, D-Ile, D-Val, D-NVal, 
D-Pro, D-Tyr, D-Phe, D-Trp, Arg, .gamma.-aminobutyric acid, 
.xi.-aminocaproic acid, --OH, NH.sub.2, N.sub.2 H.sub.3, or a mono- or 
di-substituted amide (C.sub.1 -C.sub.3); with the proviso that when X is 
H, Y is not --OH. 
Preferred peptides of the invention have the sequence H-Ile-Glu-Trp-OH, 
His-Glu-Trp-OH, H-Glu-Trp-NH.sub.2, H-Glu-Trp-Arg, Lys-Glu-Trp-OH, 
Arg-Glu-Trp-OH, H-Glu-Trp-Tyr, Lys-Glu-Trp-Tyr, H-Glu-Trp-N.sub.2 H.sub.3, 
H-Glu-Trp-Gly, or Val-Glu-Trp-OH. In a further preferred embodiment, the 
peptide of the present invention is H-Ile-Glu-Trp-OH. 
The following standard abbreviations for the amino acid residues are used 
throughout the specification: Abu--.gamma.-aminobutyric acid; 
Aca--.xi.-aminocaproic acid; Ala--alanine; Cys--cysteine; Asp--aspartic 
acid; Glu--glutamic acid; Phe--phenylalanine; Gly--glycine; 
His--histidine; Ile--isoleucine; Lys--lysine; Leu--leucine; 
Met--methionine; Asn--asparagine; Pro--proline; Gln--glutamine; 
Arg--arginine; Ser--serine; Thr--threonine; Val--valine; Trp--tryptophan; 
Tyr--tyrosine; and Orn--ornithine. 
The term "analog" includes any peptide having an amino acid residue 
sequence substantially identical to the sequence of the peptides described 
herein in which one or more residues have been conservatively substituted 
with a functionally similar residue and which displays the ability to 
mimic a peptide of the invention. Examples of conservative substitutions 
include the substitution of one non-polar (hydrophobic) residue such as 
alanine, isoleucine, valine, leucine or methionine for another, the 
substitution of one polar (hydrophilic) residue for another such as 
between arginine and lysine, between glutamine and asparagine, between 
glycine and serine, the substitution of one basic residue such as lysine, 
arginine or histidine for another, or the substitution of one acidic 
residue, such as aspartic acid or glutamic acid for another. The phrase 
"conservative substitution" also includes the use of a chemically 
derivatized residue in place of a non-derivatized residue provided that 
such polypeptide displays the requisite activity. 
The term derivatives as used herein refers to chemical derivatives of a 
subject peptide having one or more residues chemically derivatized by 
reaction of a functional side group. Thus, a "derivative" is a peptide 
that is derived from a peptide identified herein by one or more chemical 
steps. Such derivatized molecules include, for example, those molecules in 
which free amino groups have been derivatized to form amine 
hydrochlorides, P-toluene sulfoamides, benzoxycarboamides, 
T-butyloxycarboamides, thiourethane-type derivatives, 
trifluoroacetylamides, chloroaceamides, or formamides. Free carboxyl 
groups may be derivatized to form salts, methyl and ethyl esters or other 
types of esters or hydrazides. Free hydroxyl groups may be derivatized to 
form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine 
may be derivatized for form N-imbenzylhistidine. Also included as 
derivatives are those peptides which contain one or more naturally 
occurring amino acids derivatives of the 20 standard amino acids. For 
example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine 
may be substituted for lysine; 3-methylhistidine may be substituted for 
histidine; homoserine may be substituted for serine, and ornithine may be 
substituted for lysine. 
The invention further includes cyclic derivatives of the peptides of the 
invention. Cyclization allows the peptide to assume a more favourable 
conformation. Cyclization of the peptides may be achieved using techniques 
known in the art. In particular, disulphide bonds may be formed between 
two appropriately spaced components having free sulfhydryl groups. The 
bonds may be formed between side chains of amino acids, non-amino acid 
components, or a combination of the two. 
As will be discussed in more detail below, the invention also includes a 
peptide of the invention conjugated with a selected protein, or a 
selectable marker to produce fusion proteins. 
Peptides of the invention may be converted into pharmaceutical salts by 
reacting with inorganic acids including, without limitation, hydrochloric 
acid, sulfuric acid, hydrobromic acid, phosphoric acid, or organic acids 
including formic acid, acetic acid, propionic acid, glycolic acid, lactic 
acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, 
citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and 
toluenesulfonic acids. 
The peptides of the invention may be prepared by chemical synthesis using 
techniques known in the chemistry of proteins such as solid phase 
synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or 
synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic 
Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart). 
According to an embodiment of the present invention, the peptide of the 
formula (I) is synthesized by step-by-step building of the peptide chain 
beginning with the C-terminal amino acid. The process involves maximum 
blocking of functional groups, starting from an amino acid alkyl ester, 
using the method of active esters and the method of mixing anhydrides, 
preferably using a t-butyloxycarbonyl group as the amino protecting group. 
In a preferred embodiment, the method involves the blocking of the amino, 
carboxyl and other reactive side groups of the amino acid(s) which are not 
to react during the synthesis. Suitable blocking agents would be known to 
a person skilled in the art. For example, a suitable carboxy blocking 
agent would include, without limitation, ethyl, nitrobenzyl, and t-butyl. 
A suitable amino blocking agent would include, without limitation, 
carbobenzoxy, tosyl, trifluoracetyl and preferably t-butyloxycarbonyl 
(Boc). The amino acids are then coupled and the blocking agents 
subsequently removed. The peptide can optionally be further purified using 
reverse phase chromatography, preferably in acetonitrile/0.1% 
trifluoracetic acid. 
Most preferably, the C-terminal amino acid is blocked at its amino terminal 
end, preferably with t-butyloxycarbonyl and coupled to another molecule, 
such as pentafluorophenol, to form an amino acid alkyl-ester. This occurs 
by chilling the mixture of the protected amino acid and pentaflorophenol 
in ethylacetate to about -5 degrees celsius and adding 
N,N-dicyclohexylcarbodiimide. The mixture is then stirred at room 
temperature for three hours, the forming N,N-dicyclohexylurea removed by 
filtration, the remains crystallized in ethylacetate-hexane and the 
residue filtered out. The amino protected alkyl-ester amino acid is then 
coupled to another amino acid or peptide, such as Glu-Trp, in the 
pressence of dimethylformamide, to give an amino protected peptide, such 
as Boc-Ile-Glu-Trp-OH. The blocking agent, Boc, is then removed using 
conventional methods known in the art and the peptide purified using 
reverse phase HPLC chromatography. 
The peptide prepared using this process is a yellowish grey or white 
powder, soluble in water, substantially insoluble in alcohol and 
substantially insoluble in chloroform. It has a UV spectrum in the range 
of 250-300 nm with a maximum at 280.+-.2 nm. 
This process provides high yields of the product with a minimum number of 
steps, and maximum efficiency and simplicity. 
N-terminal or C-terminal fusion proteins comprising a peptide of the 
invention conjugated with other molecules such as proteins or selectable 
markers may be prepared by fusing, through recombinant techniques, the 
N-terminal or C-terminal of the peptide, and the sequence of a selected 
protein or selectable marker with a desired biological function. The 
resulting fusion proteins contain the peptide fused to the selected 
protein or marker protein as described herein. 
In the alternative, the peptides of the invention may be prepared using 
recombinant techniques. Nucleic acid molecules which encode a peptide of 
the invention may be incorporated in a known manner into an expression 
vector which ensures good expression of the peptide. Suitable expression 
vectors include but are not limited to cosmids, plasmids, or modified 
viruses so long as the vector is compatible with the host cell used. The 
expression vectors contain a nucleic acid molecule encoding a peptide of 
the invention and regulatory sequences necessary for the transcription and 
translation of the inserted protein-sequence. Regulatory sequences may be 
obtained from a variety of sources, such as bacterial, fungal, viral, 
mammalian, or insect genes (See the regulatory sequences described in 
Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic 
Press, San Diego, Calif. (1990)). The selection of regulatory sequences is 
dependent on the host cell chosen, and may be readily accomplished by one 
of ordinary skill in the art. Sequences, including an origin of 
replication, additional DNA restriction sites, enhancers, and sequences 
conferring educability of transcription may also be incorporated into the 
expression vector. 
A selectable marker gene which facilitates the selection of transformed or 
transfected host cells may also be included in the recombinant expression 
vector. Examples of selectable marker genes are genes encoding proteins 
such as G418 and hygromycin which confer resistance to certain drugs, 
.beta.-galactosidase, chloramphenicol acetyltransferase, and firefly 
luciferase. It will be appreciated that the selectable markers may be 
introduced on a separate vector from the nucleic acid. 
Genes may also be included in the recombinant expression vectors which 
encode a fusion portion which provides increased expression of the 
recombinant peptide; increased solubility of the recombinant peptide; 
and/or aid in the purification of the recombinant peptide by acting as a 
ligand in affinity purification. In particular, a proteolytic cleavage 
site may be inserted to allow separation of the recombinant peptide from 
the fusion portion after purification of the fusion protein. 
A recombinant expression vector may be introduced into a host cell to 
produce a transformant host cell. Transformant host cells include 
prokaryotic and eukaryotic cells transformed or transfected with a 
recombinant expression vector of the invention. The terms "transformed 
with", "transfected with", "transformation" and "transfection" include the 
introduction of nucleic acid (e.g. a vector) into a host cell by one of 
many techniques known in the art. Examples of methods for transforming and 
transfecting host cells may be found in Sambrook et al. (Molecular 
Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory 
press (1989)), and other laboratory textbooks. 
Suitable host cells include prokaryotic and eukaryotic host cells. In 
particular, the peptides of the invention may be expressed in bacterial 
cells such as E. coli, insect cells (using baculovirus), yeast cells or 
mammalian cells. Other suitable host cells are described in Goeddel, Gene 
Expression Technology: Methods in Enzymology 185, Academic Press, San 
Diego, Calif. (1991). 
Monoclonal or polyclonal antibodies specific for the peptides of the 
invention may be prepared using conventional methods. For example, the 
preparation of monoclonal antibodies can be carried out as described in 
Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd Ed., 
Academic Press, London, 1986. 
The peptides of the invention may be labelled using conventional methods 
with various enzymes, fluorescent materials, luminescent materials and 
radioactive materials. Suitable enzymes, fluorescent materials, 
luminescent materials, and radioactive material are well known to a person 
skilled in the art. 
III. Uses of the Peptide 
The present invention offers peptides that can be used for experimental 
purposes and in medicine. The peptides and pharmaceutical compositions 
containing the peptides have significant immunoregulatory effects and 
accordingly may be used to promote or suppress recognition and destruction 
of abnormal or mutant cell types or antigens which arise within the body. 
