WO1997049721A1 - Methodes de traitement du syndrome d'amaigrissement par administration de derives de la gonadotrophine chorionique humaine - Google Patents

Methodes de traitement du syndrome d'amaigrissement par administration de derives de la gonadotrophine chorionique humaine Download PDF

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Publication number
WO1997049721A1
WO1997049721A1 PCT/US1997/011448 US9711448W WO9749721A1 WO 1997049721 A1 WO1997049721 A1 WO 1997049721A1 US 9711448 W US9711448 W US 9711448W WO 9749721 A1 WO9749721 A1 WO 9749721A1
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Prior art keywords
hcg
seq
amino acid
protein
sequence
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PCT/US1997/011448
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English (en)
Inventor
Robert C. Gallo
Joseph Bryant
Yanto Lunardi-Iskandar
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University Of Maryland Biotechnology Institute
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Priority to AU35877/97A priority Critical patent/AU3587797A/en
Publication of WO1997049721A1 publication Critical patent/WO1997049721A1/fr
Priority to US09/220,415 priority patent/US6583109B1/en
Priority to US09/676,739 priority patent/US6620416B1/en
Priority to US09/675,776 priority patent/US6596688B1/en
Priority to US09/675,362 priority patent/US6699834B1/en
Priority to US09/677,152 priority patent/US6805882B1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed to methods of treatment and prevention of wasting syndromes by administration of human chorionic gonadotropin preparations, fractions of human chorionic gonadotropin preparations, the ⁇ -chain of human chorionic gonadotropin and peptides containing a sequence of a portion or portions of the ⁇ -chain of human chorionic gonadotropin.
  • the invention also provides pharmaceutical compositions comprising human chorionic gonadotropin preparations, fractions of human chorionic gonadotropin preparations, the j ⁇ -chain of human chorionic gonadotropin or peptides having a sequence of a portion or portions of the /3-chain of human chorionic gonadotropin.
  • Wasting syndrome is a serious clinical problem characterized by a decrease in body mass of more than 10% from baseline body weight and a disproportionate loss of body mass with respect to body fat (Weinroth et al., 1995, Infectious Agents and Disease 4.:76-94; Kotler and Grunfeld, 1995, AIDS Clin . Rev . £j5:229-275) .
  • wasting is distinguished from starvation in which higher levels of body fat than body cell mass are depleted (Kotler et al., 1985, Am J . Clin . Nutr . 4 . 2: 1255-1265; Cahill, 1970, N. Engl . J . Med . 282 :668-675) .
  • HIV infection human immunodeficiency virus (HIV) has been implicated in acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. , et al., 1983, Science 220:868-870; Gallo, R. , et al., 1984, Science 224 . : 500-503)
  • HIV infection human immunodeficiency virus (HIV) has been implicated in acquired immune deficiency syndrome (AIDS)
  • AIDS acquired immune deficiency syndrome
  • other infectious diseases sepsis, cancer, chronic cardiovascular disease and diarrhea
  • wasting is a significant factor in the mortality of patients suffering from infections or cancer.
  • body cell mass depletion has a linear relationship to time of survival in AIDS patients (Kotler et al., 1989, Am . J. Clin . Nutr . 50:444- 447) .
  • hCG Human chorionic gonadotropin
  • FSH follicle-stimulating hormone
  • LH luteinizing hormone
  • TSH thyroid-stimulating hormone
  • the ⁇ -subunits are identical among the glycoprotein hormones while the /3-subunits differ and determine the receptor binding specificity of the particular hormone (Kornyei, J.L., et al., 1993, Biol . Reprod . 4_9:1149).
  • the /3-subunits of the glycoprotein hormones exhibit a high degree of sequence similarity within the N-terminal 114 amino acids.
  • LH is the most similar to hCG with 85% sequence homology within the first 114 amino acids, and both proteins bind the same receptor.
  • hCG contains a C-terminal extension not present in the other glycoprotein 0-chains (Lapthorn, A.J., et al., 1994, Science 369:455-461) .
  • hCG like the growth factors nerve growth factor (NGF) , transforming growth factor- / 3 (TGF- ⁇ ) and platelet-derived growth factor-/? (PDGF-j ⁇ ) , is a cysteine-knot glycoprotein. Proteins containing such a cysteine-knot motif have at least three disulfide bridges, two of which join adjacent anti-parallel strands of the peptide, thus, forming a ring, and one of which joins the peptide chain through the ring.
  • NGF nerve growth factor
  • TGF- ⁇ transforming growth factor- / 3
  • PDGF-j ⁇ platelet-derived growth factor-/?
  • the present invention fulfills a need for safe and effective therapies for wasting syndrome associated with AIDS, other infectious diseases and cancer.
  • the present invention further relates to therapeutic methods and compositions for treatment and prevention of wasting syndromes based on hCG and ⁇ -hCG preparations, therapeutically and prophylactically effective fractions of a source of native hCG or native ⁇ -hCG and therapeutically and prophylactically effective proteins containing a sequence of a portion or portions (i.e., a fusion protein comprising more than one ⁇ -hCG peptide sequence, e . g . , having an amino acid sequence of one ⁇ -hCG peptide linked via a peptide bond to another /3-hCG peptide) of /3-hCG, and related derivatives and analogs.
  • a sequence of a portion or portions i.e., a fusion protein comprising more than one ⁇ -hCG peptide sequence, e . g . , having an amino acid sequence of one ⁇ -hCG peptide linked via a peptide bond to another /3-hCG peptide
  • the invention provides for treatment and prevention of wasting syndromes by administration of a therapeutic compound of the invention.
  • the therapeutic compounds of the invention include: hCG, ⁇ -hCG, therapeutically and prophylactically effective fractions of a source of hCG or ⁇ - hCG (preferably a source of native hCG or native ⁇ -hCG , i . e . a source of naturally occurring hCG or ⁇ -hCG and not recombinantly produced hCG or ⁇ -hCG) , therapeutically and prophylactically effective peptides having a sequence of a portion or portions of ⁇ -hCG (i.e.
  • a fusion protein comprising more than one /3-hCG peptide sequence either as non-contiguous or contiguous sequences, e.g. having an amino acid sequence of one ⁇ -hCG peptide linked via a peptide bond to another ⁇ -hCG peptide) , modified derivatives of hCG, 0-hCG and ⁇ -hCG peptides, and nucleic acids encoding ⁇ -hCG and therapeutically and prophylactically effective peptides having a sequence of a portion or portions of ⁇ -hCG , and derivatives and analogs of the foregoing.
  • the present invention also relates to the use of certain fractions (i.e. components of a source of hCG or /3-hCG isolated away from other components in the source of hCG or ⁇ -hCG by a separation technique known in the art) of any source of hCG or 0-hCG, such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and/or anti-Kaposi's Sarcoma activity and/or anti-wasting activity.
  • certain fractions i.e. components of a source of hCG or /3-hCG isolated away from other components in the source of hCG or ⁇ -hCG by a separation technique known in the art
  • any source of hCG or 0-hCG such as commercial hCG preparations and human (preferably early, i.e., first trimester) pregnancy urine, which fractions have anti-HIV and/or anti-Kaposi's Sarcoma activity and/or anti-wasting activity
  • the invention also provides in vi tro and in vivo assays for assessing the efficacy of therapeutics of the invention for treatment or prevention of wasting syndromes.
  • the invention also provides pharmaceutical compositions and methods of administration of Therapeutics of the invention for treatment.
  • FIGS 1A-E Effects of an hCG preparation, APLTM (Wyeth-Ayerst) , ⁇ -hCG peptides and certain fractions of hCG APLTM and early preganancy urine on weight and HIV-1 gene expression in HIV-1 transgenic mice.
  • A and
  • C Weight change in grams in individual HIV-1 transgenic mice after treatment from from day 1 to day 10 post partum is represented as a bar graph with open bars representing the weight at day 1 and solid bars representing the weight at day 10.
  • B (D) and (E) Suppression of HIV-1 gene expression in transgenic mice. The bar graph presents the level of expression in pixels, as determined by chemiluminescence assay of the HIV genes env.
  • bars 1-3 represent untreated control transgenic mice; bars 4-6 represent mice whose mothers received (subjects were administered through the mothers' milk) 200 ⁇ g circularized ⁇ -hCG peptide 44-57 (with cysteine substituted at position 44; SEQ ID NO:26) per day; bars 7-9 represent mice whose mothers received 300 IU per day hCG-APL ,M ; and bars 9-11 represent mice whose mothers received 200 ⁇ g per day of the fused ⁇ -hCG peptide 45-57:: 109-119 (SEQ ID NO:30).
  • the bars labeled "untreated” represent mice receiving only PBS; and the bars labeled “treated” or “treated with HAFc” represent mice administered 300 IU per day of hCG APLTM.
  • the set of bars labeled “1” represents mice treated with PBS alone; "2” represents mice treated with 100 ⁇ g ⁇ -hCG core peptide per day; “3” represents mice treated with 100 ⁇ g per day ⁇ -hCG; "4" represents mice treated with 200 ⁇ l per day of fraction 61 of the hCG APLTM fractionation; and "5" represents mice treated with 200 ⁇ l per day of fraction 65 of the early pregnancy urine fractionation.
  • FIGS 2A-D Effects of an hCG preparation on indicators of SIV infection in SIV-infected macaques.
  • SIV was given intravenously at a dose of 10 4 5 TCID 50 per ml.
  • SIV titer was monitored over time in months by quantifying the p27 gag protein (Organon Teknika assay) as nanograms (ng) of p27/ml of plasma from the plasma of the SIV infected macaques.
  • Treated SIV-infected macaques (indicated as Rx) were given hCG APL, 3000 IU, 2x weekly. Plasma levels of p27 gag in these treated monkeys are indicated on the graph by lines with diamonds, number (#) signs or . filled circles.
