The invention provides a novel amino acid, neo-tryptophan, as well as polypeptides containing this novel amino acid such as neurotensin analogs. In addition, the invention provides neo-tryptophan derivatives, serotonin-like neo-tryptophan derivatives, and polypeptides containing such derivatives. The invention also provides methods for making neo-tryptophan, neo-tryptophan derivatives, serotonin-like neo-tryptophan derivatives, and compositions containing these compounds. Further, the invention provides methods for inducing a neurotensin response in a mammal as well as methods for treating a mammal having a serotonin recognition molecule.

BACKGROUND
 1. Technical Field
 The invention relates to a novel amino acid, neo-tryptophan, as well as
 polypeptides containing this novel amino acid.
 2. Background Information
 Tryptophan is an essential component in human nutrition since it is not
 synthesized by the body. In addition, tryptophan is a hydrophobic amino
 acid that is part of many polypeptides.
 Polypeptides as well as many other types of compounds such as
 neurotransmitters and drugs can exert profound effects on the body. For
 example, neurotensin (NT) is a tridecapeptide that induces antinociception
 and hypothermia upon direct administration to brain. Systemic
 administration of NT does not induce these effects since NT is rapidly
 degraded by proteases and has poor blood brain barrier permeability.
 Currently, two NT receptors have been identified and cloned. The first NT
 receptor is designated NTR1, while the second is designated NTR2. Both
 NTR1 and NTR2 are G-protein coupled receptors that are expressed by
 various brain tissues.
 Serotonin (5HT) is a neurotransmitter that is essential to brain function.
 Multiple serotonin receptors and transporters have been identified and
 cloned. Briefly, de novo synthesis of serotonin from tryptophan occurs in
 the cytoplasm of a cell. Once synthesized, vesicular monoamine
 transporters package the transmitter into vesicular compartments so that
 its release can be regulated. Once released into the synapse upon proper
 stimulation, the transmitter can bind specific serotonin receptors, can be
 degraded by specific enzymes, and/or can be transported back into a cell
 by specific plasma membrane serotonin transporters and then re-packaged
 into vesicles. Thus, both serotonin receptors and transporters
 specifically recognize serotonin.
 Apomorphine is an example of a drug that also influences brain function.
 Specifically, apomorphine is a non-selective dopamine D.sub.2 /D.sub.3
 receptor agonist. At low doses, apomorphine (e.g., 25-200 .mu.g/kg)
 activates pre-synaptic receptors, while at higher doses (e.g., 600
 .mu.g/kg) it influences post-synaptic sites. Thus, the behavioral affects
 of apomorphine vary with dosage. In mice and rats, high doses of
 apomorphine cause a characteristic climbing behavior as well as oro-facial
 stereotypies such as sniffing and licking behaviors. Using these high
 doses of apomorphine, atypical neuroleptic compounds have been identified
 based on their ability to block potently the climbing behavior while
 causing little change to the sniffing and licking behaviors. Both typical
 and atypical neuroleptic compounds have been used to treat schizophrenia
 and other psychotic disorders. Atypical drugs are preferred because of
 their lower propensity to cause motor side effects (e.g., extrapyramidal
 side effects such as parkinsonism and tardive dyskinesia).
 SUMMARY
 The invention provides a novel amino acid, neo-tryptophan, as well as
 polypeptides containing neo-tryptophan. In addition, the invention
 provides neo-tryptophan derivatives and serotonin-like neo-tryptophan
 derivatives as well as compositions containing these derivatives.
 Specifically, the invention provides neurotensin (NT) polypeptide analogs
 as well as other polypeptides that contain neo-tryptophan. The invention
 also provides methods for making neo-tryptophan, neo-tryptophan
 derivatives, serotonin-like neo-tryptophan derivatives, and compositions
 containing such compounds. Further, the invention provides methods for
 inducing a neurotensin response in a mammal as well as methods for
 treating a mammal having a serotonin recognition molecule.
 One aspect of the invention features a polypeptide containing
 neo-tryptophan. The polypeptide can be substantially pure, and
 neo-tryptophan can be L-neo-tryptophan or D-neo-tryptophan. The
 polypeptide can interact with a neurotensin receptor, and can be a
 neurotensin analog with neo-tryptophan being located at amino acid
 position 11 of neurotensin. The polypeptide can be NT64D, NT64L, NT65L,
 NT66D, NT66L, NT67L, NT69L, NT69L', NT71, NT72, NT73, NT74, NT75, NT76,
 NT77, Ang1, Brdy1, or Lenk1.
 In another aspect, the invention features an amino acid that is
 neo-tryptophan. The amino acid can be substantially pure, and can be
 L-neo-tryptophan or D-neo-tryptophan.
 Another aspect of the invention features a neo-tryptophan derivative. The
 neo-tryptophan derivative can contain neo-tryptophan and a blocking group
 (e.g., Fmoc or Boc).
 Another aspect of the invention features a serotonin-like neo-tryptophan
 derivative having the following structure:
 ##STR1##
 with R.sub.1, R.sub.2, and R.sub.3 being H, OH, CH.sub.3, SH, F, NH.sub.2,
 or COOH, and A being zero, one, two, or three. For example, R.sub.1 and
 R.sub.3 each can be a hydroxyl group, R.sub.2 can be an amino group, and A
 can be zero.
 Another aspect of the invention features a method of synthesizing
 neo-tryptophan. The method includes providing 4-hydroxymethyl indole, and
 substituting the hydroxyl group of the 4-hydroxymethyl indole with a
 glycyl unit to produce neo-tryptophan. The N-1 nitrogen of the
 4-hydroxymethyl indole can be protected by a protecting group (e.g., Boc)
 that can be removed after the substitution. The method can include (a)
 providing 2-methyl-3-nitrobenzoic acid, (b) esterifying the
 2-methyl-3-nitrobenzoic acid to form an esterification product, (c)
 reacting the esterification product with N,N-dimethylformamide
 dimethylacetal to produce an enamine product, (d) performing reductive
 cyclization on the enamine product to produce a 4-substituted indole
 methyl ester, (e) protecting the indole nitrogen of the 4-substituted
 indole methyl ester with a Boc group, (f) reducing the protected
 4-substituted indole methyl ester with DIBAL to produce
 N-Boc-4-hydroxymethyl indole, (g) converting the N-Boc-4-hydroxymethyl
 indole into benzylic bromide, (h) performing SN.sub.2 displacement of the
 bromide of the benzylic bromide with a carbanion to produce diastereomeric
 bislactim products, (i) isolating one of the diastereomeric bislactim
 products, (j) hydrolyzing the isolated diastereomeric bislactim product to
 produce an aminoester product, (k) saponifying the aminoester product to
 produce an N.sup.ind -t-Boc amino acid, and (l) removing the Boc group to
 produce neo-tryptophan.
 Another aspect of the invention features a method of synthesizing a
 neo-tryptophan derivative. The method includes providing 4-hydroxymethyl
 indole having the N-1 nitrogen of protected by a protecting group, and
 substituting the hydroxyl group of the 4-hydroxymethyl indole with a
 glycyl unit to produce a neo-tryptophan derivative. The protecting group
 can include Boc. The method can include adding, after the substitution, an
 additional protecting group to the nitrogen within the glycyl unit. The
 additional protecting group can include Fmoc.
 Another aspect of the invention features a method of making a polypeptide
 containing neo-tryptophan. The method includes providing a neo-tryptophan
 derivative, and linking an amino acid residue to the neo-tryptophan
 derivative to form the polypeptide containing neo-tryptophan (e.g.,
 L-neo-tryptophan or D-neo-tryptophan). The neo-tryptophan derivative can
 contains a blocking group attached to a nitrogen atom.
 Another aspect of the invention features a method of inducing a neurotensin
 response in a mammal (e.g., human). The method includes administering an
 effective dose of a polypeptide containing neo-tryptophan to the mammal.
 The administration can be extracranial (e.g., intraperitoneal,
 intravenous, intradermal, subcutaneous, oral, or nasal). The neurotensin
 response can include antinociception, hypothermia, reduction in appetite,
 reduction in body weight, reduction in body weight gain, preventing or
 reducing catalepsy (e.g., haloperidol-induced catalepsy), and/or reducing
 an effect of a CNS stimulant such as apomorphine, amphetamine, or cocaine.
 For example, the neurotensin response can include reducing a climbing
 behavior induced by apomorphine. The neurotensin response can include an
 antipsychotic effect. For example, the polypeptide can reduce the signs or
 symptoms of schizophrenia in the mammal. The polypeptide can interact with
 a neurotensin receptor (e.g., a rat or human neurotensin receptor). The
 polypeptide can be NT64D, NT64L, NT65L, NT66D, NT66L, NT67L, NT69L,
 NT69L', NT71, NT72, NT73, NT74, NT75, NT76, or NT77.
 Another embodiment of the invention features a method of treating a mammal
 (e.g., human) having a serotonin recognition molecule. The method includes
 administering a composition to the mammal such that composition interacts
 with the serotonin recognition molecule (e.g., a serotonin receptor such
 as a 5HT.sub.2A receptor). The composition includes neo-tryptophan, a
 neo-tryptophan derivative, or a serotonin-like neo-tryptophan derivative.
 The composition can include a polypeptide.
 Another aspect of the invention features a method for screening a
 polypeptide for in vivo use. The method includes contacting a polypeptide
 containing neo-tryptophan with a protease, and determining whether or not
 the polypeptide remains intact.
 Another aspect of the invention features the use of a polypeptide
 containing neo-tryptophan in the manufacture of a medicament for treating
 a mammal.
 In another embodiment, the invention features the use of a compound in the
 manufacture of a medicament for treating a mammal. The compound contains
 neo-tryptophan, a neo-tryptophan derivative, or a serotonin-like
 neo-tryptophan derivative.
 Unless otherwise defined, all technical and scientific terms used herein
 have the same meaning as commonly understood by one of ordinary skill in
 the art to which this invention belongs. Although methods and materials
 similar or equivalent to those described herein can be used in the
 practice or testing of the present invention, suitable methods and
 materials are described below. All publications, patent applications,
 patents, and other references mentioned herein are incorporated by
 reference in their entirety. In case of conflict, the present
 specification, including definitions, will control. In addition, the
 materials, methods, and examples are illustrative only and not intended to
 be limiting.

DETAILED DESCRIPTION
 The invention provides a novel amino acid, neo-tryptophan, as well as
 polypeptides containing this novel amino acid such as neurotensin analogs.
 In addition, the invention provides neo-tryptophan derivatives,
 serotonin-like neo-tryptophan derivatives, and polypeptides containing
 such derivatives. The invention also provides methods for making
 neo-tryptophan, neo-tryptophan derivatives, serotonin-like neo-tryptophan
 derivatives, and compositions containing these compounds. Further, the
 invention provides methods for inducing a neurotensin response in a mammal
 as well as methods for treating a mammal having a serotonin recognition
 molecule.
 The invention provides a novel amino acid, neo-tryptophan. Neo-tryptophan
 can be used to create novel polypeptides having enhanced biological
 characteristics. For example, the invention provides
 neo-tryptophan-containing NT polypeptide analogs that exhibit enhanced
 biological effects as compared to NT itself. Specifically, such NT
 polypeptide analogs can induce antinociception, hypothermia, thirst,
 weight loss, appetite suppression, and weight gain reduction. In addition,
 these NT polypeptide analogs can prevent or reduce catalepsy, such as
 haloperidol-induced catalepsy, and can prevent or reduce an effect of a
 CNS stimulant such as apomorphine, amphetamine, and cocaine.
 The incorporation of neo-tryptophan into a polypeptide sequence can create
 polypeptide analogs that exhibit increased biological activity, increased
 resistance to degradation by proteases (e.g., metalloendopeptidases 24.11
 and 24.16), and increased blood brain barrier permeability. For example,
 neo-tryptophan can be used to make polypeptide analogs that interact with
 their receptors at a higher affinity than the natural polypeptide ligands.
 In addition, neo-tryptophan can be used as a novel fluorescence probe for
 spectroscopic studies since neo-tryptophan may have a unique fluorescence
 profile.
 The chemical structures for tryptophan, iso-tryptophan, tyrosine, and
 neo-tryptophan are provided in FIG. 1. Neo-tryptophan
 (2-amino-3-[1H-indolyl]propanoic acid) places the indole group of
 tryptophan in such a unique orientation in terms of steric and
 electrostatic fields that polypeptides containing neo-tryptophan provide
 novel arrangements for side chain interactions. It will be appreciated
 that the term "neo-tryptophan" includes both D-neo-tryptophan and
 L-neo-tryptophan.