In vitro, the claimed peptide was found to be 10.sup.3 times as active as 
known preparations. The utility of the peptides of the present invention 
can be seen in more detail with reference to the Examples. 
In one aspect, the invention provides a method of modulating the immune 
system and/or hemopoiesis in an animal comprising administering to the 
animal an effective amount of the peptide of the invention. In one 
embodiment, the invention provides a method of stimulating the immune 
system of an animal. In another embodiment, the invention provides a 
method of restoring hemopoiesis in an animal with impaired hemopoiesis, 
for example caused by irradiation or cytostatic agents. 
In yet another embodiment the invention provides a method of treating 
hemopoietic disorders, for example, without limitation to immune 
cytopenia, multiple myeloma, chronic lymphoid leukosis, lymphocytic 
lymphomas, lymphosarcomas and in particular .beta.-cellular lymphoid 
leukosis. 
In another embodiment the invention provides a method for treating immune 
and/or hemopoietic disorders such as cancer in an animal comprising 
administering to the animal an effective amount of a peptide of the 
invention, preferably in combination with a cytostatic agent. Most 
preferably the peptide is H-Ile-Glu-Trp-OH and the cytostatic agent is 
either oxyurea or hyperthermia. 
The peptides may be administered to animals in an effective amount. The 
term "animal" as used herein refers to any living organism in which an 
immune or hemopoietic response can be elicited, including without 
limitation to mammals, such as mice guinea pigs, rats, rabbits and humans. 
An "effective" amount is defined as an amount of the active ingredient 
i.e. peptides, effective, at dosages and for periods of time necessary to 
achieve the desired result. An effective amount of a peptide may vary 
according to factors such as the disease state, age, sex, and weight of 
the individual. Dosage regime may be altered to provide the optimum 
therapeutic response. 
The peptides of the invention can be formulated into pharmaceutical 
compositions for adminstration to subjects in a therapeutically effective 
amount and in a biologically compatible form suitable for administration 
in vivo, i.e. a form of the peptides to be administered in which any toxic 
effects are outweighed by the therapeutic effects. 
The peptides may be administered by injection (i.e., subcutaneous, 
intravenous, intramuscular, intraperitoneal, preferably intramuscular), 
oral administration, inhalation, transdermal application, topical 
administration or rectal administration, preferably by injection, 
transdermal or topical administration. Depending on the route of 
administration, the peptides in the pharmaceutical compositions may be 
coated in a material to protect them from the action of certain enzymes. A 
person skilled in the art would be familiar with the coating which would 
be suitable for delivery of the peptide to a particular site. Organic 
substances may also be included in the compositions to prolong the 
pharmacologic actions of the peptides. Examples of such organic substances 
include non-antigenic gelatin, carboxymethylcellulose, sulfonate or 
phosphate ester of alginic acid, dextran, polyethylene glycol and other 
glycols, phytic acid, polyglutamic acid, and protamine. 
The pharmaceutical compositions of the invention can be prepared by per se 
known methods for the preparation of pharmaceutically acceptable 
compositions which can be administered to subjects, such that an effective 
quantity of a peptide is combined in a mixture with a pharmaceutically 
acceptable vehicle. Examples of pharmaceutically acceptable vehicles are 
described in Remington's Pharmaceutical Sciences (Remington's 
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). 
Compositions for injection include, albeit not exclusively, the peptides in 
association with one or more pharmaceutically acceptable vehicles or 
diluents, and contained in buffered solutions with a suitable pH and 
iso-osmotic with the physiological fluids. Any pharmaceutically suitable 
diluent can be used in the composition for injections: distilled water, 
physiological or a salt solution, and/or a buffer solution. The 
composition for injections may be prepared by conventional volume-weight 
procedure. A certain amount of the peptide is diluted to the necessary 
volume with a diluent or solvent. Then the solution is filtered through 
sterilized filters, bottled or ampouled. The resultant solution is a 
stable transparent liquid, and does not contain any chemical or other 
impurities. 
In a preferred embodiment of the invention, the pharmaceutical composition 
generally contains 0.001 or 0.1% (preferably 0.001-0.05%) of one or more 
of the peptide, of the invention The percentage also depends on the 
medicinal form-solution or a solid form. A person skilled in the art would 
appreciate that the treatment regimen may vary according to the condition 
to be treated and the individual. The preferred dosage of the peptide is 
0.001-0.1 mg/kg, and more preferably 0.001-0.01 mg/kg. The petide is 
preferably administered daily and preferably for a period of 3 to 10 days. 
The preferred pharmaceutical composition is made by mixing the carrier and 
the peptide at the temperature of 40.degree.-70.degree. C. The composition 
is stable in solution for 24 months (2 years) at 70.degree. F. (20.degree. 
C.) and for 36 months at 4.degree. C. 
The composition for injections may contain any pharmaceutically suitable 
solvent, including distilled water, physiological solution, saline, or 
buffer solutions. The ratio of the peptide to vehicle is preferably 0.001 
to 0.1%. 
The solution for injections is made by the conventional 
volumetric-gravimetric method. The solvent is added to a certain 
accurately weighed amount of the peptide powder to make the necessary 
volume. The solution is filtered through a sterilizing filter, bottled or 
ampouled. The solution is a colorless transparent liquid. It is stable and 
contains no other impurities. 
Solid form preparations for oral administration can be made in the form of 
tablets, powders, capsules. It may contain a medium for the active 
substance and other additives, including dyes, aromas, etc. A person 
skilled in the art would understand that the oral compositions may be 
coated in a particular material to protect them from the action of certain 
enzymes. The peptide percentage may be from 0.001% to 0.1% and usually 
depends on the type of the composition for oral use. 
The peptide or pharmaceutical compositions of the invention can be packaged 
into lyposomes and administered in the form of topical creams or aerosols. 
Further, the peptide can be administered via active transdermal films or 
aerosols forms such as nasal sprays. 
An acute toxicity study of the peptide was carried out in compliance with 
methodical recommendations of the Pharmacological Committee of the Russian 
Federation (RF), "Requirements to Preclinical Study of General Toxic 
Action of New Pharmacological Substances", M., 1985. According to the 
results of the study, intraperitoneal injection of a 10,000-fold dose of 
the peptide did not cause an acute toxic effect. 
Thus, the new peptides are nontoxic and have a potent immunoregulatory 
effect. 
The following non-limiting examples are illustrative of the present 
invention: 
EXAMPLE 1 
This example outlines the synthesis of one peptide of the invention, 
H-Ile-Glu-Trp-OH. 
1. Preparation of Boc-Ile-OPFP 
A mixture of 46.0 g (0.2 M) Boc-Ile-OH and 40.5 g (0.22 M) 
pentafluorophenol in 100 ml ethyl acetate was cooled to -5.degree. C. To 
this was added 45.3 g (0.22 M) N,N-dicyclohexyl carbodiimide. The reaction 
mixture was stirred at room temperature for 3 h. Resulting 
dicyclohexylurea was removed by filtration. The solvents were subsequently 
evaporated under vacuum. The remaining residue was crystallized in a 
mixture of ethyl acetate-hexane. The precipitate was separated by 
filtration. Yield: 71.3 g (90%). 
2. Preparation of Boc-Ile-Glu-Trp-OH 
19.8 g (0.05 M) Boc-Ile-OPFP was dissolved in 100 ml dimethyl formamide. To 
this was added a water solution of 20 g (0.06 M) Glu-Trp and 5.0 g (0.06 
M) NaHCO.sub.3. The solution was stirred for 20 h at room temperature, 
then the solvents were removed by evaporation under vacuum. To this was 
added 200 ml ethyl acetate and 20 ml 2% sulfuric acid solution and mixed. 
The organic layer was washed with sulfuric acid solution (2.times.100 ml), 
with saturated NaCl solution to pH=7, dried over anhydrous sodium sulfate, 
and the solvent evaporated under vacuum. The residue was crystallized from 
ethyl acetate-hexane, the precipitate separated by filtration and dried 
under vacuum. Yield: 20.5 g (75%). 
3. Preparation of H-Ile-Glu-Trp-OH 
20.5 g Boc-Ile-Glu-Trp-Oh was dissolved in 150 ml formic acid, stirred for 
3.5 h at 45.degree. C. The solvent was evaporated under vacuum. 200 ml 
water was added to the residue and evaporation under vacuum was repeated. 
300 ml isopropanol and 200 ml ether was added to the residue and allowed 
to stand for 10 h. The precipitate was then filtered and dried under 
vacuum. Yield: 15.3 g (75%). 
The peptide was purified using reverse phase HPLC in acetonytrile--0.1% TFA 
(trifluoroacetic acid). Yield: 13 g (85%). 
The resultant peptide has the physical and chemical properties and 
characteristics as follows: 
Primary structure--H-Ile-Glu-Trp-OH 
Empirical formula--C.sub.24 H.sub.30 N.sub.4 H.sub.4 
Molecular weight--446.5 Da 
Appearance--yellowish-white or grey powder 
Solubility--readily soluble in water, moderately soluble in alcohol, 
insoluble in chloroform. 
UV--spectrum in the range of 250-300 nm has the maximum at 280.+-.2 nm and 
a shoulder at 287.+-.2 nm. 
EXAMPLE 2 
This example sets out physical organic data with respect to various 
embodiments of the peptides of the present invention. 
Table 1 contains Rf.sub.1 (in chloroform-methanol-32% acetic acid=60:45:20) 
and Rf.sub.2 (in butanol-pyridine-water-acetic acid=5:5:4:1) values for a 
number of compounds of the new peptide. 