  • results with the untreated SIV-infected macaques are indicated by the lines with either stars or triangles.
  • B CD4 * T cell levels were determined in cells/mm 3 in SIV-infected macaques either treated with hCG or untreated over time in months.
  • Results from the SIV-infected monkeys treated with hCG (APL) (Rx) are indicated by lines with diamonds, number (#) signs or filled circles, while results with the untreated monkeys (UnRx) are indicated by lines with stars or triangles.
  • C Change in weight in kilograms (kg) was monitored in treated and untreated SIV- infected monkeys over time in months.
  • Weight changes in the SIV-infected monkeys treated with hCG (APL) (Rx) are indicated by lines with diamonds, # signs or filled circles, while results in the untreated monkeys (UnRx) are indicated by lines with stars or triangles.
  • D Levels of CD4 * T cells were monitored in normal uninfected monkeys either treated with hCG (APL) or untreated over time in months.
  • CD4 * T cell levels in the untreated monkeys are indicated by lines with sun-like figures or squares, and the results in the treated monkeys are indicated by lines with pentagonal figures or with filled inverted triangles.
  • Figures 3A-J Effects of administration of hCG preparations on HIV-1 viral load and CD4 * T cell levels in individual patients in the clinical study described in Section 7.3 infra .
  • Figures A and B are data from patient PHOJ, C and D from patient PG1, E and F from patient PG3, G and H from patient PHVE, and I and J from patient PG17.
  • panels A, C, E, G and I viral load and CD4 * T Cell counts are plotted over time (in months) .
  • Viral load (measured by RT- PCR in panels A and G and by the Roche Amplicor test in panels C, E and I) is plotted as the logarithm of the viral load (represented by line with "X" data points) .
  • CD4* T Cell levels are plotted as CD4 * T Cells/ml (represented by line with triangle data points) .
  • Panels B, D, F, H, and J plot the dosage of hCG in IU (X 1000) per week over time in months, with the timing of other therapies indicated above the graph with a thick arrow.
  • Figure 4 The nucleotide (SEQ ID NO:l) and amino acid (SEQ ID NO:2) sequences of ⁇ -hCG .
  • Figures 5A and B Schematic depiction of the structures of (A) the linear peptide of amino acids 45-57 (SEQ ID NO:6) of the ⁇ -hCG sequence depicted in Figure 4 (SEQ ID NO:2) where the amino acid residues at positions 47 and 51 are substituted by a branch. made up of diaminobutyric acid peptide bonded to proline, and (B) the circularized peptide of amino acids 44-57 (SEQ ID NO: 12) with valine at position 44 substituted with cysteine, which protein is circularized via a disulfide bond between its amino- and carboxy-terminal cysteines.
  • amino acids are represented by their three letter amino acid code, except for the branched residues and the terminal cysteines, for which the structure is depicted.
  • FIGS 6A-F These graphs depict results from the fractionation by SUPERDEXTM 200 gel filtration of a commercial hCG preparation APLTM (Wyeth Ayerst) and early pregnancy urine.
  • A) and (D) These graphs depicts the relative amount of protein in mg/ml in each fraction identified by fraction number in the hCG APLTM fractionation (A) and early pregnancy urine fractionation (D) .
  • the fractions containing the hCG dimer and /3-core protein are identified with arrows and the labels "hCG" and " ⁇ -core” respectively.
  • B) and (E) The fractions containing the hCG dimer and /3-core protein are identified with arrows and the labels "hCG" and " ⁇ -core” respectively.
  • CD4 as a function of CD4 * T cell levels before therapy (in CD4 * T cells/ml)
  • CD4Base CD4Base
  • C Plot of linear regression analysis of the change in viral load (vlchange) as a function of weekly dose of hCG in IU (“HCGIU”) .
  • vlchange change in viral load
  • HCGIU weekly dose of hCG in IU
  • FIGS 8A and B (A) Plot of protein concentration (as mAUFS, milli absorbance units, at 280 nm) as a function of the fraction number of the hCG APLTM preparation SUPERDEXTM 200 fractionation. (B) Plot of protein concentration (as mAUFS, milli absorbance units, at 280 nm) of molecular weight markers of 670 kD, 158 kD, 44 kD, 17 kD and 1.3 kD (as indicated above the plot) as a function of fraction number of a SUPERDEXTM 200 column run under the same conditions as the fractionation plotted in panel A.
  • FIGS. 9A-E Mass spectrometry profiles of fractions 61, 63, 64, 65, and 67 in panels A-E, respectively.
  • the present invention further relates to therapeutic methods and compositions for treatment and prevention of wasting syndromes based on hCG and ⁇ -hCG preparations, therapeutically and prophylactically effective fractions of a source of hCG or /3-hCG, and therapeutically and prophylactically effective /3-hCG peptides.
  • the invention provides for treatment of wasting syndrome by administration of a therapeutic compound (termed herein "Therapeutic") of the invention.
  • Such Therapeutics of the invention include but are not limited to: hCG, ⁇ -hCG , therapeutically and prophylactically effective fractions of a source of native hCG or native /3-hCG, therapeutically and prophylactically effective /3-hCG proteins (i.e., those peptides which prevent or treat wasting syndrome) , related derivatives and analogs of hCG, ⁇ -hCG or ⁇ -hCG peptides, and nucleic acids encoding j ⁇ -hCG and ⁇ -hCG peptides, and analogs and derivatives thereof.
  • ⁇ -hCG peptides which are effective for treatment and prevention of wasting syndromes can be identified by in vitro and in vivo assays such as those described in Section 6.3, infra .
  • a therapeutic composition of the invention comprises a ⁇ -hCG peptide, the amino acid sequence of which consists of amino acid numbers 41-53, 42- 53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, or 48-56 (SEQ ID NOS:8-25 or 33-35, respectively) of Figure 4 (a portion of SEQ ID N0:2) , particularly a ⁇ -hCG peptide which consists of amino acid numbers 41-54, 45-54 or 109-119 (SEQ ID NOS: 3, 4, or 7, respectively) , most preferably of a ⁇ -hCG peptide which consists of amino acid numbers 47-53 (SEQ ID NO:5) or 45-57 (SEQ ID NO:6).
  • a therapeutic composition of the invention comprises a fusion protein comprising more than one ⁇ -hCG sequence, e . g . having an amino acid sequence of one ⁇ -hCG peptide linked via a peptide bond to another ⁇ -hCG peptide, in particular a protein, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO: 6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or an isolated protein of amino acids 47-57 (SEQ ID NO: 28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the ⁇ -hCG sequence depicted in Figure 4 (portions of SEQ ID NO:2), i.e., the amino acid sequence of which
  • the therapeutic comprises a /3-hCG peptide, the amino acid sequence of which consists of circularized (via a disulfide bond between its amino- and carboxy-terminal cysteines) 44-57 (SEQ ID NO:26) with the valine at position 44 substituted with cysteine
  • the therapeutic comprises fractions, preferably fractions of a source of hCG or ⁇ -hCG , such as commercial hCG preparations and human pregnancy urine (preferably early, i.e. first trimester), of material eluting from a gel filtration column, preferably a SUPERDEX 200TM column, with apparent molecular weights of approximately 40 kD, 15 kD or 2-3 kD as determined based on in which fractions compared native hCG dimer (77kD) and ⁇ -hCG core (10 kD) elute.
  • a source of hCG or ⁇ -hCG such as commercial hCG preparations and human pregnancy urine (preferably early, i.e. first trimester)
  • a gel filtration column preferably a SUPERDEX 200TM column
  • the invention provides for treatment or prevention of wasting syndrome by administration of a therapeutic compound (termed herein "Therapeutic”) .
  • “Therapeutics” include, but are not limited to: hCG, 0-hCG, therapeutically and prophylactically effective fractions of a source of native hCG or native ⁇ -hCG (i . e . naturally occurring hCG or ⁇ -hCG and not recombinantly produced hCG or 0-hCG) , and therapeutically and prophylactically effective ⁇ -hCG peptides, i.e., those fractions and peptides which prevent or treat wasting syndrome ⁇ e . g.
  • the methods of the invention can be used for treatment or prevention of any disease or disorder characterized by a loss of body cell mass.
  • Particular conditions that can be treated by methods of the invention include, but are not limited to, wasting associated with viral, such as HIV, bacterial or other types of infections, and sepsis; cachexia associated with cancer, chemotherapy, and radiation therapy; wasting associated with chronic cardiovascular disease; wasting caused by exposure to toxic substances; wasting associated with diarrhea and other gastrointestinal disorders.
  • a Therapeutic of the invention is administered to treat or prevent a wasting syndrome associated with HIV infection. In another preferred embodiment, a Therapeutic of the invention is administered to treat or prevent a wasting syndrome associated with cancer.
  • hCG, g-hCG AND g-hCG PEPTIDES AND DERIVATIVES Native preparations of hCG and ⁇ -hCG can be obtained from a variety of sources. Both hCG and /3-hCG are commercially available (e.g., Sigma Chemical Company) and hCG is commercially available in a form suitable for Therapeutic use in humans (e.g., from Fujisawa, Wyeth-Ayerst Laboratories (APLTM) , Organon, Inc. (PregnylTM) and Serono Laboratories, Inc. (ProfasiTM) ) .
  • APLTM Wyeth-Ayerst Laboratories
  • PregnylTM Organon, Inc.
  • ProfilesiTM Serono Laboratories, Inc.
  • one aspect of the invention relates to assaying preparations of hCG for efficacy in treatment or prevention of wasting syndrome.
  • the therapeutic effectiveness of hCG preparations can be tested by the assays in animal models described in Section 6.3 infra or by any method known in the art. It is preferable to test the hCG preparation in an animal model, such as HIV-1 transgenic mice or SIV infected monkeys, before testing the preparation in humans.