 The invention provides methods for making neo-tryptophan and neo-tryptophan
 derivatives. Specifically, any method that results in the production of
 neo-tryptophan or a neo-tryptophan derivative is within the scope of the
 invention. For example, one method within the scope of the invention
 involves substituting the hydroxyl group of 4-hydroxymethyl indole with a
 glycyl unit such that neo-tryptophan or a neo-tryptophan derivative is
 produced. In addition, FIGS. 3 and 4 provide methods that can be used to
 synthesize both D- and L-neo-tryptophan as well as neo-tryptophan
 derivatives that contain blocking groups. Briefly, these methods involve
 (a) providing 2-methyl-3-nitrobenzoic acid, (b) esterifying the
 2-methyl-3-nitrobenzoic acid to form an esterification product, (c)
 reacting the esterification product with N,N-dimethylformamide
 dimethylacetal to produce an enamine product, (d) performing reductive
 cyclization on the enamine product to produce a 4-substituted indole
 methyl ester, (e) protecting the indole nitrogen of the 4-substituted
 indole methyl ester with a Boc group, (f) reducing the protected
 4-substituted indole methyl ester with DIBAL to produce
 N-Boc-4-hydroxymethyl indole, (g) converting the N-Boc-4-hydroxymethyl
 indole into benzylic bromide, (h) performing SN.sub.2 displacement of the
 bromide of the benzylic bromide with a carbanion to produce diastereomeric
 bislactim products, (i) isolating one of the diastereomeric bislactim
 products, (j) hydrolyzing the isolated diastereomeric bislactim product to
 produce an aminoester product, (k) saponifying the aminoester product to
 produce an N.sup.ind -t-Boc amino acid, and (l) removing the Boc group to
 produce neo-tryptophan. Other methods within the scope of the invention
 can be easily devised by one skilled in the art once provided with the
 teachings disclosed herein.
 The term "neo-tryptophan derivative" as used herein refers to any compound
 that has the basic structure of neo-tryptophan. Neo-tryptophan derivatives
 include, without limitation, neo-tryptophan having an additional chemical
 group added to the glycyl group or to the indole structure. For example,
 one or both of the nitrogen atoms can be modified to contain a blocking
 group such as Fmoc or Boc. One such modification can result in a
 neo-tryptophan derivative having a Boc blocking group attached to the
 indole nitrogen atom and a Fmoc blocking group attached to the glycyl
 group nitrogen atom. Neo-tryptophan derivatives can be used during
 polypeptide synthesis reactions to produce polypeptides that contain
 neo-tryptophan.
 Any composition containing neo-tryptophan or a neo-tryptophan derivative is
 within the scope of the invention. Such compositions can include, without
 limitation, lipids, carbohydrates, amino acids, polypeptides, nucleic
 acids, peptide nucleic acids, and combinations thereof. Compositions
 containing neo-tryptophan or a neo-tryptophan derivative can be in an
 aqueous or non-aqueous form. For example, a neo-tryptophan-containing
 composition can contain water or saline.
 Any polypeptide containing neo-tryptophan or a neo-tryptophan derivative is
 within the scope of the invention. Such polypeptides can contain any
 sequence of natural or synthetic amino acids provided at least one residue
 is neo-tryptophan or a neo-tryptophan derivative. In other words, any
 amino acid residue within a polypeptide can be replaced with
 neo-tryptophan or a neo-tryptophan derivative. For example, D- or
 L-neo-tryptophan can be substituted for D- and L-isomers of the aromatic
 amino acid residues (i.e., tryptophan, tyrosine, and phenylalanine) in
 natural and synthetic polypeptides. Again, the incorporation of
 neo-tryptophan or a neo-tryptophan derivative into an amino acid sequence
 can improve a polypeptide's binding affinities, selectivity, blood brain
 barrier permeability, and/or resistance to peptidase degradation. Examples
 of polypeptides that can be modified to contain neo-tryptophan or a
 neo-tryptophan derivative include, without limitation, adrenocorticotropic
 hormone, angiotensin, bombesin, bradykinin, kalledin, calcitonin gene
 related peptide, BDNF, EGF, somatostatin, enkephalin (e.g.,
 met-enkephalin, leu-enkephalin, and their derivatives), dermorphin,
 substance P, proctolin, isotocin, vasopressin, vasotocin, luteinizing
 hormone releasing hormone, neurotensin, thyrotropin releasing hormone,
 endomorphin-1, endomorphin-2, and morphiceptin.
 The invention provides methods for making polypeptides that contain
 neo-tryptophan and neo-tryptophan derivatives. Specifically, any method
 that results in the production of a polypeptide that contains
 neo-tryptophan or a neo-tryptophan derivative is within the scope of the
 invention. For example, one method within the scope of the invention
 involves linking an amino acid residue to neo-tryptophan or a
 neo-tryptophan derivative to form a polypeptide. For the purpose of this
 invention, the term "polypeptide" includes, without limitation, dipeptides
 as well as polypeptides larger than dipeptides. In addition, polypeptides
 containing neo-tryptophan or a neo-tryptophan derivative can be
 synthesized using common polypeptide synthesis techniques with the
 substitution of neo-tryptophan or a neo-tryptophan derivative when
 appropriate (Morbeck DE et al., In: Methods: A Companion to Methods in
 Enzymology 6:191-200, Academic Press Inc., New York (1993)). For example,
 polypeptides can be synthesized using Fmoc chemistry with
 t-butyl-protected side chains, either individually on automated
 polypeptide synthesizers (ABI 430A or 431A) or simultaneously on a
 multiple polypeptide synthesizer (ACT350, Advanced Chemtech, Louisville,
 Ky.). Protocols concerning activation coupling times, amino acid
 dissolution, coupling solvents, and synthesis scale can be followed
 according to the manufacturer's instructions. Further, polypeptides
 containing neo-tryptophan or a neo-tryptophan derivative can be purified
 by, for example, reverse-phase HPLC, and then analyzed for purity by, for
 example, HPLC and mass spectrometry.
 The term "substantially pure" as used herein refers to a molecule (e.g., an
 amino acid or polypeptide) that has been separated from either the
 components that accompany that molecule in nature or the reaction products
 (e.g., by-products) from a chemical synthesis process. Typically, a
 molecule is substantially pure when it is at least 50%, 60%, 70%, 80%,
 90%, 95%, 99%, or 99.9%, by weight, free from other components or reaction
 products. Purity can be measured by any appropriate method (e.g., column
 chromatography, mass spectrometry, polyacrylamide gel electrophoresis, or
 HPLC analysis).
 The invention also provides serotonin-like neo-tryptophan derivatives (FIG.
 2). Such serotonin-like neo-tryptophan derivatives can interact with
 serotonin recognition molecules such as serotonin receptors and serotonin
 plasma membrane and vesicular transporters. For example, serotonin-like
 neo-tryptophan derivatives can be used as serotonin receptor agonists or
 antagonists, inhibitors of serotonin re-uptake by plasma membrane
 monoamine transporters, or inhibitors of monoamine packaging into
 intracellular compartments (e.g., synaptic vesicles) by vesicular
 monoamine transporters. In other words, the serotonin-like neo-tryptophan
 derivatives provided by the invention can be used to interact with
 serotonin recognition molecules in a manner such that serotonergic
 conditions such as depression, anxiety, migraine, schizophrenia, eating
 disorders, obsessive compulsive disorders, and panic disorders are
 influenced.
 The chemical structure of serotonin-like neo-tryptophan derivatives is
 depicted in FIG. 2. In one embodiment, R.sub.1 can be a hydroxyl group,
 R.sub.2 can be an amino group, and R.sub.3 can be either a hydroxyl group
 or hydrogen atom. The "X" symbol in FIG. 2 represents any chemical
 structure or modification including, without limitation, blocking groups
 such Fmoc and Boc.
 Any method can be used to synthesize a serotonin-like neo-tryptophan
 derivative. For example, neo-tryptophan can be chemically modified using
 common organic chemistry techniques such that a serotonin-like
 neo-tryptophan derivative is produced.
 In addition, the invention provides methods of inducing a neurotensin
 response in a mammal (e.g., a rodent, cow, pig, dog, cat, horse, sheep,
 goat, non-human primate, and human). These methods involve administering
 an effective dose of a polypeptide containing neo-tryptophan or a
 neo-tryptophan derivative. A neurotensin response is any biological
 response that can be attributed to NT or a NT polypeptide analog. For
 example, a neurotensin response can be a biological response that occurs
 after a ligand interacts with a receptor (e.g., NTR1 and NTR2) that binds
 NT. Examples of neurotensin responses include, without limitation,
 antinociception, hypothermia, antipsychotic effects, loss of appetite,
 body weight reduction, body weight gain reduction, and increased thirst.
 Other examples of neurotensin responses include, without limitation, the
 prevention or reduction of catalepsy, such as haloperidol-induced
 catalepsy, as well as the prevention or reduction of an effect of a CNS
 stimulant (e.g., apomorphine, amphetamine, and cocaine). An effect of a
 CNS stimulant can be, for example, the climbing behavior induced by
 apomorphine.
 The term "effective dose" as used herein refers to any amount of compound
 that induces the particular described response without inducing
 significant toxicity. For example, an effective dose of NT69L for appetite
 reduction can be that amount needed to cause the mammal to exhibit
 appetite suppression without significant toxicity. In addition, an
 effective dose of a particular compound administered to a mammal can be
 adjusted according to the mammal's response and desired outcomes.
 Significant toxicity can vary for each particular patient and depends on
 multiple factors including, without limitation, the patient's degree of
 illness, age, and tolerance to pain.
 In addition, any of the materials described herein can be administered to
 any part of the mammal's body including, without limitation, brain, spinal
 fluid, blood stream, lungs, nasal cavity, intestines, stomach, muscle
 tissues, skin, peritoneal cavity, and the like. Thus, a polypeptide
 containing neo-tryptophan can be administered by intravenous,
 intraperitoneal, intramuscular, subcutaneous, extracranial, intrathecal,
 and intradermal injection, by oral administration, by inhalation, or by
 gradual perfusion over time. For example, an aerosol preparation can be
 given to a mammal by inhalation. It is noted that the duration of
 treatment with the materials described herein can be any length of time
 from as short as one day to as long as a lifetime (e.g., many years). For
 example, a polypeptide containing neo-tryptophan can be administered at
 some frequency over a period of ten years. It is also noted that the
 frequency of treatment can be variable. For example, a polypeptide
 containing neo-tryptophan can be administered once (or twice, three times,
 etc.) daily, weekly, monthly, or yearly.
 Preparations for administration can include sterile aqueous or non-aqueous
 solutions, suspensions, and emulsions. Examples of non-aqueous solvents
 include, without limitation, propylene glycol, polyethylene glycol,
 vegetable oils, and injectable organic esters. Aqueous carriers include,
 without limitation, water as well as alcohol, saline, and buffered
 solutions. Preservatives, flavorings, and other additives such as, for
 example, antimicrobials, anti-oxidants, chelating agents, inert gases, and
 the like may also be present.
 Any polypeptide containing neo-tryptophan or a neo-tryptophan derivative
 that induces a neurotensin response can be administered to a mammal. Such
 polypeptides can be identified by, for example, monitoring any of the
 biological characteristics described herein before and after
 administration. In addition, a polypeptide that induces a neurotensin
 response can interact with a neurotensin receptor (e.g., a rat or human
 neurotensin receptor). The term "interaction" as used herein means that
 two components specifically bind each other. Typically, any compound that
 has a binding affinity for a particular compound in the sub-millimolar
 range (e.g., K.sub.d &lt;1 mM) is considered to interact with that particular
 compound. For example, a ligand that binds a receptor with an affinity
 less than 1 mM specifically interacts with that receptor. Examples of
 polypeptides that interact with a NT receptor include, without limitation,
 NT64D, NT64L, NT65L, NT66D, NT66L, NT67L, NT69L, NT69L', NT71, NT72, NT73,
 NT74, NT75, NT76, and NT77.
 The invention also provides methods for treating a mammal having a
 serotonin recognition molecule. The methods involve administering a
 composition containing neo-tryptophan, a neo-tryptophan derivative, or a
 serotonin-like neo-tryptophan derivative such that the composition
 interacts with a serotonin recognition molecule. The term "serotonin
 recognition molecule" includes receptors as well as transporters (e.g.,
 plasma membrane and vesicular transporters). The interaction between the
 composition and serotonin recognition molecule can either stimulate or
 inhibit serotonergic activity, and thus treat conditions such as
 depression, anxiety, migraine, schizophrenia, eating disorders, obsessive
 compulsive disorders, and panic disorders.
 The invention provides a method for screening a polypeptide for in vivo
 use. The method involves contacting a polypeptide with a protease and
 determining whether or not the polypeptide remains intact. The term
 "protease" as used herein refers to any polypeptide that cleaves a peptide
 bond. Any source can be used to obtain proteases. For example, biological
 samples such as blood and intestinal tissue can be used as a source of
 protease. In addition, any method can be used to determine whether or not
 a polypeptide remains intact. For example, polyacrylamide gel
 electrophoresis and HPLC analysis can be used to determine whether or not
 a polypeptide remains intact.
 The invention will be further described in the following examples, which do
 not limit the scope of the invention described in the claims.