TABLE 1 
______________________________________ 
PEPTIDE Rf.sub.1 
Rf.sub.2 
______________________________________ 
Abu-Glu-Trp-OH 0.40 0.56 
Aca-Glu-Trp-OH 0.41 0.57 
Ala-Glu-Trp-NH.sub.2 
0.40 0.51 
Arg-Glu-Trp-OH 0.26 0.48 
D-Ala-Glu-Trp-OH 0.37 0.55 
D-Ile-Glu-Trp-D-Phe 0.71 0.77 
D-Ile-Glu-Trp-OH 0.39 0.54 
D-Leu-Glu-Trp-NH.sub.2 
0.35 0.56 
D-Leu-Glu-Trp-OH 0.37 0.57 
D-NVal-Glu-Trp-OH 0.38 0.56 
D-Phe-Glu-Trp-Ala 0.69 0.76 
D-Pro-Glu-Trp-OH 0.58 0.72 
D-Trp-Glu-Trp-OH 0.47 0.56 
D-Tyr-Glu-Trp-OH 0.45 0.57 
D-Val-Glu-Trp-NH2 0.43 0.53 
Gly-Glu-Trp-Gly 0.44 0.49 
Gly-Glu-Trp-OH 0.42 0.56 
H-Glu-Trp-Abu 0.49 0.54 
H-Glu-Trp-Aca 0.51 0.56 
H-Glu-Trp-Arg 0.28 0.40 
H-Glu-Trp-D-Ala 0.61 0.70 
H-Glu-Trp-D-Ile 0.63 0.71 
H-Glu-Trp-D-Leu 0.64 0.72 
H-Glu-Trp-D-NVal 0.65 0.69 
H-Glu-Trp-D-Pro 0.66 0.69 
H-Glu-Trp-D-Trp 0.63 0.66 
H-Glu-Trp-D-Tyr 0.61 0.66 
H-Glu-Trp-D-Val 0.65 0.71 
H-Glu-Trp-Ile 0.64 0.68 
H-Glu-Trp-Gly 0.54 0.58 
H-Glu-Trp-NH.sub.2 0.42 0.55 
H-Glu-Trp-N.sub.2 H.sub.3 
0.32 0.41 
H-Glu-Trp-NVal 0.67 0.71 
H-Glu-Trp-Trp 0.64 0.67 
H-Glu-Trp-Tyr 0.62 0.66 
H-Glu-Trp-Val 0.66 0.71 
His-Glu-Trp-OH 0.31 0.58 
Ile-Glu-Trp-Phe 0.71 0.78 
Ile-Glu-Trp-OH 0.38 0.54 
Ile-Glu-Trp-Phe 0.72 0.78 
Ile-Glu-Trp-Pro 0.68 0.81 
Leu-Glu-Trp-OH 0.39 0.56 
Lys-Glu-Trp-OH 0.30 0.51 
Lys-Glu-Trp-Tyr 0.32 0.50 
NVal-Glu-Trp-OH 0.37 0.55 
Phe-Glu-Trp-NH.sub.2 
0.53 0.68 
Ile-Glu-Trp-Phe 0.71 0.78 
Ile-Glu-Trp-OH 0.38 0.54 
Ile-Glu-Trp-Phe 0.72 0.78 
Ile-Glu-Trp-Pro 0.68 0.81 
Leu-Glu-Trp-OH 0.39 0.56 
Lys-Glu-Trp-OH 0.30 0.51 
Lys-Glu-Trp-Tyr 0.32 0.50 
NVal-Glu-Trp-OH 0.37 0.55 
Phe-Glu-Trp-NH.sub.2 
0.53 0.62 
Pro-Glu-Trp-Leu 0.67 0.75 
Pro-Glu-Trp-OH 0.59 0.72 
Trp-Glu-Trp-OH 0.48 0.59 
Tyr-Glu-Trp-OH 0.46 0.58 
Val-Glu-Trp-Ala 0.61 0.71 
Val-Glu-Trp-NH.sub.2 
0.38 0.52 
Val-Glu-Trp-OH 0.36 0.51 
Val-Glu-Trp-Tyr 0.59 0.61 
______________________________________ 
EXAMPLE 3 
This example concerns the ability of a peptide of the invention to 
stimulate lymphocyte production. 
The biological action of the novel peptide was studied in guinea pigs in 
the conventional E-rosette formation test. Table 2 shows comparative data 
of the effect of thymic preparations and that of a claimed peptide of the 
present invention on E-rosette formation of lymphocytes in guinea pigs 
after trypsin treatment. 
TABLE 2 
__________________________________________________________________________ 
Treated 
After treatment with trypsin and 
Untreated with 
compound with concentration mg/ml* 
Compound 
Animal 
Trypsin 
10.sup.-6 
10.sup.-7 
10.sup.-8 
10.sup.-9 
10.sup.-10 
10.sup.-11 
10.sup.-12 
__________________________________________________________________________ 
Tymolin 
66.5 36.1 
57.0 
40.1 
37.0 
35.3 
37.4 
36.5 
34.7 
Tymozine 
66.5 36.1 
60.3 
35.4 
33.4 
39.5 
39.1 
33.7 
35.8 
Fraction 5 
Ile-Glu-Trp 
66.5 36.1 
61.4 
63.9 
64.8 
60.2 
37.5 
40.0 
34.3 
__________________________________________________________________________ 
*Each concentration was tested on 5 animals. The positive growth differs 
in relation to the control group; EPOK is 50% or higher. 
As can be seen from Table 2, it was established that in vitro use of the 
said peptide is 10.sup.3 times more active than other known compounds. 
EXAMPLE 4 
This example illustrates the immunomodulating properties of the peptide. 
The immumostimulating effect of H-Ile-Glu-Trp-OH was tested in intact 
animals and with secondary immunodeficiencies, specifically 
irradiation-induced ones. 
Females and male (CBA.times.C57BL)F1 mice, aged about 2.5 months weighing 
about 20 g, were irradiated with gamma-rays using a "Luch-1" apparatus. 
Immunological activity was assessed according to Jerne (Antibody Forming 
Cells count--AFC). T-cell count in spleen was determined by the method of 
spontaneous rosette formation with sheep erythrocytes (E-FRC). The peptide 
was injected intramuscularly. 
Mice were irradiated in a dose of 2 Gy, the peptide was injected in the 
dose of 10 .mu.g/kg according to the following scheme (to determine T-cell 
count by the method of spontaneous rosette formation): 1 time--an hour 
after the irradiation; 2 times--an hour, and a day after irradiation; 3 
times--an hour, a day, and two days after the irradiation; 4 times--an 
hour, a day, two days and three days after the irradiation. The intact 
mice recieved the peptide 3 or 4 times. The control group (2 Gy) recieved 
injections of physiological solution according to the same schedule. On 
completion of the treatment course, 10 mice from each group were immunized 
with sheep erythrocytes (SE) and 4-5 days later AFC counts were determined 
in their spleens. The rest of the mice were used to find T-cell count by 
the method of spontaneous rosette formation. The state of the organs of 
immune system (spleen and thymus) in mice with radiation immunodeficiency 
against the background of H-Ile-Glu-Trp-OH treatment was also listed by 
nucleated cell counts in thymus and spleen per mg of organ weight. The 
results are shown in Tables 3 and 4. AFC/mg Spleen weight is presented as 
an average of two experiments. 
TABLE 3 
______________________________________ 
T-Cell Count, 10.sup.3 /mg Spleen 
Group AFC/mg Spleen 
1 2 
______________________________________ 
Control 168.0 .+-. 10.0* 
35.1 .+-. 2.1 
15.8 .+-. 1.2** 
Irradiation 2 Gy 
81.1 .+-. 13.1*** 
22.2 .+-. 2.9## 
6.3 .+-. 3.2# 
2 Gy + Peptide 
-- 34.5 .+-. 1.7## 
-- 
1 Injection 
2 Gy + Peptide 
-- 35.6 .+-. 1.8## 
-- 
2 Injection 
2 Gy + Peptide 
152.4 .+-. 4.1*** 
45.3 .+-. 3.4## 
23.2 .+-. 3.7# 
3 Injection 
2 Gy + Peptide 
158.1 .+-. 7.1*** 
48.4 .+-. 2.5## 
13.0 .+-. 0.8# 
4 Injection 
Peptide 3 Injections 
621.1 .+-. 14.2* 12.7 .+-. 1.5 
Peptide 4 Injections 
676.1 .+-. 47.5* 18.7 .+-. 5.8** 
______________________________________ 
*, **, ***, ##, P &lt; 0.05 
TABLE 4 
______________________________________ 
Karyocyte Count, 10.sup.3 /mg 
Group Spleen Thymus 
______________________________________ 
Control 1100 .+-. 90 
1850 .+-. 260 
Irradiation 2 Gy 880 .+-. 50** 
1820 .+-. 250 
2 Gy + Peptide 1 Injection 
1120 .+-. 73* 
1860 .+-. 200 
2 Gy + Peptide 2 Injection 
890 .+-. 80 
1840 .+-. 380 
2 Gy + Peptide 3 Injection 
980 .+-. 80 
2590 .+-. 400 
2 Gy + Peptide 4 Injection 
1160 .+-. 100* 
2480 .+-. 280 
______________________________________ 
*P &lt; 0.05 
** Significance was calculated relative to this group. 
As data in Table 4 show, the peptide injections to irradiated mice (one and 
four injections) brought about a trustworthy increase in the karyocyte 
count in spleen per mg of organ weight and, a certain growth of the 
karyocyte count in thymus (3 and 4 injections). The data in Table 3 show 
that the number of antibody forming cells practically doubled in 
irradiated mice injected with the peptide (3 and 4 injections). T-cell 
count in spleen grew in all mice who received the peptide injections, 
especially three or four injections. 
H-Ile-Glu-Trp-OH thus has a pronounced immunostimulating effect under 
radiation immunodeficiencies, and it is most effective when injected 3-4 
times (Tables 3, 4). 
The immunostimulating effect of the peptide is observed not only in 
irradiated mice. When inducing the humoral response to SE in intact mice, 
AFC increased 5 times, the T-cell count being the same as it was [Table 
3]. Consequently, the peptide had a pronounced immunomodulating effect 
when injected both to intact and irradiated mice. 
EXAMPLE 5 
Radiotherapeutic properties of the peptide H-Ile-Glu-Trp-OH and its effect 
on the population of hemopoietic progenitors was studied. This example 
demonstrated the use of the peptide to substitute accessory T-cells in 
regulation of hemopoiesis. 
(CBA.times.C57BL)F1 female mice (n=2500), 2-months old, weighing about 20 
g, were used in the experiment. 
As is known, there is a close relationship between T-cells and hemopoietic 
progenitors. It was shown that, along with colony-forming units of spleen 
(CFU-S), thymocytes participate in colony formation of spleens of 
irradiated mice by controlling proliferation of the hemopoietic progenitor 
cells. The fact was established in a test system, which allowed a 
selective removal of accessory T-cells from bone marrow with the help of 
rabbit antimouse brain antiserum (RAMBS) and their substitution with other 
factors. Aliquots (0.1 ml) of bone marrow suspension (2.times.10.sup.7 
cells), RAMBS, and medium 199 were mixed and incubated for 1 hour at 
37.degree. C. Then the cells were washed with centrifuging, resuspended 
and injected to lethally irradiated mice. Spleen colony counts were 
notably lower as compared to control. The colony formation was restored by 
injecting the mice with additional thymocytes, along with RAMBS-treated 
bone marrow cells, which proved the participation of T-cells in colony 
formation. 
According to the aforesaid scheme, instead of thymocytes mice received the 
peptide, H-Ile-Glu-Trp-OH injections 30 minutes before the injections of 
RAMBS-treated bone marrow suspension. The results are presented in Table 
5. 