  • a preparation comprising hCG is used that contains not only the hCG heterodimer but also peptide fragments thereof, e.g., ⁇ chain peptides.
  • hCG and /3-hCG can also be purified, or preferably partially purified, from any source known to contain hCG, e.g., urine from pregnant women, using conventional techniques well-known in the art, such as affinity chromatography.
  • affinity chromatography antibodies prepared against hCG or ⁇ -hCG can be used to prepare an affinity chromatography column which can be used to purify the proteins by well-known techniques (see, e.g., Hudson & May, 1986, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdom) .
  • the /3-hCG-related proteins are preferably prepared by any chemical or enzymatic synthesis method known in the art, as described infra .
  • proteins e.g., peptides
  • derivatives the amino acid sequence of which consists of a portion of the ⁇ -hCG sequence ( ⁇ -hCG peptides) , and derivatives thereof, are used to treat or prevent wasting syndrome.
  • the portions of the /3-hCG sequence are at least 3, 5, 10, 20, or 30 amino acids.
  • peptides are preferably ⁇ -hCG peptides, or nucleic acids encoding ⁇ -hCG peptides, from amino acids 41-54, 45-54, 47-53 and 45-57 (SEQ ID NOS: 3-6, respectively) of Figure 4 (a portion of SEQ ID NO:2) .
  • a protein which contains the amino acid sequence of two or more at least 5, 7 or 10 amino acid, non-naturally contiguous portions of the jS-hCG sequence ( Figure 4 (SEQ ID NO:2)) linked by peptide bonds between the N-terminus of one portion and the C-terminus of another portion.
  • a protein is used, the amino acid sequence of which consists of amino acids 45-57 (SEQ ID NO:6) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 109-119 (SEQ ID NO:7) or linked at the N-terminus via a peptide bond to the C-terminus of amino acids 110-119 (SEQ ID NO:27); or a protein is used that has an amino acid sequence of amino acids 47-57 (SEQ ID NO: 28) linked at the C-terminus via a peptide bond to the N-terminus of amino acids 108-119 (SEQ ID NO:29) of the ⁇ -hCG sequence depicted in Figure 4 (portions of SEQ ID NO:2); i.e., the fused peptides represented as 45- 57::109-119, 110-119::45-57, or 47-57::: 108-119 (SEQ ID NOS: 30-32, respectively) .
  • fusion proteins comprising two or more such portions of the ⁇ -hCG sequence are provided; such portions may or may not be contiguous to one another (i.e., an intervening sequence may be present) .
  • Molecules comprising such portions linked by hydrocarbon linkages are also provided.
  • the peptides of the invention (i) have an amino acid sequence consisting of no more than 8 peptides of the / ⁇ -hCG sequence ( Figure 4 (SEQ ID NO:2)) and (ii) comprise amino acid numbers 47-53 (SEQ ID NO:5) of ⁇ -hCG ( Figure 4 (SEQ ID NO:2)).
  • the invention provides an isolated protein which protein (a) comprises one or more portions of the amino acid sequence of /3-hCG, a peptide consisting of said portion(s) being effective to inhibit HIV infection or replication; and (b) lacks ⁇ -hCG amino acids contiguous to said portion (s) .
  • the invention provides an isolated protein (a) comprising a 0-hCG amino acid sequence consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 45-57, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45- 54, 45-55, 45-56, 45-58, 47-54, 47-57, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65, and 48-56 (SEQ ID NO: amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 45-57, 41-53, 42-53, 43-53, 44-53, 44-57, 45-53, 46-53, 45- 54, 45-55, 45-56, 45-58, 47-54, 47-57, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40
  • Analogs of the above-mentioned proteins and peptides which have one or more amino acid substitutions forming a branched peptide e.g., by substitution with an amino acid or amino acid analog having a free amino- or carboxy-side chain that forms a peptide bond with a sequence of one or more amino acids, including but not limited to prolines
  • allowing circularization of the peptide e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally
  • sulfhydryl group for disulfide bond formation are also provided.
  • ⁇ -hCG peptides may have utility in the therapeutic methods of the invention.
  • the utility of /3-hCG peptides may be determined by the in vitro and in vivo assays described in Section 6.3 infra or by any other method known in the art.
  • peptides of less than 50, or less than 25, amino acids are provided.
  • the invention also relates to derivatives, modifications and analogs of /3-hCG peptides.
  • a purified derivative of a protein the amino acid sequence of which protein is selected from the group consisting of amino acid numbers 41-54, 45-54, 47-53, 45-57, 109-119, 41-53, 42- 53, 43-53, 44-53, 44-57, 45-53, 46-53, 45-54, 45-55, 45-56, 45-58, 47-54, 47-55, 47-56, 47-58, 48-145, 58-145, 109-145, 7-40, 46-65 or 48-56 (SEQ ID NOS: 3-25 or 33-35, respectively) as depicted in Figure 4 (a portion of SEQ ID NO: 2) is used to treat or prevent wasting syndrome.
  • ⁇ -hCG peptide derivatives can be made by altering the /3-hCG peptide sequence by substitutions, additions or deletions . that provide for therapeutically effective molecules.
  • the ⁇ -hCG peptide derivatives include peptides containing, as a primary amino acid sequence, all or part of the particular ⁇ - hCG peptide sequence including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a peptide which is functionally active.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • amino acids within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Such ⁇ -hCG peptide derivatives can be made either by chemical peptide synthesis or by recombinant production from a nucleic acid encoding the ⁇ -hCG peptide which nucleic acid has been mutated. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C. , et al., 1978, J . Biol . Chem 253:6551) , use of TAB ® linkers (Pharmacia) , etc.
  • 0-hCG peptides and analogs and derivatives of ⁇ -hCG peptides can be chemically synthesized.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., see Creighton, 1983, Proteins, Structures and Molecular
  • ⁇ -hCG peptides can also be synthesized by use of a peptide synthesizer.
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H. Freeman and Co., N.Y. , pp. 34-49) .
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the ⁇ -hCG peptide.
  • Non- classical amino acids include but are not limited to the D- isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, fluoro-amino acids, designer amino acids such as / S-methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • amino acid can be D (dextrorotary) or L (levorotary) .
  • peptides of the invention can be chemically synthesized and purified as follows: Peptides can be synthesized by employing the N- ⁇ -9-fluorenylmethyloxycarbonyl or Fmoc solid phase peptide synthesis chemistry using a Rainin Symphony Multiplex Peptide Synthesizer.
  • the standard cycle used for coupling of an amino acid to the peptide-resin growing chain generally includes: (1) washing the peptide-resin three times for 3C seconds with N,N-dimethylformamide (DMF) ; (2) removing the Fmoc protective group on the amino terminus by deprotection with 20% piperidine in DMF by two washes for 15 minutes each, during which process mixing is effected by bubbling nitrogen through the reaction vessel for one second every 10 seconds to prevent peptide-resin settling; (3) washing the peptide- resin three times for 30 seconds with DMF; (4) coupling the amino acid to the peptide resin by addition of equal volumes of a 250 mM solution of the Fmoc derivative of the appropriate amino acid and an activator mix consisting or 400 mM N-methylmorpholine and 250 mM (2-(lH-benzotriazol-1-4) ) - 1, 1, 3,3-tetramethyluronium hexafluorophosphate (HBTU) in DMF;
  • This cycle can be repeated as necessary with the appropriate amino acids in sequence to produce the desired peptide. Exceptions to this cycle program are amino acid couplings predicted to be difficult by nature of their hydrophobicity or predicted inclusion within a helical formation during synthesis. For these situations, the above cycle can be modified by repeating step 4 a second time immediately upon completion of the first 45 minute coupling step to "double couple" the amino acid of interest. Additionally, in the first coupling step in peptide synthesis, the resin can be allowed to swell for more efficient coupling by increasing the time of mixing in the initial DMF washes to three 15 minute washes rather than three 30 second washes.
  • the peptide can be cleaved from the resin as follows: (1) washing the peptide-resin three times for 30 seconds with DMF; (2) removing the Fmoc protective group on the amino terminus by washing two times for 15 minutes in 20% piperdine in DMF; (3) washing the peptide-resin three times for 30 seconds with DMF; and (4) mixing a cleavage cocktail consisting of 95% trifluoroacetic acid (TFA), 2.4% water, 2.4% phenol, and 0.2% triisopropysilane with the peptide- resin for two hours, then filtering the peptide in the cleavage cocktail away from the resin, and precipitating the peptide out of solution by addition of two volumes of ethyl ether.
  • TFA trifluoroacetic acid
  • the ether-peptide solution can be allowed to sit at -20°C for 20 manures, then centrifuged at 6,000xG for 5 minutes to pellet the peptide, and the peptide can be washed three times with ethyl ether to remove residual cleavage cocktail ingredients.
  • the final peptide product can be purified by reversed phase high pressure liquid chromatography (RP-HPLC) with the primary solvent consisting of 0.1% TFA and the eluting buffer consisting of 80% acetonitrile and 0.1% TFA. The purified peptide can then be lyophilized to a powder.
  • RP-HPLC reversed phase high pressure liquid chromatography
  • the invention provides a peptide with branched amino acids (branched peptide) , preferably a branched peptide of amino acids 45-57 (SEQ ID NO:6) with branches occurring at positions 47 and 51, respectively, instead of the Gly and Ala residues normally present.
  • diaminobutyric acid is substituted for the gly and ala residues at positions 47 and 51, respectively, and proline bonded to both diaminobutyric acid residues (45-57 branched) as shown in Figure 5A.
  • branched versions of the ⁇ -hCG peptides listed hereinabove are provided, e . g . , by substituting one or more amino acids within the ⁇ -hCG sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a "branch") .
