The present invention provides methods for improving performance (e.g. learning and/or memory) using (ADNF) polypeptides, by treating the subject prenatally or postnatally with an Activity Dependent Neurotrophic Factor (ADNF) polypeptide in an amount sufficient to improve postnatal learning and/or memory of the subject.

Description of the Invention

Methods for Improving Performance

The present invention provides a method for improving performance (e.g., learning and/or memory) in a subject. The method comprises administering to the subject, either prenatally or postnatally, an ADNF polypeptide in an amount sufficient to improve post natal performance. In particular, prenatal administration can improve spatial learning in a subject. Candidate subjects who can benefit from such post or prenatal treatment, ADNF polypeptides that can be administered, timing and modes of administration, tests to assess improvement in learning and memory, and methods for producing ADNF polypeptides are described in detail below.

Candidate Subjects for Treatment with ADNF Polypeptides

The prenatal and postnatal treatment with ADNF polypeptides has applications in many types of subjects. For example, normal subjects can benefit from the prenatal treatment of ADNF polypeptides in terms of improving their learning and memory. A normal subject or a subject with normal mental capacity refers to those whose intellectual functioning level, even without the prenatal treatment with ADNF polypeptides, is around or above average (e.g., having an IQ over 75). In the context of a fetus, a normal subject can refer to a subject who does not appear to have any mental impairment (e.g., according to an amniocentesis test) and/or risk factors for mental retardation (e.g., family history of mental retardation or a mother who consumed enough alcohol during pregnancy to cause fetal alcohol syndrome in the subject). A mother who wishes her unborn embryo or fetus to have enhanced capacity for learning and memory can be administered with ADNF polypeptides while the embryo or fetus is in utero.

Moreover, the present methods can benefit subjects whose mental ability is compromised. For example, if a fetus is diagnosed as likely having mental retardation or Down's syndrome, the fetus can be treated in utero with ADNF polypeptides so that postnatal learning and memory can be ameliorated. In a preferred embodiment, mental retardation is not caused by maternal consumption of alcohol during pregnancy. In other words, a candidate subject who has mental retardation does not have fetal alcohol syndrome (which can include a condition of usually mild to moderate, but occasionally severe, mental retardation or learning disabilities).

Severe mental retardation (defined as an IQ of 50 or less) often originates from genetic disorders. These include, e.g., Down's syndrome, fragile X syndrome, Klinefelter's syndrome, Prader-Willi syndrome and cri du chat syndrome. Many of these conditions can be diagnosed with a prenatal genetic test. For example, genetic disorders can be tested by an amniocentesis test which is typically performed between 14 and 18 weeks of pregnancy or by a chorionic villus sampling which is performed between 9 and 12 weeks of pregnancy. Prenatal treatment of the fetus with ADNF polypeptides can benefit their postnatal learning and memory.

Even without a prenatal diagnosis of genetic disorders that cause mental retardation, ADNF polypeptides can be prophylactically administered to the fetus in certain circumstances. For example, if the subject has a family history of mental retardation, the subject can be prenatally treated with ADNF polypeptides. In another example, if the subject is at a higher risk of being born with mental retardation due to infections such as rubella, meningitis, CMV, etc., the subject can be prenatally treated with ADNF polypeptides. In another example, the subject is at a higher risk of being born with certain genetic disorders, such as Down's syndrome, when the mother is older (e.g., 35 years or older). Prophylactic prenatal treatment with ADNF polypeptides can ameliorate the subject's capacity for learning and memory.

In other embodiments, the subject can be treated later in life, for example, to improve short term learning and memory. For example, certain memory and learning disorders, such as Alzheimer 's disease, may not be apparent until later in life. Other conditions that can be treated using postnatal administration of ADNF polypeptides include, neuropathology; sensory-motor problems; improving the performance of subjects impaired in cognitive tasks; improving the performance of subjects with memory deficiencies; improving the performance of normal subjects; and the like. Accordingly, embodiments of the invention in suitable formulations, can be employed for decreasing the amount of time needed to learn a cognitive, motor or perceptual task. Alternatively, invention compounds, in suitable formulations, can be employed for increasing the time for which cognitive, motor or perceptual tasks are retained. As another alternative, embodiments of the invention in suitable formulations, can be employed for decreasing the quantity and/or severity of errors made in recalling a cognitive, motor or perceptual task. Such treatment may prove especially advantageous in individuals who have suffered injury to the nervous system, or who have endured disease of the nervous system. Moreover, ADNF polypeptides can be administered to normal subjects to improve their performance (e.g., learning and memory). ADNF polypeptides can be particularly useful for an aged population in which capacity for memory (e.g. short term) has generally declined.