 EXAMPLES
 Example 1
 Synthesis of neo-tryptophan and neo-tryptophan-containing Polypeptides
 D- and L-neo-tryptophan were synthesized according to the schemes depicted
 in FIGS. 3 and 4. Polypeptides containing neo-tryptophan were synthesized
 as described elsewhere using the neo-tryptophan amino acids when
 appropriate (Morbeck DE et al., In: Methods: A Companion to Methods in
 Enzymology 6:191-200, Academic Press Inc., New York (1993)). Briefly,
 polypeptides were synthesized using Fmoc chemistry with t-butyl-protected
 side chains, either individually on automated polypeptide synthesizers
 (ABI 430A or 431A) or simultaneously on a multiple polypeptide synthesizer
 (ACT350, Advanced Chemtech, Louisville, Ky.). Protocols concerning
 activation coupling times, amino acid dissolution, coupling solvents, and
 synthesis scale were followed according to the manufacturer's
 instructions. All polypeptides were purified by reverse-phase HPLC using a
 C18 column (2.2.times.25 cm; Vydac, Hesperia, Calif.) in 0.1% TFA/water
 and a gradient of 10%-60% acetonitrile in 0.1% TFA/water. A combination of
 analytical HPLC and mass spectrometry was used to analyze polypeptide
 purity.
 The following methods were used for the convenient, multigram synthesis of
 neo-tryptophan. The Fmoc-t-Boc derivative of neo-tryptophan was readily
 incorporated into bioactive synthetic peptides using standard solid phase
 synthesis. The synthesis of neo-tryptophan featured the use of Schollkopf
 chiral auxiliary reagents for chirality induction during a key step. For
 convenience, alpha-numerical designations are used to describe specific
 compounds used in the synthesis steps depicted in FIG. 4. In addition, a
 description of these compounds and the reaction procedures are provided.
 In general terms, as depicted in FIG. 4, the enantiomeric synthesis of
 neo-tryptophan 1a and neo-tryptophan derivative 1b began with
 2-methyl-3-nitrobenzoic acid 2 which, after esterification, was followed
 by reaction with N,N-dimethylformamide dimethylacetal to furnish the
 enamine 4. Reductive cyclization using H.sub.2 /Pd-C gave the
 4-substituted indole methyl ester 5. Protection of the indole nitrogen of
 the 4-substituted indole methyl ester 5 with the tert-butoxycarbonyl (Boc)
 group and reduction of the resulting ester with DIBAL proceeded
 uneventfiilly to give N-Boc-4-hydroxymethyl indole 6. It is noted that the
 indole nitrogen was initially protected with a benzyl or carbobenzyloxy
 (Cbz) group but these groups turned out to be problematic later in the
 synthetic sequence. Specifically, the indole nitrogen benzyl group could
 not be removed with catalytic hydrogenolysis, and the Cbz group gave low
 yields due to its partial removal during the reduction of the methyl ester
 moiety with DIBAL. Next, conversion to the benzylic bromide 7 with
 phosphorus tribromide in ether was followed by the key SN.sub.2
 displacement of the bromide with the carbanion derived from commercially
 available (R)-Schollkopf reagent,
 (2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine, to provide the
 diastereomeric bislactim products 8a and 8b in 10:1 diastereomeric excess
 in 67% yield. The desired diastereomer 8a was readily isolated by flash
 silica gel chromatography and then subjected to mild acid (0.1 M
 TFA/CH.sub.3 CN) treatment that only hydrolyzed the bislactim leaving the
 Boc group unaffected. The resulting aminoester 9 was saponified to produce
 N.sup.ind -t-Boc neo-tryptophan 10. It is noted that the TFA-salt of the
 aminoester 9 was easily isolated in pure form by extraction with methylene
 chloride leaving the corresponding (D)-valine aminoester in the aqueous
 phase. Finally, the amino nitrogen was protected with
 flourenylmethoxycarbonyl (Fmoc) to form Fmoc/Boc neo-tryptophan derivative
 1b. The Fmoc/Boc neo-tryptophan derivative 1b was conveniently
 incorporated into bioactive peptides using commonly employed solid phase
 synthesis methods. For spectral, chemical, and optical characterization,
 the t-Boc group of N.sup.ind -t-Boc neo-tryptophan 10 was removed to
 furnish neo-tryptophan 1a. Following the above protocol, multigram
 synthesis of enantiopure Fmoc/Boc neo-tryptophan derivative 1b was
 achieved. Since the S-enantiomer of the Schollkopf reagent is commercially
 available, the (R)-2-amino-3-(1H-indolyl)propanoic acid was similarly
 synthesized.
 The doubly protected neo-tryptophan 1b was readily incorporated into novel
 biologically active neurotensin analogs by conventional Fmoc-related
 automated solid phase chemistry. In addition, neo-tryptophan was
 incorporated into other polypeptides having biological and therapeutic
 interest including angiotensin, bradykinin, and Leu-enkephalin (Table I).
 TABLE I
 Amino acid sequences of NT, angiotensin, bradykinin, and leu-enkephalin
 polypeptides and polypeptide analogs
 Sequence
 Polypeptide 1 2 3 4 5 6 7 8
 9 10 11 12 13
 NT p-Glu L-Leu L-Tyr L-Glu L-Asn L-Lys L-Pro
 L-Arg L-Arg L-Pro L-Tyr L-Ile L-Leu
 (SEQ ID NO: 1)
 NT(8-13)
 L-Arg L-Arg L-Pro L-Tyr L-Ile L-Leu
 (SEQ ID NO: 2)
 NT(9-13)
 L-Arg L-Pro L-Tyr L-Ile L-Leu
 (SEQ ID NO: 3)
 NTW
 L-Arg L-Arg L-Pro L-Trp L-Ile L-Leu
 (SEQ ID NO: 4)
 NT
 L-Arg L-Arg L-Pro L-Tyr tert-Leu L-Leu
 (tert-Leu)
 (SEQ ID NO: 5)
 Eisai*
 N-methyl- L-Lys L-Pro L-Trp tert-Leu L-Leu
 (SEQ ID NO: 6)
 Arg
 NT2
 D-Lys L-Arg L-Pro L-Tyr L-Ile L-Leu
 (SEQ ID NO: 7)
 NT24 "27" L-Arg
 D-Orn.sup.& L-Pro L-Tyr L-Ile L-Leu
 (SEQ ID NO: 8)
 NT34
 L-Arg L-Arg L-Pro L-3,1'- L-Ile L-Leu
 (SEQ ID NO: 9)
 Nal.sup.#
 NT64D
 L-Arg L-Arg L-Pro D-neo- L-Ile L-Leu
 (SEQ ID NO: 10)
 Trp
 NT64L
 L-Arg L-Arg L-Pro L-neo- L-Ile L-Leu
 (SEQ ID NO: 11)
 Trp
 NT65L
 L-Arg L-Arg L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 12)
 Trp
 NT66D
 D-Lys L-Arg L-Pro D-neo- tert-Leu L-Leu
 (SEQ ID NO: 13)
 Trp
 NT66L
 D-Lys L-Arg L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 14)
 Trp
 NT67L
 D-Lys L-Arg L-Pro L-neo- L-Ile L-Leu
 (SEQ ID NO: 15)
 Trp
 NT69L
 N-methyl- L-Lys L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 16)
 Arg Trp
 NT69L'
 N-methyl- L-Arg L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 17)
 Arg Trp
 NT71
 N-methyl- DAB.sup.$ L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 18)
 Arg Trp
 NT72
 D-Lys L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 19)
 Trp
 NT73
 D-Lys L-Pro L-neo- L-Ile L-Leu
 (SEQ ID NO: 20)
 Trp
 NT74
 DAB L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 21)
 Trp
 NT75
 DAB L-Pro L-neo- L-Ile L-Leu
 (SEQ ID NO: 22)
 Trp
 NT76
 L-Arg D-Orn L-Pro L-neo- L-Ile L-Leu
 (SEQ ID NO: 23)
 Trp
 NT77
 L-Arg D-Orn L-Pro L-neo- tert-Leu L-Leu
 (SEQ ID NO: 24)
 Trp
 Angiotensin Asp Arg Val Tyr Ile His Pro
 Phe
 (SEQ ID NO: 25)
 Ang1 Asp Arg Val L-neo- Ile His Pro
 Phe
 (SEQ ID NO: 26) Trp
 Bradykinin Arg Pro Pro Gly Phe Ser Pro
 Phe Arg
 (SEQ ID NO: 27)
 Brdy1 Arg Pro Pro Gly L-neo- Ser Pro
 Phe Arg
 (SEQ ID NO: 28) Trp
 Leu- Tyr Gly Gly Phe Leu
 enkephalin
 (SEQ ID NO: 29)
 Lenk1 L- Gly Gly Phe Leu
 (SEQ ID NO: 30) neo-
 Trp
 *Tsuchiya Y et al., (1989) European Patent Application 89104302.8; .sup.#
 naphthalylalanine; .sup.$ diaminobutyric acid; .sup.& D-ornithine
 The reagents and conditions used during the steps indicated in FIG. 4 can
 be summarized as follows: (a) K.sub.2 CO.sub.3, MeI, DMF, RT (100%); (b)
 N,N-dimethylformamide dimethylacetal, DMF, 120.degree. C.; (c) H.sub.2,
 10% Pd-C (cat), MeOH, RT, 50-55 Psi, benzene (67% over 2 steps); (d)
 (Boc).sub.2 O, CH.sub.3 CN, DMAP (cat), RT (100%); (e) DIBAL-H, CH.sub.2
 Cl.sub.2 /ether, -78.degree. C. (88%); (f) PBr.sub.3, ether/CH.sub.2
 Cl.sub.2 (95%); (g)
 (2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine/BuLi, THF, -78.degree.
 C., then 7 (67%); (h) 0.1 M aq. TFA, CH.sub.3 CN, RT, (100% overall); (i)
 LiOH, H.sub.2 O , THF/H.sub.2 O , RT (62%); (j) TFA/CH.sub.2 Cl.sub.2, RT;
 (k) Fmoc-Suc, 10% NaHCO.sub.3, acetone, 0.degree. C.-RT, (72%).
 The following section provides a detailed description of the chemical steps
 used to synthesize neo-tryptophan. In addition, the nuclear magnetic
 spectra (.sup.1 H, .sup.13 C) described herein were measured with a Bruker
 WH-300 instrument (.sup.1 H frequency 300 MHZ, .sup.13 C frequency 75 MHZ)
 in the solvent noted. .sup.1 H chemical shifts are expressed in parts per
 million downfield from Me.sub.4 Si used as internal standard. Melting
 points (mp.) were taken with a GallenKamp instrument and are uncorrected.
 The column chromatographic separations were performed with `J. T. Baker`
 Silica gel (40 .mu.m). Anhydrous DMF was obtained from Aldrich Chemicals.
 Tetrahydrofuran (THF) and diethyl ether were distilled over sodium
 benzophenone ketyl before use. Methylene chloride was distilled over
 calcium hydride or P.sub.2 O.sub.5. Acetonitrile was reagent grade
 obtained from E.M. SCIENCE, and was used without further drying. Ethyl
 acetate and hexane were reagent grade, and used as received. The purity of
 all compounds was shown to be &gt;95% by TLC as well as by high field .sup.1
 H NMR and .sup.13 C NMR (300 and 75 MHZ Brucker instrument). Optical
 rotations were taken with a 241-Perkin Elmer Polarimeter (Na lamp). IR
 spectra were measured with a 2020 GALAXY Series FT-IR (Mattson
 Instruments).
 With reference to FIG. 4, DMF (130 mL) was added to a well mixed
 2-methyl-3-nitrobenzoic acid 2 (50 g, 0.28 mol) and KHCO.sub.3 (84 g, 0.84
 mol) solution. Since the mixture became highly viscous, it was heated to
 40.degree. C. with manual shaking. lodomethane (79 g, 0.56 mol) was added
 via syringe after the gas evolution had ceased. The resulting orange
 colored solution was stirred for 12 hours at room temperature. The
 reaction mixture was poured into water (800 mL), and the resulting
 precipitate collected by filtration and dried over P.sub.2 O.sub.5 to give
 pure methyl 2-methyl-3-nitrobenzoate 3 (56 g, 100%) as a white solid: mp.
 64.2-65.5.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta. 8.00 (d, J=7.8 Hz,
 1H), 7.85 (d, J=8.0 Hz, 1H), 7.39 (t, J=8.0 Hz, 1H), 3.95 (s, 3H), 2.63
 (s, 3H); IR (KBr, cm.sup.-1) 1724, 1548, 1279; MS (EI): 195 (M.sup.+).
 A solution of methyl 2-methyl-3-nitrobenzoate 3 (20 g, 0.1 mol) and
 N,N-dimethylformamide dimethyl acetal (40 mL, 0.3 mol) in DMF (50 mL) was
 stirred at 120.degree. C. under nitrogen for 12 hours. The solution became
 deep red. The excess amount of N,N-dimethylformamide dimethyl acetal and
 DMF was distilled off under reduced pressure to give crude enamine 4 that
 was directly used in the next step without purification.
 The crude enamine 4 was dissolved in anhydrous benzene (250 mL). Pd/C (10%,
 2.8 g) was added to this solution, and the resulting mixture was
 hydrogenated at 55 psi. Warming was observed at the start of the reaction.
 The deep red mixture became dark gray after 12 hours at room temperature.
