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Title:  Method and pharmaceutical composition for prevention and treatment of brain damage

United States Patent:  6,680,295

Issued:  January 20, 2004

Inventors:  Arimura; Akira (New Orleans, LA)

Assignee:  The Administrators of the Tulane Educational Fund (New Orleans, LA)

Appl. No.:  809500

Filed:  May 21, 1997

PCT Filed:  September 21, 1995

PCT NO:  PCT/US95/12057

PCT PUB.NO.:  WO96/09064

PCT PUB. Date:  March 28, 1996

Abstract

The present invention relates to methods and pharmaceutical preparations for treating or preventing neuronal cell damage in the brain and other tissues in mammals, comprising administering an effective amount of a PACAP, or an agonist, analog or derivative thereof having PACAP neurotrophic activity, in a pharmaceutically acceptable carrier, in a concentration which is effective for protection of neuronal nerve cells in vivo.

SUMMARY OF THE INVENTION

The present invention relates to a method and pharmaceutical preparations for treating or preventing neuronal cell damage in mammals, comprising administering a effective amount of a PACAP, or an agonist, analog or derivative thereof having PACAP neurotrophic activity, in a pharmaceutically acceptable carrier, in a concentration which is effective for protection of neuronal nerve cells in vivo.

It has now been discovered that, unexpectedly, although PACAP is extremely effective in protecting and/or resuscitating neuronal cells, there is a rather narrow window of concentrations of PACAP which provide such results, i.e., the effectiveness of the treatment falls off rapidly both above and below that concentration range. Thus the present invention involves a method of treatment of mammalian neuronal cells in which the concentration of the PACAP compound is between about 10-15 and 10-12 M in the tissues. Even more unexpectedly, it has been discovered that within the generally effective concentration range of the PACAP pharmaceuticals of this invention, there are two sub ranges of concentration, in each of which there is a peak effectiveness, above and below which the effectiveness of the composition falls off to a significant degree. As shown in FIG. 8, the preferred concentration range for treatment with the PACAP compounds of the present invention lies between about 10-14 and about 10-12 M and another range of concentration lies between about 10-11 and about 10-9 M. The preferred concentration range for treatment is the range between about 10-14 and about 10-12 M in the tissue, which permits treatment of the subject with minimal risks of side effects from the treatment. The present discovery makes possible the use of such PACAP pharmaceuticals in extremely low concentrations to provide very substantial protection of neuronal cells, such as brain cells, from death due to transient ischemia, reperfusion, toxic substances, trauma, or other causes.

Pharmaceutical compositions in accordance with the present invention include PACAP, in either of its forms, commonly referred to as PACAP38 and PACAP27, as well as any peptide or non-peptide agonist for PACAP receptors, especially agonists for the Type I PACAP receptor. Preferably the PACAP compound is a polypeptide, or a salt or derivative thereof, which contains at least twelve amino acids joined in a sequence corresponding to a part of the sequence shown for PACAP38 in FIG. 1, and which binds to at least one receptor which binds to PACAP. As used herein a "PACAP12 agonist" refers to a polypeptide, or salt or derivative thereof, which has at least 12 amino acids corresponding in sequence to some part of the amino acid sequence of PACAP38, as shown in FIG. 1, and which binds to at least one PACAP receptor. Similarly, the terms "PACAP23 agonist" and "PACAP27 agonist" refer to polypeptides, or salts or derivatives thereof, which has at least 23 and 27 amino acids, respectively, corresponding in sequence to some part of the amino acid sequence of PACAP38, as shown in FIG. 1, and which binds to at least one PACAP receptor. Determination of the amino acid sequence of the polypeptide, and determination as to whether it binds to a PACAP receptor, are both well within the skill in the art. The ability to treat neuronal cells at such low concentrations also makes possible the administration of the PACAP compounds intravenously or otherwise into the blood, in concentrations sufficient to provide PACAP compound transfers across the blood/brain barrier sufficient to provide concentrations of the PACAP compound in contact with the neuronal cells which are effective to protect and/or resuscitate the traumatized neuronal cells.

It has also been discovered that administration of the PACAP compound is effective in protecting and/or resuscitating traumatized neuron cells up to at least 24 hours after injury.