  • Branched peptides may be prepared by any method known in the art for covalently linking any naturally occurring or synthetic amino acid to any naturally occurring or synthetic amino acid in a peptide chain which has a side chain group able to react with the amino or carboxyl group on the amino acids so as to become covalently attached to the peptide chain.
  • amino acids with a free amino side chain group such as, but not limited to, diaminobutyric acid, lysine, arginine, ornithine, diaminopropionic acid and citrulline, can be incorporated into a peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free amino side group, from that residue.
  • amino acids with a free carboxyl side chain group such as, but not limited to, glutamic acid, aspartic acid and homocitrulline
  • glutamic acid aspartic acid and homocitrulline
  • amino acids with a free carboxyl side chain group can be incorporated into the peptide so that an amino acid can form a branch therewith, for example, by forming a peptide bond to the free carboxyl side group, from that residue.
  • the amino acid forming the branch can be linked to a side chain group of an amino acid in the peptide chain by any type of covalent bond, including, but not limited to, peptide bonds, ester bonds and disulfide bonds.
  • amino acids, such as those described above, that are capable of forming a branch point are substituted for / ⁇ -hCG residues within a peptide having a j ⁇ -hCG sequence.
  • Branched peptides can be prepared by any method known in the art.
  • branched peptides can be prepared as follows: (1) the amino acid to be branched from the main peptide chain can be purchased as an N- ⁇ -tert-butyloxycarbonyl (Boc) protected amino acid pentafluorophenyl (Opfp) ester and the residue within the main chain to which this branched amino acid will be attached can be an N-Fmoc- ⁇ - ⁇ -diaminobutyric acid; (2) the coupling of the Boc protected amino acid to diaminobutyric acid can be achieved by adding 5 grams of each precursor to a flask containing 150 ml DMF, along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing the solution to mix for 24 hours; (3) the peptide can then be extracted from the 150 ml coupling reaction by mixing the reaction with 400 ml dichlormethane (DCM) and
  • DCM dichlorme
  • Branched peptides prepared by this method will have a substitution of diaminobutyric acid at the amino acid position which is branched.
  • Branched peptides containing an amino acid or amino acid analog substitution other than diaminobutyric acid can be prepared analogously to the procedure described above, using the N-F-moc coupled form of the amino acid or amino acid analog.
  • the peptide is a cyclic peptide, preferably a cyclic peptide of ⁇ -hCG amino acids 44- 57 (SEQ ID NO:26) with cysteine substituted for valine at position 44 and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 (C[V44C] 45-57) ( Figure 5B) , or a cyclic fused peptide of j ⁇ -hCG amino acids 110-119 (SEQ ID NO:27) linked at the C-terminus by a peptide bond to the N-terminus of amino acids 45-57 (SEQ ID NO:6) and circularized via a disulfide bond between the cysteine residues at positions 110 and 57.
  • the peptide is a cyclic branched peptide of ⁇ -hCG amino acids 44-57 (SEQ ID NO: 12) with cysteine substituted for valine at position 44 and circularized via a disulfide bond between the cysteine residues at positions 44 and 57 and positions 47 and 51 substituted with a diaminobutyric acid residue on which a proline is peptide bonded to its free amino sidechain.
  • Cyclization can be, for example, but not by way of limitation, via a disulfide bond between two cysteine residues or via an amide linkage.
  • disulfide bridge formation can be achieved by (1) dissolving the purified peptide at a concentration of between 0.1.-0.5 mg/ml in 0.01 M ammonium acetate, pH 7.5;
  • cysteine residues can be introduced at the amino-terminus and/or carboxy-terminus and/or internally such that the peptide to be cyclized contains two cysteine residues spaced such that the residues can form a disulfide bridge.
  • a cyclic peptide formed by an amide linkage can be obtained by, for example but not limited to, the following procedure:
  • An allyl protected amino acid such as aspartate, glutamate, asparagine or glutamine, can be incorporated into the peptide as the first amino acid, and then the remaining amino acids are coupled on.
  • the allyl protective group can be removed by a two hour mixing of the peptide-resin with a solution of tetrakistriphenylphophine palladium (0) in a solution of chloroform containing 5% acetic acid and 2.5% N-methylmorpholine.
  • the peptide resin can be washed three times with 0.5% N,N-diisopropylethylamine (DIEA) and 0.5% sodium diethyldithiocabamate in DMF.
  • DIEA N,N-diisopropylethylamine
  • the amino terminal Fmoc group on the peptide chain can be removed by two incubations for 15 minutes each in 20% piperdine in DMF, and washed three times with DMF for 30 seconds each.
  • the activator mix, N-methylmorpholine and HBTU in DMF can be brought onto the column and allowed to couple the free amino terminal end to the carboxyl group generated by removal of the allyl group to cyclize the peptide.
  • the peptide can cleaved from the resin as described in the general description of chemical peptide synthesis above and the peptide purified by reverse phase-high pressure liquid chromatography (RP-HPLC) .
  • RP-HPLC reverse phase-high pressure liquid chromatography
  • an allyl protected amino acid can be introduced into the sequence of the peptide, at the amino- terminus, carboxy-terminus or internally, such that the peptide can be cyclized.
  • ⁇ -hCG peptides can also be obtained by recombinant expression techniques. (See, e.g., Sambrook et al., 1989., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 2d Ed.
  • nucleic acid sequence encoding the ⁇ -hCG peptide is operatively linked to a promoter such that the /3-hCG peptide is produced from said sequence.
  • a vector can be introduced into a cell, within which cell the vector or a portion thereof is expressed, producing the ⁇ -hCG peptide.
  • the nucleic acid is DNA if the source of RNA polymerase is DNA-directed RNA polymerase, but the nucleic acid may also be RNA if the source of polymerase is RNA- directed RNA polymerase or if reverse transcriptase is present in the cell or provided to produce DNA from the RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in bacterial or mammalian cells.
  • Expression of the sequence encoding the 0-hCG peptide can be by any promoter known in the art to act in bacterial or mammalian cells. Such promoters can be inducible or constitutive.
  • Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310) , the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 11:787-797) , the HSV-1 (herpes simplex virus-1) thymidine kinase promoter (Wagner et al., 1981, Proc . Natl . Acad . Sci .
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al . , 1984, Cell 3J3: 639-646; Ornitz et al. , 1986, Cold Spring Harbor Symp. Quant . Biol .
  • beta-globin gene control region which is active in erythroid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 4_6, 89-94)
  • myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell _ ⁇ 8 :703-712)
  • myosin light chain- 2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314 :283-286)
  • gonadotropin releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234 : 1372-1378) .
  • the promoter element which is operatively linked to the nucleic acid encoding the ⁇ -hCG peptide can also be a bacteriophage promoter with the source of the bacteriophage RNA polymerase expressed from a gene for the RNA polymerase on a separate plasmid, e.g., under the control of an inducible promoter, for example, a nucleic acid encoding the / 3-hCG peptide operatively linked to the T7 RNA polymerase promoter with a separate plasmid encoding the T7 RNA polymerase.
  • peptides can be obtained by proteolysis of hCG or ⁇ -hCG followed by purification using standard techniques such as chromatography (e.g., HPLC), electrophoresis, etc.
  • ⁇ - hCG peptide derivatives which are differentially modified during or after synthesis, e . g . , by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
  • the peptides are acetylated at the N-terminus and/or amidated at the C-terminus. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
  • the /3-hCG peptide derivative is a chimeric, or fusion, protein comprising a functional /3-hCG peptide (or two or more portions of /3-hCG joined by peptide bond(s)) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a / 3-hCG-coding sequence joined in- frame to a coding sequence for a different protein) .
  • a nucleic acid encoding the protein comprising a / 3-hCG-coding sequence joined in- frame to a coding sequence for a different protein.
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • such a chimeric product may be made by protein synthetic techniques, e . g . , by use of a peptide synthesizer.
  • a fraction, particularly a size fraction, of a source of hCG or ⁇ -hCG active in treating or preventing wasting syndrome is used to treat or prevent wasting syndrome.
  • the utility of /3-hCG peptides and fractions of hCG and ⁇ -hCG sources may be determined by the in vitro and in vivo assays described in Section 6.3 infra or by any other method known in the art.
  • the present inventors have found that different preparations of native hCG and ⁇ -hCG have variable effects on HIV infections and Kaposi's Sarcoma both in vitro and in vivo and on wasting syndromes in animal models. Specifically, the inventors found that among the commercial preparations of (non-recombinant) hCG they investigated, hCG from Fujisawa was the most effective, hCG APLTM (Wyeth-Ayerst) the next most effective, and PREGNYLTM (Organon) the next most effective in inhibiting HIV infection and replication. A highly purified hCG preparation and recombinant /3-hCG were found not to be active against HIV infection and Kaposi's Sarcoma cell growth in vitro .
  • APLTM active hCG preparation
  • human early (i.e. first trimester) pregnancy urine have anti-HIV activity, anti-KS activity, and activity against wasting syndrome in transgenic mice, as described infra in Section 8.
  • hCG preparations and fractions of hCG preparations can be screened for efficacy in treating or preventing HIV infection by the. assays described in Sections 6.3, 7 and 8 infra or by any method known in the art.
  • the invention provides a first composition comprising one or more first components of a second composition comprising native hCG or native /3-hCG, said first components being separated from other components of the hCG sample, said first components being active to inhibit Kaposi's sarcoma, and said second composition being active to inhibit Kaposi's sarcoma, and said native hCG or native ⁇ -hCG not being purified to homogeneity in said second composition.