ADNF Polypeptides

Any suitable ADNF polypeptides can be administered in embodiments of the invention. For example, an ADNF polypeptide can be an ADNF I polypeptide, an ADNF III polypeptide, or a mixture thereof In some embodiments, ADNF polypeptides may comprise all L-amino acids, all D-amino acids, or a combination thereof. When ADNF polypeptides are to be orally administered, preferably an ADNF polypeptide comprises at least one D-amino acid within its active core site, more preferably at the N-terminus and/or the C-terminus of the active core site, and even more preferably at the entire active core site or over the length of the molecule. Alternatively, the D-amino acid can be at any suitable position in the polypeptide sequence. Since D-enatiomers of polypeptides are enzymatically more stable than their L-enatiomers, particularly in the gastrointestinal tract, an ADNF polypeptide comprising D-amino acids are particularly useful for oral administration.

In one aspect, the method comprises administering an ADNF I polypeptide that comprises an active core site having the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1). In one embodiment, the ADNF I polypeptide consists of an active core site that has an amino acid sequence of Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1). In another embodiment, the ADNF I polypeptide can comprise additional amino acids at the N-terminus and/or at the C-terminus of the active core site. For example, the ADNF I polypeptide can comprise up to 40 amino acids at the N-terminus and/or the C-terminus of the active core site. In another example, the ADNF I polypeptide can comprise up to 20 amino acids at the N-terminus and/or the C-terminus of the active core site. In yet another example, the ADNF I polypeptide can comprise up to 10 amino acids at the N-terminus and/or the C- terminus of the active core site. In yet another embodiment, the ADNF I polypeptide can be a fill length ADNF I polypeptide.

In another aspect, the method comprises administering to the subject an ADNF III polypeptide that comprises an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln(SEQ ID NO:2). In one embodiment, the ADNF I polypeptide consists of an active core site that has an amino acid sequence of Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln(SEQ ID NO:2). In another embodiment, the ADNF III polypeptide can comprise additional amino acids at the N-terminus and/or at the C-terminus of the active core site. For example, the ADNF III polypeptide can comprise up to 40 amino acids at the N-terminus and/or the C- terminus of the active core site. In another example, the ADNF III polypeptide can comprise up to 20 amino acids at the N-terminus and/or the C-terminus of the active core site. In yet another example, the ADNF III polypeptide can comprise up to 10 amino acids at the N-terminus and/or the C-terminus of the active core site. In yet another embodiment, the ADNF III polypeptide can be a full length ADNF III polypeptide.

In a preferred embodiment, the ADNF I polypeptide comprises an amino acid sequence of (R.sup.1).sub.x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R.sup.2).sub.y (SEQ ID NO:3), and the ADNF III polypeptide comprises an amino acid sequence of (R.sup.3).sub.w-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R.sup.4).sub.z (SEQ ID NO:13).

In the above formula, each of R.sup.1, R.sup.2, R.sup.3, and R.sup.4, if present, is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected. The term "independently selected" is used herein to indicate that the amino acids making up, for example, the amino acid sequence R.sup.1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R.sup.1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). This discussion pertaining to R.sup.1 is fully applicable to R.sup.2, R.sup.3, and R.sup.4.

Within the above formula for the ADNF I polypeptide, x and y are independently selected and are equal to zero or one. The term independently selected is used herein to indicate that x and y may be identical or different. For example, x and y may both be zero or, alternatively, x and y may both be one. In addition, x may be zero and y may be one or, alternatively, x may be one and y may be zero. Moreover, if x and y are both one, the amino acid sequences R.sup.1 and R.sup.2 may be the same or different. As such, the amino acid sequences R.sup.1 and R.sup.2 are independently selected. If R.sup.1 and R.sup.2 are the same, they are identical in terms of both chain length and amino acid composition. For example, both R.sup.1 and R.sup.2 may be Val-leu-Gly-Gly-Gly. If R.sup.1 and R.sup.2 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences. For example, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:4), whereas R.sup.2 may be Val-Leu-Gly-Gly. (SEQ ID NO:5). Alternatively, R.sup.1 may be Val-Leu-Gly-Gly-Gly(SEQ ID NO:4), whereas R.sup.2 may be Val-Leu-Gly-Gly-Val (SEQ ID NO:6). Alternatively, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:4), whereas R.sup.2 may be Gly-Val-Leu-Gly-Gly(SEQ ID NO:7).