Preferably the PACAP compound has the general formula:

X-PACAP[a-b] -Y

wherein X is H or a solubility effecting group such as C1-20 carboxylic acid moiety, such as formyl, acetyl, etc.; a and b are N and C terminus amino acids taken in the sequence of PACAP38 as shown in FIG. 1, and Y is H, NH2, OH, or C1-4 carboxy. For adjustment of lipophilic nature to increase the amount of PACAP compound passing the blood/brain barrier, it is preferred that X is a fatty acid moiety, preferably derived from Lauric, Myristic, Palmitic, Stearic, or Oleic acid, most preferably from Palmitic or Stearic acid. Thus expressed, PACAP38 is PACAP[1-38] -NH2, i.e., X is H, Y is the NH2 attached to the C-terminal Lysine, and the compound has the complete sequence of 1-38 amino acids of FIG. 1. The polypeptide can be substituted at either end with moieties which favorably effect the solubility in the carrier, or favorably effect the ability of the PACAP compound to transfer across the blood brain barrier without substantially adversely effecting the effectiveness of the compound. Thus X can be an organic acid or salt thereof, preferably containing only alkyl groups of C1-25, preferably C1-20, or a residue from such an acid, e.g., an ether derived from such an acid. Low molecular weight (C1-4) acids or acid residues can be used to increase the solubility of the polypeptide in the pharmaceutical composition, or in bodily fluids. Larger molecular weight moieties, such as the C12-20 long chain fatty acid residues, can be used to enhance the transferability of the PACAP compound across the blood brain/barrier. Substituents at the C-terminus of the polypeptide can also be used to enhance the solubility of the PACAP compound without deleteriously effecting its usefulness. For example, the amino (NH2) group on the C-terminal amino acid can be substituted by a hydroxyl group or a lower (C1-4) alcohol or carboxyl group.

It is also possible to make various substitutions of certain of the amino acids in the PACAP sequence, to make minor adjustments in the physical properties of the molecule without substantially effecting its usefulness in treatment of neuronal cells. For example, substitution of less reactive amino acids can provide increased stability and shelf life of the pharmaceutical composition. Thus it is possible to make one or more of the following substitutions:

        Location                   Substitute (s)
        For His at position 1      Tyr, Ala, Arg or Glu
        For Asp at position 3      Glu
        For Gly at position 4      Ala
        For Asp at position 8      Glu
        For Ser at position 9      Asn
        For Ser at position 11     Thr
        For Tyr at position 13     Leu
        For Met at position 17     Gly, Ser, Phe, Nle, Arg or Glu
        For Ala at positions 24, 25 Ser

As used in the present application, such substitutions will be referred to in brackets prior to the modified PACAP structure. For example, [Glu3,8 ]PACAP[1-27] -NH2 refer to PACAP27 wherein the asparagine at position 3 and the asparagine at position 8 have each been replaced by glutamic acid.

Suitable exemplary compositions are disclosed below.

The most preferred active ingredient of the pharmaceutical composition is PACAP 38, its salts and derivatives. The next most preferred is PACAP27, its salts and derivatives. As used herein, "PACAP27" and "PACAP38" refer to the polypeptides which have the same amino acid sequence as amino acids 1-27 and 1-38, respectively, of PACAP38, as shown in FIG. 1. Thus both PACAP38 and PACAP27 come with the scope of the terms "PACAP12 " "PACAP23 " and "PACAP27. " Other suitable PACAP type compounds include:

          1.      N.alpha. Acetyl-PACAP1-38 -NH2
                  where PACAP1-38 represents amino acids
                  1-38 of SEQ ID NO:1.
          2.      N.alpha. Acetyl-PACAP2-38 -NH2
                  where PACAP2-38 represents amino acids
                  2-38 of SEQ ID NO:1.
          3.      N.alpha. -Stearyl-PACAP1-38 -NH2
                  where PACAP1-38 represents amino acids
                  1-38 of SEQ ID NO:1.
          4.      N.alpha. -Stearyl-PACAP2-38 -NH2
                  where PACAP2-38 represents amino acids
                  2-38 of SEQ ID NO:1.
          5.      PACAP1-38 -OH
                  where PACAP1-38 represents amino acids
                  1-38 of SEQ ID NO:1.
          6.      PACAP1-30 -NH2
                  where PACAP1-30 represents amino acids
                  1-30 of SEQ ID NO:1.
          7.      PACAP2-30 -NH2
                  where PACAP2-30 represents amino acids
                  2-30 of SEQ ID NO:1.
          8.      N.alpha. -Acetyl-PACAP2-30 -NH2
                  where PACAP2-30 represents amino acids
                  2-30 of SEQ ID NO:1.
          9.      PACAP1-27 -NH2
                  where PACAP1-27 represents amino acids
                  1-27 of SEQ ID NO:1 or SEQ ID NO:2.
         10.      N.alpha. -Acetyl-PACAP1-27 -NH2
                  where PACAP1-27 represents amino acids
                  1-27 of SEQ ID NO:1 or SEQ ID NO:2.
         11.      N.alpha. -Acetyl-PACAP2-27 -NH2
                  where PACAP2-27 represents amino acids
                  2-27 of SEQ ID NO:1 or SEQ ID NO:2.
         12.      N.alpha. -Stearyl-PACAP1-27 -NH2
                  where PACAP1-27 represents amino acids
                  1-27 of SEQ ID NO:1 or SEQ ID NO:2.
         13.      N.alpha. -Stearyl-PACAP2-27 -NH2
                  where PACAP2-27 represents amino acids
                  2-27 of SEQ ID NO:1 or SEQ ID NO:2.
         14.      PACAP2-27 -NH2
                  where PACAP2-27 represents amino acids
                  2-27 of SEQ ID NO:1 or SEQ ID NO:2.
         15.      [Tyr1 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         16.      [Ala1 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         17.      [Arg1 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         18.      [Glu1 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         19.      [Glu3 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         20.      [Glu8 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         21.      [Glu3,8 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         22.      [Asn9 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         23.      [Thr11 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         24.      [Leu13 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         25.      [Ser24,25 ]PACAP1-b -NH2, b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         26.      X-[Gly17 ]PACAP1-b -NH2, X = C10-18
                  fatty acid; b = 27-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 27 to 38.
         27.      X-[Ser17 ]PACAP27-NH2, X = C10-18 fatty acid;
         28.      X-[Phe17 ]PACAP27-NH2, X = C10-18 fatty acid;
         29.      X-[Glu17 ]PACAP27-NH2, X = C10-18 fatty acid;
         30.      X-[Arg17 ]PACAP27-NH2, X = C10-18 fatty acid;
         31.      X-[Nle17 ]PACAP27-NH2, X = C10-18 fatty acid;
         32.      X-[Ala4 ]PACAP(1-23)-NH2, X = C10-18 fatty
     acid;
         33.      [Ala4, Leu13 ]PACAP1-b -NH2, b = 23-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 23 to 38.
         34.      [Leu13 ]PACAP1-b -NH2, b = 23-38
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1, where b represents
                  amino acids 23 to 38.
         35.      [Tyr1 ]PACAP1-b -NH2, b = 23-26
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1 or SEQ ID NO:2, where b represents
                  amino acids 23 to 26.
         36.      PACAP1-b -NH2, b = 23-26
                  where PACAP1-b represents amino acids
                  1-b of SEQ ID NO:1 or SEQ ID NO:2, where b represents
                  amino acids 23 to 26.
         37.      PACAP1-24 -NH2
                  where PACAP1-24 represents amino acids
                  1-24 of SEQ ID NO:1 or SEQ ID NO:2.
         38.      PACAP1-23 -OH
                  where PACAP1-23 represents amino acids
                  1-23 of SEQ ID NO:1 or SEQ ID NO:2.
         39.      PACAP2-23 -NH2
                  where PACAP2-23 represents amino acids
                  2-23 of SEQ ID NO:1 or SEQ ID NO:2.
         40.      N.alpha. -X-PACAP1-38 -NH2, X = C10-18
     fatty acid
                  where PACAP1-38 represents amino acids
                  1-38 of SEQ ID NO:1.
         41.      N.alpha. -X-PACAP2-38 -NH2, X = C10-18
     fatty acid
                  where PACAP2-38 represents amino acids
                  2-38 of SEQ ID NO:1.
         42.      N.alpha. -X-PACAP1-27 -NH2, X = C10-18
     fatty acid
                  where PACAP1-27 represents amino acids
                  1-27 of SEQ ID NO:2.
         43.      N.alpha. -X-PACAP2-27 -NH2, X = C10-18
     fatty acid
                  where PACAP2-27 represents amino acids
                  2-27 of SEQ ID NO:2.
         45.      Any peptide or non-peptide agonist (except those
                  listed above) for PACAP receptor 1 and organic and
                  inorganic salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that sustained intracerebroventricular administration of PACAP can be used to protect neuronal cells and significantly prevent neuronal cell damage and death induced by ischemia, hemorrhage, trauma, toxic substances or other causes. It is particularly effective in treating neuronal cell damage caused by transient ischemia followed by reperfusion. Unexpectedly, the present inventors found that PACAP compounds are most effective in vivo in a particular concentration range, above which the effectiveness drops off. Most surprisingly, within that effective range, there are sub ranges of concentration, within which the effectiveness peaks and drops off, and the activity of the PACAP compound in the lower sub range is higher than at higher concentrations, thus permitting use of the PACAP compounds at extremely low concentrations, in turn avoiding possible side effects and complications.