 Pd/C was filtered off over Celite, and the filtrate was concentrated under
 reduced pressure. Chromatography on silica gel (ethyl acetate/hexanes:
 30:70 v/v, Rf=0.55) afforded methyl 1H-4-indolecarboxylate 5 (11.8 g, 67%)
 as a light yellow solid: mp. 67.5-69.0.degree. C.; .sup.1 H-NMR
 (CDCl.sub.3) .delta. 8.40 (s, 1H), 7.93 (d, J=8.3 Hz, 1H), 7.60 (d, J=8.9
 Hz, 1H), 7.36 (t, J=3.0 Hz, 1H), 7.26 (t, J=3.7 Hz, 1H), 7.26-7.18 (m,
 1H), 4.0 (s, 3H): IR (KBr, cm.sup.-1) 3322, 1705, 1279; MS (ESI): 176
 (M.sup.+ +1).
 Di-tert-butyl dicarbonate (14.7 g, 67.4 mmol) and DMAP (0.2 g) was added to
 a solution of methyl 1H-4-indolecarboxylate 5 (11.8 g, 67.4 mmol) in
 acetonitrile (50 mL). The mixture was stirred at room temperature for 12
 hours. Some bubbling was observed. Solvent was removed under reduced
 pressure to give a residue that was redissolved in ethyl acetate (200 mL).
 The solution was washed sequentially with cold 1N HCl (80 mL), water (50
 mL), and brine (50 mL), and then dried (MgSO.sub.4). The solvent was
 removed under reduced pressure to give pure 1-(tert-butyl) 4-methyl
 1H-1,4-indoledicarboxylate (18.5 g, 100%) as a light yellow oil: .sup.1
 H-NMR (CDCl.sub.3) .delta. 8.41 (d, J=8.2 Hz 1H), 7.98 (d, J=7.7 Hz, 1H),
 7.71 (d, J=3.7 Hz, 1H), 7.36 (t, J=7.9 Hz, 1H), 7.28 (d, J=3.8 Hz, 1H),
 3.98 (s, 3H) 1.68 (s, 9H); .sup.13 C-NMR (CDCl.sub.3) .delta. 187.3,
 149.4, 135.9, 130.5, 127.8, 125.4, 123.5, 121.9, 119.7, 107.8, 84.1, 51.8,
 28.1; IR (KBr, cm.sup.-1) 1703, 1603, 1283, 1146; MS (ESI): 276 (M.sup.+
 +1).
 DIBAL (1.0 M in CH.sub.2 Cl.sub.2, 163 mmol) was added at -78.degree. C. in
 30 minutes under nitrogen to a solution of 1-(tert-butyl) 4-methyl
 1H-1,4-indoledicarboxylate (18.5 g, 67 mmol) in ether (150 mL). Stirring
 was continued at this temperature for another 30 minutes at which point
 the reaction was quenched with saturated citric acid at -78.degree. C. A
 precipitate immediately formed that, after warming to room temperature,
 was acidified to pH 1 with 1N HCl and extracted with ethyl acetate
 (3.times.200 mL). The combined extracts were washed sequentially with
 water (100 mL), and brine (200 mL), and then dried (MgSO.sub.4). The
 solvent was evaporated under vacuum to give a residue that was purified by
 chomatography on silica gel (ethyl acetate/hexanes: 30/30 v/v, Rf=0.45)
 yielding tert-butyl 4-(hydroxymethyl) 1H-1-indolecarboxylate 6 (14.5 g,
 88%) as a light yellow solid: mp. 62.9-64.1.degree. C. .sup.1 H-NMR
 (CDCl.sub.3) .delta. 8.09 (d, J=8.1 Hz 1H), 7.6 (d, J=3.7 Hz, 1H), 7.28
 (t, J=7.5 Hz, 1H), 7.20 (d, J=7.3 Hz, 1H), 6.69 (d, J=3.7 Hz, 1H), 4.90
 (s, 2H), 2.01 (s, 1H), 1.67 (s, 9H); .sup.13 C-NMR (CDCl.sub.3) .delta.
 149.7, 135.3, 132.7, 128.8, 125.9, 124.2, 121.2, 114.8, 105.2, 83.7, 63.4,
 28.1; IR (KBr, cm.sup.-1) 3364, 1732, 1130; MS (ESI): 248 (M.sup.+ +1).
 PBr.sub.3 (2.4 mL. 25.8 mmol) was added dropwise at 0.degree. C. under
 nitrogen to a stirred solution of tert-butyl 4-(hydroxymethyl)
 1H-1-indolecarboxylate 6 (6.0 g, 24.3 mmol) in ether (80 mL) and CH.sub.2
 Cl.sub.2 (20 mL) under nitrogen. The reaction was completed 30 minutes
 after the addition. The mixture was poured into a cold aqueous NaHCO.sub.3
 solution (100 mL) and extracted with ethyl acetate (3.times.80 mL). The
 combined extract was washed sequentially with water (80 mL), and brine (80
 mL), then dried (MgSO.sub.4), filtered, and finally concentrated to
 provide tert-butyl 4-(bromomethyl)-1H-1-indolecarboxylate 7 (6.3 g, 84%)
 as an oil which was immediately taken to the next step.
 n-BuLi (2.5 M in hexane) was added dropwise via syringe to a solution of
 (2R)-2-isopropyl-3,6-dimethoxy-1, 5-dihydropyrazine (3.8 g, 20.4 mmol) in
 THF (70 mL) under nitrogen at -78.degree. C. The carbanion was allowed to
 form for 10 minutes at the same temperature, at which point a solution of
 tert-butyl 4-(bromomethyl)-1H-1-indolecarboxylate 7 in THF (40 mL) was
 added in a dropwise fashion. The reaction proceeded to completion in one
 hour at -78.degree. C. Saturated aqueous NH.sub.4 Cl (80 mL) was added at
 -78.degree. C., and the THF was evaporated under reduced pressure. The
 aqueous phase was extracted with ethyl acetate (3.times.80 mL). The
 combined extracts were washed with brine (100 mL), dried (MgSO.sub.4), and
 concentrated. The residue was purified on silica gel column (ethyl
 acetate/hexanes: 5/95 then 10/90 v/v, Rf=0.60 for the major product) to
 afford tert-butyl
 4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-1H-1
 -indolecarboxylate 8a (5.8 g, 67%) as a colorless oil:
 [.alpha.].sub.D.sup.25 =+26.7 (c=14.8 mg/mL, CHCl.sub.3); .sup.1 H-NMR
 (CDCl.sub.3) .delta. 7.98 (d, J=8.2 Hz 1H), 7.54 (s, 3H), 7.17 (t, J=7.8
 Hz, 1H), 6.96 (d, J=7.3 Hz, 1H), 6.69 (d, J=4.0 Hz, 1H), 4.45-4.38 (m,
 1H), 3.69 (s, 3H), 3.61 (s, 3H), 3.45-3.20 (m, 3H), 2.15-2.05 (m, 1H),
 1.67 (s, 9H), 0.91 (d, J=6.8 Hz, 3H), 0.58 (d, J=6.8 Hz, 3H); .sup.13
 C-NMR (CDCl.sub.3) .delta.163.7, 162.2, 149.8, 134.9, 131.0, 130.0, 125.0,
 124.0, 121.8, 113.3, 106.5, 83.4, 60.1, 56.7, 52.3, 52.1, 36.9, 31.1,
 28.2, 18.9, 16.4; IR (KBr, cm.sup.-1) 1734, 1696, 1346, 1128; MS (ESI):
 414 (M.sup.+ +1).
 TFA (0.15 N, 24 mmol) was added to a solution of tert-butyl
 4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-1H-1
 -indolecarboxylate 8a (3.5 g, 8.6 mmol) in acetonitrile (95 mL). The
 mixture was purged with nitrogen and stirred for 12 hours at room
 temperature. The acetonitrile was evaporated, and the water phase
 extracted with CH.sub.2 Cl.sub.2 (5.times.60 mL). The combined extract was
 washed sequentially with water (3.times.100 mL), and brine (80 mL), and
 then dried (MgSO.sub.4). Filtration and evaporation of the solvent left
 tert-butyl 4-[(2S)-2-amino-3-methoxy-3-oxopropyl]-1H-1-indolecarboxylate 9
 (2.7 g, 98%) as a colorless oil: [.alpha.].sub.D.sup.25 =+17.6 (c=12.8
 mg/mL, CHCl.sub.3); .sup.1 H-NMR (CDCl.sub.3) .delta. 8.06 (d, J=8.3 Hz
 1H), 7.61 (d, J=3.7 Hz, 1H), 7.26 (t, J=7.6 Hz, 1H), 7.06 (d, J=3.7 Hz,
 1H), 3.84 (dd, J=5.1, 8.1 Hz, 1H), 3.70 (s, 3H), 3.36 (dd, J=5.1, 13.5 Hz,
 1H), 3.06 (dd, J=8.2, 13.6 Hz, 1H), 1.67 (s, 9H), 1.46 (s 2H); .sup.13
 C-NMR (CDCl.sub.3) .delta. 175.4, 149.7, 135.2, 130.2, 129.5, 125.8,
 124.3, 114.0, 105.3, 83.7,55.5, 52.0, 38.5, 28.1; IR (neat, cm.sup.-1)
 3383, 1732, 1346,1155; MS (ESI): 319 (M.sup.+ +1).
 LiOH.H.sub.2 O (980 mg, 26 mmol) dissolved in H.sub.2 O (100 mL) at room
 temperature was added to a solution of tert-butyl
 4-[(2S)-2-amino-3-methoxy-3-oxopropyl]-1H-1-indolecarboxylate 9 (2.7 g,
 8.5 mmol) in THF (200 mL). The reaction was (close monitoring by TLC)
 judged complete after 10 minutes. After neutralizing with 1N HCl (30 mL),
 the THF and most of the water were evaporated in vacuo. The precipitated
 product N.sup.ind -t-Boc neo-tryptophan
 ((2S)-2-amino-3-[1-(tert-butoxycarbonyl)-1H-4-indolyl]propanoic acid) 10
 (1.8 g, 62%) was collected by filtration, and dried over P.sub.2 O.sub.5
 under high vacuum: mp. 169.5-171.2.degree. C. (dec).
 [.alpha.].sub.D.sup.25 =-9.85 (c=6.6 mg/mL, EtOH); .sup.1 H-NMR
 (DMSO-d.sub.6) .delta. 7.89 (d, J=8.2 Hz 1H), 7.69 (d, J=3.8 Hz, 1H), 7.27
 (t, J=7.7 Hz, 1H), 7.14 (d, J=7.5 Hz, 1H), 6.96 (d, J=3.8 Hz, 1H), 3.99
 (t, J=6.6 Hz, 1H), 3.45-3.26 (m, 2H), 1.63 (s, 9H); .sup.13 C-NMR
 (DMSO-d.sub.6) .delta. 170.3, 149.1, 134.6, 130.0. 128.0, 126.0, 124.3,
 123.8, 113.7, 105.8, 83.8, 53.3, 33.5, 27.6; IR (KBr, cm.sup.-1) 3432,
 3179, 1734, 1603, 1051; MS (ESI): 305 (M.sup.+ +1).
 A mixture of N.sup.ind -t-Boc neo-tryptophan 10 (1.8 g, 5.28 mmol) in 10%
 aqueous NaHCO.sub.3 (30 mL) was stirred for one hour at room temperature.
 After adding a solution of Fmoc-Suc (1.9 g, 5.55 mmol) in acetone (30 mL)
 to this mixture, the resulting mixture was stirred for 12 hours at room
 temperature. Acetone was evaporate under reduced pressure. The aqueous
 phase was acidified to pH 5 with 1N HCl, and extracted with ethyl acetate
 (3.times.60 mL). The combined extracts were washed with brine (80 mL),
 then dried (Na.sub.2 SO.sub.4), and concentrated. The resulting residue
 was purified on silica gel (MeOH/CH.sub.2 Cl.sub.2 : 5/95 v/v. Rf=0.3) as
 a white solid yielding the Fmoc/Boc derivative of neo-tryptophan
 ((2S)-3-[1-(tert-butoxycarbonyl)-1H-4-indolyl]-2-{[(9H-fluorenylmethoxy)ca
 rbonyl]amino} propanoic acid) 1b: mp. 92.1-93.8.degree. C.
 [.alpha.].sub.D.sup.25 =+5.5 (c=3.6 mg/mL, HCCl.sub.3); .sup.1 H-NMR
 (DMSO-d.sub.6) .delta. 7.93 (d, J=8.2 Hz 1H), 7.87 (d, J=7.6 Hz, 2H), 7.78
 (d, J=8.7 Hz, 1H), 7.67 (d, J=6.0 Hz, 1H), 7.59 (dd, J=7.5 10.6 Hz, 2H),
 7.45-7.36 (m, 2H), 7.36-7.19 (m, 3H), 7.15 (d, J=7.2 Hz, 1H), 6.85 (d,
 J=3.6 Hz, 1H), 4.34-4.23 (m, 1H), 4.23-4.10 (m, 2H), 3.41-3.32 (m, 2H),
 3.20-3.08 (m, 1H), 1.62 (s, 9H); .sup.13 C-NMR (DMSO-d.sub.6) .delta.