Similarly, w and z are independently selected and are equal to zero or one within the above formula for the ADNF III polypeptide. The term independently selected is used herein to indicate that w and z may be identical or different. For example, w and z may both be zero or, alternatively, w and z may both be one. In addition, w may be zero and z may be one or, alternatively, w may be one and z may be zero. Moreover, if w and z are both one, the amino acid sequences R.sup.3 and R.sup.4 may be the same or different. As such, the amino acid sequences R.sup.3 and R.sup.4 are independently selected. If R.sup.3 and R.sup.4 are the same, they are identical in terms of both chain length and amino acid composition. For example, both R.sup.3 and R.sup.4 may be Leu-Gly-Leu-Gly-Gly (SEQ ID NO:8). If R.sup.3 and R.sup.4 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences. For example, R.sup.3 may be Leu-Gly-Leu-Gly-Gly(SEQ ID NO:8), whereas R.sup.4 may be Leu-Gly-Leu-Gly(SEQ ID NO:9). Alternatively, R.sup.3 may be Leu-Gly-Leu-Gly-Gly (SEQ ID NO:8), whereas R.sup.4 may be Leu-Gly-Leu-Gly-Leu (SEQ ID NO:10).

Within the scope, certain ADNF I and ADNF III polypeptides are preferred, namely those in which x, y, w, and z are all zero (i.e., SALLRSIPA; SEQ ID NO:1 and NAPVSIPQ; SEQ ID NO:2, respectively). Equally preferred are ADNF I polypeptides in which x is one; R.sup.1 is Val-Leu-Gly-Gly-Gly (SEQ ID NO:4; and y is zero. Also equally preferred are ADNF I polypeptides in which x is one; R.sup.1 is Val-Glu-Glu-Gly-Ile-Val-Leu-Gly-Gly-Gly (SEQ ID NO:11); and y is zero. Also equally preferred are ADNF III polypeptides in which w is one; R.sup.3 is Gly-Gly; and z is zero. Also equally preferred are ADNF III polypeptides in which w is one; R.sup.3 is Leu-Gly-Gly; z is one; and R.sup.4 is Gln-Ser. Also equally preferred are ADNF III polypeptides in which w is one; R.sup.3 is Leu-Gly-Leu-Gly-Gly-(SEQ ID NO:8); z is one; and R.sup.4 is Gln-Ser. Also equally preferred are ADNF III polypeptides in which w is one; R.sup.3 is Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly (SEQ ID NO:12); z is one; and R.sup.4 is Gln-Ser. Additional amino acids can be added to both the N-terminus and the C-terminus of these active sites (SALLRSIPA; SEQ ID NO:1 or NAPVSIPQ; SEQ ID NO:2) without loss of biological activity as evidenced by the fact that the intact ADNF I or ADNF III growth factors exhibit extraordinary biological activity. See, U.S. Ser. No. 08/324,297, filed Oct. 17, 1994 (also published as WO96/11948) for the description of ADNF I polypeptides; and U.S. Ser. No. 60/037,404 filed Feb. 27, 1997 and U.S. Ser. No. 60/059,621 filed, Sep. 23, 1997 (also published as WO98/35042) for the description of ADNF III polypeptides, all of which are incorporated herein by reference.

In yet another aspect the method comprises administering to the subject a mixture of an ADNF I polypeptide and an ADNF III polypeptide. Any one or more of the ADNF I polypeptides described herein can be mixed with any one or more of the ADNF III polypeptides described herein. A mixture of an ADNF I polypepdde and an ADNF Ill polypeptide can be a blend of two or more of these polypeptides. A mixture of an ADNF I polypeptide and an ADNF III polypeptide can also refer to one or more of ADNF I polypeptides that are coupled (directly or indirectly) to one or more of ADNF III polypeptides. For example an ADNF I polypeptide can be covalently linked to an ADNF III polypeptide. A mixture of ADNF I polypeptides and ADNF III polypeptides can be prepared as a single composition and can be administered to a subject. Alternatively, an ADNF I polypeptide and an ADNF III polypeptide can be prepared as separate compositions. The separate compositions can then be administered simultaneously or sequentially to the subject. Furthermore, different proportions of an ADNF I polypeptide and an ADNF III polypeptide can be administered to a subject. For example, the subject can be administered with ADNF polypeptides, wherein the ratio of an ADNF I polypeptide and an ADNF III polypeptide can be in the range of 1:100 to 100:1, 1:10 to 10:1, or 1:2 to 2:1.