TABLE 5 
______________________________________ 
RAMBS Preparation 
The Number of Mice 
Average Colony Count 
______________________________________ 
- - 19 10,7 .+-. 0,7 
+ - 23 2,3 .+-. 0,5 
+ thymocytes 
22 6,0 .+-. 0,6 
+ the peptide 
20 6,3 .+-. 0,4 
______________________________________ 
The peptide was shown to increase colony yields considerably (2-3 times as 
compared to RAMBS-treated bone marrow), and acted practically at the same 
level as thymocytes. The same experiment was conducted using the peptide 
H-Glu-Trp-OH ("Thymogen") instead of H-Ile-Glu-Trp-OH. This resulted in an 
average colony count of 2.9+/-0.3. Thus Thymogen does not posess such 
activity. 
EXAMPLE 6 
The next example concerns the effect of a peptide of the invention on 
hemopoiesis after exposure to ionising radiation. The present inventors 
studied the ability of a peptide of the invention to weaken the harmful 
effects of ionising radiation. 
For this purpose, known methods using exogenous spleen colonies were 
applied. A suspension of intact marrow cells was irradiated in the dose of 
1 Gy. Different doses of the peptide were injected intravenously (i.v.), 
intraperitoneally (i.p.), or intramuscularly (i.m.) to lethally irradiated 
recipients an hour after the injections of irradiated bone marrow. 
Colonies were counted on day 9. 350 mice were used as test animals. The 
data for each group are the average of three tests. 
The data obtained is contained in Table 6 and shows that the peptide can 
reduce the detrimental effect of radiation on hemopoietic progenitor 
cells, i.e. the peptide is not only an immunostimulator but has also a 
definite effect on the initial stages of hemopoiesis. 
TABLE 6 
______________________________________ 
Irradiated 
Drug Dose Colony 
Dose mcg/Kg i.v. i.p. i.m. Count 
______________________________________ 
- - - - - 10.6 .+-. 0.4 
1 Gy - - - - 3.4 .+-. 0.5** 
1 Gy 2.5 + - - 6.0 .+-. 0.7* 
1 Gy 10 + - - 8.7 .+-. 0.4* 
1 Gy 25 + - - 7.6 .+-. 0.4* 
1 Gy 50 + - - 7.1 .+-. 0.5* 
1 Gy 100 + - - 6.0 .+-. 0.5* 
1 Gy 200 + - - 5.3 .+-. 0.7* 
1 Gy 500 + - - 4.2 .+-. 0.3 
1 Gy 10 - + - 8.2 .+-. 0.7* 
1 Gy 10 - - + 10.6 .+-. 0.8* 
3 Gy - - - - 0.8 .+-. 0.2** 
3 Gy 10 + - - 2.3 .+-. 0.3* 
______________________________________ 
From the data of Table 6, it can be seen that IM administration of the 
peptide was more effective in reconstituting CFU-S colonies than IV or IP 
administration. A further study of the effect of the peptide, using 
different methods of administration, on the dynamics of reconstitution of 
CFU-S, Nucleated blood Cells (NC) in the femoral bone marrow and 
peritheric blood (mm) of irradiated mice (4 Gy) was conducted. 
Donor mice were irradiated with .gamma.-rays in the dose of 4 Gy. Then they 
received the petide i.v. or i.m. 1, 2, 3, or 4 times, once a day, starting 
from the 1 first hour of the 3rd day after the irradiation (10 .mu.g/kg 
per injection). The mice were observed for 14-16 days. At different 
intervals after the irradiation 10 mice were taken from each group to 
determine CFU-S in the bone marrow. The mice were killed, cell suspensions 
were prepared from the bone marrow and injected to lethally irradiated 
recipients. NC were counted in the femoral bone marrow and peritheric 
blood, and the lymphocyte percentage was found. 
Test animals were 1110 mice. 
The study revealed that i.v. injection of H-Ile-Glu-Trp-OH 1, 3, or 4 times 
had helped better reconstitution of the CFU-S pool by day 14 only. While 
IM administration of the drug (especially 1, 2, 3 times) positively 
influenced the reconstitution of the CFU-S pool earlier, beginning from 
day 8 after the irradiation, but it had no effect on the NC content in the 
bone marrow, which points to an increase, as compared to control, of the 
hemopoietic progenitor percentage as a result of the H-Ile-Glu-Trp-OH 
treatment of irradiated mice. Each group consisted of 10 mice. 
EXAMPLE 7 
The example illustrates the ability of the peptide to eliminate the 
cytostatic effect of irradiation (in the dose of 4 Gy) on stem and mature 
cells of the hemopoietic system in the test of regeneration of the 
karyocyte counts and CFU-S population in femoral bone marrow and nucleated 
blood cells (1 mm.sup.3). 
Irradiated mice (4 Gy) received intramuscular injections of the peptide in 
the dose of 10 .mu.g/kg according to 2 schemes. 
a) Scheme 1: mice received the peptide immediately after irradiation, 
daily. The mice were divided into groups: a control group--irradiated mice 
(4 Gy), and test groups--irradiated mice (4 Gy) received the peptide 1, 2, 
3, and 4 times daily. Blood samples were taken four hours after the last 
peptide injection and on the 7th, 10th, and 16th day after the 
irradiation. Determined were total count of nucleated blood cells (NC) and 
lymphocyte percentage of them; nucleated cells and the CFU-S population in 
femoral bone marrow were counted on the 4th, 8th, and the 14th day. The 
results are presented in Tables 7, 8 and FIG. 1. 
TABLE 7 
______________________________________ 
Karyocyte Count in blood .times. 10.sup.3 /lmm.sup.3 on 
Group Day 4 Day 7 Day 10 Day 16 
______________________________________ 
Control (4 Gy) 
1.33 .+-. 0.2 
5.30 .+-. 0.9 
6.00 .+-. 0.1 
3.67 .+-. 0.7* 
Peptide 1 Time 
0.80 .+-. 0.5 
3.53 .+-. 0.5 
4.30 .+-. 0.3 
4.49 .+-. 0.8 
Peptide 2 Times 
1.30 .+-. 0.2 
3.64 .+-. 0.6 
6.42 .+-. 0.7 
5.96 .+-. 1.0 
Peptide 3 Times 
1.19 .+-. 0.1 
5.58 .+-. 0.7 
4.18 .+-. 0.8 
6.32 .+-. 0.7* 
Peptide 4 Times 
1.64 .+-. 0.2 
4.14 .+-. 0.8 
4.90 .+-. 0.7 
7.32 .+-. 1.0* 
Intact: NC-6900 .+-. 25 
Lymphocytes: 4533 .+-. 213.2 (65.7 .+-. 3.3) 
______________________________________ 
*p &lt; 0.05 
Survival 100% 
TABLE 8 
______________________________________ 
Lymphocytes, % (absolute) in blood on 
Group Day 4 Day 7 Day 10 Day 16 
______________________________________ 
Control 20.0 .+-. 1.2 
21.4 .+-. 1.9 
18.8 .+-. 1.2 
29.6 .+-. 3.2 
(266) (1130) (1080) (1064) 
Peptide 1 Time 
29.0 .+-. 2.5 
38.8 .+-. 3.1 
26.3 .+-. 2.9 
53.6 .+-. 6.0 
(232) (1365) (1118) (2412) 
Peptide 2 Times 
36.0 .+-. 1.7 
34.8 .+-. 2.1 
25.5 .+-. 2.7 
44.8 .+-. 6.2 
(468) (1260) (1664) (2682) 
Peptide 3 Times 
36.0 .+-. 1.3 
27.1 .+-. 1.9 
29.5 .+-. 3.6 
44.8 .+-. 4.80* 
(432) (1512) (1233) (2853) 
Peptide 4 Times 
39.0 .+-. 2.9 
25.6 .+-. 3.7 
30.7 .+-. 6.5 
38.5 .+-. 1.40 
(639) (1025) (1475) (2847) 
______________________________________ 
According to the data in Table 7, up to the 16th day after the irradiation, 
in none of the test groups did the karyocyte count in blood exceed the 
control level. On day 16, in all test groups the karyocyte counts were 
higher than in control. Table 8 shows that in all test mice the lymphocyte 
percentage was higher, and the absolute number of lymphocytes grew faster, 
than in control throughout the whole observation period. In those mice, 
who received the peptide more than once, the lymphocytes count in blood at 
the time of the maximum depletion of the pool of mature cells (on day 4 
after the irradiation) was much higher than in control. 
This pool, after it goes through cell division and maturation from stem 
cells to the mature functioning ones, which usually lasts about 8 days, 
appears in blood. This is observed as an increasing karyocyte count on day 
16. Thus, treatment of irradiated mice with the peptide started 
immediately after the irradiation, i.e. before the bone marrow depleted, 
led to a greater survival of the remaining hemopoietic progenitors. 
Treatment according the scheme did not affect the cellular composition of 
bone marrow, but it markedly stimulated regeneration of the CFU-S 
population (FIG. 1). This example illustrates the stimulating effect of 
intramuscular injections of the peptide of regeneration of the CFU-S 
population in the bone marrow of irradiated mice (4 Gy). 
From each group at least five mice were killed, their bone marrow was taken 
out and used to prepare the cell suspension, which was injected to 
lethally irradiated mice. After 9 days, CFU-S was counted in irradiated (4 
Gy) donors receiving the peptide by counting the colonies grown on spleen. 
The results are shown in FIG. 1. It was noted that with intramuscular 
administration of the peptide the CFU-S count in irradiated mice bone 
marrow was steadily growing starting from the 4th day, leaving the control 
CFU-S far behind. 
b) According to scheme 2, the peptide injections were made starting from 
the 3rd day after the irradiation, at the same time of maximum depletion 
of the pool of mature nucleated cells and the bone marrow. The groups of 
mice were formed in the same way as in the previous scheme. Blood samples 
were taken 4 hours after the last injection i.e. on day 6, as well as on 
days 11, 14, 18, 21. Tables 9 and 10 present the data on karyocyte and 
lymphocyte counts in blood (per 1 mm.sup.3) of mice treated according to 
scheme 2. 