 173.4, 156.0, 149.2, 143.9, 140.8, 134.6, 130.7, 129.9, 127.8, 127.2
 126.0, 125.4, 124.3, 123.6, 120.2, 113.3, 105.8, 84.0, 65.8, 55.2, 46.6,
 34.2, 27.8; IR(KBr, cm.sup.-1) 3308, 1703, 1346, 1128; MS (ESI): 563
 (M+K.sup.+), 549 (M+Na.sup.+).
 A solution of N.sup.ind -t-Boc neo-tryptophan 10 (10 mg, 0.03 mmol) in TFA
 (1 mL) and CH.sub.2 Cl.sub.2 (2 mL) was stirred for 90 minutes. Solvent
 was evaporated under reduced pressure. The residue was purified on reverse
 phase HPLC on a Vydak C.sub.8 column (15-20 .mu.m particle size,
 250.times.22 mm i.d) using a gradient of 10% B to 90% B in 30 minutes
 (buffer A: 0.1% TFA in H.sub.2 O; buffer B: 80% CH.sub.3 CN in buffer A;
 UV detection at .lambda..sub.max 220 nm; Flow rate 8 mL/min) to give
 neo-tryptophan ((2S)-2-amino-3-(1H-4-indolyl)propanoic acid) 1a as a
 trifluoroacetate salt: mp. 110.0-111.8.degree. C. [.alpha.].sub.D.sup.25
 =+31.8 (c=1.1 mg/mL, H.sub.2 O); .sup.1 H-NMR (DMSO-d.sub.6) .delta. 11.22
 (s, 1H), 8.27 (s, 3H), 7.42-7.34 (m, 2H), 7.04 (t, J=7.6 Hz 1H), 6.87 (d,
 J=7.1 Hz, 1H), 6.35 (s, 1H) 4.17 (s, 1H), 3.41-3.26 (m, 2H); IR (KBr,
 cm.sup.-1) 3399, 1736; MS (ESI): 205 (M.sup.+ +1).
 Example 2
 Neurotensin Receptor Binding Properties of neo-tryptophan-containing
 Polypeptides
 CHO-K1 cells were stably transfected with nucleic acid encoding either the
 human NTR1 or the rat NTR1, and cultured in 150 mm petri plates with 35 mL
 of Dulbecco-modified Eagle's medium containing 100 .mu.M minimal essential
 medium nonessential amino acids (GIBCO) supplemented with 5% (v/v)
 FetalClone II bovine serum product (Hyclone Labs, Logan, Utah). CHO cells
 (subculture 9-19) were harvested at confluency. Briefly, the medium from
 each plate was removed by aspiration, and the cells washed with 6 mL of 50
 mM Tris-HCl (pH 7.4) and resuspended in 5-10 mL of Tris-HCl by scraping
 the cells with a rubber spatula. The resuspended cells were placed into a
 centrifuge tube and collected by centrifugation at 300.times.g for five
 minutes at 4.degree. C. in a GPR centrifuge (Beckman Instruments,
 Fullerton, Calif.). The cellular pellet (in 50 mM Tris-HCl, 1 mM EDTA, pH
 7.4) was stored at -180.degree. C. until radioligand binding was
 performed.
 For binding assays, crude membranal preparations were prepared by
 centrifugation of the cellular pellet at 35,600.times.g for ten minutes.
 The supernatant was decanted and discarded, and the cellular pellet was
 resuspended in 2 mL of Tris-HCl, 1 mM EDTA (pH 7.4) followed by
 homogenization with a Brinkmann Polytron at setting 6 for ten seconds.
 Centrifugation was repeated as above and the supernatant was decanted and
 discarded. The resulting final cellular pellet was resuspended in 50 mM
 Tris-HCl, 1 mM EDTA, 0.1% bovine serum albumin, and 0.2 mM bacitracin.
 Polypeptide concentration of the membranal preparation was estimated by
 the method of Lowry et al. (J. Biol. Chem. 193:265-275 (1951)) using
 bovine serum albumin as a standard.
 A Biomek 1000 robotic workstation was used for all pipetting steps in the
 radioligand assays as previously described (Cusack and Richelson, J.
 Recept. Res. 13:123-34 (1993)). Competition binding assays with [.sup.3
 H]NT (1 nM), varying concentrations of unlabeled NT, and polypeptide
 analogs were carried out with membranal preparations from the appropriate
 cell lines. Nonspecific binding was determined with 1 .mu.M unlabeled NT
 in assay tubes with a total volume of 1 mL. Incubation was at 20.degree.
 C. for 30 minutes. Each reaction was terminated by addition of cold 0.9%
 NaCl (5.times.1.5 mL) followed by rapid filtration through a GF/B filter
 strip that had been pretreated with 0.2% polyethylenimine. Details of
 binding assays are described elsewhere (Cusack et al., Mol. Pharmacol.
 44:1036-1040 (1993)). The data were analyzed using the LIGAND program
 (Munson and Rodbard, Anal. Biochem. 107:220-239 (1980)). The values
 presented for K.sub.d are expressed as the geometric means .+-.SEM
 (Fleming et al., J. Pharmacol. Exp. Ther. 182:339-345 (1972) and DeLean et
 al., Mol. Pharmacol. 21:5-16 (1982)).
 Radioligand binding assays were performed using various NT analogs. In each
 case, the equilibrium dissociation constant (K.sub.d) was derived for both
 human NTRl and rat NTR.sub.1 (Table II). All polypeptides tested had a
 Hill Coefficient close to unity, indicating that binding was to a single
 class of receptors. Substituting L-neo-tryptophan for Tyr.sup.11 in NT
 (8-13) resulted in the most potent compound (NT64L) tested at the human
 receptor, and nearly the most potent tested at the rat receptor. In fact,
 the binding affinity of NT64L was in the range of that found for [L-3,
 1'-Nal.sup.11 ]NT(8-13) (NT34) at the rat receptor. NT72, a pentapeptide,
 was found to be the least potent at both receptors. While substituting
 D-Lys for L-Arg.sup.8 in NT64L resulted in a polypeptide (NT67L)
 exhibiting greater resistance to peptidase degradation than NT64L, the
 NT67L polypeptide exhibited a binding affinity (K.sub.d =0.61 nM) about
 six fold lower at the human receptor than that exhibited by NT64L (K.sub.d
 =0.09 nM). In addition, steric factors appear to influence NT receptor
 binding since the results revealed a more than 30 fold reduction in
 binding affinity for NT64D, which contains the D-isomer of neo-tryptophan,
 and for NTW, which contains the natural isomer of tryptophan, when
 compared to NT64L.
 TABLE II
 Comparison of binding affinity for NT analogs at human and
 rat NT receptors.
 K.sub.d [nM]
 Polypeptide hNTR rNTR
 NT64L 0.09 .+-. 0.01 (3) 0.10 .+-. 0.01 (5)
 NT(8-13) 0.14 .+-. 0.01 (4) 0.16 .+-. 0.01 (3)
 NT65L 0.32 .+-. 0.01 (5) 0.075 .+-. 0.004 (3)
 NT67L 0.61 .+-. 0.06 (3) 0.21 .+-. .02 (10)
 NT2 1.0 .+-. 0.1 (6) 0.8 .+-. 0.1 (3)
 NT69L 1.55 .+-. 0.09 (5) 0.82 .+-. 0.07 (4)
 NT71 1.8 .+-. 0.1 (4) 0.22 .+-. 0.03 (8)
 NT(1-13) 1.97 .+-. 0.07 (130) 2.39 .+-. 0.08 (99)
 NTW 3.2 .+-. 0.3 (3) 0.34 .+-. 0.03 (3)
 NT64D 3.3 .+-. 0.4 (3) 3.8 .+-. 0.4 (3)
 NT66L 3.7 .+-. 0.4 (3) 0.85 .+-. 0.09 (14)
 NT34 5.8 .+-. 0.6 (4) 0.046 .+-. 0.003 (3)
 NT(tert-Leu) 13.2 .+-. 0.5 (4) 19.8 .+-. 0.4 (4)
 NT(9-13) 30 .+-. 2 (3) 46 .+-. 4 (3)
 NT75 34 .+-. 1 (5) 10.0 .+-. 0.4 (5)
 NT73 45 .+-. 3 (3) 32 .+-. 6 (6)
 Eisai 95 .+-. 9 (12) 5.4 .+-. 0.6 (8)
 NT66D 210 .+-. 20 (10) 77 .+-. 9 (8)
 NT74 360 .+-. 10 (3) 160 .+-. 40 (3)
 NT72 640 (2) 270 .+-. 30 (5)
 Values are geometric mean .+-. SEM, n value is in parenthesis; K.sub.d =
 equilibrium dissociation constant in CHO-K1 membranes; n.d. = no data.
 In general, substitution of L-Ile.sup.12 with tert-Leu, a substitute for
 the natural amino acid leucine, lowered the binding affinity of the NT
 analogs when compared to their counterparts containing L-Ile.sup.12. For
 example, NT(tert-Leu) was about 100 fold less potent at both the human
 (K.sub.d =13.2 nM) and rat (K.sub.d =19.8 nM) receptors than was NT(8-13)
 (K.sub.d =0.14 nM and 0.16 nM at human and rat receptors, respectively). A
 smaller decrease (3 fold) in binding affinity resulted when L-Ile.sup.12
 was replaced with tert-Leu as observed between NT64L and NT65L.
 The Eisai compound was found to be almost 20 fold weaker at the human NT
 receptor when compared to its binding affinity at the rat receptor.
 Substituting L-Trp.sup.11 in Eisai with L-neo-tryptophan to give NT69L
 resulted in a 60 fold increase in binding affinity at the human receptor,
 but only a 6 fold increase at the rat receptor.
 Other modifications included substitutions in the sequence of NT(9-13).
 Briefly, NT(9-13) was found to have low affinity for NT receptors. In
 fact, NT(9-13) was over 200 fold weaker at the human and rat receptors
 than was NT(8-13). Of the pentapeptides tested (NT72, NT73, NT74, and
 NT75), NT75 was found to be the most potent (K.sub.d =34 nM and 10 nM at
 the hNTR1 and rNTR1, respectively). These pentapeptides, however, were not
 more potent than NT(9-13). Again, substitution of Ile.sup.12 with tert-Leu
 caused a several fold reduction in binding affinity as observed when NT75
 is compared to NT74, and when NT73 is compared to NT72.
 The K.sub.d values obtained using the human NT receptor for the various NT
 analogs were plotted against the respective K.sub.d values obtained using
 the rat receptor (FIG. 5). This analysis revealed a strong correlation
 (R=0.88, P&lt;0.0001) between the binding affinity at human and rat NT
 receptors. In addition, the line of identity (dotted line having slope=1)
 revealed that most NT analogs have a higher binding affinity for the rat
 receptor than for the human receptor, while some have a similar binding
 affinity for both receptors. No NT analog exhibited a higher binding
 affinity for human NTR1 than for rat NTR1.
 In summary, every tested NT analog containing L-neo-tryptophan exhibited an
 increase in binding affinity over similar NT analogs (e.g., NTW vs. NT64L,
 NT2 vs. NT67L, and Eisai vs. NT69L). Thus, the addition of
 L-neo-tryptophan contributed significantly to increasing the potency of NT
 analogs.
 Example 3
 PI Turnover Properties of neo-tryptophan-containing Polypeptides
 To measure PI turnover, intact CHO-K1 cells were harvested at about 80%
 confluency. Cells were detached from the petri plates by removal of
 culture medium followed by incubation of the cellular monolayer for 20
 minutes at 37.degree. C. with gentle shaking in a modified Puck's D.sub.1
 solution containing 2 mM EGTA. The details of assaying the relative
 changes in PI turnover in intact cells using a radioactively labeled
 precursor was described elsewhere (Pfenning and Richelson, In: Methods in
 Neurotransmitter Receptor Analysis Eds: Yamamura HI, SJ Enna and MJ Kuhar,
 pp. 147-175, Raven Press, New York (1990)). Briefly, intact CHO cells were
 prelabeled with D-myo-[.sup.3 H]inositol (18.3 Ci/mmol) in the presence of
 lithium chloride (final concentration, 10 mM). Cells were then stimulated
 with NT or the appropriate NT analog, and the amount of [.sup.3 H]inositol
 1-phosphate ([.sup.3 H]IP.sub.1) produced by the cells isolated
 chromatographically on Dowex 1-X8 (200-400 mesh). For the experiments
 described herein, the stimulation time was 30 minutes, and the number of
 CHO cells per assay tube was 1.5.times.10.sup.5. The presented EC.sub.50
 values are expressed as the geometric means .+-.SEM (Fleming et al., J.
 Pharmacol. Exp. Ther. 182:339-345 (1972) and DeLean et al., Mol.
 Pharmacol. 21:5-16 (1982)).
 Each tested NT analog was finctionally coupled to PI turnover as determined
 using intact CHO-K1 cells (Table III). The most potent NT analog tested at
 the human NTR1 was NT67L (EC.sub.50 =0.83 nM). Substituting Ile.sup.12 in
 NT67L with tert-Leu to give NT66L did not improve potency at the human
 NTR1 (EC.sub.50 =10 nM). For NT69L, an EC.sub.50 of 2.3 nM and 1.3 nM at
 the hNTR1 and rNTR1, respectively, was observed.