In yet another aspect, other ADNF polypeptide (including their alleles, polymorphic variants, species homologs and subsequences thereof) can be used to enhance performance.

Various parameters can be measured to determine if an ADNF polypeptide or a mixture of ADNF polypeptides improves performance of a subject. For example, the degree of learning deficits can be compared between the control (e.g. untreated with ADNF polypeptides) and a group pretreated with ADNF polypeptides. Learning deficits can be assessed using, for example, a Morris water maze (see, e.g., the Example section). If any one or more of these parameters are changed for the group treated with ADNF polypeptides by, e.g., about 10%, optionally at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, etc., compared to control, then these ADNF polypeptides can be advantageously used in the present invention.

Administration and Pharmaceutical Compositions

ADNF polypeptides and nucleic acids encoding ADNF polypeptides can be prenatally or postnatally administered to the subject using any suitable methods known in the art. For example, ADNF polypeptides or nucleic acids can be formulated as pharmaceutical compositions with a pharmaceutically acceptable diluent, carrier or excipient. Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences (17th ed. 1985)), which is incorporated herein by reference. A brief review of methods for drug delivery is also described in, e.g., Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference. In addition, the pharmaceutical compositions comprising peptides and proteins are described in, e.g., Therapeutic Peptides and Proteins Formulations, Processing, and Delivery Systems, by Banga, Technomic Publishing Company, Inc., Lancaster, Pa. (1995).

In one embodiment, ADNF polypeptides are formulated for oral administration, e.g., to the subject, or for prenatal administration, to the subject 's mother. In this embodiment, it is preferred that ADNF polypeptides comprising all D-amino acids are used. A pharmaceutically acceptable nontoxic composition is formed by incorporating any of normally employed excipients, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%. Furthermore, to improve oral absorption of ADNF polypeptides, various carrier systems, such as nanoparticles, microparticles, liposomes, phospholipids, emulsions, erythrocytes, etc. can be used. The oral agents comprising ADNF polypeptides of the invention can be in any suitable form for oral administration, such as liquid, tablets, capsules, or the like. The oral formulations can be further coated or treated to prevent or reduce dissolution in stomach. See, e.g., Therapeutic Peptides and Proteins, Formulation, Processing, and Delivery Systems, by A. K. Banga, Technomic Publishing Company, Inc., 1995.

Furthermore, the ADNF polypeptides can be formulated for parenteral, topical, nasal, sublingual, gavage, or local administration. For example, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly, or intranasally. Thus, the invention provides compositions for parenteral administration that comprise a solution of a mixture of ADNF polypeptides, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used including, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or they may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. In one embodiment, a nucleic acid encoding an ADNF polypeptide is administered as a naked DNA.

For aerosol administration, ADNF polypeptides are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intrinasal delivery.

For solid compositions, conventional nontoxic solid carriers may be used. Solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

The present invention also provides for therapeutic compositions or medicaments comprising a mixture of one or more of the ADNF I and ADNF III polypeptides described herein above in mixture with a pharmaceutically acceptable excipient, wherein the amount of a mixture the ADNF I and ADNF III polypeptide is sufficient to provide a desirable therapeutic effect.

Small polypeptides including SALLRSIPA (SEQ ID NO:1) and NAPVSIPQ (SEQ ID NO:2) cross the blood brain barrier. For longer polypeptides that do not the cross blood brain barrier, methods of administering proteins to the brain are well known. For example, proteins, polypeptides, other compounds and cells can be delivered to the mammalian brain via intracerebroventricular (ICV) injection or via a cannula (see, e.g., Motta & Martini, Proc. Soc. Exp. Biol. Med. 168:62-64 (1981); Peterson et al., Biochem. Pharamacol. 31:2807-2810 (1982); Rzepczynski et al., Metab. Brain Dis. 3:211-216 (1988); Leibowitz et al., Brain Res. Bull. 21:905-912 (1988); Sramka et al., Stereotact. Funct. Neurosurg. 58:79-83 (1992); Peng et al., Brain Res. 632:57-67 (1993); Chem et al., Exp. Neurol. 125:72-81 (1994); Nikkhah et al., Neuroscience 63:57-72 (1994); Anderson et al., J. Comp. Neurol. 357:296-317 (1995); and Brecknell & Fawcett, Exp. Neurol. 138:338-344 (1996)). In particular, cannulas can be used to administer neurotrophic factors to mammals (see, e.g., Motta & Martini, Proc. Soc. Exp. Biol. Med. 168:62-64 (1981) (neurotensin); Peng et al., Brain Res. 632:57-67 (1993) (NGF); Anderson et al., J. Comp. Neurol. 357:296-317 (1995) (BDNF, NGF, neurotrophin-3).