Accordingly, the present invention provides a method for the treatment and prevention of neuronal cell damage and death, preferably brain cell damage and death, in vitro and/or in vivo, induced by various causes comprising administrating to neuronal cells or to a mammal in need thereof an effective amount of a PACAP or an agonist thereof, which interacts with specific Type I PACAP receptors. Preferably, the in vivo mode of administration is intraventricular or intravenous.

While not wishing to be bound by theory, the neurotrophic action of PACAP appears to take place at two different sites. PACAP-stimulates proliferation of sympathetic neuroblast and neurite outgrowth in PC12 cells directly, but it requires nanomolar concentration. Cytoprotective action of PACAP on chick embryos also requires nanomolar concentration of the peptide, much higher than the effective concentrations discovered in connection with the present invention. That type of cytoprotective action is believed to be mediated via or through adenylate cyclase activation by PACAP.

Applicant has also shown that when neuronal cells are plated on a feeder layer of astrocytes, addition of HIV envelope glycoprotein gp120 results in significant cell death. Yet, when small concentration of PACAP, e.g., 10-13 M, was added to the culture, the gp120-induced neuronal cell death was completely prevented. In such cases, higher concentrations of PACAP have been shown to be less effective. Since this cytoprotective action is difficult to demonstrate in neuron cultures in the absence of astrocytes, this cytoprotective action is considered to be mediated through astrocytes, which express an extremely high affinity Type I PACAP receptors. PACAP at such a low concentration does not stimulate adenylate cyclase or phospholipase C, and as a result, the cytoprotective action may be mediated by one or more other second messengers, most likely by alteration of intracellular Ca2+. PACAP which has been found to increase intracellular Ca2+ of cultured pancreatic beta cells at 10-13 M (Yada et al., J. Biol. Chem., in press 1994). Based on this evidence it is believed that a subtype of Type I PACAP receptors with an extremely high affinity may be expressed on astrocytes in vivo under certain conditions, such as ischemia-induced brain injury. As discussed above, astrocytes with Type I PACAP receptors begin appearing 2 days after ischemia and the number increases over 7 days after ischemia. Applicants' in vitro examination of gp120-induced brain cell death suggests that the interaction of PACAP with these newly expressed receptors in astrocytes may stimulate synthesis and release of a neurosurvival factor that prevents neuronal cell death.

In accordance with the method of the present invention, PACAP may be used in the treatment and prevention of neuronal cell damage resulting from ischemia/reperfusion, trauma, hemorrhage, infection and exposure to toxic substances. PACAP can also be used for 1) treatment of congestive heart failure of neonate, through its inotropic action and stimulation of adrenalin secretion in vivo; 2) treatment of neuropathy, such as diabetic neuropathy; 3) treatment of spinal cord injury; 4) treatment of ischemia/reperfusion induced lung injury (cAMP has been shown to prevent such injury, and PACAP is a potent stimulater for cAMP production); 5) treatment of ischemia/reperfusion induced cardiac injury; 6) treatment of gastric and intestinal ulcer (PACAP regulates production of various growth factors which are known to prevent ulcer); 7) stimulation of neonatal and prenatal brain development; 8) protection of transplanted neural cells in the brain; 9) treatment of certain male infertility; 10) improvement of brain circulation; 11) treatment of shock, such as the condition resulting from exposure to bacteria toxin.