TABLE 9 
__________________________________________________________________________ 
Karyocyte Count in Blood .times. 10.sup.3 on: 
Group Day 3 
Day 7 Day 11 
Day 14 
Day 18 
Day 21 
__________________________________________________________________________ 
(Treatment According to Scheme 2) 
Control 1.2 .+-. 0.1 
1.38 .+-. 0.1* 
1.85 .+-. 0.4* 
2.46 .+-. 0.3 
2.33 .+-. 0.2 
2.02 .+-. 0.1 
Peptide 1 Time 
1.64 .+-. 0.2 
1.88 .+-. 0.3 
1.50 .+-. 0.2 
2.62 .+-. 0.2 
1.63 .+-. 0.2 
Peptide 2 Times 
1.33 .+-. 0.2 
2.03 .+-. 0.3 
1.60 .+-. 0.2 
1.67 .+-. 0.1 
2.70 .+-. 0.2 
Peptide 3 Times 
2.22 .+-. 0.3 
1.94 .+-. 0.2 
1.50 .+-. 0.2 
2.07 .+-. 0.2 
2.45 .+-. 0.4 
Peptide 4 Times 
2.71 .+-. 0.4* 
3.03 .+-. 0.5* 
1.17 .+-. 0.1 
2.48 .+-. 0.1 
1.66 .+-. 0.2 
__________________________________________________________________________ 
*P &lt; 0.05 
TABLE 10 
__________________________________________________________________________ 
Lymphocyte in Blood, % (Absolute) on: 
Group Day 3 
Day 7 
Day 11 
Day 14 
Day 18 
Day 21 
__________________________________________________________________________ 
(Treatment According to Scheme 2) 
Control 24.4 .+-. 1.7 
29.0 .+-. 1.4 
27.4 .+-. 1.1 
29.7 .+-. 3.2 
30.9 .+-. 3.1 
29.5 .+-. 3.1 
(400) 
(499) 
(731) 
(730) 
(596) 
Peptide 1 Time 
45.1 .+-. 3.9 
49.2 .+-. 3.8 
43.3 .+-. 3.2 
30.6 .+-. 2.9 
45.6 .+-. 3.9 
(232) 
(1365) 
(1118) 
(2412) 
(743) 
Peptide 2 Times 
52.7 .+-. 2.9 
45.1 .+-. 5.4 
38.8 .+-. 2.2 
42.1 .+-. 1.4 
45.2 .+-. 2.3 
(701) 
(915) 
(619) 
(703) 
(1220) 
Peptide 3 Times 
43.6 .+-. 2.1 
46.3 .+-. 5.4 
47.7 .+-. 6.1 
35.5 .+-. 1.4 
52.5 .+-. 3.2 
(968) 
(892) 
(744) 
(7353) 
(1289) 
Peptide 4 Times 
45.6 .+-. 2.5 
42.6 .+-. 3.6 
44.0 .+-. 3.1 
45.1 .+-. 1.8 
47.5 .+-. 5.3 
(1231) 
(1406) 
(515) 
(1116) 
(789) 
__________________________________________________________________________ 
*- p &lt; 0.05 
It was shown that karyocyte counts were of the same order in control and 
test groups throughout the observation period, save those mice who 
received the peptide 4 times: on days 6 and 11 (the phase of abortive 
elevation) the number of nucleated cells in them was higher than in 
control. Interestingly, on day 14 the content of these cells decreased 
considerably, especially in the group which received 4 peptide injections. 
Probably, the peptide accelerates maturation of the CFU-S, which survived 
irradiation, their pool exhausts faster and by the 14th day no mature 
cells come in blood from this pool, while the abortive elevation of 
karyocyte count still lasts in control mice. Throughout the observation 
period, the contribution of lymphocytes to karyocytes was notably higher 
in the peptide- treated mice than in control. Absolute lymphocyte count in 
test mice was higher as compared to control in the period of the abortive 
elevation (the 6th-11th day after irradiation) and on day 21 in 
restoration period. During the second depletion (the 14th-18th day), the 
lymphocyte counts were equal in test and control groups. 
The data in Table 11 reflect the process of bone marrow regeneration in 
control and test irradiated (4 Gy) mice treated with peptide according to 
scheme 2. The regeneration was equally intensive in all animal groups till 
the 11th day, from day 14 to day 18 the nucleated cells counts were 
considerably higher in the bone marrow of test mice. 
TABLE 11 
__________________________________________________________________________ 
Karyocyte Count in Femoral Bone Marrow .times. 10.sup.6 on: 
Group Day 3 
Day 7 Day 11 
Day 14 
Day 18 
Day 21 
__________________________________________________________________________ 
(Treatment According to Scheme 2) 
Control 9.3 .+-. 0.8 
16.8 .+-. 1.0 
21.8 .+-. 1.5 
13.4 .+-. 1.9 
13.7 .+-. 1.5 
20.6 .+-. 0.9 
Peptide 1 Time 
16.4 .+-. 0.3 
17.9 .+-. 1.8 
18.7 .+-. 1.1 
18.0 .+-. 0.4 
22.4 .+-. 2.2 
Peptide 2 Times 
14.9 .+-. 1.7 
18.6 .+-. 2.3 
14.1 .+-. 1.7 
17.2 .+-. 1.0 
18.9 .+-. 0.6 
Peptide 3 Times 
12.25 .+-. 1.3 
18.5 .+-. 2.9 
16.9 .+-. 0.8 
16.9 .+-. 0.8 
19.5 .+-. 1.6 
Peptide 4 Times 
2.71 .+-. 2.7 
20.3 .+-. 1.3 
18.9 .+-. 0.2 
17.7 .+-. 1.2 
20.0 .+-. 1.8 
__________________________________________________________________________ 
TABLE 12 
______________________________________ 
CFU-S (absolute) in blood on: 
Group Day 3 Day 7 Day 11 Day 14 
______________________________________ 
4 Gy 297 .+-. 36.3 
378 .+-. 53 
937 .+-. 91 
844 .+-. 94 
N 1 Time 459 .+-. 26 
858 .+-. 67 
1197 .+-. 75 
N 2 Time 298 .+-. 27 
846 .+-. 44 
902 .+-. 56 
N 3 Time 305 .+-. 33 
1077 .+-. 56 
1166 .+-. 68 
N 4 Time 431 .+-. 48 
1147 .+-. 55 
1434 .+-. 89 
______________________________________ 
Table 12 illustrates that reconsitution of CFU-S Population in Irradiated 
(4 Gy) and Irradiated (4 Gy) and H-Ile-Glu-Trp-OH (N)-Treated Mice The 
peptide had a positive effect on the size of the CFU-S population on the 
11th day in those mice who received the peptide for 3 or 4 days, and by 
the 14th day the CFU-S counts in all test groups were greater than in 
control [see Table 12]. The results are shown in FIG. 2. From day 14 till 
day 18 a considerable growth of NC count was recorded in the bone marrow 
of treated mice [see Table 11]. 
EXAMPLE 8 
The effect of the peptide on erythropoiesis in irradiated mice (4 Gy) was 
also studied. The mice were irradiated and treated with the peptide as in 
Example 7. 
Erythrocyte counts did not differ in test and control groups. In treated 
mice, hemoglobin level restored faster and become normal by the 11th day, 
while in control--by day 14. Table 13 presents the results. 
TABLE 13 
__________________________________________________________________________ 
Hemoglobin Content (r/dl) in blood on 
Group Day 3 
Day 7 Day 11 
Day 14 
Day 18 
Day 21 
__________________________________________________________________________ 
(Treatment According to Scheme 2) 
Control 7.8 .+-. 0.9 
8.4 .+-. 0.7 
9.9 .+-. 0.3 
12.0 .+-. 0.4 
12.1 .+-. 0.1 
12.2 .+-. 0.4 
Peptide 1 Time 
9.4 .+-. 0.8 
12.6 .+-. 0.1 
11.8 .+-. 0.5 
12.1 .+-. 0.1 
12.1 .+-. 0.4 
Peptide 2 Times 
9.6 .+-. 0.4 
12.0 .+-. 0.1 
10.8 .+-. 0.8 
11.5 .+-. 0.6 
11.7 .+-. 0.6 
Peptide 3 Times 
10.15 .+-. 0.8 
11.9 .+-. 0.4 
12.1 .+-. 0.2 
11.7 .+-. 0.2 
11.9 .+-. 0.3 
Peptide 4 Times 
14.4 .+-. 0.9 
12.2 .+-. 0.1 
12.0 .+-. 0.2 
11.7 .+-. 0.4 
12.0 .+-. 0.6 
__________________________________________________________________________ 
The four-injection treatment was most effective in this case. 
EXAMPLE 9 
In the next series of experiments, the effect of H-Ile-Glu-Trp-OH on 
formation of endogenous spleen colonies in irradiated mice (6 Gy) was 
studied. The results of the study reflect the response of hemopoietic 
progenitors to ionising radiation in the body. 
Test mice were irradiated in the dose of 6 Gy Beginning from the first hour 
after the irradiation, the mice received once daily IM injections of 10 
.mu.g/kg H-Ile-Glu-Trp-OH. Injections were made 1, 2, 3, or 4 times. The 
mice were killed on Day 9, spleens were taken out and fixed in Buene's 
solution, the colonies were counted. 110 mice were used in the test. 
TABLE 14 
______________________________________ 
Group Colony P 
______________________________________ 
6 Gy 2.4 .+-. 03 
N 1 times 3.3 .+-. 0.6 
N 2 times 4.4 .+-. 0.5 
&lt;0.05 
N 3 times 5.0 .+-. 0.7 
&lt;0.05 
N 4 times 5.2 .+-. 0.8 
&lt;0.05 
______________________________________ 
The data in the Table 14 show that H-Ile-Glu-Trp-OH significantly increased 
the yield of endogenous spleen colonies after irradiation with the dose of 
6 Gy in all administration schemes except that with one injection. To put 
it differently, in this experimental system the peptide reduced the 
destructive effect of ionising radiation on the CFU-S pool. 
EXAMPLE 10 
The effect of H-Ile-Glu-Trp-OH on hemopoiesis: reconstitution of 
granulocytes and lymphocytes in cytostatic-treated mice was studied. Also 
studied was the correcting effect of H-Ile-Glu-Trp-OH in a 
cytostatic-induced cytopenia of the hemopoietic organs. 
Used as a cytostatic, Cytosine-Arabinoside (CA) was intraperitoneally 
injected to donor mice once in the dose of 20 mg/kg. The peptide was given 
according to the following scheme: 10 .mu.g/kg once daily, three 
injections starting from the first hour after CA. Intact mice received 
H-Ile-Glu-Trp-OH in the same doses and according to the same schedule. On 
day 8 after the cytostatic injection, determined were karyocyte count in 
the donor bone marrow and the content of hemopoietic progenitors of 
different polipotencies: committed CFU-S-8 and CFU-S-12. CFU-8 are 
committed stem cells which later undergo differentiation. CFU-12 are the 
polypotent cells, from which stem cells can be recruited (i.e. they 
maintain the pool of stem cells). This is a slowly proliferating cell 
population, mainly in G phase. That is why it is important to evaluate the 
effect of a preparation on the pools of both committed (CFU-8) and 
polypotent (CFU- 12) cells. To this end, bone marrow was extracted from 
donor's femoral bones and injected to lethally irradiated recipients. 
After the cell injections, every test group was divided into 2 subgroups: 
some animals were killed on day 8, the other on day 12. Spleens were taken 
out and the colonies were counted. The tests were carried out on 105 mice. 