Alternatively, longer ADNF polypeptides that do not cross blood brain barrier can be coupled with a material which assists the ADNF polypeptide to cross the blood brain barrier and to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as ADNF polypeptides across a cell membrane.

For example, "membrane translocation polypeptides" have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the hydrophobic domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:14255-14258 (1995)).

Examples of peptide sequences which can be linked to a ADNF polypeptide of the invention, for facilitating uptake of ADNF polypeptides into cells, include, but are not limited to: an 11 animo acid peptide of the tat protein of HHV (see Schwarze et al., Science 285:1569-1572 (1999)); a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to ADNF polypeptides.

Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called "binary toxins"): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used in attempts to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991); Donnelly et al., Proc. Nat'l Acad. Sci. USA 90:3530-3534 (1993); Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., Proc. Nat'l Acad. Sci. USA 89:10277-10281 (1992); and Novak et al., J. Biol. Chem. 267:17186-17193 1992)).

Such subsequences can be used to translocate ADNF polypeptides across a cell membrane. ADNF polypeptides can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the ADNF polypeptides and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.

The ADNF polypeptides and nucleic acids encoding ADNF polypeptides can also be introduced into an animal cell, preferably a mammalian cell, via a liposomes and liposome derivatives such as immunoliposomes and lipid:nucleic acid complexes. The term "liposome" refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase. The aqueous phase typically contains the compound to be delivered to the cell, i.e., an ADNF polypeptide.

The liposome fuses with the plasma membrane, thereby releasing the ADNF polypeptides into the cytosol. Alternatively, the liposome is phagocytosed or taken up by the cell in a transport vesicle. Once in the endosome or phagosome, the liposome either degrades or fuses with the membrane of the transport vesicle and releases its contents.

In current methods of drug delivery via liposomes, the liposome ultimately becomes permeable and releases the encapsulated compound (in this case, an ADNF polypeptide) at the target tissue or cell. For systemic or tissue specific delivery, this can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Alternatively, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Nat'l Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989)). When liposomes are endocytosed by a target cell, for example, they become destabilized and release their contents. This destabilization is termed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basis of many "fusogenic" systems.

Such liposomes typically comprise an ADNF polypeptide and a lipid component, e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen). A variety of methods are available for preparing liposomes as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91/17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., Proc. Nat'l Acad. Sci. USA 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., Proc. Nat'l Acad. Sci USA 85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles and ether-fusion methods, all of which are well known in the art.

In certain embodiments of the present invention, it is desirable to target the liposomes of the invention using targeting moieties that are specific to a particular cell type, tissue, and the like. Targeting of liposomes using a variety of targeting moieties (e.g., ligands, receptors, and monoclonal antibodies) has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044). Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporation into liposomes lipid components, e.g., phosphatidylethanoiamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid derivatized bleomycin. Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen et al., J. Biol. Chem., 265:16337-16342 (1990) and Leonetti et al., Proc. Nat'l Acad. Sci. USA 87:2448-2451 (1990).

Alternatively, nucleic acids encoding ADNF can also be used to provide a therapeutic dose of ADNF polypeptides. These nucleic acids can be inserted into any of a number of well-known vectors for the transfection of target cells and organisms. For example, nucleic acids are delivered as DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in, e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam.TM. and Lipofectin.TM.. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

In therapeutic applications, a mixture of ADNF I and ADNF III polypeptides of the invention are administered to a patient in an amount sufficient to improve a subject's performance (e.g., learning and/or memory). An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, for example, the particular ADNF I or ADNF III polypeptide employed, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. For example, for the improvement of performance (e.g., learning and memory), an amount of ADNF I or ADNF III polypeptides falling within the range of a 1 .mu.g to 50 .mu.g, preferably 1 .mu.g to 10 .mu.g dose given intranasally once a day per mouse (e.g., in the evening) would be a therapeutically effective amount. This dose is based on the average body weight of a mouse. Therefore, an appropriate dose can be extrapolated for a human body.