  • the invention provides a composition comprising components which have been separated from other components of the native hCG or native 0-hCG sample by sizing column chromatography, preferably where the components elute from a gel filtration, preferably a
  • SUPERDEXTM 200 sizing column with an apparent approximate molecular weight of 40 kD, 14 kD or 2-3 kD as determined relative to the elution of a native hCG heterodimer, having a molecular weight of 77 kD, and a ⁇ -hCG core protein ( ⁇ -hCG amino acids 6-40 linked via a disulfide bond to ⁇ -hCG amino acids 55-92, as depicted in Figure 4 (SEQ ID NO:2)), having a molecular weight of 10 kD.
  • hCG human immunodeficiency virus
  • proteins proteins
  • ⁇ -hCG having anti-HIV, anti-KS and/or anti-wasting activity
  • the source of hCG of ⁇ -hCG can be fractionated using any technique available in the art for the separation and isolation of molecules, for example but not limited to, sizing chromatography, ion-exchange chromatography, affinity chromatography, etc.
  • urine can be prepared for fractionation as follows:
  • Urine is collected and stored either frozen or refrigerated for not more than two (2) days. Then, sodium azide is then added at a concentration of 1 gram/liter and the sample is stored frozen until sufficient material is collected for the fractionation. At this point, the urine is thawed over night, the pH adjusted to 7.2 to 7.4 with sodium hydroxide and then centrifuged to remove any precipitate (alternatively, the precipitate can be allowed to sediment, e.g., for 1 hour at room temperature, approximately 75% of the supernatant is decanted, the remainder of the supernatant and the precipitate is centrifuged to pellet the precipitate, and the supernatant decanted and added to the first volume of decanted supernatant) .
  • the precipitate can be allowed to sediment, e.g., for 1 hour at room temperature, approximately 75% of the supernatant is decanted, the remainder of the supernatant and the precipitate is centrifuged to pellet the precipitate, and the supernatant
  • the urine is then filtered through, e.g., a 45 micron filter to remove any remaining particulate matter.
  • the urine is concentrated using any concentration method available in the art which does not remove higher molecular weight material, e.g., material larger than 3,000 daltons in molecular weight; for example, the material may be concentrated using a Pellicon (Millipore) filtration system with a membrane filter cassette having a molecular weight cut off of 3,000 daltons. Concentration with the Pellicon filtration system using the 3,000 molecular weight membrane filter cut off concentrates 30 liters of urine to 500 ml (i.e., a 60-fold concentration) overnight.
  • Pellicon Micropore
  • the concentrate can then be passed over a column containing a large volume of Sephadex G25 resin in 0.05 M ammonium bicarbonate (for example, 250 ml of the concentrate can be passed over a column of approximately 1.7 liters, washing the column with 25% ethanol between runs to remove adsorbed lipids and glycoprotein) .
  • the resulting desalted and delipidated urine concentrate is then lyophilized.
  • the lyophilized urine material or commercial hCG preparation (or any source of hCG or 0-hCG) is resuspended in either phosphate buffered saline (PBS— 30 mM sodium phosphate buffer, pH 8.3) or in 0.10 M ammonium bicarbonate at a concentration and in a volume appropriate for the column upon which the sample will be loaded, for example, but not limited to 0.5 grams of protein in 6 ml (i.e., approximately 83 mg/ml) . It is within the skill of the skilled artisan to determine the concentration and volume of the sample to be subjected to fractionation.
  • PBS phosphate buffered saline
  • ammonium bicarbonate 0.10 M ammonium bicarbonate
  • the sample can then be fractionated by any method known in the art for the separation of proteins.
  • a preferred method is high resolution gel filtration on a Pharmacia pre- packed SUPERDEXTM 200 column (26/60) by HPLC using any available HPLC apparatus, e.g., with a Hewlett Packard 1050 HPLC equipped with a photodiode array detector.
  • the resuspended sample is loaded onto the column in 30 mM phosphate buffer, pH 8.3, and the material can then be eluted from the column with 30 mM sodium phosphate buffer, pH 7.0; 2M NaCl.
  • Fractionation can also be performed using other types of chromatography matrices e.g., heparin, DEAE- cellulose.
  • the column chromatography can also be run using any method available in the art, e.g., standard gravity flow or low pressure chromatography, FPLC, or reverse phase HPLC.
  • any method known in the art such as but not limited to, those described in sections 6.3, 7, and 8 infra , can be used to determine which fractions have anti-HIV activity and/or anti-KS activity and/or a pro-hematopoietic effect.
  • the apparent molecular weight of material in the fractions can be determined by the relative elution of those fractions compared with the elution of specific hCG and ⁇ -hCG species having a known molecular weight or with the elution of known protein size markers.
  • proteins elute from a sizing column as a function of their molecular weight.
  • the elution of, for example, hCG and the ⁇ -hCG core protein can be determined by assaying the column chromatography fractions for the presence of hCG and the j ⁇ -hCG core protein, or any hCG or /3-hCG species, by any immunoassay technique available in the art, such as radioimmunoassays (either liquid or solid phase) , enzyme- linked assays or ELISA assays.
  • Antibodies can be generated against hCG or the /3-hCG core protein by any method known in the art. Preparation of monoclonal antibodies against hCG and ⁇ -hCG species have been described in the art, see, e .g . , O'Connor et al., 1994, Endocrine Reviews 15:650-683; Krichevsky et al, 1991, Endocrinology 128:1255-1264; and Krichevsky et al. , 1988, Endorcrinology 123:584-593.
  • hCG human immunological response
  • hCG human immunological response
  • Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete) , mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) , and the EBV-hybridoma technique to produce human monoclonal antibodies Colde et al. , 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) .
  • Monoclonal cells lines can then be screened for binding to the particular hCG or /3-hCG species using the purified species in any type of immunoassay available in the art (see, e . g . , Erlich et al., 1985, Am. J . Reprod Immunol . Microbiol . .8:48) .
  • the fractions can then be assayed for the presence of the hCG or ⁇ -hCG species using a monoclonal antibody specific for the hCG or ⁇ -hCG species.
  • the assay can be performed by any method known in the art.
  • an immunoradiometric assay can be used (Krichevsky et al., 1988, Endocrinology 123:584-593) . Briefly, the IRMA assay is performed by adsorbing an antibody against the hCG or j ⁇ -hCG species onto the surface of wells of a microtiter plate by incubation in a coating buffer (0.2 M sodium bicarbonate, pH 9.5) overnight at 4°C. The residual non- specific binding sites are blocked by the addition of a 1% bovine serum albumin solution (with 0.1% sodium azide) to the wells for 3 hours at room temperature, and the wells of the microtiter plate are then washed with deionized water.
  • a coating buffer 0.2 M sodium bicarbonate, pH 9.5
  • nucleic acids comprising a sequence encoding hCG, ⁇ -hCG or a 0-hCG peptide or a protein derivative thereof, are administered for treatment or prevention of wasting syndrome, by way of gene therapy.
  • Gene therapy refers to therapy performed by the administration of a nucleic acid to a subject.
  • the nucleic acid produces its encoded protein that mediates a therapeutic effect by preventing or treating wasting syndrome.
  • any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
  • a nucleic acid encoding a /3-hCG peptide is part of an expression vector that produces the ⁇ -hCG peptide in a suitable host.
  • a nucleic acid has a promoter operably linked to the nucleic acid sequence, coding for the ⁇ -hCG peptide, said promoter being inducible or constitutive, and, optionally, tissue- specific.
  • a nucleic acid molecule is used in which the ⁇ -hCG sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the hCG nucleic acid (Roller and Smithies, 1989, Proc . Natl . Acad . Sci . USA 8_6:8932-8935; Zijlstra et al., 1989, Nature 342:435-438) .
  • Delivery of the nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then administered to the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
  • the nucleic acid is directly administered in vivo , where it is expressed to produce the encoded product.
  • This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating with lipids or cell-surface receptors or transfecting agents encapsulation in liposomes, microparticles, or microcapsules, or by administering it in linkage to a peptide which is known to enter the cell or nucleus, e.g., by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see e.g., Wu and Wu, 1987, J . Biol . Chem .
  • the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications W092/06180 dated April 16, 1992 (Wu . et al.); W092/22635 dated December 23, 1992 (Wilson et al.); WO92/20316 dated November 26, 1992 (Findeis et al.);
  • a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
  • the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc . Natl . Acad . Sci . USA ⁇ 6:8932-8935; Zijlstra et al., 1989, Nature 342:435-438) .
  • a viral vector that contains the nucleic acid sequence encoding ⁇ -hCG or a ⁇ -hCG peptide is used.
  • a retroviral vector can be used (see Miller et al., 1993, Meth . Enzymol . 217:581-599) . These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome. Retroviral vectors are maintained in infected cells by integration into genomic sites upon cell division. The nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
  • retroviral vectors More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6_:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
  • Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin . Invest . £1:644-651; Kiem et al., 1994, Blood 8_3:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4_:129-141; and Grossman and Wilson, 1993, Curr. Opin . in Genetics and Devel . 1:110-114.
  • Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus- based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 1:499-503 present a review of adenovirus-based gene therapy.
  • Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc . Soc . Exp. Biol . Med . 204 :289-300. )
  • Herpes viruses are other viruses that can also be used.
  • Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
  • the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
  • the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
  • introduction can be carried out by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
  • Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth . Enzymol . 217:599-618; Cohen et al., 1993, Meth. Enzymol .
  • recombinant cells can be delivered to a patient by various methods known in the art.
  • epithelial cells are injected, e.g., subcutaneously.
  • recombinant skin cells may be applied as a skin graft onto the patient.
  • Recombinant blood cells e.g., hematopoietic stem or progenitor cells
  • Recombinant blood cells are preferably administered intravenously.
  • the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
  • a nucleic acid sequence coding for hCG, ⁇ -hCG or 0-hCG peptide is introduced into the cells such that it is expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
  • stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention.