TABLE 15 
______________________________________ 
Number of Day 8 COUNT 
GROUP Mice Karyocytes .times. 10.sup.6 
CFU-S-8 CFU-S-12 
______________________________________ 
Control 24 25.0 .+-. 1.2 
2674 .+-. 150 
2841 .+-. 188 
Control + N 
20 23.0 .+-. 0.9 
2805 .+-. 232 
2930 .+-. 152 
CA 24 17.5 .+-. 0.8* 
1270 .+-. 53* 
1244 .+-. 71* 
CA + N 24 22.0 .+-. 0.7 
1644 .+-. 154 
2488 .+-. 203 
______________________________________ 
*- Significance was calculated relative to this group 
P &lt; 0.05 
The result of the effect of H-Ile-Glu-Trp-OH on reconstitution of CFU-S-8 
and CFU-S-12 and karyocyte count after cytostatic treatment are shown in 
Table 15. The cytostatic CA lowered the karyocyte count in the bone marrow 
and halved CFU-S-8 and CFU-S-12 counts as compared to control. With the 
H-Ile-Glu-Trp-OH therapy, by day 8 the karyocyte count had been completely 
reconstituted, the CFU-S-8 population had grown significantly, the 
CFU-S-12 pool had reached the values of the intact control. Noteworthy is 
that H-Ile-Glu-Trp-OH most effectively reconstituted the compartment of 
polipotent progenitors (CFU-S-12) able to originate not only all lines of 
hemopoiesis but also immune cells. 
So, H-Ile-Glu-Trp-OH was found to help restore hemopoiesis after exposure 
to such factors as ionising radiation and cytostatics. 
EXAMPLE 11 
The influence of the oxyurea on the tumor cells, treated by synthetic 
H-Ile-Glu-Trp-OH was studied. 
Various cytostatics (preparations which stop the proliferation of cells) 
are included in the traditional schemes of treatment of any malignant 
tumors. One of them is the oxyurea (OU). 
The goal of this series of experiments was to examine the response of the 
thymoma EL-4 cells to action of OU after their incubation with 
H-Ile-Glu-Trp-OH. 
The Thymoma EL-4 cells were extracted from the abdomen cavity of C57B1 mice 
at the 7th day after transplantation. The suspension was washed by 
centrifugation in the medium RPMI. Then the cells were resuspended in the 
growth medium (RPMI+10% of embryo serum+0.1% penicillin/streptomycin) and 
were split in culture flasks 5 ml each (10 ml cells per flask). 2 mg of 
the preparation were added (20 .mu.l) to each probe. After 18 hours of 
incubation (37 degrees Celsius CO.sub.2), OU was added to the half of 
probes (1 mg per 5 ml). Incubation was done for 1 more hour in the same 
conditions and then cells were washed out by centrifugation in the medium 
RPMI. The precipitated cells were resuspended in calculation 1 ml per 0.2 
ml of RPMI medium and then transplanted to the mice C57B1 under the back 
skin. 
The taking of the measurements of the size of the tumor was started after 
the knot became visible. The tumor was measured every day until it was 
impossible to measure. The survival ability of the mice was examined 
during 60 days after transplantation. Mice that survived after this period 
were considered to be cured. Each experimental group consisted of 6 
animals. 
The treatment of the intact tumor cells (control) with OU causes 
approximately a 12-day delay in the tumor growth (FIG. 3). During the 
action of cytostatics on the thymoma cells, incubated with 
H-Ile-Glu-Trp-OH, the depression of the tumor growth on the 12th day is 
most definite (the dimensions of the tumor are smaller than those of the 
group not treated with OU). 
The incubation of thymoma EL-4 cells with any of the tested peptides caused 
some delay in growth of the tumor (registered in mm squared) in comparison 
with the control. This effect was more definite before the 10th day of 
growth, then the rate of the tumor growth was comparable with control. 
The results of the survival experiments on mice are shown in FIG. 3 and 
Table 16. 
TABLE 16 
______________________________________ 
Influence of Immunomodulators on the Survival and Average 
Lifespan of Mice with Thymomo EL-4 
Number of 
Survived % of Average of 
Group Mice From 6 
Survival Lifetime 
______________________________________ 
Control 0/6 0 21.0-.+-. 1.3 
Control + OU 0/6 0 26.0-.+-. 1.1 
H-Ile-Glu-Trp-OH 
1/6 16.6 24.5-.+-. 1.0 
H-Ile-Glu-Trp-OH + OU 
2/6 33.3 41.2-.+-. 16.0 
______________________________________ 
The result in Table 16 and FIG. 3 show the increase in lifetime of 5 days 
of mice with tumor, treated by OU. 
Incubation of the tumor cells with H-Ile-Glu-Trp-OH extended the lifetime 
of animals by 4 days and in combination with cytostatics by 15 days. On 
average the lifetime of the animals treated by H-Ile-Glu-Trp-OH and OU was 
20 days longer than the control group. Only in these groups were surviving 
animals registered after 60 days. 
Therefore, a positive effect on the dynamic of growth of tumor (delay) and 
the lifetime extension of the experimental animals was found by incubation 
of the Thymoma cells with synthetic immunomodulators. Immunostimulants 
(H-Ile-Glu-Trp-OH) can potentiate the damaging action of cytostatic on the 
Thymoma cells. 
EXAMPLE 12 
At the present time hyperthermia is widely used in the treatment of 
malignant tumors, and not only locally, but during autologic 
transplantation of the bone marrow for the therapy of leukosis (when it is 
not possible to find an identical donor). The influence of 
H-Ile-Glu-Trp-OH on the growth of tumor cells EL-4, treated by 
hyperthermia, was examined. 
Incubation of the tumor cells with the tested drugs were performed the same 
way as described above in the experiments with OU. After 18-hour 
incubation, the cells were washed in the medium RPMI, and then resuspended 
in the concentration of 5 ml/1 ml in plastic test tubes of total volume of 
2 ml each. Then the cells were heated for 1 hour at 43 degrees Celsius, 
and transplanted under the back skin of the mice C57B1. The growth of the 
tumor was examined the same way as described above. 
TABLE 17 
______________________________________ 
Influence of Heating and H-Ile-Glu-Trp-OH on Tumor Size - 
EL-4 (13th day) 
Number Tumor size in 
Group Of Mice mm squared 
______________________________________ 
Control 6 230.1 
Control + Heating 6 10.8 
H-Ile-Glu-Trp-OH 6 188.7 
H-Ile-Glu-Trp-OH + Heating 
6 No Tumor 
______________________________________ 
The results indicated that after heating of the intact Thymoma EL-4 cells 
and preliminary treatment in vitro with H-Ile-Glu-Trp-OH, followed by 
heating on the 21st day, no tumor formation was observed in any of the 
mice in that group. The mice were observered for 25 days. 
EXAMPLE 13 
Action of H-Ile-Glu-Trp-OH on the proliferation of a mixed lymphocyte 
culture (MLC) was studied. 
This experimental system is an in vitro the analog of the Graft Versus Host 
Disease reaction. 
In the present series of experiments the reaction H-2d, anti H-2b was 
examined. The new combination responder-stimulator. Each variant was made 
in a triplet. Microcultures were incubated for 4 days, then 3H-thymidine 
was added; then the mixture was incubated for 16 more hours, and after its 
transfer to the filters, the amount of 3H-thymidine was determined. 
H-Ile-Glu-Trp-OH was added at the beginning of the incubation in different 
concentrations. The results are presented as indexes of stimulation of 
proliferation. 
The results are presented in FIG. 4. The results show that H-Ile-Glu-Trp-OH 
in concentrations 1, 10, and 20 .mu.g/ml produces stimulation of 
proliferation of the allogenic lymphocytes, while in concentrations of 0.1 
.mu.g/ml it exhibited negligible inhibition of the proliferation. 
EXAMPLE 14 
The use of H-Ile-Glu-Trp-OH in treatment of patients with hemopoietic 
diseases was studied. 
The clinical study of H-Ile-Glu-Trp-OH was continued in groups of patients 
with pronounced secondary pathological changes in their immune system. The 
primary task of the first stage of the investigation was to observe the 
preparation tolerance, to reveal toxic, allergic and other reactions, 
hemogram dynamics and biochemical indices. Further investigation, along 
with further monitoring of the major clinical and laboratory parameters, 
included evaluation of the immunological status of a patient before and 
after the immunopeptide treatment. 
The effect of the immunopeptides on hemopoietic status was assessed on the 
basis of the dynamics of a number of immunological tests characterising 
both humoral and cellular immunity. 
Materials And Methods 
The study was continued in groups of patients with immune cytopenias, 
multiple myeloma, chronic lymphoid leukosis, lymphocytic lymphomas, 
lymphosarcomas (55 patients). Special attention was paid to the group of 
patients with P-cellular lymphoid leukosis. The peculiarity of the 
leukemic B-lymphocyte clone and its relatively vast representation in the 
peripheral blood of patients with a pronounced secondary immune deficiency 
makes B-cellular chronic Lymphoid leukosis a unique model for clinical 
studies of the effect of an immunomodulator. 
Research Methods 
Immune phenotyping is the most reliable method to determine the changes of 
ratios of immunocompetent cells in the peripheral blood and 
lymphocyte-containing organs. The method allows detection of 
subpopulations by the presence of cellular differentiating antigen 
structures on lymphocyte surface. For more trustworthy results, some 
patients were examined using a flow-type cytoflourimeter and monoclonal 
antibodies. Taking into account the contingent of the patients, the main 
surface structures to be examined were the following: CD45++CD14-, 
CD45+CD14-, CD45+CD14+, CD3+CD19-, CD3-CD19+, CD4+CD8-, CD4-CD8+, CD3+HLA- 
DR+, CD3+CD16,56+, CD34+, CDS+. 
The dynamics of CD4+CD8- and CD4-CD8+ (T-helpers and T-suppressers) and the 
dynamics of other necessary CD-markers were determined in the course of 
treatment with H-Ile-Glu-Trp-OH. A special attention was paid to 
CD45++CD14- and CD45+CD14 as they characterize the proportion between 
normal and leukemic lymphocytes in chronic lymphoid leukosis and 
illustrate the influence of the immunoactive peptide on the proportion. 
These peculiarities connected with the characteristics of the disease 
allow investigation of the molecular mechanism of its genesis. In the case 
of an immune cytopenia most important are the quantitative ratios of 
CD45++CD14- and CD45+CD14- to CD4-CD8+, their reliable values can be 
obtained with the help of a cytofluorimeter. 
The other clinical and instrumentally methods of investigation and 
observation remained the same as earlier: 
Before instituting the treatment, examination was carried out including 
case history, clinical, laboratory and instrumental investigations. 