ADNF polypeptides can be prenatally administered to the subject directly or indirectly through the subject's mother. ADNF polypeptides can be administered at any time during the pregnancy. Preferably, ADNF polypeptides are administered to the subject during the first trimester (i.e., first 12 weeks) of the pregnancy when organs and the nervous system of the subject are actively developing. More preferably, ADNF polypeptides are administered during the time of neural tube development (which begins around 22 days post-conception) and prior to its closure. ADNF polypeptides can be administered as a single dose, preferably during the critical period of neural tube development, or can be administered as multiple doses throughout the pregnancy.

Tests for Measuring Improved Learning and/or Memory

Various parameters can be measured to determine if ADNF polypeptides improve performance (e.g., learning and memory) in vivo. For example, the hidden platform test of the Morris water maze, which is described in the example section below, can be used to test spatial learning and memory. Generally, mice that are treated with ADNF polypeptides and control mice (that are not treated with ADNF polypeptides) are trained to escape the swimming task by learning the position of a hidden platform and climbing on it. The time it takes them to complete this task is defined as the escape latency. This test can be conducted one or more times daily for a number of days. One parameter that is indicative of improved learning and memory is the reduction in latency in escaping the swimming task by climbing onto the hidden platform (see the example section below). See, also, methods described in Gozes et al., Proc. Natl. Acad. Sci. USA 93:427-432 (1996), incorporated herein by reference. Animals treated with suitable ADNF polypeptides show improvement in their leaning and memory capacities compared to the controls that are not treated with ADNF polypeptides. Embodiments of the invention are not limited by examples of the test used to measure performance. Any suitable test methods can be used to measure performance, such as learning and memory.

Other methods known in the art can be used in human subjects to determine if an ADNF polypeptide or a combination of ADNF polypeptides improves performance (e.g., learning and memory) in vivo. For example, these methods include assessment of memory or learning over time by the Randt Memory Test (Randt et al., Clin. Neuropsychol., 1980, 2:184), Wechsler Memory Scale (J. Psych. 19:87-95 (1945), Forward Digit Span test (Craik, Age Differences in Human Memory, in: Handbook of the Psychology of Aging, Birren, J., and Schaie, K. (Eds.), New York, Van Nostrand (1977), Mini-Mental State Exam (Folstein et al., J. of Psych. Res. 12:189-192 (1975), or California Verbal Learning Test (CVLT). See, also, U.S. Pat. No. 6,030,968. In these tests, factors unrelated to effects of ADNF polypeptides (e.g., anxiety, fatigue, anger, depression, confusion, or vigor) are controlled for. See, U.S. Pat. No. 5,063,206. Methods for assessing and controlling for subjective factors is known in the art and determined by such standard clinical tests such as the BECK Depression Scale, Spielberger Trait State Anxiety test, and POMS test (Profile of Mood State).

Spatial learning can also be tested in human. For example, a subject can be asked to draw a picture, and then the picture is taken away. The subject is then asked to draw the same picture from memory. The latter picture drawn by the subject reflects a degree of spatial learning in the subject.

Various parameters can be measured to determine if ADNF polypeptides improve learning and memory of a subject. For example, the degree of learning and memory improvement can be compared between the control (e.g. untreated with ADNF polypeptides) and a group pretreated with ADNF polypeptides. Learning and memory improvement can be assessed using, for example, a Morris water maze for rodents (see, e.g., the Example section) or any suitable tests such as those described above for humans. If any one or more of these parameters are changed for the group treated with ADNF polypeptides by, e.g., about 10%, optionally at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, etc., compared to control, then it can be said that the ADNF polypeptides improved learning and memory of the subject. Alternatively, statistical analysis using ANOVA for continuous variables, Mann-Whitney U for nonparametic data, Chi square for categorical variables or Fisher 's exact test with p<0.05 is considered significant.

Methods for Production of ADNF Polypeptides

Recombinant Methods for Production of ADNF Polypeptides

Cloning and Isolation of ADNF Nucleic Acids

Several specific nucleic acids encoding ADNF polypeptides are described herein. See, also, e.g., Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996), Brenneman, J. Pharm. Exp. Ther. 285:619-627 (1998), and Bassan et al., J. Neurochem 72:1283-1293 (1999), the teachings of which are hereby incorporated in their entirety by reference. These nucleic acids can be made using standard recombinant or synthetic techniques. Given the nucleic acids of the present invention, one of skill can construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids that encode the same ADNF polypeptides. Cloning methodologies to accomplish these ends, and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd ed. 1989) and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

In addition, product information from manufacturers of biological reagents and experimental equipment also provide information useful in known biological methods. Such manufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources known to one of skill.