  • the invention further provides methods of testing the efficacy of Therapeutics of the invention for treatment or prevention of wasting syndrome.
  • the Therapeutics of the invention are preferably tested in an animal model prior to use in humans.
  • Specific embodiments provide a method for screening a preparation comprising hCG or an hCG a chain or hCG ⁇ chain or a derivative of hCG or of said alpha or beta chain or a fraction of native hCG or native 0-hCG, for anti-wasting activity comprising assaying said preparation for the ability to promote weight gain in an animal model that exhibits a wasting syndrome.
  • the hCG preparation is screened by a method comprising measuring the weight of an offspring of an HIV-1 transgenic mouse, which offspring has been exposed to the preparation; and comparing the weight of the offspring which has been exposed to the preparation with the weight of an offspring not so exposed, wherein a greater weight in said exposed offspring indicates that the preparation has anti-wasting activity.
  • the hCG preparation is screened by a method comprising measuring the change in weight of an SIV infected monkey which has been exposed to the preparation; and comparing the change in weight of the monkey which has been exposed to the preparation to the change in weight of a monkey which has not been so exposed, wherein a greater weight increase or smaller weight loss in said exposed monkey indicates that the preparation has anti-wasting activity.
  • a Therapeutic of the invention can be assayed in mice transgenic for HIV-l, e.g., mice which have integrated molecular clone pNL4-3 containing 7.4 kb of the HIV-l proviral genome deleted in the gag and pol genes
  • mice exhibit cachexia and growth retardation (Franks, R.R. , et al., 1995, Pediatric Res . 17:56-63) .
  • a Therapeutic which reverses the cachexia and growth retardation in the HIV transgenic mice is predicted to have utility for treatment or prevention of wasting syndromes.
  • the efficacy of Therapeutics of the invention can also be assayed in SIV infected rhesus monkeys (see Letrin, N.L., and King, N.W. , 1990, J. AIDS 1:1023-1040), particularly rhesus monkeys infected with SIV mac251 , which SIV strain induces a syndrome in experimentally infected monkeys which is very similar to human AIDS and results in weight loss in the infected monkeys (Kestler, H. , et al., 1990, Science 248:1109-1112) .
  • monkeys are infected with cell free SIV mac251 , for example, with virus at a titer of 10 4 - 5 TCID S0 /ml and SIV infection is monitored by the appearance of SIV p27 antigen in PBMCs.
  • An increase in the weight of infected monkeys indicates that the Therapeutic has utility in the treatment of wasting syndrome.
  • Compounds for use in therapy can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used.
  • the utility of the Therapeutic can be determined in human subjects. Improvement in wasting syndrome, i . e . and increase in body cell mass, can be assessed by any well known clinical techniques available in the art. For example but not limited to, measuring body weight, determination of total body potassium content, determination of intracellular water volume, bioelectrical impedance analysis, anthropometries and determination of total body nitrogen content (see, e . g . , Kotler, D.P. et al., 1985, Am. J . Clin . Nutr . 11:1255-65; Ott, M. et al., 1993, Am . J . Clin . Nutr . 52:15-19; Miller, T.L. et al., 1993, Am . J . Clin . Nutr . 5J7:588-592) . Therapeutics, the administration of which increases body weight or cell mass, should have utility in treatment of wasting syndrome.
  • the invention provides methods of treatment and prevention by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the subject is preferably an animal, including, but not limited to, animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • the subject. is a human not afflicted with a cancer which secretes hCG or hCG fragments and, more particularly, not afflicted with Kaposi's Sarcoma.
  • a Therapeutic of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the Therapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a Therapeutic nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved, for example and not by way of limitation, by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the Therapeutic can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ijbid.).
  • a liposome see Langer, Science 249:1527-1533 (1990); Treat et al. , in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ijbid.).
  • the Therapeutic can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra ; Sefton, CRC Crit . Ref . Biomed . Eng. 14_:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N . Engl . J. Med . 321:574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984) ; Ranger and Peppas, J . Macromol . Sci . Rev . Macromol . Chem . 2_3:61 (1983) ; see also Levy et al., Science 228: 190 (1985) ; During et al., Ann. Neurol . 1 . 5:351 (1989); Howard et al., J. Neurosurg. 21:105 (1989)) .
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see , e . g. , Goodson, in Medical Applications of Controlled Release, supra , vol. 2, pp. 115-138 (1984)).
  • Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)) .
  • the nucleic acid can be administered by gene therapy methods as described supra Section 6.2.4.
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals» and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the Therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the. composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the Therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. For example but not by way of limitation, therapy of at least 15,000 I.U. and up to 45,000 I.U. hCG weekly was shown to be effective and well tolerated in humans. Weekly doses of 6,000 I.U. in monkeys and 300-500 I.U. in mice were also shown to be effective.
  • Suitable doses of a 0-hCG peptide predicted to be effective for treatment of wasting syndrome in a human include, but are not limited to, 1 to 1000 micrograms of peptide per week, administered, for example but not limited to, intramuscularly, subcutaneously or intravenously. Effective doses may be extrapolated from dose-response curves derived from or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • hCG Human Chorionic Gonadotropin
  • ⁇ and ⁇ which associate noncovalently to form a heterodimer which embodies its hormonal activity.
  • some partially purified preparations of hCG, ⁇ - hCG, and / S-hCG peptides prevent wasting associated with HIV infection. Specifically, in the presence of some preparations of hCG, ⁇ -hCG , or ⁇ -hCG peptide, the fatal wasting of homozygous transgenic mouse offspring is reversed.
  • the HIV-l transgenic mice used for this study contain 7.4 kb of foreign DNA, including 5.1 kb of the HIV-l proviral genome deleted in the gag and pol genes and 2.3 kb of vector (Dickie et al., 1991, Virology 185:109-119).
  • the birth weights of mice homozygous for the HIV-l transgene are normal, but soon the mice uniformly display severe growth retardation (Figure 1A) , cachexia, and early mortality from expression of HIV-l genes (env and regulatory genes) which are highly expressed shortly after birth in homozygotes (Franks et al., 1995, Pediatric Res . 3_7:56-63) ( Figure IB).
  • mice develop severe hyperkeratotic skin lesions with marked expression of gpl20 and nef genes (Kopp et al., 1993, AIDS Res . Hum . Retroviruses £:267-275; Vasli et al., 1994, AIDS Res . Hum . Retroviruses 10:1241-1250).
  • hCG preparations were administered a commercial preparation of native hCG (APLTM, Wyeth Ayerst) (300-500 I.U.) , and the mothers of other HIV-l transgenic mice received other commercial native hCG preparations (PREGNYLTM and Sigma (G10) , ⁇ -chain preparations and partially purified native ⁇ -hCG and recombinant ⁇ -hCG preparations (all Sigma) (50-100 ⁇ g) .
  • heterozygous transgenic mothers of 6 homozygous transgenic mice were given 10 ⁇ g of /?-hCG peptide 45-57 (SEQ ID NO:6) where the amino acid residues at positions 47 and 51 are substituted by a branch, where the branches are made up of diaminobutyric acid and proline (branched j ⁇ -hCG 45-57) (prepared by Dr. N. Ambulos, UMAB) subcutaneously, daily for 10 days.
  • Other studies were carried out with other /3-hCG peptides (see Table 1) .
  • Heterozygous transgenic mothers were treated with the hCG preparation subcutaneously twice weekly. Pups received hCG via the mother's milk. Blood levels of hCG in the mother and pups were monitored and ranged from 5 IU/ml to over 150 IU/ml over a 96 hour time period (data not shown) .
  • RNA was reverse transcribed into cDNA using random hexamer primers and MMTV reverse transcriptase (Life Technologies, MD) in a final volume of 30 ⁇ l.
  • MMTV reverse transcriptase (Life Technologies, MD)
  • One tenth of the cDNA reactions were used for PCR amplification of various HIV gene sequences (env, tat, rev, nef and vif) .
  • GPHD glyceraldehyde 3-phosphate dehydrogenase
  • PCR product was resolved by electrophoresis through 2% agarose gels and processed for Southern hybridization using FITC-labeled oligonucleotide probes complementary to internal sequences of the amplicons. Detection was performed by chemiluminescence (Amersham) and relative mRNA levels determined by densitometry after normalization with GAPDH mRNA levels.
  • the hCG (APLTM) treatments resulted in marked down regulation of HIV-l gene expression in skin biopsies as determined by the RT-PCR technique ( Figure ID) .
  • the 25 cycles of amplification employed in these experiments readily detected abundantly expressed genes (e.g., env and rev) while the tat gene in treated animals was at low levels, and more readily detected with more cycles of amplification (not shown) .
  • Other skin biopsies were examined for HIV viral proteins using mouse monoclonal antibodies against gpl20 and Nef by an immunostaining technique. A marked decrease in viral proteins occurred after 2 weeks treatment and no detectable HIV proteins were found after 30 days of hCG treatment (not shown) and the hyperkeratosis of the skin regressed. When the treatment was halted, reappearance of viral protein expression occurred after 2 weeks (not shown) .
  • the "HIV: in vitro” column reports results from assays of the inhibition of HIV-l replication in vitro (HIV-l strains and HIV-l primary isolates) as described infra sections 7.4 and 7.6.
  • the "HIV transgenic mice” column reports data from the inhibition of HIV RNA and protein expression in HIV-l transgenic mice as described in section 7.1.
  • Column 5 provides data on the relative increase of hematopoietic colony cell number in vitro clonogenic assays as described in section 7.8.