 TABLE III
 Comparison of PI turnover results for NT analogs at human and
 rat NT receptors.
 PI Turnover EC.sub.50 [nM]
 Polypeptide hNTR rNTR
 NT64L 2.8 .+-. 0.5 (4) 2.3 .+-. 0.2 (3)
 NT(8-13) 1.5 .+-. 0.1 (3) n.d.
 NT67L 0.83 .+-. 0.09 (3) 13 .+-. 2 (3)
 NT69L 2.3 .+-. 0.5 (3) 1.34 .+-. 0.02 (3)
 NT(1-13) 5.0 .+-. 0.3 (39) 6.8 .+-. 0.4 (10)
 NTW 110 .+-. 20 (5) n.d.
 NT66L 10 .+-. 2 (4) 1.9 .+-. 0.4 (3)
 NT34 130 .+-. 20 (3) 2.8 .+-. 0.2 (4)
 NT(9-13) 380 .+-. 50 (3) n.d.
 Eisai 300 .+-. 20 (4) 3.1 .+-. 0.4 (3)
 Values are geometric mean .+-. SEM, n value is in parenthesis; EC.sub.50 =
 concentration of compound needed to stimulate 50% of maximum PI response
 in intact CHO-K1 cells; n.d. = no data.
 In general, the EC.sub.50 values observed for the various tested analogs
 were similar at rat NTR1 (in the 1-13 nM range). At the human NT receptor,
 however, the EC.sub.50 values were quite different (in the 1-300 nM range
 for the hexapeptides tested). These results may reflect the size of the
 binding site at the human NTR1 and the conformation of the NT
 analog-receptor complex, with the human receptor being much less tolerant
 of size and steric changes of the ligand. In addition, the Eisai analog
 was nearly 100 fold weaker at the human NT receptor than at the rat NT
 receptor (EC.sub.50 of 300 nM at human NTR1, and 3.1 nM at rat NTR1). This
 difference was less striking than that found for the respective affinities
 in radioligand binding experiments with membranal preparations from these
 cells (Table II). In addition, comparing results obtained using the Eisai
 analog to those obtained using NT69L revealed an improvement for NT69L in
 the potency at PI turnover of about 130 fold at human NTR1 and about two
 fold at rat NTR1. Thus, these results indicate that the addition of
 L-neo-Trp to NT analogs significantly influences their pharmacology and
 biochemistry at human NTR1.
 Example 4
 Degradation Properties of neo-tryptophan-containing Polypeptides
 The following experiments determined the stability of the novel
 polypeptides containing neo-tryptophan in rat and human plasma as well as
 rat intestinal preparations. Whole blood was collected into tubes
 containing heparin (200 units/mL), and placed on ice. Samples were spun at
 500.times.g for ten minutes. The supernatant was recovered and frozen at
 -20.degree. C. overnight. The samples were thawed at room temperature and
 spun at 500.times.g for ten minutes. After recovery, the supernatant was
 filtered through 0.2 .mu.m syringe filter.
 For degradation studies, ultrapure water that had been filtered twice
 through 0.02 .mu.m filters was used. Each polypeptide to be tested was
 resuspended in this water at a concentration of 1 mg/mL. The filtered
 plasma was diluted 1:1 with the filtered H.sub.2 O (50% v/v). Then, 500
 .mu.L of diluted plasma was combined with 500 .mu.L of the polypeptide
 solution (1 mg/nL), and vortexed. The final concentration of polypeptide
 was 0.5 mg/nl in 25% plasma (v/v).
 An initial time point was taken at 0.degree. C. before placing the
 plasma/polypeptide sample in a 37.degree. C. water bath. At each time
 point, 50 .mu.L of the plasma/polypeptide sample was removed and combined
 with 1.050 mL of filtered H.sub.2 O, representing a 1:22 dilution. Thus,
 the final amount of material injected into the HPLC represents 22.7 .mu.g
 of polypeptide in 1.14% plasma. Peak area was recorded at each time point
 and compared to the plasma/polypeptide sample at zero time at 0.degree. C.
 Values are expressed as the percent of peak area at zero time.
 The HPLC conditions were as follows: C-18 column; flow rate equal to 3
 mL/minute; and the gradient equal to 10-90% B in 50 minutes, where A=TFA,
 0.1% and B=TFA 0.1% in 80% acetonitrile.
 For intestinal preparations, rats were sacrificed by decapitation, and the
 small intestines removed. The tissue was washed with ice-cold PBS (10 mM,
 pH 7.4). About 2 mg of intestinal tissue was added to about 10 mL of PBS
 and homogenized using a Brinkman polytron homogenizer (setting 6). After
 homogenization, 10 mL of PBS was added to 10 mL of the intestinal
 homogenate. The mixture was then centrifuged at 500.times.g for 15 minutes
 at 4.degree. C. The supematant was removed and stored at -20.degree. C.
 until testing. Immediately before incubation with the polypeptide, the
 intestinal preparation was filtered through a 0.2 .mu.m filter. Testing
 procedures were similar to those for the plasma tests.
 For the degradation studies, data were plotted in linear form as a semi-log
 plot with SigmaPlot for Windows Version 4.00. Linear regression on these
 plots provided the parameters used to calculate the half-time (t.sub.1/2)
 for degradation of each polypeptide. The correlation coefficient (R) for
 the linear regression was taken as a measure of the goodness-of-fit.
 L-neo-tryptophan provided increased resistance to peptidases. The half-life
 of NT2 in human plasma was determined to be about 0.85 hours (Table IV).
 Substituting L-Tyr.sup.11 of NT2 with L-neo-tryptophan resulted in a
 polypeptide (NT67L) having a half-life in human plasma of about 96 hours.
 In addition, substituting L-Trp.sup.11 of Eisai with L-neo-tryptophan
 resulted in a polypeptide (NT69L) having a longer half-life in human
 plasma. These results demonstrate that the substitution of animo acid
 residues with neo-tryptophan can produce polypeptides having increases
 resistance to degradation.
 TABLE IV
 Half-life values for Neurotensin (NT) and NT analogs.
 Plasma Rat Intestinal
 Human Rat Preparation
 half-time half-time half-time
 Polypeptide (hours) R (hours) R (hours) R
 NT 1.9 0.99 5.4 1.0 0.04 1.00
 NT(8-13) n.d. 0.22 0.98 n.d.
 NT2 0.85 0.71 n.d. n.d.
 NT64L 1.4 0.96 n.d. n.d.
 NT65L 1.1 1.00 n.d. n.d.
 NT66L 170 0.94 350 0.94 n.d.
 NT67L 96 0.97 130 0.95 0.77 0.94
 NT69L 500 0.90 250 0.88 4.1 0.91
 NT71 n.d. 260 0.72 5.1 0.98
 NT72 3000 0.19 n.d. 54 0.99
 NT73 300 0.58 n.d. 0.83 0.99
 NT74 n.d. 110 1.00 n.d.
 NT75 n.d. 1.6 0.93 0.27 0.96
 Eisai 460 0.91 n.d. 22 0.96
 These degradation studies also revealed some difference between human and
 rat plasma. For example, NT had a half-life in human plasma of about
 one-third that observed in rat plasma. In general, however, the half-life
 times observed for the tested analogs correlated reasonably well between
 the human and rat plasma samples (slope=0.76, R=0.97, df=3, P=0.03). In
 human plasma, NT, NT2, NT64L, and NT65L were the most rapidly degraded.
 The remaining analogs tested all had significantly longer half-life times
 in human plasma. In addition, all hexapeptide NT analogs containing a
 substitution for Arg.sup.8 and all pentapeptide NT analogs containing a
 substitution for Arg.sup.9 were found to be substantially resistant to
 degradation. Similar results were obtained using the rat plasma.
 NT72 was found to be the most resistant to degradation by rat intestinal
 proteases, while NT67L, an analog relatively stable in plasma, was
 degraded very rapidly by rat intestinal proteases. Interestingly, NT69L
 was less stable in the rat intestinal preparation than was the Eisai
 analog. Again, NT69L was found to be more resistant to human plasma
 proteases than the Eisai analog. Substituting Ile.sup.12 of NT73 with
 tert-Leu resulted in a polypeptide analog (NT72) exhibiting a substantial
 increase of stability in the intestinal preparation when compared to NT73.
 In general, these results demonstrate that NT analogs having unnatural
 amino acid substitutions at positions 8 and 12 for the hexapeptides and
 positions 9 and 12 for the pentapeptides are more resistant to degradation
 in plasma and in the rat intestinal preparation than NT analogs not having
 such substitutions.
 Example 5
 Antinociceptive and Hypothermic Properties of neo-tryptophan-containing
 Polypeptides
 Male Sprague Dawley rats (Harlan, Prattville, Ala.; 150-250 g) were house
 in a temperature controlled room with a 12 hour light/dark cycle, and
 given water and standard rat chow. Testing occurred during the light
 cycle. Hot plate measurements were performed to assess antinociception,
 while body temperature measurements were taken to assess hypothermia.
 The baseline hot plate measurements and body temperatures were determined
 immediately prior to each experiment. Briefly, the hot plate was performed
 to determine pain sensitivity. Thirty minutes after administration of the
 test compound, the rat was placed on the hot plate and latency was
 measured. Hot plate measurements were taken on a metal surface
 (15.times.20 cm) maintained at a temperature of 52.0.+-.0.15.degree. C.
 (Al-Rodhan et al., Brain Res. 557: 227-235 (1991), and Jensen and Yaksh,
 Brain Research 372:301-312 (1986)). The latency between the time the rat
 was placed on the surface and the time it licked either of its hind paws
 was measured. Failure to respond in 30 seconds resulted in ending of the
 trial and assignment of that latency. Animals were removed immediately
 after responding or at the cutoff latency. Hot plate tests were scored as
 the percent of maximum possible effect (% MPE) and calculated using the
 following equation: % MPE=[(post-drug latency-pre-drug
 latency)/(cut-off-pre-drug latency)].times.100; where the cut-off was 30
 seconds. Immediately after completion of the hot plate test, body
 temperature measurements were taken using a digital thermistor probe
 inserted into the rectum about 2-4 cm.
 For these behavioral and physiological measurements, data were tested for
 significance with a student's t-test and p&lt;0.05 was considered
 significant. Preference for the t-test instead of the ANOVA was given due
 to the reasons cited elsewhere (O'Brien PC, Biometrics 39:787 (1983)).
 For intraperitoneal (ip) delivery, the test compound was injected into the
 intraperitoneal cavity while control rats received an equal volume of
 saline (0.9% NaCl). For nasal delivery, the test compound was dissolved in
 4 .mu.L of sterile saline, and the rats lightly anaesthetized with
 CO.sub.2. The rats then were held in a vertical position while 2 .mu.L of
 the test compound were delivered to each nostril using a polyethylene gel
 loading tip attached to a Gilson P20 pipettor. The rats remained in a
 vertical position until it was clear that all the liquid had been inhaled
 into the nostril. After inhalation, the rats were allowed to recover from
 the anesthesia which usually occurred within one minute. For subcutaneous
 (sc) delivery, the test compound was injected into the fold of skin at the
 back of the neck. Injection volumes were about 100 .mu.L. For oral
 deliver, a gavage device attached to a syringe that extended into their
 stomachs was used to ensure complete delivery of the test compound. The
 volume delivered was about 0.3 mL.
 The following methods were used to deliver the test compounds directly into
 brain. Under sterile conditions, the rats were stereotaxically implanted
 with stainless steel guide cannulae (26 gauge) into the periaqueductal
 gray (PAG) region of the rat brainstem under sodium pentobarbital
 anesthesia (50 mg/kg, ip) as described in detail elsewhere (Jensen and
 Yaksh, Brain Res. 372:301-312 (1986), and Al-Rodhan, Brain Res.
 557:227-235 (1991)). The coordinates used for PAG cannulations are -5.6 mm
 posterior from bregma, 1.0 mm lateral from bregma, and 5.5 mm down from
 the dura. The guide cannula was pre-measured to be 5.5 mm (Plastics One,
 Roanoke, Va.) and the internal cannula was ordered to fit below the
 pedestal with a 2.0 mm projection. The guide cannula was then fixed to the
 skull using a stainless steel screw (1/8 inch) and cranioplastic cement. A
 stainless steel stilette was then placed in each guide to keep it patent
 and free of debris. Immediately after surgery, the animals were allowed to
 recover before returning them to an individual housing cage. All
 injections began 5-7 days after surgery. If any problem, such as an
 infection, was observed with an animal after cannulation, then the animal
 was euthanized immediately by decapitation.
 Intraperitoneal administration of NT64L (1 mg/kg) did not induce
 antinociception as measured by the hot plate test or hypothermia. When
 injected into the PAG, however, NT64L (18 nmol) induced both
 antinociception and hypothermia. Specifically, rats receiving NT64L
 exhibited a peak % MPE value of 76% at 30 minutes, and a peak body
 temperature reduction of 2.1.degree. C. at 30 minutes. NT and NT24 "27"
 also did not induce antinociception or hypothermia upon intraperitoneal
 administration. After PAG administration, rats receiving NT (18 nmol)
 exhibited a peak % MPE value of 80% at 30 minutes, and a peak body
 temperature reduction of 1.8.degree. C. at 30 minutes, while rats
 receiving NT24 "27" (18 nmol) exhibited a peak % MPE value of 20%, and a
 peak body temperature reduction of 1.1.degree. C. at 30 minutes.