Mammalian subjects which can be treated with the PACAP compounds and compositions in accordance with the present invention include, for example, cattle, swine, sheep, monkeys, dogs, cats, rodents, such as laboratory animals, and humans. Further, PACAP stimulates growth and differentiation of neuronal and glial cells and cell lines in vitro. Any cell line which possesses PACAP receptors can be stimulated by PACAP in growth and differentiation.

Synthesis of PACAP38, PACAP27 and their related peptides.

PACAP38, PACAP27, PACAP their agonist analogs, precursors and salts are prepared in a manner which will be apparent to the skilled in the art. The peptides were synthesized by solid phase techniques using an automated peptide synthesizer (Beckman 990B). 4-methyl benzhydrylamine resin and PAM-resin were employed for the synthese of C-terminal amide form peptides and C-terminal free form peptides, respectively. The peptide chain was elongated on the resin with the use of N.alpha. -Boc-amino acid derivatives such as:

Boc-Lys(Cl-Z)-OH, Boc-Asn-OH, Boc-Val-OH, Boc-Arg(Tos)-OH, Boc-Gin-OH, Boc-Tyr(Br-Z)-OH, Boc-Leu-OH, Boc-Ala-OH, Boc-Met-OH, Boc-Ser(Bzl)-OH, Boc-Asp(OBzl)-OH, Boc-Thr(Bzl)-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-His(Tos)-OH, Boc-Glu(OBzl)-OH and Boc-Nle-OH

These N.alpha. Boc amino acid derivatives were successively introduced to the peptide chain in the presence of diisopropylcarbodiimide in dichloromethane with the exception of Boc-Asn-OH and Boc-Glyn-OH which were coupled in the presence of 1-hydroxybenzotriazole as a catalyst in DMF. The completed, protected peptide resins (90.025 mmol each) were treated with 20 mL of anhydrous hydrogen fluoride containing 10% anisole and 100 mg of dithiothreitol for 45 min at 0oC. After removal of the hydrogen fluoride under a stream of nitrogen, the free peptides were precipitated with either ether or ethyl acetate, filtered, and extracted with 2M AcOH. After lyophilization, the crude peptides were obtained. The crude peptides were purified by gel filtration on a column of SEPHADEX G-50 fine (2.5x100 cm) using 2M AcOH containing 0.02% .beta.-mercaptoethanol as an eluent, followed by preparative reverse phase HPLC column (1.5x50 cm) of Vydac C-18 silica (15-20 mm particle size), which was eluted with a linear gradient of 10-35% acetonitrile in 0.1 % TFA at a flow rate of 3 mL/min.

The purity of each purified material was confirmed by analytical reverse phase HPLC, amino acid analyses, sequencing and FABMS.

Further information on preparation of the materials referred to in this application is disclosed, for example, in U.S. Pat. Nos. 5,198,542, 5,128,242, A. Sakiyama et al., Pep. Chem. 1991:215 (1991), and C. Kitada et al., Pep. Chem. 1990:239 (1991), the disclosures of which are hereby incorporated by reference.

PACAP or its agonists may be intracerebroventricularly administrated to a host in need thereof utilizing a variety of means known to the skilled artisan including, for example, an infusion system such as The SynchromMed Infusion System with Catheter Access Port (Medtronic Neurological, Minneapolis, Minn.). Suitable compositions for direct administration to the brain include the polypeptide in a carrier such as spinal fluid, artificial spinal fluid, or a combination of physiological saline, Ringer's solution, glucose (e.g., 3-7%, preferably about 5% by weight), and an isotonic phosphate buffer (pH of about 7). Artificial cerebrospinal fluid, comprises about 128 mM NaCl, 2.6 mM KCl, 1.3 mM CaCl2, 20 mM NaHCO3, 1.3 mM Na2 HPO4, pH 7.35 and contains 0.1% bovine serum albumin. For iv infusion 0.9% saline containing 0.1% bovine serum albumin may be used. Bovine serum albumin is used for protection of loss of the peptide due to adsorption. Bovine serum albumin can be replaced by any other inert protein such as human serum albumin and gelatin.