The nucleic acid compositions of this invention, whether RNA, cDNA, genomic DNA or a hybrid of the various mixtures, are isolated from biological sources, such as astrocyte, neuroblastoma cells, or fibroblasts, or synthesized in vitro. The nucleic acids of the invention are present in transformed or transfected cells, in transformed or transfected cell lysates, or in a partially purified or substantially pure form.

In vitro amplification techniques suitable for amplifying sequences for use as molecular probes or generating nucleic acid fragments for subsequent subcloning are known. Examples of techniques sufficient to direct persons of skill through such in vitro amplification methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Q.beta.-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), are found in Berger, Sambrook et al. and Ausubel et al., all supra, as well as in U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al., eds., 1990); Arnheim & Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research 3:81-94 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173 (1989); Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874 (1990); Lomell et al., J. Clin. Chem 35:1826 (1989); Landegren et al., Science 241:1077-1080 (1988); Van Brunt, Biotechnology 8:291-294 (1990); Wu & Wallace, Gene 4:560 (1989); Barringer et al., Gene 89:117 (1990); and Sooknanan & Malek, Biotechnology 13:563-564 (1995). Improved methods of cloning in vitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleic acids are summarized in Cheng et al., Nature 369:684-685 (1994) and the references cited therein. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase.

Oligonucleotides for use as probes, for example, with in vitro ADNF nucleic acid amplification methods, or for use as nucleic acid probes to detect ADNF nucleic acids, are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetrahedron Letts. 22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g. as described in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168 (1984). Oligonucleotides can also be custom made and ordered from a variety of commercial sources known to those of skill in the art. Purification of oligonucleotides, where necessary, is typically performed by either native acrylamide gel electrophoresis, or by anion-exchange HPLC as described in Pearson & Regnier, J. Chrom. 255:137-149 (1983). The sequence of the synthetic oligonucleotides can be verified using the chemical degradation method of Maxam & Gilbert, in Methods in Enzymology 65:499-560 (Grossman & Moldave, eds., 1980).

One of skill will recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see, Giliman & Smith, Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987); and Sambrook et al., Molecular Cloning--A Laboratory Manual (2nd ed. 1989)).

Recombinant Expression of ADNF Polypeptides

In one embodiment, the polypeptides, or subsequences thereof, are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid sequence that encodes the protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the protein in a host cell, isolating the expressed protein and, if required, renaturing the protein.

Once a nucleic acid encoding an ADNF polypeptide of the invention is isolated and cloned, the nucleic acid is optionally expressed in recombinantly engineered cells known to those of skill in the art. Examples of such cells include, but are not limited to, bacteria, yeast, plant, filamentous fungi, insect (especially employing baculoviral vectors) and mammalian cells. The recombinant nucleic acids are operably linked to appropriate control sequences for expression in the selected host. For E. coli, example control sequences include the T7, trp, or lambda promoters, a ribosome binding site and, preferably, a transcription termination signal. For eukaryotic cells, the control sequences typically include a promoter and, preferably, an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

If desired, recombinant nucleic acids can be constructed to encode a fusion polypeptide comprising an ADNF polypeptide. For example, a nucleic acid encoding an ADNF I polypeptide can be linked to a nucleic acid encoding an ADNF III polypeptide to provide a mixture of ADNF polypeptides. In another example, a nucleic acid encoding an ADNF polypeptide (e.g., an ADNF I polypeptide, an ADNF III polypeptide, or a fusion ADNF I/ADNF III polypeptide) can be linked with another nucleic acid, such as a portion of HIV tat nucleic acid, which facilitates the delivery of the ADNF III polypeptide into tissues. In yet another example, a nucleic acid encoding an ADNF polypeptide can be linked to nucleic acids that encode affinity tags to facilitate protein purification protocol. An ADNF nucleic acid and a heterologous polynucleotide sequence can be modified to facilitate their fusion and subsequent expression of fusion polypeptides. For example, the 3' stop codon of the ADNF polynucleotide sequence can be substituted with an in frame linker sequence, which may provide restriction sites and/or cleavage sites.

The plasmids of the invention can be transferred into the chosen host cell by well-known methods. Such methods include, for example, the calcium chloride transformation method for E. coli and the calcium phosphate treatment or electroporation methods for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo, and hyg genes.