 Intraperitoneal administration of NT66D, NT66L, NT67L, NT69L, NT71, NT72,
 NT73, and Eisai did induce antinociception and hypothermia (Table V).
 TABLE V
 Antinociception and hypothermia after intraperitoneal
 administration of NT analogs.
 1 mg/kg 5 mg/kg
 Peak BT Peak % Peak BT Peak % ED.sub.50 ED50
 Polypeptide change MPE change MPE BT % MPE
 Eisai -2.9 @ 80% @ n.d. n.d. 0.26 mg/kg 0.42 mg/kg
 60 min 90 min @30 min @ 30 min
 0.12 mg/kg 0.08 mg/kg
 @90 min @ 90 min
 NT66D* -1.0 @ 58% @ n.d. n.d. n.d. n.d.
 30 min 30 min
 NT66L -3.0 @ 100% @ -5.0 @ 100% @ 0.45 mg/kg 0.04 mg/kg
 240 min 120 min 120 min 330 min @30 min @ 30 min
 0.18 mg/kg 0.02 mg/kg
 @ 60 min @ 60 min
 NT67L -1.8 @ 70% @ n.d. n.d. n.d. n.d.
 120 min 90 min
 NT69L -5.3 @ 100% @ n.d. n.d. 0.4 mg/kg 0.3 mg/kg
 300 min 300 min @ 90 min @ 90 min
 NT71 -2.0 @ 70% @ -2.4 @ 79% @ n.d. n.d.
 40 min 60 min 90 min 180 min
 NT72 n.d. n.d. -2.8 @ 100% @ n.d. n.d.
 180 min 120 min
 NT73 n.d. n.d. -0.9 @ 40% @ n.d. n.d.
 90 min 120 min
 *NT66D was administered at a dose of 0.5 mg/kg instead of 1 mg/kg.
 NT69L had potent and long lasting behavioral effects. Specifically, NT69L
 given intraperitoneally to rats at a dose of 1 mg/kg induced a significant
 reduction in body temperature, reaching a peak of -5.3.degree. C. This
 peak reduction of body temperature was reached about 90 minutes after
 administration and remained significant up to 300 minutes after treatment.
 In addition, NT69L produced significant and long lasting antinociception.
 Specifically, a peak % MPE value of 100% was observed for up to about 200
 minutes after treatment. Analysis of the time course for NT69L-induced
 antinociception and hypothermia revealed that the peak antinociception
 effect (100% MPE) remained for almost three hours after administration
 while the hypothermia effect began to recover from the peak value of
 -5.degree. C. at about 90 minutes. One possible explanation for these time
 course differences is the existence of different NT receptor subtypes that
 subserve these two different behavioral responses.
 The results from a dose response analysis using NT69L indicated that the
 ED.sub.50 value for body temperature lowering (hypothermia) was 0.4 mg/kg
 at 90 minutes, while the ED.sub.50 value for % MPE (antinociception) was
 0.3 mg/kg at 90 minutes. For NT66L, the ED.sub.50 value was found to be
 0.45 mg/kg at 30 minutes for hypothermia, and 0.04 mg/kg at 30 minutes for
 antinociception. For Eisai, the ED.sub.50 value was found to be 0.26 mg/kg
 at 30 minutes for hypothermia, and 0.42 mg/kg at 30 minutes for
 antinociception. Although the ED.sub.50 values for Eisai and the
 neo-tryptophan-containing NT analogs (NT66L and NT69L) may appear similar
 in degree, their effects were found to be very different. For hypothermia,
 animals treated with the Eisai compound exhibited a peak reduction in body
 temperature of about 3.degree. C. at about one hour. NT69L induced a
 5.3.degree. C. reduction in body temperature that was maintained for up to
 five hours. For antinociception, animals treated with the Eisai compound
 reached peak of about 80% MPE at 90 minutes. This peak level, however,
 started to drop shortly thereafter, returning to baseline levels at about
 six hours after administration. The antinociception induced by NT69L,
 however, was maintained at a significant level for up to five hours.
 The effectiveness of NT analogs administered by several routes also was
 analyzed. For NT69L, the antinociception and hypothermia results observed
 after subcutaneous administration were similar to those obtained after
 intraperitoneal administration with the exception that the observed
 effects after subcutaneous administration appeared to lag behind the
 effects observed after intraperitoneal administration. Nasal
 administration of NT69L also produced antinociception and hypothermia.
 Specifically, NT69L given nasally to rats at a dose of 5 mg/kg induced a
 reduction in body temperature, reaching a peak of -1.4.degree. C. at 30
 minutes, and induced antinociception with a peak % MPE value of 70% at 60
 minutes. Thus, the nasal administration of NT69L appeared to induce
 antinociception more effectively than hypothermia. In addition, oral
 administration of NT69L (20 mg/kg) induced a reduction in body
 temperature, reaching a peak of -0.63.degree. C. at 60 minutes, and
 induced antinociception with a peak % MPE value of 11% at 30 minutes.
 While the hypothermia response was significant after oral administration
 of NT69L, the antinociceptive effect was not. Oral administration of
 NT66L, however, induced significant antinociception and hypothermia.
 Specifically, oral administration of NT66L (20 mg/kg) induced a reduction
 in body temperature, reaching a peak of -1.4.degree. C. at 30 minutes, and
 induced antinociception with a peak %MPE value of 40% at 60 minutes.
 Example 6
 Interactions Between Brain Receptors and neo-tryptophan-containing
 Polypeptide
 In a radiolabeled competitive binding assay, NT64L, NT66L, and NT67L were
 found to compete with labeled ketanserin for binding at the 5HT.sub.2A
 receptor in human brain tissue (Table VI). Specifically, NT64L had a
 K.sub.d of 6.6 .mu.M at this receptor in a competition binding assay using
 [.sup.3 H]ketanserin as the radioligand. In addition, NT2, NT(8-13), and
 NT(9-13) were found to compete with labeled ketanserin for binding at the
 5HT.sub.2A receptor in human brain tissue. L-neo-tryptophan itself,
 however, did not compete with labeled ketanserin for binding at the
 5HT.sub.2A receptor. These results indicate that NT analogs can interact
 with serotonin recognition molecules.
 TABLE VI
 Binding affinities for serotonin receptors.
 Kd [nM]
 Human Brain Tissue
 Compound 5HT.sub.2A
 NT69L 34600
 NT66L 8700
 NT67L 7400
 NT(9-13) 6340
 NT2 6800
 L-neo-Trp &gt;100000
 NT64L 6600
 NT(8-13) 4400
 Serotonin 680
 Haloperidol 61
 Clozapine 9.1
 Additional radiolabeled competitive binding assays were designed to assess
 the interaction of NT analogs and other ligands with other various types
 of receptors such as adrenergic and dopamine receptors (Table VII). These
 studies revealed that NT67L can interact with adrenergic .alpha.1
 receptors having a K.sub.d value of 6.9 .mu.M. In addition, these studies
 revealed that L-neo-tryptophan itself does not bind to human NT receptors
 from CHO cells.
 TABLE VII
 Comparison of binding at different receptors.
 K.sub.d (nM)
 Human brain tissue
 Com- adrenergic Musca- CHO cells
 pound .alpha.1 .alpha.2 rinic Dopamine rNTR hNTR
 NT66L 38000 n.d. n.d. 56000 0.85 3.7
 NT67L 6900 &gt; &gt; &gt;100000 0.21 0.61
 100000 100000
 NT69L n.d. 58600 n.d. 13300 0.82 1.55
 L-neo- n.d. n.d. n.d. n.d. n.d. &gt;100000
 Trp
 Sero- n.d. n.d. n.d. n.d. &gt;100000 &gt;100000
 tonin
 Halo- 17 600 &gt;10000 2.6 &gt;100000 &gt;100000
 peridol
 Cloza- 19 16 8.5 211 &gt;100000 &gt;100000
 pine
 Example 7
 Neo-tryptophan-containing Polypeptides and CNS Stimulants
 Apomorphine was used to assess the ability of NT analogs to act as
 neuroleptics. Briefly, male Sprague-Dawley rats were pretreated
 intraperitoneally with either NT69L or saline only. Thirty minutes
 following pretreatment, the rats received a subcutaneous injection of
 apomorphine (600 .mu.g/kg). Occasionally, the effectiveness of NT69L was
 assessed by measuring antinociception and hypothermia just prior to
 apomorphine administration. Control animals included rats receiving NT69L
 followed by saline only, and rats receiving no treatment. The apomorphine
 was dissolved in oxygen-free boiled 0.9% NaCl solution containing 0.1%
 ascorbic acid and 0.1% metabisulfite to prevent oxidation. The volume of
 injection was 1 mL/kg under the loose skin at the back of the animal's
 neck. Immediately following injection, the rats were placed in cages for
 observation. About 5-10 minutes later, behavioral monitoring was initiated
 and lasted for one hour. Climbing episodes were measured by observing the
 number of times the rat moved up and down in a vertical position during a
 two minute observation period. Statistical analysis was done using the
 Student's t-test with P&lt;0.05 being considered significant. Graphs and
 ED.sub.50 data were generated using GraphPad Prism.RTM. software (version
 2.01, GraphPad Software, Inc., San Diego, Calif.).
 Rats receiving saline followed by apomorphine exhibited distinctive
 climbing, sniffing, and licking behaviors that lasted for about 60 minutes
 after treatment. Pretreatment with NT69L (1 mg/kg) 30 minutes before the
 apomorphine injection caused a long-lasting blockade of the climbing
 behavior. This NT69L pretreatment, however, did not influence the sniffing
 and licking behaviors induced by apomorphine. Control animals receiving no
 treatment or NT69L without apomorphine did no exhibit any of these
 behaviors. The ED.sub.50 as determined by non-linear regression analysis
 for NT69L at 75 minutes after injection of NT69L (45 minutes after
 injection of apomorphine) was 16 .mu.g/kg (95% confidence interval; 6.1 to
 44 .mu.g/kg; R=0.94). At no dose did NT69L affect the oro-facial
 stereotypies. Thus, NT69L was found to be an extremely potent compound
 capable of preventing the climbing behavior induced by apomorphine. These
 results indicate that NT69L may have clinical effects similar to those of
 an atypical neuroleptic. The site of the blockade of apomorphine-induced
 climbing by NT69L does not appear to be dopamine, serotonin, or adrenergic
 receptors since NT69L was found to have a weak affinity for dopamine
 D.sub.2 receptors as well as 5HT.sub.2A and .alpha..sub.2 -adrenergic
 receptors (Table VII).
 At 5 mg/kg (ip), a dose of NT69L that completely blocked the effects of the
 apomorphine-induced climbing behavior, NT69L caused a large reduction in
 body temperature. Specifically, a change in body temperature of about
 -3.degree. C. was observed 30 minutes after injection, while a change of
 about -4.degree. C. was observed at 77 minutes after injection. At 90
 minutes, the ED.sub.50 for NT69L-induced body temperature lowering was 390
 .mu.g/kg (95% confidence interval; 110 to 1400 .mu.g/kg, R=0.98).
 Injection of apomorphine alone caused a more modest reduction of body
 temperature than did NT69L. The hypothermic effects observed in animals
 receiving NT69L alone were not different from those observed in animals
 receiving the combination of NT69L and apomorphine. Thus, the effects of
 apomorphine and NT69L on body temperature were not additive. The fact that
 there was no additive effects of these compounds on hypothermia indicates
 a common site of action or a "ceiling" effect on body temperature
 lowering. In other words, NT69L may cause the maximum body temperature
 lowering possible in the animals.
 The following experiments were performed to assess the ability of NT69L to
 cause and influence catalepsy. Male Sprague-Dawley rats (150-250 g) or
 CD-1 mice (24-26 g) were housed in a temperature-controlled room in groups
 of five with free access to food and water. The animals were kept on a 12
 hour light/dark cycle, and all tests were performed during the light
 cycle. The test for catalepsy is well established and quite simple
 (Munkvad et al., Brain Behav. Evol. 1:89-100(1968)). Animals received one
 of three compounds intraperitoneally: NT69L, a typical neuroleptic
 (haloperidol), or an atypical neuroleptic (clozapine). Thirty minutes
 after injection, the animals were tested for catalepsy. For rats, the test
 involved simply placing the animal's fore paws on a suspended metal bar 10
 mm in diameter, 11 cm above the counter. The time elapsed until the
 animal's fore paws touch the counter was recorded. If the rat did not drop
 to the counter top by four minutes, the rat was removed from the bar. For
 mice, catalepsy was scored based on the time that the mice maintained a
 fixed rearing posture against the side of the cage (Adams et al., Proc.
 Natl. Acad. Sci. USA 94:12157-12161 (1997)). The cut-off time was 20
 seconds, and each animal was tested three times for each time point.