The amount of PACAP compound to be administered is sufficient to achieve a concentration in the tissue to be treated of from about 10-15 to about 10-12 M, more preferably from about 10-14 to about 10-12 M. In in vivo studies on laboratory rats, it was found that 10-13 M was achieved and maintained by intracerebroventricular ("icv")infusion of about 16 ng/kg body weight/hour for seven days. Thus the dosage ranges corresponding to the above concentration ranges are from about 0.16 ng/kg body weight/hour to 160 ng/kg body weight/hour, preferably either from 1.6 ng/kg body weight/hour to 160 ng/kg body weight/hour. Thus for a 300 g. rat, the optimal dosage for icv infusion was about 1 pmol/.mu.l/hr for a seven day course of treatment, which is equal to a rate of 3.33 pmol (15.7 ng)/3.33 .mu.l/kg/h. In a human patient weighing 70 kg, the infusion rate is about 233 pmol (1.1 .mu.g)/233 .mu.l/h. Thus, when an infusion rate of 233 .mu.l/h is used the concentration of PACAP is 1 nmol/ml (4.5 .mu.g/ml) for icv infusion. An infusion system, such as a SynchroMed Infusion System can deliver solution at a range between 4 .mu.l and 900 .mu.l/h. An infusion rate of 50-500 .mu.l/h is preferred. Suitable concentrations of the PACAP compound in the pharmaceutically acceptable carrier for icv infusion are prepared according to the infusion rate. For example, when one wishes to infuse 1.1 .mu.g/hour and an infusion rate of 50 .mu.l/h is chosen, the concentration of PACAP is 1.1 .mu.g/50 .mu.l or 22 .mu.g/ml. When 500 .mu.l/h is chosen, a 2.2 .mu.g/ml solution is prepared. Accordingly, suitable concentrations of the PACAP compound in pharmaceutically acceptable carrier for icv infusion at a rate of 1.1 .mu.g/hour are from about 1.2 .mu.g/ml to about 300 .mu.g/ml, preferably from about 2.2 .mu.g/ml to about 22 .mu.g/ml. Suitable concentration ranges for other infusion rates can be likewise calculated.

As shown in Example 7, PACAP administered icv is not evenly distributed throughout the brain tissues. For example, administration of 125 I labelled PACAP into the right lateral ventricle resulted in four times greater CPM in the right hippocampus than in the left. Thus, in order to achieve the desired concentration of PACAP in a specific brain tissue or region (e.g., left or right side), the amount of PACAP administered must be adjusted accordingly.

The administration of PACAP is not limited to the intracerebroventricular route. Despite the existence of blood-brain-barrier, a small portion of intravenously administered PACAP is transported into the brain. Banks, et al., Journal of Pharmacology and Experimental Therapeutics, 267: No. 2 690-696 (1993). PACAP38 is transported into the brain in a saturated manner. Therefore, it is also possible to administer PACAP and its analogs by a prolonged intravenous or even subcutaneous infusion to attain the optimal concentration of PACAP in the brain tissues for attaining the similar cytoprotective effect. The percentage of the amount of circulating PACAP which enters into the brain can be precisely calculated. Banks et al., supra. Analogs of PACAP with a greater lipophilic nature, such as PACAP analogs with fatty acid residue at the N-terminus, for example: N.alpha. -stearyl-[Nle17 ]PACAP1-38 -NH2 or N.alpha. -stearyl-[Nle17 ]PACAP2-38 -NH2 may be more preferable for such modes of administration.