Necessary morphological investigations were performed to verify the 
diagnosis. There were also the results of hemogram dynamics before and 
after each treatment course of H-Ile-Glu-Trp-OH. After these courses the 
biochemical indices and urine tests were examined again. X-ray, ultrasonic 
and morphological examinations were repeated on indication. 
The incidence rate of infectious inflammatory diseases in patients treated 
with H-Ile-Glu-Trp-OH was further recorded. 
For patients on cytostatic therapy, the following were recorded: the date 
of the beginning of a next course, the necessity to prolong the interval 
between courses in connection with unsatisfactory hemogram indices, as 
well as the cases of dosage adjustments. 
Humoral immunity was also investigated: the content of different types of 
immunoglobulins, protein fractions, antibodies to thyroid, and the Coombs 
test (the latter was performed in the case of haemolytic anaemia). 
H-Ile-Glu-Trp-OH Treatment Protocol 
One ml daily, intranasally, once or twice in divided doses, for 5 days. The 
course was repeated after a 3-week interval. To those patients who 
received a cytostatic mono- or polychemotherapy, H-Ile-Glu-Trp-OH was 
administered from the first day of the break in the cytostatic treatment. 
Treatment Results-H-Ile-Glu-Trp-OH 
In the period of a prolonged administration of H-Ile-Glu-Trp-OH (5-6 
courses) no complaints of the patients were noted. There were no toxic 
reactions, skin rash, disturbances of gastrointestinal function, central 
or peripheral nervous system, vascular, muscular or other adverse 
reactions. 
Physical investigations of patients before and after the treatment revealed 
no such changes of the somatic status, which could be ascribed to the 
action of the test preparation. 
The dynamics of biochemical indices--bilirubin, alanine and asparaginic 
transaminases, alkaline phosphatase, lactate dehydrogenase, glucose, 
carbamide, creatinine, uric acid, total protein, protein fractions, 
electrolytes--before and after 1-3 treatment courses revealed no changes 
associated with H-Ile-Glu-Trp-OH administration. We failed to reveal any 
changes in AP dynamics and ECG caused by H-Ile-Glu-Trp-OH. Noteworthy is 
that we observed no toxic manifestations, deviations of biochemical 
indices, or shifts on the part of the cardiovascular system not only after 
the first course but also after a series of H-Ile-Glu-Trp-OH courses. For 
this reason, these indices are not presented in detail for each group. 
Examination of hemograms for a longer observation period showed little 
difference from the results presented in the previous report. APcer 
treatment with H-Ile-Glu-Trp-OH,the same positive dynamics was noted as in 
the case of its administration to patients with depression of granulocytic 
cell line. 
Humoral status was evaluated--detection of protein fractions and 
immunoglobulins in patients' sera. No dynamics of these indices was found. 
It should be mentioned that the repeated detection of protein fractions 
and serum immunoglobulins was performed 1-1.5 months after the treatment, 
because in that period there was a probability of immunopeptidedependent 
stimulation of immunoglobulin production. 
Cellular immunity studies showed the most conspicuous results in patients 
with chronic lymphoid leukosis. Below are cited the examples of indices 
dynamics in patients who did not receive steroid and cytostatic therapy 
before (at least 6 months) and during treatment with H-Ile-Glu-Trp-OH. 
Patient M., born in 1925. Chronic lymphoid leukosis since 1990 Treatment: 
COPP--6 courses, M-2-6 courses with Thymogen after the courses till 1995 
with positive effect. No further treatment with cytostatics. Since October 
1996--has been receiving H-Ile-Glu-Trp-OH. The dynamics of indices before 
and after H-Ile-Glu-Trp-OH treatment course: leukocytes 7.9-8.6 blnA, 
Lymphocytes 41-42% (3.24-3.62 bln/1). 
TABLE 18 
__________________________________________________________________________ 
Absolute 
% of Cells Absolute 
Cell Phenotype 
% of Cells, 
% of Cells, 
Count, 
after H-Ile-Glu-Trp-OH 
Count, 
Subpopulation 
Normal 
Test Sample 
bln/l 
Treatment bln/l 
__________________________________________________________________________ 
CD45++CD14- 28.7 2.27 43.2 3.715 
lymphocytes 
CD45+CD14- 60.0 47.7 
granulocytes 
CD45+CD14+ 4.5 4.5 
monocytes 
CD3+CD19- 
58.2-84.3 
41.9 35.6 
T-lymphocytes 
CD3-CD19+ 
7.1-23.3 
59.0 61.2 
B-lymphocytes 
CD4+CD8- 
31.4-63.8 
15.8 11.6 
T-helpers 
CD4-CD8+ 
18.9-47.9 
32.9 30.6 
T-suppressers 
CD4+/CD8+ 
0.6-3.0 
0.5 0.4 
ratio 
CD3+HLA-DR+ 
3.5-25.9 
7.4 
activated 
T-lymphoc. 
CD3-CD16,56+ 
5.4-33.5 
11.6 
Nat. Killers 
CD34+ 0.4 
stem cells 
CD5+ 95.5 
T-cell. marker 
__________________________________________________________________________ 
According to the presented data (patient M.), before the beginning of the 
treatment course the lymphocyte count found by surface markers did not 
correspond to that obtained in the stained smear. At the same time, the 
total count of B- and T-lymphocytes (CD3+CD19 and CD3-CD19+) was in line 
with the number of cells having the CD5+ marker. It shows that CD5+ 
markers are carried not only by T-lymphocytes, but also by B-lymphocytes, 
which, as a rule, occurs in B-cellular chronic lymphoid leukosis. Thus, a 
complete clinical and haematological remission was not observed in the 
patient. After the course of H-Ile-Glu-Trp-OH the number of 
CD45++CD14--lymphocytes (supposedly, non-leukaemia) in blood grew 
significantly, while the relative and absolute lymphocyte counts 
determined morphologically and in smears of peripheral blood were the 
same. The ratio of CD4+CD8- and CD4-CD8+ (helpers and suppressers) 
remained as before. 
Patient S., born in 1926. Chronic lymphoid leukosis since 1985. Was not 
treated with cytostatics. Since October 1996--has been receiving monthly 
courses of H-Ile-Glu-Trp-OH. The dynamics of indices before and after the 
H-Ile-Glu-Trp-OH treatment course: leukocytes 57.2-67.5 bin/1, lymphocytes 
84-86% (48.04-58.06 bln/1). 
TABLE 19 
__________________________________________________________________________ 
Absolute 
% of Cells Absolute 
Cell Phenotype 
% of Cells, 
% of Cells, 
Count, 
after H-Ile-Glu-Trp-OH 
Count, 
Subpopulation 
Normal 
Test Sample 
bln/l 
Treatment bln/l 
__________________________________________________________________________ 
CD45++CD14- 5.9 3.37 13.5 9.112 
lymphocytes 
CD45+CD14- 83.7 78.0 
granulocytes 
CD45+CD14+ 0.7 0.8 
monocytes 
CD3+CD19- 
62.8-85.0 
61.3 52.0 
T-lymphocytes 1.7* 2.6* 
CD3-CD19+ 
7.1-23.3 
15.8 40.0 
B-lymphocytes 90.9* 96.9* 
CD4+CD8- 
31.4-63.8 
22.1 27.1 
T-helpers 1.4* 1.6* 
CD4-CD8+ 
18.9-47.9 
44.8 36.5 
T-suppressers 0.7* 1.9* 
CD4+/CD8+ 
0.7-3.3 
0.5 0.7 
ratio 2.0* 0.5* 
CD3+HLA-DR+ 
2.8-17.3 
4.2 16.0 
activated 0.2* 0.5* 
T-lymphoc. 
CD3-CD16,56+ 
4.8-26.7 
16.0 
Nat. Killers 0.3* 
CD34+ 0.1 
stem cells 
CD5+ 78.9 
T-cell. marker 
78.9* 
__________________________________________________________________________ 
*% of cells in the CD45+CD14- subpopulation (leukemic lymphocytes and 
granulocytes, the latter being in small quantities in chronic lymphoid 
leukosis) 
The data in Table 19 show a discrepancy between the morphologically 
obtained typical pattern of lymphoid leukosis and the small lymphocyte 
count by the CD45++CD14 fraction. For clarity, we presented ratios in the 
CD45+CD14- fraction, where the majority of leukaemia lymphocytes were. The 
diagnosis of B-cellular lymphoid leukosis was not doubted since most of 
the B-lymphocytes carry the CD5+ marker. After the treatment with 
H-Ile-Glu-Trp-OH, the count of lymphocytes belonging to the population of 
conventionally normal lymphocytes doubled. The ratio of CD4+CD8- and 
CD4-CD8+ (helpers and suppressers) had a tendency to normalisation and 
overcoming of the predominance of suppressers characteristic of B-cellular 
lymphoid leukosis. 
Patient 0., born in 1929. Chronic lymphoid leukosis since 1996. Was not 
treated with cytostatics. H-Ile-Glu-Trp-OH treatment--since Feburary 1997. 
The dynamics of indices before and after the treatment course: leukocytes 
17.3-20.8 blnd, Lymphocytes 65-74%(11.2-15.4 blnd). 
TABLE 20 
__________________________________________________________________________ 
Absolute 
% of Cells Absolute 
Cell Phenotype 
% of Cells, 
% of Cells, 
Count, 
after H-Ile-Glu-Trp-OH 
Count, 
Subpopulation 
Normal 
Test Sample 
bln/l 
Treatment bln/l 
__________________________________________________________________________ 
CD45++CD14- 9.1 1.54 28 5.8 
lymphocytes 
CD45+CD14- 83.3 63.8 
granulocytes 
CD45+CD14+ 2.5 3.6 
monocytes 
CD3+CD19- 
58.2-84.3 
6.9* 
T-lymphocytes 
CD3-CD19+ 
7.1-23.3 
88.8* 
B-lymphocytes 
CD4+CD8- 
31.4-63.8 
3.5* 8.2* 
T-helpers 
CD4-CD8+ 
18.9-47.9 
5.2* 8.1* 
T-suppressers 
CD4+/CD8+ 
0.6-3.0 
0.7* 1.0* 
ratio 
CD3+HLA-DR+ 
3.5-25.9 
1.0* 
activated 
T-lymphoc. 
CD3-CD16,56+ 
5.4-33.5 
3.7* 
Nat. Killers 
CD34+ --* 
stem cells 
CD5+ 95.0* 
T-cell. marker 
__________________________________________________________________________ 
*% of cells in mixed population of CD45++CD14- and CD45+CD14- (we failed 
to isolate the population of normal lymphocytes). 