 NT69L at a dose that induced significant hypothermia (5 mg/kg; ip) had no
 effect on catalepsy scores in rats. At the same dose, NT69L caused no
 catalepsy in mice. As expected, however, haloperidol caused profound
 catalepsy at a dose (1 mg/kg) that had minimal effect on body temperature.
 In addition, the haloperidol administration did not influence the body
 temperature lowering effect of NT69L when both drugs were given to the
 same animal. The haloperidol-induced catalepsy was observed at 30 minutes
 and lasted for at least six hours. The ED.sub.50 value for
 haloperidol-induced catalepsy was found to be 130 .mu.g/kg (95% confidence
 interval: 280 to 520 .mu.g/kg, R=0.998) at 30 minutes; 260 .mu.g/kg (95%
 confidence interval: 130 to 530 .mu.g/kg, R=1.00) at 60 minutes, and 310
 .mu.g/kg (95% confidence interval: 42 to 2300 .mu.g/kg, R=1.00) at 180
 minutes.
 When animals were injected with NT69L (5 mg/kg; ip) 30 minutes before
 injecting haloperidol (1 mg/kg; ip), the rats did not exhibit significant
 cataleptic behavior. When animals were injected with NT69L (5 mg/kg; ip)
 30 minutes after injecting haloperidol (1 mg/kg; ip), the moderate
 catalepsy observed 30 minutes after the haloperidol treatment was reversed
 by 30 minutes after NT69L treatment. From the 30 minute time point after
 NT69L treatment, the rats no longer exhibited catalepsy, and remained
 non-cataleptic for up to four hours from the time of haloperidol
 injection. These results indicate that NT69L given before haloperidol
 blocks haloperidol-induced catalepsy, while NT69L given after haloperidol
 reverses the catalepsy induced by haloperidol.
 To determine the ED.sub.50 value of NT69L for the reversal of the
 cataleptic effects induced by haloperidol, animals were given haloperidol
 (120 .mu.g/kg; ip) followed by varying doses of NT69L (ip) 40 minutes
 later. Catalepsy was scored at 130 minutes. The ED.sub.50 value for NT69L
 at reversing the effects of haloperidol was found to be 260 .mu.g/kg (95%
 confidence interval: 180 to 370 .mu.g/kg, R=1.00). This ED.sub.50 value
 was not statistically different (two-tailed t-test, t=0.67, df=6, P=0.53)
 from the ED.sub.50 value (390 .mu.g/kg) determined for NT69L-induced
 hypothermia at 90 minutes. The ED.sub.50 value for the blockade by NT69L
 of the apomorphine-induced climbing behavior was significantly lower than
 the ED.sub.50 values both for reversing haloperidol-induced catalepsy
 (t=2.74, df=15, P=0.0152) and hypothermia (t=3.88, df=17, P=0.0012).
 Clozapine (a classical, atypical neuroleptic) was also tested for its
 ability to affect catalepsy caused by haloperidol. At a dosage of 20 mg/kg
 (ip), clozapine did not cause catalepsy. When haloperidol (1 mg/kg; ip)
 was injected 30 minutes before clozapine (20 mg/kg; ip), the animals
 exhibited cataleptic effects similar to those observed in animals
 receiving haloperidol alone. Pretreatment with clozapine followed by
 haloperidol, however, modulated the cataleptic effects of haloperidol.
 This modulation was not statistically significant. In addition, the change
 in body temperature for the animals receiving treatment with clozapine
 and/or haloperidol was in the range of -1.degree. C. to -2.degree. C. This
 temperature reduction range is markedly less than that observed in animal
 treated with NT69L alone.
 These results indicate that NT69L, but not clozapine, completely prevents
 catalepsy when given before haloperidol. These results also indicate that
 NT69L, but not clozapine, reverses haloperidol's cataleptic effects when
 given after haloperidol. Thus, NT69L may have neuroleptic properties in
 humans and may be useful in the treatment of extrapyramidal side effects
 caused by neuroleptics such as the irreversible tardive dyskinesia.
 Example 8
 Chronic Treatment with a neo-tryptophan-containing Polypeptide
 The effect of chronic injection of NT69L was tested. Rats were injected
 with NT69L (1 mg/kg; ip) daily, and tested for antinociception and
 hypothermia. Antinociception was measured using the hot plate test
 described herein. Rats exhibited 100% MPE and about 4 degrees body
 temperature lowering after the first injection. After the second injection
 on day 2, however, no analgesic effect was observed and the body
 temperature was lowered only 1.5 degrees. After the third injection on day
 3, there was still no analgesic effect and no body temperature lowering
 was detected. In addition, the rats exhibited catalepsy when injected with
 haloperidol after the third and fourth days of NT69L injection.
 To check if the rats had developed tolerance to NT69L, the rats were
 challenged with five times (5.times.) the dose of NT69L (5 mg/kg; ip)
 after four days of NT69L treatment at 1 mg/kg (ip). The rats exhibited a
 reduction in body temperature comparable to the reduction normally
 observed in naive rats injected with NT69L (1 mg/kg; ip) for the first
 time. The haloperidol-induced cataleptic effect, however, was not reversed
 upon administration of the 5.times. dose of NT69L to the chronically
 treated rats (four day treatment with 1 mg/kg NT69L; ip). In addition, no
 analgesic effect was observed in the chronically treated rats after
 challenge with the 5.times. dose of NT69L.
 The effect of chronic treatment with NT69L on the number of NT binding
 sites within brain was assessed. Rats were treated daily with NT69L (1
 mg/kg; ip) for four days. On day five, the rats were treated with 5 mg/kg
 (ip) NT69L, tested behaviorally, and then sacrificed. The PAG and rest of
 brain were dissected from the animal and used in NT binding analysis.
 Briefly, homogenates were prepared from freshly obtained PAG and the rest
 of brain of rats according to Goedert et al. (Brain Res. 304, 71-81
 (1984)) with the following modifications: the assay buffer contained the
 peptidase inhibitors 1,10 phenanthroline (1 .mu.M) and aprotonin (5
 mg/ml). For PAG and rest of brain binding assays, tissues were incubated
 with 0.3 nM [.sup.125 I]NT (NEN, Boston, Mass.) at room temperature for 30
 minutes. Total and nonspecific binding was measured and the binding sites
 were normalized to polypeptide concentrations by BCA protein determination
 (Pierce Chemical Co., Rockford, Ill.).
 Brain tissues from control rats exhibited more [.sup.125 I]NT binding than
 brain tissues from rats chronically treated with NT69L. Specifically, the
 PAG tissue from control rats contained 2.34 dpm/.mu.g protein (n=2), while
 the same tissue from NT69L-treated rats contained 1.89 dpm/.mu.g protein
 (n=5). Likewise, the rest of brain tissue from control rats contained 3.1
 dpm/.mu.g protein (n=2), while the same tissue from NT69L-treated rats
 contained 2.1 dpm/.mu.g protein (n=5). These results represent about a 20
 to 30 percent reduction in [.sup.125 I]NT binding for brain tissue from
 NT69L-treated animals.
 Example 9
 Weight Loss Properties of neo-tryptophan-containing Polypeptides
 Two groups of male Sprague-Dawley rats (small and large) and one group of
 genetically obese Zucker rats were used to study the influence of NT69L on
 various aspects of body weight. The group of small Sprague-Dawley rats
 weighed about 270 g at the beginning of the study, while the group of
 large Sprague-Dawley rats weighted about 400 g. Under normal conditions,
 the small rats exhibit steady growth, and the large rats do not. All
 animals were individually housed in a room with a 12 hour light/dark
 cycle. The rats had free access to commercial hard rat chow pellets and
 tap water. During the study, the rats received 100 .mu.L of either saline
 only or 1 .mu.g/kg, 10 .mu.g/kg, or 1 mg/kg of NT69L on days 1, 2, 7, 8,
 11, and 12. Food intake (g), water consumption (mL), and body weight (g)
 were recorded daily for 15 days. The results were presented as mean
 .+-.SEM, or as % of original weight. The data were compared by variance
 analysis (unpaired or paired Student's t test) and Rank sum test.
 NT69L caused a significant (P&lt;0.001) reduction in body weight gain when
 injected (ip) into small Sprague-Dawley rats at a dose of 1 .mu.g/kg and
 10 .mu.g/kg. The reduction in body weight gain was greatest one day after
 injection of NT69L. In addition, small rats failed to make-up for the
 reduction in body weight gain even after the NT69L administration was
 discontinued. Specifically, small rats receiving saline only exhibited a
 29% increase in body weight by the end of 15 days, while small rats
 receiving 1 .mu.g/kg NT69L exhibited only an 9.0% increase from their
 original body weight at day 15. Small rats receiving 10 .mu.g/kg NT69L
 exhibited an 8.4% increase from their original body weight at day 15. Food
 intake for the small rats was significantly (P&lt;0.003) less than the food
 intake observed for saline treated control animals throughout the
 experiment, indicating that the observed reduction in body weight gain
 after injection of NT69L is attributable in part to less food intake.
 The large Sprague-Dawley rats injected (ip) with 1 mg/kg, but not with 1
 .mu.g/kg or 10 .mu.g/kg, exhibited a significant reduction in body weight
 (P&lt;0.003). Specifically, the large rats receiving 1 mg/kg NT69L exhibited
 a 3.0% reduction in their original body weight, while saline treated
 control animals exhibited a 2.4% increase in their original body weight by
 day 15. In addition, food intake for the large rats was significantly
 (P&lt;0.003) less than the food intake observed for saline treated control
 animals during the one to two days post NT69L injection. These results
 indicate that the observed reduction in body weight in NT69L-treated
 animals is attributable in part to less food intake.
 The genetically obese Zucker rats injected (ip) with NT69L (1 mg/kg) also
 exhibited a significant reduction in weight gain (P&lt;0.009). Specifically,
 NT69L-treated animals exhibited a 25% increase in their original body
 weight, while saline-treated control animals exhibited a 31% increase in
 their original body weight by day 15. Again, food intake by NT69L-treated
 animals was significantly reduced (P&lt;0.01) during the one to two days post
 NT69L injection as compared to the food intake of saline-treated control
 animals. These results demonstrate the potent effect of NT69L on (1) body
 weight gain reduction, (2) body weight loss, and (3) appetite when
 injected (ip) for two consecutive days at four to five day intervals.
 The effect of NT69L on blood hormone levels was assessed. Briefly,
 Sprague-Dawley rats were injected (ip) with either saline (n=20) or 1
 mg/kg NT69L (n=20). Five NT69L-treated rats and five control rats were
 sacrificed by decapitation at one, four, eight, and twenty-four hours
 post-injection. Brains were harvested and dissected on ice. The different
 brain sections were kept on dry ice for HPLC analysis. In addition, blood
 was collected in cold centrifuge tubes with heparin and kept on ice. After
 collection, the blood was centrifuged at 2500 rpm for ten minutes, and the
 plasma was collected and stored at -20.degree. C. until analysis. Glucose,
 thyroxine (T4), thyroid stimulating hormone (TSH), and corticosterone
 levels were determined by enzyme assay or RIA.
 Rats treated with NT69L (1 mg/kg; ip) exhibited a significant increase in
 blood glucose (P&lt;0.005) and corticosterone (P&lt;0.001) levels as compared to
 the level observed in saline-treated controls. Specifically, NT69L-treated
 animals had a glucose level of 221 mg/dL and a corticosterone level of
 24.6 .mu.g/dL, while saline-treated animals had a glucose level of 130
 mg/dL and a corticosterone level of 6.1 .mu.g/dL at one hour post
 injection. In addition, rats treated with NT69L (1 mg/kg; ip) exhibited a
 significant reduction in TSH (P&lt;0.001) and T4 (P&lt;0.02) levels as compared
 to the level observed in saline-treated controls. Specifically,
 NT69L-treated animals had a TSH level of 0.9 mIU/L and a T4 level of 1.8
 .mu.g/dL, while saline-treated animals had a TSH level of 7.65 mIU/L and a
 T4 level of 2.6 .mu.g/dL at one hour post injection. By 24 hours
 post-injection, the levels of blood glucose, corticosterone, TSH, and T4
 had returned to the levels observed in control animals, indicating that
 the hyperglycemia as well as the inhibitory effect of thyroid function due
 to NT69L were only transitory. The Zucker rats exhibited a similar
 increase in blood glucose (310 mg/dL for the NT69L-treated vs. 140 mg/dL
 for the control) and corticosterone (19.7 .mu.g/dL for the NT69L-treated
 vs. 10.9 .mu.g/dL for the control) levels, and a reduction in TSH (0.43
 mIU/L for the NT69L-treated vs. 1.9 mIU/L for the control) and T4 (1.03
 .mu.g/dL for the NT69L-treated vs. 1.47 .mu.g/dL for the control) levels
 one hour after injection (ip) of NT69L (1 mg/kg).
 Other Embodiments
 It is to be understood that while the invention has been described in
 conjunction with the detailed description thereof, the foregoing
 description is intended to illustrate and not limit the scope of the
 invention, which is defined by the scope of the appended claims. Other
 aspects, advantages, and modifications are within the scope of the
 following claims.