Compositions for intravenous administration are preferably prepared by forming a solution of the PACAP compound in a water-soluble solvent (e.g. physiological saline, Ringer's solution). In tests with laboratory rats, it was determined that in order to achieve a concentration of 10-13 M in the brain, it was necessary to inject into the bloodstream about 1.7 pmol/h (or 7.7 ng per hour) of PACAP compound. This dose corresponds to 25.5 ng/kg/h. Thus, the dosage ranges corresponding to the above concentration ranges from about 0.25 ng/kg body weight/hour to 25 .mu.g/kg body weight/hour, preferably from 0.25 ng/kg body weight/hour to about 2.5 .mu.g/kg body weight/hour, most preferably from about 2.5 ng/kg body weight/hour to about 250 ng/kg body weight/hour. Based on the above a human patient weighing 70 kg, the optimal IV dose would be about 1.8 .mu.g/h. Using an ordinary infusion pump, solution can be infused at, for example, 100 .mu.l/h, and thus, 1.8 .mu.g PACAP is dissolved in 100 .mu.l to make 18 .mu.g/ml in 0.9% saline, 5% glucose solution or a solution for IV infusion which contains various salts (these solutions are commercially available and used to meet patients' conditions.) Infusion rates can be modified to a rate faster or slower than 100 .mu.l/h, and the concentrations of PACAP should be adjusted accordingly. For example, when the infusion rate is 1000 .mu.l/h, the concentration of PACAP is 1.8 .mu.g/ml.

The amount of PACAP in a pharmaceutical composition for intravenous administration is 10 to 100,000 times the amount that is effective at the active region, preferably 100 to 10,000 times and most preferably 500 to 5,000 times.

As desired, additives such as a dissolution aid (e.g.sodium salicylate, sodium acetate), buffer (e.g. sodium citrate, glycerine), isotonizing agent (e.g. glucose, invert sugar), stabilizer (e.g. human serum albumin, polyethylene glycol), preservatives (e.g. benzyl alcohol, phenol), or analgesics (e.g. benzalkonium chloride, procaine hydrochloride).

Compositions for administration of PACAP include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes and may be prepared by any methods well known in the art of pharmacy.

Such methods include the step of bringing into association the ingredients to be administered with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

The amount of PACAP in a composition for parenteral administration (e.g., suppository, sublingual tablet, nasal application) is 100 to 1,0000,000 times, the amount that is effective at the active region, preferably 1,000 to 100,000 times and most preferably 5,000 to 50,000 times.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion or packed in liposomes and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing water. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Compositions suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the compound and a pharmaceutically acceptable carrier. A suitable topical delivery system is a transdermal patch containing the ingredient to be administered.

Sublingual tablets can be prepared by using binders (e.g. hydroxypropylcellulose, hydroxypropylmethylcellulose, polyethylene glycol), disintegrating agent (e.g. starch, carboxymethylcellulose calcium,), lubricant (e.g. magnesium stearate, talc)

Compositions suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, polyethylene glycol 600, cocoa butter or a salicylate.

Compositions suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 microns. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tables of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable for oral administration may include flavoring agents.

As the PACAP compound or an agonist for Type I PACAP receptor sites are extremely low in toxicity, compositions comprising these compounds are extremely low in toxicity.

Claim 1 of 8 Claims

What is claimed is:

1. A method of preventing or attenuating neuronal cell death comprising continuously administering to a subject in need thereof a compound selected from the group consisting of:

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH2 (SEQ ID NO:3),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-OH (SEQ ID NO:4),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-NH2 (SEQ ID NO:5),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-OH (SEQ ID NO:6),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-NH2 (SEQ ID NO:7),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-OH (SEQ ID NO:8),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-NH2 (SEQ ID NO:9),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-OH (SEQ ID NO:10),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-NH2 (SEQ ID NO:11),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-OH (SEQ ID NO:12),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-NH2 (SEQ ID NO:13),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-OH (SEQ ID NO:14),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-NH2 (SEQ ID NO:15),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala- Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-OH (SEQ ID NO:16),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -NH2 (SEQ ID NO:17),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -OH (SEQ ID NO:18),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-NH2 (SEQ ID NO:19),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-OH (SEQ ID NO:20),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-NH2 (SEQ ID NO:21),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-OH (SEQ ID NO:22),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-Asn-NH2 (SEQ ID NO:23),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-Asn-OH (SEQ ID NO:24),

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-Asn-Lys-NH2 (SEQ ID NO:25),

and

X-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Xaa-Ala-V al-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg -Val-Lys-Asn-Lys-OH (SEQ ID NO:26),

wherein:

X is NHR, where R is H or a solubility effecting group having the acyl group CH3 (CH2)nCO where n=0-24; and

Xaa is Met, Gly, Ser, Phe, Nle, Arg or Glu,

and wherein the compound is continuously administered by intravenous infusion at 0.0556 pmol/kg body weight/hour to 5.56 nmol/kg body weight/hour.




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