The data in Table 21, as in the previous cases, show the lymphocyte 
predominance in peripheral blood smears and their small quantity in the 
CD45++CD14- fraction, which is common to B-cellular chronic lymphoid 
leukosis. This fraction is a graphic example of the growth of cells with 
CD45++CD14- markers after treatment with H-Ile-Glu-Trp-OH-1: their number 
grew three times. An increase and normalisation of helper/suppresser ratio 
was also noted. 
Patient B., born in 1926. Chronic lymphoid leukosis since 1984. Did not 
receive cytostatics during the treatment. The dynamics of indices before 
and after treatment with H-Ile-Glu-Trp-OH: leukocytes 23,0-26.6 bin/1, 
Lymphocytes 85-82% (18.7-21.8 bln/1). 
TABLE 22 
__________________________________________________________________________ 
Absolute 
% of Cells Absolute 
Cell Phenotype 
% of Cells, 
% of Cells, 
Count, 
after H-Ile-Glu-Trp-OH 
Count, 
Subpopulation 
Normal 
Test Sample 
bln/l 
Treatment bln/l 
__________________________________________________________________________ 
CD45++CD14- 11.3 2.5 9.5 2.52 
lymphocytes 
CD45+CD14- 82.7 86.4 
granulocytes 
CD45+CD14+ 1.8 2.1 
monocytes 
CD3+CD19- 
62.8-85.0 
8.0* 10.6* 
T-lymphocytes 
CD3-CD19+ 
7.1-23.3 
89.0* 85.3* 
B-lymphocytes 
CD4+CD8- 
31.4-63.8 
5.9* 6.4* 
T-helpers 
CD4-CD8+ 
18.9-47.9 
4.4* 3.8* 
T-suppressers 
CD4+/CD8+ 
0.7-3.3 
1.3* 1.7* 
ratio 
CD3+HLA-DR+ 
2.8-17.3 
1.5* 1.31* 
activated 
T-lymphoc. 
CD3-CD16,56+ 
4.8-26.7 
2.5* 
Nat. Killers 
CD34+ 0* 
stem cells 
CD5+ 93.1* 
T-cell. marker 
__________________________________________________________________________ 
In this case, there was also a discrepancy between the lymphocyte number 
found in the morphological examination and that in the CD45++CD14- 
fraction. After the treatment the ratio improved significantly. 
In those cases when patients with chronic lymphoid leukosis were treated 
with H-Ile-Glu-Trp-OH, there was a distinct tendency towards increase of 
CD45++CD14- cells characteristic of normal lymphocytes. At the same time, 
there was a relative decrease in the number of cells in the CD45+CD14- 
fraction, the majority of which are leukaemia lymphocytes. 
The findings suggest that H-Ile-Glu-Trp-OH helps increase the lymphocyte 
fraction with the markers, which allow assigning them to healthy cells 
CD45++CD14-. Besides, there is a tendency to an increase and normalisation 
of the T-helper/T-suppresser ratio. 
EXAMPLE 15 
To establish the safety of use of the peptides, toxicity tests were 
performed. Toxicity tests where performed in accordance Pharmacological 
Committee Russian Federation (RF) "guidelines for the pre-clinical study 
of general toxic activity of new pharmacological compounds". M., 1985. 
I. Acute Toxicity 
Acute toxicity was studied in 120 mice (60 males, 60 females). Ld.sub.50 
was not reached with the dose 10000 times as high as the therapeutic one. 
The preferred therapeutic dose is 0.001-0.1 mg/kg. 
Acute toxicity was studied in 20 guinea pigs and 6 dogs. No toxic effects 
were observed. The drug has a wide therapeutic range. 
II. Chronic Toxicity 
The pathomorphologic investigation of animals (with histologic 
investigation of organs) failed to find any pathologic processes in the 
subject animals. 
II.1 The H-Ile-Glu-Trp-OH effect on the composition and properties of the 
peritheric blood, the morphological state of the internals. 
The drug was injected once daily for 30 days. 
The test animals were 20 rabbits and 40 guinea pigs. The results of the 
test showed that H-Ile-Glu-Trp-OH did not cause any pathologic changes in 
the body or peritheric blood. 
II.2 The effect of H-Ile-Glu-Trp-OH on the cardio-vascular and respiratory 
systems. 
The effects of H-Ile-Glu-Trp-OH on ECG and arterial pressure were studied 
in guinea pigs and cats. 
The test was conducted in 20 guinea pigs. 
The influence of H-Ile-Glu-Trp-OH on arterial pressure was studied in 10 
cats. No significant difference from control was found both in ECG 
readings and arterial pressure. Therefore, the drug did not cause any 
disorders in functioning of the cardio-vascular or respiratory systems. 
II.3. The effect of H-Ile-Glu-Trp-OH on liver function. 
The tests were performed in 20 rabbits, who received injections of 0.01% 
H-Ile-Glu-Trp-OH solution for 30 days. H-Ile-Glu-Trp-OH was shown not to 
have any toxic effect on the liver function. 
II.4. The effect of H-Ile-Glu-Trp-OH on kidney function 
The anti-diuretic activity of H-Ile-Glu-Trp-OH was studied in 40 white 
mice. H-Ile-Glu-Trp-OH was shown not to have any anti-diuretic activity. 
II.4. Local irritating effect of H-Ile-Glu-Trp-OH 
The local irritating effect of H-Ile-Glu-Trp-OH was studied in 20 guinea 
pigs and 5 rabbits. H-Ile-Glu-Trp-OH was shown to have no local irritating 
effect. 
II.5. Allergenicity study of H-Ile-Glu-Trp-OH 
The study was performed in 12 rabbits, who received subcutaneous injections 
of 0.01% H-Ile-Glu-Trp-OH solution for 5 days. The control animals were 
injected with horse serum. No macroscopic reaction was observed on the 
H-Ile-Glu-Trp-OH injection sites. 
In the chronic toxicity study, no side effects were observed with the doses 
10-100 times as large as the therapeutic ones. The general state of the 
animals, behaviour, motor function, cardio-vascular, respiratory activity, 
liver and kidney functions were within the range of physiologic 
fluctuations. 
The pathomorphologic investigation of animals (with histologic 
investigation of organs) failed to find any pathologic processes in the 
body. 
The absence of side effects from H-Ile-Glu-Trp-OH administration in animals 
makes it possible to recommend the drug for clinical testing. 
III. This example describes the toxicity study of the peptide. 
To determine the peptide toxicity, male mice received single injections of 
the peptide in the dose of 0.15 g per kg body weight, which was 15,000 
times as large as the therapeutic dose with favourable effect in 
post-irradiation regeneration of hemopoietic progenitors. The second group 
of mice received daily peptide injections in the dose of 10 .mu.g/kg (the 
therapeutical dose) for 5 days. Survival of the animals was observed for 
30 days. Not an animal died in the first group or in the second one. On 
the 14-th day after the peptide injection 10 mice from each group were 
killed to determine the condition of their cellular systems. Nucleated 
cells were counted in spleen and thymus (per mg of organ weight), bone 
marrow, blood (per ml). Erythrocyte count and hemoglobin level were 
determined. The results are presented in tables 22 and 23. 
TABLE 22 
______________________________________ 
karyocyte count 
spleen thymus bone marrow .times. 
group weight (10.sup.3 /mg) 
(10.sup.3 /mg) 
10.sup.7 /femur 
______________________________________ 
control 21-22.5 1100 .+-. 90* 
1850 .+-. 260** 
2.1 .+-. 0.18 
peptide 21-22.0 2280 .+-. 30* 
3030 .+-. 250** 
2.39 .+-. 0.25 
(0.15 g/kg) 
peptide 21-22.5 1760 .+-. 100 
2780 .+-. 340 
1.94 .+-. 0.2 
(10 mcg/kg) .times. 
5 injections) 
______________________________________ 
*, **= statistically reliable 
TABLE 23 
______________________________________ 
karyocytes 
karyocytes erythrocyte 
hemoglobin 
group (10.sup.3) 
%/absolute (10.sup.3) 
g/dl 
______________________________________ 
control 7.1 .+-. 0.9 
57.4 .+-. 2.8 
8600 .+-. 500 
11.8 .+-. 0.3 
4075 
peptide 5.4 .+-. 0.7 
62.0 .+-. 3.4 
8100 .+-. 600 
10.8 .+-. 0.7 
(0.15 g/kg) 3348 
peptide 10.1 .+-. 0.8 
58.6 .+-. 3.6 
8400 .+-. 200 
11.5 .+-. 0.2 
(10 mcg/kg .times. 
5 injections) 
______________________________________ 
as the data show, 14 days after the injection of the large single dose of 
the peptide the karyocyte count in spleen and thymus went up. Five 
injections of the therapeutical dose caused by trustworthy elevation of 
nucleated cells count in thymus per mg thymus. The rest of determined 
indices in both test groups did not differ from control. Thus, the peptide 
dose as large as 15,000 times the therapeutic dose did not cause death of 
mice or toxic effects as regards the cells of blood, spleen, thymus, bone 
marrow. The same is also true for long (for 5 days) peptide adminstration. 
It should be mentioned that throughout the experiment no weight losses 
were recorded in mice. Moreover, their average weight grew by 2 g by the 
end of the study, the same as in control. 
Thus, LD.sub.50 of the peptide was not found, since even doses 15,000 as 
large as the therapeutical one neither killed mice, nor produced any side 
effects. 
Noteworthy is that no toxic or allergic reactions were observed either 
during the study of the H-Ile-Glu-Trp-OH specific and general 
pharmacological activity or during the 1-6 month observation period after 
the study. 
The study of the specific and general pharmacological activity of 
H-Ile-Glu-Trp-OH showed H-Ile-Glu-Trp-OH to have a pronounced stimulating 
effect on cellular and humoral immune response and non-specific resistance 
of the organism. 
In the study of specific and general pharmacological activity, the test 
animals were mice, guinea pigs, rats, rabbits. Some tests were performed 
using lymphocyte or monocyte cultures of the peritheric blood of donors 
and patients with different diseases accompanied by immunity disorders. 
H-Ile-Glu-Trp-OH may be recommended for clinical tests in patients with 
different immunodeficiency states as a means of normalisation of the 
immunologic reactivity of the organism. H-Ile-Glu-Trp-OH parenteral 
(intramuscular or subcutaneous) doses of preferably, 0.001-0.01 mg/kg 
produce a marked immunoregulatory effect when administered, preferably, 
daily for, preferably, 3-10 days. 
Having illustrated and described the principles of the invention in a 
preferred embodiment, it should be appreciated to those skilled in the art 
that the invention can be modified in arrangement and detail without 
departure from such principles. We claim all modifications coming within 
the scope of the following claims. 
All publications, patents and patent applications referred to herein are 
incorporated by reference in their entirety to the same extent as if each 
individual publication, patent or patent application was specifically and 
individually indicated to be incorporated by reference in its entirety.