  • the commercial hCG preparations tested were APLTM (Wyeth Ayerst) , PREGNYLTM (Organon) , ORGANON (a highly purified preparation obtained from Organon) , PROFASITM (Serono) , Goldline, STERISTM, and Shein, and two preparations from Sigma, Sigma 1 (GHO) and
  • the hCG preparations CR127 and CR1XY17V are highly purified hCG preparations and CR1XY17B is a mixture of highly purified ⁇ -hCG and ⁇ -hCG , all three preparations were obtained from the National Institute of Child Health and Human Development (NICHD) at the National Institute of Health (NIH) and the rhCG is recombinant hCG expressed in a mouse cell line (Sigma) .
  • NICHD National Institute of Child Health and Human Development
  • NIH National Institute of Health
  • rhCG is recombinant hCG expressed in a mouse cell line (Sigma) .
  • ⁇ hCG and /ShCG are purified native subunits (Sigma) ; “r ⁇ hCG” and “r ⁇ hCG” all the recombinant subunits expressed in mouse cells (Sigma) ; and ⁇ fpl769A is purified, native ⁇ subunit (NICHD, NIH) .
  • the peptide "scrambled Al” has the sequence Cys-Val-Ala-Gln-Pro- Gly-Pro-Gln-Val-Leu-Leu-Val-Leu-Cys (SEQ ID NO:36) and "Scrambled A2" has the sequence Cys-Val-Ala-Gln-Gly-Val-Leu- Pro-Ala-Leu-Pro-Gln-Val-Val-Cys (SEQ ID NO: 37).
  • "Scrambled Al/B” has the sequence ⁇ -hCG amino acids 45-57 (SEQ ID NO:6) and 109-119 (SEQ ID NO:7) which has been scrambled.
  • Trimers is a mixture of tripeptides from the ⁇ -hCG sequence of amino acids 45-57: Leu-Gln-Gly, Leu-Gin-Pro, Gln-Gly-Val, Gln-Pro-Val, Gln-Val-Leu, Val-Leu-Pro, Leu-Pro-Ala, Leu-Pro- Pro, Pro-Ala-Leu, Pro-Pro-Leu, Ala-Leu-Pro, Pro-Leu-Pro, Leu- Pro-Gln, Pro-Gln-Val, Gln-Val-Val, and Val-Val-Cys (SEQ ID NOS: 38-53, respectively) .
  • Peptides were synthesized by Dr. N. Ambulos (University of Maryland Biomedicine Center) , Becham (CA) or Peptide Technologies Corp. (Gaithersburg, MD) .
  • SIV and HIV-l are similar in many of their biological and physical properties including their genomic structure.
  • SIV mac251 unlike several other SIV isolates, induces a syndrome in experimentally infected rhesus macaques that is similar to human AIDS (Kestler et al., 1990, Science 248:1109-1112) .
  • the protocol involved intralesional administration of 500 IU hCG (PREGNYLTM) to 4 lesions for 2 weeks, followed by subcutaneous administration of 2,500 IU hCG (PREGNYLTM) 5 days per week for 4 to 6 weeks. Additional systemic intramuscular or subcutaneous hCG treatment with either PREGNYLTM, APLTM, or STERISTM (one patient) was provided as ongoing therapy in some patients or as part of compassionate use protocols.
  • AIDS patients treated with hCG therapy were tested for increases in CD4* T cell levels (in numbers of cells per mm 3 ) and decrease in viral load by one of the following assays for determining viral load: NASBA (Louache, et al., 1992, Blood 180:2991-2999; Geller, et al., 1985, Ar ch s . Path . Lab . Met .
  • hCG APLTM With-Ayerst
  • hCG APLTM With-Ayerst
  • Both human early pregnancy urine and the APLTM (Wyeth-Ayerst) hCG commercial preparation were subjected to fractionation.
  • human early pregnancy urine 5 liters of urine were collected from women in the first trimester of pregnancy. Twenty-four hour collections were stored frozen or refrigerated for up to 2 days.
  • sodium azide was added at 1 g/liter and the urine frozen until five liters had been collected. At this time, all the urine was thawed overnight, and the pH was adjusted to 7.2-7.4 with NaOH, which causes some precipitation.
  • the precipitate was allowed to sediment for 1 hour at room temperature, most of the supernatant decanted and the remaining supernatant centrifuged to remove any additional precipitate with that supernatant being added to the first supernatant decanted.
  • the urine was concentrated with a Pellicon (Millipore) filtration system using a membrane cassette with a 3,000 MW cut off, which concentrates the urine approximately 60 to 80 fold.
  • the urine was desalted and delipidated by passing 500 ml of the material at a time through a Sephadex G25 column with a volume of 1.7 liters in 0.05 M ammonium bicarbonate (the column was washed between runs with 25% ethanol to remove absorbed lipids and glycoprotein) .
  • the material was lyophilized and stored for further fractionation.
  • the urinary material was then reconstituted in 6 ml of 30 mM sodium phosphate buffer, pH 8.3 fractionation.
  • the lyophilized hCG preparation from eleven vials was resuspended in 6 ml of 30 mM sodium phosphate buffer, pH 8.3 and filtered twice through the 0.45 ⁇ m particle filter.
  • the prepared sample was then loaded onto a pre-packed SUPERDEXTM 200 HiLoad Column (Pharmacia 26 mm 2 x 60 cm) in the 30 mM sodium phosphate buffer, pH 8.3 and then eluted from the column with a solution containing 30 mM sodium phosphate buffer, pH 7.0 and 2 M NaCl.
  • the column flow rate was 1 ml/minute (due to the viscosity of the hCG APLTM material; this flow rate scheme was also used for the urine material) ; after the first 10 minutes, the flow rate was 2 ml/minute.
  • the column was run on a Hewlett Packard 1050 HPLC equipped with a photodiode array detector. Four ml fractions were collected and frozen until further analysis.
  • the protein concentration in each fraction was determined by amino acid analysis.
  • a 50 ⁇ l aliquot of alternate column fractions was processed for anlysis by hydrolysis in vapors of 6N HCl with 0.1% phenol at 110°C for 24 hours in a Waters Associates Pico-Tag Workstation (Waters, Milford, MA) .
  • An internal standard, norleucine was added to all fraction samples before hydrolysis to correct for any losses during hydrolysis or liquid transfer.
  • the hydrolyzed samples were then analyzed on a Beckman Instruments 6300 amino acid analyzer and the data was collected on the PE Nelson Data System (Perkin-Elmer) and transformed using PE Nelson Turbochrome software.
  • Mass spectrometry did indicate that some peptides separated at anomalous positions, showing that they were being carried by other proteins to earlier elution positions in some cases, or interacting with the column matrix and eluting much later than their molecular size would indicate.
  • 3,000 and 6,000 molecular weight materials eluted from the gel filtration column with material of 14,000 molecular weight while 11,000 molecular weight material eluted with material of approximately 1,000-2,000 molecular weight, hCG and hCG- related molecules eluted at their expected positions.
  • the fractions were then tested for anti-HIV, anti-KS and pro-hematopoietic activities in vitro.
  • the HIV-l HID viral strain was used to infect PBMCs and PM-1 cells (derived from the HUT-78 T-cell lymphoma cell line) at 10 3 TCID 50 /ml.
  • the infected cells were incubated for three days in 100 IU/ml of the hCG APLTM or ⁇ -hCG C-Sigma preparations; 50-100 ⁇ l per ml of the hCG APLTM or early pregnancy urine fractions; 50 ⁇ g/ml / S-core protein or ⁇ -hCG preparation; 200 IU/ml of the highly purified CR127 hCG preparation; or 100 ⁇ l/ml of the circularized j ⁇ -hCG peptide 44-57 (with cysteine substituted at position 44; SEQ ID NO:26). Viral levels were determined by measurement of p24 antigen.
  • the KS Y-l cells were obtained from mononuclear cells isolated from pleural effusion of an AIDS patient with KS in the lungs. After the depletion of T lymphocytes, monocytes/macrophages and fibroblasts using monoclonal antibodies against CD2, CD3, CD4, CD8, CD10 and CD14 membrane antigens and baby rabbit complement, the cells were cultured in the absence of exogenous growth factors to select for transformed cells. Immunological characterization of the KS Y-l cells showed that CD34, CD31 and endoglin were expressed.
  • Clonogenic assays were performed by seeding the KS Y-l or KS-SLK cells in methylcellulose (0.8%, v/v), incubating the cells for 10 days in the presence or absence of the test substance and then counting the number of well- formed colonies of triplicate wells formed after seeding with 5 X 10 4 cells.
  • the cells were incubated in 200 IU/ml of commercial hCG preparations; 50 ⁇ l/ml of certain fractions from the hCG preparation of early pregnancy urine fractionation; or 100 ⁇ g/ml ⁇ - and ⁇ -hCG chains, ⁇ -hCG core protein, ⁇ -hCG peptides or LH (leuteinizing hormone) .
  • mice were administered 300 IU hCG APLTM by osmotic pump or 200 IU hCG APLTM by slow release; 200 ⁇ l of certain fractions of hCG commercial preparation or of early pregnancy urine; 200 ⁇ g of the cyclized / S-hCG peptide of amino acids 44-57 (with cysteine substituted at position 44; SEQ ID NO:26) or the fused peptide of amino acids 45-57:: 109-119 (SEQ ID NO: 30); or 100 ⁇ g ⁇ -hCG core peptide or the ⁇ -hCG sub unit per day, and the pups were dosed through the mother's milk.
  • fractions of both the APLTM hCG preparation and the human early pregnancy urine were assayed for inhibition of HIV-l HID replication in PBMCs and PM-1 cells as described above.
  • Many of the APLTM hCG preparation fractions exhibited significant inhibition of HIV-l HID replication ( Figure 6C) .
  • fractions containing material of approximately 70 kD to approximately 2-3 kD exhibited HIV-l inhibitory activity.