Once expressed, the recombinant ADNF polypeptides or naturally occurring can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Polypeptide Purification (1982); Deutscher, Methods in Enzynology Vol. 182: Guide to Polypeptide Purification (1990)). Once purified, partially or to homogeneity as desired, the ADNF polypeptides may then be used, e.g., to improve learning and memory in a subject. See, also, e.g., Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996), Brenneman et al., J. Pharm. Exp. Ther. 285:619-627 (1998), and Bassan et al. J. Neurochem 72:1283-1293 (1999), the teachings of which are hereby incorporated in their entirety by reference

Synthesis of ADNF Polypeptides

In addition to the foregoing recombinant techniques, the ADNF polypeptides of the invention are optionally synthetically prepared via a wide variety of well-known techniques. Polypeptides of relatively short size are typically synthesized in solution or on a solid support in accordance with conventional techniques (see, e.g., Merrifield, Am. Chem. Soc. 85:2149-2154 (1963)). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis (2nd ed. 1984)). Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are described by Barany & Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156 (1963); and Stewart et al., Solid Phase Peptide Synthesis (2nd ed. 1984).

After chemical synthesis, biological expression or purification, the polypeptide(s) may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it is helpful to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing polypeptides and inducing re-folding are well known to those of skill in the art (see Debinsid et al., J. Biol. Chem. 268:14065-14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:581-585 (1993); and Buchner et al., Anal. Biochem. 205:263-270 (1992)). Debinski et al., for example, describe the denaturation and reduction of inclusion body polypeptides in guanidine-DTE. The polypeptide is then refolded in a redox buffer containing oxidized glutathione and L-arginine.

One of skill will recognize that modifications can be made to the polypeptides without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion polypeptide. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

Conservative Modifications of the ADNF Nucleic Acids and Polypeptides

One of skill will appreciate that many conservative variations of the ADNF nucleic acid and polypeptide sequences provided herein yield functionally identical products. For example, due to the degeneracy of the genetic code, "silent substitutions" (i.e., substitutions of a nucleic acid sequence that do not result in an alteration in an encoded polypeptide) are an implied feature of every nucleic acid sequence that encodes an amino acid. Similarly, "conservative amino acid substitutions," in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties (see the definitions section, supra), are also readily identified as being highly similar to a disclosed amino acid sequence, or to a disclosed nucleic acid sequence that encodes an amino acid. Such conservatively substituted variations of each explicitly listed nucleic acid and amino acid sequences are a feature of the present invention.

One of skill will recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see Giliman & Smith, Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)). For example, alanine scanning can be used to determine conservatively modified variants for SALLRSIPA (SEQ ID NO:1) or NAPVSIPQ (SEQ ID NO:2) (i.e., by substituting each amino acid one by one with an alanine or other small neutral amino acid and assay for activity as described herein).

Polypeptide sequences can also be altered by changing the corresponding nucleic acid sequence and expressing the polypeptide. Polypeptide sequences are also optionally generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide (see, Merrifield, supra, and Stewart & Young, supra).

More particularly, it will be readily apparent to those of ordinary skill in the art that the ADNF polypeptides of the present invention can readily be screened for their performance enhancing effect using various assays (e.g., Morris watermaze assay).

Using these assays, one of ordinary skill in the art can readily prepare a large number of ADNF polypeptides in accordance with the teachings of the present invention and, in turn, screen them using the foregoing assay to find ADNF polypeptides, in addition to those set forth herein, which possess the neuroprotective/neurotrophic activity of the intact ADNF growth factor. For instance, using ADNF III-8 (i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln;(SEQ ID NO:2) as a starting point, one can systematically add, for example, Gly-, Gly-Gly-, Leu-Gly-Gly- to the N-terminus of ADNF III-8 and, in turn, screen each of these ADNF III polypeptides in the foregoing assay to determine whether they possess neuroprotective/neurotrophic activity. In doing so, it will be found that additional amino acids can be added to both the N-terminus and the C-terminus of the newly discovered active site, i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2), without loss of biological activity as evidenced by the fact that the intact ADNF III growth factor exhibits extraordinary biological activity. This discussion also applies to ADNF I polypeptides.
 

Claim 1 of 22 Claims

1. A method for improving learning and memory in a subject, the method comprising the step of administering postnatally to the subject in an amount sufficient to improve postnatal learning and memory of the subject, a mixture of: (a) an Activity Dependent Neurotrophic Factor (ADNF) I polypeptide comprising an active core site having the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1); and (b) an Activity Dependent Neurotrophic Factor (ADNF) III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2); and assessing learning and memory of the post-natal subject, wherein the active core site of at least one of the ADNF I polypeptide and the ADNF III polypeptide comprises all D-amino acids.

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