  • fractions effecting the highest percent inhibition of HIV-l replication were fractions 62, 63, 65, and 73, with the three main peaks of activity eluting with apparent molecular weights of approximately 40 kD, approximately 15 kD, and approximately 2-3 kD, as determined by comparison with the elution of native hCG (77 kD) and ⁇ - core protein (10 kD) .
  • the fractions of human early pregnancy urine were also assayed for ability to inhibit HIV-l HID replication in the PBMCs and the PM-1 cells. Again, several fractions had at least some HIV-l replication-inhibitory activity. Fractions 64 and 67 caused more than twice the inhibition of HIV-l HID replication than any of the other fractions ( Figure 6F) . There were approximately two peaks of activity eluting from the gel filtration column with apparent molecular weights of approximately 15 kD and 3 kD, as determined by comparison with the elution of native hCG (77 D) and / ⁇ -core protein (10 kD) identified by immunoassay.
  • phenol had no effect on HIV-l replication, demonstrating that the anti-HIV activity of the APLTM hCG is not due to the presence of phenol in the APLTM hCG preparation, and purified ⁇ -hCG core protein (the peptide of amino acids to 6-40 of ⁇ -hCG linked via a disulfide bond to the peptide of amino acids 55-92 of ⁇ -hCG as depicted in Figure 4 (SEQ ID NO:2)) was also found not to inhibit HIV-l replication (data not shown) .
  • FIG. 6B depicts the results of assays of the APLTM hCG fractions for inhibition of KS Y-l cell growth.
  • a fraction containing material about the same size as the 0-hCG core protein exhibited the highest level of inhibition; however, purified /3-hCG core was found not to inhibit KS cell growth (data not shown) .
  • Figure IE presents results of administration of 200 ⁇ l of fraction 61 of the APLTM hCG fractionation (bar 4) and fraction 65 of the early pregnancy urine fractionation (bar 5) .
  • These two fractions both of which are within the peak of anti-HIV and anti-KS activity that contains material with an apparent molecular weight of approximately 15 kD, significantly suppressed HIV-I gene expression in the HIV-I transgenic mice (as measured in the skin and the kidney) in comparison to PBS alone (bar 1) , ⁇ - hCG core peptide (bar 2) and ⁇ -hCG subunit (bar 3).
  • the above-described experiments demonstrate that the factor(s) responsible for the anti-HIV and anti-KS activities can be further isolated from the hCG preparations by gel filtration on a SUPERDEXTM 200 gel filtration column.
  • the factor(s) were fractionated from both the commercial APLTM hCG preparation and urine from women in early pregnancy (first trimester) .
  • the fractions of highest anti-HIV and anti-KS activity contained material eluting from the gel filtration column with an apparent molecular weights of approximately 40 kD, 15 kD and 2-3 kD.
  • certain active fractions contained material of approximately the size of the ⁇ -hCG core protein (-10 kD) , purified / S-hCG core protein was found to have neither anti-HIV nor anti-KS activity.
  • GGT CCC AAG GAC CAC CCC TTG ACC TGT GAT GAC CCC CGC TTC CAG GAC 436 Gly Pro Lys Asp His Pro Leu Thr Cys Asp Asp Pro Arg Phe Gin Asp 105 110 115 5 TCC TCT TCC TCA AAG GCC CCT CCC CCC AGC CTT CCA AGC CCA TCC CGA 484 Ser Ser Ser Lys Ala Pro Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg 120 . 125 130
  • Leu Pro Gly Pro Ser Asp Thr Pro lie Leu Pro Gin 135 140 ⁇ 145
  • MOLECULE TYPE protein

Abstract

La présente invention concerne des méthodes thérapeutiques ou préventives du syndrome d'amaigrissement par administration de gonadotrophine chorionique humaine, de β-gonadotrophine chorionique, d'une peptide contenant une séquence d'une portion de β-gonadotrophine chorionique humaine ou une fraction d'une source de gonadotrophine chorionique humaine endogène ou de β-gonadotrophine chorionique. Dans un mode de réalisation préféré, l'invention concerne l'utilisation de peptides de β-gonadotrophine chorionique humaine pour le traitement et la prévention du syndrome d'amaigrissement. En outre, l'invention concerne des techniques pour la mise en oeuvre de préparations particulières de gonadotrophine chorionique humaine dans le traitement du syndrome d'amaigrissement. Enfin, l'invention propose des compositions pharmaceutiques et des méthodes d'administration desdites compositions.
PCT/US1997/011448 1996-06-24 1997-06-24 Methodes de traitement du syndrome d'amaigrissement par administration de derives de la gonadotrophine chorionique humaine WO1997049721A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
AU35877/97A AU3587797A (en) 1996-06-24 1997-06-24 Methods of treatment of wasting syndrome based on administration of derivatives of human chorionic gonadotropin
US09/220,415 US6583109B1 (en) 1997-06-24 1998-12-24 Therapeutic polypeptides from β-hCG and derivatives
US09/676,739 US6620416B1 (en) 1997-06-24 2000-09-29 Method for treating HIV
US09/675,776 US6596688B1 (en) 1997-06-24 2000-09-29 Method for promoting hematopoiesis
US09/675,362 US6699834B1 (en) 1997-06-24 2000-09-29 Method for treating cancer
US09/677,152 US6805882B1 (en) 1997-06-24 2000-10-02 Therapeutic fractions of sources of HCG

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US66967596A 1996-06-24 1996-06-24
US08/669,675 1996-06-24
US70993396A 1996-09-09 1996-09-09
US08/709,933 1996-09-09

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1138692A1 (fr) * 2000-03-29 2001-10-04 Erasmus Universiteit Rotterdam Fragments de gonadotrophine chorionique humaine comme immunorégulateurs
FR2817752A1 (fr) * 2000-12-07 2002-06-14 Georges Dreux Medicament a base de gonadotropine chorionique extraite d'urine de femme enceinte, et de serum de jument gravide
US6844315B2 (en) 1998-05-20 2005-01-18 Erasmus Universiteit Rotterdam Immunoregulator
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USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
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US7402322B2 (en) 1998-05-20 2008-07-22 Biotempt B.V. Methods of treatment for septic shock with urine extract
US6921751B1 (en) 1998-05-20 2005-07-26 Erasmus Universiteit Rotterdam Immunoregulator
US7365155B2 (en) 2000-03-29 2008-04-29 Biotempt B.V. Immunoregulator
EP1887016A1 (fr) * 2000-03-29 2008-02-13 Biotempt B.V. Fragments de chorione gonadotropine humaine comme immunorégulateur
USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
SG127739A1 (en) * 2000-03-29 2006-12-29 Univ Erasmus Use of immuno-regulators
EP1138692A1 (fr) * 2000-03-29 2001-10-04 Erasmus Universiteit Rotterdam Fragments de gonadotrophine chorionique humaine comme immunorégulateurs
USRE43140E1 (en) 2000-03-29 2012-01-24 Biotempt B.V. Immunoregulator
JP2004503468A (ja) * 2000-03-29 2004-02-05 エラスムス　ユニフェルシテイト　ロッテルダム 免疫調節物
USRE43309E1 (en) 2000-03-29 2012-04-10 Biotempt B.V. Immunoregulatory compositions
WO2001072831A3 (fr) * 2000-03-29 2002-04-25 Univ Erasmus Immunoregulateur
US7576174B2 (en) 2000-03-29 2009-08-18 Biotempt B.V. Compositions capable of reducing elevated blood urea concentration
US7202348B2 (en) * 2000-04-20 2007-04-10 The University Of Arkansas For Medical Sciences Monoclonal antibody antagonists for treating medical problems associated with d-amphetamine-like drugs
FR2817752A1 (fr) * 2000-12-07 2002-06-14 Georges Dreux Medicament a base de gonadotropine chorionique extraite d'urine de femme enceinte, et de serum de jument gravide
US7358330B2 (en) 2001-03-29 2008-04-15 Biotempt B.V. Immunoregulatory compositions
US7175679B2 (en) 2001-03-29 2007-02-13 Biotempt B.V. Oligopeptide treatment of NF-κB mediated inflammation
AU2002328028B2 (en) * 2001-10-04 2008-09-25 Biotempt B.V. Gene regulatory peptides
US7524820B1 (en) 2001-10-04 2009-04-28 Biotempt B.V. Compounds of therapeutic value in the treatment of multiple sclerosis and other diseases wherein foamy cells are involved in the disease etiology
EP2060585A2 (fr) 2001-10-04 2009-05-20 Biotempt B.V. Peptides régulant des gènes
EP2036924A2 (fr) 2001-10-04 2009-03-18 Biotempt B.V. Peptides régulant des gènes
EP2060585A3 (fr) * 2001-10-04 2010-01-06 Biotempt B.V. Peptides régulant des gènes
US7501391B2 (en) 2001-12-21 2009-03-10 Biotempt B.V. Treatment of transplant survival
US7517529B2 (en) 2003-04-08 2009-04-14 Biotempt B.V. Treatment of type I diabetes
WO2007004869A2 (fr) 2005-07-05 2007-01-11 Biotempt B.V. Traitement de tumeurs
WO2009043450A3 (fr) * 2007-09-11 2009-07-30 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009043451A3 (fr) * 2007-09-11 2009-07-30 Mondobiotech Lab Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009043451A2 (fr) * 2007-09-11 2009-04-09 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
EP2109054A1 (fr) 2008-04-09 2009-10-14 Biotempt B.V. Procédés pour identifier biologiquement des peptides actifs et prédire leur fonction
EP2490021A1 (fr) 2011-02-18 2012-08-22 Biotempt B.V. Modulateurs de signalisation PRR et GPCR
WO2012112048A1 (fr) 2011-02-18 2012-08-23 Biotempt B.V. Modulateurs de la signalisation par prr et par gpcr

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